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Dr. AC. Tangavelou [Biodiversity, Plant Taxonomy] Bio-Science Research Foundation, India.

Vaclav Vetvicka [Immunomodulators and Breast Cancer] University of Louisville, Kentucky.

Nasroallah Moradi Kor [Animal Science] Razi University of Agricultural Sciences and Natural Resources, Iran

José F. González-Maya [Conservation Biology] Laboratorio de ecología y conservación de fauna Silvestre, Instituto de Ecología, UNAM, México.

T. Badal Singh [plant tissue culture] Panjab University, India

Dr. Kalyan Chakraborti [Agriculture, Pomology, horticulture] AICRP on Sub-Tropical Fruits, Bidhan Chandra Krishi Viswavidyalaya, Kalyani, Nadia, West Bengal, India. Dr. Monanjali Bandyopadhyay [Farmlore, Traditional and indigenous practices, Ethno botany] V. C., Vidyasagar University, Midnapore. M.Sugumaran [Phytochemistry] Adhiparasakthi College of Pharmacy, Melmaruvathur, Kancheepuram District. Prashanth N S [Public health, Medicine] Institute of Public Health, Bangalore. Tariq Aftab Department of Botany, Aligarh Muslim University, Aligarh, India. Manzoor Ahmad Shah Department of Botany, University of Kashmir, Srinagar, India. Syampungani Stephen School of Natural Resources, Copperbelt University, Kitwe, Zambia. Iheanyi Omezuruike OKONKO Department of Biochemistry & Microbiology, Lead City University, Ibadan, Nigeria. Sharangouda Patil Toxicology Laboratory, Bioenergetics & Environmental Sciences Division, National Institue of Animal Nutrition and Physiology (NIANP, ICAR), Adugodi, Bangalore. Jayapal Nandyal, Kurnool, Andrapradesh, India. T.S. Pathan [Aquatic toxicology and Fish biology] Department of Zoology, Kalikadevi Senior College, Shirur, India. Aparna Sarkar [Physiology and biochemistry] Amity Institute of Physiotherapy, Amity campus, Noida, INDIA. Dr. Amit Bandyopadhyay [Sports & Exercise Physiology] Department of Physiology, University of Calcutta, Kolkata, INDIA . Maruthi [Plant Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. Veeranna [Biotechnology] Dept of Biotechnology, SDM College (Autonomous), Ujire Dakshina Kannada, India. RAVI [Biotechnology & Bioinformatics] Department of Botany, Government Arts College, Coimbatore, India. Sadanand Mallappa Yamakanamardi [Zoology] Department of Zoology, University of Mysore, Mysore, India. Anoop Das [Ornithologist] Research Department of Zoology, MES Mampad College, Kerala, India.

Dr. Satish Ambadas Bhalerao [Environmental Botany] Wilson College, Mumbai Rafael Gomez Kosky [Plant Biotechnology] Instituto de Biotecnología de las Plantas, Universidad Central de Las Villas Eudriano Costa [Aquatic Bioecology] IOUSP - Instituto Oceanográfico da Universidade de São Paulo, Brasil M. Bubesh Guptha [Wildlife Biologist] Wildlife Management Circle (WLMC), India Rajib Roychowdhury [Plant science] Centre for biotechnology visva-bharati, India. Dr. S.M.Gopinath [Environmental Biotechnology] Acharya Institute of Technology, Bangalore. Dr. U.S. Mahadeva Rao [Bio Chemistry] Universiti Sultan Zainal Abidin, Malaysia. Hérida Regina Nunes Salgado [Pharmacist] Unesp - Universidade Estadual Paulista, Brazil Mandava Venkata Basaveswara Rao [Chemistry] Krishna University, India. Dr. Mostafa Mohamed Rady [Agricultural Sciences] Fayoum University, Egypt. Dr. Hazim Jabbar Shah Ali [Poultry Science] College of Agriculture, University of Baghdad , Iraq. Danial Kahrizi [Plant Biotechnology, Plant Breeding,Genetics] Agronomy and Plant Breeding Dept., Razi University, Iran Dr. Houhun LI [Systematics of Microlepidoptera, Zoogeography, Coevolution, Forest protection] College of Life Sciences, Nankai University, China. María de la Concepción García Aguilar [Biology] Center for Scientific Research and Higher Education of Ensenada, B. C., Mexico Fernando Reboredo [Archaeobotany, Forestry, Ecophysiology] New University of Lisbon, Caparica, Portugal Dr. Pritam Chattopadhyay [Agricultural Biotech, Food Biotech, Plant Biotech] Visva-Bharati (a Central University), India

Table of Contents (Volume 3 - Issue 3) Serial No

Accession No

RA0328

Title of the article An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India.

Page No 852-860

Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. 2

RA0327

Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms.

861-869

Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC 3 RA0167

Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats.

870-875

Akpa GN, Alphonsus C and Usman N. 4

RA0245

Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India.

876-885

Samit Roy Choudhury and Basant Kumar Agarwala. 5

RA0329

RA0332

Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Renju GL and Muraleedhara Kurup G. Identification of animal Pasteurellosis by PCR assay.

886-894

895-899

Venkatesan PS, Deecaraman M and Vijayalakshmi. 7

RA0345

Source of light emission in a luminous mycelium of the fungus Panellus stipticus.

900-905

Puzyr Alexey, Burov Andrey and Bondar Vladimir. 8

RA0274

Local peopleâ&#x20AC;&#x2122;s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India.

906-910

Lakshmi Kodoth and Ramamoorthy D. 9

RA0318

Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria.

911-921

Nwanyanwu CE and Abu GO. 10

RA0317

Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria.

922-931

Nwanyanwu CE, Nweke CO, Orji JC and Opurum CC. 11

RA0337

Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN.

932-939

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India Authors:

ABSTRACT:

Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, North-east India is a part of Indo-Burma hotspot and among the richest bird Joydeb Majumder and zones in India. Tripura lies in the border of Indo-Burma global biodiversity hotspot Basant Kumar Agarwala.

area but is poorly covered by ornithological works. Avifauna of Tripura state is known by 277 species but there is lack of information about their distribution, particularly in and around Agartala city, which is the capital of Tripura state and is a tourist Institution: destination along the border of Bangladesh for its natural landscapes, inland water Ecology & Biodiversity species, and strong presence of green flora. With a view to enhance its value for Laboratories, Department of tourist attraction and naturalists, a study was conducted to record the species of birds Zoology, Tripura University, that occur in and around the City. In the present study 73 bird species were recorded Suryamaninagar-799 022, from Agartala city and its adjacent areas belonging to 41 families and 14 orders. Tripura, India. Corresponding author: Basant Kumar Agarwala.

Email: bagarwala00@gmail.com

Web Address:

http://jresearchbiology.com/ documents/RA0328.pdf.

Keywords: Avifauna, biodiversity hotspot, Agartala, Tripura, north-east India .

Article Citation: Partha Pratim Bhattacharjee, Rahul Lodh, Dipten Laskar, Joydeb Majumder and Basant Kumar Agarwala. An ornithological survey in the vicinity of Agartala city of Tripura state, north-eastern India. Journal of Research in Biology (2013) 3(3): 852-860 Dates: Received: 28 Jan 2013

Accepted: 15 Feb 2013

Published: 10 Apr 2013

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology An International Scientific Research Journal

852-860 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Bhattacharjee et al., 2013 little is known regarding the bird species found in the

INTRODUCTION Avifauna contributes most significantly to the

vicinity of Agartala city, situated by international

diversity of terrestrial vertebrates, which have a special

boundary of Bangladesh.

role in conservation of biodiversity of a particular area

STUDY SITES

(Daniels,

1994).

indicator

Agartala city is situated in the western region of

of environmental changes as they respond in the minute

Tripura state with the latitude of 23°45' North and

change

composition

longitude of 91°45' East and an average elevation of

(Robert et al., 2001). Indian subcontinent harbour nearly

20.36 m above sea level. It is the capital town of Tripura

1300 species of birds, which is more than 13% of total

with a mix of urban and semi urban complex and a rich

bird species of the world (Grimmet et al., 2004), and

green cover. Forests and farms adjoin the town on three

more than 60% of Indian birds are found in north-east

sides, and therefore, it is also called ‘Green City’. The

India (Choudhury, 2010). North-east India is one of the

total city area is 62.02 km2 and is delimited on the west

most significant biodiversity hotspots of the world and

side by international boundary with Bangladesh.

among the richest bird zones in India because of

Climatic condition is of tropical monsoon type with an

convergence of the Indo-Malayan, Indo-Chinese and

average annual rainfall of 220 cm. Average minimum

Indian biogeographical realms. As a result, it is unique in

and maximum temperature recorded in the region are

providing an abundance of habitats that harbour diverse

6.8°C in January and 37.70°C in June, respectively.

Birds are very good

habitat

structure

and

biota with a high degree of endemism (Chatterjee et al.,

Present study was carried out in eleven

2006; Narwade et al., 2011). Tripura (22°56´- 24°32´ N

different sites (viz., College Tilla lake area, Golbazar,

and 91°10´- 92°21´ E, with an area of 10,490 km ) is a

Pratapgarh, Dashamighat, Arundhutinagar, Shanmura,

small state of north-east India bounded by Bangladesh

Bhubanban, Barjala, Jagannath Bari lake area, G B Bazar

on three sides and with Assam and Mizoram on the other

and Nandannagar) (Table 1, Figure 1) covering different

side. It lies in the border of Indo-Burma global

sides of Agartala city and its adjacent areas.

biodiversity hotspot area (Myers et al., 2000) but very

poorly

covered

ornithological

works

METHODOLOGY

(Choudhury, 2010). Although avifaunal checklist for Tripura state listed 277 species (Choudhury, 2010) but

The

study

sites

were

visited

fortnightly

throughout the study period from 2009-2011. Data on

Table 1: Geo-coordinate details of the study sites Sl. No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 853

Sites College Tilla lake area Golbazar Pratapgarh Dashamighat Arundhutinagar Shanmura Bhubanban Barjala Jagannath Bari lake area G B Bazar Nandannagar

Coordinates 23°49'35.45" N; 91°17'42.28" E 23°49'38.30" N ; 91°16'57.15" E 23°49'08.98" N ; 91°17'17.10" E 23°49'46.34" N ; 91°15'51.45" E 23°49'01.44" N ; 91°16'21.68" E 23°50'51.98" N ; 91°16'07.20" E 23°51'56.50" N; 91°15'73.70" E 23°52'05.05" N ; 91°16'32.13" E 23°50'05.43" N; 91°16'53.70" E 23°51'33.74" N ; 91°17'33.97" E 23°51'43.68" N ; 91°17'57.00" E

Altitude (m) 17 16 16 16 31 15 21 23 14 27 28

Journal of Research in Biology (2013) 3(3): 852-860

Bhattacharjee et al., 2013

Figure 1. Showing the study sites in and around Agartala City. present bird species were collected by direct observations

(2002) recorded 259 species of birds, belonging to

with the help of binoculars (VISTA LE 8 X 40). Almost

56 families and 16 orders. Recently Choudhury (2010)

all the species mentioned in the checklist were

recorded 277 species of birds, in the annotated checklist

photographed. For this purpose, digital cameras of Canon

from Tripura, but avifaunal diversity of Agartala city is

Power shot SX 200 IS (12 X Digital zoom), Cannon SRL

not yet available. Out of 14 orders. Passeriformes was

EOS 50D and Panasonic Lumix DMC FZ 40 were used.

found

In lake areas birds were observed from the bank,

Coraciiformes with 3 families and Pelecaniformes and

peripheral areas, and urban areas were surveyed on foot

Piciformes with 2 families each. Dominance of

regularly. Farm and forested areas in the vicinity of the

Passeriformes was also recorded by Choudhury (2010)

city were surveyed to record the assemblages of different

and Majumdar et al., (2002) from the state and from

bird species. Most of visits were made in morning and

Nagpur district of central India (Chinchkhede and Kedar,

afternoon time when birds are most active. Identification

2012). The resident birds such as Pond heron, Cattle

of birds was based on the field guides produced by Ali

egret, Lapwing, Blue rock pigeon, Spotted dove,

and Ripley (1995), Ali (1996 and 2002) and Grimmett et

Parakeets, Asian koel, Kingfisher, Bee eater, Lineated

al., (2003).

barbet, Woodpecker, Bush lark, Bulbul, Shrike, Robin,

dominant

with

families

followed

Status of the birds was classified as C-Common,

Tailorbird, Cinereous tit, Sunbird, Sparrow, Starling,

MC-Most common, NC-Not common, S-Singleton,

Myna, Oriole, Black drongo and Crow etc were found

W- Winter visitor.

regularly throughout the study period. Little Cormorant, Asian Openbill-Stork, Black headed Ibis, Lesser

RESULTS AND DISCUSSION

Whistling Duck, Crested Serpent Eagle, Red Junglefowl,

In the present study 73 bird species were

Red Collared Dove, Yellow-footed Green Pigeon, Brown

recorded from Agartala city and its adjacent areas

Fish Owl, Asian Palm Swift, Indian Roller, Coppersmith

belonging to 41 families and 14 orders (Table 2, Plate 1

Barbet, Orange headed Thrush, Blue Rock Thrush,

and 2). There is no authentic information about the

White-rumped Shama, Scarlet-backed Flower pecker,

avifauna of Tripura except that by Blyth (1845, 1846),

Tricoloured Munia etc were found less common in this

Ali and Ripley (1968-74) and International Waterfowl

study. Common Sandpiper and Black headed Ibis were

and Wetlands Research Bureau on Asian Waterfowl

observed during the winter season only in the paddy

Census, 1989 (Scott and Rose 1989). Majumdar et al.,

fields of peripheral areas of the city. Common Hoopoe

Journal of Research in Biology (2013) 3(3): 852-860

854

Bhattacharjee et al., 2013 Table 2: List of birds in and around Agartala city during 2009-2011 Sl. No.

Common name

Scientific name

Status IUCN

Abundance

Phalacrocorax niger Vieillot, 1817

Cormorants [Phalacrocoracidae] 1.

Little Cormorant Herons & Egrets [Ardeidae]

Indian Pond Heron

Ardeola grayii (Sykes, 1832)

Cattle Egret

Bubulcus ibis (Linnaeus, 1758)

Median Egret

Mesophoyx intermedia (Wagler, 1827)

Storks [Ciconiidae] 5.

Asian Openbill-Stork

Anastomus oscitans Boddaert, 1783

Black headed Ibis

Threskiornis melanocephalus (Latham, 1790)

W, NC

Dendrocygna javanica (Horsfield, 1821)

Ducks [Anatidae] 7.

Lesser Whistling Duck Hawks & Eagles [Accipitridae]

Crested Serpent Eagle

Spilornis cheela Latham, 1790

Black Kite

Milvus migrans (Boddaert, 1783)

Gallus gallus (Linnaeus, 1758)

Amaurornis phoenicurus Pennant, 1769

Vanellus indicus (Boddaert, 1783)

Actitis hypoleucos (Linnaeus, 1758)

W, C

Pheasants [Phasianidae] 10.

Red Junglefowl Rails & Coots [Rallidae]

11.

White-breasted Waterhen Lapwings [Charadriidae]

12.

Red-wattled Lapwing Sandpipers [Scolopacidae]

13.

Common Sandpiper Pigeons & Doves [Columbidae]

14.

Blue Rock Pigeon

Columba livia Gmelin, 1789

15.

Spotted Dove

Streptopelia chinensis (Scopoli, 1768)

16.

Red Collared Dove

Streptopelia tranquebarica (Hermann, 1804)

17.

Orange-breasted Green Pigeon

Treron bicinctus (Jerdon, 1840)

18.

Yellow-footed Green Pigeon

Treron phoenicoptera (Latham, 1790)

Parakeets [Psittacidae] 19.

Rose-ringed Parakeet

Psittacula krameri (Scopoli, 1769)

20.

Red-breasted Parakeet

Psittacula alexandri (Linnaeus, 1758)

Cuckoos & Coucals [Cuculidae] 21.

Asian Koel

Eudynamys scolopaceus (Linnaeus, 1758)

22.

Greater Coucal

Centropus sinensis (Stephens, 1815)

Owls [Strigidae] 23.

Collared Scops Owl

Otus lettia Hodgson, 1836

24.

Spotted Owlet

Athene brama (Temminck, 1821)

Brown Fish Owl

Bubo zeylonensis (Gmelin, 1788) LC NC Journal of Research in Biology (2013) 3(3): 852-860

25. 855

Bhattacharjee et al., 2013 Swifts [Apodidae] 26.

Asian Palm Swift

Cypsiurus balasiensis Gray, 1829

27.

House Swift

Apus affinis (J E Gray, 1830)

Kingfishers [Alcedinidae] 28.

Common Kingfisher

Alcedo atthis (Linnaeus, 1758)

29.

Stork-billed Kingfisher

Halcyon capensis (Linnaeus, 1766)

30.

White-throated Kingfisher

Halcyon smyrnensis (Linnaeus, 1758)

Merops orientalis Latham, 1802

Coracias benghalensis (Linnaeus, 1758)

Upupa epops Linnaeus, 1758

Bee-eaters [Meropidae] 31.

Little Green Bee-eater Rollers [Coraciidae]

32.

Indian Roller Hoopoe [Upupidae]

33.

Common Hoopoe Barbets [Capitonidae]

34.

Lineated Barbet

Megalaima lineata (Vieillot, 1816)

35.

Coppersmith Barbet

Megalaima haemacephala Muller, 1776

Woodpeckers [Picidae] 36.

Rufous Woodpecker

Celeus brachyurus (Vieillot, 1818)

37.

Greater Flameback

Chrysocolaptes lucidus (Scopoli, 1786)

38.

Fulvous-breasted Woodpecker

Dendrocopos macei (Vieillot, 1818)

Mirafra cantillans Blyth, 1844

Larks [Alaudidae] 39.

Singing bush lark

Pipits & Wagtails [Motacillidae] 40.

Paddy field Pipit

Anthus rufulus Vieillot, 1818

41.

White Wagtail

Motacilla alba Linnaeus, 1758

W,C

Bulbuls [Pycnonotidae] 42.

Red-whiskered Bulbul

Pycnonotus jocosus (Linnaeus, 1758)

43.

Red-vented Bulbul

Pycnonotus cafer (Linnaeus, 1766)

Aegithina tiphia (Linnaeus, 1758)

Loras [Irenidae] 44.

Common Lora Shrikes [Laniidae]

45.

Brown Shrike

Lanius cristatus Linnaeus, 1758

W, MC

46.

Grey-backed Shrike

Lanius tephronotus (Vigors, 1831)

W, C

Zoothera citrina (Latham, 1790)

Thrushes [Turdidae] 47.

Orange headed Thrush Flycatchers [Muscicapidae]

48.

Blue Rock Thrush

Monticola solitarius (Linnaeus, 1758)

49.

White-rumped Shama

Copsychus malabaricus (Scopoli, 1786)

50.

Oriental Magpie Robin

Copsychus saularis (Linnaeus, 1758)

Journal of Research in Biology (2013) 3(3): 852-860

856

Bhattacharjee et al., 2013 Babblers [Timaliidae] 51.

Rufous-necked Laughingthrush

Garrulax ruficollis (Jardine & Selby, 1838)

Orthotomus sutorius (Pennant, 1769)

Culicicapa ceylonensis (Swainson, 1820)

W, C

Parus cinereus Vieillot, 1818

Dicaeum cruentatum (Linnaeus, 1758)

Warblers [Sylviidae] 52.

Common Tailorbird Flycatchers [Stenostiridae]

53.

Grey-headed Canaryflycatcher Tits [Paridae]

54.

Cinereous Tit Flowerpeckers [Dicaeidae]

55.

Scarlet-backed Flowerpecker Sunbirds [Nectariniidae]

56.

Ruby-cheeked Sunbird

Anthreptes singalensis (Gmelin, 1788)

57.

Purple-rumped Sunbird

Nectarinia zeylonica (Linnaeus, 1766)

58.

Purple Sunbird

Cinnyris asiaticus Latham, 1790

Zosterops palpebrosus (Temminck, 1824)

White-eyes [Zosteropidae] 59.

Oriental White-eye Munias [Estrildidae]

60.

Scaly-breasted Munia

Lonchura punctulata (Linnaeus, 1758)

61.

Tricoloured Munia

Lonchura malacca (Linnaeus, 1766)

Passer domesticus (Linnaeus, 1758)

Ploceus philippinus (Linnaeus, 1766)

Sparrows [Passerinae] 62.

House Sparrow Weavers [Ploceidae]

63.

Baya Weaver Starlings & Mynas [Sturnidae]

64.

Chestnut-tailed Starling

Sturnus malabaricus (Gmelin, 1789)

65.

Asian Pied Starling

Gracupica contra (Linnaeus, 1758)

66.

Common Myna

Acridotheres tristis (Linnaeus, 1766)

67.

Jungle Myna

Acridotheres fuscus (Wagler, 1827)

Oriolus xanthornus (Linnaeus, 1758)

Orioles [Oriolidae] 68.

Black-hooded Oriole Drongos [Dicruridae]

69.

Black Drongo

Dicrurus macrocercus (Vieillot, 1817)

70.

Greater racket-tailed Drongo

Dicrurus paradiseus Linnaeus, 1766

Crows & Treepie [Corvidae] 71.

Rufous Treepie

Dendrocitta vagabunda (Latham, 1790)

72.

House Crow

Corvus splendens Vieillot, 1817

73.

Jungle Crow

Corvus macrorhynchos Wagler, 1827

Abbreviations: Status: LC = least concern; NT = near threatened; C = common; MC = most common; NC = not common; S = singleton; W = winter visitor. 857

Journal of Research in Biology (2013) 3(3): 852-860

Bhattacharjee et al., 2013

Plate 1. A-Lineated barbet, B-Cattle egret, C-Red-wattled Lapwing, D-Common Hoope, E-Little cormorant, F-Stripe-breasted woodpecker, G-Rufous woodpecker, H-White throated kingfisher, I-Yellow footed green pegion, J-Asian open bill stork, K-Chestnut-tailed starling, L-Collared scops owl.

Plate 2: M-Asian Koel, N-Crested serpent eagle, O-Common Tailorbird, P-Cinereous Tit, Q-Emerald dove, R-Little green bee-eater, S-Grey-baked shrike, T-Indian pond heron, U-Red collared dove, V-White-rumped shama, W-Singing bush lark, X-Black headed ibis. Journal of Research in Biology (2013) 3(3): 852-860

858

Bhattacharjee et al., 2013 and Collared Scops Owl were sighted only once in the

Ali S and Ripley SD. 1995. A pictorial guide to the

two years study. Brown Shrike, Grey-backed Shrike,

birds of Indian Subcontinent. Bombay Natural History

Grey-headed Canary-flycatcher were observed in the

Society, Mumbai.

winter season only (Table 2), which corroborates with the findings of Choudhury (2010) and Majumdar et al., (2002).

BirdLife International 2009: IUCN 2011. IUCN Red List

Threatened

Species.

Version

2011.2.

<www.iucnredlist.org>. Downloaded on 18 June 2012.

CONCLUSION The present avifaunal survey of Agartala city and its adjacent areas revealed 73 bird species which is very important as it is the first ornithological record of the city and will give a baseline data for future study. Rich bird diversity is influenced by the topographical location of the city and adjacent areas of Bangladesh.

Blyth E. 1845. Notices and descriptions of various new or little known species of birds, Journal of the Asiatic Society of Bengal, 14: 546-602. Blyth E. 1846. Notices and descriptions of various new or little known species of birds, Journal of the Asiatic Society of Bengal, 15: 1-54.

Expansion of the city by construction activities,

Chatterjee S, Saikia A, Dutta P, Ghosh D, Pangging

reducing forest and farm areas with population pressure,

G and Goswami AK. 2006. Biodiversity significance of

filling of pond and lake areas, dumping of wastes and

north east India: WWF-India. New Delhi. pp-71.

garbage in the low lands, use of chemical pesticides in agricultural fields and hunting of birds are the major threats to the avifaunal diversity here which needs proper conservation management practices.

Chinchkhede KH and Kedar GT. 2012. Avifaunal diversity of Koradi lake in Nagpur district of central India. Journal of Research in Biology, 2: 70-76. Choudhury A. 2010. Recent ornithological records from

ACKNOWLEDGEMENT Authors are thankful to Mr. Dipankar Kishore Sinha for his constant services, tireless field assistance and in capturing photographs during the study. REFERENCES

Tripura, north-eastern India, with an annotated checklist. Indian BIRDS 6(3): 66-74. Daniels

RJR.

1994.

A landscape

approach

conservation of birds. Journal of Bioscience 19(4): 503509.

Ali S. 1996. The book of Indian birds, Twelfth Revised

Grimmet R, Inskip T and Islam MZ. 2004. Birds of

Edition, Bombay Natural History Society Oxford

Northern India. Christopher Helm A and C Bleak

University Press, Mumbai.

Publishers Ltd. London.

Ali S. 2002. The book of Indian birds, Thirteenth

Grimmett R, Inskipp C and Inskipp T. 2003. Pocket

Revised Edition, Bombay Natural History Society

Guide to the Birds of the Indian Subcontinent, Oxford

Oxford University Press, Mumbai.

University Press, New Delhi.

Ali S and Ripley SD. 1968-74. Pakistan 10 vols.,

Majumdar N, Ray CS and Datta BK. 2002. Aves. In:

Oxford University Press, Bombay.

Fauna of Tripura (Part 1) (Vertebrates). State Fauna Series 7, pp. 47-158 (Ed.: Director 2002). Kolkata: Zoological Survey of India.

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Bhattacharjee et al., 2013 Myers N, Russell A, Mittermelert C, Mittermelert G, Gustavo AB and Fonseca KJ. 2000. Biodiversity hotspots for conservation priorities. Nature 403: 853858. Narwade S, Kalra M, Jagdish R, Varier D, Satpute S, Khan N, Talukdar G, Mathur VB, Vasudevan K, Pundir DS, Chavan V and Sood R. 2011. Literature based species occurrence data of birds of North-East India. In: Smith V, Penev L (Eds) e-Infrastructures for data publishing in biodiversity science. ZooKeys 150: 407-417. Robert A Fimbel, John AG and Robinson G. 2001. The Cutting Edge: Conserving Wildlife in Logged Tropical Forest. Scott DA and Rose PM. 1989. Asian Waterfowl Census. The International Waterfowl and Wetlands Research Bureau, 43-46.

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Journal of Research in Biology (2013) 3(3): 852-860

860

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Authors: Ujowundu CO1, Nwaogu LA1, Igwe KO1, Ujowundu FN1, Belonwu DC2.

Institution: 1. Department of Biochemistry, Federal University Technology Owerri, Nigeria. 2. Departmentof Biochemistry, University of Portharcourt, Nigeria.

ABSTRACT: The phytochemical, proximate, mineral and vitamin contents of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms were investigated. Plant samples were harvested from Polluted Environment (PE) at Izombe in Oguta Local Government Area- an oil drilling and gas flaring environment. The results obtained were compared to identical vegetables harvested from Eziobodo in Owerri West Local Government Area, designated as Unpolluted Environment (UPE). Our result showed that A. esculentus and P. mildbraedii have excellent nutritional value, which can confer biochemical and physiological advantage to humans. The quantitative proximate composition showed that the carbohydrate and ash contents of samples harvested from PE differed significantly (P<0.05) from samples obtained from unpolluted environment. The protein, crude fibre, moisture and total fat contents of samples from PE differed non significantly (P<0.05) when compared with samples obtained from UPE. The phytochemical contents of A. esculentus and P. mildbraedii were significantly higher in samples from UPE than in samples from PE. The mineral and vitamin contents were also determined. The concentration of nutritionally important macro and micro elements indicates that the two vegetable samples studied are rich sources of minerals and, therefore, can be used to improve the diet of both humans and livestock. This study also showed that environmental pollutants emanating from the activities of oil and gas industries can impact negatively on some important chemical and nutritive compositions of edible vegetables.

Corresponding author: Ujowundu CO.

Keywords: Oil and gas, Pollution, Phytochemicals, Vitamins, Oha, Okra.

Email: ujowundu@yahoo.com

Article Citation: Ujowundu CO, Nwaogu LA, Igwe KO, Ujowundu FN, Belonwu DC. Evaluation of the Impact of Oil and Gas Pollutants on the Chemical Composition of Abelmoschus esculentus Moench and Pterocarpus mildbraedii Harms. Journal of Research in Biology (2013) 3(3): 861-869

Web Address:

Dates: Received: 16 Jan 2013

http://jresearchbiology.com/ documents/RA0327.pdf.

Accepted: 09 Feb 2013

Published: 11 Apr 2013

Journal of Research in Biology An International Scientific Research Journal

861-869 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Ujowundu et al., 2013 pollution.

INTRODUCTION Nigeria, a major producer of crude oil, benefits

The most glaring sight in gas production flow

as well as suffers from the positive and negative effects

station

the

ten-meter-high

flame

that

burns

of crude oil drilling and gas flaring (Adeniye et al.,

continuously from vertical pipes at many facilities owned

1983). Gas flaring is the unscientific burning of excess

by oil companies. These vertical pipes are fed with gas

hydrocarbons gathered in an oil/gas production flow

given off during production. Gas flaring, for about four

station. Gas flaring is a major source of pollution in

decades has contributed to the high pollution level, and

Nigeria's Niger Delta because it is the preferred means of

the ecosystem of Izombe may have been impacted

disposing waste gas associated with oil exploitation in

negatively. A good example of such negative effect is

that region by many multinational oil companies that

high soil acidity that creates chemical and biological

operate its fields. Gas flaring releases carbon monoxide,

conditions which may be harmful to the soil and plants

oxides of sulphur and nitrogen, hydrocarbons, soot and

(Nwaugo et al., 2006). One of these conditions is the

heavy metals (Coker, 2007; Ikoro, 2003). These

reduction in the capacity of plants to absorb cations

pollutants actually interfere with growth and survival of

(Wild et al., 2005). The higher acidic nature of soil is

living organisms in such environment especially in

attributable to high concentrations of sulphur dioxide and

plants. It pollutes seedlings and fruits of plants which in

particulates from gases flared into the atmosphere which

turn have a devastating effect on humans who consume

is washed back to the soil as acid rain.

them. Such effects include respiratory or cardiovascular

This study has two objectives, first to contribute

diseases. This study used Abelmoschus esculentus

to the knowledge of nutritional and antinutritional

Moench and leaves of Pterocarpus mildbraedii Harms,

composition of A. esculentus Moench and P. mildbraedii

two commonly consumed indigenous vegetables to

Harms. Secondly, to evaluate the effect of environmental

evaluate the biochemical effects of these environmental

pollution resulting from crude oil exploration and

pollutants.

exploitation and other industrial processes within the

Abelmoschus esculentus Moench

(local name,

area of study. The arable nature and vast land mass of

Okra) and leaves of Pterocarpus mildbraedii Harms

Izombe, confers it the status of food basket of Imo State,

(local name, Oha) are vegetables commonly consumed

Nigeria.

as a source of food and medication for their high content of nutrients and phytochemicals and mainly used for

MATERIALS AND METHODS

soup preparation. Consumption of vegetables provide

Collection and preparation of plant samples

taste, palatability, increases appetite and provides fiber

Samples

esculentus

Moench

and

for digestion and to prevent constipation. They play key

P. mildbraedii Harms were obtained at Izombe, in Oguta

roles in neutralizing acids produced during digestion of

Local Government Area and at Eziobodo, in Owerri

proteins and fatty foods and also provide valuable

West Local Government Area both in Imo state, Nigeria

roughages which helps in movement of food in the

and identified by a plant taxonomist in the Federal

intestine. Some of these vegetables possess the ability to

University of Technology, Owerri (FUTO). Izombe is a

reduce or reverse so many disease conditions and

rainforest

disorders such as those which require a reduced intake of

industries specialized in crude oil exploration and

glucose (diabetes) (Mcdowell, 2001: Ogbonnia et al.,

exploitation. Flaring of gases constitutes the major

2008). These vegetables can be deeply affected by

method of waste gas disposal at these oil fields. Situate

862

ecosystem,

which

hosts

multinational

Journal of Research in Biology (2013) 3(3): 861-869

Ujowundu et al., 2013 to these oil fields are communities of indigenous

1998). Saponin, alkaloid and flavonoid were done by

inhabitants whose occupation are subsistence and semi-

method

commercial farming. Eziobodo is also within the

spectrophotometric method as described by Griffiths and

rainforest region of Nigeria, occupied by indigenous and

Thomas (1981) was used for determining phytate

FUTO students population. It has no known industrial

content. Determinations were done in triplicates and

activities,

results were expressed as averages of percent values on

except

few

automobiles

that

convey

described

Harborne,

inhabitants in and out of the village. Samples were sorted

dry weight basis.

by removing extraneous materials, spoilt and unhealthy

Evaluation of vitamins content

(1973).

The

ones. After washing, okro samples were carefully sliced.

Retinol, ascorbic acid and α-tocopherol contents

The samples were oven dried, macerated, sieved and

in the samples were determined using the method of

properly stored.

Association of Vitamin Chemist as described by Kirk

Evaluation of proximate composition

and Sawyer (1998).

The method described by James (1995) and

Evaluation of mineral content

Onwuka (2005) were used to determine crude fiber. Fat

Some mineral contents were determined by

content was determined by the method of Min and Boft

atomic absorption spectrophotometer (James, 1995). The

(2003). Moisture content was determined by the method

dry samples were burnt to ashes to remove all organic

of AOAC (1990). The sample’s total protein content was

materials leaving inorganic ash. The resulting ash was

determined by microkjeldhal method described by

dissolved in 10 ml of 2 M HCl solution and diluted to

James (1995). Protein concentration was obtained by

100 ml with distilled water in a volumetric flask. The

determining total nitrogen and multiplied by the

mixture was filtered and the resulting extract was used

factor- 6.25. Carbohydrate contents was calculated using

for the specific evaluation of copper, zinc and iron.

the arithmetical difference method described by Pearson

Sodium, potassium, calcium and magnesium were

(1976) and James (1995).

determined with the aid of Jaway digital flame

Evaluation of phytochemical content

photometer. Phosphorus was determined as phosphate by

Tannin content of samples were determined by Folin-Denis colorimetric method (Kirk and Sawyer,

the vanadomolybdate colorimetric method (Pearson, 1976)

Table 1: Proximate composition (%) of Abelmoschus esculentus Moench Environment of sampling

Carbo- hydrates

Crude Protein

Polluted

32.47±2.22a

14.28±0.30a

13.78±0.40a

Unpolluted

37.94±1.78b

12.67±0.07a

7.99±0.16b

Ash

Crude Fibre

Moisture

Total Fat

22.13±1.40a

10.52±0.89a

6.82±0.90a

21.83±0.23a

12.71±1.71a

6.85±0.03a

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 2: Proximate Composition (%) of Pterocarpus mildbraedii Harms Environment of sampling

Carbo- hydrates

Crude Protein

Polluted

29.38±1.24c

8.06±1.43c

19.40±0.57c

17.65±0.79c

22.37±1.18c

3.14±0.33c

Unpolluted

34.82±0.30d

9.67±0.07c

12.05±0.18d

16.58±0.21c

23.67±0.29c

3.21±0.06c

Ash

Crude Fibre

Moisture

Total Fat

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Journal of Research in Biology (2013) 3(3): 861-869

863

Ujowundu et al., 2013 PE were higher than that of A. esculentus from PE. The

Statistical analysis Da t a

obt a i n ed

wer e

expr ess ed

highest concentrations of phytochemicals were observed

means± standard deviation. Statistical Package for the

in flavonoids (0.54±0.02%) and tannin (1.83±0.01%)

Social Sciences (SPSS) was used for the Analysis of

from A. esculentus and P. mildbraedii respectively from

Variance (ANOVA) for the test of significant difference

PE. However, alkaloids and tannins contents were

between means (P<0.05).

highest in A. esculentus and P. mildbraedii respectively from UPE. Vitamin contents were presented in tables 5 and

RESULTS The proximate contents of A. esculentus and

6. Vitamin A concentration in A. esculentus and

P. mildbraedii are presented in Tables 1 and 2

respectively.

375.48±0.18

Results

obtained

from

unpolluted

mildbraedii

were

627.59±0.47

mg/100g respectively,

mg/100g

and

indicating the

environment (UPE) showed that A. esculentus have

highest vitamin content in the samples. Also, vitamin C

higher content of carbohydrate, protein, fibre and total

and B5 contents in samples from UPE were significantly

fat compared to P. mildbraedii. However, higher ash

higher (P<0.05) when compared to samples from PE.

and moisture content were observed in P. mildbraedii

Similarly P. mildbraedii have significantly higher value

when

UPE.

of vitamin B5 (189.33±2.31 mg/100g) compared to

Carbohydrate content in samples obtained from polluted

A. esculentus from UPE. In A. esculentus (table 5), all

environment (PE) were significantly (P<0.05) lower than

the vitamins determined were significantly (P<0.05)

the UPE. But the ash contents were significantly higher

lower except vitamin B5, vitamin B9 and vitamin E in

in samples from PE. The protein, crude fiber, moisture

samples from PE. Also, samples of P. mildbraedii from

and total fat contents of the samples showed no

PE when compared with samples from UPE showed

significant difference.

significantly lower content in all the vitamins (table 6)

compared

esculentus

from

Results of phytochemical analysis are presented

except in vitamin B2, vitamin B3 and vitamin E.

in tables 3 and 4. The concentrations of phytochemicals

The mineral contents are shown in tables 7 and 8.

were significantly higher (P<0.05) in samples from PE.

The concentrations of copper, iron, zinc and lead in

Also, the phytochemical contents of P. mildbraedii from

A. esculentus from PE were significantly higher (P<0.05)

Table 3: Phytochemical Composition (%) of Abelmoschus esculentus Moench Environment of sampling

Saponins

Tannins

Phytates

Alkaloids

Phenols

Flavonoids

Polluted

0.33±0.03a

0.37±0.01a

0.23±0.01a

0.43±0.01a

0.26±0.04a

0.54±0.02a

Unpolluted

0.12±0.05b

0.20±0.01b

0.09±0.01b

0.27±0.09b

0.11±0.00b

0.24±0.02b

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Table 4: Phytochemical Composition (%) of Pterocarpus mildbraedii Harms Vegetables Environment of sampling

Saponins

Tannins

Phytates

Alkaloids

Phenols

Flavonoids

Polluted

0.41±0.02c

1.83±0.00c

0.37±0.01c

0.57±0.01c

0.44±0.02c

0.63±0.01c

Unpolluted

0.23±0.08d

1.56±0.14d

0.15±0.01d

0.28±0.12d

0.35±0.00d

0.45±0.08d

Values (mean ± SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different. 864

Journal of Research in Biology (2013) 3(3): 861-869

0.02±0.02a 0.08±0.01b

592.78±19.69a

627.59±0.47b

Polluted

Unpolluted

0.08±0.01b

0.05±0.01a

Vitamin B2

1.12±0.01b

0.83±0.10a

Vitamin B3

23.33±2.31a

21.00±2.52a

Vitamin B5

0.81±0.10b

0.58±0.02a

Vitamin B6

0.73±0.08a

0.67±0.11a

Vitamin B9

0.07±0.02c 0.12±0.00d

302.57±7.01c

375.48±0.18d

Polluted

Unpolluted

0.06±0.01c

0.04±0.01c

Vitamin B2

0.57±0.02c

0.65±0.09c

Vitamin B3

189.33±2.31d

175.22±6.97c

Vitamin B5

0.59±0.05d

0.25±0.08c

Vitamin B6

0.89±0.02d

0.53±0.17c

Vitamin B9

Journal of Research in Biology (2013) 3(3): 861-869 70.23±2.02a 82.83±2.32b

22.17±1.39a

39.60±1.39b

Polluted

Unpolluted

68.28±0.46a

57.35±0.59a

Phosphorus

7.47±0.23a

6.34±0.46a

Sodium

130.40±6.55b

107.63±4.45a

Potassium

0.06±0.01b

0.42±0.15a

Copper

0.74±0.25b

0.95±0.26a

Iron

64.38±3.95c 77.48±2.32d

48.67±4.38c

52.80±2.40c

Polluted

Unpolluted

413.89±12.48c

409.89±0.43c

Phosphorus

21.13±0.12d

17.16±1.71c

Sodium

284.27±2.44d

250.73±4.29c

Potassium

0.04±0.00d

0.34±0.13c

Copper

0.58±0.09d

0.77±0.18c

Iron

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Calcium

Magnesium

Environment of sampling

Table 8: Mineral Content (mg/100g) of Pterocarpus mildbraedii Harms Vegetables.

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Calcium

Magnesium

Environment of sampling

Table 7: Mineral Content (mg/100g) of Abelmoschus esculentus Moench.

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Vitamin B1

Vitamin A

Environment of sampling

Table 6: Vitamin Content (mg/100g) of Pterocarpus mildbraedii Harms Vegetables

Values (mean + SD of triplicate determinations) with different superscripts per column are significantly (P<0.05) different.

Vitamin B1

Vitamin A

Environment of sampling

Table 5: Vitamin Content (mg/100g) of Abelmoschus esculentus Moench

0.16±0.02d

0.34±0.06c

Zinc

0.32±0.07b

0.48±0.14a

Zinc

99.15±2.69d

85.29±1.79c

Vitamin C

78.03±1.02b

68.41±2.31a

Vitamin C

0.19±0.13d

0.50±0.22c

Lead

0.35±0.10b

0.65±0.07a

Lead

2.27±0.17c

1.97±0.18c

Vitamin E

1.88±0.17a

1.52±0.02a

Vitamin E

Ujowundu et al., 2013

865

Ujowundu et al., 2013 when compared to samples from UPE. Results presented

those from UPE but were not significantly different,

in table 8 showed that P. mildbraedii from UPE are

which indicates that they were not adversely affected by

excellent

the pollution.

source

phosphorus

(413.89±12.48),

potassium (284.27±2.44), calcium (77.48±2.32) and

The

phytochemical

results

indicate

that

magnesium (52.80±2.40). Also, the concentration of

A. esculentus and P. mildbraedii are good sources of

minerals in P. mildbraedii from PE and UPE were

these beneficial chemicals. They have antioxidative,

significantly different in all except in magnesium and

hypocholesterolemic, chemoprotective and antibacterial

phosphorus.

properties (Price et al., 1987; Enechi and Odonwodo, 2003; Okwu, 2004). Both vegetables are rich in

DISCUSSION

alkaloids, flavonoids and tannins which indicates that

Gas flaring and other oil and gas activities for

they have diuretic, antispasmodic, anti-inflammatory and

about four decades have contributed to pollution in

analgesic effects (Owoyele et al., 2002; Nobre-Junior,

Oguta, which have impacted on the ecosystem. Soots

2007 ; Alisi et al., 2011). Comparatively, P. mildbraedii

were seen on vegetation within the communities around

had higher content of the phytochemicals studied. Also,

the flaring site. Plants growing in such environment have

significantly higher amount of phytochemicals were

over the years taken in varying doses of pollutants which

observed in vegetables obtained from PE. The increase

invariably may affect the nutritional and chemical

can be linked to their role in oxidative stress in plants.

contents.

Phytochemicals are secondary metabolite of plants,

Our result showed that A. esculentus had better

known

exhibit

diverse

pharmacological

and

nutritional value than P. mildbraedii with respect to

biochemical effects on living organisms. It has been

protein and carbohydrate contents. Also, A. esculentus

reported that certain phytochemicals play important role

and P. mildbraedii showed higher values in proximate

in antioxidant defense systems of vegetative plants

contents (except in protein) than A. hybridus as reported

(Ugochukwu and Babady, 2003). Pollution by gas flaring

by Nwaogu et al., (2006). Carbohydrates provide energy

is taught to generate free radicals in surrounding

to cells in the body, particularly to the brain, a

environment. Thus, it is expected that plants may

carbohydrate dependent organ in the body. (Nelson and

increase synthesis of antioxidant defense compounds.

Cox, 2005). These vegetables can supplement the daily

These vegetables showed significantly high

energy intake of humans (Bingham, 1998; Effiong et al.,

amount of vitamins especially vitamins A, B1, B2, B5, B6

2009). The crude fibre content, indicates that the

and C in samples from

vegetables are good sources of fibre, thus making them

to samples from PE. These vitamins are involved in

veritable source of roughage. The concentrations of

intermediary metabolism of both plants and animals

carbohydrate were significantly reduced while ash

acting as part or whole coenzyme to some specific

contents were increased in plants from polluted

enzyme system and playing important role in both

environment when compared to plants from unpolluted

enzyme and non enzyme oxidative stress defense

environment (UPE). The reduced carbohydrate can be

systems. The high concentrations of vitamins A and C

attributed to the effect of air pollutants as reported by

will contribute significantly to the daily requirements in

Farzana (2005), in which he affirmed that it reduces

view of the reports of Murray (1998). Vitamin C

photosynthesis in chloroplasts. The contents of protein,

maintains

crude fiber and fat in samples from PE were lower than

circulation in the arteries of smokers. The most important

866

blood

vessel

UPE

flexibility

when compared

and

improves

Journal of Research in Biology (2013) 3(3): 861-869

Ujowundu et al., 2013 benefit of vitamins A and C is their involvement in free

phosphorus are important and indispensable for the

radical scavenging processes (Trumbo et al., 2004;

synthesis of strong bones and teeth, kidney function and

Nwaogu et al., 2011). These chemically active radicals

cell growth (Uddoh, 1988; Brody, 1994). Phosphorus

are byproducts of many normal biochemical processes.

and magnesium are also important in the regulation of

Their numbers are increased by environmental assaults

acid-alkaline balance in the body (Fallon, 2001).

such as chemicals and toxins. The lower concentrations

The mineral contents, like Mg, Ca, P, S and K in

of these vitamins in samples from PE suggest an inability

vegetables from PE have significantly (P<0.05) reduced

of the plants to synthesize these vitamins in sufficiently

value compared to vegetables obtained from UPE. The

large amount for their metabolic functions. Oxidative

release of pollutants such as oxides of sulphur and

stress caused by gas flaring in Oguta community can

nitrogen, hydrocarbons and other volatile organic

interfere with the synthetic mechanisms of the plants in

carbons can create chemical and biological conditions

the environment (Farzana, 2005).

which may be harmful to plants and soil microorganisms.

Som e

One of such conditions is the reduction in the capacity of

A. esculentus Moench and P. mildbraedii Harms are

plants to absorb cations (Wild et al., 2000). Crops grown

comparable

for

in soil with low mineral contents exhibit various forms of

2006)

mineral deficiency. In plants, potassium is an essential

2010),

nutrient and has an important role in the synthesis of

Commelina nudiflora and Boerhavia diffusa (Ujowundu

amino acids and proteins (Malik, 1982). Ca and Mg play

et al., 2008). The values obtained for the minerals

significant role in photosynthesis, carbohydrate and

indicates that the samples are good sources of mineral

nucleic acids metabolism (Russel, 1973). The reduced

and are of great nutritional importance. In animals,

content of these minerals will definitely affect these

potassium and sodium are important electrolytes.

important plant processes. Lead is yet to record any

Potassium is a major intracellular cation. Sodium is

physiological role in the biological system and are

involved in the regulation of acid-base equilibrium,

known to be extremely toxic even at the slightest

protection against dehydration and maintenance of

concentration. Their presence in the samples calls for

osmotic pressure in living system. It plays a role in the

serious concern

Amaranthus Mucuna

the

mineral

higher

hybridus

utilis

than

contents

that

(Nwaogu

(Ujowundu

reported et

al., al.,

normal irritability of muscles and cell permeability (Schwart,

1975).

Copper

(Cu)

for

and P. mildbraedii Harms are good sources of nutrients

haemoglobin synthesis, normal bone formation and the

and their consumption should be encouraged. Improved

maintenance of myelin within the nervous system

information on these plants will contribute to the

(Passmore and Eastwood, 1986). In animals, the

awareness of their nutritive value, especially in this time

manifestations of copper deficiency include; anaemia,

of increased food insecurity. Also, gas flaring showed

hypo-pigmentation,

keritinization,

negative effects on these plants, which could affect

abnormal bone formation with spontaneous reproductive

animals that consume them. Similarly, the adverse health

and heart failure (Williams, 1982). In humans, it has

consequences on the inhabitants around the gas flare

been established that occurrence of Cu absorption

site are of great concern. Communities around such

disorder in after partial gastetomycin leads to severe

environment should be enlightened on the inherent

malnutrition just as when protein is severely deficient in

dangers. Oil and gas industries should be compelled to

the diet; as in kwashiorkor (Davies, 1972). Calcium and

upgrade their waste disposal technologies, with emphasis

defective

wool

essential

This study has shown that A. esculentus Moench

Journal of Research in Biology (2013) 3(3): 861-869

867

Ujowundu et al., 2013 in gas disposal. This will reduce the detrimental effects on the health and well-being of inhabitants of Izombe in Oguta Local Government Area of Imo State. REFERENCES Adeniye EO, Olu-Sule R and Anyanye A. 1983. Environmental and Socio-economic Impacts of Oil Spillage in the Petroleum Producing Areas in Nigeria. The Petroleum industry and the Nigerian Environmental proceedings of the 1983 International oil seminar, NNPC 3:130-135. Alisi CS, Nwaogu LA, Ibegbulem CO and Ujowundu CO. 2011. Antimicrobial Action of Methanol Extract of Chromolaena Odorata-Linn is Logistic and Exerted by Inhibition of Dehydrogenase Enzymes. Journal of Research in Biology 1(3): 209-216.

pollution and climate in sindh, Pakistan. Digital-verlg GmbH, 41:3-15. Harborne JB. 1973. Phytochemical methods: a guide to modern techniques of plant analysis. Chapman and Hall Publishers 3(1):5-29. Ikoro NJ. 2003. The Socio-economic Implications of Gas flaring in Nigeria. Du-France Communications, Yenagoa, Bayelsa 4(13):35-47. James CS. 1995. Analytical chemistry of foods. Backie Academic Journal 198:125-181. Kirk RS and Sawyer R. 1998. Pearson’s Composition and Analysis of Food. Addison Linsle y Longman Limited 9:15-28. Malik CP and Srivastava AK. 1982. Text book of plant physiology. New Delhi: Ludhiana.

AOAC. 1990. Method of analyses of association of official analytical chemists. Journal of the Association of Analytical Chemists 25:516-524.

Mcdowell IF. 2001. Folate, homocysteine, endothelial function and cardiovascular disease, what is the link. Biomedical Pharmacoether 55(8):425-433.

Bingham S. 1998. Nutrition: A consumer’s guide to food eating. London: Transworld Publishers. 123-127.

Murray MJ. 1998. High quality vitamins, minerals and special supplements. Jama 279:1200-1205.

Brody T. 1994. Nutritional Biochemistry, San Diego, CA: Academic Press.

Nelson DC and Cox MM. 2005. Integration and Control of Metabolic Processes. Lehninger Principles of Biochemitsry (4th ed) Worths Publishers. 780-783.

Coker KA. 2007. Ludwig’s Applied Process Design for Chemical and Petrochemical Plants. Gulf Professional Publishing 1(4):732-737. Davies ITJ. 1972. Copper Absorption. The Clinical Significance of the Essential Biological Metals William Heine, Mann Medical Books Ltd, London 47. Effiong BN, Sanni A and Fakunle JO. 2009. Phytochemical and chemical composition of Combretum zenkeri. Human Science 86:301-307. Enechi OC and Odonwodo I. 2003. Assessment of the Phytochemical and Nutrient composition of Pulverized Root of Cissus quadrangularis. J. Biol. Res. Biotechnol., 1(1):63-68. Fallon S and Enig MG. 2001. Nourishing Traditions: The Cookbook that Challenges Policitally Correct Nutrition and the Diet Dictocrats. 40-45. Farzana P. 2005. Response of plant metabolism on air 868

Nobre-Junior HV. 2007. Chemoprotective actions of tannins. Journal of Herbs, Spices and Medicinal Plants 13(2):48-55. Nwaogu LA, Igwe CU, Ujowundu CO, Arukwe U, Ihejirika CE and Iweke AV. 2011. Biochemical changes in tissues of albino rats following subchronic exposure to crude oil. J. Res. Biol., 8: 617-623 Nwaogu LA, Ujowundu CO and Mgbemena AI. 2006. Studies on the nutritional and phytochemical composition of Amaranthus hybridus Leaves. Bio- Res., 4: 28-31. Nwaugo VO, Onyeagba RA and Nwachukwu NC. 2006. Effect of Gas Flaring on Soil Microbial Spectrum in parts of Niger Delta Area of Nigeria. Annual Journal of Biotechnology 5(19):1824-1826. Ogbonnia S, Odimegwu J and Enwuru VN. 2008. Clinical manifestation of diabetes. Department of Journal of Research in Biology (2013) 3(3): 861-869

Ujowundu et al., 2013 Pharmacognosy, Nordestgaard 22(6):35-42. Okwu DE. 2004. Phytochemicals and Vitamin content of Indigenous Spices of South Eastern Nigeria. J. Sust. Agric. Environ., 6(1):30-37. Onwuka SK. 2005. Cortical metal and crude protein levels of certain vegetables. Brain Talk Communities 3:15-23. Owoyele BV, Oguntoye SO, Dare K, Ogunbiyi BA and Aruboula EA. 2002. Analgelsic, anti-inflammatory and antipyretic activities of flavonoid, alkaloid and tannin fractions of Chromolagna odorata. Journal of Medicinal Plants 2(9):219-225.

Anti-Nutritive Properties of Boerhavia diffusa and Commelina nudiflora Leaves. Pak. J. Nutr. 7(1): 90-92. Wild E, Dent J, Barber JC, Thomas GO and Jones KC. 2005. Real Time Visualization and Quantification of Polycyclic Aromatic Hydrocarbon (PAH) Photodegradation on and within Plant Leavesâ&#x20AC;? Environmental Science Technology 39(1):268-273. Williams DM. 1982. Clinical significance of copper deficiency and toxicity in the World population. In the clinical Bio-chemical and nutritional aspects of trace elements. Prasad, A.S., New York, 177.

Passmore R and Eastwood MA. 1986. In Davison Passmore R., Human Nutrition and Dietetics, Churchill Livingstone, London. 124-126. Pearson D. 1976. Chemical analyses of food. Church hill, Livingstone. 7:72-73. Price KR, Johnson IT, Fenwick GR. 1987. The chemistry and biological significance of saponins in food and feeding stuffs. CRC. Crit. Rev. Food Sci. Nutr., 26:27-135. Russel EW. 1973. Soil conditions and plant growth. Supergene Zone, M. Nedra, (in Russian). 19. Schwart MK. 1975. Role of trace elements in cancer. Cancer Res., 35:3481-3484. Trumbo P, Schlicker S and Yates AA. 2004. Roles vitamin A and C play as antioxidants. Science Metabolism 30:253-258. Uddoh CK. 1988. Nutrition: Macmillan Publishers Ltd. London and Basingstoke, 71-97,109. Ugochukwu NH and Babady NE. 2003. Antioxidant effects on Gongonemia catifolium in hepatocytes of rat model of non insulin dependent diabetes mellitus. Filoterapia 173(7-8):612-618. Ujowundu CO, Okafor OE, Agha NC, Nwaogu LA, Igwe KO and Igwe CU. 2010. Phytochemical and Chemical Composition of Combretum zenkeri Leaves. Journal of Medicinal Plants Research 4(10):965-968. Ujowundu CO, Igwe CU, Enemor VHA, Nwaogu LA and Okafor OE. 2008. Nutritive and Journal of Research in Biology (2013) 3(3): 861-869

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869

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Authors: Akpa GN, Alphonsus C and Usman N.

Institution: Animal Science Department, Ahmadu Bello University, Zaria, Nigeria.

ABSTRACT: This study was conducted to evaluate the effect of haemoglobin (Hb) types, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats. Ninety four (94) goats were sampled from two locations: Dei-dei and Gwagwalada grazing reserved, Abuja. Data were collected on adaptive characteristics {heart rate (HR) and rectal temperature(RT) and adaptive coefficient (AC) was calculated from the HR and RT} and blood biochemical characteristics{ haemoglobin (Hb) types, Hb-concentration (Hb-conc), Potassium concentration (K-conc) and albumin concentration (alb-conc)}. The effects of haemoglobin type, sex and age on the adaptive and blood biochemical characteristics of the goats was analyzed by general linear model (GLM) procedure of SAS. The results showed that the mean RT of the sampled goats was 38.9째C with very minimal variations (CV=0.5). The mean HR of the goats was 76.1bpm, with min and max HR of 70 and 80bpm. The mean albumin, Hb and K concentration were 38.4g/l, 8.9g/dl and 4.0Mmol/l, respectively. The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05) except Hb concentration. Higher HR was observed in goats with Hb AA and AB. Age and sex had significant effect (P<0.05; P<0.01) on HR, AC and albumin concentration of the goats. Although there was no trend in the variation of HR and AC with age, but HR and AC were higher in the older goats than the younger, however the albumin concentration significantly decreased with progressive increase in age of the goats.

Corresponding author: Alphonsus C.

Keywords: Adaptive coefficient, heart rate, rectal temperature, blood biochemical characteristics.

Email: mcdyems@gmail.com

Article Citation: Akpa GN, Alphonsus C and Usman N. Effect of age, sex and hemoglobin type on adaptive and blood biochemical characteristics in Red Sokoto Goats Journal of Research in Biology (2013) 3(3): 870-875

Web Address:

Dates: Received: 15 Dec 2011

http://jresearchbiology.com/ documents/RA0167.pdf.

Accepted: 01 Jan 2012

Published: 16 Apr 2013

Journal of Research in Biology An International Scientific Research Journal

870-875 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Akpa et al.,2013 INTRODUCTION

related to farm animals. Comprehensive reviews have

In recent years, advances in the field of

appeared under the authorship of Alderson, 1992,

biotechnology have opened up a completely new area at

Derman and Noakes, 1994, Tambuwal et al., (2002),

molecular levels with the introduction of techniques such

Otoikhian et al., 2009, chukwuka et al., 2010,

as routine electrophoresis employed for detection of

Opara et al., 2010, Gurcan et al., 2010 and a lot of

polymorphism at protein and enzyme loci as well as

others. Research results reported in this paper is intended

other serological and immunogenetic procedures for the

to supplement data reviewed by authors listed.

measurement of variations (Salako et al., 2007). Data

Eventually, accumulated data will permit specific

obtain from this type of study could be useful as genetic

recommendation on breeding, feeding and management

markers for important economic characteristics and

of farm animals.

could aid significantly in selection of superior animals for breeding purposes. Haemoglobin typing is very important as

This study therefore aimed at studying the effects of Haemoglobin type, sex and age on adaptive and blood biochemical characteristics of Red Sokoto goats.

different Hb types may have selective advantage in different geographical regions (Ndamukong, 1995).

MATERIALS AND METHODS

Economic pressures of various kinds are forcing the

Location

production of livestock into climatic environments that

The study was conducted at the Federal Capital

are increasingly more remote from the considered ideal

Territory Abuja, located within the Northern guinea

for optimal production and feed utilization.

Savanna zone of Nigeria. It is laying between latitudes

Thermal stress, which is one of the major factors

8.25째 and 9.0째 N of the equator and Longitude 6.45째 and

that affect the productivity of many farm animals can be

7.39째 E of the Greenwich Meridian. (Presentation

reflected in an easily observable changes in pulse rate,

respiration rate, and rectal temperature, although the

Data collection

whole body reacts to thermal stress by an elaborate series

Ninety four (94) goats were sampled from two

of chain reactions (Ahmed, 2004). The most obvious

locations: Dei-dei and Gwagwalada grazing reserve,

index of thermal stress is body temperature response.

Abuja. Data were collected on the adaptive and blood

Deviation from normal rectal temperature indicates that

biochemical traits. The adaptive traits were Heart rate

the animal is under stress, that its homeothermic

(HR), Rectal temperature(RT) and Adaptive coefficient

mechanisms are overtaxed Ahmed, 2004).

(AC) while the blood biochemical characteristics were

Adaptive characteristics of animals serve as a

haemoglobin (Hb) types, Hb-concentration (Hb-conc),

key to managing any livestock operation. Adaptive traits

Potassium

concentration

such as rectal temperature, heart rate, and flank

concentration (alb-conc).

(K-conc)

and

albumin

movement have been documented to have some significant effect on genetic variations. Every normal

METHODS OF MEASUREMENTS

animal has a range of individual adaptive traits in

Adaptive Traits

relation to a specific physiological pattern.

Heart Rate (HR)

Because study of environmental physiology

Heart rate was taken by placing stethoscope on

involves so many variables and scientific discipline,

femoral artery of the hind limb of the goat to count the

much is being published on this subject especially as

number of beat per minute.

871

Journal of Research in Biology (2013) 3(3): 870-875

Akpa et al.,2013 the Rectal Temperature (RT) and Heart Rate (HR) as

Rectal Temperature (RT) This was taken using clinical thermometer which was inserted into the rectum of the goat and left for

thus Adaptive coefficient (AC) = (RT/38.33) + (HR/23.00)

45-50 seconds. It was then removed and the temperature

The effects of haemoglobin types, sex and age on

level was read. The values read were recorded, and the

the adaptive and blood biochemical characteristics of the

process was repeated for the other goats.

goats were determined by general linear model (GLM)

Blood Biochemical Characteristics

procedure of SAS, (2005).

Blood samples were taken from each of the experimental goats through the jugular vein. 5 mls of the blood was taken from each goat, from which 2 mls was put

into heparinized vacutainer tubes

containing

RESULTS AND DISCUSSION Rectal Temperature (RT) is directly affected by the surrounding and ambient temperature, and high

anticoagulant ethylene diamine tetra acetic acid (EDTA).

ambient

temperature

has

negative

effect

The remaining 3 mls of the blood was put into sterile

productivity of the animal. Chukwuka et al., (2010)

vacutainer tubes (without anticoagulant). The samples

reported that negative effect of high ambient temperature

were labeled accordingly. The blood samples in the

is direct in the form of stress suffered by the animal and

sterile vacutainer tube were centrifuged in order to have

the diversion of energy from the purpose of production to

a clear layer of serum. This serum was pipetted into

regulation of body temperature and indirectly by

another sterile bottle and store in a refrigerator. The

affecting the availability of feed resources upon which

blood samples were taken to the Haematological

production is dependent. In this study, the mean RT of

laboratory of Ahmadu Bello Teaching Hospital from

the goats was 38.9°C with minimum and maximum body

where the analysis of blood biochemical characteristics

temperature of 38.1 and 39.4 °C (Table 1). These values

was carried out.

were within the reference range of previous study

A spectrometer with wavelength capability of

of goats in thermal neutral condition (Otoikhian et al.,

600-650 nm (Zenway 5041 colorimeter) was used

2009) and this indicate that the goats used for this

to analyzed for the albumin concentration, while the

research showed no clinical signs of stress during the

research period. The body temperature of the goats

concentration

flame

was

photometer

analysed

410.

using

Corning

Electrophoresis

and

exhibited minimal variations (CV=0.5%), thus implying

Cyanmethaemoglobin method was used to analyzed for

that goats are homoeothermic animals, they can maintain

Hb-types and Hb-conc, respectively.

near constant body temperature under wide range of

Data Analysis

environmental conditions.

The adaptability of the goats were measured by

The Heart Rate (HR) is the pulse that helps to

determining the adaptive coefficient from the values of

know the beating rate of the heart which is measured

Table 1: Summary Statistics of the measured characteristics in Red Sokoto Goats Characteristics Rectal Temperature (°C) Heart Rate (bpm) Adaptive coefficient Albumin concentration (g/l) Hemoglobin concentration (g/dl) Potassium concentration (Mmol/l) Journal of Research in Biology (2013) 3(3): 870-875

N 94 94 94 94 94 94

Mean±SE 38.9±0.02 76.1±0.39 4.3±0.02 38.4±0.34 8.9±0.16 4.0±0.06

CV(%) 0.5 3.8 5.0 8.5 17.1 14.7

Min 38.1 70.0 4.1 31.0 4.0 3.0

Max 39.4 81.0 4.6 48.0 12.7 5.8 872

Akpa et al.,2013 in

beats

per

minute

stethoscope

amount of K-concentration is fairly high at intracellular

(Otoikhian et al., 2009). The mean HR of the goats used

membranes (Gurcan et al., 2010). The values of

in this study was 76.1bpm, with the min and max HR of

K-concentration reported by Opara et al., (2010) for

70 and 80bpm. This is slightly higher than the range of

WAD bucks and does were 17.8 and 6.9mmol/l,

70-75bpm reported by Derman and Noaks (1994) in

respectively. These values were higher than the mean

goats. The minor difference observed in the values of the

value of 4.0mmol/l observed in this study; this is

HR may be explained by differences in geographical

probably due to differences in breed and physiological

conditions,

conditions of the sampled animals. Researchers had

season

(bpm)

climate

using

and

physiological

conditions of the sample goats.

identified the existence of different type of K in different

Rectal temperature and heart rate have been

species of animals, and that in sheep for instance, there

shown to be good indicators of the thermal stress and

are two types of K which is high and low K with the

may be used to assess thermal adversity of the

low K

environment (Al- Haidary, 2004).

(Soysal et al., 2003). Also Gurcan et al., (2010) reported

The Adaptive Coefficient (AC) (which is the function of RT and HR) signifies the level of adaptability

type

dominant

over

the high

K type

a range of 4.23 to 11.69mmol/l for low K type in animals.

of the goats to the environments varied significantly

The concentration of albumin in this study

(P<0.05) with Hb types, sex and age of the goats. The

(38.4g/dl) was slightly higher than the 34.5g/dl reported

goats with Hb AA and AB had higher AC than those

by Opara et al., (2010).

with BB and AC; likewise the bucks had higher AC than the does.

The variation of Hb type with adaptive and blood biochemical characteristics was significant (P<0.05)

Potassium is one of the intracellular elements

except Hb-concentration (Table 2). The relationship

that regulate the intracellular density of the cell. The

between Hb types and HR can be linked to the different

Table 2: Effect of hemoglobin type on adaptive and blood biochemical characteristics Hemoglobin Type AA BB AB AC SEM Rectal Temperature (째C) 39.0a 39.0a 39.0a 38.9b 0.02 Heart Rate (bpm) 76.6a 75.3b 76.2a 75.1b 0.40 Adaptive coefficient 4.4a 4.3b 4.4a 4.3b 0.02 Albumin concentration (g/l) 37.6b 39.2a 38.6a 38.8a 0.34 Hemoglobin concentration (g/dl) 9.1 8.6 8.8 8.6 0.16 Potassium concentration (Mmol/l) 3.8b 3.9a 4.0ab 4.4a 0.06 Number of observations 30 11 41 12 94 ab : means within the same row with different superscripts differ significantly(P<0.05); ns:not significant; Characteristics

LOS * * * * ns *

Table 3: Effect of Sex on adaptive and blood biochemical characteristics Sex Buck Doe SEM LOS Rectal Temperature (째C) 39.0 39.0 0.03 ns Heart Rate (bpm) 78.5a 75.0b 0.49 ** Adaptive coefficient 4.4a 4.3b 0.02 ** a Albumin concentration (g/l) 39.8 37.7b 0.49 ** Hemoglobin concentration (g/dl) 9.1 8.7 0.26 ns Potassium concentration (Mmol/l) 3.9 4.0 0.08 ns Number of observations 30 64 94 ab : means within the same row with different superscripts differ significantly(P<0.01); ns:not significant; Characteristics

873

Journal of Research in Biology (2013) 3(3): 870-875

Akpa et al.,2013 Table 4: Effect of Age on adaptive and blood biochemical characteristics Age of goat 12mon 18mon 24mon 30mon SEM LOS Rectal Temperature (oC) 39.0 39.0 39.0 39.0 0.02 ns Heart Rate (bpm) 77.1b 74.4c 74.4c 78.5a 0.37 * Adaptive coefficient 4.4b 4.3c 4.3c 4.5a 0.02 * Albumin concentration (g/l) 39.2a 37.4b 36.7c 36.5c 0.32 * Hemoglobin concentration (g/dl) 9.1 8.7 8.3 8.5 0.16 ns Potassium concentration (Mmol/l) 4.0 4.0 4.0 4.0 0.06 ns Number of observations 55 25 12 2 94 ab : means within the same row with different superscripts differ significantly(P<0.05); ns:not significant; Characteristics

levels of

oxygen carrying capacity of the different

albumin concentration significantly decreased with

Hb types. In this study, higher HR was observed in goats

progressive increase in age of the goats. The observed

with Hb AA and AB, and Hb A is known to be the

significant influence of age on albumin concentration

haemoglobin allele with highest affinity for oxygen. This

is in line with the earlier report of Huisman et al., (1959)

Piccione et al., (2009) and Opara et al., (2010) who

who relates the preponderance of Hb A to it greater

reported non-significant effect of age on albumin

affinity to oxygen. This could also explain the high

concentration of WAD goats.

variance

with

the

earlier

studies

adaptive coefficient observed on goats with Hb types AA and AB since adaptive coefficient is a function of HR and RT. The variation of HR,

CONCLUSION The mean body temperature (38.9째C) of the

AC and Albumin

goats used was within the reference normal range for

concentration with sex was highly significant (P<0.01;

goats in thermal neutral condition and this indicates that

Table 3). The RT of the buck and does were similar

the goats showed no clinical signs of stress during the

however, the HR was higher in bucks (78.5bpm) than the

research period.

does (75.0bpm) this is probably due to the high sexual activity of the bucks. There was no significant (P>0.05) difference between the bucks and does in Hb and K concentration. This is contrary to the study of opera et al., (2010) who reported significant differences between WAD bucks and does in there Hb and K concentration. This is probably due to differences in breeds and location of the animal, Hb type had been reported to vary with breed and location (Ndamukong, 1995, Abdussamad et al., 2004 Essien et al., 2011) Age significantly (P<0.05) influence HR, AC and albumin concentration but had no significant influence on the RT, Hb and K concentration (Table 4). Although there was no trend in the variation of HR and AC with age, but it was observed that the HR AC was higher in the older goats than the younger, however the Journal of Research in Biology (2013) 3(3): 870-875

The albumin concentration, heart rate and adaptive coefficient of the goats had clear variation based on differences in haemoglobin type, sex and age of the animals. REFERENCES Abdussamad AM, Esievo KAN, Akpa GN. 2004. Haemoglobin types in the Nigerian Zebu and their crosses in Zaria. Proceedings of the Nigerian Society for Animal Production (NSAP). 29-31. Alderson GLH. 1992. genetic conservation of domestic livestock. CAD International, Wallingford, UK. 242. AL-Haidary. 2004. Physiological Responses of Naimey Sheep to Heat Stress Challenge under Semi-Arid Environments. International Journal of Agriculture &Biology 06(2):307-309. 874

Akpa et al.,2013 Chukwuka OK, Okoli IC, Okeudo NJ, Opara MN, Herbert U, Obguewu IP and Ekenyem BU. 2010. Reproductive potentials of West African Dwarf sheep and goats. A Review, Research Journal of Vetrenary Sciences 3(2):86-10. Derman KD and Noakes TD. 1994. Comparative aspect of exercise physiology. In: Hodgson, D.R., Rose, R.J (Eds). The Athletic horse: Principle and practice of Equine Sports Medicine. Philadelphia. W.B. Saunders. 13-25. Essien IC, Akpa GN, Barje PP, Alphonsus C. 2011. Haemoglobin types in Bunaji cattle and their Friesian crosses in Shika, Zaria-Nigeria. Afri. J. Anim. Biomed. Sci., 6(1):112-116. Gurcan EK, Erbas C and Ozden C. 2010. Biochemical polymorphism of erythrocyte, Potassium and glutathione protein: the relationship with some blood parameters in Kivircik sheep breed. African Journal of Agricultural Research 2(10):1022-1027. Huisman THJ, Van Der Helm HJ, Visser HKA and Van Vilet G. 1959. Symposium on abnormal Haemoglobin. Blackwell Scientific Publication. 181.

Medicine, University of Messina, Italy. Presentation Copyright @Falling Rain Genomics, Inc 1996-2010. SAS. 2005. user guide for personal computers, statistical programme 9.01 windows version.(SAS Institute Inc. Cary, NC). Salako AE, Ijadunola TO and Agbesola YO. 2007. Haemoglobin polymorphism in Nigerian indigenous Small Ruminant population-preliminary investigation. African Journal of Biotechnology 6(22):2636-2638. Soysal ML, Gurcan EK, Ozkan E. 2003. Turkiye de yetistirilen cesitli koyun Irklrinda trim kan potasyum konsan trasyonu polimorfizmi vizerine arastirmalar. GAP III Tarim Kongresi, sanliurfa. Tambuwal FM, Agala BM and Bangana A. 2002. Haematological and Biochemical values of apparently healthy Red Sokoto goats. Proceeding of 27th Annual Conference, Nigerian Society of Animal Production (NSAP).FUTA, Akure, Nigeria. 50-53.

Ndamukong. 1995. Haemoglobin polymorphism in Grassland dwarf sheep and goats of the North West province of Cameroon. Bulletin of Animal Health and Production in Africa. 43:53-56. Opara MN, Udeyi N and Okoli IC. 2010. Haematological parameters and blood chemistry of apparently healthy West African Dwarf (WAD) goats in Owerri, South-eastern Nigeria. Tropical Animal Health and Welfare Research group. New York Science Journal 3(8). Otoikhian CSO, Orheruata MA, Imaseuen JA and Akporhuarho OP. 2009. Physiological response of local (West African Dwarf) and adapted Switzerland (White Bornu) goat breed to varied climatic conditions in South-south Nigeria. African Journal of General Agriculture. 5(1):1-6. Piccione G, Casella S, Lutri L, Vazzana I, Ferrantelli V, Caola G. 2009. Reference values of some Haematological, Haematochemical and electrophoretic parameters in the Girgentana goats. Faculty of Vetrinary 875

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Journal of Research in Biology (2013) 3(3): 870-875

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India Authors: ABSTRACT: Samit Roy Choudhury and Butterflies of the family Pieridae are common in tropical parts of the world. Basant Kumar Agarwala* They are considered as major pollinators as well as pests of economically important plants. Catopsilia pomona is a dominant pierid butterfly found in association with wild plants of Tripura, northeast India. It is abundant throughout the year. Present study was conducted to document the eco-biology of Catopsilia pomona with special reference to its life table attributes in the state of Tripura. Survival rates of life cycle Institution: stages in the semi-natural as well as in the field were the maximum during the wet Ecology & Biodiversity Laboratories, Department of and hot season. Mortality (k value) of different life cycle stages as a proportion of Zoology, Tripura University, individuals dying during development varied from 0.16 to 0.46 in different seasons. Results suggested that abiotic factors and mortality factors of egg significantly Suryamaninagar- 799022, influenced the survival rate of C. pomona population. This butterfly depends on three Tripura, India. species of Cassia plants, all shrubs, for their oviposition and larval development in the environment of Tripura. Corresponding author: Basant Kumar Agarwala

Keywords: Catopsilia pomona butterfly, Pieridae, eco-biology, life table, Tripura, north east India.

Email: bagarwala00@gmail.com

Article Citation: Samit Roy Choudhury and Basant Kumar Agarwala. Eco-biology of Common Emigrant Catopsilia pomona Fabricius (Lepidoptera: Pieridae) with special reference to its life table attributes in Tripura, India. Journal of Research in Biology (2013) 3(3): 876-885

Phone No: 0091 381 237 9083/9123 Web Address:

http://jresearchbiology.com/ documents/RA0245.pdf.

Dates: Received: 22 May 2012

Accepted: 28 May 2012

Published: 17 Apr 2013

Journal of Research in biology An International Scientific Research Journal

876-885 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Roy Choudhury and Agarwala, 2013 INTRODUCTION

plants of north east India, including Tripura. However,

Host selection for survival, development and

information on life table and host selections are available

reproduction in majority of insects often vary in space

on other pierid species that feed and oviposit on crop

and time (van Nouhuys et al., 2003; Nylin et al., 2009)

plants (Chew, 1995). C. pomona, a dominant pierid

which, in turn, depends on the availability (minimum

butterfly, is found throughout the year in the state of

density per unit area) of closely related host plant species

Tripura (Agarwala et al., 2010; Majumder et al., 2011;

(Thorsteinson, 1960), and trade off between host

Roy Choudhury et al., 2011). It prefers green and moist

preference by females for oviposition and larval

lands, pasture lands, farms, and edge of drains, moist

performance of insects (van Nouhuys et al., 2003).

deciduous forests, hillocks, and semi-arid areas with high

However, adult butterflies and their caterpillars show

abundance of grasses, small herbs and shrubs i.e.

preference for certain host plants for tender shoots,

secondary type of vegetation (Atluri et al., 2004).

pollen and nectar as food source. Thus, butterfly

Reported larval host plants of common emigrant

diversity of a particular habitat generally reflects the

comprise of Cassia fistula L., C. sophera L.,

overall plant diversity of that habitat. Butterflies are

C. occidentalis L., C. tora L., C. siamea (Lam.) Irwin et

essential component of any natural ecosystem. Their

Barneby, Butea frondosa, and Bauhinia racemosa L.

value as indicators of biotope quality is being recognized

(Kunte, 2000; Atluri et al., 2004). Among these plants

because of their sensitivity to minor changes in micro-

C. fistula, C. tora, C. occidentalis, C. sophera, and

habitat, climatic conditions as well as seasonal changes

(Kremen, 1992; Murugesan and Muthusamy, 2011).

(Anonymous, 2004; Danish et al., 2011; Harshal et al.,

They are considered as ideal subject for ecological

2011; Singh and Dubey, 2012), and C. siamea is used in

studies of terrestrial landscapes (Thomas and Malorie,

social forestry (Atluri et al., 2004, Borikar et al., 2009).

1985).

Hence, it is very important to document the seasonal

racemosa are important as medicinal

plants

North eastern region of India is blessed with

occurrence and its host plant preference for oviposition

vegetation rich landscapes that support diverse butterfly

and larval development of C. Pomona. With this view,

fauna and other insects (Alfred et al., 2002). The state of

the present study was conducted to know the eco-biology

Tripura, being a part of this region, also contains large

of Catopsilia pomona with special reference to its life

number of butterfly species which is evident from

history attributes in the state of Tripura.

infrequent records of these taxa (Mandal et al., 2002;

Study site

Agarwala et al., 2010; Majumder et al., 2011; Roy

Present study was conducted in Trishna Wildlife

Choudhury et al., 2011). Butterflies of the family

Sanctuary of south Tripura district (23°26.137’ N,

Pieridae are common in tropical parts of the world and

91°28.184’ E: 51-82 m asl), having an area of about

are considered as major pollinators of crop plants

194.7 sq. km. Study location is characterized by patches

(Borges et al., 2003), and a few of them are also

of secondary moist deciduous forests and surrounded by

considered as pests of economically important plants

swamp areas. Forest patches are rich in sal trees, garjan

(Anonymous, 2007; Capinera, 2008). Despite their

trees, bamboo bushes, herbs, shrubs and climbers.

common occurrence, there is a lack of substantial study

Trishna sanctuary is known by 230 tree species, 110

on the ecology, seasonal abundance, host preference and

species of shrubs, 400 species of herbs, and 150 species

life history of the most common pierid species

of climbers (Economic review of Tripura, 2008-2009).

Catopsilia pomona F. found in association with wild

Among the known host plants of C. pomona, the study

877

Journal of Research in Biology (2013) 3(3): 876-885

Roy Choudhury and Agarwala, 2013 area contains three species of Cassia only viz.

plastic tags. Thus, sixty plants from three species were

Cassia sophera, C. tora and C. occidentalis which are

selected from transects. Ovipositing females were

considered to be the preferred hosts of larvae. Some part

followed in the selected host plants for recording number

of the study area is used for rubber cultivation and paddy

of eggs laid per female per leaf. Binoculars were used to

cultivation (Figure 1). The area has a tropical climate,

observe the females from a distance (about 2 m) without

with cold weather from November to February. Average

disturbing them. The same host plant was also observed

daily temperature varies from the minimum of 6.8째C in

for presence of larvae. All the females seen ovipositing

January to the maximum of 37.7째C in June. The area

on the selected host plants was recorded during the

receives, on an average, 3353.4 mm rainfall annually.

transect walk. Two transects were walked in two consecutive days in a week. Ten apical leaves were observed within a selected plant for egg and larval counts

MATERIALS AND METHODS Field

census

eggs,

larvae

and

oviposition

preference of C. pomona

which were made between 8.00 AM to 12.00 noon local time. When a female was found to either laying eggs or

Prior to the study a reconnaissance survey was

seen perching near a host plant, halt was made for

made in the Trishna study area to locate the available

approx. 8 to10 minutes, and then move to the subsequent

host plants distribution of C. pomona. Walk census for

host plants along the transect. Different host plants

leaves of host plants containing eggs and larvae were

selected by females for oviposition were recorded,

held at an interval of 7-days from March 2007 to

photographed,

February 2008. For this, two line transects (approx. 1 km

comparing with the herbarium deposited in the gallery of

long and 5 m wide) were set up in the study area. Thirty

Plant

host plants, 10 plants each of C. sophera, C. tora and

Department of Botany, Tripura University.

collected

Taxonomy

and

later

identified

Biodiversity

Laboratories,

C. occidentalis, were randomly selected for the study along the length of transects and were marked with

Figure 1. Geographical map of Trishna and landscape of the Study area. Journal of Research in Biology (2013) 3(3): 876-885

878

Roy Choudhury and Agarwala, 2013 Larval

host range

and seasonal

variation in

Survival rate and K-factor analysis An age-specific life table was constructed

development Leaves of the host plant species found to contain

following the method of Stiling (2002). To prepare the

freshly laid eggs of C. pomona in field were brought to

life table, records were made on the larval durations and

the field station (3 km from the study area), and

survival rate at each developmental stage i.e. eggs to

transferred individually to 10 cm diameter paired Petri

emergence of adults from pupae. For this purpose,

dishes lined with corrugated papers. These were fed with

409 eggs and 317 eggs of C. pomona were studied in

surplus quantity of tender leaves of respective host plants

natural (in field) and in controlled conditions (ambient

from which they were actually collected. Twenty

condition of field station), respectively. Meteorological

replicates were used for each host plant species. Food

data of Trishna study area were collected from the

was changed every 24 hrs intervals. Petri dishes were

records maintained by the Department of Agriculture,

cleaned at the time of food change. These were observed

Govt. of Tripura at Arundhuti Nagar, Agartala.

twice in a day at 11 am and again at 5 pm to record the

Data analysis

incubation period of eggs, developmental time of larvae,

Field data on proportion of host plants used by

and pupae. Mortality in development, if any, was also

C. pomona for laying of eggs and distribution of eggs per

recorded. This was simultaneously done on each host

leaf of the different host species during a year were used

plant, once in five different seasons to record the

to draw population curves. For this purpose, weekly data

seasonal variation, if any. Experiments were set up at the

were pooled on monthly basis. Developmental time from

field station (Temp: 18Â°C ~ 27Â°C, RH: 45~75%, and

egg to the eclosion of pupae on different host plants and

L: D: 16:8h) i.e. in the controlled environment.

between different seasons was subjected to one-way

Larval development in field

analysis of variation (ANOVA). Mean values of

Selected plants with freshly laid eggs and

development time on different host plant species and

subsequent developmental stages were provided with

between different seasons were compared by Tukeyâ&#x20AC;&#x2122;s

coloured tags and these were numbered for easy

multiple comparison test. Differences in development

identification. Individual eggs, larvae and pupae were

time recorded in field and in field station were compared

followed daily, and the disappearance of individuals or

by Students t-test. A significance level of 0.05 was used

those that failed to develop in to the next stage at

to reject the null hypothesis. Field data on distribution of

different life stages were recorded. Larvae were found to

eggs on different host plant species were subjected to

be slightly sluggish and females laid solitary eggs,

regression analysis to reveal the relationship between

usually one on each leaf. The study was repeated once in

oviposition preference and host utilization. Based on the

different seasons.

life table data, survival rate and K factor value that closely mirrors the overall population mortality was

Table 1. Oviposition preference of C. pomona females on different host plants in the study area Host plant

No. of leaves observed

No. of larvae counted

Mean (+SEM) no of larvae/ leaf

C. sophera

4800

984

0.21 + 0.01

C.occidentalis

4800

563

0.12 + 0. 02

C. tora

4800

647

0.13 + 0.01

879

ANOVA

F = 6.909 , df = 2,14397, P = 0.0001

No. of eggs counted

Mean (+SEM) no of eggs/ leaf

1237

0.26+0.02

899

0.19+0.03

816

0.17+0.02

ANOVA

F = 5.26, df = 2,14397, P = 0.006

Journal of Research in Biology (2013) 3(3): 876-885

Roy Choudhury and Agarwala, 2013 Table 2. Development time (in days) of C. pomona on different host plant species Month

Mean + SEM value (days) C. sophera 1

C. occidentalis 1

C. tora

March

24.50 + 0.26 a

24.75 + 0.25 a

24.74 + 0.33 1 a

May

20.67 + 0.31 2 a

20.92 + 0.42 2 a

August

18.92 + 0.23 3 a

19.42 + 0.63 3 a

19.00 + 0.28 3 a

October

21.17 + 0.24 2 a

21.33 + 0.28 2 a

21.25 + 0.25 2 a

December

30.67 + 0.47 4 a

30.83 + 0.41 4 a

31.00 + 0.41 4 a

Dissimilar numbers following means in a column denote significant difference and similar letters accompanying means show no difference between them by Tukeyâ&#x20AC;&#x2122;s multiple comparison range test at 5% significant level. determined. At each life stage, number of deaths

found with one or more eggs. Between the three host

(k value) was calculated as under: k = log Nt - log Nt+1,

plant species, common emigrant females selected the

where Nt is the density of the population before it is

highest proportion of C. sophera for oviposition during

subjected to the mortality and Nt+1 is the density

hot and wet months, and the maximum was recorded in

afterward. Total generational mortality factor K is

the month of August (Figure 2). In comparison,

determined as the sum of the individual mortality factors

distribution pattern of eggs on C. occidentalis plants

k at egg, larval and pupal stage of the C. pomona species

showed marked difference from the distribution of eggs

(Stilling, 2002). For interpretation of colleted data, the

on C. sophera. Higher proportion of this host plant

year was divided in to five seasons: spring (March,

species was recorded during dry and cooler months, and

April), summer (May, June), rain (July, September),

the maximum was recorded in the month of January

autumn (October, November), and winter (December-

(69.47%) (Figure. 2). In case of C. tora, the trend of egg

February). To determine the relationship between

distribution was found to be nearly similar to that of

successful development (%) of C. pomona eggs and

C. sophera but the proportion of host use was found to

climatic factors in the study area regression analysis was

be much lower than C. sophera (Figure. 2). Occurrence

carried out. Origin 7 software (www.originlab.com) was

of eggs showed that 4800 leaves each of C. sophera,

used for the analysis of data.

C. occidentalis and C. tora that were surveyed during the year, contained 1237, 899 and 816 eggs, respectively

RESULTS

(mean + SEM: C. sophera: 0.26+0.02 eggs per leaf,

Egg abundance and oviposition preference

C. occidentalis: 0.19+0. 03 eggs per leaf and C. tora:

Females of C. pomona laid solitary eggs at edges

0.17+0.02 eggs per leaf, ANOVA: F = 5.26, df = 2,

and on undersides of tender or young leaves (one egg/

14397, P = 0.006) (Table 1).

leaf/female) of C. sophera, C. occidentalis and C. tora

Larval host range

plants throughout the year (Table 1, Figure. 2). In the 2

Larvae of C. pomona were found to feed on

year-round census of 10000 m (1000 m long x 5 m wide

tender leaves of the three host plant species, viz.

x 2 transects @ 1 ha) which represents less than 0.5% of

C. sophera, C. occidentalis and C. tora. Higher

the study area (19.47 ha), 52.54% to 85.07% of

proportion of C. sophera plants were used as food and

C. sophera plants, 21.31% to 69.47% of C. occidentalis

maximum was recorded in the hot and wet month of

plants and 23.88% to 56.52% of C. tora plants were

August (26.70%). Incidence of larvae on C. occidentalis

Journal of Research in Biology (2013) 3(3): 876-885

880

Proportion of host plants selected for oviposition

Roy Choudhury and Agarwala, 2013

C. occidentalis C. sophera C. tora

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

Months

Figure 2. Proportion of host plants of three Cassia Figure 3: Mean number of larvae of C. pomona species recorded with eggs of C. pomona in different recorded on different host plants. months of the year in the study area. 0.13 + 0.01 larva per leaf, ANOVA: F = 6.909, df = 2,14397, P = 0.0001) (Table 1). Developmental time and seasonal variation Developmental time of different immature stages (egg to pupae) of C. pomona was found to vary in different seasons but did not show difference in any one season between different host species (Figure 4). Development time was recorded to be the longest at lower

temperature

and

lower

relative

humidity

corresponding to the month of December (controlled condition: average temperature=18째C, average relative Figure 4. Development time (in days) of C. pomona on different larval host plants in different months of a humidity=51.33%) and shortest at higher temperature year. Similar alphabets accompanying bars denote no and higher relative humidity in August (average significant difference between the mean values in that temperature=27.91째C, average relative humidity month. =77.07%) (Table 2). plants was recorded to be the highest in January

Survival rate and K factor analysis

(20.61%) and lowest in August (1.64%), respectively. In

Results showed that in field about 30% of the

case of C. tora host, the highest proportion was recorded

eggs deposited by C. pomona developed in to pupae

in the month of June (17.24%) and the lowest in the

during the months of July and August (average

month of January (5.33%) (Figure. 3). Occurrence of

temperature 31.09째C, average humidity 70%, mean

larvae showed that 4800 leaves each of C. sophera,

rainfall 7.45 cm). Developmental success was limited to

C. occidentalis and C. tora that were surveyed during the

13.04% in the month of December (average temperature

year, contained 984, 563 and 647 larvae, respectively

19.330C, average humidity 51%, rainfall 0 cm).

(mean + SEM: C. sophera: 0.21 + 0.01 larva per leaf,

Regression analysis of survival rate showed positive

C. occidentalis: 0.12 + 0. 02 larva per leaf and C. tora:

correlations with average temperature (y =1.08 + 0.87x,

881

Journal of Research in Biology (2013) 3(3): 876-885

Roy Choudhury and Agarwala, 2013 Number of eggs that developed successfully in fields (24.03+1.46; n=240) and in semi natural condition (76.36+0.90; n=240) showed significant difference (t =30.54, df =478, P =0.000). K-value analysis showed maximum mortality in development (0.46) in the month of December and minimum (0.16) in the month of September. K - values of the eggs recorded in different seasons were found to be very high (0.21) and very low (0.09), respectively, during these two months. Analyses showed that mortality in the egg stage influenced the total K value the most (Figures. 6a, b). DISCUSSION Natural populations of phytophagous insects including butterflies frequently encounter wide choice of host plants of differing suitability (Badeness et al., 2004; Dennis et al., 2006). The dominant strategy among herbivorous insects involves specialization on a set of closely related plants that will maximize offspring survival and fitness (Ward and Spalding, 1993; Gibbs et al., 2006), and also to the phenological characteristics of host plants. It is evident from the present study that C. pomona butterflies utilize three species of Cassia for oviposition and larval development in Trishna study area. Among these host plants, maximum number of C. pomona eggs were found in C. sophera with higher proportion recorded during hot and wet months, and lowest in dry and cooler months of the year. During dry and cool months, females choose C. occidentalis in higher proportion for oviposition followed by C. tora. This might be due to the availability of more young Figure 5. Regression analysis between successful development (%) of C. pomona eggs and climatic factors: (a) average temperature (oC), (b) average relative humidity (%), and (c) mean rainfall (cm).

leaves in C. occidentalis and C. tora compared to C. sophera in dry and cooler months of the year. Results indicated that common emigrants preferred C. sophera than the two other host plants but utilized three hosts

r =0.74)

(Figure

5a),

average

relative

humidity

throughout the year depending on the host plant

(y = 3.87 + 0.33x, r = 0.52) (Figure 5b), and with mean

phenology, and made the larval host range wider.

rainfall (y =20.07 + 1.64x, r = 0.64) (Figure 4c). Journal of Research in Biology (2013) 3(3): 876-885

Patterns of host use have several effects on butterfly 882

Roy Choudhury and Agarwala, 2013 relation to host plants in time and space (Begon et al., 1996; Scheirs and Bryn, 2002). Population of C.

pomona

showed strong

relationships with climatic factors. They took longer time for development in the dry and cooler months when the suitable habitat for oviposition and larval development were minimum than in wet and hot months. But, developmental time on the different host plants did not differ during a particular season that suggested possible qualitative similarity between host plants. However, several studies showed that ovipositing females of phytophagous butterflies typically show a preference for host plants that are capable of supporting fast larval growth (Thompson, 1988a, b, c; Janz et al., 1994). Climatic factors are well known for their significant influence on population dynamics of animal communities (Leonard et al. 1998). Analysis of K-value in this study has revealed that the average temperature, the average relative humidity and the mean rainfall showed strong positive relationships with survival rate of C. pomona. In the present study no biotic factors such as parasites, predators were noticed which can also Figure 6. Key- factor analysis of development of C. Pomona: (a) mortality in developmental stages expressed as k values, (b) regression fit of mortality in egg stage (k1) to the total K value.

influence the population dynamics of C. pomona butterfly. CONCLUSION

population dynamics (Hanski and Singer, 2001). Food

Results revealed that C. pomona females

plant-insect herbivore association is based on resource

occurred and laid eggs throughout the year on three host

size and optimal synchronization of their respective

plant species of Cassia. It preferred C. sophera host over

life-cycles. If resource size in time and given space is

C. occidentalis and C. tora for oviposition and larval

large, insects will show monophagism. In comparison, if

development. Pattern of egg distribution i.e. oviposition

resource size is short and patchy, then insect herbivores

was found to be linked with host plant phenology. Egg

are generally polyphagous or oligophagous (Price, 1997;

mortality

Dixon, 1998; Nylin et al., 2009). In this study,

determination of survival rate. The k-value of egg

availability of taxonomically closely related Cassia

mortality (k1) and total mortality factor (K) showed

plants in time and given area under study on which

strong positive relationship.

was

the

major

influencing

factor

C. pomona successfully completed their life history attributes might widen their host range. This finding is in conformity with the optimisation theory of species in 883

ACKNOWLEDGEMENT Authors are thankful to the Head, Department of Journal of Research in Biology (2013) 3(3): 876-885

Roy Choudhury and Agarwala, 2013 Zoology, Tripura University for the laboratory facilities.

Properties. Journal of Natural Product Plant Resources. 1(1): 101-118.

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Journal of Research in Biology (2013) 3(3): 876-885

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema Authors: Renju GL and Muraleedhara Kurup G.

ABSTRACT:

Even though role of lycopene (all-trans) in controlling inflammation was reported, lycopene (cis and all-trans 40:60) isolated from green algae Chlorella marina was not reported so far. In this present study inflammation was induced in male Sprague dawley rats and edema was produced acutely by injecting 0.1 ml of carrageenan into the plantar region of the right hind paw of the rats subcutaneously. Intra peritoneal administration of algal lycopene (AL) at the dose of 10 mg/kg b.wt Institution: Department of Biochemistry, showed maximum (83%) inhibition on paw edema. The anti- inflammatory effect was University of Kerala, significantly (P< 0.05) higher in rats fed with algal lycopene when compared to the Trivandrum, India. standard drug voveran (71%) and all- trans tomato lycopene (TL) (63%). Carrageenan induced rats showed elevated levels of cyclooxygenase (COX) and lipoxygenase (LOX) activities in monocytes. Myeloperoxidase (MPO) in serum, C- reactive protein (CRP) and ceruloplasmin activity in plasma was also high in carrageenan induced rats when compared to normal. Lycopene from Chlorella marina showed significant effect in Corresponding author: reducing the above parameters to that of the standard drug while tomato lycopene Muraleedhara Kurup G. showed less effect when compared to algal lycopene. Therefore algal lycopene from Chlorella marina would be recommended for the treatment of anti-inflammatory disorders. Email: gmkbio@gmail.com.

Phone: +919447251408. Fax: 91-471 2308078. Web Address:

Keywords: Microalgae, Chlorella marina, lycopene, anti-inflammation.

Article Citation: Renju GL and Muraleedhara Kurup G. Anti-inflammatory activity of lycopene isolated from Chlorella marina on carrageenan-induced rat paw edema. Journal of Research in Biology (2013) 3(3): 886-894 Dates: Received: 02 Feb 2013

Accepted: 22 Feb 2013

Published: 23 Apr 2013

http://jresearchbiology.com/ documents/RA0329.pdf. This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology An International Scientific Research Journal

886-894 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Renju and Kurup., 2013 INTRODUCTION

was that algal lycopene contain cis-configuration

Inflammation is a response which protects and

(5-cis, 9-cis, 13- cis and 15-cis). Recently it has been

heals the host tissue after infection or injury. (Nathan,

reported that the cis form of lycopene is more

2002). However, it is frequent that the inflammatory

biologically active than the trans form (Stahl and Sies,

response to several insults erroneously leads to the

1996).

damaging of normal tissues. Prostaglandin-E2 is generated from arachidonic acid by the enzyme

MATERIALS AND METHODS

cyclooxygenase (COX) at sites of inflammation in

Chemicals

substantial amounts and can mediate many of the

Lycopene,

carrageenan,

linoleic

acid,

pathologic features of inflammation (Serhan and Levy,

Histopaque, arachidonic acid other fine chemicals were

2003). One of the early cellular events in inflammation is

purchased from Sigma, St. Louis, MO, USA. Diclofenac

the margination of leukocytes, primarily neutrophils and

sodium (Voveran) was obtained from Novartis, India.

this can be measured by myeloperoxidase activity

Salt and vitamin mixtures were purchased from Merck,

(Goulet et al., 1994).

Germany. All other chemicals and reagents were

Currently, non steroidal anti-inflammatory drugs

purchased from Sisco Research Laboratory Pvt.Ltd

(NSAIDs) were used for inflammatory diseases. Even

(SRL), India, and were of analytical grade.

though this drugs transiently suppresses inflammation,

Algal source

but their long term use cause ulceration in the

Marine algae Chlorella marina Butcher was

gastrointestinal tract and renal morbidity (James and

collected from the Vizhinjam coast of Kerala, located at

Hawkey, 2003). However research focused on finding

Latitude 08° 22’ North Longitude. 76° 59’ East on the

newer drugs with pharmacological actions without side

south west coast of India and was cultured under

effects.

laboratory conditions. The microalgae were identified by Several antioxidants have been reported to

the botanist (Dr. G. Valsaladevi, Department of botany)

have anti-inflammatory and anti-arthritic activities

and a voucher specimen (No. KUBH 5812) has been

(Maxwell et al., 2006). In the present study a culturable

deposited in the Department of Botany, University of

marine edible algae Chlorella marina was selected to

Kerala, India.

evaluate the anti-inflammatory activity of lycopene.

Culture medium

Generally tomatoes are the source of lycopene, but it has

Walne’s medium (1970) was used as a basal

many disadvantages (Shi and Le mague, 2000). The

medium for the cultivation of Chlorella marina. 5 g /L

content of lycopene in tomato is very less and the

glucose was added to the basal medium. Flasks were

configuration of lycopene is all-trans. Even though

incubated at 25°C with continuous illumination. The pH

lycopene from algae has been reported (Ishikawa and

was adjusted to 7.5. Nicotine (10 µM/ L) was sterilized

Abe, 2004), no attempt has been made so far for the

by autoclaving and was added to 5 days old cultures for

commercialization of algal lycopene. It can be seen that

the production of lycopene.

marine sources especially algae are the least exploited

Biomass harvest

for their bioactive molecules (Pinky and Goswai, 2012).

Chlorella marina cells were grown in suspension

Work in our laboratory has shown that the lycopene

cultures up to 30 to 40 days. The cells were harvested at

content in algae is comparatively high, when compared

stationary phase by withdrawing the cultures in 50 ml

to tomato lycopene. The most interesting observation

polypropylene tubes and centrifuged at 5000 rpm for

887

Journal of Research in Biology (2013) 3(3): 886-894

Renju and Kurup., 2013 10 minutes. Removed the medium and the pellets were

concentrations of different fractions. All samples were

freeze dried, weighed and stored under nitrogen at -20°C.

injected in duplicate.

Isolation of lycopene from Chlorella marina (AL) and

Experimental animals Male Sprague Dawley rats with the average body

analysis Harvested

biomass

(5g

dry

weight)

was

weight of 150- 200 g of the same breed were selected for

suspended with 5 ml of 80% cold acetone and kept

the study. These animals were housed in the department

overnight under 4°C for better and easy recovery of

animal house and provided standard pellet diet and water

carotenoids. The mixtures were vortexed for 2 minutes

ad libitum and maintained with temperature at 25 ± 1°C,

and centrifuged at 5000 rpm for 20 minutes. After

humidity (55-60%) and photoperiod (12:12 h) light and

repeated extractions (4 times), the supernatants were

dark cycle. Experimental procedures conducted on rats

pooled and the colorless cell pellets were discarded. The

were approved by the Animal Experiment Committee

extracts were dried over anhydrous sodium sulphate and

(218/CPCSEA) for animal care of Kerala University

reduced to a minimum volume by evaporating the

according to Government of Indian law on animal use

solvents using N2 stream. The crude extracts were kept

and care.

for further separation of carotenoids in amber colored

Induction

containers under nitrogen at -20°C. All operations were

induced rat paw edema

acute

inflammation-Carrageenan

done at subdued light under nitrogen atmosphere. The

Carrageenan-induced rat paw edema assay was

absorbance in the solvent phase was quantified by

conducted according to the procedure as described by

spectrophotometric method at 470 nm as described by

Winter et al., (1962). Five groups of six

Lichtenthaler (1987).

treated as AL and TL with doses 10 mg/kg and reference

Isolation of all-trans lycopene from tomato (TL)

drug

Voveran,

Diclofenac

sodium

rats were preparation

Tomatoes obtained from the local market,

(20 mg/kg) were given orally and intraperitoneally (i.p),

Trivandrum, India were used. The all-trans lycopene

1 h before the injection of carrageenan. Control rats were

from tomato was extracted and evaluated according to

given 0.1 ml 1% carrageenan.

the procedure of Fish et al., (2002).

induced by 0.1 ml, 1% carrageenan suspension in

Determination of lycopene by HPLC

0.9% NaCl solution was injected into the right hind paw

Inflammation was

Lycopene extracted from algal cells and tomatoes

after 1 hour. The volume of the right paw was measured

were determined by HPLC method at 450 nm as

by paw edema meter before and after injection in

described by Shaish et al., (1992). HPLC analysis of

the third and fifth hour. The paw edema and inhibition

lycopenes were performed using a silia chrom® column

was calculated by the equation: Activity= 100 - (100 ×

(250 x 4mm + 5 x 4, NCLIOSIL 100-5-C18 5.0µm),

average drug treated/average for control).

K 1001 type pump and the UV detector type of K 2600,

Treatment Protocol and Experimental Design in

Germany. Elution was performed isocratically with

Acute Inflammation

methanol: acetonitrile (9:1) v/v at a flow rate of -1

Edema was induced on rat right hind paw by

1 ml min . A UV detector with a wavelength of 450 nm

aponeurosis injection of 0.1ml of 1% carrageenan in

was employed. Lycopene (95%) obtained from Sigma

0.9% saline. The experimental groups consisted of 30

chemicals were used as standard. The retention time was

rats were divided in to five groups.

recorded and peak areas of standards and tests were

Group I: control (received saline only),

noted on each run and used for calculation of

Group II: carrageenan alone

Journal of Research in Biology (2013) 3(3): 886-894

888

Renju and Kurup., 2013 Group III: carrageenan + algal lycopene (AL groups,

carrageenan-induced paw edema. Algal lycopene showed

10 mg/kg i.p)

maximum edema inhibition compared to all-trans tomato

Group IV: carrageenan + tomato lycopene (TL groups,

lycopene and drug. AL exhibited 70% and 83% edema

10 mg/kg i.p)

inhibition at third/fifth hours, respectively. This effect

Group V: carrageenan + Voveran (VOV groups,

was comparable to the reference drug Voveran which

20 mg/kg i.p.)

exerted 54% and 71% edema inhibition at third and fifth

At the end of third hour, the animals were

hour, respectively. TL showed 51% and 63% edema

sacrificed by euthanasia. Blood was removed to ice cold

inhibition at third and fifth hour after carrageenan

containers for various biochemical analyses.

induction (Figure. 1).

Activity

Cyclooxygenase

Lipooxygenase

(LOX)

(COX)

Peripheral

and

COX activity in PBMC was significantly

Blood

(p<0.05) increased in carrageenan treated rats when compared to control rats (Figure. 2). Treatment with AL

Mononuclear Cells (PBMC) Mononuclear cells were isolated the procedure

showed significant (p<0.05) decrease in COX activity

described by Radhika et al., (2007). Cox activity was

when

measured by the method of Shimizu et al., (1984).

Prostaglandin is formed by the interaction of two distinct

15-LOX activity was determined

but related enzymes, COX-1 and COX-2 and plays an

by the method of

compared

carrageenan

induced

rats.

Axelrod et al., (1981).

important role in promoting the signs and symptoms

Biochemical analysis

inflammation

(Otterness

and

Bliven,

1985;

Serum myeloperoxidase (MPO) activity was

Ibegbulem et al., 2012). The activity of COX in PBMC

measured by Mullane et al., (1985). CRP in plasma was

was decreased (p<0.05) in AL treated group when

determined by using Immunoturbidometric kit (Diasys

compared to TL and voveran treated group. Reduction of

Diagnostics, Germany). Ceruloplasmin was estimated by

paw swelling and decreased activity of COX showed the

the method of Ravin (1961). Protein was determined by

immunological

the methods of Lowry et al., (1951).

lycopene. These results showed the anti-inflammatory

Statistical analysis

potential of the AL.

protection

rendered

by the

algal

The Statistical package for social sciences

The activity of 5-LOX and 15-LOX in PBMC

(SPSS/PC+), version 11.5 (SPSS Inc; Chicago. IL, USA)

was significantly (p<0.05) increased in carrageenan

was used to analyze the results for statistical significance

induced

using one-way ANOVA followed by Duncanâ&#x20AC;&#x2122;s test.

(Figure.3 and 4). Algal lycopene treatment significantly

P value < 0.05 was considered as significant.

reduced (p<0.05) in 5-LOX and 15-LOX activity, when

rats

when

compared

normal

rats

compared to CII rats. The effect was significantly higher RESULTS AND DISCUSSION

(p<0.05)

than

and

drug

treated

groups.

Sub plantar injection of carrageenan into the foot

Lipoxygenases are a family of key enzymes in the

of rats caused a time-dependent increase in paw volume.

biosynthesis of leukotrienes that are postulated to play an

The localized inflammatory response as evidenced

important role in the pathophysiology of several

visually by the edema reached a maximum intensity at

inflammatory diseases (Henderson, 1994; Yamamoto,

third hour after carrageenan induction and this maximal

1992). In the normal situation, cellular leukotriene

effect was seen until the fifth hour. Administration of AL

production is suppressed by selenium dependent

and TL has showed significant effects in decreasing

peroxidases

889

(Werz

al.,

1997).

receiving

Journal of Research in Biology (2013) 3(3): 886-894

Renju and Kurup., 2013 inflammatory stimuli, leukotriene production is elicited

Table 1 also shows the variations in serum CRP

through the arachidonic acid cascade, causing micro

and ceruloplasmin level in the test animals compared to

vascular injury, vasoconstriction and production of

control. Serum CRP and ceruloplasmin levels were

pro-inflammatory cytokines (Peskar, 1991). Studies have

significantly increased (p<0.05) in carrageenan induced

shown that LOX and leukotrienes have a profound role

rats when compared to normal rats. Supplementation

in carrageenan-induced inflammation (Henderson, 1994;

with AL significantly decreased (p<0.05) the serum

Gamache et al., 1986). In the carrageenan-induced

CRP and ceruloplasmin levels when compared to

inflammation

reduced

carrageenan induced rats. The levels of CRP and

carrageenan-induced 5-LOX and 15-LOX activities in

ceruloplasmin were decreased significantly (p<0.05),

mononuclear cells, indicating decreased leukotriene

when compared to TL and Voveran treated groups.

production and hence a protective effect.

C-reactive protein is an acute phase protein that has been

model,

significantly

MPO activity in serum was significantly

identified as an important biomarker for various

increased (p<0.05) in carrageenan induced rats when

inflammatory, degenerative, and neoplastic diseases.

compared to normal group (Table 1). Treatment with AL

Elevated levels of CRP have been found in the blood

showed significant decrease (p<0.05) in MPO activity

during virtually all diseases associated with active

when compared to carrageenan induced rats. The MPO

inflammation or tissue destruction, particularly in

activity was significantly decreased when compared to

patients with rheumatoid arthritis (Pepys and Hirschfield,

TL and drug treated groups. The activity of MPO is a

2003; Kushner, 1991). In our study the increased levels

marker of neutrophil infiltration (Bradley, 1982), and was found to be significantly increased in the paw tissue of carrageenan-induced rats. AL significantly decreased (p<0.05) the elevated MPO activity, an indicator of neutrophil in inflamed paws, suggesting that inhibition of neutrophil infiltration might be another mechanism by which AL achieves its anti-inflammatory effect.

Figure 1: Effect of algal lycopene on carrageenaninduced paw edema in normal and experimental rats.

Journal of Research in Biology (2013) 3(3): 886-894

Figure 2: Effect of algal lycopene on activity of COX in PBMC of normal and experimental rats COX activity is expressed as an optical density increase (OD increase) per mg protein per minute. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05.

890

Renju and Kurup., 2013 of CRP level was found to be significantly decreased in

compared to TL and standard drug Voveran might be

algal lycopene treatment when compared to TL and

having a protective response against free radical

Voveran treatments.

mediated lipidperoxidation.

The serum protein, ceruloplasmin is a powerful

Lycopene

from

edible

marine

microalgae

free radical scavenger that oxidizes iron from the ferrous

C. marina showed higher anti-inflammatory activity than

to ferric state. Ceruloplasmin levels increase under

all-trans tomato lycopene and standard drug Voveran.

conditions leading to the generation of oxygen products

These effects might be due to the presence of two

such as the superoxide radical and hydrogen peroxides

isomeric form of lycopene (cis and all-trans) in the

(Revnic,

microalgae.

1995).

Serum

ceruloplasmin

level

was

Reports

available

indicate

that

the

significantly increased in carrageenan induced rats when

cis-lycopene has a high antioxidant potential when

compared to normal rats. Treatment with AL showed

compared to all-trans lycopene (Stahl and Sies 1992;

significant

Clinton et al., 1996). Algal lycopene isolated from

decrease

the

concentration

ceruloplasmin. The increased levels of ceruloplasmin in carrageenan

induced

rats

could

C. marina could reduce cell influx, oedema formation

decreased

significantly on treatment with algal lycopene when

Figure 3: Effect of algal lycopene on activity of 5- LOX in PBMC of normal and experimental rats 5-LOX activity is expressed as an optical density increase (OD increase) per mg protein per minute. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05.

891

Figure4: Effect of algal lycopene on activity of 15- LOX in PBMC of normal and experimental rats 15-LOX activity is expressed as an optical density increase (OD increase) per mg protein per minute. Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05. Journal of Research in Biology (2013) 3(3): 886-894

Renju and Kurup., 2013 Table 1: Levels of CRP, Ceruloplasmin in plasma and MPO in serum of experimental animals. Groups

MPO (µm/min/mg)

CRP (mg/ml)

Ceruloplasmin (mg/dl)

Control

5.85 ± 0.37

22.0 ± 1.24

0.10 ± 0.006

CII

20.52 ± 1.11a

97.71 ± 3.80a

0.34 ± 0.014a

7.45 ± 0.37bc

45.94 ± 2.01bc

0.19 ± 0.007bc

13.75 ±0.48bcd

80.64 ±3.18bcd

0.28 ± 0.016bcd

VOV

9.74± 0.39b

56.89 ± 2.42b

0.22 ± 0.010b

Values are expressed as mean ± SEM of six rats in each group. a – Statistical difference of Control group with CII group when p < 0.05. b – Statistical difference of CII group with group AL, TL and VOV when p < 0.05. c – Statistical difference of VOV group with group AL and group TL when p < 0.05. d –Statistical difference of TL group with AL when p <0.05.

Table 4: Statistical table of CRP in one way ANOVA followed by Duncan’s test CeruloplasSum of Mean min Squares df Square Between .196 4 .049 Groups Within .021 25 .001 Groups Total .217 29 Where df is degrees of freedom, F is F- ratio.

F 59.148

and release of mediators associated with inflammatory condition, and therefore has the potential to be used as an anti-inflammatory agent. Further studies are in progress to evaluate the molecular mechanism of its anti-inflammatory activity. ACKNOWLEDGEMENT We express gratitude to Dr. Anantha Lekshmi, Veterinary

Doctor,

Department

Biochemistry,

University of Kerala, Kariavattom, India for helping us with the animal experiments.

Table 2: Statistical table of Myeloperoxidase in one way ANOVA followed by Duncan’s test

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MPO Between Groups Within Groups Total

Sum of Squares

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206.911

56.842

2.274

884.485

F 91.002

Rothstein

Total

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1083.082

22067.705

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Journal of Research in Biology (2013) 3(3): 886-894

894

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Identification of Animal Pasteurellosis by PCR Assay Authors: Venkatesan PS, Deecaraman M and Vijayalakshmi M.

Institution: Department of IBT, Dr. M.G.R. Educational & Research Institute, Department of IBT, Maduravoyal, Chennai - 600095.

ABSTRACT: Diagnosis of pasteurellosis has become difficult, as there are five different capsular types and 16 somatic types. Molecular techniques like PCR are adapted nowadays for rapid and accurate diagnosis in early stage of the disease and also it provides useful information for epidemiological studies. The present study was conducted to study the efficiency of polymerase chain reaction (PCR) in the identification of P. multocida isolates and evaluation of different PCR methods viz., (i) PCR using genomic DNA (ii) PCR using culture lysate and (iii) PCR by colony touch method. In the present study P. multocida specific PCR was performed by using KMT1SP6 and KMT1T7 oligos. These oligos amplified the genomic DNA from P. multocida isolates only. All the three methods produced PCR amplified product at 460 bp and colony touch method was found to be the best method.

Corresponding author: Venkatesan PS.

Keywords: Culture lysate, genomic DNA, Pasteurella multocida, PCR .

Email: venkyvet74@gmail.com

Article Citation: Venkatesan PS, Deecaraman M and Vijayalakshmi. Identification of animal Pasteurellosis by PCR assay. Journal of Research in Biology (2013) 3(3): 895-899

Web Address:

Dates: Received: 04 Feb 2013

http://jresearchbiology.com/ documents/RA0332.pdf.

Accepted: 05 Mar 2013

Published: 30 Apr 2013

Journal of Research in Biology An International Scientific Research Journal

895-899 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Venkatesan et al., 2013 incubated at 37°C with 5-10 % CO2 for 24-48 h. Plates

INTRODUCTION The various forms of pasteurellosis caused by

were examined for colonies, the suspected colonies were

Pasteurella multocida are the major health problem for

subjected to grams staining, and biochemical test as

livestock

per standard techniques. Standard vaccine strain of

pasteurellosis has become difficult, as there are five

P. multocida P52 (B:2) was taken as reference strain.

different capsular types and 16 somatic types. Molecular

Pathogenicity test in mice were carried out for all the

techniques like PCR are adapted nowadays for rapid and

fifteen isolates and PCR was performed for all the

accurate diagnosis in early stage of the disease and also it

isolates.

provides useful information for epidemiological studies.

Isolation and Purification of Genomic DNA

population

worldwide.

Diagnosis

Pasteurellosis has high impact on economic status of Indian

farmers.

rate

resuspended in 100 µl of 10x Tris-EDTA (TE) buffer

reported

(pH 8.3) with 10 mg of lysozyme and were incubated at

6.4 per lakh population during 1974-86, resulting in

37°C for 1.5 h. Bacterial cultures were treated with 10 µl

losses

of proteinase K (10 mg/ml) and incubated at 50°C

haemorrhagic

The

overall

septicaemia

exceeding

ten

(HS)

incidence

A 900 µl cell suspension of each sample were

was

million

rupees

annually

(Dutta et al., 1990; singh et al., 1996).

for 1 h. The nucleic acid was extracted with

Isolation and identification of P. multocida from

phenol-chloroform-isoamyl alcohol followed by ethanol

specimens like fresh tissues or heart blood followed by

precipitation as per the method of Sambrook et al.,

the performance of various biochemical and serological

(1989) and Sachithanandam et al., (2011).

methods have been used to study P. multocida. These

PCR Using Culture Lysate

include catalase, indole, oxidase and sugar fermentation

One Milliliter of 18 h broth culture or take few

tests. Due to time consuming procedure and limitations

freshly grown pure colonies from blood agar plate and

of these methods, molecular techniques like polymerase

suspend in 500 µl sterile distilled water and centrifuge at

chain reaction (PCR) were adapted nowadays. PCR has

4000 g for 1 minute and collect the pellet. The pellet was

advantages over the conventional techniques in rapidity,

washed with sterile distilled water, resuspended in 100 µl

sensitivity and specificity to identify the P. multocida.

sterile distilled water and boiled for 10 min. The samples

The present study was conducted to assess the efficiency

were centrifuged to sediment cell debris and 10 µl of the

of PCR in the identification of P. multocida from poultry

supernatant was used in the PCR reaction.

and ruminants and to evaluate the different methods in

PCR Using Colony Touch Method

PCR assay viz. PCR using genomic DNA, PCR using culture lysate and PCR by colony touch method.

A single pure colony grown on agar plates was used to perform PCR. A pipette tip was lightly touched onto a colony and then suspend in PCR amplification

MATERIALS AND METHODS

mixture.

Isolation and Identification of P. multocida

PCR Technique

Fifty two samples were collected from various

The species-specific primers KMT1SP6 and

geographical areas of Tamil Nadu, India. Specimens

KMT1T7 designed by Townsend et al., (1998) were used

such as heart blood swab, liver, spleen and long bones

in this study to amplify the gene sequences in

collected from various animals, were streaked directly

P. multocida.

onto 5% sheep blood agar and Pasteurella multocida

Primers 1 KMT1SP6 5’-GCT GTA AAC GAA CTC

selective agar as reported earlier (Moore et al., 1974) and

GCC AC- 3’

896

Journal of Research in Biology (2013) 3(3): 895-899

Venkatesan et al., 2013 Primers 2 KMT1T7 5’- ATC CGC TAT TTA CCC AGT

Finland).

GG-3’

The biochemical tests were carried out as per the PCR mixture was prepared using PCR kit

standard procedure followed in Arun kumar et al., (2012)

obtained from FINNZYME, Finland. The 50 µl of reaction mixture was prepared with 10 µl template DNA,

RESULTS

10ng of each primers, 200 µM concentration of each

Out of total collection of 52 suspected samples,

dNTPs, 10x PCR buffer and 1 unit Taq DNA

procured from cattle sheep, goat and poultry, 15 samples

polymerase. PCR amplification was carried out in an

were confirmed as P. multocida based on biochemical

automated thermal cycler (Perkin Elmer Gene AMP PCR

tests (Table 1) and PCR. All the P. multocida isolates

system 2400) with the following thermal programme.

were pathogenic to mice and dies within 24 h. PCR was

Initial denaturising at 95°C for 4 min followed by

performed for all the 15 isolates by 3 methods viz.,

30 cycles of denaturising at 95°C for 1 min., annealing at

colony touch method, culture lysate and with genomic

55°C for 1 min., extension at 72°C for 1 min. and final

DNA. P52 strain of P. multocida, obtained from the

extension at 72°C for 9 min, were carried out. After

Institute of veterinary preventive medicine (IVPM)

amplification,

Ranipet, Tamil Nadu, taken as a positive control

PCR

products

were

checked

1.5% agarose gel electrophoresis along with the standard

and

the

following

bacteria

molecular weight marker (Lambda DNA Hind III digest

Clostridium

chauvoei,

and ϕ X 174 DNA Hae III digest; FINNZYME,

Salmonella

typhimurium,

Escherichia

Salmonella Bacillus

coli,

enteritidis, anthracis,

Table 1. Biochemical Profiles for the Identification of Pasteurella multocida Isolates Tests Hemolysis on Blood agar Growth on MacConkey agar Motility Gelatin Liquefaction Methyl Red Test H2S (Hydrogen sulphide) Catalase Oxidase Nitrate Reduction Indole Lysine Decarboxylase Ornithine Decarboxylase Urease Pyrase Esculin Hydrolysis VT (Voges Proskaeur Phenylalanine β-Galactosidase (ONPG) β-Glucuronidase α-Galactosidase β-Xylosidase N-acetyl β-D-glucosaminedase

Name of the Isolates D1P

D2P

+ + + + -

+ + -

+ + + + -

+ + -

+ : Positive, - : Negative,

Journal of Research in Biology (2013) 3(3): 895-899

897

Venkatesan et al., 2013 D1P

D 2P

460bp→

Figure 1: Pasteurella multocida – specific PCR (PM-PCR) assay These figures illustrate fragments specifically amplified by PCR in all the P. multocida isolates by means of the primers KMT1SP6 and KMT1T7. Variation in the intensity of the amplified product was observed, due to variation in DNA concentration of each sample. D1P, D2P, FP, GP, HP, KP, LP, NP, OP, AS, CS, TS, YS, BG, and MC are the names of P. multocida isolates. Staphylococcus aureus and Klebsiella spp. used as

reference strain P52, but no amplified product was

negative controls. The expected amplification size of

noticed among the negative controls. It is concluded that

460 bp was obtained in all the 15 isolates. PCR

the primers were highly specific to P. multocida isolated

amplification was noticed at approximately 460 bp by all

from various sources. The above result agrees with the

the three methods and in all the 15 isolates as like that of

previous reports of earlier workers (Townsend et al.,

positive control (figure 1). No amplification product was

1998; Hunt et al., 2000; Miflin and Blackall, 2000; OIE

observed in negative controls (figure 1). Molecular

manual, 2000; Dutta et al., 2001). In this study the

weight of PCR product was estimated based on the

amplified product of approximately 460 bp was observed

standard molecular weight marker.

using three different methods viz. colony touch method, culture lysate method and purified genomic DNA

DISCUSSION

method (figure 1). The intensity of the amplified PCR

The 15 isolates of P.multocida collected from

product varies (figure 1), due to the variation in DNA

different places and sources of origin produced

concentrations. Townsend et al., (1998) reported that

approximately 460 bp amplified product as that of

PCR using colony touch method produced amplification

898

Journal of Research in Biology (2013) 3(3): 895-899

Venkatesan et al., 2013 product approximately at 460 bp and the intensity of the amplified product varied due to inconsistency of the DNA concentration. Dabo et al., (2000) reported that the boiled cell extract method has the advantages of simplicity and rapidity in

the

identification

P. multocida isolates. Since the PCR amplified product of 460 bp was noticed in all samples of poultry and ruminants, using oligos KMT1SP6 and KMT1T7, the oligos are considered as specific to P. multocida affecting all

species

of poultry and ruminants.

Considering the cost and time involved in the preparation and purification of genomic DNA, the colony touch method has advantages of simplicity and rapidity for epidemiological surveys involving large number of P. multocida isolates. PCR using colony touch method would be an adaptable easy to perform method in regional laboratories for rapid diagnosis of HS and FC from field cases without the need to obtain pure culture and extensive biochemical and serological tests. ACKNOWLEDGEMENT The authors thank the Head, department of microbiology, Madras Veterinary College, Chennai, for providing the facilities to carry out this work. REFERENCE Arun Kumar JM, Lakshmi A, Sangeetha Rani V and Sailaja B. 2012. Isolation and characterization of feather degrading bacteria from poultry waste. Journal of Research in Biology. 2(7): 676-682. Blackall PJ and Miflin JK. 2000. Identification and typing of Pasteuruella multocida; A Review. Avian Pathology 29(4):271-287. Dabo SM, Confer A and Lu YS. 2000. Single primer polymerase chain reaction fingerprinting for Pasteurella multocida isolates from laboratory rabbits. American Journal of Veterinary Research 61(3):305-309. Dutta J, Rathore BS, Mullick SG, Singh R and Sharma GC. 1990. Epidemiological studies on

Journal of Research in Biology (2013) 3(3): 895-899

occurrence of haemorrhagic septicaemia in India. Indian Veterinary Journal 67(10): 893-899. Dutta TK, singh VP and Kumar AA. 2001. Rapid and Specific diagnosis of animal pasteurellosis by using PCR assay. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases 22(1):43-46 Hunt ML, Alder B and Townsend KM. 2000. The molecular biology of Pasteurella multocida. Veterinary Microbiology 72(1-2):3-5. Manual of Standards for diagnostics tests and vaccines. 2000. Office International Des Epizootics Manual, France. 446-456. Sachithanandam V, Mohan PM, Dhivya P, Muruganandam N, Baskaran R, Chaaithanya IK and Vijayachari P. 2011. DNA barcoding, phylogenetic relationships and speciation of Genus: Plectropomus in Andaman coast. Journal of research in Biology. 1( 3): 179-183. Sambrook J, Fritisch EF and Mamiatis T. 1989. Molecular cloning: a laboratory manual, Cold spring harbor press, Plainview, N.Y. 2nd ed. 3. Singh VP, Kumar AA, srivastava SK and Rathore BS. 1996. Significance of Haemorrhagic Septicaemia in Asia: India. International workshop on diagnosis and control of Haemorrhagic Septicaemia. Bali, Indonasia. May 28-30. Townsend KM, Frost AJ, Lee CW, Papadimitrion JM and Dawkins HJS. 1998. Development of PCR assays for species and type specific identification of Pasteurella mutlocida isolates. Journal of Clinical Microbiolgy 36(4):1096- 1100. Submit your articles online at www.jresearchbiology.com Advantages

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.

899

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Source of light emission in a luminous mycelium of the fungus

Journal of Research in Biology

Panellus stipticus Authors: Puzyr Alexey, Burov Andrey and Bondar Vladimir.

ABSTRACT:

Mechanism of bioluminescence and light-emitting sources in higher fungi remain as an open question for a long time. We investigated the mycelium of cultivated luminous Panellus stipticus using confocal microscopy. No excitation light was imposed on the sample. Two types of sources of bioluminescence and their Institution: location were determined in the substrate mycelium. One were small 0.1-3 Âľm local 1. Institute of Biophysics SB formations disposed on the surface of hyphae, the other - relatively vast areas in bulk RAS, Krasnoyarsk. of the nutrient medium. No luminescence signal was recorded inside the hyphae. This may mean that the components of luminescent reaction are spatially separated within 2. Special DesignTechnology Bureau "Nauka" the cells, or the intracellular conditions block the reaction. The origin and formation of KSC SB RAS, Krasnoyarsk. the light-emitting structures are discussed. 3. Institute of Biophysics SB Keywords: RAS, Siberian Federal Bioluminescence, University Krasnoyarsk. microscopy.

Corresponding author: Burov Andrey. Email: andrey@icm.krasn.ru

Panellus

stipticus,

luminous

mycelium,

confocal

Article Citation: Puzyr Alexey, Burov Andrey and Bondar Vladimir. Source of light emission in a luminous mycelium of the fungus Panellus stipticus. Journal of Research in Biology (2013) 3(3): 900-905 Dates: Received: 02 Apr 2013

Accepted: 27 Apr 2013

Published: 06 May 2013

Web Address:

http://jresearchbiology.com/ documents/RA0345.pdf. This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

Journal of Research in Biology An International Scientific Research Journal

900-905 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Puzyr et al., 2013 widely

INTRODUCTION Bioluminescence in fungal cells, which involves the emission of light generated by a chemical reaction, has long attracted attention of scientists (Harvey, 1952;

used

investigate

non-luminous

fungi

(Riquelme and Bartnicki-Garcia, 2008; Roberson et al., 2011; Steinberg and Schuster, 2011). In this report the mycelium of luminous

Shimomura, 2006; Desjardin et al., 2008). Researchers

Panellus

studying bioluminescence of fungi focus their efforts on

microscopy to determine and localize the source of light

three key areas: (i) methods of cultivation under

emission. In our opinion it is important to find in

laboratory conditions and characteristics of the light

luminous fungi structures (or formations), which are the

emission (Weitz et al., 2001; Prasher et al., 2012; Dao,

light-emitting sources, and their location. On the one

2009; Mori et al., 2011), (ii) the molecular organization

hand, this can provide additional knowledge about

of luminescence system and light emission mechanism

morphology of luminous fungi, on the other - might give

(Shimomura,

insight into molecular-cellular organization of fungal

2006;

Airth

and

McElroy,

1959;

Kamzolkina et al., 1983; Oliveira and Stevani, 2009;

stipticus

was

studied

using

confocal

luminescent system and mechanism of light emission.

Bondar et al., 2011), (iii) - application of fungal luminescence in analytical techniques (Weitz et al., 2002; Mendes and Stevani, 2010).

MATERIALS AND METHODS In this work we studied the culture of

There has been little research conducted to

Panellus stipticus luminous fungus (Bull:Fr.) Karst.,

determine sources of luminescent light in the fungal

IBSO 2301 (Figure 1). The mycelium was grown in

structures. To the best of our knowledge, only the

plastic Petri dishes at temperature 22°С on a commercial

mycelium of Panus stipticus and Armillaria fusipes,

nutrient medium Potato Dextrose Agar (HiMedia

growing on agar were investigated for light source

Laboratories Pvt., India), or on richer medium containing

detection (Berliner and Hovnanian, 1963). The used

in 1 liter: 10 g of glucose, 5 g of peptone, 3 g of yeast

photographic process allowed to record light from a

extract, 3 g of malt extract, 20 g of agar-agar. The

single hypha.

specimens exhibiting the highest light intensity were

However, a low resolution of the technique

selected for the experiments.

limited by the emulsion grain size denied localizing the

For confocal microscopy, a confocal laser

source of light. The authors of this, obviously, pioneer

scanning microscope (LSM-780 NLO, Carl Zeiss,

work, suggested that the light was emitted over the entire

Gottingen, Germany) equipped with a high sensitivity

cell. Given the size of the objects under study, such

GaAsP was used. Bioluminescence was recorded in the

research should employ methods of microscopic

accumulation mode with the 491–631 nm filter. The laser

investigations. Calleja and Reynolds, who studied

was turned off (laser power = 0.0%) so that no excitation

Panus stipticus and Armillaria mellea by optical

light was imposed on the sample. This was done to avoid

microscope with EMI 4-stage image intensifier tube,

fungal autofluorescence - emission of light by biological

came to the conclusion that light emission in an

substances such as flavins, lipofuscins and porphyrins

individual hypha was limited to a segment removed from

when excited by ultraviolet, violet, or blue light (Zizka

the apical point (Calleja and Reynolds, 1970). Absence

and Gabriel, 2008).

of later works related to structural and morphological

Images were processed using ZEN 2010 software

studies of mycelium of luminous fungi with microscopy

(version 6.0; Carl Zeiss). To prepare a specimen for

is astonishing as all known microscopic methods are

microscopy a fragment of agar with mycelium was cut

901

Journal of Research in Biology (2013) 3(3): 900-905

Puzyr et al., 2013

Figure 1 View of culture of Panellus stipticus (IBSO 2301) growing on agar in natural light (A) and in the dark (B).

out and transferred to the cover glass.

of hyphae could be assumed, finding of luminescent areas in the agar came as a surprise. It is uncontroversial

RESULTS AND DISCUSSION

that the recorded bioluminescent signals result from the

Figure 2 shows a 3D projection of the mycelium

interaction of mixing light components synthesized by

by producing a Z-stack with 82 sections, 0.208 μm thick

the fungal cells. Luminescent signals were recorded by

each. No bioluminescence was detected from the aerial

the confocal microscope only when these components

mycelium. The light emission was recorded from the

were outside the cells. No bioluminescence inside

surface of specimen to a depth of ~ 16 μm with

hyphae may mean that inside the cells the components of

maximum intensity localized at the depth of Z= 6-8 μm

luminescent reaction are spatially separated and do not

where the main body of mycelium was located. Only

interact with each other, or the intracellular conditions

isolated signals were detected at Z=8-16 μm that

(pH, oxygen concentration, presence of inhibitors, etc.)

confirmed that the agar did not contribute to the observed

block the reaction. One could argue that the surface of glowing

bioluminescence. Two types of sources emitting luminescent

structures should be either hydrophobic or they have a

signals could be distinguished. One light source were

membrane enclosing the internal volume. Only under

small 0.1-3 µm local formations, associated with the

these

substrate hyphae, the other – vast areas in bulk of agar

luminescent reaction do not mix with the water phase

(Figure 3). Light intensity recorded in the agar was much

contained within the nutrient medium. This suggestion is

higher than that of the local sites in the area of hyphae.

based on the sharp boundaries exhibiting by both small

The use of the larger magnification (Figure 4) and bright

local formations on the walls of hyphae and vast areas in

field microscopy (Figure 5a) suggests that the local

the nutrient agar (Figure 5b).

conditions

components

necessary

for

the

luminous sites are cellular excretions located on the

So far it is not clear whether the luminous

hyphae surface while vast luminescent areas are formed

structures containing components necessary for the

by their aggregation in agar.

emission are formed within the fungal hypha or on/in

While presence of luminous sites on the surface

their surface. In the first case it requires a transport system providing for the mechanism excreting the

Journal of Research in Biology (2013) 3(3): 900-905

902

Puzyr et al., 2013

Figure 2 Fragment of 3D pattern of bioluminescence produced by P. stipticus.

20Âľm 5Âľm

Figure 3 Confocal luminescence image of the P. stipticus mycelium.

Figure 4 Confocal luminescence image of an individual hyphae.

forming structures outside the cell. This is plausible

are moved on the outside surface of the hyphae by

because the Golgi apparatus, that synthesizes secretory

a mechanism analogous to the mechanism of transport

vesicles containing products of vital functions and

via the Golgi complex. They can be also assumed to

excretes them from the cell, is well known. In the second

form directly on/in outside surface of the hyphae by

case on/in the wall cell there should exist structural

structural elements of the cell possessing secretory

elements performing specialized secretory function.

function. Such enclosed structures make possible to

On the basis of the results above we hypothesize

concentrate the necessary components within a small

the following. Cells of P. stipticus synthesize and

volume. Separation of luminous structures from the

localize the components required for bioluminescence in

surface of hyphae and their subsequent diffusion into the

structures which can originate within the cell and then

bulk of the nutrient medium produce the vast

903

Journal of Research in Biology (2013) 3(3): 900-905

Puzyr et al., 2013

Figure 5 Confocal luminescence (A), bright field (B) and overlay (C) images of the substrate. Scale bar = 20 μm. areas of luminescence in the agar.

Program of the Government of Russian Federation «Measures to Attract Leading Scientists to Russian Educational Institutions» (grant No 11. G34.31.058); by

CONCLUSION Confocal microscopy due to its high resolution

the Program of SB RAS (project No 71).

and ability to record low light signals offers new opportunities in investigation of fungal bioluminescence

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Berliner

and

Hovnanian

HP.

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Autophotography of luminescent fungi. J Bacteriol. 86 (2):339-341.

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Bondar VS, Puzyr AP, Purtov KV, Medvedeva SYe,

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Rodicheva EK, Gitelson JI. 2011. The luminescent

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Doklady Biochem Biophys.;438(1):138-140. Calleja GB, Reynolds GT. 1970. The oscillatory nature

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S.E.

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Armillariella mellea. Dokl Akad Nauk SSSR.;271:750752. Mendes LF and Stevani CV. 2010. Evaluation of metal toxicity by a modified method based on the fungus Gerronema

viridilucens

bioluminescence

agar

medium. Environ Toxicol Chem. ;29:320-326. Mori K, Kojima S, Maki S, Hirano T, Niwa H. 2011. Bioluminescence characteristics of the fruiting body of Mycena chlorophos. Luminescence. 26(6): 604-10. Oliveira AG and Stevani CV. 2009. The enzymatic nature of fungal bioluminescence. Photochem Photobiol Sci. 8(10):1416-21. Prasher IB, Chandel VC, Ahluwalia AS. 2012. Influence of culture conditions on mycelial growth and luminescence of Panellus stipticus (bull.) P. Karst. J Res Biol. 2(3):152-9. Riquelme M and Bartnicki-Garcia S. 2008. Advances in understanding hyphal morphogenesis: ontogeny, phylogeny and cellular localization of chitin synthases. Fungal Biol. Rev.;22(2):56-70. Roberson RW, Saucedo E, Maclean D, Propster J, Unger B, Oneil TA, Parvanehgohar K, Cavanaugh C, Steinberg G, Schuster M. 2011. The dynamic fungal cell. Fungal Biol. Rev.;25(1):14â&#x20AC;&#x201C;37. Shimomura O. Bioluminescence: chemical principles

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and methods. Singapore: World Scientific, 2006.

Advantages

Weitz HJ, Ballard AL, Campbell CD, Killham K. 2001. The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi. FEMS Microbiol Lett. 202(2):165-170.

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright

Weitz HJ, Colin D, Campbell CD, Killham K. 2002.

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Development of a novel, bioluminescence-based, fungal

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905

Journal of Research in Biology (2013) 3(3): 900-905

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Local peopleâ&#x20AC;&#x2122;s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Authors: Lakshmi Kodoth and Ramamoorthy D.

Institution: Department of Ecology & Environmental Sciences, Pondicherry University, Puducherry.

Corresponding author: Lakshmi Kodoth.

ABSTRACT Studies in mangrove ecosystem are often focused on biological or ecological criteria and interdependency between mangroves and people is normally neglected. The situation is similar in Tamil Nadu; India which has a coastline of about 950 km. One of the major mangrove forests in Tamil Nadu is situated in Pichavaram, Cuddalore district. The present study was carried out in the seventeen hamlets, which are directly or indirectly dependent on the Pichavaram mangrove wetlands for their livelihood and survival. These seventeen hamlets consist of over 2600 households many of whom derive their principal income from fishing and related activities. Individual surveys were carried out for 10% of the households in each of the selected hamlets. Semi-structured questionnaires were used for surveys to study the attitude and perception of the community on the conservation and importance of mangrove wetlands and resources. The study was conducted to assess the awareness, attitudes and views of people dependent on the mangrove ecosystem towards conservation issues and development options. It was observed that a large percentage of the sampled population showed a positive inclination towards conservation of the ecosystem and were well aware of their responsibility towards it.

Keywords: Email: Mangrove ecosystem, Livelihood, Attitudes, Conservation, Development. lakshmi.kodoth@gmail.com

Web Address:

http://jresearchbiology.com/ documents/RA0274.pdf.

Article Citation: Lakshmi Kodoth and Ramamoorthy D. Local peopleâ&#x20AC;&#x2122;s attitude towards conservation and development around Pichavaram mangrove ecosystem, Tamil Nadu, India. Journal of Research in Biology (2013) 3(3): 906-910 Dates: Received: 08 Aug 2012

Accepted: 26 Aug 2012

Published: 06 May 2013

Journal of Research in Biology An International Scientific Research Journal

906-910 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Kodoth and Ramamoorthy, 2013 INTRODUCTION

The Pichavaram mangrove wetland is located in

The Mangrove ecosystem has been studied

the northern extreme of Cauvery delta between the

extensively by scientists more in the ecological and

Vellar and Coleroon estuaries (figure 1). Geographically,

biological sense. During the 1980s and early 1990s, more

it is located between 79°47’E longitude and 11°27’N

attention was given to research involving the human

latitude. The Pichavaram mangrove forests have an area

interactions with the forested wetlands (FAO, 1985;

of about 1,350 ha, which are colonised by 13 true

Hamilton et al., 1989; FAO, 1994; Cormier-Salem,

mangrove species. Rhizophora Sp and Avicennia Sp are

1999). Mangrove wetlands are a dominant feature of the

the predominant mangrove species present in the

intertidal areas of the tropical and subtropical regions in

Pichavaram mangrove forests. Pichavaram mangrove

between 25°N and 25°S latitudes. The mangrove

wetland is also rich in its fishery resources (figure 2).

ecosystem provides a number of ecological services:

Annually about 245 tons of fishery resources are

provision of plant and animal products (Macnae, 1974;

harvested from this mangrove wetland, of which prawns

Rasolofo, 1997; Spaninks and Beukering, 1997),

alone contribute 85% of the catch (Selvam et al., 2003).

sediment trapping and nutrient uptake and transformation

Methods

(Furukawa et al., 1997; Hussain and Badola, 2008), they

People belonging to 17 hamlets surrounding the

provide detritus food for the aquatic fauna, harbour migratory and aquatic birds, serve as spawning ground for fishes, mussels and prawns. They also act as a natural shield against storms and tidal waves (Kathiresan and Rajendran, 2005). The coastal communities are largely dependent on the mangrove forests for firewood, timber, honey, fodder and for its fishery resources. Most coastal communities in the tropics are significantly dependent on the harvest of marine and coastal resources for sustaining their livelihoods (Kunstadter et al., 1986). The majority of people living near the mangrove areas derive their

Figure 1 Glimpse of the Pichavaram mangrove forest

income predominantly from fishing and related activities. Hence, the present study was carried out as it is essential to understand people’s attitude and perception towards the mangrove ecosystem as they derive their livelihood from it; it helps us in formulating better policies and enhances the developmental plan for the ecosystem. Study Area India has a coastline of 7,516 km of which Tamil Nadu has about 950 km. Extensive mangrove wetlands are located in two places namely, i) in Pichavaram, Cuddalore district and ii) Muthupet in Thivarur and Tanjore districts. 907

Figure 2 Fishing in the mangrove backwaters Journal of Research in Biology (2013) 3(3): 906-910

Kodoth and Ramamoorthy, 2013 Table 1: Attitude of people towards Pichavaram Mangrove Ecosystem and conservation (n= 324) Questions Are you aware that Pichavaram Mangrove Ecosystem is declared as Protected area? Do you feel any sense of responsibility for the protection of the ecosystem? Do you feel your rights have been violated after the declaration of PA? Do you face any problem because of PA? Are you in favour of the implementation of an ecodevelopment project? Would you like to co-operate with the forest department with regard to the ecodevelopment project?

Yes (%)

No (%)

Don’t Know (%)

13.8

2.2

11.9

80.5

7.6

15.8

78.6

5.6

76.7

15.3

Pichavaram mangroves wetland were selected for survey.

RESULTS AND DISCUSSION

For each selected hamlet 10% of the households were

Assess the awareness and views towards conservation

picked up randomly for the household survey. Using

The results (Table 1) showed that majority of the

semi-structured questionnaires, information on the

respondents i.e.

91%

(n=324) were

aware that

demography, land use, income and occupational pattern

Pichavaram mangrove was as declared protected area

as well as local dependence on the mangrove resources

and this awareness was gained largely because of,

were gathered (Badola and Hussain, 2003; Glaser,2003) .

NGO’s working in that area and the forest department.

Few open ended questions were also included to

An overwhelming percentage (84%) of the local

determine the attitude and perception of villagers

population felt responsible towards the protection of the

towards development and conservation issues. A total of

mangrove ecosystem and another 76.7% are in favour of

324 households were surveyed. The responses we got

eco-development projects in the area. Out of the 324

were mostly in terms of yes, no and we don’t know.

respondents, 67% of the people are willing to cooperate with the forest department for the same. Only a small

Table 2: View of respondents towards Eco-Development itiatives (n = 324) Views Want through govt. initiative Want through Community initiative Want through NGO initiative

Frequency

Percentage

23.1

145

44.7

104

because of the protected area status if the ecosystem. When questioned regarding their views on eco-development initiatives and its implementation, a majority of the respondents (44.7%) were in favour of the community led initiatives. 32% felt that NGO’s should take lead in eco-development and the rest 23% felt

Table 3: View of local people towards various management alternatives (n = 324) Management Alternatives Forests should be cut and land used for other purposes Current situation of protecting the forests is good Increase in mangrove plantations needed

percentage of people feel their rights being violated

that the government should take up

eco-developmental projects by itself ( Table 2). The importance of the mangrove forests to the

Responses (%)

local population was emphasized when a majority of

0.6

people were against cutting down of the forests. A

28.4 71

majority of the respondents (71%) felt that more mangrove plantations need to be carried out, while 28.4% felt that the present conditions of the mangrove forests were good (Table 3).

Journal of Research in Biology (2013) 3(3): 906-910

908

Kodoth and Ramamoorthy, 2013 Table 4: View of local people towards various developmental options (n = 324) Queries Are you in favour of developing eco tourism in the area Are you in favour of shrimp farms Has Shrimp farms been useful to you? (n=14)

Yes (%) 76 8 47

No (%) 16 83 46

Don’t know(%) 8 9 7

The findings in this study are similar to that of

highest ranking to contribution of mangroves towards

the study in Bitarkanika mangrove ecosystem in Orissa

fishing. 63% gave agriculture as their second preference.

(Badola and Hussain, 2003) which shows that the

Incase of ranking ecological functions performed by

villagers are well aware of their responsibility to the

the Pichavaram mangrove ecosystem, 77.8% of the

ecosystem and willing to participate in the conservation

responses favoured Tsunami/cyclone mitigation. 67.2%

efforts of both the government and NGO’s.

gave second preference to nutrient cycling (Table 6). The results show that the respondents were aware

Developmental Options Recently, eco tourism has been promoted to a

of both the direct and indirect benefits of the mangrove

large extent in Pichavaram mangrove forests. Majority of

ecosystem. It is evident from the results that people value

the respondents (76%) were in favour of developing eco

the uses or function which more beneficial to them in

tourism as it will improve job opportunities for the local

their day today lives.

population. Shrimp farms are not favoured in the area as 83% of the responses were against setting up of such

CONCLUSION

farms. This is primarily due to the fact that shrimp farms

The results showed that in general people have a

in the area are the reason for increase in salinity of the

positive attitude towards conservation and are aware of

canal water (Table 4).

their responsibility in sustaining these mangrove forests.

Ecological functions and values identified by local

The socio economic and market conditions influence the

community

people’s

attitude

towards

the

resources.

Eco

The respondents were given a list of ecological

developmental plans were in favour with the local

functions to find out how much they were aware of the

population since it will be helpful in formulating

functions and its direct or indirect importance in their

sustainable policies for ecosystem. The promotion of eco

livelihoods.

tourism in the area had a largely positive response hence

Table 5 shows ranking of use values, 76% gave Table 5: Ranking of the use values in Percentage (n=324) Use values Fishing Agriculture Tourism

Rank 1 (%) 76 26 35

909

Inclusion of the local people in decision making process can lead to successful management of the Pichavaram

Rank 2 (%)

Rank 3 (%)

mangrove ecosystem.

18 63 56

6 11 9

REFERENCE

Table 6: Percentage ranking of various functions (n=324) Ecological functions Fish Aesthetic Tsunami/cyclone mitigation Nutrient

it should be capitalised on to improve local economy.

Rank 1 (%) 59.4 38

Rank 2 (%) 34.3 59

Rank 3 (%) 6.3 3

77.8

22.2

32.2

67.2

0.6

Badola R and Hussain SA. 2003. Valuation of the Bhitarkanika mangrove ecosystem for ecological security and sustainable resource use. Study report. Wildlife Institute of India, Dehra Dun.

Journal of Research in Biology (2013) 3(3): 906-910

Kodoth and Ramamoorthy, 2013 Cormier-Salem MC. 1999. The Mangrove: an area to be clearedâ&#x20AC;Śfor social scientists. Hydrobiologia. 413: 135-142. FAO. 1985. Mangrove management in Thailand, Malaysia and Indonesia. FAO Environment Paper 4, Food and Agriculture Organization of the United Nations, Rome.

Fishery Commission. Publication IOFCDev. 74:34-35. Rasolofo MV. 1997. Use of mangroves by traditional fishermen in Madagascar. Mangroves Salt Marshes. 1:243-253. Selvam V, Ravichandran KK, Gnanappazham L and Navamuniyammal

2003.

Assessment

community-based restoration of Pichavaram mangrove

FAO. 1994. Mangrove forest management guidelines.

wetland using remote sensing data. Current Science.

FAO Forestry Paper 117, Food and Agriculture

85:6,794-798.

Organization of the United Nations, Rome.

Spaninks F and Beukering PV. 1997. Economic

Glaser M. 2003. Interrelations between mangrove

Valuation of Mangrove Ecosystems: Potential and

ecosystem, local economy and social sustainability in

Limitations. CREED Working 14.

Caete Estuary, North Brazil. Wetland Ecology and Management. 11:265-272. Furukawa K, Wolanski E and Mueller H. 1997. Currents and sediment transport in mangrove forests. Estuarine, Coastal and Shelf Science. 44:301-310. Hamilton LS, Dixon JA and Miller GO. 1989. Mangrove forests: an undervalued resource of the land and of the sea. In: Borgese EM, Ginsburg N, Morgan JR. (Eds.), Ocean Yearbook 8. University of Chicago Press, Chicago. 254-288. Hussain SA and Badola R. 2008. Valuing mangrove ecosystem services: linking nutrient retention function of mangrove forests to enhanced agroecosystem production. Wetlands Ecology and Management. 16:441-450. Kathiresan K and Rajendran N. 2005. Coastal

Submit your articles online at www.jresearchbiology.com

mangrove forests mitigated tsunami. Estuarine, Coastal

Advantages

and Shelf Science. 65:601-606. Kunstadter P, Bird ECF and Sabhasri S. (Eds.). 1986. Man in the Mangroves. United Nations University, Tokyo. Macnae W. 1974. Mangrove forest and fisheries. FAO/

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright submit@jresearchbiology.com www.jresearchbiology.com/Submit.php.

UNDP Indian Ocean Fishery Programme. Indian Ocean Journal of Research in Biology (2013) 3(3): 906-910

910

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria Authors: Nwanyanwu CE 1* Abu GO2.

ABSTRACT:

2.Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria.

Assessments on biodegradation at low and high doses of phenol by bacterial strains indigenous to Okrika River in Niger Delta of Nigeria were carried out. Growth at low dose of 0.01 Âľg/l phenol showed that highest and lowest cell density values of OD540nm of 0.15 and 0.09 in Pseudomonas sp. SD1 and Citrobacter sp. RW1 while at 1.0 Âľg/l phenol concentration the highest cell density values of OD 540nm of 0.28 was observed in Staphylococcus sp. RW2. The highest specific growth rate of 0.019 h-1 at 500 mg/l of phenol was obtained for Pseudomonas sp. SD1 while Citrobacter sp. RW1 had the lowest specific growth rate of 0.014 h-1 at 500 mg/l of phenol. The specific phenol degradation rate ranges from 55.35 to 130.98 mg/(L.h.OD). The order of specific phenol consumption rate at 1000 mg/l by the organisms is: Bacillus sp. SD3>Pseudomonas sp. SD1>Citrobacter sp. RW1>Staphylococcus sp. RW2. Citrobacter sp. RW1 exhibited highest growth yield in 250 mg/l of phenol with the growth yield of 6.24 (x 10-4 A540 unit.l/mg). The results showed that the test organisms might be the most suitable bacterial strains for removal of phenols at low and high doses in phenolic polluted media.

Corresponding author: Nwanyanwu CE.

Keywords: Biodegradation, phenol, bacteria, Okrika River.

Email:

Article Citation: Nwanyanwu CE and Abu GO. Biodegradation of phenol at low and high doses by bacterial strains indigenous to Okrika River in the Niger Delta of Nigeria. Journal of Research in Biology (2013) 3(3): 911-921

Institution: 1.Department of Microbiology, Federal university of Technology, P.M.B.1526, Owerri, Nigeria.

cnwanyanwu2000@yahoo.com

Web Address:

http://jresearchbiology.com/ documents/RA0318.pdf.

Dates: Received: 26 Dec 2012

Accepted: 17 Jan 2013

Published: 06 May 2013

Journal of Research in biology An International Scientific Research Journal

911-921| JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Nwanyanwu and Abu, 2013 (2000) and Polymenakou and Stephanou (2005).

INTRODUCTION Contamination of aquatic environment brought

Microorganisms indigenous to aquatic environment are

about by the discharge of wastewater resulting from

crucial for the biodegradation of organic matter and the

anthropogenic activities clearly continues to be a major

cycling of nutrients, while these microorganisms are

environmental issue. Effluents are very important

susceptible to toxic pollutants from industrial effluent

sources of chemicals entering aquatic ecosystems. They

discharges, especially petroleum refinery. Therefore,

may contain hundreds, or even thousands of chemicals,

perturbations of aquatic microbial communities could

but only a few of them are responsible for effluent

have consequences for the higher trophic levels and for

toxicity (Tisler et al., 1999). High strength wastewaters

the overall aquatic environment.

have been reported to be associated with chemical

The composition of effluents from petroleum

processing industries. Wastewaters generated from these

refineries varies according to their origin, storage and

processing

oil

treatments as these wastewaters are enriched with

refineries, coke-processing plants, etc contain a large

different pollutants. Phenol and its derivatives along with

number of organic and inorganic pollutants at high

other organic and inorganic compounds is one of the

concentrations that exhibit adverse effect on the

most common contaminants present in refinery effluents

environments when released (Papadimitriou et al., 2009).

(Jena et al., 2005) which renders refinery effluents its

The presence of high level of these contaminants formed

toxic nature. Phenols as constituents of industrial

the major pollutant in the water body as a result of

effluents may remain in water body for much longer

continuous discharge of effluents by industries into the

period if it is continually or consistently released into the

ecosystem. In water these pollutants of the discharged

aquatic environments from sources thereby increasing its

effluent sorbs onto particulate materials and if not

elevation in the environment. The toxic nature of phenol

degraded eventually end up in sediments. As an ultimate

and its derivatives to microbial cells is well documented

respiratory of most xenobiotic contaminants that enter

(Kahru et al., 2002; Keweloh et al., 1990). Owing to

water bodies, sediments act as both carrier and sources of

toxic nature of phenol, its contact with microorganisms

contaminants in aquatic environment (Akan et al., 2010).

always results in the decrease of microbial enzyme

Thus, the contaminated sediments may represent a

activity (Nwanyanwu and Abu, 2011) as well as leading

continual threat of recontamination of the aquatic

to death of organisms at higher concentration.

industries

such

petrochemical,

environment as the adsorbed pollutants if not degraded,

A large number of microbial genera possess the

in turn lead to the exposure of aquatic life to organic

capability to degrade organic pollutants. Among the

pollutants such as phenol (Mort and Dean-Ross, 1994).

bacterial genera implicated in the degradation of phenol

On the other hand, the release of contaminants from

include

sediments could increase the amount of toxic compounds

species etc. The ability of organisms to degradation

in the waters making them more available to organisms

phenol and other toxicants is related to adaptation of the

and affecting their life cycles, reproduction, metabolism

microorganisms to the compound of concern and

and physiology. Microorganisms being ubiquitous in

adaptation is associated with synthesis of new enzymes

nature exploit many carbon and energy sources in its

capable of transformation of the toxicant to harmless

niche for growth. Several species of micro-organisms

substances (Jaromir and Wirgiliusz, 2007). The resultant

inhabiting hostile ecological niche have been reported by

effect of biodegradation of phenol and other organic

Colwell and Walker (1977), Atlas (1981), Heinaru et al.

compounds is growth as the organic pollutants are used

912

Pseudomonas,

Bacillus,

Corynebacterium

Journal of Research in Biology (2013) 3(3): 911-921

Nwanyanwu and Abu, 2013 as the source of carbon and energy.

0.4 at 540 nm and used as inocula.

This research assessed the growth and utilization of phenol at low and high doses by bacterial strains

Assay for isolates growth in very low concentrations of phenol

indigenous to Okrika River in the Niger Delta of Nigeria.

The ability of the isolates to grow and utilize phenol at low concentrations (0-1.0 Âľg/l) was assessed in

MATERIALS AND METHODS

sterile Bushnell Haas (BH) mineral salt broth medium.

Chemical reagents

The

assay

was

carried

out

described

All chemical reagents used in the study were of

Nwanyanwu et al., (2012) with little modification. The

analytical grade and were obtained from sigma chemical

medium without agar was used instead for the assay.

company, St Louis Missouri, USA, BDH chemicals,

After inoculation of the flasks, growth profile of the

Poole, England and HACH chemical company.

organisms was monitored by the optical density

Sample collection and analysis

(OD540nm) on daily basis.

The Okrika River is a small tidal river that empties into Bonny estuary in Niger Delta of Nigeria.

Growth and biodegradation of phenol at high concentration

The River is highly polluted as a result of effluent

Degradation of phenol at high concentration by

discharges from Port Harcourt petroleum refinery

the organisms was carried out in sterile BH medium

industry sited along its bank (IAIA09 Conference

contained

Proceeding, 2009). Sediment and water samples were

supplemented with aliquot of sterile phenol (2000 mg/l)

collected from the river as described by Nweke et al.,

to bring the final phenol concentrations in the flasks to

(2007) and the samples analyzed within few hours of

250, 500, 750 and 1000 mg/l. The flasks after inoculation

collection. The results of the physicochemical analysis of

with the test organisms were incubated at 30oC in an

the samples are as shown in Table 1.

incubator.

Isolation and identification of bacterial strains

withdrawn to determine cell growth and phenol

Erlenmeyer

flasks.

The

predetermined time,

flasks

were

samples were

The bacterial strains used in this work were

concentration. Controls, one without phenol and another

isolated from the samples by spreading one tenth of

without cells in BH medium were set up. At

decimally diluted sediment suspension and water

predetermined time, samples were removed and used to

samples on mineral salt agar-phenol (2.5 mM) medium

measure for cell growth (optical density, OD540nm) and

and the isolated organisms identified as described elsewhere (Nwanyanwu et al., 2012). The isolates were

Table 1: Physicochemical characteristics of Okrika River

designated according to their sources (RW for River water, SD for sediment) and were then maintained on nutrient agar slants. Preparation of inoculum The bacterial strains used for the assay were grown in 100 ml of sterile nutrient broth media for 48 h. The turbid culture medium were harvested, washed and suspended in deionized distilled water then followed

standardization

spectrophotometrically

the

optical

suspensions density

Journal of Research in Biology (2013) 3(3): 911-921

Parameter/unit pH Elect. conduc. (Âľscm-1) Oil and grease (mg/l) BOD (mg/l) COD (mg/l) PO4 (mg/l) SO4 (mg/l) Phenol (mg/l) Zn (mg/l) Cu (mg/l) Pb (mg/l)

Sample source Water Sediment 8.90 6.90 364 615 16.0 103.0 8.16 84.0 0.15 0.90 118 117 6.1 15.5 0.03 3.48 <0.01 0.06 <0.01 <0.01 913

Nwanyanwu and Abu, 2013 Table 2: Yield factor (Y) of biomass after growth at different initial phenol concentrations 

-4

Yield factor, Y(x 10 A540 unitsa. l/mg) Phenol concentration (mg/l)

Bacteria

250 500 Citrobacter sp. RW1 6.24 4.46 Staphylococcus sp.RW2 4.96 3.80 Pseudomonas sp. SD1 4.96 3.80 Bacillus sp. SD3 3.28 4.46 a A540 units = optical density at 540 nm

750 2.69 3.28 3.28 2.69

1000 3.11 3.00 3.00 3.11

Analytical methods growth

was

determined

spectrophotometrically while phenol was analyzed by photometric method using 4-aminoantipyrine as the colouring agent and measuring the absorbance at 500 nm (Folsom et al., 1990).

growth

rate

(µ)

for

each

concentration of phenol was calculated from the slope of linear logarithmic plots of optical density against time as expressed in equation 1 (Gokulakrishnan and Gummadi, 2006):

with the OD at 540 nm. RESULTS AND DISCUSSION The phenol content of Okrika River water and the River water and sediment were 16.0 and 103.0 mg/l respectively (Table 1). This level of oil and grease as well as phenol in the River water and sediment were much higher than the previously reported levels of 10.56 and 15.23 mg/l (oil and grease) and 5.13 and 16.0 mg/l (phenol) (Otokunefor and Obiukwu, 2005). This indicated that these compounds have accumulated

Figure 1 shows the growth of the test organisms in low concentration of phenol amended mineral salt medium. All the organisms showed progressive growth in low phenol concentration medium. Highest growth of the organisms was observed in phenol concentration of observed in 0.01 µg/l. Among the test organisms,

Staphylococcus sp. RW2 showed the highest growth in

Specific degradation rate

0.1 and 1.0 µg/l of phenol with optical density (OD) was

values of 0.23 and 0.28 respectively while Citrobacter

determined through the relationship of equation 2 (Loh

sp. RW1 showed the least growth in all the low

and Wang, 1998):

concentrations (0.01, 0.1 and 1.0 µg/l) of phenol

specific

the change in substrate concentration dS. X was replaced

1.0 µg/l followed by 0.1 µg/l. The least growth was

In X 2 X 1 t 2 t1 The

Where dX is the change in cell biomass related to

of the river.

Specific growth rate specific



in Okrika River over time and pose the major pollutants

Data Analysis The

sediment were 6.1 and 15.5 mg/l while oil and grease of

phenol residue (4-amino antipyrine) in cell free samples. Cell

dX dS

d Ph / dt X

degradation

rate

(Qs)

amended medium with OD values of 0.09, 0.11 and 0.13

Where: [Ph] denotes phenol

respectively. Growth of microorganisms especially concentration

bacterial species at phenol concentration as low as

(mg/l), t denotes incubation time (h) and X denotes cell

microgram per litre have been reported by many authors.

concentration (optical density, OD540 nm).

Chesney et al., (1985) have reported growth of water

Yield factor

microorganism in water sample supplemented with 0.001

Yield factor (Y) of the biomass was calculated using equation 3 (Bajaj et al., 2009): 914

to 1.0 µg/ml of phenol. Also Goldstein et al., (1985) have reported the growth of Pseudomonas sp. in a Journal of Research in Biology (2013) 3(3): 911-921

Absorbance (A540 nm)

Nwanyanwu and Abu, 2013

Time (h) Figure 1: Growth profile of the bacteria in mineral salt medium fortified with phenol concentrations medium amended with 1.0 and 10.0 Âľg/l concentration of

concentrations of 500, 750 and 1000 mg/l was degraded

2, 4-dichlorophenol. Pahm and Alexander (1993) found

completely within 96, 132 and 156 h by Pseudomonas

that Pseudomonas sp. K, Flavobacterium sp. M4,

sp. SD1 while same concentrations of phenol was

Flavobacterium sp. M1 and Pseudomonas sp. SP3 grown

degraded completely within 108, 144 and 180 h by other

in p-nitrophenol (PNP) of concentration of 0.1 Âľg/l

test organisms. Time-dependent degradation of organic

reached a total viable count of 105 and 106 cells/ml.

compounds has been reported to be linked with

Figures 2 and 3 showed typical profiles of cell

concentration of the organic compound as observed by

and

high

many authors (Colwell and Walker, 1977; Kotresha and

concentrations by bacterial strains of Okrika River

Vidyasagar, 2008). This may be due to changes in the

ranging from 250 to 1000 mg/l. The lag phase of the

transport mechanism of the substrate across the cell

organisms in phenol fortified medium was short. The

membrane in response to high phenol concentration

short in lag phase period depends on the pre-exposure of

hence diminished capacity to catabolize phenol. This is

the organism. Phenol was completely utilized by the

in line with the reports of Gilbert and Brown (1978),

isolates

Keweloh et al., (1990), Collins and Daugulis (1997) and

growth

biodegradation

within

180

phenol

incubation.

Phenol

Journal of Research in Biology (2013) 3(3): 911-921

915

Absorbance (A540 nm)

Phenol (mg/l)

Nwanyanwu and Abu, 2013

Time (h) Figure 2: Biodegradation and cell growth profile of planktonic bacteria of Okrika River in high phenol concentrations

Nwanyanwu and Abu (2011) who observed the toxic

the medium progressively decreased. This may be due to

effect of phenol at the membrane level, thereby

high phenol concentration made available more carbon to

disrupting the activity of enzymes in phenol-utilizing

the organism for growth. Pseudomonas sp. SD1

bacteria. Also, Joseph and Joseph (1999) and Ye and

degraded 1000 mg/l of phenol in 160 h with a cell

Shen (2004) reported that phenol toxicity depends on the

biomass (OD540nm) of 0.363.

sensitivity as well as source of organism.

The dependence of specific growth rate on

The growth profiles of the pure cultures expressed

phenol concentration is shown in Figure 4. From this

as optical density and phenol residues at different initial

plot, the specific growth rate increased with increase in

concentrations are shown in figures 2 and 3. The cells

the initial phenol concentration upto 250 mg/l and then a

gradually increase in number as the phenol residues of

progress decrease started with increase in phenol

916

Journal of Research in Biology (2013) 3(3): 911-921

Absorbance (A540 nm)

Phenol (mg/l)

Nwanyanwu and Abu, 2013

Time (h) Figure 3: Biodegradation and cell growth profile of sediment bacteria of Okrika River in high phenol concentrations

concentration. In the present study, at 500 mg/l of phenol

concentration and then started decreasing to a constant

concentration, the specific growth rate of Pseudomonas

(0.057 h-1) at 600 and 700 mg/l of phenol. This trend

sp. SD1 is increased (highest µ =0.017 h -1). For

suggested that the phenol is an inhibitory substrate. Thus

concentration higher than 500 mg/l, the specific growth

the parameter has been found to be a strong function of

rate of Pseudomonas sp. SD1 decreases and became

initial phenol concentration. At 250 and 500 mg/l, the

-1

almost constant at 750 mg/l (µ = 0.011 h ) and

highest specific growth rate values of 0.026 and 0.017 h -1

1000 mg/l (µ = 0.011 h -1) of phenol. This is quite similar

were observed in Citrobacter sp. RW1 and Pseudomonas

to the result obtained by Dey and Mukherjee (2010) who

sp. SD1 respectively while the lowest specific growth

-1

observed increase in specific growth rate (0.093 h ) of

rate of 0.016 and 0.014 h -1 at the same concentration of

mixed microbial culture up to 300 mg/l of initial phenol

phenol was observed in Pseudomonas sp. SD1 and

Journal of Research in Biology (2013) 3(3): 911-921

917

Specific growth rate (h-1)

Specific degradation rate (mg/(L.h.OD))

Nwanyanwu and Abu, 2013

Phenol, So (mg/l) Phenol, So (mg/l)

Figure 5: Specific degradation rate at different initial phenol concentrations by the bacterial strains

Figure 4: Specific growth rate of the organisms at different initial phenol concentrations Citrobacter sp. RW1 respectively.

However, the

concentration decreases. This is in line with the work of

growth rates of the test organisms are similar

Cho et al., (2000) who observed an increase in specific

degradation rate as phenol concentration increases in

that

Pseudomonas

aeruginosa

and

Pseudomonas pseudomallei degrading phenol in saline

their

assessment

influence

phenol

solutions (Afzal et al., 2007).

biodegradation of p-nitrophenol by freely suspended and

The specific rate of phenol degradation of the

immobilized Nocardioides sp. NSP41. Agarry and

organisms is depicted in figure 5. The specific

Solomon (2008) also made similar reports in their work

degradation rate (Qs), was estimated by correlating

on kinetics of batch microbial degradation of phenols by

phenol

indigenous Pseudomonas fluorescence.

concentration

versus

culture

time

using

regression technique in Microsoft Excel to obtain the

Table 2 shows the growth yield of the test

equation of best fit of the degradation curve. The

organisms expressed as absorbance, A at 540nm unit litre

correlation were differentiated with respect to time and

of cells produced per mg of phenol substrate utilized.

then divided by the cell mass (Loh and Wang, 1998).

The growth yield varied among the test organisms

The specific degradation (consumption) rate of a

ranging from 2. 69 to 6.24 (x 10-4A540 units. l/mg). High

compound was suggested to be a measure of microbe

growth yield were obtained at low concentration of

performance. The highest specific consumption rate of

toxicant (phenol) while low values of growth yield were

phenol was observed in Bacillus sp. SD3 with specific

obtained at high phenol concentration. At 250 mg/l

degradation rate value of 130.98 mg/(L.h.OD) at

highest and lowest growth yield were observed in

1000 mg/l while Staphylococcus sp. RW2 showed the

Citrobacter sp. RW1 and Bacilllus sp. SD3 with cell

least specific consumption rate of phenol with a specific

yield coefficients of 6.24 and 3.28 (x 10-4A540 units.l/mg)

degradation rate value of 99.83 mg/(L.h.OD) at the same

respectively. The higher value of Y observed in

concentration. The organisms in this work showed a

Citrobacter sp. RW1 indicate that phenol was degraded

robust decrease in specific degradation rate as the phenol

very efficiently by the organism. All the growth yields

918

Journal of Research in Biology (2013) 3(3): 911-921

Nwanyanwu and Abu, 2013 reported here were lower than those reported by other

Mineralizing Microorganisms in Fresh Water. Appl.

authors. Yield coefficients of 0.14 and 0.16 have been

Environ. Microbiol. 49: 15 – 18.

reported (Bajaj et al., 2009). The yield coefficients reported by Yoong et al., (1997) are 0.16 and 0.27. As Citrobacter sp. RW1, Staphylococcus sp. RW2, Pseudomonas sp. SD1and Bacillus sp. SD3 shown high specific

phenol

consumption

rate,

they

have

Cho YG, Rhee SK, Lee ST. 2000. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and

immobilized

Nocardioides

sp.

NSP41.

Biodegradation 11(1): 21 – 28.

demonstrated strong potential to utilize and grow in

Collins LD and Daugulis AJ. 1997. Characteristics and

phenol of low and high phenol concentrations of upto

optimization of a two-phase partitioning bioreactor for

1000 mg/l. This indicated that these strains have great

the

potential for application in the treatment of phenolic

Biotechnol. 48(1):18 - 22.

wastewater and in the bioremediation of phenol impacted media.

biodegradation

phenol.

Appl.

Micobiol.

Colwell RR and Walker J.D. 1977. Ecological Aspects of Microbial Degradation of Petroleum in the Marine Environment. Crit. Rev. Microbiol. 5(4): 423 - 445.

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Folsom BR, Chapman PJ, Pritchard PH. 1990.

Characteristics of phenol biodegradation in saline

Phenol

solutions by monocultures of Pseudomonas aeruginosa

Pseudomonas cepcia GA: Kinetics and interaction

and Pseudomonas pseudomallei. J. Hazard. Mat. 149(1):

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1279 – 1285.

Agarry SE and Solomon BO. 2008. Kinetics of batch

Gilbert P and Brown MRW. 1978. Influence of growth

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Pseudomonas fluorescence. Int. J. Environ. Sci. Tech. 5

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Akan JC, Abdulrahman FI, Sodipo OA, Ochanya AE

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and Askira YK. 2010. Heavy metals in sediments from

Gokulakrishnan S and Gummadi SN. 2006. Kinetics

River Ngada, Maiduguri Metropolis, Borno State,

of cell growth and caffeine utilization by Pseudomonas

Nigeria. J. Environ. Chem. Ecotoxicol. 2(9): 131 - 140.

sp. GSC 1182. Proc. Biochem. 41: 1417 – 1421.

Atlas RM. 1981. Microbial degradation of petroleum

Goldstein RM, Mallory LM, Alexander M. 1985.

hydrocarbons:

Reasons for Possible Failure of Inoculation to enhance

environmental

perspective.

Microbiology Rev. 45(1): 180 – 209. Bajaj M., Gallert C and Winter J. 2009. Phenol

biodegradation. Appl. Environ. Microbiol. 50(4): 977 – 983.

degradation kinetics of an aerobic mixed culture.

Heinaru E, Truu J, Stottmeister U, Heinaru A. 2000.

Biochem. Eng. J. 46(2): 205 – 209.

Three types of phenol and p-cresol catabolism in phenol

Chesney RH, Sollitti P, Rubin HE. 1985. Incorporation of Phenol Carbon at Trace Concentrations by PhenolJournal of Research in Biology (2013) 3(3): 911-921

and p-cresol-degrading bacteria isolated from River water continuously polluted with phenolic compounds. FEMS Microbiol. Ecol. 31(3): 195 – 205. 919

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phenolic compounds by sulphate reducing bacteria from

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contaminated sediments. Microb. Ecol. 28: 67 – 77.

Harcourt Oil Refinery Company (PHRC), Nigeria. Impact Assessment and Human Well-Being 29th Annual Conference of the International Association for Impact Assessment, 16-22 May 2009, Accra International Conference Center, Accra, Ghana (www.iaia.org) Jaromir

and

Wirgiliusz

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phenol. J. Res. Biol. 3: 167 - 177

viability responses of refinery effluent bacteria after exposure to phenol stress. J. Res. Biol. 1(8): 594 - 602 Nweke co, Alisi CS, Okolo JC and Nwanyanwu CE.

Roy GK and Meikap BC. 2005.

Development and comparative study of a semi-fluidized bed bioreactor for treatment of wastewater from process industries. Proc. Plant Eng. 23(1): 70 ‐ 75

2007. Toxicity of zinc to heterotrophic bacteria from a tropical river sediment. Appl. Ecol. Environ. Res. 5(1): 123-132 Otokunefor TV and Obiukwu C. 2005. Impact of

Joseph V and Joseph A. 1999. Acclimation of algal species following exposure to phenol. Bull. Environ. Contam. Toxicol. 62(1): 87 - 92. Kahru A, Maloverjan A, Sillak H. and Pollumaa L. 2002. The toxicity and fate of phenolic pollutants in the contaminated soils associated with the oil-shale industry. Environ Sci. Pollut Res. 1: 27 – 33.

refinery effluent on the physicochemical properties of a water body in the Niger Delta. Appl. Ecol. Environ. Res. 3 (1): 61 - 72. Pahm MA and Alexander M. 1993. Selecting inocula for the biodegradation of organic compounds at low concentrations. Microb. Ecol. 25(3): 275 – 286. Papadimitriou CA, Samaras P, Sakellaropoulos GP.

Keweloh H, Weyrauch G, Rehm HJ. 1990. Phenolinduced membrane changes in free and immobilized Escherichia coli. Appl. Microbiol. Biotechnol. 33(1): 66 71.

2009. Comparative study of phenol and cyanide containing wastewater in CSTR and SBR activated sludge reactors. Biores. Technol. 100: 31 – 37. Polymenakou PN and Stephanou EG. 2005. Effect of

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Jena HM,

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Journal of Research in Biology (2013) 3(3): 911-921

921

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria Authors: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC.

Institution: Department of Microbiology, Federal University of Technology, P.M.B. 1526, Owerri, Nigeria.

Corresponding author: Nwanyanwu CE.

ABSTRACT: Bacterial isolates from petroleum refinery effluent were evaluated for growth in increasing doses of phenol and heavy metal ions. All the test organisms were able to grow in mineral salt medium with phenol concentration of 15.0 mM (â&#x2030;&#x2C6; 1412.0 mg/l) except Pseudomonas sp. RBD3. Aeromonas sp. RBD4, Staphylococcus sp. RBD5 and Pseudomonas sp. RBD10 showed the highest tolerance to 15.0 mM of phenol followed by Corynebacterium sp. RBD7 while Escherichia coli RBD2 and Citrobacter sp. RBD8 showed the least tolerance to 15.0 mM of phenol. The minimum inhibitory concentrations (MICs) ranged from 1.0 mM for mercury and 4.5 mM for chromium, nickel, lead and copper. The bacterial strains were most susceptible to mercury toxicity. Viable counts of the organism on mineral salt-phenol agar showed a typical growth pattern for inhibitory substrate. The threshold concentration is 0.5 mM for Bacillus sp. RBD1, Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp. RBD11 and Escherichia coli RBD12 and 1.0 mM for Pseudomonas sp. RBD3, Aeromonas sp. RBD4, Staphylococcus sp. RBD5, Corynebacterium sp. RBD7 and Corynebacterium sp. RBD10. The results suggest that microorganisms isolated from petroleum refinery effluent are potentially useful for detoxification of phenol impacted systems in the presence of heavy metals.

Keywords: Phenol, heavy metals, refinery effluent bacteria. cnwanyanwu2000@yahoo.com Email:

Web Address: http://jresearchbiology.com/ documents/RA0317.pdf.

Article Citation: Nwanyanwu CE, Nweke CO, Orji JC, Opurum CC. Phenol and Heavy Metal Tolerance Among Petroleum Refinery Effluent Bacteria. Journal of Research in Biology (2013) 3(3): 922-931 Dates: Received: 24 Dec 2012

Accepted: 09 Jan 2013

Published: 10 May 2013

Journal of Research in Biology An International Scientific Research Journal

922-931 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Nwanyanwu et al., 2013 Discharge of these metals into natural waters at

INTRODUCTION Petroleum refinery effluents are wastes liquids

increased concentration in refining operations can have

that resulted from the refining of crude oil in petroleum

severe toxicological effects on aquatic environment and

refinery. The effluents are composed of oil and grease

humans. Heavy metals as well as phenol are known to

along with many other toxic organic and inorganic

compounds (Diyaâ&#x20AC;&#x2122;uddeen et al., 2011). Among the toxic

wastewaters

components of these effluents are heavy metals. Heavy

microorganisms. These metals are in the form of

metals

iron,

inorganic and metallo-organic compounds while phenol

manganese, zinc, etc. They usually form complexes with

appears to be a soluble component of the industrial

different non metal donor atoms which account for their

effluents (Nwanyanwu and Abu, 2010; Hernandez et al.,

participation in various microbial metabolisms in the

1998). These environmental pollutants which are

environment (Kamnev, 2003). Some of these heavy

environmentally mobile tend to accumulate in organisms,

metals

iron

and become persistent because of their chemical stability

manganese, zinc, etc. are required in trace amount by

or poor biodegradability (Emoyan et al., 2005).

microorganisms at low concentration as nutrients, since

Contamination of wastewater with high concentration of

they provide vital co-factors for metalloproteins and

heavy

enzymes and are known as essential metals while others

the

such as cadmium, mercury, lead, etc have no

(Otokunefor and Obiukwu, 2005). It is obvious that

physiological functions and are known as nonessential

heavy metals are one of the toxic contaminants in

metals (Sevgi et al., 2010). At high concentration both

wastewaters and causes disorder in biological wastewater

essential and nonessential heavy metals exert an

treatment

inhibitory action on microorganisms by impairing the

ubiquitous in nature have been reported to be found in

essential functional groups as well as modifying the

inhospitable habitats

active conformation of biological molecules. This results

effluents, coke effluents, etc (El-Sayed et al., 2003;

in reduction of microbial activity leading to increased lag

Hidalgo et al., 2002) as the effluents are characterized by

phase as well as slow growth rate (Aleem et al., 2003).

the presence of phenols, metal derivatives, surface active

It is expected that petroleum refinery effluents

substances and other chemicals (Suleimanov,1995).

will contain some of these metals in reasonable quantity

Bruins et al., (2000) in their work reported that

as well as aromatic compounds such as phenols. Organic

organisms in such inhospitable environment must have

and inorganic mixed pollutants are known to be

developed metal resistance systems in an attempt to

commonly present in industrial effluents and also other

protect sensitive cellular components. On the other hand,

contaminated sites. In this case, apart from affecting the

utilization of phenol and other pollutant is enhanced by

viability of the microbiota, the metal activity may have

adaptation and production of appropriate enzymes by

synergistic effect on biodegradation processes of the

organisms

aromatic compounds. Thus studies related to the

(Nwanyanwu et al., 2012).

include

such

cobalt,

chromium,

nickel,

harmful

pollutants that

metals

numbers

have

caused of

bacteria

(Saâ&#x20AC;&#x2122;idi,

for

emanating

the

2010). such

from

negative

significant in

industrial

effects

decrease

biological

removal

system

Microorganisms as

being

petroleum refinery

the

toxicants

association of the bacterial tolerance properties to metals

This study investigated the tolerance to heavy

and degradation of phenolic compounds may be relevant

metals and phenol by bacterial population in petroleum

to applications in bioremediation processes (Silva et al.,

refinery effluent.

2007). 923

Journal of Research in Biology (2013) 3(3): 922-931

Nwanyanwu et al., 2013 MATERIALS AND METHODS

allowed to cool at room temperature (28±2oC).

Sample collection

Thereafter, 0.1 ml aliquot of cell suspensions were

Petroleum oil refinery effluent was collected

seeded into the tubes and incubated at 30oC for 96 h. The

from Biological treatment plant unit (Rotary biodisk,

final concentrations of phenol in the tubes ranged from

RBD) of Port Harcourt oil refinery complex and

0.1-100 mM. Controls included cells in mineral salt

transported to the laboratory for physicochemical

medium without phenol and mineral salt medium

analysis which includes pH, total dissolved solids,

supplemented

biological oxygen demand (BOD), chemical oxygen

Development of turbid culture depicted tolerance to

demand (COD), phosphate (PO4), nitrate (NO3), oil and

phenol stress. Isolates that exhibited phenol tolerance

grease, phenol, electrical conductivity and heavy metals

from 5.0 mM and above were used for further phenol and

content. The methods used for the analysis were as

heavy metal toxicity assay.

with

phenol

but

without

cells.

shown elsewhere (Nwanyanwu et al., 2012). Microbiological analysis Microbiological counts were estimated by plating 0.1 ml of the 102 - 106 decimally diluted effluent samples

Table1: Physicochemical and microbiological analyses of biological treatment unit of petroleum refinery wastewater

in physiological saline on appropriate agar plates. Total

Parameter/ unit

Value

heterotrophic bacterial count was done on nutrient agar

8.18

plates while phenol-utilizing bacterial count was done on

Elect. conduct (µs/cm)

485

phenol-agar medium of Hill and Robinson (1975). The

Oil and grease (mg/l)

15.0

TDS (mg/l)

250

BOD (mg/l)

8.0

COD (mg/l)

76.0

Phenol (mg/l)

13.6

PO42- (mg/l)

0.14

inoculated plates were incubated for 24 h at 30oC for the heterotrophic bacterial count and 72 h for phenolutilizing bacteria count. Isolation and identification of bacterial strains The discrete bacterial colonies that developed on phenol-agar biochemically

medium and

were

purified,

identified

characterized described

Nwanyanwu et al., (2012).

NO3 (mg/l)

1.20

Metal concentration 2+

Preparation of inoculum The organisms were grown in nutrient broth

Zn (mg/l)

0.02

Cu2+ (mg/l)

<0.02

medium contained in Erlenmeyer flasks (100 ml) at

Cr (mg/l)

0.05

28±2oC for 48 h. Thereafter, the cells were harvested and

Pb3+ (mg/l)

<0.01

washed in sterile deionized distilled water. The cell

Ni (mg/l)

0.02

suspensions were standardized by adjusting the turbidity

Cd2+ (mg/l)

<0.01 Microbial load

to an optical density of 0.1 at A540. Screening of isolates for phenol tolerance Into 5.0 ml mineral salt broth medium contained in 15.0 ml screw capped glass culture tubes were added aliquots of phenol stock solution (200 mM). The tubes o

were sterilized by autoclaving at 121 C for 15min and Journal of Research in Biology (2013) 3(3): 922-931

THBC (CFU/ml)

2.52 x 108

TPUBC (CFU/ml)

1.14 x 108

% TPUBC (%)

45.24

THBC = Total Heterotrophic bacterial count; TPUBC = Total phenol-utilizing bacterial count 924

Nwanyanwu et al., 2013 properties of the petroleum refinery effluent are shown in

Growth on phenol-mineral salt agar The isolates were tested for their ability to grow

Table 1. Phenol-utilizing bacteria represented 45.24% of

on mineral salt agar medium (MSM) amended with

the microbial load of biodisk effluent. The high

increasing phenol concentrations. An aliquot (100 µl) of

population of phenol-utilizing bacteria obtained could be

decimally diluted standardized inoculum of each isolate

related to natural selection and adaptation to phenol at

in physiological saline was spread plated onto surface

the unit. The concentration of heavy metals in the

phenol

effluent present in the effluent may be as a result of

concentrations. Control included cells in MSM plates

physicochemical treatment (oxidation and reduction,

MSM

plates

with

2.0-20

without phenol. The culture was incubated at 30 C for

chemical precipitation, etc) given to the raw wastewater

72 h (Kahru et al, 2002). The number of the colony that

before been channeled into the biological treatment unit.

developed was enumerated as colony forming unit per ml (CFU/ml). Minimum

The result of screen test for phenol tolerance is shown in Table 2. With the exception of Pseudomonas

inhibitory

concentration

(MIC)

sp. RBD3 that tolerated phenol up to 10 mM, all the organisms are able to tolerate phenol stress up to

determination Stock solutions of Cd, Zn, Hg, Cu, Pb, Ni, Co

15.0 mM. The growth of the isolates in the medium with

and Cr as salts of CdCl2, ZnSO4, HgCl2, CuSO4, PbCl2,

phenol concentrations above 10.0 mM may be attributed

Ni(NO3)2, CoCl2.6H2O and K2Cr2O7 were prepared in

to previous exposure to phenolic raw wastewater influent

deionized distilled water. All the chemicals used were

into the biological treatment unit (RBD). This is in line

analytical reagent grade.

with the report of Santos et al., (2001) in which they

The minimum inhibitory concentrations (MIC) of

related the growth of Trichosporom sp. in phenolic

eight heavy metal ions at which no growth was observed

amended medium of 10.0 mM concentration to previous

were determined at pH 7.2 against each bacterial isolate

phenolic wastewater shock load from stainless steel

using tube dilution method (Hassen et al., 1998) with

industry. Moreso, the tolerance of the organisms to high

little modifications. Graded concentrations of each heavy

concentration of phenol (15.0 mM) may be the ease with

metal ranging from 0.05 mM to 10.0 mM were prepared

which the isolates open the phenol ring for its subsequent

in tryptone soy broth (TSB) contained in screw capped

uptake as carbon and energy source (Ajaz et al., 2004).

culture tubes. The supplemented TSB-heavy metal

Gurujeyalakshmi and Oriel (1989) in their work have

medium was sterilized by autoclaving at 121 C for o

reported that Bacillus stearothermophilus strain BR219

15 min. On cooling to room temperature (28±2 C), the

could grow on phenol at levels up to 15 mM. In contrast,

tubes were seeded with 100 µl of the bacterial

growth inhibition of Bacillus,

suspension and incubated at 30oC for 72 h. Inoculated

Citrobacter species at phenol concentration above

medium free of heavy metal ions and uninoculated

1.0

medium with metal ions served as positive and negative

(Obiukwu and Abu, 2011). Janke et al., (1981) reported

controls respectively. The MIC of the metal to the test

inhibition

isolates is the lowest concentration that totally inhibited

Pseudomonas species at 0.25 mM phenol concentration.

growth of the organisms.

Yang and Humphrey (1975) found that the growth of

925

physicochemical

has of

been

reported

phenol

Pseudomonas

putida

hydroxylase

was

many

authors

activity

strongly

inhibited

above phenol concentration of 0.5 mM. Buswell and

RESULTS AND DISCUSSION The

Pseudomonas and

and

microbiological

Twomey

(1975)

reported

that

growth

Journal of Research in Biology (2013) 3(3): 922-931

Nwanyanwu et al., 2013 Table 2: Phenol tolerance of the test isolates in different concentrations of phenol

Bacteria

Growth in mineral salt broth with added phenol Phenol concentration (mM) 0.2 0.5 1 2 5 10 15 20 50 + + + + + + + -

0.1 +

Bacillus sp. RBD1

100 -

Escherichia coli RBD 2

Pseudomonas sp. RBD 3

Aeromonas sp. RBD 4

Staphylococcus sp. RBD 5

Bacillus sp. RBD 6

Corynebacterium sp. RBD7

Citrobacter sp. RBD8

Streptococcus sp. RBD9

Pseudomonas sp. RBD10

Corynebacterium sp. RBD11

Escherichia coli RBD12

+ = growth ; - = no growth

Bacillus stearothemophilus was inhibited at phenol

of Pseudomonas sp. RBD3, Aeromonas sp. RBD4,

concentration above 5.0 mM.

Staphylococcus sp. RBD5, Corynebacterium sp. RBD7

The effect of increasing doses of phenol

and Corynebacterium sp. RBD10 with a total viable

(0.05 - 15.0 mM) on the population of the test organisms

count of 8.4 x 106, 7.5 x 106, 7.2 x 106 and

are shown in Figure 1. Generally, the viable counts

8.4 x 106 CFU/ml respectively, were stimulated.

increased with the concentration of phenol until a certain

Thereafter,

concentration when the growth of the organisms was

decreased as the phenol concentration increases. This

inhibited. The growth of the organisms on phenol

growth pattern is typical of in an inhibitory substrate like

followed a substrate inhibition pattern. Increasing phenol

phenol. The inhibition of bacterial growth by phenol is

concentration resulted in decrease in microbial growth

well-documented. However, some bacteria are more

and eventually very minimal growth was detected at the

tolerant to phenol than others. For instance, the growth

highest phenol concentration (15.0 mM) in all the test

inhibition constant (Ki) for bacteria degrading phenol

organisms.

have

The

growth

Bacillus

sp.

RBD1,

been

the

total

reported

viable

counts

54.1mg/l

progressively

(0.57

mM)

Escherichia coli RBD2, Bacillus sp. RBD6, Citrobacter

(Monteiro et al., 2000), 129.79 mg/l (1.379 mM) (Kumar

sp. RBD8, Streptococcus sp. RBD9, Pseudomonas sp.

et al., 2005), 2434.7 mg/l (25.87 mM) (Arutchelvan et

RBD11 and Escherichia coli RBD12 with a total viable

al., 2006) and 7.818 mM (Wei et al., 2008). In this study,

count of 7.1 x 10 , 8.0 x 10 , 7.2 x 10 , 7.8 x10 , 6

all the test organisms tolerated phenol up to 10.0 mM

7.5 x 10 , 8.8 x 10 , 7.4 x 10 and 7.4 x 10 CFU/ml

(≈ 941 mg/l) and with the exception of Pseudomonas sp.

respectively were stimulated at phenol concentrations up

RBD 3, all the bacterial strains tolerated 15 mM

to 0.5 mM (≈ 47.06 mg/l). Similarly, at phenol

(≈ 1412 mg/l). This is in line with the report of Worden

concentration up to 1.0 mM (≈ 94.11 mg/l), the growth

et al., (1991) that Bacillus stearothermophilus BR219

Journal of Research in Biology (2013) 3(3): 922-931

926

Total Viable Count (x 106 CFU/ml)

Nwanyanwu et al., 2013

10 8

Pseudomonas sp. RBD3

6 4

2 0 10 Citrobacter sp. RBD8

8 6 4

2 0

Phenol (mM) Figure 1: Growth of bacteria on mineral salt agar medium supplemented with increasing doses of phenol. tolerated phenol concentration of 15.0 mM. Similarly,

phenol concentration (Hossein and Hill, 2006; Kotturi et

Corynebacterium species was reported to resist 15 mM

al, 1991). Li and Humphrey (1989) as well as

phenol while Staphylococcus, Corynebacterium, Bacillus

Gurujeyalakshmi and Oriel (1989) have reported

and Proteus were found to resist 10 mM of phenol

microbial

(Ajaz et al., 2004). However, many authors have

concentrations of 2.0 mM and 0.25 mM respectively.

growth

inhibition

relatively

low

reported inhibition of microorganisms at such high 927

Journal of Research in Biology (2013) 3(3): 922-931

Nwanyanwuet al., 2013 Table 3: Minimal inhibitory concentrations of heavy metals MIC of metal (mM) Organism

Bacillus sp. RBD1

3.5

2.0

1.5

4.0

4.5

3.5

2.0

4.0

Escherichia coli RBD2

3.5

2.5

1.0

4.0

3.0

4.0

2.5

3.5

Pseudomonas sp. RBD3

4.0

3.0

1.5

4.5

3.0

4.5

3.0

4.5

Aeromonas sp. RBD4

3.5

3.0

1.0

3.0

4.0

2.0

4.0

Staphylococcus sp. RBD5

4.0

2.0

1.0

3.5

3.0

4.0

3.0

4.0

Bacillus sp. RBD6

3.0

2.5

1.5

3.5

4.0

3.0

4.0

Corynebacterium sp. RBD7

3.0

2.0

1.5

3.0

3.5

2.5

3.5

Citrobacter sp. RBD8

3.5

1.5

1.0

2.5

3.0

2.0

2.5

Streptococcus sp. RBD9

4.0

2.0

1.0

3.5

2.5

4.0

3.0

2.5

Pseudomonas sp. RBD10

3.5

3.0

1.5

4.5

4.0

4.5

3.5

4.0

Corynebacterium sp. RBD11

2.5

2.0

1.0

4.0

3.0

4.0

4.5

Escherichia coli RBD12

3.0

1.5

1.0

3.0

3.5

2.5

3.0

The tolerance levels of refinery wastewater

reported for cadmium, chromium, lead, cobalt, mercury

phenol-utilizing bacteria to heavy metals expressed as

and copper respectively (Nweke et al., 2006a). These

minimal inhibitory concentrations (MIC) are shown in

reported MICs in most cases corroborates the values

Table 3. The test isolates in this study showed similar

observed in this study. The MIC in growth inhibition

trend of susceptibilities to heavy metal ions based on

assay is analogous to the concentration of metal ion

minimal inhibitory assay. The high MIC values obtained

that exhibited 100 % inhibition in dehydrogenase

in the study may be as a result of long term exposure of

activity assay. Thus, the MIC of zinc against river

the organisms to metal ions in the refinery effluent.

water planktonic bacteria have been reported as

Highest MIC values were exhibited in Chromium,

1.558

Copper and Nickel while the least MIC was shown in

2.469 ± 0.045 mM and 1.328 ± 0.094 mM for

mercury among the isolates with a maximum value of

Escherichia, Proteus, Micrococcus and Pseudomonas

>3.0

mM.

species respectively (Nweke et al., 2006b). Likewise, the

Pseudomonas sp. RBD3 showed maximum MICs value

concentration of zinc that gave 100% inhibition of

range of 1.5 - 4.5 mM whilst Escherichia coli RBD12

dehydrogenase activity in sediment Bacillus and

showed minimum MICs value range of 1.0 - 3.5 mM in

Arthrobacter species are 1.442 ± 0.062 mM and

all the metals tested. The MICs are higher than that

1.199 ± 0.042 mM respectively (Nweke et al., 2007).

reported by El-Deeb (2009) for some phenol-degrading

Also, Hassen et al., (1998) have reported MIC values of

bacteria. However, the MIC values are similar to the

0.1, 0.8, 1.5, 1.6 and 1.8 mM for Mercury, Cobalt, Zinc

values

and Cadmium, Copper and Chromium respectively on

and

reported

minimum

elsewhere

value

(Nieto

<2.0

al.,

1989,

0.037

mM,

0.068

Pseudomonas

of metal ranging from 0.5 - 2.5 mM, 1.25 - 2.5 mM,

Staphylococcus

5.0 - 12.0 mM, 1.0 - 1.25 mM, 0.25 - 1.0 mM and

Bacillus thurieniensis. Hassen et al., (1998) in their work

1.25 - 5.0 mM against hydrocarbon-utilizing bacteria was

reported

Journal of Research in Biology (2013) 3(3): 922-931

aureus, mM

Citrobacter

mM,

Nweke et al., 2006a, Akinbowale et al., 2007). The MIC

3.0

aeruginosa,

1.283

Streptococcus

chromium

the

freundii, sp. MIC

and for 928

Nwanyanwu et al., 2013 Pseudomonas aeruginosa S8 and Citrobacter freundii

Akinbowale OL, Haihong P, Peter G, Barton MD.

S24. The variation in the tolerance of heavy metal could

2007. Antibiotic and heavy metal resistance in motile

be attributed to the bacterial strain involved, assay

aeromonads and pseudomonads from rainbow trout

technique or culture conditions. However, the study has

(Oncorhynchus mykiss) farms in Australia. Inter. J.

proved that heavy metals such as mercury, zinc and lead

Antimicrob. Agents 30: 177â&#x20AC;&#x201C;182

do indeed have toxic effect on bacteria. Although it may vary from one species to another, there is no doubt that heavy metals do inhibit bacterial growth. Metals

toxic

contaminants

various

environmental sites have been reported to have adversely

Aleem A, Isar J, Malik A. 2003. Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chroococcum isolated from rhizospheric soil .Biores. Technol., 86: 7 - 13.

affected potential biodegradation processes occurring in

Alves de Lima A., Pereira MP, Filho RGS, Hofer E.

the environment (Said and Lewis, 1991). Amor et al.,

2007. Utilization of phenol in the presence of heavy

(2001) reported that the level of metal inhibition of

metals by metal-tolerant nonfermentative gram-negative

microbial growth depends on concentration as well as

bacteria isolated from wastewater. Rev. Latinoam.

nature of the metal and the type of microbial species.

Microbiol., 49 (3 - 4): 68 -73.

Sandrin and Maier (2003) reported that metals such as copper, zinc, cadmium, chromium, nickel, mercury and lead are known to inhibit biodegradation of organic pollutants by microorganisms. Phenol biodegradation have also been reported to be inhibited by metals (Nakamura

and Sawada,

2000;

Alves

Amor L, Kennes C, Veiga MC. 2001. Kinetics of inhibitionin

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hydrocarbons in the presence of heavy metals. Biores. Technol., 78: 181 - 185.

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Arutchelvan V, Kanakasabai V, Elangovan R,

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Nagarajan S and Muralikrishnan V. 2006. Kinetics of

behaviour of heavy metals, the effluents from petroleum

high strength phenol degradation using Bacillus brevis, J.

refinery industries could constitute enriched media to

Haz. Mat., B129(1-3): 216 - 222.

propagate and spread microbial populations which are resistant to metallic ions. Thus, microorganisms isolated from petroleum refinery effluent having combined abilities to grow in high concentration of phenol medium

Bruins MR, Kapil S, Oehme FW. 2000. Microbial resistance to metals in the environment. Ecotoxicol. Environ. Saf., 45:198 - 207.

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Journal of Research in Biology (2013) 3(3): 922-931

Journal of Research in Biology

An International Scientific Research Journal

Original Research

Journal of Research in Biology

Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats Authors: Nwankpa P1, Ezekwe AS1, Ibegbulem CO3 and Egwurugwu JN2.

Institution: 1. Department of Medical Biochemistry Imo State University, Owerri, Nigeria 2. Department of Physiology, Imo State University, Owerri, Nigeria 3. Department of Biochemistry, Federal University of Technology Owerri, Nigeria.

ABSTRACT: Haematological indices provide an insight about the internal environment of a given organism. In this present study, the possible anti-haemototxic effect of Chromolaena odorata on Salmonellae typhi â&#x20AC;&#x201C; induced haematotoxicity in rats were investigated. The experimental animals were divided into three groups. Group A received only food and water (control). Group B and C received in addition to food and water, single dose of stock Salmonellae typhi at a dose of 106cfu/ml. The animals in group B and C were allowed to be infected with Salmonellae typhi for 7 days and confirmed by widal test, after which group C was treated with 750mg/kg body weight/ day ethanolic extract of Chromolaena odorata for 10 days. The result showed a significant (p < 0.05) decrease in Red Blood Cells (RBC) count, packed cell volume (PCV), haemoglobin (Hb), mean corpuscular haemoglobin (MCH), Mean Corpuscular haemoglobin Concentration (MCHC), neutrophil and increase in platelet, total White Blood Cell (WBC) and lymphocytes in animals infected with Salmonellae typhi when compared to the control non-infected group. Treatment of animals in group C with ethanolic extract of Chromolaena odorata showed a significant (P < 0.05) increase in mean values of RBC count, PCV, Hb, MCH, MCV, MCHC and decrease in platelets, WBC and lymphocytes when compared to the group infested with Salmonellae typhi only. The results above suggest the anti-haematotoxic potential of ethanolic extract of Chromolaena odorata in Salmonellae typhi infested rats.

Corresponding author: Promise Nwankpa

Keywords: Salmonellae typhi, Chromolaena odorata, Blood cells, Anti-haematotoxic, Rats.

Web Address:

Article Citation: Nwankpa P, Ezekwe AS, Ibegbulem CO and Egwurugwu JN. Effect of Chromolaena odorata leaf extract on haematological profiles in Salmonellae typhi infested Wistar rats. Journal of Research in Biology (2013) 3(3): 932-939

http://jresearchbiology.com/ documents/RA0337.pdf.

Dates: Received: 15 Feb 2013

Accepted: 05 Mar 2013

Published: 11 May 2013

Journal of Research in Biology An International Scientific Research Journal

932-939 | JRB | 2013 | Vol 3 | No 3

www.jresearchbiology.com

Nwankpa et al., 2013 INTRODUCTION

of animals may be influenced adversely by diabetic

Enteric fever, also called typhoid fever caused by

condition (Edet et al., 2011), phenylhydrazine (Sanni

the bacterium Salmonellae typhi, is an acute life

et al., 2005), some anti-retroviral drugs (Kayode et al.,

threatening febrile ailment (Kotton, 2007). Typhoid fever

2011) and aqueous leaf extract of Ocimum gratissimum

is distributed worldwide and prevalent throughout the

(Obianime et al., 2011).

tropics where it is the commonest cause of fever

Chromolaena odorata (known as siam weed,

(Wilcocks and Manson-Bahr, 1972). Literature reports

independent weed, killer weed) is a perennial shrub

have shown that two million cases of typhoid and 200

which grow in rainforest, grassland and arid bushvelds

thousand related deaths occur worldwide each year

(Timbilla and Braimah, 2002). The leaves of the plant

(Steinberg et al., 2004). One challenge of development in

has been reported to be widely used as herbal remedy for

developing countries, is the provision of portable water

the treatment of various ailments. Available reports have

for the populace as poor sanitary condition and hygiene

shown a decotion of the leaf extract effective in the

has been reported to increase the prevalence of

treatment of malaria and cough (Suksamran et al., 2004).

Salmonellae typhi infection with reduced incidence in

Akah

developed countries (Kotton, 2007). Available reports

anti-inflammatory property of the leaf extract while

indicate that typhoid infection is the leading cause of

Thang et al., (2005) has shown the stimulation of

morbidity and mortality in a developing country like

granular tissue and re-epithelization of the epithelial

Nigeria where water carriage method of sewage disposal

tissue during wound healing. Recently Nwankpa et al.,

is inefficient (Crump et al., 2004). Salmonellae typhi

(2012) reported the antioxidative effect of ethanolic leaf

infection causes gastroenteritis which symptoms include

extract of Chromolaena odorata in rats. Other medicinal

nausea, vomiting and diarrhea (Parry et al., 2002). The

uses including anti-hypertensive, anti-diarrhoeal and

affected organs include spleen, liver and other tissues

diuretic has been reported (Iwu, 1993).

(1990) has reported the haemostatic and

which habor the bacterium before entering the blood

In rural communities in Nigeria, the use of

(Jones and Falkow, 1996). During metabolism, bacterial

Chromolaena odorata for treating Salmonellae typhi

cells, release chemical toxins which interactions damage

infection is common but the effect of the plant on

the tissue of the host organism. This tends to disrupt the

haematological indices in typhoid fever is not known.

blood components or blood forming tissues.

This study was therefore designed to assess the effect of

Blood is one of the specialized body fluid responsible for the transportation of nutrients, oxygen,

Chromolaena odorata on haematological profiles in Salmonellae typhi infested rats.

hormones and other metabolites to the bodyâ&#x20AC;&#x2122;s cell and metabolic waste products away from those cells to sites

MATERIALS AND METHODS

of elimination. It is known to be the most important body

Plant Material: The Chromolaena odorata leaves were

fluid that regulates various vital functions of the body

collected from a natural habitat in Owerri and

such as excretion, respiration, circulation, osmotic and

authenticated by professor S.C. Okeke, a taxonomist at

temperature balance etc. Mammalian circulation of blood

the department of Plant Science and Biotechnology, Imo

transports specific nutrients, gases, metabolic products

State University Owerri, Nigeria. The voucher specimen

and hormones between different tissues and organs

was kept in the university herbarium for references.

(Baynes and Dominiczak, 2005). Literature reports

Preparation of Extract: Large quantities of fresh leaves

indicated that haematological profiles of different species

of Chromolaena odorata, washed free of sand and

933

Journal of Research in Biology (2013) 3(3): 932-939

Nwankpa et al., 2013 debris, were dried under shade at room temperature at

with Salmonellae typhi and serve to monitor successful

27°C for 3 weeks. Electric blender was used to

induction of typhoid.

homogenize the dried leaves to a powder form. A 700g

Group B: The rats in this group served as control. They

of the powder macerated in 1.1 litres of 80% (v/v)

were fed with rat chow and had free access to water.

ethanol were allowed to stand for 24 hours. A chess clot

Single dose of Salmonellae typhi at106cfu/ml was orally

was used to filter the mixture and the filtrate

administered to rats in this group but were not treated

concentrated in vacuo at 37-40°C to 10% its original

with the plant extract.

volume using a rotary evaporator. The concentrate was

Group C: The rats in this group were fed with

evaporated in a water bath at 40°C to a solid residue, the

rat chow and had access to water. Single dose

extract. The extract was dissolved in 100ml of 10%

of Salmonellae tysphi at 106cfu/ml were orally

ethanol to an approximate concentration used for the

administered to the rats in this group. After 7 days

experiment.

of infection, 750 mg/kg ethanolic leaf extract of

Salmonellae typhi: The stock Salmonellae typhi was

Chromolaena odorata were orally administered to the

procured from Federal College of Veterinary and

animals daily for 10 days.

Medical

Collection and preparation of blood samples for

Laboratory Technology of the National

Veterinary Research Institute Vom, Jos, Plateau State,

analysis

Nigeria. Nutrient agar plate, cesteine lactose electrolyte

At the end of the treatment, the animals were

deficient plate (DCA) was used to sub-culture the micro-

fasted for 24 hours, re-weighed and sacrificed under

organism which was incubated at 37°C for 24 hours and

chloroform anesthesia. By cardiac puncture, blood

examined for growth. The stock sample used for the

sample was collected from each animal with a sterile

experiment was prepared as culture slants using

syringe and needle, in EDTA anti coagulated bottle. The

McCartney bottle and nutrient agar. Salmonellae typhi

anti-coagulated

from the sub-cultured medium was aseptically incubated

haematological analyses which were carried out within

for 18 hours at 37°C.

24 hours of sample collection.

Animals: Albino Wistar rats of both sexes weighing

Haematological analysis

blood

samples

were

used

for

between 150-200g were obtained from the animal house

Full blood counts such as packed cell volume

of Faculty of Medicine, Imo State University Owerri,

(PCV), Haemoglobin (Hb), Red Blood Cell (RBC), Total

Nigeria. They were maintained at room temperature and

White Blood Cells (TWBC), Platelet count, differential

acclimatized for 12 days to daily handling. They were

white

fed ad-libitum with commercial rat chow (Product of

eosinophils, neutrophils) and red cell indices including

Pfizer Nigeria Ltd) and had free access to water.

Mean

Induction of typhoid: Each rat was orally administered

Corpuscular Volume (MCV), Mean Cell Haemoglobin

blood

cell

Corpuscular

(like

lymphocytes,

Haemoglobin

monocytes,

(MCH),

Mean

with 1ml of Salmonellae typhi at a dose of 10 cfu/ml to

Concentration (MCHC) were estimated using the

induce typhoid (Kirby, 1960).

Sysmex® Automated Haematology Analyzer KX-2IN,

Experimental design: Twenty - four albino Wistar rats

Sysmex Corporation, Kobe, Japan.

were used for the study. They were randomly assigned

Statistical analysis

into 3 groups. Each group has 8 rats.

Data generated were statistically analysed by

Group A: The rats in this group were fed with rat chow

one-way analysis of variance (ANOVA) of the SPSS

and had free access to water. They were not administered

statistical programme of Microsoft Excel. Values were

Journal of Research in Biology (2013) 3(3): 932-939

934

Nwankpa et al., 2013 declared significantly different at p<0.05.

Salmonellae typhi infested group (Table 2). However the results of this study showed no significant (P > 0.05) difference in RBC, Hb, PCV, MCV, MCH, MCHC,

RESULTS AND DISCUSSION Table

and

shows

the

effect

platelets, WBC, and lymphocytes in Salmonellae typhi

Salmonellae typhi infection and subsequent treatment

infested

with ethanolic leaf extract of Chromolaena odorata on

compared to the non-infested rats (Table 1 and 2).

haematological parameters in rats. The results showed a

rats

treated

Haematological

with

Chromolaena

indices

provide

odorata relevant

significant (P < 0.05) decrease in Red Blood Cells (RBC)

information regarding the internal milieu of an organism.

count, haemoglobin (Hb), Packed Cell Volume (PCV),

Nutritional, environmental and microbial infection are

Mean

among several other factors which have been reported to

Corpuscular

have adverse effects on the haematological profiles of

Haemoglobin Concentration (MCHC) and percentage

most organisms. Vitamin B12 and folic acid deficiency

nuetrophil levels in Salmonellae typhi infested rats

(Jee et al., 2005, Murray et al., 2007) and exposure to

compared to the non-infested group (Table 1 and 2).

environmental pollutants such as carbondisulphide,

On the contrary, the total White Blood Cell (WBC),

insecticide, hexane, gasoline vapour, nitrocellulose

platelets and lymphocyte levels in rats infested with

thinner has been reported (Dhembara and Pandhe, 2000;

Salmonellae typhi showed a significant (P < 0.05)

Uboh et al., 2007; 2009; 2012 and Savithri et al., 2010).

increase compared to the non-infested group (Table 2).

Bacterial infection in living cells release toxins which

Treatment of the rats in group C with ethanolic leaf

metabolism results to increase in release of free radical

extract of Chromolaena odorata showed a significant

species with attendant damage to the cells (Stipanuk,

(P < 0.05) increase in RBC count, Hb, PCV, MCH,

2000). In this study, Salmonellae typhi infection

MCV,

levels

significantly decreases the level of RBC, PCV, Hb,

compared to the Salmonellae typhi infested non-treated

MCH, MCV, MCHC, neutrophils and increases the level

group (Table 1 and 2) while treatment of rats in group C

of WBC and lymphocytes. The observation made in this

with ethanolic leaf extract of Chromolaena odorata

study agrees with the report of Wilcocks and Manson-

showed a significant (P < 0.05) decrease in platelets,

Bahr (1972) in Salmonellae typhi infection and Kumar

WBC and lymphocyte levels compared to the non-treated

and Kuttan (2005) on cyclophosphamide induced

Corpuscular

Haemoglobin

Volume

MCHC

and

(MCV),

(MCH),

Mean

percentage

neutrophil

Table 1: Effect of Chromolaena odorata on mean values of red blood cells, packed cell volume, hemoglobin and red cell indices in both experimental and control groups. Group A B

Treatment Negative control/water Salmonellae typhi (Positive control) Salmonellae typhi + Chromolaena odorata

RBC X1012/L

Hb (g/dL)

PCV (%)

MCV (fL)

MCH (pg)

MCHC (g/dL)

3.69 ± 0.21

14.43 ± 0.65

44.33 ± 2.13

63.12 ± 1.60

17.19 ± 1.12

31.27 ± 1.20

1.62 ± 0.03a

10.09 ± 0.71a

33.26 ± 2.14a

54.85 ± 1.55a

12.52 ± 1.30a

24.12 ± 1.23a

3.49 ± 0.05bc

14.15 ± 0.79bc

43.40 ± 2.34bc

61.95 ± 1.32bc

16.55 ± 1.02bc

30.12 ± 1.33bc

Mean ± SD (n = 8) a Significantly different compared with negative control (P < 0.05). b Significantly different compared with Salmonellae typhi (positive control) (P < 0.05). c No significant difference compared with negative control (P > 0.05). 935

Journal of Research in Biology (2013) 3(3): 932-939

Nwankpa et al., 2013 Table 2: Effect of CO on mean values of platelets, total white blood cells and differential cell counts in both experimental and control groups Group A B C

Platelets X103μL-1

Treatment Negative control/ water Salmonellae typhi (Positive control) Salmonellae typhi + Chromolaena odorata

TWBC X103μL-1

Lymphocytes (%)

Neutrophils (%)

Eosinophils

(%)

Monocytes (%)

855.18 ± 2.11

16.24 ± 0.78

70.11 ± 2.01

20.19 ± 1.15

1.98 ± 0.6

2.51 ± 0.11

880.13 ± 1.5a

25.85 ± 1.16a

82.14 ± 2.11a

11.56 ± 0.87a

3.20 ± 1.10

2.90 ± 0.55

858.82 ± 1.46bc

17.14 ± 1.21bc

72.18 ± 1.88bc

19.26 ± 1.11bc

2.10 ± 0.80

2.6 ± 0.52

with Salmonellae typhi.

infection may be explained by the interaction of the

Ethanolic extract of Chromolaena odorata

bacteria or its toxins with the blood forming tissues/

significantly increased the level of RBC, Hb, PCV,

organs which may inhibit the rate at which some specific

MCV, MCH and MCHC thereby reducing and

or generalized haemopoeitic committed stem cells are

ameliorating

synthesized by the tissues. Some reports have shown that

Salmonellae typhi infection. The observed increase in

hexane,

induced

RBC, Hb, and PCV may be explained by the role of

haematotoxic effect is associated with the interaction of

Chromolaena odorata extract in reversing bone marrow

their metabolites with the haematopoeitic tissues and

depression with attendant improvement in erythrocyte

cause depression in their haematopoeitic activities

membrane stability through the antioxidant potential of

(Synder and Hedli, 1996; Kumar and Kuttan, 2005).

the plant extract, thus reducing haemolysis (Krause and

Increase in total white blood cells and lymphocytes as

Mahan, 1984; Naaz et al., 2007, Nwankpa et al., 2012).

well as decrease in neutrophils seen in this study is

The improvement on the haematopoetic activities of the

consistent with the reports on effect of insecticides and

tissues and/or maintenance of red blood cell membrane

pesticides such as fenvalerate, lindane, aldrin among

integrity relieves the anaemic condition observed in

others, on total white blood cells and the differential

Salmonellae typhi infection.

cyclophosphamide

and

benzene

the

anaemic

condition

induced

counts in experimental animals (Synder and Hedli, 1996;

Consequently, increase in RBC count on

Kumar et al., 1996; Savithri et al., 2010). This may be

administration of Chromolaena odorata leaf extract

explained by increased lymphopoeisis and/or enhanced

translates to an increase in MCV while increase in Hb

release of lymphocytes from lymph myeloid tissue (Das

translates, to an increase in MCH and MCHC.

and Mukherjee, 2003). This response may be a direct

Furthermore, inhibition of microbial growth by the plant

stimulatory effect of toxic substance on lymphoid tissue/

extract has been reported. Okigbo and Ajalie (2005) and

pollutant induces tissue damage and disturbance of the

Alisi et al., (2011) showed that Chromolaena odorata

non-specific immune system leading to increase in

leaf extract possess antibacterial activity which inhibit

production of leukocytes. Neutrophils are known to be

the growth of Salmonellae typhi in cells. Decrease in

involved in the phagocytosis of foreign substances in the

total white blood cell, lymphocytes and attendant

body during which some of them are ruptured. This may

increase in neutrophils on administration of the plant

explain the decrease in neutrophil count on infection

extract may be explained by the inhibition of growth of

Journal of Research in Biology (2013) 3(3): 932-939

936

Nwankpa et al., 2013 Salmonellae typhi in the cell. The inhibition of growth of the microorganism lead to the destruction of excess WBC and lymphyocytes released by the cell in response to bacterial infection (Nancy et al., 2005). Conversely, increase in neutrophil count on administration of the plant extract may be explained by reduced phagocytosis of the microbial cell consequent upon drastic reduction in the growth of microbial cell.

insecticides. J. Exp. Zool., India. 3:41-44. Edet EE, Akpanabiatu MI, Uboh FE, Edet TE, Eno AE, Itam EH and Umoh B. 2011. Gongronema latifolium crude leaf extract reverses alterations in haemotological indices and weight-loss in diabetic rats. J. Pharmacol. Toxicol., 6(2):174-181. Iwu NM. 1993. Handbook of African Medical Plants. CRC Press London.

CONCLUSION This study has established the anti-haematotoxic p ot en t i a l

et h a n ol i c

l ea f

ext r a ct

Chromolaena odorata against Salmonellae typhi induced haematotoxicity in rats.

Jee LH, Masroor F and Kang JC. 2005. Responses of cypermethrin-induced

stress

haematological

parameters of Korean rockfish, Sebastes schlegeli (Hilgendorf) Aquacult. Res., 36(9):898-905. Jones BD and Falkow S. 1996. Salmonellosis: Host

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