Nusantara Bioscience vol. 3, no, 2, July 2011 by Nusantara Bioscience

Vulpes vulpes photo by AFN Abd. Rabou

| Nus Biosci | vol. 3 | no. 2 | pp. 59‐103| July 2011 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

| Nus Biosci | vol. 3 | no. 2 | pp. 59‐103 | July 2011 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia (sugiyarto_ys@yahoo.com) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia (jrwitono@yahoo.com) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia (janung4@yahoo.com.au) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia (pattiselannofreddy@yahoo.com) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India (pmkrai@hotmail.com) Biomedical Sciences, Alfiono, Sebelas Maret University, Surakarta, Indonesia (afieagp@yahoo.com), Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia (iyahya@uns.ac.id), Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia (cecep_kusmana@ipb.ac.id) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia (lufehakim@yahoo.com) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia (nnsutarno@yahoo.com), Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia (gadis@limnologi.lipi.go.id) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt (shokry_1@yahoo.com) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia (charis@ukdw.ac.id) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia (ajamsari@yahoo.com) Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China (xiuyunzh@yahoo.com.cn) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia (pudjiwi@yahoo.com) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta (unsjournals@gmail.com) Associated Editor (English Editor), Wiryono, State University of Bengkulu (wiryonogood@yahoo.com), Technical Editor, Ari Pitoyo, Sebelas Maret University Surakarta (aripitoyo@yahoo.co.id) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia (nusbiosci@gmail.com)

PUBLISHER: Society for Indonesian Biodiversity CO-PUBLISHER: School of Graduates, Sebelas Maret University Surakarta

FIRST PUBLISHED: 2009

ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbiosci@gmail.com

ONLINE: biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

ISSN: 2087-3948 (print. ISSN: 2087-3956 (electronic.

Vol. 3, No. 2, Pp. 59-63 July 2011

A biogenic approach for green synthesis of silver nanoparticles using extract of Foeniculum vulgare and its activity against Staphylococcus aureus and Escherichia coli SHITAL BONDEâ&#x2122;Ľ Department of Biotechnology, SGB Amravati University, Amravati 444602, Maharashtra, India. Tel: +91-721-2662207/8, Extension-267. Fax: +91 721 2660949, 2662135, â&#x2122;Ľemail: shitalbonde@gmail.com Manuscript received: 10 May 2011. Revision accepted: 7 June 2011.

Abstract. Bonde S. 2011. A biogenic approach for green synthesis of silver nanoparticles using extract of Foeniculum vulgare and its activity against Staphylococcus aureus and Escherichia coli. Nusantara Bioscience 3: 59-63. We report green synthesis of silver nanoparticles from extract of Foeniculum vulgare (fennel, saunf). The synthesis of silver nanoparticles was detected by changing color from green to brown after treatment with AgNO3 (1mM) and the UV-visible spectrophotometer analysis showed the absorbance peak at about 427 nm, which indicates the synthesis of silver nanoparticles. Nanoparticle Tracking and Analysis (NTA) by LM-20 was used for multi-parameter analysis, allowing for characterization of particle size and particle distribution of silver nanoparticles synthesized from extract of F. vulgare. NTA revealed the polydispersed nanoparticles in the range of 18-83 nm. Phytosynthesized silver nanoparticles showed antibacterial activity against the Staphylococcus aureus (ATCC-25923) and Escherichia coli (ATCC-39403). The silver nanoparticles also demonstrated remarkable antibacterial activity against two human pathogenic bacteria when used in combination with commercially available antibiotics. The bactericidal activity of the standard antibiotics was significantly enhanced in presence of silver nanoparticles against pathogenic bacteria, viz. E. coli-JM-103 (ATCC-39403) and S. aureus (ATCC-25923). Silver nanoparticles in combination with vancomycin showed maximum activity against E. coli (increase in fold area 5.76. and followed by S. aureus (1.08) and Gentamicin showed the maximum activity S. aureus (2.6) while E. coli (0.96). The approach of phytosynthesized silver nanoparticles using F. vulgare appears to be cost efficient, eco-friendly and easy alternative to conventional methods of synthesis. Key words: Foeniculum vulgare, silver nanoparticles, LM20, antibacterial.

Abstrak. Bonde S. 2011. Pendekatan biogenik untuk sintesis nanopartikel perak menggunakan ekstrak Foeniculum vulgare dan aktivitasnya terhadap Staphylococcus aureus dan Escherichia coli. Nusantara Bioscience 3: 59-63. Kami melaporkan sintesis nanopartikel perak dari ekstrak Foeniculum vulgare (adas). Sintesis nanopartikel perak terdeteksi dengan mengubah warna dari hijau sampai coklat setelah perlakuan dengan AgNO 3 (1mm); dan analisis spektrofotometer UV-vis menunjukkan puncak absorbansi pada sekitar 427 nm, yang menunjukkan sintesis nanopartikel perak. Analisis Pelacakan Nanopartikel (NTA) oleh LM-20 digunakan untuk analisis multi-parameter, memungkinkan untuk karakterisasi ukuran partikel dan distribusi partikel nanopartikel perak yang disintesis dari ekstrak F. vulgare. NTA mengungkapkan nanopartikel tersebar di kisaran 18-83 nm. Fitosintesis nanopartikel perak menunjukkan aktivitas antibakteri terhadap Staphylococcus aureus (ATCC-25923) dan Escherichia coli (ATCC-39403). Perak nanopartikel juga menunjukkan aktivitas antibakteri yang luar biasa terhadap dua bakteri patogen manusia apabila digunakan dalam kombinasi dengan antibiotik yang tersedia secara komersial. Aktivitas bakterisida antibiotik standar secara signifikan ditingkatkan dengan adanya nanopartikel perak terhadap bakteri patogen, yaitu: E. coli-JM-103 (ATCC-39403) and S. aureus (ATCC-25923). Nanopartikel perak yang dikombinasi dengan vankomisin menunjukkan aktivitas maksimal terhadap E. coli (kenaikan berlipat 5,76) dan diikuti oleh S. aureus (1,08); dan gentamisin menunjukkan aktivitas maksimum S. aureus (2,6) sedangkan E. coli (0,96). Pendekatan fitosintesis nanopartikel perak menggunakan F. vulgare tampaknya memerlukan biaya yang efisien, ramah lingkungan dan merupakan alternatif mudah untuk metode sintesis konvensional. Kata kunci: Foeniculum vulgare, nanopartikel perak, LM20, antibakteri.

INTRODUCTION Phytosynthesis at present seems to be the biological method of much interest (Safaepour et al. 2009). Phytosynthesis is better compared to microorganisms because the later suffer from various problems like culture maintenance and cost effectiveness during the scale up process. Various plants have been successfully used for the synthesis of biogenic metal nanoparticles (Singh et al. 2011).

The rapid synthesis of silver nanoparticles by biological method using plant extracts of Pinus, Persimmon, Ginkgo, Magnolia and Platanus were used and compared for their extracellularly metallic silver nanoparticles (Song et al. 2008) and the utilization of Azadirachta indica (neem). (Shankar et al. 2004), Medicago sativa (alfalfa), Aloe vera (Chandran et al. 2006), Emblica officinalis (amla). (Amkamwar et al. 2005), Capsicum annuum (Li et al. 2007), Cinnamomum

3 (2): 59-63, July 2011

camphora (Huang et al. 2007), Gliricidia sepium Jacq. (Raut et al. 2009), Carrica papaya (Mude et al. 2009), Opuntia ficus-indica (Gade et al. 2010), Murraya koenigii (Bonde et al. 2010), Ocimum sanctum (Mallikarjum et al. 2011), Saururus chinenis (Nagajyoti et al. 2011), and microorganisms (Duran et al. 2005; Bhainsa et al. 2006) has been reported fennel (Foeniculum vulgare) is an important crop plant with medicinal value being carminative. Its fruits are used as a digestive adjuvant having antimicrobial activity (He and Huang 2011). The multi-drug resistant pathogens are responsible for causing death worldwide (Bandow et al. 2003; Wright et al. 2005) and hence there is a pressing need for the development of novel antimicrobial agents. Reports suggest that silver nanoparticles can be used effectively against multi-drug resistant bacteria (Ingle et al. 2008) due to their small size and relatively large surface area in comparison to their volume makes easy to interact with substances and increases their antibacterial efficacy. Silver nanoparticles are the new generation of antimicrobials (Rai et al. 2009) and it can be used in many antimicrobial preparations. Gade et al. (2008. and Ingle et al. (2008) reported the antibacterial activity of silver nanoparticles synthesized by fungi. Antibacterial activity of silver nanoparticles synthesized by leaf broth of Gliricidia sepium was reported by Raut et al. (2009). Duran et al. (2007) successfully developed silver nanoparticle impregnated wound dressings and textile fabrics which can be used for burnt patients. Silver nanoparticles are also used for the preparation of surgical masks (Li et al. 2006). In the present study, F. vulgare was used for the synthesis of silver nanoparticles and the activity of synthesized silver nanoparticles was evaluated against S. aureus and E. coli in combination with commercially available five antibiotics viz. gentamicin, oxacillin, vancomycin, ampicillin and amoxycillin, to study synergistic effect, if any.

MATERIALS AND METHODS The test plant The young and healthy leaves of Foeniculum vulgare were collected from the field of Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, Maharashtra State, India. Test bacteria Escherichia coli-JM-103 (ATCC-39403) and Staphylococcus aureus (ATCC-25923) were used to evaluate the activity of silver nanoparticles in combination with standard antibiotics (viz. gentamicin, oxacillin, vancomycin, ampicillin and amoxycillin, purchased from Himedia). Extraction The leaves of F. vulgare (20 g) were washed twice in tap water and rinsed thrice in distilled water. Then surface sterilized by HgCl2 (0.1%) for 1 min, cut into small pieces and crushed with 100 mL of sterilized distilled water in an Omni mixer. Later, crude extract was filtered through muslin cloth and centrifuged at 10,000 rpm for 15 min to

obtain clear leaf extract which was later used for the synthesis of silver nanoparticles. Synthesis of silver nanoparticles For the synthesis of silver nanoparticles leaf extract was challenged with AgNO3 (1mM) solution and incubated at room temprature. Control (without treatment with AgNO3. (1mM) i.e. only leaf exrtact) was also maintained. After the reduction of aqueous silver ions into silver nanoparticles, residual silver ions (unreacted silver ions) were removed from the reaction mixture by centrifugation and presence of unreacted silver ions were detected by the treatment with sodium chloride (NaCl) as a result white precipitate of silver chloride (AgCl) was formed after reacting with Ag+ ion. Triplicates of each treatment were maintained. Detection and characterization of silver nanoparticles Visual observation. After treatment of leaf extract with AgNO3 (1mM), the colour change of the reaction mixture was visually observed. UV-Vis spectrophotometric analysis. The aliquotes of reaction mixture were subjected to the meausurement of absorbance by UV-visible spectrophotometer (Perkin Elmer, Labda-25. at a resolution of 1 nm from 250 to 800 nm for the detection of silver nanoparticles. NanoSight LM-20 analysis. Liquid sample of silver nanoparticles at the concentration range of 107-109/mL were introduced into a scattering cell through which a laser beam (approx. 40 mW at k = 635 nm) was passed. Particles present within the path of the laser beam were observed via a dedicated non-microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK. having CCD camera. The motion of the particles in the field of view (approx. 100 X 100 μm) was recorded (at 30 fps) and the subsequent video and images were analyzed. Each particle visible in the image is individually but simultaneously traced from frame to frame, the mean square displacement is determined by analytical programme. Particle size measurement. Particle sizing experiments were carried out by means of laser diffractometry, using Zetasizer nano series (Malvern) Measurements were taken in the range range between 0.1-1000µm. Assessment of antibacterial activity The disc diffusion method was used to evaluate the antibacterial potential of silver nanoparticles and combined effect of silver nanoparticles with five antibiotics against two human pathogenic bacteria: E. coli-JM-103 (ATCC39403) and S. aureus (ATCC-25923) grown on nutrient agar plates. The overnight grown bacterial culture having 105 CFU/mL was used to assess the activity. The test bacterial cultures were inoculated on to solidified agar plates. The different standard antibiotic discs (viz. gentamicin, oxacillin, vancomycin, ampicillin and amoxycillin) purchased from Hi-Media, Mumbai were used. To evaluate the combined effects, each standard antibiotic disc impregnated with 20 µL solution of silver nanoparticles was placed on to the agar surface inoculated with test bacteria. The plates were then incubated at 370C for 24 hours. After incubation, the zones of inhibition were

BONDE-Synthesis of silver nanoparticles using extract of Foeniculum vulgare

measured and its activities were evaluated by calculating the increase in fold-area. The assays were performed in triplicate. Assessment of increase in fold area The increase in fold area was assessed by calculating the mean surface area of the inhibition zone of each tested antibiotic. The fold increase area of different antibiotics was calculated by using the equation calculated as (b2-a2) â &#x201E; a2, where a and b are the inhibition zones for A and B, respectively. In the same way, c and d.

Huang et al. (2007) and Li et al. (2007) reported the similar results, observed that when sun dried leaf extract of Cinnamomum camphora and Capsicum annuum were challenged with aqueous silver ions, the reaction mixture containing silver nanoparticles showed the absorption peak at about 427 nm due to the excitation of plasmon resonance vibration.

RESULTS AND DISCUSSION The synthesis of silver nanoparticles by leaf extract of F. vulgare was performed in the present study. On treatment of leaf extract with AgNO3 (1mM) and incubated in dark at room temperature, within 1 hour of the reaction, colour changes from green to brown (Figure 1) indicating the synthesis of silver nanoparticles. It is an efficient and rapid method of synthesis which corroborate with the results obtained by other researchers who worked with different plant systems (Shankar et al. 2004; Chandran et al. 2006; Li et al. 2007; Mallikarjun et al. 2011). Colour change was due to the excitation of surface plasmon vibrations in the metal nanoparticles (Ahmad et al. 2003).

Figure 1. Control (left) and silver (right) nanoparticles synthesized from Foeniculum vulgare

UV-Vis spectrophotometric analysis has proved to be a very useful technique for the analysis of nanoparticles. In order to verify the synthesis of silver nanoparticles, the test samples were subjected to UV-Vis spectrophotometric analysis. The test samples (leaf extract treated with 1mM silver nitrate. were collected in aliquots from the reaction mixture and analyzed to record their absorbance by UV-Vis spectrophotometer. This analysis showed the sharp absorbance at around 427 nm in the form of peak (Figure 2), which was specific for silver nanoparticles (Shankar et al. 2004; Chandran et al. 2006; Elumalai et al. 2010).

Figure 2. UV-Visible spectroscopy of synthesized silver nanoparticles from Foeniculum vulgare

Here we demonstrated Nanoparticle Tracking and Analysis (NTA) to measure the dispersion characteristics i.e. size and size distribution on their brownian motion in suspension. NTA allows individual nanoparticles in a suspension to be microscopically visualized and their brownian motion to be separately but simultaneously analyzed and from which the particle size distribution can be obtained on a particle-by-particle basis which enables separation of particle population by size and intensity. The NTA showed particle populations by size and intensity. These results correlates the results obtained by MontesBurgos and group (Montes-Burgos et al. 2010). Total Concentration of silver nanoparticles synthesized by leaf extract of F. vulgare was found to be 7.6 particles/frame, 3.35X108 particles/mL. The size of silver particle analyzed by NTA was in the range of 18-83 nm. Statistical distribution of silver nanoparticles using LM 20, mean: 82nm, mode: 83 nm. SD: 25 nm. Distribution of Particle Size/Concentration of Ag NPs was showed in Figure 3a and the particle populations of Ag NPs using NanoSight LM-20 were studied (Figure 3a and 3b). Particle size determination of the formulated nanoparticles was shown under different categories like size distribution by volume, by intensity (Figure 4). First and second peaks, the average diameter of the particles were found to be 127, 100% and width 37.25 nm. The formed silver nanoparticles are well distributed with respect to volume and intensity is an indication of the formation of well built silver nanoparticles and their monodispersity.

Figure 3a. Particle Size / Concentration of Ag NPs

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Figure 3b. Particle populations of Ag NPs using NanoSight LM20.

Figure 4. Particle size distribution of Ag-NPs by intensity with Zeta Analyzer. Table 1. Comparison in increase in fold area zone of activity of different antibiotics against S. aureus, and E. coli (in absence and in presence of silver nanoparticles (AgNPs) at concentration of 20µl/disc. (Where, Ab= Antibiotics, Ab+ Ag-NPs= Antibiotics + silver nanoparticles) Staphylococcus aureus Ab Ab+ *Increase Antibiotics (A) Ag-NPs in fold (B) area Gentamicin 19 ± 0.3 36±0.1 2.6±0.2 Oxacillin 24± 0.2 29±0.3 0.32±0.3 Vancomycin 18 ± 0.4 26±0.4 1.08±0.2 Ampicillin 27 ± 0.1 31±0.2 0.31±0.1 Amoxycillin 29 ± 0.2 32±0.1 0.21±0.3 Note: Inhibition zone in mm

Escherichia coli Ab+ *Increase Ab Ag-NPs in fold (C) (D) area 20±0.3 28±0.4 0.96 28±0.5 30±0.1 0.14 10±0.1 26±0.2 5.76 21±0.2 24±0.4 0.31 19±0.2 25±0.3 0.73

We report synthesis of silver nanoparticles by F. vulgare for the first time as there is no report of synthesis of silver nanoparticles by this plant. Moreover, the phytosynthesized silver nanoparticles were used for the evaluation of their antibacterial efficacy in combination with commercially available antibiotics. From the present study, it was observed that the efficacy of silver nanoparticles against test bacteria (E. coli and S. aureus) was increased when assessed in combination of antibiotics. It is evidenced by the data provided in Table 1, which showed increase in activity fold area for each antibiotic. The bactericidal activity of the standard antibiotics was significantly increased in presence of silver nanoparticles against pathogenic bacteria, viz. E. coli-JM-103 (ATCC39403) and S. aureus (ATCC-25923) Silver nanoparticles in combination with Vancomycin showed maximum activity against E. coli (increase in fold area 5.76) and S. aureus (1.08) and gentamicin showed the maximum activity S. aureus (2.6) while E. coli (0.96). While other antibiotics did not show significant inhibitory activity against the test bacteria. These findings support the report by Birla et al. 2009 year, who reported that the activity of commercially available antibiotics with silver nanoparticles synthesized by a

BONDE-Synthesis of silver nanoparticles using extract of Foeniculum vulgare

fungus Phoma glomerata was more against Gram-negative bacteria compared to the Gram-positive bacteria and also Savitthramma et al. (2011) report the silver nanoparticles of Boswellia avalifoliota showed maximum inhibition of E. coli. The green method of synthesis is safer than other. There are three types of antimicrobial mechanisms observed by Song et al. (2006), i.e. (i) Plasmolysis, cytoplasm of bacteria separated from bacterial cell wall, was observed in Gram negative bacteria and Gram positive bacteria, (ii) inhibited cell wall synthesis and (iii) induces metabolic disturbances to pathogenic bacteria.

CONCLUSION The present study included the bioreduction Ag + ions by plant F. vulgare and its antibacterial activity. The study reveals that plant species are good and fast rate source of synthesis and the antibacterial efficacy against S. aureus and E. coli confirmed that the silver nanoparticles are capable of rendering antibacterial efficacy and strengthen the medicinal value of plants. Phytosynthesis of silver nanoparticles are most convenient, easily scale up and ecofriendly.

ACKNOWLEDGEMENTS The author is grateful to Prof. M.K. Rai and A.K. Gade for their guidance and support. Author wishes to thank S.C. Gaikwad for helping in analysis of LM20, and to Funds for Infrastructure Science and Technology (FIST. for providing computer facility, Department of Biotechnology, S.G.B.A.U. Amravati and Prof. Nelson Duran, Department of Biological Chemistry, University of Campinas, Brazil for providing Zeta analyzer instrument facility.

REFERENCES Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R. 2003. Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Coll Surf B 28: 313-318. Amkamwar B, Damle C, Ahmad A, Sastry M. 2005. Biosynthesis of gold and silver nanoparticles using Emblica officinalis fruit extract, their phase transfer and transmetallation in an organic solution. J Nanosci Nanotechnol 5: 1665-1671. Bandow JE, Brotz H, Leichert LIO, Labischinski H, Hecker M. 2003. Proteomic approach to understanding antibiotic action. Antim Agents Chem 47: 948-955. Bhainsa KC, D'Souza SF. 2006. Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids Surf B Biointerfaces 47: 160-164. Birla SS, Tiwari VV, Gade AK, Ingle AP, Yadav AP, Rai MK. 2009. Fabrication of silver nanoparticles by Phoma glomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Lett App Microbiol 48: 173-179. Bonde SR, Rathod DP, Ingle AP, Ade RB, Gade AK, Rai MK. 2010. Murraya koenigii mediated synthesis of silver nanoparticles and its activity against three human pathogenic bacteria. J Exp Nanosci (in press).

Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. 2006. Synthesis of gold nanotriangles and silver nanotriangles using Aloe vera plant extract. Biotech Prog 22: 577-579. Duran N, Marcarto PD, De Souza GIH, Alves OL, Esposito E. 2007. Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment. J Biomed Nano 3: 203208. Duran N, Marcato PD, Alves OL, Souza GI, Esposito E. 2005. Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J Nanobiotechnol 3: 8. Elumalai EK, Prasad TN, Kambala , Nagaiyothi PC, David E. 2010. Green synthesis of silver nanoparticle using Euphorbia hirta L and their antifungal activities. Arch Appl Sci Res 2 (6): 76-81 Gade AK, Bonde P, Ingle AP, Marcato PD, Duran N, Rai, MK. 2008. Exploitation of Aspergillus niger for synthesis of silver nanoparticles. J Biobased Mat Bioengineer 2: 243-247. Gade AK, Gaikwad SC, Tiwari V, Yadav A, Ingle AP, Rai MK. 2010. Biofabrication of silver nanoparticles by Opuntia ficus-indica: In vitro antibacterial activity and study of the mechanism involved in the synthesis. Curr Nanosci 6: 370-375. He W, Huang B. 2011. A review of chemistry and bioactivities of a medicinal spice: Foeniculum vulgare. J Med Pl Res 5 (16): 35953600 Huang J, Chen C, He N, Hong J, Lu Y, Qingbiao L, Shao W, Sun D, Wang Y, Wang XH, Yiang X. 2007. Biosynthesis of silver and gold nanoparticles by novel sun dried Cinnamomum camphora leaf. Nanotechnol 18: 105-106. Ingle A, Gade A, Pierrat S, Sonnichsen C, Rai M. 2008. Mycosynthesis of silver nanoparticles using the fungus Fusarium acuminatum and its activity against some human pathogenic bacteria. Curr Nanosci 4: 141-144. Li LP, Song QW, Newton E. 2006. Antimicrobial effects of surgical masks coated with 869 nanoparticles. J Hosp Infect 62: 58-63. Li S, Qui L, Shen Y, Xie A, Yu X, Zhang L, Zhang Q. 2007. Green synthesis of silver nanoparticles using Capsicum annum L. extract. Green Chem 9: 852-858. Mallikarjun K, Narsimha G, Dillip GR, Praveen B, Shreedhar B, Lakshmi S, Reddy VS, Raju DP. 2011. Green synthesis of silver nanoparticles using Ocimum leaf extract and their characterization. Digest J Nanomat Biostruct 6 (1): 181-186. Mude N, Ingle A, Gade A, Rai M. 2009. Synthesis of silver nanoparticles using callus extract of Carica papaya-A first report. J Pl Biochem Biotechnol 18: 83-86. Nagajyoti PC, Prasad TN, Shreekanth VM, Lee KD. 2011. Biofabrication of silver nanoparticles using leaf of Saururus chinenis. Digest J Nanomat Biostruct 6 (1): 121-133. Rai MK, Yadav AP, Gade AK. 2009. Silver nanoparticles as a new generation of antimicrobials .Biotech Adv 27 (1): 76-82. Raut RW, Lakkakula JR, Kolekar NS, Mendhulkar VD, Kashid SB. 2009. Phytosynthesis of silver nanoparticle using Gliricidia sepium (Jacq.). Curr Nanosci 5: 117-122. Safaepour M,Shahaverdi AR, Shaverdi HR, Khorramizadeh MR, Gohari AR. 2009. Green Synthesis of small silver nanoparticles using geraniol and its cytotoxicity against Fibrosarcoma-Wehi 164. Avicenna J Med Biotech 1 (2): 111-115. Savittramma N, Rau LM, Suvarnalatha P. 2011. Evaluation of antibacterial efficacy of biological synthesized silver nanoparticles using stem barks of Boswellia avalifoliolata Bal. and Henry and Shorea tumbuggaia Roxb. J Biol Sci 11 (1): 39-45. Shankar SS, Rai A, Ahmad A, Sastry MJ. 2004. Rapid synthesis of Au, Ag, and bimetallic Au core-Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J Colloid Interface Sci 275: 496-502. Singh C, Sharma V, Naik PK, Khandelwal V, Singh H. 2011. A green biogenic approach for synthesis of gold and silver nanoparticles using Zingiber officinale. Digest J Nanomat Biostruct 6 (2): 535-542. Song H.Y, Ko KK, Oh IH, Lee BT. 2006. Fabrication of silver nanoparticles and their antimicrobial mechanisms. Eur Cells Mater 11 (1): 58. Song JY, Beom SK. 2008. Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioproc Biosyst Engineer 32 (1): 79-84. Wright GD. 2005. Bacterial resistance to antibiotics: Enzymatic degradation and modifications. Adv Drug Del Rev 57: 1451-1470.

ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 64-67 July 2011

Oral squamous cell carcinoma patients which human papilloma virus infection: a case control study in Muwardi Hospital Surakarta, Central Java, Indonesia ADI PRAYITNO1,♥, ELYANA AZNAR2, POERNOMO2, SUHARTONO TAAT PUTRA2 1

Department of Dental and Oral Disease, Faculty of Medicine, Sebelas Maret University (UNS), Jl. Ir. Sutami 36A, Surakarta 57126, Central Java, Indonesia. Tel. +62-271-664178. Fax. +62-271-637400. ♥Email: drgadiprayitno@yahoo.com 2 Faculty of Medicine, Airlangga University, Surabaya 60131, East Java, Indonesia. Manuscript received: 27 February 2011. Revision accepted: 4 June 2011.

Abstract. Prayitno A, Aznar E, Poernomo, Putra ST. 2011. Oral squamous cell carcinoma patients which human papilloma virus infection: a case control study in Muwardi Hospital Surakarta, Central Java, Indonesia. Nusantara Bioscience 3: 64-67. Annual incidence rates for oral and pharyngeal cancer are estimated at 25 cases per 100,000 in developing countries. Human papilloma virus (HPV) was implicated in pathogenesis of Oral Squamous Cell Carcinoma (OSCC). Aims of this research were to know the incidence of OSSC patient which realized HPV infection. Women OSCC (15) and Benign Oral Squamous Cells (BOSC) (40) tissue biopsy frozen sections were from Departement of Oral and Dental, Muwardi Hospital in Surakarta from January to December 2007. Tissue was cut into two parts. To ascertain the type of neoplasm was subsequently stains with HE. To amplify the L1-HPV gene for 450bp long. The collected data was analyzed by Chi Square Test. The result of this experiment showed nine patients from 40 patients BOSC identified have HPV infections (9/40 = 23%). Eleven patient from 15 patient OSCC identified have HPV infections (11/15 = 73%). From Chi Square analysis have significant differences between BOSC and OSCC. HPV is a factor for OSCC pathogenesis. Key words: developing countries, HPV, pathogenesis, OSCC, Moewardi Hospital.

Abstrak. Prayitno A, Aznar E, Poernomo, Putra ST. 2011. Pasien oral squamous cell carcinoma dengan infeksi virus papiloma manusia: studi kasus kontrol di Rumah Sakit Muwardi Surakarta, Jawa Tengah, Indonesia. Nusantara Bioscience 3: 64-67. Tingkat insiden tahunan untuk kanker mulut dan faring diperkirakan mencapai 25 kasus per 100.000 di negara-negara berkembang. Human papilloma virus (HPV) terlibat dalam patogenesis Oral Squamous Cell Carcinoma (OSCC). Tujuan penelitian ini adalah untuk mengetahui kejadian pasien OSSC yang mengalami infeksi HPV. Irisan beku biopsi jaringan OSCC (15) dan Benign Oral Squamous Cells (BOSC) (40) wanita diperoleh dari Bahian Mulut dan Gigi Rumah Sakit Muwardi Surakarta, dari bulan Januari sampai Desember 2007. Jaringan dipotong menjadi dua bagian. Untuk memastikan jenis neoplasma, maka diwarnai dengan HE. Untuk gen L1-HPV digunakan penanda berukuran 450bp. Data yang dikumpulkan dianalisis dengan Chi Square Test. Hasil penelitian ini menunjukkan sembilan pasien dari 40 pasien BOSC diidentifikasi mengalami infeksi HPV (9/40 = 23%). Sebelas pasien dari 15 pasien OSCC diidentifikasi mengalami infeksi HPV (11/15 = 73%). Dari analisis Chi Square terdapat perbedaan yang signifikan antara BOSC dan OSCC. HPV merupakan faktor patogenesis OSCC. Kata kunci: negara berkembang, HPV, patogenesis, OSCC, Rumah sakit Muwardi.

INTRODUCTION Factor that known implicated as a potential cock and or promoter cancer were tobacco, alcohol, radiation of sunrise, ionization radiation, carcinogen related work, environment pollutant, medicines, nutrition and infectious agent. Another factor is life in village, social-economic factor, age, gender and response immune mechanism. Information about another factor was little. The followed factor is periodontal disease chronic, bed oral hygiene, diseases of tooth, sharp of set teeth, electrogalvanism and edentulism. Another researcher found that human papilloma virus (HPV), especially 16 and 18 type, implicated in oral squamous cell carcinoma (OSCC) pathogenesis (Bsoul et al. 2005). Risk Factors (account for 75% of cases) are tobacco abuse confers 6 fold risk, smokers represent 90% of oral cancer

patients, alcohol abuse or heavy use, combined risk of heavy alcohol and tobacco use (women: 100 fold risk of oral cancer, men: 38 fold risk of oral cancer), other risks are sunlight exposure, poor dentition and viral infection (HSV, HPV) (Ravi and Yadav 2006; Ord et al. 2007). The prevalence of oral cancer is also on the increase in Africa. Annual incidence rates for oral and pharyngeal cancer are estimated at 25 cases per 100,000 in developing countries. The rapid urbanisation and increasing access to, and utilization of tobacco in its various forms as well as alcohol, is leading to an increase in the incidence of oral pre-cancer and cancer. Epidemiology of oral cancer are squamous cell represents 90% of oral cavity tumors, incidence increases with age and oral cancer is 9th most common cancer (represents 3% of cancers in men and represents 2% of cancers in women). The aims of this

PRAYITNO et al. – OSCC with HPV infection

research were to know the incidence of OSSC patient which realized HPV infection.

450 bp

MATERIALS AND METHODS Kind of this research is observasional - cross sectional and the design of this research is post test only control group design. Ethical clearance was done by dr Muwardi Distric Hospital Surakarta team and sign at August 5, 2008. All patients was womens Fourty biopsy frozen section of Benign Oral Squamous Cells (BOSC) tissue patient and fiveteen biopsy frozen section of Oral Squamous Cell Carcinoma (OSCC) tissue patients collected from Oral and Dental Clinic of Muwardi Hospital in Surakarta, Central Java, Indonesia from January to December 2007. Parrafin blocks were made from cutting I, which was subsequently stains with Haematoxyline Eosine (HE) to ascertain the type of neoplasm. Cutting II was subjected to DNA isolation. Dioxyribonucleic Acid (DNA) isolation was made by Schmits (1994) with some modifications. Cut up to 25 mg of tissue into small pieces, place in 1.5 mL a microfuge tube volume, and add 200 ul of DNA extraction buffer. Add 20 µL of Proteinase K stock solution, mix by vortexing, and incubate at 550 C overnight. The DNA isolation results were subjected to Polymerase Chains Reaction (PCR) to amplify L1-HPV for fixed the HPV. Diagnose related HPV infections are made by Schmits (1994) and McMillan and Fowler (1998). PCRmethod with some modifications (25 µL microfuge tube Ready To Go PCR Bead (Amersham Pharmacia Biotech) mixed with 2 µL HPV consensus primers (MY09: 5’GC(A/C)CAGGG(A/T)CATAA(C/T)AATGC3’ and MY11: 5’CGTCC(A/C)A(A/G)(A/G)GGA(A/T)ACTATC3’) (Cybergene AB) and 2 µL DNA template. PCR protocol for both amplifications are 940C for 50 seconds, 590C for 50 seconds, 720C for 50 seconds and 40C soak. The amplification very conserved region of HPV the L-1 gene that present in all HPV subtypes produced 450 bp long. The collected data was analyzed by Chi Square Test (SPSS for Windows 15).

Figure 2. Polymerase Chains Reaction (PCR) L1-HPV gene in BOSC tissue frozen section, amplified 450 bp long.

450 bp

Figure 3. Polymerase Chains Reaction (PCR) L1-HPV gene in OSCC tissue frozen section, amplified 450 bp long.

Tabel 1. The data women patient with OSCC (Malignant) and BOSC (Benign). Malignant Age No. (years)

Benign Age Age Age No. No. No. (years) (years) (years)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

39 53 69 60 40 45 42 73 50 45 43 50 21 56 70

30 40 45 31 58 20 19 27 27 40 15 55 57 60 67

Mean = 50

RESULTS AND DISCUSSION The result of this experiment showed in Figure 1-3 and Table 1-2.

Figure 1. Histopathology view from HE stain of OSCC.

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

15 54 40 27 27 19 28 67 31 45 32 55 65 54 61

31 32 33 34 35 36 37 38 39 40

47 45 49 51 51 53 43 44 41 70

Mean = 42.6

Table 2. Result of the experiment in table 2X2

OSCC BOSC

HPV positive HPV negative

Total

11 (73%) 9 (23%) 20

15 40 55

4 (27%) 31 (77%) 35

The result of this experiment showed mean for BOSC was 42.6years and OSCC was 50 years. And nine patient from 40 patient BOSC identified have HPV infections (9/40 = 23%). Eleven patient from 15 patient OSCC identified have HPV infections (11/15 = 73%). Dental caries and periodontal disease are generally considered to be the major oral health problems around the world. In developing countries of Africa, these appear to be neither as common nor of the same order of severity as in

3 (2): 64-67, July 2011

the developed world. An epidemiological description of a given health problem usually includes its prevalence, severity (morbidity, mortality) and age-adjusted distribution in the population. Oral diseases known to exist in each community must be assessed in this way in order to develop programmes appropriate to community needs. Based on this form of analysis, the most prominent oral health problems in Africa amongst low socio-economic communities include Noma, ANUG (Acute Necrotising Ulcerative Gingivitis), oral cancer, the oral manifestations of HIV and AIDS, oro-facial trauma, and dental caries. The highest global prevalence of HIV and AIDS is found in Africa. Studies have shown that the oral manifestations of HIV/AIDS are common. Candida infections, necrotizing gingivitis and oral hairy leukoplakia are the most common. The prevalence of oral cancer is also on the increase in Africa. Annual incidence rates for oral and pharyngeal cancer are estimated at 25 cases per 100,000 in developing countries. The rapid urbanisation and increasing access to, and utilisation of tobacco in its various forms as well as alcohol, is leading too (WHO/AFRO 2008). In the case of high risk HPV infection and under favorable conditions, the viral genome is integrated into the host genome which is the necessary event for the keratinocytes immortality. During this process of integration the circular form of viral genome breaks at the level of the E1 and E2 regions, never at the level of the E6 or E7 region. Different studies have shown that the integrated part of the genome corresponds to E1, E6 and E7 while the regions from E2 to E5 are lost and are not transcribed in the tumours. The loss of E2 during this process of integration produces the loss of E6 and E7 control. Therefore, the sequences E6 and E7 are directly involved in the cellular cycle by inhibiting the normal functions of p53 and pRb respectively. The protein p53 is known as the "genome's guard" and in the case of DNA damage, the p53 can provoke the arrest of cellular division and assure the time necessary for DNA repair. If damage can’t be repaired, p53 is able to induce the programmed cellular death and prevent the propagation of DNA damage in subsequent generations of cells. In the case of other types of tumors p53 is usually mutated and acts as a real oncogene. In the case of HPV infection, E6 suppresses the properties of p53 gene product achieving the functional equivalent of the two hits required to knock out both alleles of a tumor suppressor gene. The mutations of p53 are normally not found. The E7 protein interacts with retinoblastoma protein (pRb), which is the crucial factor for the cellular cycle control. This interaction causes the release of the transcription factor E2F, which is now free to act and can stimulate the cellular division. E7 is also able to bind and inactivate the protein kinase inhibitors p21 and p27 and can interact with different proteins whose significance has still not been determined. E6 and E7 can cooperate with cellular oncoproteins like ras and myc which enables the virus to act at the level of growth factors and cellular and nuclear metabolism producing oncogenic cells. E6 and E7 can provoke directly DNA mutations of the host cell, probably by causing alterations of DNA repair mechanisms. This means that certain types of HPV are able

to cause malignant lesions even without the action of other cofactors (González Intxaurraga et al. 2002). Because Xt (df=1; p<0.01) < X2 (68.59) that showed in Table 3, so that have significant differences between BOSC and OSCC. Table 3. Chi Square data analysis of table 1. k.b c.1.r.1 c.2.r.1 c.1.r.2 c.2.r.2

O 11 4 9 31

E 14.5 25.4 5.4 9.5

12.25 424.36 12.96 462.25

(O-E)2 E 0.84 16.70 2.40 48.65

X2 Xt (df = 1; p<0,01)

68.59 6.63

(O-E) -3.5 -20.6 3.6 21.5

(O-E)2

Cancer is widely perceived as a heterogeneous group of disorders with markedly different biological properties, which are caused by a series of clonally selected genetic changes in key tumour-suppressor genes and oncogenes. However, recent data suggest that cancer has a fundamentally common basis that is grounded in a polyclonal epigenetic disruption of progenitor cells, mediated by “tumour-progenitor genes”. Furthermore, tumour cell heterogeneity is due in part to epigenetic variation in progenitor cells, and epigenetic plasticity together with genetic lesions drives tumour progression. This crucial early role for epigenetic alterations in cancer is in addition to epigenetic alterations that can substitute for genetic variation later in tumour progression. Therefore, non-neoplastic but epigenetically disrupted progenitor cells might be a crucial target for cancer risk assessment and chemoprevention (Bsoul et al. 2005; Feinberg et al. 2006). The inducible transcription of heat shock genes is the response to a plethora of stress signals, including (i) environmental stresses, (ii) nonstress conditions, and (iii) pathophysiology and disease states (e.g. HPV). Although changes in heat shock protein (Hsp) expression are associated with certain diseases, these observations leave open the question of whether this is an adaptation to the particular pathophysiological state, a reflection of the suboptimal cellular environment associated with the disease, or serves to warn other cells and tissues of imminent danger (Morimoto 1998). In the face of injury or stress with the use of various mechanisms for anticipated, including systems of proteins called molecular chaperones. The typical function of a chaperone is to assist a nascent polypeptide chain to attain a functional conformation as a new protein and then to assist the protein's arrival at the site in the cell where the protein carries out its functions. It has become increasingly clear that disruption of chaperoning mechanisms contributes to aging and disease. This review outlines the involvement of defective chaperones in senescence and in several diseases. Since chaperones are ubiquitous, their deficiencies and defects are bound to affect diverse tissues and, hence, to be of interest to those in internal medicine, ophthalmology, neurology, immunology, endocrinology, pediatrics, and

P PRAYITNO et al. â&#x20AC;&#x201C; OSCC witth HPV infectioon

ggerontology. Only O a fractioon of chaperonnes are encodded in g genes that are inducible by stressors and thus belong to t the large class of o stress protteins. OSCC was includeed in d distress cell, so to make possible p distuurbance in prrotein f folding proceess (Fan and Neff 2000; Rho et al. 2002; 2 M Martin 2004; Bonnet B et al. 2007). 2 The accum mulation of protein misffolded in distress c condition to be b the result of o an increase of toxic functtions, w which are often o accomppanied by Hsp70 H and other c chaperones. N matter how No w toxicity is generated, eithher by s soluble forms or insoluble fibrils f of the disease d proteinns, the identification of protein aggregates, including Hsp70, H inside or arounnd dead cells has tempted many m researchhers to m manipulate thhe level of Hssp70 to exam mine whether overe expression off chaperones would reduuce the extennt of a aberrant agggregation, thhereby suppressing diisease p phenotypes orr delaying thhe onset of the t diseases (Ellis 1 1996; Li et all. 2000; Muchhowski et al. 2000; Soto 2003; 2 M Morishima. 20005; Butler annd Loh 2006; Park et al. 2007; 2 G Gruschus 20088; Garyali et al. a 2009).

CONC CLUSION The result of this experriment showedd that from BOSC B ppatient identified have 233% HPV inffections and from O OSCC patientt identified haave 73% HPV V infections. There T h have significaant differencess between BO OSC and OSC CC in H HPV infectionn. The concluusion is HPV V as a significcance f factor for OSC CC pathogenesis.

WLEDGMENT TS ACKNOW We thankss to acknowleedge to Airlanngga Universiity in Surabaya for chance S c to studdy about canceer and so thannks to a acknowledge to Sebelas Maret M Universitty in Surakartta for h PhD grand. Biomedicaal Laboratory of Sebelas Maret his M U University for his laborattory facilitiess. We thankss AO S Suryanata annd Tri Darm mani for theiir cooperatioon in c collecting sam mple. We also thank Prof. Widya W Asmara and P Prof. Noerhayyati Suripto forr their inspirattion.

REFE ERENCES Bonnet S, Archeer SL, Allaluniis-Turner J, Harromy A, Beauliieu C, B Thompson R, Lee CT, Lopascchuk GD, Puttaguunta L, Bonnet S, S et al. 2007. A mitoochondria-K+ channnel axis is supprressed in cancer and its

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normalization n promotes apoptosis and inhibits canccer growth. Canccer Cell C 11: 37-51. Bsoull SA, Huber MA A, Terezalmy G. 22005. Squamous cell carcinoma of th he oral tissues: a comprehensive review for oral heealthcare providerrs. J Contemp Dent Pract P 6 (4): 1-16. hanisms of the p553 Butlerr JS, Loh SN. 20006. Folding and misfolding mech DNA D binding dom main at physioloogical temperature. Protein Sci 15 1 (11): 2457-2465. c Induuction of chaperronins synthesis in Ellis JR. 1996. The chaperonins, reesponse to stress.. Academic Press. San Diego, CA.. Fan Z, Z Neff RA. 2000. Susceptibility oof ATP-sensitive K+ channels to ceell sttress through mediation m of phhosphoinositides as examined by b photoirradiation. p J Physiol 529 (Pt 3): 707-721. Feinb berg PA, Ohlssonn R, Henikoff S S. 2006. The epiigenetic progenitor origin o of human caancer. Nat Rev G Genet 7: 21-33. Garyaali P, Siwach P, Singh PK, Puri R R, Mittal S, Seng gupta S, Parihar R, R Ganesh G S. 2009. The malin-laforiin complex supp presses the cellullar to oxicity of misfoldded proteins by ppromoting their deegradation througgh th he ubiquitin-proteeasome system. H Human Mol Geneet 18 (4): 688-7000. Gonzaales-Intxaurraga MA, Stankovic R R, Sorli R, Trevisan G. 2002. HP PV and carcinogenesis. Acta Dermatovvenerol 11: 1-8. Gruscchus JM. 2008. Do amyloid oliggomers act as trraps for misfoldeed proteins? p a hypothhesis. Amyloid 155 (3): 160-165. Li CY Y, Lee JS, Ko YG, Kim JI, S Seo JS. 2000. Heat H shock proteein 70 7 inhibits apopttosis downstream m of cytochrom me c release annd upstream u of casppase-3 activationn. J Biol Chem 275 (33): 2566525671. 2 Martin n J. 2004. Chhaperonin functiion â&#x20AC;&#x201C; effects of o crowding annd confinement. J Mool Recognit 7: 4665-472. N 1998. Typing oof HPV isolates by b PCR: molecullar McMillan N, Fowler N. pproach toward vaccine v developm ment for viral infeection. Icro-Unescco ap Training T Course, At Inter University Center for Biiotecnology, Gajaah Mada M University, Yogyakarta, Indoonesia, Novembeer 1998. Morim moto RI. 1998. Regulation R of the hheat shock transccriptional responsse: cross talk betweeen a family oof heat shock factors, f molecullar n regulatorrs. Gen Develop 12: 1 3788-3796. chaperones, and negative 70: more than an a Morisshima M. 2005. Control of celll fate by HSP7 evanescent meetinng. J Biochem 1377 (4): 449-453 howski PJ, Schafffar G, Sittler A, W Wanker EE, Hayeer-Hartl MK, Harrtl Much FU. F 2000. Hsp70 and Hsp40 chapperones can inhibit self-assembly of polyglutamine p prooteins into amylooid-like fibrils. Proc P Natl Acad Sci S USA U 97:1589-15994 Ord RA, R Nikitakis NG G, Sauk JJ. 2007. Why screening works. Universiity of o Maryland. Baltiimore Maryland. Park SH, Bolender N,, Eisele F, Kostoova Z, Takeuchi J, J Coffino P, Woolf DH. D 2007. The cytoplasmic c Hspp70 chaperone machinery m subjeccts misfolded m and enddoplasmic reticullum import-incom mpetent proteins to degradation d via thhe ubiquitin-protteasome system. Mol Biol Cell 18 1 (1): 153-165. Ravi M, Yadav S. 2006. Oral squuamous cell carrcinoma: etiologgy, pathogenesis p and prognostic value of genomic allterations. Indian J Cancer C 43 (2): 60--66. Rho HW, H Choi MJ, Lee L JN, Park JW,, Kim JS, Park BH, B Sohn HS, Kiim HR. H 2002. Cytotooxic mechanism m of vibrio vulniificus cytolysin in CPAE C cells. Life Sci S 70: 1923-1934. Schm mits HL. 1994. Sppecies diagnosticss protocols: PCR R and other nucleeic acid methods. In: Clapp JP (ed). M Methods in moleccular biology. Vool. 50. Humana Presss Inc., Totowa, NJJ. n misfolding in Soto C. 2003. Unnfolding the rrole of protein neurodegenerative n e diseases. Nat Reev Neurosci 4 (1)): 49-60. WHO O/AFRO. 2008. Oral health in the African Region. A Regionnal Strategy S 1999-20008. WHO Repoort office for Africa. A Brazavillle, Congo. C

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 68-72 July 2011

Multiple shoot formation in Gloriosa superba: A rare and endangered Indian medicinal plant RAVINDRA ADE, MAHENDRA RAI♥ Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444602, Maharashtra, India, Tel.: +91-721-2662208-9, ext. 267, Fax.: +91-721-2662135, 2660949, ♥e-mail: pmkrai@hotmail.com Manuscript received: 6 April 2011. Revision accepted: 24 July 2011.

Abstract. Ade R, Rai M. 2011. Multiple shoot formation in Gloriosa superba: A rare and endangered Indian medicinal plant. Nusantara Bioscience 3: 68-72. We report in vitro growth of callus and multiple shoots and standardized the culture conditions for Gloriosa superba. The main aim of the present study was to evaluate the effect of different growth media with different hormonal concentrations on callus induction and multiple shoot formation in Gloriosa superba. The results indicated that MS medium supplemented with 2,4-D 4.52 μM and BAP 13.30 μM promoted the formation of the maximum number of shoots compared to IAA, IBA and with Gamborg B5 medium supplemented with kinetin, IBA and BAP were found to be superior. Key words: callus induction, Gloriosa superba, multiple shoots, kinetin.

Abstrak. Ade R, Rai M. 2011. Pembentukan pucuk ganda pada Gloriosa superba: Sebuah tanaman obat dari India yang langka dan hampir punah. Nusantara Bioscience 3: 68-72. Kami melaporkan pertumbuhan in vitro kalus dan pucuk ganda, serta standarisasi kondisi budibudaya untuk Gloriosa superba. Tujuan utama penelitian ini adalah untuk menguji pengaruh mediapertumbuhan yang berbeda dengan konsentrasi hormon yang berbeda terhadap induksi kalus dan pembentukan pucuk ganda pada Gloriosa superba. Hasilnya menunjukkan bahwa medium MS yang ditambah dengan 2,4-D 4,52 μM dan BAP 13,30 μM lebih unggul untuk meningkatkan pembentukan jumlah pucuk terbanyak dibandingkan dengan IAA, IBA dan media Gamborg B5 yang ditambah dengan kinetin, IBA dan BAP. Kata kunci: induksi kalus, Gloriosa superba, pucuk ganda,kinetin.

INTRODUCTION Gloriosa superba L. (Glory lily), a perennial tuberous climbing herb is widely distributed in tropical and subtropical parts of India including foothills of Himalayas (Kapoor 2001). It is known by different names in India, such as Kalihari, Agnishikha, Languliata and Nangulika. The plant thrives from arid Bundelkhand to the humid Assam valley, India. It is one of the most important medicinal plants of Asia and Africa (Sivakumar and Krishnamurthy 2000, Jana and Shekhawat 2011). Almost all parts of it find diverse medicinal usage (Kapoor 2001). It has been a well-known plant in Indian Ayurveda and pharmacological industries as well (Asolkar et al. 1992). It is used in ayurvedic medicine as abortifacient, anti-gout, antileprotic, antipyretic, thermogenic and also anticancerous agent. It also provides relief to the swollen joints and in gout (Narein et al. 1996). The tubers have long been used as alternative source of colchicine and gloriosin (Sivakumar et al. 2003a; 2003b, Jana and Shekhawat 2011). It contains alkaloids like colchicine (C22H25O6N) and its derivative like gloriosin and colchicocide (C27H33O11N) along with Benzoic acid, Salicylic acid, sterols and resinous substances and therefore, the demand of this plant

is increasing day by day. The present cost of colchicine is about US $ 318/5g (www. sigmaaldrich.com). Food and drug administration of US approves colchicine for acute gout, mediterenian fever in July 2009. FDA approved Colcrys Pvt. Ltd., USA to treat acute flairs in patient with gout, recurrent and painful form of arthritis and patient with familial mediterenian fever, an inherited inflammatory disorder. These all medications contain colchicine as active ingredients (FDA 2009). Its tubers are thermogenic, antipyretic, antihelmintic, purgative, anti-inflammatory and antileprotic. The tubers and seeds contain the maximum colchicine (0.7-0.9%) (Fennei and Van Staden 1989; Rajagopal and Kandhasamy 2009). In conventional propagation method seeds and rhizome or tubers can propagate Gloriosa. The major problem in the regeneration of Gloriosa superba is dormancy of tuber. The germination capacity of seeds is very low (about 0.01%). Another basic problem is presence of hard seedcoat. The fungal contamination is associated with the seeds and tubers, such as Curvularia lunata, and tuber-rot caused by Sclerotium species (Prota 2010). Though it has been originated in our country, its germplasm is very rare and far to meet the present increasing demands from pharmacological industries all over the world. (http://database.prota.org/dbtw-wpd/exec/

ADE & RAI - Multiple shoot formation in Gloriosa superba

dbtwpab). The demands of its seeds in pharma industries at home and abroad has been rapidly increasing (Sivakumar 2000). Due to its diverse usage in medicine and overexploitation for colchicine in industries and excessive use of planting material i.e. seeds and tubers for local purpose, susceptibility towards many pests, and excessive collection in habitats for medicinal purposes have pushed this taxon to endangered. Due to overexploitation and unscientific collection, G. superba is included in the world record of endangered plants i.e. Red Data Book by International Union for Conservation of Nature (IUCN) (Sivakumar and Krishnamurthy 2000). In order to provide enough plant material for commercial exploitation, the cultivation-using corm is not sufficient. Thus, mass clonal multiplication through tissue culture is urgently needed not only to conserve this taxon but also to meet the tremendous demands of this medicinal plant as a source of colchicine. In the present study, we have developed an effective protocol for the rapid propagation and thus regeneration of this medicinal, ornamental and commercially useful plant.

callusing and multiple shoots were recorded at regular intervals. Results were analyzed statistically.

RESULTS AND DISCUSSION In the present study, we have tried different growth media for the maximum induction of calli and multiple shoots formation. It includes Murashige and Skoog, Gamborg’s B5, Nitsch, White’s and Chu’s N6 media having different hormonal combinations of auxins (2,4-D, IAA and IBA) and cytokinins, (BA, BAP and BAP). Murashige and Skoog’s medium was found to be suitable for the induction of calli and multiple shoot formation followed by Gamborg’s medium, while all other media showed no response (Table 1). Table 1. Effect of different media on the growth of callus and formation of multiple shoots of Gloriosa superba Hormonal combination Culture medium

MATERIALS AND METHODS Germplasm Germplasm of Gloriosa superba L. was collected from Warud, Melghat and Chikhaldara of district Amravati, Central India and planted in Garden of Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati, India (Figure 1A).

Ex-plants Leaves, non-dormant corm bud (Figure 1B), auxillary bud, nodal portion and seeds

Surface sterilization of ex-plants Large ex-plants were cut into 1-2 cm pieces with fine scalpel and were washed under running tap water 3-4 times and rinsed in double distilled water 4 to 5 times in hood. The surface sterilization was carried out by using mercuric chloride 0.2% (HgCl2) for delicate explants (leaf base, and axillary buds) for 3 minutes and 1% mercuric chloride (HgCl2) for tubers and seeds for 3-4 minuets respectively. Later, rinsed in sterile double distilled water 4 to 5 times and then dried on the sterile petridish containing sterile blotting paper. Growth media and culture conditions Murashige and Skoog, Gamborg’s B5, Nitsch medium, White’s medium, Chu’s N6 and Coconut water were used. The solidifying agent was Phytagel (0.25%) and 5.8 pH was maintained. High quality chemicals of Hi-media, Mumbai were used. All the treatments were replicated thrice with 10 culture tubes in each set. The cultures were maintained at 25±20C with 16 hours illumination at 15002000 lux and a relative humidity of 60%. Initiation of

Murashige and Skoog’s medium Gamborg’s B5 medium Nitsch,s medium White’s medium Chu N6 medium

2,4-D

Callus and multiple shoots Cytokinin observed (%) BAP 70 %

IBA

BAP

Auxin

50%

2,4-D, IAA, IBA BA, BAP No response 2,4-D, IAA, IBA BAP No response 2,4-D, IAA, IBA BAP No response

Number of calli and multiple shoots formed per explant were recorded at regular interval. The MS medium when fortified with auxins and cytokinin at different concentrations showed variation in number of calli and shoots formation (Table 2). Initiation of callus and multiple shoot formation was noted after 30-35 days of inoculations (Figure 1C, D, E, F, G, H, I, J, K). Table 2. Effect of 2,4-D, BAP with MS medium on callus and multiple shoot formation in Gloriosa superba

Treatment (µM/L) MS+BAP (4.40)+2,4-D (4.52) MS+BAP (8.90)+2,4-D (4.52) MS+BAP (13.30)+2,4-D (4.52) MS+BAP (17.80)+2,4-D (4.52) MS+BAP (22.20)+2,4-D (4.52) MS+BAP (4.40)+2,4-D (4.52)+ADS (5.28) MS+BAP (8.90)+2,4-D (4.52)+ADS (10.57) MS+BAP (13.30)+2,4-D (4.52)+ADS (13.86) MS+BAP (17.80)+2,4-D (4.52) +ADS (21.14) MS+BAP (22.20)+2,4-D (4.52)+ADS (26.43) Note: *Each value is the average± SD

Number of multiple shoots* 11.00 ± 1.04 15.00 ± 0.30 21.00 ± 1.05 03.86 ± 0.30 06.00 ± 0.91 11.00 ± 1.57 09.00 ± 0.26 05.00 ± 1.05 04.00 ± 0.80 04.00 ± 0.70

3 (2): 68-72, July 2011

Figure: 1. A. Two month mature plant of G. Superba, B. “V” shape macro tubers of G. superba, C. Initiation of callus, D, E. Callus, F, G. Initiation of multiple shoot, H,I,J,K. Multiple shoots formation in Gloriosa superba

ADE & RAI - Multiple shoot formation in Gloriosa superba

Shivkumar and Krishnamurthy (2000) reported that MS medium with combination of BAP (7.77µM) and ADS (5.44 µM) showed the maximum formation of shoots. Finnie and Van Staden (2004) observed that MS basal medium with only 2,4-D showed the callus formation. Jadhav and Hegde (2001) also reported that the callus formation occurs at 2,4-D (18.08µM) + Kn 23.20µM + CH (10 mg/l)+ CW (20%). On the contrary, in the present study, we have found that the MS medium with 2,4-D (4.52µM) and BAP (13.30µM ) showed the maximum response in case of calli and shoot formation (Figure 2), while MS medium with 2,4-D (4.52µM) and BAP (17.80µM ) showed the minimum response (average no. of calli and shoot formation=3.86). On the other hand, MS medium having hormonal combination of BAP, 2,4-D and ADS did not show any significant response as compared to BAP and 2,4 D combination used above (Figure 3).

Samarajeewa et al. (1993) reported the callus and multiple shoots on Gamborg’s B5 medium with kinetin 0.46µM along with BAP and IAA 0.04-0.28µM/L the explants were non-dormant corm bud. In the present study, we have tried Gamborg’s B5 along with BAP and IBA (Table 3). Initially there was no response at lower concentration but on increase of concentration better response was observed at BAP (13.30 µM/L) +IBA (9.80 µM/L) (Figure 4). To assess the effect of IBA and BAP with Gamborg B5 medium in combination with NAA after inoculation with all necessary culture conditions such as photoperiod 16 hour at 25 ± 20C, the maximum response with respect to initiation of callus was observed at the concentration of 4.90µM/L and 17.80µM/L respectively, while the other combinations of BAP, IBA and NAA (Figure 5) were not suitable for growth of callus (Table 3).

Table 3. Effect of Gamborg’s B5 growth medium supplemented with BAP, IBA and NAA on calli and multiple shoot formation in G. superba.

No. of Shoots

Hormonal treatment (µM/L) 15

0 BAP (4.40)+ 2,4D (4.52)

BAP (8.90)+ 2,4D (4.52)

BAP (13.30)+ 2,4D (4.52)

BAP (17.80) + 2,4D (4.52)

BAP (22.20) + 2,4D (4.52)

Different Conc. of BAP & 2,4D (µM/l)

Figure 2. Effect of different conc. of BAP and 2.4-D with MS medium on multiple shoot formation

B5+BAP (4.40)+IBA (9.80) B5+BAP (8.90)+IBA (9.80) B5+BAP (13.30)+IBA (9.80) B5+BAP (17.80)+IBA (9.80) B5+BAP (22.20)+IBA (9.80) B5+BAP (4.40)+IBA (4.90)+NAA (5.37) B5+BAP (8.90)+IBA (4.90)+NAA (10.74) B5+BAP (13.30)+IBA (4.90)+NAA (16.11) B5+BAP (17.80)+IBA (4.90)+NAA (21.48) B5+BAP (22.20)+IBA (4.90)+NAA (26.85) Note: *Each value is the Average± SD

No. of callus induction and multiple shoot* 09.00 ± 0.36 11.00 ± 1.04 18.33 ± 0.92 15.02 ± 0.36 17.00 ± 0.96 14.10 ± 0.45 14.00 ± 0.87 18.00 ± 0.60 No response No response

No. of Shoots

0 BAP (4.40)+ 2,4D BAP (8.90)+ 2,4D BAP (13.30)+ BAP (17.80) + BAP (22.20) + (4.52) + ADS (4.52) + ADS 2,4D (4.52) + ADS 2,4D (4.52) + ADS 2,4D (4.52) + ADS (5.28) (10.57) (13.86) (21.14) (26.43)

Different Conc. of BAP+2,4D+ADS

Figure 3. Effect of combination of BAP and 2.4-D and ADS with MS medium on multiple shoot formation

0 BAP (4.40) + IBA (9.80)

BAP (8.90) + IBA (9.80)

BAP (13.30) + IBA (9.80)

BAP (17.80) + IBA (9.80)

BAP (22.20) + IBA (9.80)

Different Conc. of BAP & IBA (µM/l)

Figure 4. Effect of different conc. of BAP and IBA with Gomborg’s B5 medium on multiple shoot formation

3 (2): 68-72, July 2011

REFERENCES

N o. of Sh oots

0 BAP (4.40) + IBA BAP (8.90) + IBA BAP (13.30) + (4.90) + NAA (4.90) + NAA IBA (4.90) + (5.37) (10.74) NAA (16.11)

BAP (17.80) + IBA (4.90) + NAA (21.48)

BAP (22.20) + IBA (4.90) + NAA (26.85)

Different Conc. of BAP, IBA & NAA

Figure 5. Effect of different conc. of BAP, IBA and NAA with Gomborg’s B5 medium on multiple shoot formation

The calli were independently sub-cultured for maximum shooting in MS medium with varying concentrations of Kinetin and BA ranging from 2.32 17.80µM/L. But profuse shooting was noted in 2,4-D 9.05 and BAP 17.80µM/L after 20 days sub-culturing. Differential response of tissue to any growth hormone depends upon their endogenous level in the explant used for the initiation of culture. Thus, there is need of optimum concentration of the growth hormone at which the response of culture initiation and growth of tissue is maximum.

CONCLUSION It can be concluded that Murashige and Skoog’s (MS) and Gamborg’s B5 media were found to be ideal for the induction of callus and multiple shoot formation as compared to White’s medium, N6 and Nitsch media, which did not give any response. Required culture conditions such as photoperiod 16 hours at 1500-2000 lux, humidity 60% and temperatures 25±20C were maintained.

Asolkar LV, Kakkar KK, Chakare OJ. 1992. Second supplement to Glossary of Indian Medicinal plant with Active principles. part-I) (AK). Publications and Information Directorate, Council of Scientific and Industrial Research, New Delhi. Custers JBM, Bergervoet JHW. 1994. Micropropagation of Gloriosa: towards a practical protocol. J Sci Hort 4: 323-34. Dvorakova S, Sedmera P, Potesilova H, Sandavy F, Simanek V. 1984. Alkaloids of Gloriosa superba L. Collect. Czech Chem Comm 6: 1536-1542. Finnie JF and Van Staden J. 1994. Micropropagation of Gloriosa and Sandersonia. J Plant Cell Tiss Organ Cult 19: 151-158. Finnie JF, Van Staden J. 1989. In vitro propagation of Sandersonia and Gloriosa. J Plant Cell Tiss Organ Cult 19: 151-158. Finnie JF, Van Staden J. 1991. Isolation of colchicines from Sandersonia auracaceae and Gloriosa superba variation in Alkaloid level of plant grown in vivo. J Plant Physiol 318: 691-695. Food and Drug Administration (FDA), USA. 2009. Newsletter. Gamborg OL, Miller RA, Ojima K. 1968. Nutrient requirement of suspension culture of soybean root cells. Exp Cell 50: 150-158. Jadhav SY, Hegde BA. 2001. Somatic embryogenesis and plant regeneration in Gloriosa L. Indian J Exp Biol 9: 943-946. Jana S, Shekhawat GS. 2011. Critical review on medicinally potent plant species: Gloriosa superba. Fitoterapia 82 (3):293-301. Kapoor LD. 2001. Traditional uses of medicinal plant In: Ayurvedic Medicinal Plant. CRC Press, New Delhi. Le-Roux LG, Robbertse P J. 1997. Aspects relating to seed production in Gloriosa superba L. South African J Bot 4: 191-199. Murashige TF, Skoog F. 1962. A revised medium for rapid growth and bioassays with Tobacco tissue culture. Physiol Plant 15: 473-497. Narain P. 1981. A case of terminal chromosome deletion in Gloriosa superba L. Curr Sci 6: 285-286. Prota [Plant Resources of Tropical Africa]. 2010. www.prota.org Rai MK. 1987. Ethnomedicinal studies of Patalkot and Tamia (Chhindwara): plants used as tonic: Ancient Sci Life 2: 119-121. Rajagopal C, Kandhasamy R. 2009. Genetic variability of kazhappai kizhangu (Gloriosa superba L.) in Tamil Nadu. J Trop Agric 47 (1-2): 77-79 Samarajeewa PK, Dassanayake M.D, Jayawardena SDG. 1993. Clonal Propagation of Gloriosa superba. Indian J Exp Biol 31: 719-720. Senanyake N, Karalliedde L. 1986. Acute poisoning in Shri Lanka, an overview. Ceylon Med J 2: 61-71. Sivakumar G, Krishnamurthy KV, Rajendran TD. 2003a. Embryoidogenesis and plant regeneration form leaf tissue of Gloriosa superba. Planta Med 69: 479-481. Sivakumar G, Krishnamurthy KV, Rajendran TD. 2003b. In vitro corm production in Gloriosa superba L. Ayurvedic Medicinal Plant. J Hort Sci Biotech 78: 450-453. Sivakumar G, Krishnamurthy KV. 2000. Micropropagation of Gloriosa superba L. an endangered species of Asia and Africa. Curr Sci 78: 1-10. Sivakumar G, Krishnamurthy KV. 2002. Gloriosa superba L. a very useful medicinal plant. In: Singh VK, Govil JN, Hashmi S, Singh G, (eds). Series Recent Progress in Medicinal Plants, vol. 7, Ethnomedicine and Pharmacognosy, Part II. Sci Techn Publ, Texas. Somani VJ, John CK, Thengane RJ. 1989. In vitro propagation and corm formation in Gloriosa superba. Indian J Exp Biol 27: 578-579. www.sigmaaldrich.com

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 73-81 July 2011

Corals differential susceptibilities to bleaching along the Red Sea Coast, Egypt MOHAMMED SHOKRY AHMED AMMAR1,â&#x2122;Ľ, AHMED HAMED OBUID-ALLAH2, MONTASER ALY MAHMOUD AL-HAMMADY3 1

National Institute of Oceanography and Fisheries (NIOF), P.O. Box 182, Suez, Egypt. Tel. +20 11 1072982, Fax. +20 623360016, E-mail: shokry_1@yahoo.com. 2 Department of Zoology, Faculty of Science, Assiut University, Assiut, Egypt 3 National Institute of Oceanography and Fisheries (NIOF), Hurghada, Egypt Manuscript received: 5 May 2011. Revision accepted: 16 July 2011.

Abstract. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2011. Corals differential susceptibilities to bleaching along the Red Sea Coast, Egypt. Nusantara Bioscience 3: 73-81. Coral bleaching was studied at four sites in four widely geographically separated areas. Three of these sites are subjected to different human activities and the fourth one is considered as a control site. Data were collected by using SCUBA diving equipments and the line transects method. A total of 3940 coral colonies, representing 62 species in 21genera and 10 families, were recorded on transects on the reefs of four studied sites.20.11% of all corals were affected by bleaching: 5.4% were moderately affected; 2.7% severely affected and 12.007% were dead. Overall, there were differences in the proportion of colonies affected by bleaching between the studied sites. Ras El-Behar, the site impacted by petroleum oil, has the maximum average proportion of moderately, severely bleached and dead colonies. While, the lowest average proportions of severely bleached colonies and dead colonies were found at Kalawy bay. Surprisingly, coral reef taxa at El-Hamraween harbor showed high resistance to bleaching probably because of having a new different clade of Symbiodinium which can withstand sea water temperature. Species with highest susceptibilities to bleaching in areas of oil pollution, increased sedimentation and heavy load of phosphate are Stylophora pistillata, Acropora granulosa and Montipora meandrina, respectively while species with lowest susceptibilities are Fungia fungites, Alveopora daedalea and Millepora dichotoma, respectively. Key words: coral, bleaching, eutrophication, oil pollution, sedimentation, Red Sea.

Abstrak. Ammar MSA, Obuid-Allah AH, Al-Hammady MAM. 2011. Kerentanan diferensial karang terhadap pemutihan di sepanjang Pantai Laut Merah, Mesir. Nusantara Bioscience 3: 73-81. Pemutihan karang dipelajari di empat lokasi pada empat wilayah geografis yang terpisah secara luas. Tiga dari lokasi ini dipengaruhi aktivitas manusia yang berbeda dan lokasi yang keempat dianggap sebagai lokasi kontrol. Data dikumpulkan dengan menggunakan peralatan menyelam SCUBA dan metode garis transek. Sebanyak 3940 koloni karang, yang mewakili 62 spesies dari 21genera dan 10 famili, tercatat pada transek yang mewakili empat lokasi terumbu karang yang dipelajari. 20.11% dari semua karang mengalami pemutihan: 5,4% cukup terpengaruh; 2,7% terkena dampak parah, dan 12,007% mati. Secara keseluruhan, terdapat perbedaan proporsi koloni yang dipengaruhi oleh pemutihan antara masing-masing lokasi yang dipelajari. Ras El-Behar, lokasi yang dipengaruhi oleh minyak bumi, memiliki proporsi merata antara koloni yang cukup, parah dan mati. Sementara, proporsi rata-rata terendah dari koloni yang parah dan mati ditemukan di teluk Kalawy. Mengejutkannya, terumbu karang di pelabuhan El-Hamraween menunjukkan resistensi yang tinggi terhadap pemutihan kemungkinan karena memiliki kelompok baru Symbiodinium yang berbeda dan dapat menahan suhu air laut. Spesies dengan kerentanan tertinggi untuk pemutihan di lokasi yang mengalami pencemaran minyak, peningkatan sedimentasi dan fosfat secara berturut-turut adalah Stylophora pistillata, Acropora granulosa dan Montipora meandrina, sementara spesies dengan kerentanan terendah secara berturut-turut adalah Fungia fungites, Alveopora daedalea dan Millepora dichotoma. Key words: karang, pemutihan, eutrofikasi, polusi minyak, sedimentasi, Laut Merah.

INTRODUCTION Coral reefs are subject to extensive anthropogenic damage which is associated mainly with urbanization and coastal development (Wielgus et al. 2004; Hagedorn et al. 2010; Ammar 2011). Dramatic reversible decline in coral reef health has been reported from every part of the world. Between 50% and 70% of all coral reefs are under direct threat from human activities (Wilkinson 1999). In addition, coral reefs have experienced unprecedented levels of

bleaching, disease and mortality during the last three decades (Kramer 2003; Ammar 2009; Miller et al. 2011). Various stressors have led directly or interactively to coral reef decline, and may be linked to global changes in climate, land use or human activities in coastal areas (Yee et al, 2008; Miller et al. 2011). Carriquiry and Horta Puga (2010) and Ammar et al. (2007) reported that, eutrophication, increased sedimentation flowing from disturbed terrestrial environment, mining and oil pollution are the main causes of reef destruction. Coral bleaching is

3 (2): 73-81, July 2011

yet another major contributing factor to decline of coral reefs (Obura, 2005; Manzello et al., 2007; Baker et al., 2008). Coral bleaching results in the breakdown of a mutualistic symbiosis that is essential for the survival of corals, since the polyp receives a substantial part of its energy from the zooxanthellae (Douglas, 2003), and any disruption of this relationship will affect photosynthetic potential, coral growth and reproductive output; and might lead to eventually killing corals (Baker et al, 2008). Miller et al. (2011) found that, bleaching and associated mortality is an extreme threat to the persistence of coral populations in the projected warming regime of the next few decades. Most coral reef biologists do agree that coral reefs are changing and will exist in the near future but they will not be the ‘‘coral reefs’’ we have come to know in many parts of the world (Knowlton 2001). It is essential for the coral reef community to work together for the common good of the ecosystem. Time is short, as are finances, to conduct the integrative studies required to understand the range of acclimative capabilities that reef corals has in the face of continued environmental change, and to potentially predict which reefs will remain. This could be possible by monitoring different coral areas after a tough program of decreasing the global CO2 transmission.

In this study, we examined the initial bleaching response of corals to test the predictions of coral taxa that vary in their susceptibility to bleaching. We also aimed to study the relative bleaching susceptibilities of taxa to different impacts, outlining the degree of harm of each impact, the possibility of recovery and putting the possible scientific solutions.

MATERIALS AND METHODS Study area A preliminary visual survey of the Egyptian Red Sea coast, using snorkelling and SCUBA led to selection of four sites at four widely geographically separated areas along the western coast of the Red Sea (Figure 1). Which are Ras El-Behar (northern Hurghada), Middle reef (Hurghada), Kalawy bay (Safaga) and El-Hamraween harbor (El-Quseir). Three of these sites are subjected to different human activities and the fourth one is considered as a control site. Ras El-Behar (Site 1) lies at the northern part of the Red Sea, at a distance of about 60 km northern to Hurghada city, between latitudes 27o 43’ 12” N and 27o

Figure 1. Location map of the studied site: 1. Ras El-Behar (northern Hurghada), 2. Middle reef (Hurghada), 3.Kalawy bay (Safaga) and 4. El-Hamraween harbor (El-Quseir).

AMMAR et al. - Coral bleaching of Red Sea coast, Egypt

43’ 51” N and longitudes 33o 33’ 12” E and 33o 33’ 04” E. This site is impacted by petroleum oil pollution coming from the nearby petroleum fields and oil tankers. Furthermore, the commercial fishing activities are impacting the same site. Middle reef (Site 2) is located 200 m off shore between the northern reef and crescent reef, directly in front of National Institute of Oceanography and Fisheries (NIOF). This location is about 5 km northern to Hurghada city, between longitudes 27o 17.13’ N and 27 o 17.09’ N and latitudes 33 o 46.43’ E and 33 o 46.47’ E.The middle reef is situated in the area that has been subjected to land filling which is associated with high sedimentation rate El-Hamraween harbor (Site 3) is located about 60 km southern of Safaga, 20 km northern of El-Qusier City and about 120 from the Capital City of the Red Sea governorate (Hurghada). It is dominated between latitudes 26o 15’ 02” N and 26o15’17” N and longitudes 34o 12’ 07” E and 34o 12’ 00” E. The site is impacted by heavy load of phosphate due to preparation and shipment operations of phosphate are in El-Hamraween harbor (Al-Hammady 2011). Kalawy area (site 4) lies between latitudes 26o30’32” N and 26o 30’ 35” N and longitudes 34o 03’ 59” E and 34o 04’ 00” E. It lies about 30 km south Safaga City. This site is a pristine area having no source of pollution beside being difficult to be accessed by fishermen because of the heavy wave breaking in addition to the tough patrolling in the area, does not subject to any impact and thus it is considered as a control site in this study. Bleaching monitoring Coral bleaching have been detected at the studied sites using visual estimation, and Line Intercepted Transect (LIT) method (Wilkinson and Baker 1997). Bleaching was initially scored on a scale of 1 to 6 following the scheme of Gleason and Wellington (1993). However, we found that a four-point scale was more useful for comparing bleaching severity at our study sites, and was less prone to observer basis in allocating colonies to bleaching categories. The following categories were used: (i) unbleached = no visible loss of color; (ii) moderate = 1-50% of colony affected, or entire colony pale but not white; (iii) Severe= 51-100% of colony with strong pigment loss; (iv) dead = recently dead. Recently dead hard corals were recognized by an absence of living tissue and minimal algal overgrowth, and were considered to be bleaching fatalities. To estimate the percentage cover of bleached coral and other categories at each site, a 20 m long graded tap transect were surveyed on each reef flat and every other depth zone. A total of 12 transects were surveyed. Transects were laid down along the depth contour, parallel to the shore. The length of bleached coral colonies and other categories underlaying the transect were measured (the intercepted length) and the number of coral colonies was also counted. Percent cover of each category was calculated as follow: percent cover = (intercepted length/transect length) × 100. All colonies were identified to species or genus level, according to Wallace (1999) and Veron (2000) by using digital underwater photos.

RESULTS AND DISCUSSION Results Of the 3940 studied coral colonies, 20.11% of all corals were affected by bleaching: 5.4% were moderately affected; 2.7% severely affected and 12.007% were dead (Table 1). Overall, there were differences in the proportion of colonies affected by bleaching between the studied sites. The maximum average proportion of moderately bleached colonies was 12.62% and recorded at Ras El-Behar (the site was impacted by oil pollution and fishing activities), while the minimum proportion was 2.01% and recorded at Kalawy bay (pristine site). Moreover, the highest average proportions of severely bleached colonies (5.74%) and dead colonies (25.33%) were also found at site 1, while, the lowest average proportions of severely bleached colonies (1.101%) and dead colonies (3.59%) were found at site 4. The weighted average proportion of unbleached colonies recorded are 56.28%, 79.73%, 90.19% and 93.28% in site 1, site 2, site 3 and site 4 respectively. Table 1. Weighing average proportions of unbleached, moderately bleached, severely bleached and dead colonies at the studied sites. Unbleached Moderate Severe Dead Location (%) (%) (%) (%) Site 1 56.28 12.62 5.74 25.33 Site 2 79.73 4.92 3.35 12.009 Site 3 90.19 2.06 0.64 7.102 Site 4 93.28 2.01 1.101 3.59 Total average 79.87 5.4025 2.70525 12.00775

Many of the abundant taxa varied in their bleaching response according to their site. The five branching common acroporids varied between sites; Acropora clathrata (Brook 1891) was more susceptible to bleaching at Ras El-Behar (71% unbleached) than at Kalawy bay (99% unbleached), Acropora granulose (Milne Edwards and Haime 1860) was more susceptible to bleaching at Middle reef-NIOF (54% unbleached) than at Kalawy bay (72% unbleached), Acropora humilis (Dana 1846) was more susceptible to bleaching at Ras El-Behar (64% unbleached) than at El-Hamraween harbor and Middle reef-NIOF (92 and 91% unbleached respectively),Acropora hyacinthus (Dana 1846) was more moderately affected at Ras El-Behar (23% moderately bleached) than at ElHamraween harbor and Middle reef-NIOF (5% and 7% moderately bleached respectively), and Acropora pharaonis (Edwards and Haime 1860) was consistently more severely affected at Middle reef-NIOF (26% bleached) than at Kalawy bay (1% bleached). Stylophora pistillata (Esper 1797) was the another common branching coral varied in bleaching response between sites, Stylophora pistillata was 33% dead at Ras El-Behar, 17% dead at El-Hamraween harbour and 10% at Middle reefNIOF. There were different effects of different sites on bleaching responses of some coral massive form. Galaxea fascicularis (Linnaeus 1758) and Porites solida (Forskal 1775) were more susceptible to bleaching at Middle reefNIOF (79% and 77% unbleached respectively) than at Kalawy bay (100% unbleached). Favia pallida (Dana 1846) was other massive coral recorded 50% dead in Ras El-Behar and 1% dead in Middle reef-NIOF.

3 (2): 73-81, July 2011

Table 2. Species susceptibilities to bleaching in Red Sea coastal area of Egypt; Site: 1. Ras El-Behar (northern Hurghada), 2. Middle reef (Hurghada), 3.Kalawy bay (Safaga) and 4. (El-Quseir). Data were the percent of colonies in each of four bleaching categories (Unbleached: no visible loss of color, moderate: 1-50% of colony bleached or entire colony pale, severe: > 50% of colony bleached and dead: absence of living tissue and algal overgrowth) for all taxa which recorded on survey transects.

Category

Species name n

Branching hard corals Acropora brueggmanni Acropora clathrata Acropora corymbosa Acropora cytheraea Acropora digitefera Acropora eurystoma Acropora formosa Acropora granulosa Acropora hemperchi Acropora humilis Acropora hyacinthus Acropora nasuta Acropora nobilis Acropora pharaonis Acropora squarrosa Acropora valenciennesi Lobophyllia corymbosa Lobophyllia hemperichi Pocillopora damicornis Pocillopora verrucosa Seriatopora hystrix Stylophora pistillata Weighing average Massive hard corals

Ctenactis ecchinata Echinopora lamellosa Favia amicorum Favia pallida Favia helianthoides Favia pallida Favites persi Favia speciosa

Ras El-Behar (site 1) Bleached Un blea- Mode- SeveDead ched rate re (%) (%) (%) (%)

Middle reef of Hurghada (site 2) Bleached Un blea- Moden Severe Dead ched rate (%) (%) (%) (%)

* 29 * * 13 * * 24 * 31 42 9 * 12 17 * * * * * 2 823

* 71 * * 82 * * 50 * 64 51 67 * 83 81 * * * * * 100 49 51.85

* 22 * * 3 * * 16 * 21 23 4 * 6 7 * * * * * 0 13 13.57

* 5 * * 4 * * 23 * 7 5 2 * 1 3 * * * * * 0 5 5.36

* 2 * * 11 * * 11 * 8 21 27 * 10 9 * * * * * 0 33 29.21

* * * * * * 27

* * 4 * * 2 * *

* * 100 * * 50 * *

* * 0 * * 0 * *

* * 0 * * 50 * *

* * 16 * 16 7 * *

* 21 43 * * 33 12 * 67 * * 9 19 54

* * * * * * 93

Kalawy bay (site 3) Bleached Un blea- Mode- SeveDead ched rate re (%) (%) (%) (%)

El-Hamraween harbor (site 4) Bleached Un blea- ModeSevere ched rate Dead (%) (%) (%) (%)

* * * * * * 1 17 * * 91 0 78 7 * * * * 74 2 79 2 * * 79 0 * * * * 81 2 91 2 59 16 74.28 6.4

* * * * * * 1 8 * 0 3 * * 2 3 * 0 * * 0 2 15 4.45

* * * * * * 5 21 * 9 12 * * 22 16 * 21 * * 17 5 10 14.86

* * * 34 18 9 9 * 10 129 7 * * * 21 * 2 * 76 189 * 7

* * * 94 96 90 93 * 100 92 88 * * * 90 * 79 * 95 81 * 65 88.23

* * * 2 1 1 1 * 0 0 5 * * * 0 * 0 * 3 2 * 11 1.6

* * * 0 0 2 1 * 0 0 2 * * * 0 * 0 * 1 0 * 7 0.32

* * * 4 3 7 5 * 0 8 5 * * * 10 * 21 * 1 17 * 17 9.83

5 16 8 * * 5 * 19 15 * 36 8 27 4 * 11 * 21 23 42 * 44

98 99 100 * * 100 * 72 92 * 81 100 97 99 * 100 * 84 99 96 * 73 88.86

0 0 0 * * 0 * 13 1 * 6 0 0 0 * 0 * 0 0 2 * 9 3.37

0 0 0 * * 0 * 4 0 * 3 0 0 0 * 0 * 0 0 1 * 7 1.88

2 1 0 * * 0 * 11 7 * 10 0 3 1 * 0 * 16 1 1 * 11 5.88

* * 89 * 88 98 * *

* * 2 * 1 0 * *

* * 6 * 9 1 * *

* * 16 12 * * * *

* * 89 97 * * * *

* * 3 1 * * * *

* * 2 0 * * * *

* * 6 2 * * * *

6 10 * * * * 6 26

100 100 * * * * 100 100

0 0 * * * * 0 0

* * 3 * 2 1 * *

AMMAR et al. - Coral bleaching of Red Sea coast, Egypt

Favia stelligera Galaxea fascicularis Goniastraea palauensis Goniastrea retiformis Goniopora somaliensis Leptoria phrygia Montipora circumvallata Montipora edwardsi Montipora gracilis Montipora meandrina Montipora monasteriata Montipora spongiosa Montipora stilosa Montipora verrucosa Pavona cactus Pavona yabei Platygyra daedalea Platygyra sinensis Porites compressa Porites lobata Porites lutea Porites rus Porites solida Porites undulata Weighing average

21 * * * * 6 14 * * 47 * 29 * * * * 23 * * * * * * *

79 * * * * 90 79 * * 66 * 67 * * * * 64 * * * * * * * 70.69

7 * * * * 2 5 * * 11 * 12 * * * * 8 * * * * * * * 8.75

5 * * * * 0 4 * * 16 * 12 * * * * 9 * * * * * * * 10.05

9 * * * * 8 12 * * 7 * 9 * * * * 19 * * * * * * * 10.49

28 683 * * * 29 14 4 5 32 * 11 * * 18 6 63 * * * * * 8 *

90 79 * * * 92 70 98 77 72 * 92 * * 79 91 91 * * * * * 77 * 80.9

1 5 * * * 1 2 0 6 13 * 0 * * 8 2 1 * * * * * 4 * 4.55

1 3 * * * 1 0 0 5 9 * 1 * * 2 0 0 * * * * * 3 * 2.7

8 13 * * * 6 28 2 12 6 * 7 * * 11 7 8 * * * * * 16 * 11.83

* * 18 * 15 * * * * 16 * * * * * * * * 19 * 30 22 61 43

* * 99 * 90 * * * * 75 * * * * * * * * 100 * 97 99 91 96 93.59

* * 0 * 3 * * * * 12 * * * * * * * * 0 * 2 0 5 1 2.79

* * 0 * 1 * * * * 7 * * * * * * * * 0 * 1 0 2 0 1.23

* * 1 * 6 * * * * 6 * * * * * * * * 0 * 0 1 2 3 2.36

11 5 * 7 * * * * * * 13 * 7 19 * * 5 6 * 16 * * 9 *

100 100 * 100 * * * * * * 99 * 99 100 * * 98 100 * 96 * * 100 * 99.35

0 0 * 0 * * * * * * 0 * 0 0 * * 1 0 * 1 * * 0 * 0.14

0 0 * 0 * * * * * * 0 * 0 0 * * 0 0 * 0 * * 0 * 0

0 0 * 0 * * * * * * 1 * 1 0 * * 1 0 * 3 * * 0 * 0.5

Encrusting

Alveopora daedalea Echinopora gemmacea Echinopora lamellosa Hydnophora microconos Weighing average

2 54 13 8

100 84 77 90 83.85

0 11 4 0 8.38

0 2 7 4 3

0 3 12 6 4.75

4 6 23 12

100 94 93 90 92.95

0 0 2 1 1.28

0 0 1 1 0.78

0 6 4 8 4.98

6 * 9 1

100 * 95 94 96.81

0 * 2 1 1.18

0 * 1 3 0.75

0 * 2 2 1.25

* * * * *

Solitary

Fungia fungites Fungia klunzingeri Fungia fungites Fungia repanda Fungia scutaria Weighing average

4 * * 1 *

100 * * 100 * 100

0 * * 0 * 0

25 18 * 25 11

89 90 * 64 88 81.17

4 6 * 7 2 5.12

0 1 * 23 2 7.78

7 4 * 6 8 6.13

* * 4 21 *

* * 100 78 * 81.25

* * 0 8 * 6.72

* * 0 2 * 1.68

* * 0 12 * 10.08

* * * * * *

* *

15 11

3.35

12.009

100 100 100 90.19

0 0 0 2.06

0 0 0 0.64

0 49 0 6 0 7.102

100 100 100 93.28

0 0 0 2.01

0 0 0 1.101

0 0 0 3.59

Millepora dichotoma Millepora platyphylla Weighing average Total Note: n= number of colonies; * = n.a.

Hydrocorals

56.28 12.62 5.74 25.33

79.73 4.93

3 (2): 73-81, July 2011

Figure 2. Patterns of bleaching at site 1 (Ras El-Behar). A. Dead colonies of Stylophora pistillata; B. Severe bleached colonies of Stylophora pistillata; C. Moderately bleached colonies of Stylophora pistillata; D. Severe bleached colony of Acropora spp. intercepted under the Line Intercepted Transect; E. Moderately bleached colony of Montipora spp.

Figure 3. Patterns of bleaching at site 2 (Middle Reef-NIOF). A. Galaxea fascicularis; B.Moderately bleached colony of Montipora spp.; C. and D. Moderately bleached colony of Acropora spp.

Figure 4. Patterns of bleaching at site 3 (Al Hamraween harbor). A. Moderately bleached colony of Porites spp.; B. Moderately bleached colony of Acropora spp.; C. Moderately bleached colony of Pocillopora damicornis.

AMMAR et al. - Coral bleaching of Red Sea coast, Egypt

C Recently dead

D Moderately bleanced

Moderately bleanced Partially dead

Figure 5. Patterns of bleaching at site 4 (Kalawy bay). A. Severely bleached colony of Stylophora pistillata; B. Moderately bleached and dead colony of Acropora spp.; C. Colony of Pocillopora verrucosa having unbleached, moderately bleached and dead branches; D. unbleached colony of Millepora dichotoma.

Stylophora pistillata, the only abundant species, with unusual big and thick branches, was the most susceptible to bleaching at site 1 (Figure 2), of which 33% of colonies dead, 5% severely bleached and 13% moderately bleached. However, over 49% of colonies remained unbleached at the time of the survey. Acroporids were also highly susceptible to bleaching; Acropora granulosa was the most affected, with 11% of colonies dead, 23% of colonies severely bleached and 16% moderately bleached. A number of taxa appeared resistant to bleaching, 100% of colonies of Seriatopora hystrix (Dana 1846), Favia amicorum (Edwards and Haime 1850), Alveopora daedalea (Dana 1846), Fungia fungites (Linnaeus 1758) and Fungia repanda (Dana 1846) were unbleached, although these corals were the least abundant species at site 1. Leptoria phyrigia (Milne Edwards and Haime 1860) and Hydnophor amicroconos (Lamark 1816) were less susceptible to bleaching, more than 90% of colonies of these species apparently unaffected by bleaching (Table 2). The massive coral Galaxea fascicularis, the most abundant species at site 2 (Figure 3), was the Least resistance to bleaching, of which 13% of colonies were dead, 3% severely bleached and 5% moderately bleached. However, over 79% of coral colonies remained unbleached at the time of the survey. Acroporids were also low resistance to bleaching (Figure 3). Acropora granulose was the worst affected, with 21% of colonies have been dead, 8% severely bleached and 17% moderately bleached. However, over 54% of coral colonies remained unbleached. Only one species could be considered as being unaffected by bleaching; Alveopora daedalea (100% unbleached). A number of taxa appeared particularly resistant to bleaching. Less than 20% of colonies of Pocillopora verrucosa (Ellis and Solander 1786), Favia amicorum (Edwards and Haime 1850), Favia helianthoides (Edwards and Haime 1850), Fungia scutaria (Lamark 1801) and Fungia fungites (Linnaeus 1758) showed signs of bleaching with very few colonies severely bleached or dead (Table 3). Two taxa relatively being affected by bleaching at site 3, which were Stylophora pistillata and Montipora meandrina (Ehrenberg 1834). Stylophora pistillata, the none expectedly expressing rare distribution as it is well known in literatures

as an opportunistic species, was the most susceptible to bleaching, of which 17% of colonies have dead, 7% severely bleached and 11% moderately bleached. However, over 65% of colonies remained unbleached at the time of the survey. Montipora meandrina was also highly susceptible to bleaching with over 12% of colonies moderately bleached and 75% unbleached. The branching coral Pocillopora verrucosa, the most abundant species at site 3 (Figure 4), was the worst affected of which 17% of colonies was dead, on the other hand, 81% was unaffected (Table 4). Relatively, two species also being affected by bleaching at site 4; Acropora granulosa and Stylophora pistillata. On the other hand, 18 species could be considered as being unaffected by bleaching (100% unbleached) (Table 5). In general, the scleractinian corals with branching growth form morphologies suffer higher rates of bleaching than species with massive, encrusting and solitary morphologies at the four study sites. Discussion Our results show that, there were differences in the proportion of colonies affected by bleaching between the studied sites. Spatial variation in the severity of bleaching may be driven by variation between sites in environmental conditions that trigger the bleaching event (Manzello et al., 2007). The maximum average proportion of moderately and severely bleached and dead colonies at Ras El-Behar (site 1) is probably attributed to oil pollution, the prominent stressor in that site. This agrees with the laboratory finding of Frisch et al. (2007) who assessed the effects of clove oil solution on colonies of Pocillopora damicornis, finding that, corals treated with high concentrations (50 ppt) of clove oil solution died immediately. Carriquiry and Horta Puga (2010) reported that, oil pollution is the main causes of reef destruction. Ammar et al. (2007) found that, Ras El-Behar is characterized by the paucity of coral species and relatively high abundance of algae and sea urchin Diadema setosum. Fishing activities is another important factor that may play an important role in increasing bleaching susceptibilities at site 1. Reef damage due to dynamite fishing, recent and old, is frequently encountered on Egyptian Red Sea reefs (Ammar 1998; Ammar and

3 (2): 73-81, July 2011

Madkour, 2011). Mac Manus et al. (1997), suggested that, approximately 1.4%/year of the hermatypic coral cover may have been lost due to blasting fishing, 0.4%/year to cyanide fishing, and 0.03%/year to coral-grabbing anchors. Moreover, Ammar and Mohammed (2006) observed that, moderate diving and low fishing are the main reasons for decreasing the percentage cover of coral community at Tobia Kebir-Safaga-Red Sea. There was a difference in the susceptibility to bleaching stress among the corals at the studied species. Bhagooli and Hidaka, 2004) confirmed this result. They found that, Montipora spp. were highly susceptible to coral bleaching, inconsistent, bleaching of coral species on the same reef suggests that there is a range of tolerance within species (Celliers and Schleyer 2002). Statt et al. (2006) recorded significant variation in intra-specific bleaching and ascribed this phenomenon to difference in symbiont clade composition. While, bleached corals lose most of their symbiotic dinoflagellates and appear white after relatively short high-temperature exposures, adjacent colonies of the same, or another, species may display normal coloration for weeks or even months living in the same conditions on the same reef (Baghooli and Hidaka, 2004). In fact, several factors can apparently determine whether a coral colony being more susceptible to bleaching than the others. One such characteristic is tissue thickness (Ainsworth et al., 2008). Corals from genera such as Porites that have thicker tissues and appear more robust to thermal stress than corals from genera such as Acropora which has thinner tissues (Loya et al. 2001).Although the previous result seems to depend more on the host rather than on the Symbiodinium, no actual study related the tissue thickness with the clade of Symbiodinium. Thermal stress and tissue thickness also interact as shown by Manzello et al. ( 2007) and Fitt et al. (2000), who documented steady decreases in tissue biomass and symbiont density during summer months, and interpreted it as an increase in metabolic demand and subsequent use of stored energy reserves. Fitt et al. (2009) concluded that, P. cylindrica contains a heat resistant C15Symbiodinium and critical host proteins are present at higher concentrations than observed for S. pistillata, the combination of which provides greater protection from bleaching conditions of high temperature in the light. Size can also play a role in determining patterns of mortality on bleached reefs as discussed by Baker et al. (2008), Small juvenile colonies of some species can survive better than large, mature colonies (Loya et al. 2001; Riegl 2002; van Woesik et al. 2004).This could explain the the high rate of mortality among Stylophora pistillata having unusual thick and big branches at site 1. Millepora spp. among species that reported the least susceptibility to bleaching (100% unbleached) among the 104 taxa included in our surveys, the least susceptibility of this hydrocoral genus has been reported by Gleason and Wellington (1993). He found that, Millepora did not show any evidence of bleaching other than mild paling during the 1991 bleaching event in Moorea, French Polynesia. However, Millepora with most susceptibility to bleaching has been reported from bleaching episodes elsewhere

(Glynn and de Weerdt 1991). Species of Millepora suffered particularly rapid mortality in the eastern Pacific during the 1982-1983 bleaching episode (Manzello et al., 2007). On the other hand, Ammar and Emara (2004) reported that, Millepora dichotoma prefers clear water and cannot tolerate excessive sediments. Ammar (2004) also reported that, Millepora sp. prefers highly illuminated sites and has a strong skeletal density to tolerate strong waves. The least susceptibilities among coral taxa at ElHamraween harbor can be explained by phosphorus enrichment at this site. This result is consistent with the result of Ammar et al. (2007). Who recorded that, the presence of sulphur beside phosphorus at El-Hamraween harbor (Site 3) may have beneficial synergetic effect that may lead to flourishing corals. Faxneld, et al. (2010) found no physiological effects from nitrate enrichmentalone, which is in accordance with Ferrier-PagĂ¨s et al. (2001), who found no effects on zooxanthellae density or photosynthesiswith nitrate enrichment (2 ÂľM). The present surveys demonstrated that, the scleractinian corals with branching growth form morphologies generally suffer from higher rates of bleaching than species with massive, encrusting and solitary morphologies at the four study sites. Surveys conducted over a broad range of habitats, biogeographic regions and different sea warming events have confirmed our finding that scleractinian corals with branching colony morphologies generally suffer higher rates of bleaching than species with massive and encrusting morphologies (Loya et al. 2001; McClanahan and Maina 2003). Field observations supporting differential mortality of branching and massive species have been supported experimentally for five species in the eastern Pacific (Hueerkamp et al. 2001). Massive species of Porites and Diploastrea are frequently among the survivors (McClanahan and Maina 2003; Schuhmacher et al. 2005). Favia spp. often survives, as do non-branching species in the family Agariciidae (Loya et al. 2001).

CONCLUSION AND RECOMMENDATIONS Local anthropogenic stressors (e.g. fishing activity and oil pollution) expressed a higher bleaching event than areas subjected to eutrophication stressors. Bleaching susceptibility has extraordinarily decreased with increased phosphate level in contrast to most experimental literatures. Therefore, review of various techniques dealing with the effect of phosphates on corals is necessary. Stylophora pistillata, the only abundant species at site 1 (with oil pollution), was the most susceptible to bleaching in that site. The massive coral Galaxea fascicularis, the most abundant species at site 2, was the most susceptible to bleaching. Some other species like Acropora granulosa, Stylophora pistillata, Montipora meandrina, Fungia repanda and other species have higher bleaching. Tough control, public awareness and continuous shore patrolling to the activities of oil pollution and fishing activities at the vicinity of site 1 are urgent. Review of various techniques, dealing with the effect of phosphate on

AMMAR et al. - Coral bleaching of Red Sea coast, Egypt

corals, and taking care of their precision is necessary because indications of the field work are different from many of the reviewed experimental literatures. Site 4 is already virgin, and needs more guarding to keep that site always pristine. However, it could be also protected from any possible cause of rising sea temperature like installing desalination plants or any other industrial installation, urbanization and coastal development.

ACKNOWLEDGEMENTS This work is a part of the PhD of the student Montaser Aly Mahmoud Al-Hammady.

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Ferrier-PagĂ¨s C, Schoelzke V, Jaubert J, Muscatine L, Hoegh-Guldberg O. 2001. Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment. Exp Mar Biol Ecol J 259: 249-261. Fitt WK, Gates RD, Hoegh-Guldberg O, Bythell JC, Jatkar A, Grottoli G, Gomez M, Fisher P, Lajuenesse TC, Pantos O, Iglesias-Prieto R, Franklin DJ, Rodrigues LJ, Torregiani JM, van Woesik R, Lesser MP. 2009. Response of two species of Indo-Pacific corals, Porites cylindrica and Stylophora pistillata, to short-term thermal stress: The host does matter in determining the tolerance of corals to bleaching. J Exp Mar Biol Ecol 373: 102-110. Fitt WK, McFarland FK, Warner ME. 2000. Seasonal patterns of tissue biomass and densities of symbiotic dinoflagellates in reef corals and relation to coral bleaching. Limnol Oceanogr 45 (3): 677-685. Frisch AJ, Ulstrup KE, Hobbs JA. 2007. The effects of clove oil on coral: An experimental evaluation using Pocillopora damicornis (Linnaeus). J Exp Mar Biol Ecol 345 (2): 101-109. Gleason DF, Wellington GM. 1993. Ultraviolet radiation and coral bleaching. Nature 365: 836-838. Glynn PW, de Weerdt WH. 1991. Elimination of two reef-building hydrocorals following the 1982-83 El Nino warming event. Science 253: 69-71 Hagedorn M, Carter VL, Leong JC, Kleinhans FW. 2010. Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium). Cryobiol 60: 147-158. Knowlton N. 2001. The future of coral reefs. Proc Natl Acad Sci USA 98: 5419-5425. Kramer PA. 2003. Synthesis of coral reef health indicators for the Western Atlantic: results of the AGRRA program (1997-2000). Atoll Res Bull 496: 1-57. Loya Y, Sakai K, Yamazato K, Nakano H, Sambali H, van Woesik R. 2001. Coral bleaching: the winners and the losers. Ecol Lett 4: 122-131. Manzello DP, Berkelmans R, Hendee JC. 2007. Coral bleaching indices and thresholds for the Florida Reef Tract, Bahams, and St. Croix, Us Virgin Islands. Mar Pollut Bull 54 (12): 1923-1931 Mc Manus J, Rodolfo B, Reyes JR. 1997. Effects of Some destructive fishing methods on coral cover and potential rates of recovery. Environ Manag 21 (1): 69-78 McClanahan TR, Maina J. 2003. Response of coral assemblages to the interaction between natural temperature variation and rare warmwater events. Ecosystems 6: 551-563. Miller MW, Piniak GA, Williams DE. 2011. Coral mass bleaching and reef temperatures at Navassa Island, 2006. Estuar Coast Shelf Sci 91: 42-50 Obura DO. 2005. Resilience and climate change: lessons from coral reefs and bleaching in the Western Indian Ocean. Est Coast Shelf Sci 63 (3): 353-372 Richmond RH. 1997. Reproduction and recruitment in corals. In: Birkeland C (ed) Life and death of coral reefs. Chapman and Hall, New York. Riegl B. 2002. Effects of the 1996 and 1998 positive sea-surface temperature anomalies on corals, coral diseases and fish in the Arabian Gulf (Dubai, UAE). Mar Biol 140: 29-40. Schuhmacher H, Loch K, Loch W, See WR. 2005. The after math of coral bleaching on a Maldivian reef a quantitative study. Facies 51: 80-92. Statt M, Carter D, Hoegh-Guldberg O. 2006. The evolutionary history of Symbiodinium and sclerectinian hosts. Symbiosis, diversity and effect of climate change. Persp. Pl Ecol Evol Sys 8 (1): 23-43 Van Woesik R., Irikawa A, Loya Y. 2004. Coral bleaching: signs of change in southern Japan. In: Rosenberg E, Loya Y (eds) Coral health and disease. Springer, Berlin. Veron JEN . 2000. Corals of the world. Vol. 3. Australian Institute of Marine Science, Townsville, Australia. Wallace CC ( 1999. Staghorn corals of the world: A revision of the coral genus Acropora. CSIRO Publishing, Canberra, Australia. Wielgus J, Chadwick-Furman NE, Dubinsky Z. 2004. Coral cover and partial mortyality on anthropogenically impacted coral reefs at Eilat, northern Red Sea. Mar Poll Bull 48: 249-253. Wilkinson C, Baker V. 1997. Survey manual for tropical marine resources. 2nd ed. Australian Institute of Marine Science, Townsville. Wilkinson CR. 1999. Global and local threats to coral reef functioning and existence: review and prediction. Mar Fresh W Res 50: 867-878. Yee SH, Santavy DL, Barron MG. 2008. Comparing environmental enfluences on coral bleaching across and within species using clustered binomial regression. Ecol Mod 218 (1-2): 162-174.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 82-91 July 2011

The Palestinian mammalian fauna acquired by the zoological gardens in the Gaza Strip ABDEL FATTAH N. ABD RABOUâ&#x2122;Ľ Department of Biology, Faculty of Science, Islamic University of Gaza, P.O.Box 108, Gaza Strip, Palestine, Tel. 00970-8-2860700, Fax. 00970-82860800, â&#x2122;Ľemail: arabou@iugaza.edu.ps Manuscript received: 14 June 2011. Revision accepted: 15 July 2011.

Abstract. Abd Rabou AFN. 2011. The Palestinian mammalian fauna acquired by the zoological gardens in the Gaza Strip. Nusantara Bioscience 3: 82-91. The Gaza Strip, which is an arid strip of the Palestinian land along the southeastern Mediterranean, harbors a considerable number of mammalian fauna due to its eco-geo-strategic position. Prior to 2006, the establishment of zoological gardens in the Gaza Strip was a sort of imagination due to Israeli constraints. These constraints were nurtured by the total Israeli destruction and demolition of the Rafah and Gaza private zoological gardens in 2004 and 2009 respectively, using heavy tanks and bulldozers. The establishment of many zoological gardens following the Israeli evacuation from the Gaza Strip in late 2005 encouraged wildlife trading. Hence, the current study comes to document the Palestinian mammalian faunistic species acquired by the zoological gardens in the Gaza Strip through frequent visits to Gaza zoological gardens and meetings with local people, wildlife hunters and zoo owners. A total number of 17 Palestinian mammalian faunistic species belonging to 12 families and 5 orders was encountered in the zoological gardens throughout the study period. The encountered species represent a good mix of the families and sizes of mammals generally found in other parts of Palestine. Order Carnivora represents 52.94% of the caged mammals, while the orders Rodentia, Lagomorpha, Artiodactyla and Insectivora represent 47.06%. The study documented the first sight of the Greater Egyptian Gerbil Gerbillus pyramidis in the Gaza Strip. Local hunting, tunnel trade and delivery were the lonely sources of the mammals encountered in the zoological gardens. The economic deprivation under the current Israeli blockade and the poor implementation of environmental laws and legislations concerning wildlife protection have made wildlife trading as a common practice. Finally, The author recommends to improving the management process of Gaza zoological gardens under the care of the governmental authorities and the cooperation of the different parties in the Gaza Strip to enhance public ecological awareness to protect and conserve wildlife; especially mammals. Key words: mammals, carnivores, zoological gardens, wildlife hunting, tunnel trade, Gaza, Palestine. Abstrak. Abd Rabou AFN. 2011. Fauna mamalia Palestina yang dipelihara kebun binatang-kebun binatang di Jalur Gaza. Nusantara Bioscience 3: 82-91. Jalur Gaza, yang merupakan jalur tanah kering bagian negara Palestina di sepanjang tepi Laut Tengah bagian tenggara, menjadi tempat tinggal sejumlah hewan mamalia karena posisi eko-geo-strategisnya. Sebelum tahun 2006, pendirian kebun binatang di Jalur Gaza hanyalah sebuah impian karena larangan Israel. Hal ini tampak dari penghancuran dan pembongkaran seluruh kebun binatang swasta di Rafah dan Gaza oleh Israel, secara berturut-turut pada tahun 2004 dan 2009, menggunakan tank dan buldoser. Pembentukan kebun binatang banyak dilakukan setelah penarikan mundur Israel dari Jalur Gaza pada akhir tahun 2005, didorong oleh maraknya perdagangan satwa liar. Penelitian ini dilakukan untuk mendokumentasikan jenis-jenis hewan mamalia Palestina yang dipelihara oleh kebun binatang di Jalur Gaza dengan cara sering berkunjung ke kebun binatang serta pertemuan dengan masyarakat setempat, pemburu satwa liar dan pemilik kebun binatang. Sebanyak 17 jenis hewan mamalia Palestina dari 12 suku dan 5 bangsa ditemukan di kebun binatang selama periode penelitian. Jenis-jenis yang ditemukan mewakili keragaman suku dan kekayaan mamalia yang juga umum ditemukan di bagian lain Palestina. Bangsa karnivora mewakili 52,94% dari mamalia yang dipelihara, sementara bangsa Rodentia, Lagomorpha, Artiodactyla dan Insectivora mewakili 47,06%. Studi ini mendokumentasikan untuk pertamakalinya keberadaan Gerbillus pyramidis di Jalur Gaza. Perburuan lokal, perdagangan melalui terowongan dan pengiriman merupakan sumber asal mamalia di kebun binatang. Kemunduran ekonomi akibat blokade Israel dan buruknya implementasi hukum lingkungan dan peraturan perundang-undangan tentang perlindungan satwa liar telah menyebabkan perdagangan satwa liar menjadi praktik yang umum dilakukan. Penulis menyarankan adanya peningkatan manajemen kebun binatang-kebun binatang di Gaza dengan bimbingan otoritas pemerintah dan kerjasama dari para pihak untuk meningkatkan kesadaran ekologi masyarakat dalam melindungi dan melestarikan satwa liar, terutama mamalia. Kata kunci: mamalia, karnivora, kebun binatang, perburuan satwa liar, perdagangan melalui terowongan, Gaza, Palestina.

INTRODUCTION Mammals are one of the most diversified of all creatures on earth today. They range from tiny bat species to the largest Blue Whales. The overall mammalian species known worldwide is about 4,500 species (Boitani and

Bartoli 1983). Globally, mammals are threatened by destruction and degradation of ecological habitats, caused by a variety of factors including agricultural intensification, urbanization, pollution and climate change. Human disturbance, overexploitation of natural resources and invasive species are also major threats (Temple and

ABD RABOU – The Palestinian mammalian in the Gaza zoological gardens

Cuttelod 2008). The small geographic area of Palestine harbors a rich fauna and flora because of its strategic location at the juncture of the three continents (Asia, Africa and Europe) and because of the large variety of its unusual topography that creates diverse ecological habitats, niches and climates (Qumsiyeh 1996). An approximate number of 120 mammalian species of the 4,500 species known worldwide inhabit Palestine (Yom-Tov 1988; PIALES 1996; Ali Shtayeh and Hamad 1997; PCBS 2000). Qumsiyeh (1996) pointed out that most Palestinian mammalian fauna have Palearctic affinities and a few have affinities to the Ethiopian and Oriental realms. Zoological gardens (zoos) are institutions or facilities in which animals are confined within enclosures, displayed to the public, and in which they may also be bred. The history of modern zoological gardens, however, started some 200 years ago with the creation of the first public zoological garden. Since that time, large numbers of zoological gardens have been established in all parts of the world (IUDZG/CBSG of IUCN/SSC 1993). Globally, zoological gardens are known to offer great opportunities for entertainment and education, and to contribute to wildlife conservation and promote scientific research, especially for environmentalists and conservationists, as the rate of extinction of wild life increases. During the Israeli occupation era, which started in 1967, no zoological gardens have been established in the Gaza Strip, with the exception of the Rafah private zoological garden which was established in 1999 and was by far considered as the first zoological garden in the Gaza Strip and the only place for entertainment in Rafah city that kids could escape the tense atmosphere. There is a total understanding among the Gazans that the Israeli occupation prohibited the establishment of such animal collections or zoological gardens in the Gaza Strip, in an attempt to impose what could be termed as “ecological illiteracy” among the Palestinians. This thought was nurtured by the total Israeli destruction and demolition of the Rafah zoological garden with the ground using heavy tanks and bulldozers in 21 May 2004, i.e. after 5 years of its establishment. All the existing zoo animals were either being killed or escaped. The targeted mammalian set included Wolves, Jackals, Foxes, Wild Cats, Mongooses, Raccoons, Badgers, Gazelles, Deer, Ibexes and Kangaroos (Figure 1). After the Israeli evacuation of the colonies in the Gaza Strip in late 2005, local hunting of wildlife was reestablished. The establishment of local private zoological gardens or animal collections in the Gaza Strip encouraged wildlife trading (Abd Rabou 2009a) especially for mammalian fauna. About eight private zoological gardens have been established since 2006 in the five governorates of the Gaza Strip (North Gaza, Gaza, Middle, Khan Younis and Rafah). However, many of these gardens have been closed in the last two years due to intrinsic factors. The area of these zoological gardens ranges from 1.5 to 8 dunums (dunum = 1000 square meters). Apart from the judgment on their own merits, these zoological gardens were found to be (i) good private

projects providing revenues to their owners especially in the current times where the Israeli blockade was and is still imposed on the Gazans and the unemployment crisis which dominates the majority of the Gazan people, (ii) a good contributor to the knowledge of the Palestinians about their biodiversity items; especially vertebrate fauna, and (iii) a progressive challenging practice against the Israelis who prevented the establishment of such zoological gardens during its occupation era to the Gaza Strip which started in 1967. In non-war times of the Gaza Strip, these zoological gardens usually attract thousands of visitors a day, mainly families and pre-school and school children (Figure 2). It is worth mentioning that the main zoological garden of the Gaza city was attacked by Israeli soldiers and tanks during the Israeli War of December 2008 – January 2009. According to local media reports, about 400 zoo animals were lost during that attack. The few zoo animals which survived the Israeli onslaught were left to starve just like the Gazans themselves. The repeated Israeli attack to zoological gardens of the Gaza Strip ensured a programmed policy adopted by the Israeli occupation aimed at destroying such zoological gardens in order to disseminate its strategy of “ecological illiteracy” among the Palestinians. The Israeli policy of vegetation clearance of many agro-ecosystems in the Gaza Strip further deteriorates wildlife ecology. For example, UNEP (2003) documented the destruction by Israeli forces of vast agricultural areas in Beit Hanoun, North Gaza. This largescale modification of the landscape clearly would impact habitats, perhaps increase desertification, and at least effect human interactions with the remaining wildlife habitats. Survey studies on the occurrence of mammals in various ecosystems are fundamental to the conservation, research and management of wildlife populations. Accordingly, an extensive work on mammals; particularly carnivores, was carried out in different important Middle East and North Africa countries. Amr et al. (1987) studied a set of Jordanian mammals with particular emphasis on the distribution of the endangered species Gazella gazella. Amr and Disi (1988) described the Jordanian mammals acquired by the Jordan University Natural History Museum. Hatough and Disi (1991) worked on the large mammalian species of Jordan in terms of history, distribution and conservation, while Qumsiyeh et al. (1993) and Bunaian et al. (2001) worked on the local status and conservation strategies of the Jordanian carnivore species. In Syria, Masseti (2009) outlined the local occurrence and recent distribution of 15 carnivore species. In Turkey, the distributional records of 5 carnivore species were noted by Ozkurt et al. (1998). In Arabia, extensive work was taken place on mammalian species; particularly carnivores (Gasperetti et al. 1985; Nader 1989, 1990, 1996). Seddon et al. (1997) and Masseti (2010) have modest works carried out on the mammalian species occurring in the Harrat alHarrah Protected Area and the Farasan Archipelago of the Saudi Arabia respectively. Finally, Al-Jumaily (1998) reviewed the various mammals of the Republic of Yemen. There has been a few studies of mammals in the Gaza Strip (Abd Rabou 2005, 2009a, 2011; Yassin et al. 2006; Abd Rabou et al. 2007a; Abu Taleb 2008). Collectively,

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Figures: 1. Palestinian children look at a raccoon killed during the Israeli destruction of the Rafah private zoo (21 May 2004); 2. Gaza zoological gardens attract thousands of visitors; mainly school children a day

these studies revealed the occurrence of more than 20 mammalian species in the Gaza Strip. Many of which, especially carnivores, were found to enter the area through gaps or ground burrows at the political borders separating the Gaza Strip from Israel. A substantial interest was paid to the wild mammals caged in the zoological gardens of the Gaza Strip, where new species that were unknown to happen before in the Gaza Strip have been seen there. Accordingly, the current study comes to document the Palestinian mammalian faunistic species acquired by the zoological gardens in the Gaza Strip, which will benefit the Palestinians in the sense that they will be familiar with their mammalian fauna.

MATERIALS AND METHODS The study area The Gaza Strip (31°25′N, 34°20′E) is a 365 km2 arid strip of the Palestinian land along the southeastern Mediterranean. It represents the northern link between the Sinai and the Negev deserts. About 1.7 million residents, of whom the majority are United Nations-registered refugees, are living in the five governorates of the Gaza Strip (North Gaza, Gaza, Middle, Khan Younis and Rafah). The annual rainfall ranges from 200 mm in the south to 400 mm in the north. Three dry to semi-dry wadis (valleys) dissect the Gaza Strip. They are, from north to south, Wadi Beit Hanoun, Wadi Gaza and Wadi Al-Salqa. Procedures Frequent visits were carried out to the eight local zoological gardens of the Gaza Strip during a three-year study (October 2007 until September 2010) in order to record and determine the caged Palestinian mammalian species. Observations and interviews with tens of local people, wildlife hunters and zoo owners were taken place in Gaza zoological gardens and hunting sites as well. The intreviews involved different questions such as the sources

and costs of zoo animals, hunting sites and means, reasons of hunting and other aspects of zoo manangement. Digital cameras have been used throughout the study period and photos were taken for documentary and confirmatory purposes. Identification of mammalian species acquired by the zoological gardens follows published keys and guidebooks (Boitani and Bartoli 1983; Gross 1987; Harrison and Bates 1991; Qumsiyeh 1996; Hoath 2003).

RESULTS AND DISCUSSION Sources of zoo mammalian fauna The current study revealed that the mammalian fauna acquired by the zoological gardens of the Gaza Strip had three sources as follows: Local hunting: It was found that the skilled local hunters used to prepare their own hunting tools mainly the metal live traps which include the leghold or foothold trap and wire cage traps (called locally as Maltash) with different sizes in the field by night to hunt mammals. The leghold trap is usually set on an animal trail. Compared to wire cage traps, the leghold traps are known among Palestinians for breaking animals' legs and leaving them in pain often for prolonged periods of time. Other hunting tools such as ground mist nets and spare guns were used as well. Introduction from Egypt: Due to the reluctance of the Israeli occupation for any legal import of zoo animals, it was found that large mammals were commonly illegally introduced from Egypt through the earth tunnels connecting the Gaza Strip with the Egyptian territories. It is worth mentioning that these earth tunnels have been intensively established as a result of the Israeli blockade imposed on the Gaza Strip since 2006. The tunnel trade provides the Gazans with most of their needs including zoo animals such as lions, tigers, wolves, foxes, hyenas, deer, llamas, monkeys, ostriches, pheasants, peafowl, Flamingos, cranes, crocodiles and pythons.

ABD RABOU â&#x20AC;&#x201C; The Palestinian mammalian in the Gaza zoological gardens

Delivery: According to zoo owners, many of the caged pairs of zoo mammals were known to reproduce and to give birth to babies, e.g. lions, foxes, jackals and monkeys. Some of these babies were sold later to other zoological gardens in the Gaza Strip. Zoo mammalian fauna in the Gaza Strip A total number of 17 mammalian species belonging to 12 families and 5 orders was encountered in the zoological gardens of the Gaza Strip throughout the study period. Order Carnivora represents 52.94% of the caged mammals, while the orders Rodentia, Lagomorpha, Artiodactyla and Insectivora represent 47.06% (Table 1). Herewith is a brief description of each recorded mammalian fauna in the zoological gardens of the Gaza Strip. Some documentary photos concerning certain mammalian species are also provided. Table 1. Mammalian fauna documented at the zoological gardens of the Gaza Strip Family

Common name

Order Carnivora Canidae Golden Jackal Grey Wolf Red Fox Felidae Wild Cat Jungle Cat Herpestidae Egyptian Mongoose Mustelidae Marbled Polecat Common Badger Hyaenidae Striped Hyena Order Rodentia Gerbillidae Greater Egyptian Gerbil Hystricidae Indian Crested Porcupine Spalacidae Palestine Mole-rat Order Lagomorpha Leopridae Cape Hare Order Artiodactyla Bovidae Dorcas Gazelle Camelidae Dromedary Camel Order Insectivora Erinacedidae Long-eared Hedgehog Ethiopian Hedgehog

Scientific name Canis aureus Canis lupus Vulpes vulpes Felis silvestris Felis chaus Herpestes ichneumon Vormela peregusna Meles meles Hyaena hyaena Gerbillus pyramidis Hystrix indica Spalax leucodon ehrenbergi Lepus capensis Gazella dorcas Camelus dromedarius Hemiechinus auritus Paraechinus aethiopicus

Golden Jackal Canis aureus (Linnaeus 1758) The Golden Jackal has a golden reddish-yellow fur with a white mark on the throat. The animal seems to be found in all climatic areas of Palestine including the open and woody areas. The species feeds on a variety of preys including rodents, hares and domestic sheep. Locals of eastern Gaza claimed that the Golden Jackals comes at night from Israel to attack their animal husbandries and to prey on their domestic animals such as chicken, rabbits and young cattle. Due to the great similarities between the

Golden Jackal and the Grey Wolf, most of the Palestinians including some zoo owners can not distinguish between the two species and they may give them the same local names (Wawi or Deeb) . As many as 24 individuals of the Golden Jackal were encountered in Gaza zoological gardens (Figure 3). Depending on frequent meetings with zoo owners and local hunters, trapped animals were apparently sold for approximately U.S.$150-200 per live specimen. Wire cage and leghold traps are commonly used for hunting the Golden Jackal along with other mammals in the eastern parts of the Gaza Strip where fruit orchards and animal husbandries are common. Besides, the Golden Jackal was known to enter the Gaza Strip from Israel through burrows or gaps found beneath the political borders separating the two entities. Grey Wolf Canis lupus (Linnaeus 1758) The Grey Wolf is the largest member of the Canidae family and it inhabits different parts of Palestine especially the mountainous regions. It primarily feeds on domestic animals, ungulates, hares, rodents, birds and reptiles such as lizards and snakes. Wolves are generally nocturnal but may sometimes hunt during the day. In spite of the debate concerning the occurrence of the Grey Wolf in the Gaza Strip, all the recent existing individuals (N = 13) of the species (Figure 4) in Gaza zoological gardens were known to be illegally imported from Egypt through the earth tunnels joining the Gaza Strip with the Egyptian territories. Prior to 2002, the animal was legally imported from Israel to be caged in the Rafah zoological garden. Finally, the free movement of the Grey Wolf in the Negev Desert suggest large home ranges (Afik 1983) and this may explain the occasional movement of the species across borders from the Negev Desert into the Gaza Strip. Red Fox Vulpus vulpus (Linnaeus 1758) The Red Fox is the most widely distributed carnivore in the world and is an adaptable species found in a variety of ecological habitats including that of Palestine and its neighboring countries (Bunaian et al. 2001 and Hoath 2003). The Red Fox is the largest fox in the genus Vulpus in Palestine. It is commonly found caged in Gaza zoological gardens (N = 17) (Figure 5). As many as six individuals were found losing one of their legs due to their capture using leghold traps. Meetings with both zoo owners and hunters revealed that the healthy Red Fox was sold for approximately U.S.$100. Moreover, the poor care admitted to the caged injured individuals resulted in their death. The Red Fox is commonly hunted using the same methods applied for the Golden Jackal in the open areas of the eastern parts of the Gaza Strip where most animals husbandries are present. In parallel with that, many zoo owners claimed that they imported many individuals of the Red Fox from Egypt via the tunnel trade. Wild Cat Felis silvestris (Schreber 1777) The Wild Cat is a common felid species in Palestine. It feeds on small animals including rodents, lizards and insects. Due to the great similarities between the Wild Cat

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and the Domestic Cat, most Palestinians in the Gaza Strip are unable to distinguish between them and they could not exclude their interbreeding as well. Palestinians inhabiting remote areas in the Gaza Strip including Wadi Gaza seem to be familiar with the species in question. They claimed that they poisoned or killed the species many times due to their opportunistic attack to their poultry husbandries. Meetings with many zoo owners revealed that the Wild Cat (N = 4) was opportunistically caged for short periods of time with no further details. They claimed that Wild Cats are more aggressive than Domestic ones. Meanwhile, caging of the Domestic Cat Felis catus (Linnaeus 1758) was a common practice in all Gaza zoological gardens. The scientific thought of Wild Cats as being as an ancestor of Domestic Cats was totally accepted by the Palestinian community. The species is considered by the way as the most common and the very desired vertebrate pet in the Gaza Strip. Jungle Cat Felis chaus (Schreber 1777) The Jungle Cat – as its name displays – is a riparian feline associated with dense vegetation encompassing water bodies and courses. It feeds on small animals including rodents, lizards, birds and rodents. What characterize the species are its grizzled gray color and its ears, which have black tufted tips. With regard to the Palestinian carnivore species, the Jungle or Swamp Cat (Figure 6) is the most occurring species in Gaza zoological gardens (N = 38). Meetings with both hunters and zoo owners indicated that the individual Jungle Cat is sold at U.S.$70-100. In the Gaza Strip, the species is usually hunted along the eastern borders using wire cage traps. This belt of land is rich in cultivated and/or irrigated areas that are not far from the Gazan residential dwellings (perhaps hunting rodents that also increased in those areas, see Abu Baker et al. 2003) and in this it maybe exceptional to other carnivores in the Gaza Strip. Along with other carnivores, locals usually treat the Jungle Cats as pests and they were known to get rid of many of them through direct killing or poisoning. Egyptian Mongoose Herpestes ichneumon (Linnaeus 1758) The Egyptian Mongoose is a common mammal in Palestine. In the Gaza Strip, the species prevails the eastern parts where many citrus, almond and olive orchards in addition to other agricultural fields are found. Locally, the animal is unloved by Gazans and they usually called it “Nims” or “Nisnas” and sometimes "Abu Al-Khesiat". The author confirmed the presence of the Egyptian Mongoose among clumps of the small stands of the Common Reed Phragmites australis and Tamarisk Tamarix nilotica shrubs along the Wadi Gaza Nature Reserve in the middle of the Gaza Strip. To prevent the attack of the animal to their domestic poultry, many locals were found to kill the Mongoose using different means including rodenticides. As many as 26 individuals of the species were recoded in Gaza zoological gardens and all of them were hunted locally using wire cage traps. Hunters and zoo keepers stated that the Egyptian Mongoose was sold at U.S. $ 30-50 per live specimen.

Marbled Polecat Vormela peregusna (Guldenstaedt 1770) The Marbled Polecat is the smallest and rarest among the carnivore species occurring in the Gaza Strip though it happens in diverse ecological habitats in Palestine. The animal is colorful in terms of having variegated brown and yellow patches and stripes of irregular shape on the back. It seems to feed on small rodents. Only eight specimens were encountered in few zoological gardens throughout the survey period (Figure 7). Zoo owners claimed that they bought the animal with U.S. $10-30 from local people capturing it by hand in cultivated fields or in the wild. However, most of the zoo specimens were able to escape from their cages due to their very low body weights that does not exceed 0.75 kilograms as estimated by zoo keepers. Common Badger Meles meles (Linnaeus 1758) The Common Badger is a burrowing nocturnal animal found in the hilly, cultivated and woody parts of the Holy Land which includes Palestine and Jordan (Qumsiyeh 1996 and Abu baker and Amr 2002) . It feeds upon rodents, reptiles and insects. It builds extensive burrow systems using its well-clawed front legs. What characterizes the animal is the presence of a median white stripe on the head. No previous records indicating that the animal was found in the Gaza Strip as an isolated geographical area. Nowadays, no Badgers are caged in Gaza zoological gardens, and the only sight of the Common Badger (Figure 8) was made in the Rafah private zoological garden during 2002-2004. According to the Rafah zoo owner, the specimen was imported from Israel at the beginning of the zoo establishment in 1999. It is worth mentioning that the Israeli tanks and bulldozers attacked the Rafah private zoological garden in 21 May 2004, and demolished it with the ground and most of the existing zoo animals including the Common Badger either escaped or died. Striped Hyena Hyaena hyaena (Linnaeus 1758) The Striped Hyena is the only contemporary Hyena in Palestine. Although it is common in various ecological habitats in the West Bank of Palestine, the species was not known to occur in the Gaza Strip since at least the last 7-8 decades as told be aged inhabitants. The animal is named by the Palestinians as “Abu El-Fatayes”; a name indicating that the species is a “scavenger”, feeding on carcasses or dead animals, in spite of its ability to kill large preys including domestic animals. Depending on stories drawn by aged Palestinians, the Striped Hyena was familiar in straying near their villages, rural dwellings and agricultural orchards searching for food and water. By its powerful jaws, the Striped Hyena can crack large bones. Different types of animal bones are usually encountered at the entrance of Hyena den (see Kerbis-Peterhans and Horwwttz 1992; Qumsiyeh 1996; Qarqaz et al. 2004; AbiSaid and Marrouche 2007) and this bone collection is an actual sign of a Hyena den identification as indicated by aged Palestinians. As many as 7 individuals of the Striped Hyena were encountered in the Gaza zoological gardens. All caged Hyenas were introduced from Egypt through tunnel trade.

ABD RABOU – The Palestinian mammalian in the Gaza zoological gardens

Although the species is unloved by the Palestinian community, the caged species seemed to attract zoo visitors perhaps due to its strange physical appearance as well as its fear stories and films drawn by the locals. In parallel, one live caged specimen of the Spotted Hyena Crocuta crocuta (Erxleben 1777) was noticed in Khan Younis zoological garden. Although it is not a Palestinian carnivore, this imported specimen may give zoo visitors the opportunity to know more about Hyenas and their diversity in the wild of both Asia and Africa. Greater Egyptian Gerbil Gerbillus pyramidis (Geoffroy 1825) The Greater Egyptian Gerbil is the largest hairy-footed Gerbillus in the area. Although the upper color of the Gerbil varies from orange-brown to tawny grey, the belly seems white. This nocturnal rodent is distributed mainly in North Africa, Sinai Peninsula and Palestine in areas rich in cultivated and shrubby sandy soils and dunes. No recent scientific records indicated the occurrence of the Greater Egyptian Gerbil in the Gaza Strip (see Abd Rabou et al. 2007a). In a sudden event taken place in 2008, the author encountered 7 individuals of the species in a wire cage in a zoological garden in North Gaza (Figure 9). This was the first sight of the Greater Egyptian Gerbil in the Gaza Strip. Thence, many farmers confirmed the occurrence of the species in the cultivated sandy areas covering the western coastal belt of the Gaza Strip. Indian Crested Porcupine Hystrix indica (Kerr 1792) The Indian Crested Porcupine is the largest rodent species inhabiting the different ecological habitats of Palestine. It is a herbivore; feeding mostly on plant materials including roots and bulbs. The dorsal surface is covered with quills ranging from 25-35 centimeters in length. These quills are a good defense strategy for the animal when disturbed by enemies. Although it was considered as a disappeared animal in the Gaza Strip (Abd Rabou 2005; Abd Rabou et al. 2007a), recent meetings and discussions with locals inhabiting the eastern belt of the Gaza Strip revealed that the Indian Crested Porcupine is still found and is rarely seen. The presence of residual quills – picked up by the author himself and many other inhabitants in the area – may confirm such an occurrence of the species. The Indian Crested Porcupine seemed to be found in all Gaza zoological gardens (N = 28). Such cages may harbor 3-6 individuals. No evidence indicating that the species was hunted in the Gaza Strip. Nevertheless, all the encountered individuals seemed to be introduced from Egypt through the tunnel trade. Successful deliveries of the Indian Crested Porcupine were documented in Gaza zoological gardens as well (Figure 10). One funny and risky aspect of the animals, attracting the eyes and hearts of zoo visitors, is their defense strategy, where they are known to charge backwards towards visitors who disturbing them. Cages of Porcupines usually contain quills of different sizes and different developmental stages that may promote many zoo visitors to request them from zoo keepers.

Palestine Mole-rat Spalax leucodon ehrenbergi (Nehring 1898) The Palestine Mole-rat is a subterranean mammalian species having atrophied eyes. It is considered as an actual pest for farmers due to its continual feeding upon roots, bulbs and tubers of the agricultural crops. In open fields, it is very difficult to see the animal outside because it spends its life building tunnels and burrows. As a result, the earth heaps made by the Palestine Mole-rat are commonly seen by the Palestinians in a variety of agricultural and open fields. The powerful incisors of the animal are used as major tools for its digging behavior (Figure 11). Although it is unusual to find local people trapping or holding the Palestine Mole-rat, it was a good opportunity by the author to attend a striking event of a farmer bringing a live specimen to a zoological garden in North Gaza. Zoo keepers find it is impossible to cage live specimens of the species for long due to the animal’s digging behavior and ultimate death. In stead, as many as 3 specimens of the species were found stuffed in formalin solutions at few zoological gardens in Gaza. Cape Hare Lepus capensis (Linnaeus 1758) The Cape or Brown Hare is a common and well-known mammalian species throughout Palestine. It inhabits various ecological habitats extending from the rocky mountains and deserts to coastal plains. It feeds on a variety of plant forms extending from grasses to shrubs. Currently, the Cape Hare is facing the danger of disappearance in the Gaza Strip due to over-hunting, overpopulation and habitat destruction and modification. The use of ground mist nets, leghold metal traps and sometimes spare guns to hunt the Cape Hare is a common practice in the Gaza Strip. Locals claimed that the Cape Hare is mainly hunted for its delicious meat. Other causes of hunting including game, sport and trade could not be excluded as well. Single Cape Hares are occasionally seen by the Palestinian community in vegetated areas and sand dunes of the Gaza Strip. Live specimens of the Cape Hare have never been encountered by the author in Gaza zoological gardens. In contrast, many zoo owners claimed that Cape Hares (N = 4) were bought and caged in their zoological gardens, but they did not stay for long because of their ultimate death. Two specimens of the Cape Hare were found poorly stuffed at two of the Gaza zoological gardens. Dorcas Gazelle Gazella dorcas (Linnaeus 1758) Two species of Gazelles occur in Palestine; they are the Dorcas gazelle and the closely related Mountain Gazelle Gazella gazella. The Dorcas Gazelle is widespread in a different variety of ecological habitats in Palestine especially the arid and semi-arid environments. According to Qumsiyeh (1996), hunting and human activities have diminished the distribution of the species to desert and subdesert areas where the disturbance is negligible. Before the Israeli settlement evacuation from the Gaza Strip in late 2005, there were signs of the roaming of the Dorcas gazelle near the northern borders of the Gaza Strip with Israel, where many residual forests were present. As many as 8

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Figures: 3. The Golden Jackal Canis aureus; 4. The Grey Wolf Canis lupus; 5. The Red Fox Vulpus vulpus; 6. The Jungle Cat Felis chaus; 7. The Marbled Polecat Vormela peregusna; 8. The only sight of the Common Badger Meles meles was made in the Rafah private zoological garden before its demolition by the Israeli tanks and bulldozers (21 May 2004); 9. Caged individuals of the Greater Egyptian Gerbil Gerbillus pyramidis; 10. A caged juvenile Indian Crested Porcupine Hystrix indica; 11. The Palestine Mole-rat Spalax leucodon ehrenbergi; 12. The Dorcas Gazelle Gazella dorcas; 13. The Domestic Camel Camelus dromedarius ranging freely in the Gaza Strip; 14. The Long-eared Hedgehog Hemiechinus auritus

ABD RABOU â&#x20AC;&#x201C; The Palestinian mammalian in the Gaza zoological gardens

individuals of the Dorcas gazelle were encountered in Gaza zoological gardens (Figure 12). They were imported from Egypt through tunnel trade. They are usually kept in the same cages harboring the imported Red Deer Cervus elaphus (Linnaeus 1758), which exists in tens in Gaza zoological gardens. Dromedary Camel Camelus dromedarius (Linnaeus 1758) The Dromedary or Arabian Camel is no longer exists in the wild in Palestine and neighboring countries, as it was probably domesticated some 3500 years ago in the Arabia (Qumsiyeh 1996). It is a common scene for the Palestinians to find or watch herds of the Domestic Camels ranging freely in the Gaza Strip (Figure 13). In Gaza zoological gardens as well as in the beach belt of the Gaza Strip, the Dromedary Camel is commonly seen used as a recreation facility and a source of income for many subsistent Bedouin families. The meat and milk are consumed on a large scale especially among the Bedouin families inhabiting the Gaza Strip and the Negev desert of Palestine. The milk of Camels is said to have many healthful properties and as a result it is commonly used as a cultural cure for different illnesses. As many as 11 individuals of the Dromedary Camel were encountered in Gaza zoological gardens. Along with the Dromedary Camel, many zoological gardens were found to import â&#x20AC;&#x201C; through the tunnel trade â&#x20AC;&#x201C; the Llama Lama glama (Linnaeus 1758), which belongs to the Camelidae family, in order to enrich their zoo animals and at the same time to show the characteristics of this wonderful mammal which lacks the hump compared to the Dromedary Camel. Long-eared Hedgehog Hemiechinus auritus (Gmelin 1770) The Long-eared Hedgehog inhabits different agroecosystems in Palestine. Singles of this species are seldom encountered by the author in the vegetated areas of the Gaza Strip (Figure 14). What characterize this species, compared to the Ethiopian Hedgehog Paraechinus aethiopicus, are its relatively long ears and its relatively small size. The two Hedgehog species of the Gaza Strip are known to be sometimes eaten by some locals, especially the Bedouin families, living in the margins. Thus, the two species are falling under an actual threat. They are usually caught either using ground mist nets or easily by hands. As many as 5 live specimens of the Long-eared Hedgehog were encountered in Gaza zoological gardens. Two specimens were also seen preserved in formalin solution. Ethiopian Hedgehog Paraechinus aethiopicus (Ehrenberg 1832) The Ethiopian or Desert Hedgehog seems to resemble the Long-eared Hedgehog in terms of its living conditions, as it may live in desert areas as well. Only 4 live specimens were encountered in Gaza zoological gardens. Discussion Our study was built on an earlier work in which Abd Rabou (2005, 2009a) and Abd Rabou et al. (2007a) reported about 20 mammalian species, some of them are included in the present study. The present study revealed

that the Gaza Strip (an area rarely surveyed by zoologists) is similar to other areas of Palestine in having a significant number of mammalian fauna. The 17 species we report in Gaza zoological gardens represent a good mix of the families and sizes of mammals generally found in other parts of Palestine (Qumsiyeh 1996) and the other neighboring countries of the Middle East (Serhal 1985; Yom-Tov 1988; Nader 1990; Harrison and Bates 1991; Hatough and Disi 1991; Amr et al. 1996; Seddon et al. 1997; Mendelssohn and Yom-Tov 1999; Amr 2000; Hoath 2003; Masseti 2004, 2009). This comes in accordance with Boitani and Bartoli (1983) who stated that about 78 % of the mammalian families of the Palearctic realm are shared with the Ethiopian realm and 70 % with the Oriental realm. The surprising diversity including the nine carnivore species (a total number of 16 carnivore species occurs in Palestine as a geographically diverse area) of our study could be attributed to ecosystem and landscape diversity combined with human development that supports the occurrence of such species. Our study does raise serious questions about the future of the Gaza Strip. The Gaza Strip is located at the southern portion of the Palestine coast along the Mediterranean Sea and it harbors a variety of wildlife including terrestrial and aquatic forms as indicated by ongoing studies (Abd Rabou 2005, 2009a, 2011; Yassin et al. 2006; Abd Rabou et al. 2007a, b, c; and this study). The ever-increasing human impact on the existing natural resources in the Gaza Strip has caused the destruction of populations of many species including Gazelles, Porcupines, and large carnivores. There is strong evidence that the economic deprivation, political instability and lack of wildlife protection have made wildlife trading a common practice. With a per capita income for the minority with jobs (a mere 30% of the population) of less than U.S. $ 1000 per year, a trapped carnivore selling at U.S. $ 50-100 makes a compelling argument to families in crisis. Hunting of wild mammals for food in the Gaza Strip is restricted to Hares and Hedgehogs and does not involve carnivores, which are hunted for other reasons (Abd Rabou 2005, 2009a; Abd Rabou et al. 2007a). Poisoning and poaching of mammals and other wildlife species by the Palestinian farmers and ranchers trying to protect their income is a common practice. In few field visit, dead Jackals (N = 1) and Egyptian Mongooses (N = 6) in addition to a considerable number of Domestic or Feral Cats Felis catus were noted by the author. Most of these dead animals were considered as vertebrate pests causing harm to livestock and husbandry animals and as a result, they were probably poisoned or killed by farmers and local people. The same situation was recorded in a neighboring country like Jordan where Abu Baker et al. (2003) pointed out that many mammalian fauna including the Jungle Cat, Red Fox and Wild Boar Sus scrofa (Linnaeus 1758) were regarded as vertebrate pests and they were found to be poisoned by the Jordanian villagers and farmers because of their attack to livestock and fruit trees. In Israel, the Golden Jackal Canis aureus was poisoned due to its predation on livestock (Yom-Tov et al. 1995).

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The lack and poor implementation of environmental laws and legislations in the Occupied Palestinian Territories was reflected on practicing wildlife hunting in the Gaza Strip. The situation concerning wildlife protection in the Gaza Strip is deteriorating day by day in an alarming fashion, where the populations of some wildlife species could be declined to levels that species may go extinct. In Israel, all wildlife species have legal protection, though illegal hunting and poaching of mammals and other wildlife elements were documented to be practiced by Thai workers for food purposes (Yom-Tov 2003). The withdrawal of the Israeli army and its settlements from the Gaza Strip in August 2005 further exacerbated the problem because of accessibility to the 40% of the land mass of Gaza that was previously forbidden to Palestinians. Nowadays, the situation is still risky especially near the borders with Israel; many hunters were mistaken for resistance fighters and killed (Personal Communications and Local Media Reports). The establishment of many zoological gardens in the Gaza Strip in the last few years was also directly related to the isolation of the Gaza Strip (zoological gardens provide a venue for recreational activities and a revenue for their owners). Combined with poor resources to take care of and feed existing captured animals, this further promoted more wildlife hunting. The import of zoo animals; particularly mammals, through the tunnels joining the Gaza Strip with the Egyptian territories could be justified because of the Israeli blockade imposed on the Gaza Strip since 2006, where all crossing points of the Gaza Strip with Israel and Egypt are tightly closed (Abd Rabou 2009b). The political, economic and psychological pressures imposed on the Palestinians living in the Gaza Strip during the ongoing Israeli blockade era promoted the establishment of private zoological gardens. From a recreational point of view, the local supply of zoological gardens with wildlife species hunted in the Gaza Strip can not satisfy the desire of the Palestinians to know more about wild animals. Hence, such a tunnel trade was necessary to close the gap through importing key mammals from outside. In addition to the import of the mammalian species that have a wider distributional range through Palestine and the neighboring Middle East and North Africa countries, other non-Palestinian mammals have been introduced. These included the Lion Panthera leo, Tiger Panthera tigris, Spotted Hyena Crocuta crocuta, Fennec Fox Vulpes zerda, Savanna Monkey Cercopithecus aethiop, Llama Lama glama, Red Deer Cervus elaphus and many others. The veterinary treatment of zoo mammals in the Gaza Strip is poorly applied, and as a result, many individual mammals have passed away during the last four years. The health status of zoo animals usually varies with different factors such as management, feeding, environment, sanitation and seasonal variation (Shrikhande et al. 2008). In the light of this fact, the one capital point should be taken into consideration by zoo keepers regarding better zoo management is that zoo animals; particularly mammals, need to be housed according to the species social system. The quality of the nutrition and housing provided for the mammals at the Gaza zoological gardens

was in part reflected by the good general conditions of the caged mammalian fauna. In the Republic of Yemen, De Haas van Dorsser et al. (2003) stated that animals of the Sanaâ&#x20AC;&#x2122;a and Taiâ&#x20AC;&#x2122;z zoological gardens had reasonable diets and housing, but overstocking, poor handing and lack of preventive medicine were found to compromise the welfare of these zoo animals. Besides, deliveries among Palestinian and non-Palestinian mammals were recorded in Gaza zoological gardens e.g. Golden Jackals, Lions, Savanna Monkeys and Red Deer. Such deliveries are considered as an important source of zoo animals to the other zoological gardens in the Gaza Strip. Scientific research on the developmental stages and the breeding behavior of certain zoo mammalian species could be achieved as well. From an educational point of view, zoological gardens were known as important sources of biological knowledge. They played an important role in the development of descriptive biological sciences in the 19th Century (Prince 2001). This may be translated in advanced research in Gaza zoological gardens concerning the biology, ecology and behavior of wildlife.

CONCLUSION In conclusion, common political, economic and social factors in addition to the poor implementation of environmental laws and legislations promoted wildlife hunting and trading in the Gaza Strip. Although, this could be considered as a negative sign by scientific parties, especially ecologists and conservationists, such a hunting of key mammalian species could be a good tool in enriching Gaza zoological gardens with the demanded zoo animals. This could be rationalized for Gazans who are living under conditions having no similarities in other parts of the world. This is simply because of the Israeli occupation who spared no effort to destroy the total Palestinian environment under the claimed umbrella of security needs of the Israel State. In spite of this painful dilemma, the author recommends to improving the management process of Gaza zoological gardens under the care of the governmental authorities and the cooperation of the different parties in the Gaza Strip to enhance public ecological awareness to protect and conserve wildlife; especially mammalian fauna.

ACKNOWLEDGEMENTS The author would like to thank Prof. Dr. Mohammad R. Al-Agha (Department of Environment and Earth Sciences, Faculty of Science, Islamic University of Gaza, Palestine) who is now the Minister of Agriculture for his valuable inputs to the manuscript of this modest study. My deepest thanks go to zoo owners who made my times in Gaza zoological gardens enjoyable and my work could not have been fruitful without their company and assistance. The thanks reach local wildlife hunters and local people of the Gaza Strip who spared no effort in enriching the current study with the information needed.

ABD RABOU – The Palestinian mammalian in the Gaza zoological gardens

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ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 92-97 July 2011

Six hitherto unreported Basidiomycetic macrofungi from Kashmir Himalayas SHAUKET AHMED PALA♥, ABDUL HAMID WANI♥♥, MOHMAD YAQUB BHAT Section of Mycology and Plant Pathology, Department of Botany, Faculty of Biological Sciences, University of Kashmir, Hazratbal, Srinagar 190006, Jammu and Kashmir , India. Tel.: +99- 9419010336; Fax.: +99-942421357; email: ♥sapala29@gmail.com, ♥♥ahamidwani@yahoo.com

Manuscript received: 20 June 2011. Revision accepted: 7 July 2011.

ABSTRACT Pala SA, Wani AH, Bhat MY. 2011. Six hitherto unreported Basidiomycetic macrofungi from Kashmir Himalayas. Nusantara Bioscience 3: 92-97. The Kashmir valley located in the north extreme of the India lies between 33020’ and 34054’ N latitude and 730 55’ and 75035’ E longitude. The forests constituting more than 20% of the geographical area harbors diverse macrofungal species due to their wide variability in climate altitude and nature of species constituting them. The mushroom flora of the Kashmir Valley has not been documented completely until now. In this backdrop, a systematic survey for exploration and inventorization of macrofungal species of Western Kashmir Himalaya was undertaken during the year 2009-2010. During the study six species viz. Agrocybe molesta, Coprinus plicatilis, Inonotus hispidus, Paxillus involutus, Psathyrella candolleana and Russula fragilis were identified first time from the Kashmir. Key words: Kashmir Himalayas, wild macrofungi, edible, medicinal.

Pala SA, Wani AH, Bhat MY. 2011. Enam makrofungi Basidiomycetes yang sampai sekarang belum dilaporkan dari Kashmir Himalaya. Nusantara Bioscience 3: 92-97. Lembah Kashmir terletak di ujung utara India, antara 33o20' dan 34o54' LU, serta 73o55' dan 75o35' BT. Hutan di kawasan ini mencakup lebih dari 20% luas wilayah geografis, menjadi tempat tinggal beragam spesies makrofungi karena sangat beragamnya iklim di ketinggian dan sifat alamiah dari spesies-spesies penusunnya. Flora jamur di Lembah Kashmir belum pernah didokumentasikan secara lengkap sampai sekarang. Oleh karena itu, sebuah survei sistematika untuk mengeksplorasi dan menginventarisasi spesies macrofungi dari bagian barat Kashmir Himalaya dilakukan selama tahun 2009-2010. Selama penelitian, enam spesies yaitu: Agrocybe molesta, Coprinus plicatilis, Inonotus hispidus, Paxillus involutus, Psathyrella candolleana dan Russula fragilis diidentifikasi untuk pertama kalinya dari Kashmir. Kata kunci: Kashmir Himalaya, macrofungi liar, dimakan, obat.

INTRODUCTION Mushrooms have been fascinating man due to their unusual characters like sudden appearance in isolated places in groups, rings and in different geometrical shapes since the time immemorial. Mushrooms have been existing on earth prior to humans and has been used as food by man since the hunting and gathering period of human history (Cook 1977). Mushrooms belong to the kingdom fungi, which constitutes the most diverse group of organisms after insects on this biosphere. Defining the exact number of fungi on the earth has always been a point of discussion and several studies have been focused on enumerating the world’s fungal diversity (Crous 2006). The number of existing fungi Worldwide has been estimated to 1.5 million species (Hawksworth 2004) and about 15,000 of them are mushrooms of which about 7,000 are known to possess varying degrees of edibility and more than 3,000 species may considered prime edible and 2,000 species have been

suggested having medicinal importance (Chang and Miles 2004). Only a fraction of total fungal wealth has been subjected to scientific scrutiny and mycologists continue to unravel the unexplored and hidden wealth, as many macrofungi are becoming extinct or facing threat of extinction because of habitat destruction and global climate change (Swapana et al. 2008). Jammu and Kashmir, possess a prime place in the variety and galaxy of macro-fungi due to wide agroclimatic variations, diverse physiography and undulating topography, but understanding of the macro-fungal flora of the Kashmir is still in an exploratory or pioneer stage and undoubtfully there are many more species to be recorded (Watling and Abrahim 1992). Watling and Gregony (1980) recorded 119 taxa of macro-fungi from Kashmir. The list has been extended to 145 species (Beig et al. 2008), 150 species (Dar et al. 2009a) from Kashmir and 250 from whole Jammu and Kashmir state (Dar et al. 2009b). Four new species viz. Russula aurea, Russula atropurpurea, Suiillus variegates and Boletus rhodoxanthus has been

PALA et al. â&#x20AC;&#x201C; Basidiomycetiic macrofungi of Western Kashhmir Himalaya

aadded to the list (Dar et al. 2010). Wani W et al. (22010) from r reported nine species of morels m and pseudomorels p S Southern Kashhmir Himalayyas. The preseent communiccation d describes the general disstribution, brief morpholoogical d description, m macro and miccroscopic detaails and edibility of s newly repported speciess of macro-fuungi from Kasshmir six H Himalayas. M MATERIALS S AND METH HODS Regular fieeld trips weree carried to diifferent placess/sites o Western Kaashmir Himallayas of Jamm of mu and Kashm mir

9 93

Statee, India, nam mely: Uri, Guulmarg (Baraamula district ), Yusm marg, Doodhppathri, Chadooora (Budgam m district) annd Kellaar (Pulwamaa district) forrests represen nting differennt habittats like conniferous foressts, deciduou us forests annd grassslands (Figurre 1). These field trips were w organizeed acco ording to the method giveen by Halling (1996). Thhe sporo ocarps were carefully uprrooted by dig gging them ouut from m the soil withh the help of a fork and weere individuallly wrap pped in aluminnum foil and bbrought to thee laboratory foor furth her studies. Standard S methhod of collectiion, preservation, macro annd micrroscopic studiies were folllowed (Kumaar et al. 19900; Atri,, et al. 2003)) and the shaape, size and color of fressh

5 6

Figure 1. Reseaarch sites in Western Kashmirr Himalayas of Jammu and Kaashmir State, Inndia. 1. Uri, 2. Gulmarg (Baraamula district), 3. F Y Yusmarg (Osem marg), 4. Doodhhpathri (Dudhpaathri), 5. Chadooora (Budgam district) d and 6. Kellar K (Pulwam ma district)

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specimen were recorded before collection and preservation. The spore prints were taken according to the guidelines given by Kuo (2001). The morphology of spores such as , such as shape and size of spores were recorded under the microscope. Reagents used for preparation of spore slides were 3% KOH, cotton blue, lactophenol and Melzer’s reagent. Photographs were taken in field using Cyber shot Sony 10.1 megapixel camera. The fungal specimens were also preserved in formalin for herbarium purposes and deposited in fungal collection of KASH Herbarium of Plant Taxonomy, Division of Botany, Faculty of Biological Sciences, University of Kashmir, India. The identification of the specimens was carried out by making use of taxonomic keys, field manuals (Lakhanpal 1996, 1997; Atri et al. 2000, 2003) and taking the help of mushroom experts like Prof. N.S Atri, Prof. T.N Lakhanpal, and Dr. R.C Upadhya (Indian Council of Agriculture Research, Himachal Pradesh, India). RESULTS AND DISCUSSION During the year 2009-2010 an extensive survey was carried out to different places/sites of Western Kashmir Himalaya to unravel the macrofungal wealth. In the survey a large number of macrofungi belonging to different taxa were witnessed, but six species were first time seen in the Kashmir. The macroscopic and microscopic studies along with photographs of these six species are given below: Agrocybe molesta (Lasch) Singer Synonym(s): Pholiota dura (Bolt. ex Fr.) Kummer, Agrocybe dura (Bolt. ex Fr.) Sing. English name: Bearded field cap Local name: Maedan haddur Description: Cap: 3-7 cm across, convex expanding to almost flat, creamy white to yellowish brown, sometimes developing cracks in age and often with whitish partial veil remnants on the margin. Gills: Adnate, pale at first latter becoming brown to dark brown or purple-brown. Stipe: 4-7 cm in length, 5-1 cm thick, cylindrical, smooth to finely hairy; white; with a thin ring (but the ring often disappears). Flesh: Thick, firm, whitish. Spores: Ovoidellipsoid, smooth, 9-12 x 6-8 um2; Spore print choclate brown. Habitat and habit: Saprobic, growing singly in grass at roadsides or in meadows. Season: Early spring to summer, occasional. Edibility: Inedible Site of collection: Kellar, Yusmarg Accession number: SH.KASH-28788 Coprinus plicatilis (Curt. & Fr.) Fr. Synonym(s): Parasola plicatilis (Curtis) Redhead, Vilgalys & Hopple English name: Fragile brittlegill Local name: Vangan haddur Description: Cap: 2-4 cm in diameter, initially ovoid but latter on flattens and appears like a small umbrella. The upper surface of the cap is characteristically sulcate

(characteristic grooves on the upper surface), and a depression in the centre. Color changes from grey brown to silver blue. Gills: Free, initially clay pink then grey, finally black, hardly deliquescing. Stipe: 2-5 cm in length, 0.2-0.3 cm thick, cylindrical, hollow internally and white in color. Spores: Elliptical, smooth, 7-10 x 5-6 um2; Spore print black. Habit and habitat: Saprobe, growing solitary in grass lawns. Season: Spring and rainy summer. Edibility: Edible Site of collection: Gulmarg, Kellar Accession number: SH.KASH-28781 Inonotus hispidus (Bull.) P. Karst. Synonym(s): Boletus velutinus With. Inonotus hirsutus (Scop.) Murrill, Polyporus hispidus (Bull.) Fr., Phaeoporus hispidus (Bull.) J. Schröt. English name: Pleated inkcap Local name: Neeji haddur Description: Fruiting body: 7-22 cm across, 4-8 cm thick, bracket or semicircle or kidney shaped, upper surface hairy, rust redish yellow in color, when mature an iron rust color, and when finally old and dead it becomes black. Pores: Circular to angular, small, 2-3 per mm, olive colored. Water drops coming out of the pores when fruiting body is young. Flesh: Thick, woody, reddish brown Spores: Subglobose, smooth, 9-12 x 7-10 um2; Spores print brown. Habit and habitat: Parasitic on apple and walnut trees; growing usually alone but occasionally fusing with others into overlapping groups. Infection occurs at a branch stub or pruning wound Season: Summer Edibility: Edible Site of collection: Chadoora, Doodhpathri Accession number: SH.KASH-28792 Paxillus involutus (Batsch) Fr. Synonym(s): Agaricus adscendibus Bolton, Agaricus contiguus Bull., Agaricus involutus Batsch, Omphalia involuta (Batsch) Gray English name: Shaggy bracket Local name: Chunth lashe Description: Cap: 5-11 cm wide, convex with a depression in the centre, inrolled margins, olive-brown in color, smooth, and sticky when wet. Gills: Decurrent, close, narrow, brownish yellow, darkens when bruised. Gills can be peeled easily from the cap. Stipe: 2-6 cm long, 1-2 cm thick, equal or tapers towards the base, smooth, cap colored. Flesh: Thick, yellowish brown darkens when bruised. Spores: Ellipsoid, smooth, 7-8 x 6-7 um2; Spore print brown Habit and habitat: Mycorrhizal, growing alone or scattered in hardwoods Season: Summer Edibility: Inedible Site of collection: Kellar Accession number: SH.KASH-28799

PALA et al. â&#x20AC;&#x201C; Basidiomycetic macrofungi of Western Kashmir Himalaya

Figure 1. Basidiomycetic macrofungi collected from wild in Western Kashmir. A. Agrocybe molesta, B. Coprinus plicatilis, C. Inonotus hispidus, D. Paxillus involutus, E. Psathyrella candolleana, F. Russula fragilis

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Psathyrella candolleana (Fr.) Maire Synonym(s): Agaricus appendiculatus Bull., Agaricus candolleanus Fr., Hypholoma candolleanum (Fr.) QuĂŠl., Psathyra candolleana (Fr.) G. Bertrand, Psathyrella microlepidota Orton English name: Poison pax Local name: Zaher haddur Description: Cap: 3-7 cm diameter, rounded-conical when young, but broadly convex to flat at maturity, light brown initially with scattered small white scales, when young margin adorned with hanging veil remnants. Margins often split at maturity. Gills: Adenate to nearly free, crowded, whitish at first, later grayish or grayish purple and finally dark brown. Stipe: 4-11 cm long, 2-6 mm thick, equal, hollow, fragile, white, without any ring. Flesh: White, vrey thin, fragile Spore: Ellipsoid to ovoid, smooth, nonamloid, with an apical germ pore, 6-9 x 4-5 um2; Spore print dark brown. Habit and habitat: Saprobic; growing scattered or gregariously in lawns or pastures, on recently dead hardwood trees, their roots, stumps or debris. Season: Late spring and early autumn. Edibility: Edible. Site of collection: Kellar, Uri Accession number: SH.KASH-28784 Russula fragilis (Pers.) Fr. Synonym(s): Agaricus fragilis L., Bolbitius vitellinus (L.) J. Favre English name: Pale brittlestem Local name: Veri haddur Description: Cap: 3-7 cm in diameter, initially convex but becomes flat with a depression in the centre at maturity, color purplish with a dark centre but sometimes color fades with rain. The cuticle can be easily peeled and the margins of the cap split at maturity. Gills: Annexed, moderately spaced, white in color but changes color on bruising. Stipe: 4-6 in length, 0.7-1.5 cm thick, cylindrical or club shaped centrally attached with cap, fragile and white in color. Flesh: white and fragile. Spores: Spherical but echinate with 7.5-9 x 6-8 um2 in size; Spore print is white. Habit and habitat: Ectomycorrhizal, generally scattered in both coniferous and broad leaved trees. Season: Summer Edibility: Inedible Site of collection: Chadoora, Chrarishrief Accession number: SH.KASH-28801 Discussion Several authors have described the taxonomy of mushrooms from various regions of the world but an analysis reveals that 60% of the newly described fungi are from tropics including mushrooms and up to 55% of the mushroom species have proved to be undescribed (Hawksworth 2001). Except a dozen of species cultivated on large scale, all the macrofungal species grow in natural habitat and their harvest is being undertaken for the benefit in different countries including India. Nowadays anthropogenic activity has made countries all over the world to show serious concern about the dwindling

biodiversity being last at the rate never known before. The 1991 red list for the former republic of Germany for instance lists 1,037 species of threatened large fungi constituting 35% of the all the larger fungi (Cherfas 1991). Therefore, exploration, systematics and conservation of wild mushrooms have received more attention in the present day world. Different researchers have contributed to the study of mushroom flora of Himalayas and have reported more than 250 species from the Himalayan state Jammu and Kashmir (Abrahim and Kaul 1985, 1988; Abrahim and Kachroo 1989; Watling and Abrahim 1986, 1992; Lakhanpal 1996, 1997; Atri et al. 2000; Beigh 2008; Dar et al. 2009a,b, 2010; Wani et al. 2010). However, the ecological data available on some of the genera is still not enough. Watling and Abrahim (1992) reported 77 mycorrhizal mushroom species containing many species of Russula genus from different regions of the Kashmir. Dar et al. (2009a,b, 2010) reported many species of genus Russula from Kashmir. The six species of macrofungi mentioned above has been already reported from other parts of the world and are known to possess varying degrees nutritional and medicinal values. Agocybe molesta an edible species of macrofungi is widely distributed in U.S.A and other parts of the world (Kuo 2006) and is known to have valuable antioxidant potential. Coprinus plicatilis an edible mushroom commonly growing in America, Europe and Asia is also known to possess medicinal importance (Boa 2004). P. candolleana possessantibacterial activity against various gram positive bacteria like Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium and Candida albicans (Colletto et al. 1999) and also posses antitumor properties (Ohtsuka et al. 1973). Although Innonotus ispidus, Paxillus involutus and R. fragilis are inedible but possess varying degrees of medicinal properties. Antivral, antibacterial, cytotoxic and anticancer propertyies has been observed in I. ispidus (Ali et al. 2003; Al-Fatimi 2005; Zan et al. 2011). P. involutus forming ectomycorrhizal relationships with a number of coniferous and deciduous tree species and has been found markedly increasing the resistance of the host plants to pathogenic strains of the Fusarium oxysporum (Duchesne et al. 1988). R. fragilis forms mycorrhizal relationship with a wide variety of plants is common in temperate zones of Asia, Europe and north America (Phillips 2006). It has been proved that R. fragilis had proteins with inherent antimicrobial properties against a number of pathogens (Hearst et al. 2010). CONCLUSION Since the wild fungi play an important role to maintain the health of forests besides their medicinal importance and nutritional value in most of the cases, therefore it becomes quite necessary to explore, document and conserve this natural wealth. The present communication reports the six species of macrofungus, viz. Agrocybe molesta (Lasch) Singer, Coprinus plicatilis (Curt. & Fr.) Fr., Innonotus

PALA et al. â&#x20AC;&#x201C; Basidiomycetic macrofungi of Western Kashmir Himalaya

hispidus (Bull.) P. Karst., Paxillus involutus (Batsch) Fr., Psathyrella candolleana (Fr.) Maire and Russula fragilis (Pers.) Fr., which are first time from the Kashmir.

ACKNOWLEDGEMENTS The authors are highly thankful to Prof. N.S Atri (Department of Botany, Punjabi University, Patiala, India) and Prof. T.N Lakhanpal (Department of Biosciences, Himachal Pradesh University, Shimla India.) for providing every kind of help in identification of these six species. REFERENCES Abrahim SP, Kachroo JL. 1989. Larger fungi from Kashmir, India VI: The genus Amanita. Mycologia Neotropica Aplicada 2: 41-51. Abrahim SP, Kaul TN. 1985. Larger fungi from Kashmir-III. Kavaka 13: 77-81. Abrahim SP, Kaul TN. 1988. Larger fungi from Kashmir-III. Mycologia Neotropica Aplicada 1: 55-70. Al-Fatimi M, Wurster M, Kreisel H, Lindequist U. 2005. Antimicrobial, cytotoxic and antioxidant activity of selected basidiomycetes from Yemen. Die Pharmazie, Intl J Pharm Sci 60 (10): 776-780. Ali NA, Mothana RA, Lesnau A, Pilgrim H, Lindequist U. 2003. Antiviral activity of Inonotus hispidus. Fitoterpia 74 (4): 483-485. Atri NS, Kaur A, Kaur H. 2003. Wild mushrooms collection and identification. Mushroom Res 14: 56-59. Atri NS, Kaur A, Saini SS. 2000. Taxonomic studies on Agaricus from Punjab plains. Indian J Mushroom 18: 6-14. Beigh MA, Dar GH, Ganai NA, Khan NA. 2008. Mycorrhizal biodiversity in Kashmir forests and some new records of macro-fungi from J & K state. Appl Biol Res 10: 26-30. Boa ER. 2004. Wild edible fungi: A global overview of their use and importance. Non-Wood Forest Products 17. FAO, Rome. Chang ST, Miles PG. 2004. Mushrooms: Cultivation, nutritional value, medicinal effect, and environmental impact. 2nd ed. CRC Press, New York. Cherfas J. 1991. Disappearing mushrooms: Another mass extinction. Science 254: 1458. Cook RC. 1977. Fungi, man and his environment. Longman, London. Crous PW. 2006. How many species of fungi are there in tip of Africa. Stud Mycol 55: 13. Dar GH, Beig MA, Ganai NA. 2009a. Hitherto unrecorded macro-fungi from India. Appl Biol Res 11 (2): 59-62.

Dar GH, Beig MA, Qazi NA, Ganai NA. 2009b. Hitherto unreported Agaricales from Jammu and Kashmir. J Mycol Pl Pathol 39 (1): 3537. Dar GH, Ganai NA, Beigh MA, Ahanger FA,, Sofi TA. 2010. Biodiversity of macro-fungi from conifer dominated forests of Kashmir, India. J Mycol Pl Pathol 40 (2): 169-171. Duchesne LC, Peterson RL, Ellis BE (1988). Pine root exudates stimulates the synthesis of antifungal compounds by the ectomycorrhizal fungus Paxillus involutus. New Phytol 108 (4): 471-76. Halling RE. 1996. Recommendations for collecting mushrooms for scientific study. In: Alexiades MN, Sheldon JW (eds.) Selected guidelines for ethnobotinical research: A field manual. New York Botanical Garden Press, Bronx. Hawksworth DL. 2001. The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycol Res 105: 1422-32. Hawksworth DL. 2004. Fungal diversity and its implifications for genetic resource collections. Stud Mycol 50: 19. Hearst M, Nelson D, McCollum G, Ballard LM, Millar C, Moore M, McClean S, Moore JE, Rao J. 2010. Antimicrobial properties of protein extracts from wild mushroom fungi and native plant species against hospital pathogens. J Pharmacog Phytother 2 (8) :103-107. Kumar A, Bhatt RP, Lakhanpal TN. 1990. The Amanitaceae of India. Bishan Singh, Mahendra Pal Singh, Dheradun, India. Kuo M. 2001. Making spore prints. www.bluewillopages.com/ mushroomexpert/herbarium.html. Kuo M. 2006. Agrocybe molesta. www.mushroomexpert.com/ agrocybe_molesta.html. Lakhanpal TN. 1996. Mushrooms of Indian Boletaceae. In: Mukherji KG (ed). Studies in Cryptogamic Botany. Vol. I. APH Publishing Corp. New Delhi. Lakhanpal TN. 1997. Diversity of mushroom mycoflora in the NorthWest Himalaya. In: Sati SC, Saxena J, Dubey RC (eds) Recent researches in ecology, environment and pollution. Today and Tomorrowâ&#x20AC;&#x2122;s, New Delhi. Ohtsuka S, Ueno S, Yoshikumi C, Hirose, F, Ohmura Y, Wada T, Fujii T, Takahaashi E. 1973. Polysaccharides having an anticarcinogenic effect and method of producing them from species of Basidiomycetes. UK Patent 1331513. Phillips R. 2006. Mushrooms. Pan MacMillan, London. Swapana S, Syed A, Krishnappa M. 2008. Diversity of macrofungi in semi evergreen and moist deciduous forests of Shimoga District, Karnatka, India. J Mycol Pl Pathol 38 (1):21-26 Walting,R, Gregory NM. 1980. Larger fungi from Kashmir. Nova Hedwigia 32:494-564. Wani AH, Pala SA, Boda RH, Mir RA. 2010. Morels in Southern Kashmir Himalaya. J. Mycol Pl. Pathol 40 (4):540-546. Watling R, Abrahim SP. 1986. Bolbitaceae of Kashmir with special reference to the genus Agrocybe . Nova Hedwigia 42: 387-415. Watling R, Abrahim SP. 1992. Ectomycorrhizal fungi of Kashmir Forests. Mycorrhiza 2:81-87. Zan LF, Qin JC, Zhang YM, Yao YH, Bao HY, Li X. 2011. Antioxidant hispidin derivatives from medicinal mushroom Inonotus hispidus. Chem Pharm Bull 59 (6):770-76.

ISSN: 2087-3948 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 2, Pp. 98-103 July 2011

The knowledge of Bengkulu University’s forestry students of tree diversity in their campus WIRYONO1,♥, STEFFANIE NURLIANA2,♥♥ 1

Department of Forestry, Faculty of Agriculture, University of Bengkulu. Jl. Raya Kandang Limun, Bengkulu 38371A, Bengkulu, Indonesia. Tel.: +62736-21170; Fax.: +62-736-21290; ♥email: wiryonogood@yahoo.com. 2 Department of Biology, Faculty of Mathematics and Natural Sciences, University of Bengkulu, Bengkulu 38371A, Bengkulu, Indonesia. ♥♥email: steffanienurliana@yahoo.com Manuscript received: 8 June 2011. Revision accepted: 25 June 2011.

Abstract. Wiryono, Nurliana S. 2011. The knowledge of Bengkulu University’s forestry students of tree diversity in their campus. Nusantara Bioscience 3: 98-103. Indonesia is rich in plant diversity which has provided daily human needs for millennia. Knowledge of diverse plants and their uses is part of ecological knowledge essential for the survival of human. However, rapid deforestation has reduced plant diversity and caused the loss of traditional ecological knowledge. Furthermore, the increased availability of electronic entertainment has alienated young people from nature, causing further loss of ecological knowledge. The objective of this study was to know the ability of Bengkulu University’s forestry students to identify trees growing in the campus by local names and their genera. Knowing the name of trees growing in our environment is an indicator of concern for biodiversity. Results showed that forestry students had low ability to identify trees by local names and even lower by genera. Second-semester students could identify fewer trees than the higher-semester students, and the knowledge was not affected by student’s gender or profession of students’ parents. This low appreciation of plant diversity among young generation will have negative implication for biodiversity conservation efforts. Students should be brought closer to nature by increasing outdoor education. Key words: concern for biodiversity, botanical knowledge, forestry students.

Abstrak. Wiryono, Nurliana S. 2011. Pengetahuan mahasiswa kehutanan Universitas Bengkulu terhadap keragaman pohon di kampusnya. Nusantara Bioscience 3: 98-103. Indonesia kaya akan keanekaragaman tumbuhan yang telah memenuhi kebutuhan manusia sehari-hari selama ribuan tahun. Pengetahuan tentang tumbuhan yang beragam dan kegunaan mereka adalah bagian dari pengetahuan ekologi penting untuk kelangsungan hidup manusia. Namun, deforestasi yang cepat telah mengurangi keanekaragaman tumbuhan dan menyebabkan hilangnya pengetahuan ekologi tradisional. Selanjutnya, peningkatan ketersediaan hiburan elektronik telah mengasingkan kaum muda dari alam, menyebabkan hilangnya pengetahuan ekologi lebih banyak lagi. Tujuan penelitian ini adalah untuk mengetahui kemampuan mahasiswa kehutanan Universitas Bengkulu untuk mengidentifikasi pohon yang tumbuh di kampus dengan nama lokal dan genus. Mengetahui nama pohon yang tumbuh di lingkungan merupakan indikator kepedulian terhadap keanekaragaman hayati. Hasil penelitian menunjukkan bahwa mahasiswa kehutanan memiliki kemampuan yang rendah untuk mengidentifikasi pohon dengan nama lokal dan bahkan lebih rendah lagi dengan nama genus. Mahasiswa semester kedua dapat mengidentifikasi pohon lebih sedikit dibanding mahasiswa dengan semester yang lebih tinggi, dan pengetahuan itu tidak terpengaruh oleh jenis kelamin atau profesi orang tua. Hal ini menunjukkan rendahnya apresiasi keanekaragaman tumbuhan di kalangan generasi muda yang akan memiliki implikasi negatif bagi upaya konservasi keanekaragaman hayati. Mahasiswa harus dibawa lebih dekat dengan alam dengan meningkatkan pendidikan di luar ruangan. Kata kunci: kepedulian terhadap keanekaragaman hayati, botani pengetahuan, mahasiswa kehutanan.

INTRODUCTION Having vast tropical rain forest, Indonesia is rich in plant diversity which provides economic, ecological and cultural benefits to human. People in rural areas have good knowledge of local plants and utilize them to fulfill their daily need. Rural communities in Kandang Village, Bengkulu, used 113 species (Sunesi and Wiryono 2007), in Enggano Island, Bengkulu, 99 species (Arianto 2008), in villages near Gunung Halimun National Park, West Java 243 species (Rahayu and Hirada 2004), in Kabaena Island, Central Sulawesi 65 species (Rahayu and Rugayah 2010).

Our plant diversity, however, is threatened by rapid deforestation occurring in Indonesia with a rate between one and two million hectares per year (FWI/GFW 2001; Mas’ud et al. 2007). In the last 30 years, much of speciesrich tropical rain forests outside Java have been replaced by monoculture plantations. The loss of natural forest in the tropic has not only reduced plant diversity but also caused the loss of people’s knowledge of plants and their uses (Ramires 2007). The knowledge of plants and their uses is an essential part of ecological knowledge which is acquired by societies through long and intensive interaction with nature in search for food and other needs (Pilgrim et al. 2008). For millennia human has relied on plant diversity

WIRYONO & NURLIANA – The knowledge of forestry students to tree diversity

for fulfilling their daily need, so the loss of plant diversity and the consequent loss of ecological knowledge threats the survival of human (Aiona et al. 2007). Conserving plant diversity and local knowledge of plant uses is, therefore, essential for the survival of human. Conservation of plant diversity, however, will not succeed unless people appreciate plant diversity. Unfortunately, economic development has a negative impact of reducing direct contact between people and nature, resulting in lower appreciation of plant diversity. A study in South Sulawesi showed negative correlation between income and knowledge of plant uses among villagers (Pilgrim et al., 2007). Furthermore, the increased availability of electronic entertainment in developed countries has shifted the love of nature among the people into the love of electronic entertainment (Pergams and Zaradic 2006, 2008). The low familiarity with nature among young generation is reflected in the low ability of students to identify plants in their surrounding (Wagner 2008; O’Brien 2010). The alienation of young students from nature may also occur in Indonesia because of the increased accessibility to electronic entertainment and the disappearance of natural vegetation. The objective of this study was to know the ability of Bengkulu University’s forestry students to identify trees in the campus. Their knowledge of tree names in their environment is an indicator of their concern for plant diversity, a prerequisite for the success of biodiversity conservation efforts.

MATERIALS AND METHODS Site study This study was conducted in May, 2011 in the campus of University of Bengkulu, in Bengkulu City, Indonesia. This year, University of Bengkulu’s campus ranked fourth as the best green campus in Indonesia. More than one hundred species of trees are found in the campus (Arianto and Susatya 2009). Some trees are native species growing naturally, but many more are introduced species artificially planted in managed landscape. Respondents Eighty three forestry students of Bengkulu University (50% of all forestry students) who were available during the period of study were interviewed to identify photographs of 50 species of trees found in the campus of Bengkulu University. The use of photographs to test the knowledge of plant names have been done in other studies (Setalaphruk and Price 2007; Pilgrim et al. 2008). The respondents consisted of male and female students, between 19 and 23 years old. They came from Bengkulu and the surrounding provinces, and only one came from Java. Selection of tree species The selection of tree species was based on their abundance and frequency either in the university campus or Bengkulu city. Most of the selected species are abundant or

frequently found. There was an exception, though. Neem tree (Azadirachta indica) is not abundant and only occasionally found, but it was selected because it is often used as traditional herbal medicine for malaria, a prevalent disease in Bengkulu. The selected species are not all indigenous in Bengkulu or even in Indonesia, but most of them have been grown in Indonesia for centuries. For example, Mangifera indica was originally from IndoBurma and introduced to many South East Asia countries 1500 years ago (211.114.21.20/tropicalplant/index.jsp). The objective of this study was not to test the students’ knowledge of indigenous species but to know their concern for plant diversity as indicated by their ability to identify trees in their environment. If students are not interested in plants we can assume that they are not interested in traditional ecology either. Exotic conifers were not selected because Indonesians call them cemara (casuarinas). Only Casuarina equisetifolia was selected because it is an ubiquitous and abundant species in Bengkulu city’s beach forest, the most well known tourist destination in Bengkulu city. Of the closely related species that have similar Indonesian names, only one was selected. For example, Michelia alba (white cempaka) and Michelia champaca (yellow cempaka) were represented by M. alba, while Acacia mangium (broad leaf akasia) and Acacia auriculiformis (narrow leaf akasia) were represented by Acacia mangium because M. alba and A. mangium are more abundant than their closely related species. Data collection Each selected species was photographed, showing its easily recognizable features, and in some cases its location in the campus. During interview most students recognized where the trees are located. Several photos were downloaded from the internet (www.natureloveyou.sg and www.hear.org/starr/images/?o=plants) to provide better pictures. To ensure that the photographs were recognizable, pictures of each species were shown to several faculty members who know the species before they were used during interview. Then, the pictures were put in an album to be shown to respondents. Each species was represented by two or more photos, except for coconut (Cocos nucifera) and mast tree (Polyalthia longifolia), each of which was represented only by one photograph because of their distinctive architectures. Each respondent was asked to mention the local name and the genus of each species. For Michelia alba the respondent was considered correct if he or she mentioned just cempaka and for Acacia mangium, mentioning akasia was considered correct. The question on genera was asked because forestry students from the second semester have taken botanical courses and are expected to be familiar with scientific names. Data analyses Data were tabulated, and the mean and standard deviation were calculated based on gender, length of study and profession of parents. The percentage of students correctly identifying the trees by their local names and genera were also calculated.

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RESULTS AND DISCUSSION The ability of students to identify trees University of Bengkulu’s forestry students could identify between 10 to 40 tree species (out of 50) by their local names, with an average of 24.7. The ability of students to identify the genera of trees was much lower, ranging from 0 to 16 genera with an average of 5.6. The ability to identify trees varied greatly among students from the same semester, same gender and parent’s profession as shown by the relatively large number of standard deviation compared to the mean (Tables 1). No statistical test was conducted, but the data showed that the second semester students could identify fewer trees than those of the higher semesters, both by their local names and by their genera (Table 1). Table 1. The ability of students to identify trees correctly, based on length of study, gender, and profession of students’ parents The average number and SD of trees identified correctly by students By Local names By Genera Length of study (semester) Second Fourth Sixth Eighth Tenth or higher

18.9 + 5.6 24.1 + 4.8 28.4 + 7.3 26.9 + 4.4 28.7 + 5.1

1.4 + 1.3 4.6 + 3.0 6.5 + 5.9 9.6 + 4.0 8.3 + 3.9

Gender Male Female

25.4 + 7.4 23.6 + 5.0

6.0 + 5.3 5.0 + 3.7

Profession of students’ parents Government officials* 24.6 + 6.5 6.5 + 5.2 4.0 + 3.6 Farmers 24.0 + 7.2 Entrepreneurs 26.2 + 6.8 6.2 + 5.1 Note: *This category consisted mostly of civil servants, but also included three persons who were a policeman, a soldier and a retired person.

It is understandable that the second-semester students knew fewer tree species than their seniors because the seniors had taken more courses that require tree identification in the field such as dendrology (a course that trains student in tree identification), forest ecology, forest inventory and silviculture. But it is disappointing that even senior students could identify, on the average, only less than 60% of trees by their local names, and only 12% by their genera. They had taken field works requiring tree identification and, in several occasions, were involved in planting and maintenance of the campus trees. Their relatively low ability to identify trees suggests that they have little interest in studying tree names in their environment although during interviewed, 60% of respondents said so. Most of them recognized the trees in the photos which they could not identify the name. With the increase of computer availability and internet access in campus, students may spend more time with the computer than interacting with trees in the campus’ park (Table 2). In the U.S. and Japan, there was evidence for a fundamental and pervasive shift away from nature-based recreation, most likely caused by the increase of electronic

entertainment (Pergams and Zaradic 2008). In general, technologically oriented societies has drastically lost practical knowledge of nature (Atran et al. 2004). The result of this study is similar to those in similar studies in the United States. Wagner (2008) found that college students in South Carolina had little ability to name plant species in their environment. In another study, Atran et al. (2004) found that American students from Northwestern University identified tree and bird species only at the life-form level (‘tree’, ‘bird’), while people of Itza’ Maya, native to Guatemala who practice agriculture, hunting and fishing, could identify plant and animal species at more specific levels. Alienation from nature is one plausible reason for the low ability of young generation to identify trees in their environment. If interaction with nature remains high, the knowledge of plants among the youth can be maintained. In a small village in Thailand, where people still practiced hunting and gathering wild food, the children maintained ability to identify wild species of plants and animals used as food (Setalaphruk and Price 2007). In the US, a group of elementary school students could identify only 33.7 ± 6.8% of 60 plants presented in the slide show, but after short botanical activities outdoor, the same students could identify 55.3 ± 15.6% (Cooper 2008). Increased interaction with nature apparently increased the ability of those children to identify plant species. Another plausible reason for the low ability to identify trees among forestry students is the lack of field guides for tree identification. Serious books such as Backer and Bakhuizen van den Brink (1963), and van Steenis et al. (1981) are available in libraries, but these books are not easy to use. Good knowledge of plant morphology is needed to use these books. But this drawback can be overcome by the availability of websites providing photographs of plants and their names. Any student interested in trees may access these websites and will be able to identify many trees usually found in parks and streets. Profession of parents didn’t affect the ability of Bengkulu University’s forestry to identify tree species (Tables 1). It was assumed that students from farming background had better knowledge of plants. If the knowledge of plants is an indicator of intensity of interaction with nature, then the results of this study implied that students from farmer families did not experience more intensive with interaction with nature than students with other backgrounds. Another possible reason is that most farmers have monoculture plantation (rice, oil palm or rubber), so their children have little experience with various tree species. The knowledge of plants among forestry students was not affected by gender either. In the community of Tzotzil Maya in the Highlands of Chiapas, Mexico, women had better appreciation of tree species than men. Apparently the effect of ongoing cultural changes has led men, but not women, away from intimate contact with nature (Atran et al. 2004). In Way Kambas, Lampung, Sumatra, male respondents had better score in identifying wild life species than the females (Nylus et al. 2003). What affects knowledge of nature is certainly not gender itself, but the intensity of interaction with nature.

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Table 4. The percentage of trees correctly identified by students

Indonesian names

Kelapa Nangka Belimbing Durian Mangga Alpukat Rambutan Jengkol

Scientific names

Manggis Sirsat Kedondong Sawo Jati Akasia/ mangium Cemara laut

Cocos nucifera L. Artocarpus heterophyllus Lamk Averrhoa carambola L. Durio zibethinus Murr. Mangifera indica L. Persea americana Mill Nephelium lappaceum L. Pithecellobium jiringa (Jack.) Prain ex King Garcinia mangostana L. Anona muricata L. Spondias dulcis Soland ex Park. Achras zapota L. Tectona grandis L.f Acacia mangium Willd. Casuarina equisetifolia J.R.& G.Forst.

Kapok randu Pace, mengkudu Jarak pagar Lamtoro Jambu bol Blimbing wuluh/besi Melinjo Sengon

Percentages of students correctly identify Local Genera names 100 16 99 10 99 1 99 65 99 45 99 6 96 4 93 6 92 86 86 84 80 78 77

7 4 1 0 40 75 20

Ceiba pentandra (L.) Gaertn. Morinda citrifolia L. Jatropha curcas L. Leucaena leucocephala (Lamk) de Wit. Syzygium malaccense (L.) M. & P. Averrhoa bilimbi L. Gnetum gnemon L. Parasierianthes falcataria (L.) Nielsen

77 77 69 66 65 63 61 60

25 0 2 2 0 0 28 28

Jambu monyet/mete Beringin Kersen, cheri Mahoni Cempaka putih Sungkai Ketapang

Anacardium occidentale L. Ficus benjamina L. Muntingia calabura L. Swietenia macrophylla King Michelia alba D.C. Peronema canescens Jack. Terminalia catappa L.

60 54 51 51 47 46 43

0 29 0 27 19 19 23

Flamboyan Asam jawa Kalpataru Kayu gadis Kemiri Johar Pulai

Delonix regia (Bojor ex Hook.) Rafin Tamarindus indica L. Hura crepitans L. Cinnamomum parthenoxylon (Jack) Meissn Aleurites moluccana (L.) Willd. Cassia siamea Lamk. Alstonia scholaris (L.) R. Br.

35 34 29 25 20 20 19

12 1 2 13 2 2 14

Laban Trembesi Waru Glodogan tiang Angsana Kendidai Bunga tanjung Matoa Nilau Saga, adenanthera Balik angin Krei payung/ filisium Mimba

Vitex pinnata L. Samanea saman (Jacq.) Merr. Hibiscus tiliaceus L. Polyalthia longifolia (Sonnerat) Thwait. Pterocarpus indicus Willd. Bridelia monoica (Lour.) Merr. Mimusops elengi L. Pometia pinnata J.R. & G. Frost. Commersonia bartramia (L.) Merr Adenanthera pavonina L. Mallotus paniculatus (Lamk.) M.A. Filicium decipiens (W&A) Thwait. Azadirachta indica A.Juss.

7 6 6 5 4 4 2 2 2 1 1 0 0

6 1 2 0 7 0 0 0 1 0 0 0 0

Origin of species

Coastal regions of tropical Asia and Pacific Probably in Ghats, western India Not clear, either tropical America or South East Asia From Sri Lanka to New Guinea India Indo-Burma Central America Untracaeable South East Asia Maybe Malay Peninsula Tropical America From Melanesia through Polynesia South America India, Myanmar, Laos The Moluccas, New Guinea, Northern Australia South East Asia, northern, southern Australia, Melanesia, Polynesia Tropical America South East Asia Central America Central America Malaysia, Vietnam, Indonesia Not clear, either tropical America or South East Asia South East Asia, north to Assam, east to Fiji The Moluccas, New Guinea, the Bismarck Archipelago, Solomon Island South America South, South East Asia, Solomon Islands, Australia Tropical America Central and South America Cultivated in tropical and subtropical countries Indonesia, Malaysia India, South East Asia, Northern Australia, Polynesia Madagascar Maybe Africa America South East Asia Tropical Asia to Polynesia Burma and Thailand South Asia, South East Asia, Northern Australia, Solomon Islands South East Asia South America Tropical Asia and Africa India and Sri Lanka South East Asia, Northern Australia, pacific South east Asia Asia and Pacific South East Asia, Fiji, Samoa Malaysia, Indonesia, new Guinea, Australia South Asia, South east Asia, Solomon Islands South, South Eeast Asia, Northern Australia Sri Lanka Indo-Pakistan Subcontinent

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Identifiableness of trees Coconut (Cocos nucifera) was the most easily identified species by its local name. Although coconut was represented only by a photograph of the whole tree from a distance, all students correctly identified it by its Indonesian name. Some factors may be responsible for the high familiarity of students with coconut. First, this species is widely distributed across the country especially in lowland areas near the beach such as Bengkulu city. Second, its extremely large fruits are distinctive among palm trees’ fruits. Third, it is a versatile species. Almost all parts of this species have direct benefit to man. As most people hold anthropocentric view of nature, we can easily appreciate the value of a species if it has direct use values (Callicot 2005). Other species which could be identified by more than 80% of students were mostly fruit trees Personal experiences in handling and eating fruits enable students to identify fruit trees correctly. Eight three percents of respondents said they had experience of harvesting fruits. Non fruit tree species correctly identified by 80% respondents was teak (Tectona grandis). Although it is not native to Sumatra, teak has been widely planted in Sumatera and is mentioned in many forestry textbooks because this species produces high quality wood which can be used for many purposes (Soerianegara and Lemmens, 1994). Students could identify teak from its extremely large leaves and its architecture. No students could identify two species, neem tree (Azadirachta indica) and fern tree (Filicium decipiens). It is understandable that they could not identify A. indica because this species is not common in campus or in Bengkulu city, but it is disappointing that students didn’t able to identify Filicum either. Although it is not native to Indonesia, Filicium has been introduced to Indonesia for many decades as ornamental and shade trees (Backer and Bakhuizen van den Brink 1963, 1965, 1968) and has distinctive leaves. In University of Bengkulu’s campus, Filicium is abundant. Rain tree (Samanea saman) which is now favored by Indonesian President to be planted nation-wide and is found in great number in campus were identified only by 6 % students. Native to tropical America S. saman was introduced in Java in 1878 (Becker and van De Brink, 1963) and has been distributed across the country. Other species found very frequently in campus and along the main streets of Bengkulu city, angsana tree (Pterocarpus indicus) and Spanish cherry (Mimusops elengi), even got lower score, 4% and 2% respectively. Ironically, indigenous tree species, Bridelia monoica, Mallotus paniculatus and Commersonia bartramia, were identified only by less than 5% students. These three species are pioneers which grow naturally in open areas in the campus as well as outside. This data indicate that students have little interaction with natural vegetation. In the U.S., college students (Wagner 2008) and elementary students (Cooper 2008) could identify fewer wild plants than the planted ones in a managed landscape.

Implication for biodiversity conservation Knowing the names of plants is just the elementary level of ecological literacy. To survive in nature, a community must know more than just the names of plants but also their ecology, nutritional values, pharmaceutical values and other characteristics relevant to human needs. While old people in rural areas maintained good knowledge of local plants, the young generation who are alienated with nature may not inherit this essential knowledge. The loss of familiarity with nature will impair the community’s ability to interact with the environment sustainably (Atran et al. 2004). With rapid deforestation and other habitat degradation, it is imperative that we conserve biodiversity for the sustainability of human life. Ecological literacy is essential for the success of conservation effort (Pilgrim et al. 2008). The low ability of forestry students to identify trees in their surrounding is, therefore, a discouraging sign for biodiversity conservation because this low ability is an indicator of low ecological literacy. It is likely that students from other departments, especially social sciences, know tree names even less than forestry students because, unlike forestry students, they don’t get courses requiring tree identification and are not involved in the planting and maintenance of trees in the campus. To prevent the loss of ecological knowledge among young generation, we must bring back students closer to nature through increasing outdoor education. Researches indicate that students participated in well planned outdoor activities related to biodiversity returned home with more positive attitude toward environment (Dillon et al. 2006). To give more comprehensive understanding of biodiversity, we can integrate traditional ecological knowledge (TEK) into the mainstream scientific ecology courses (Kimmer 2002). Unlike conventional scientific ecological knowledge which is supposed to be value free, TEK is value laden, including environmental ethic. The integration of TEK will bring new ecological insight and cultural framework for environmental problem solving such as biodiversity conservation.

CONCLUSION The low ability of forestry students to identify trees in their environment is a clear indicator of diminishing interaction with nature among young generation. To ensure the success of biodiversity conservation efforts students must be brought back closer to nature through increasing outdoor education. Unless young generation have good appreciation of biodiversity, we cannot prevent further loss of biodiversity which may endanger our survival on the rapidly changing earth.

ACKNOWLEDGEMENTS In addition to using our own photos, we also used several photos from the internet. We offer gratitude to Mr. Kwan (www.natureloveyou.sg) and Ms. Kim Starr and Mr.

WIRYONO & NURLIANA – The knowledge of forestry students to tree diversity

Forest Starr (www.hear.org/starr/images/?o=plants) for allowing the public use their photos for non commercial purposes.

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| Nus Biosci | vol. 3 | no. 2 | pp. 59‐103 | July 2011 | ISSN 2087‐3948 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

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The knowledge of Bengkulu University’s forestry students of tree diversity in their campus WIRYONO, STEFFANIE NURLIANA

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Society for Indonesia Biodiversity Sebelas Maret University Surakarta

Published three times in one year PRINTED IN INDONESIA

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