Advance winter 2016

Applied Molecular Solutions

Winter 2016

Contents 2 Advance shorts 4 Future-proofing the Petrel 7 Molecular analysis assists

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News from around Unitec

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Molecular analysis of the Westland Black Petrel's diet is aiding in the conservation of the small population of rare birds

restoration project

A study of the native carabid's diet is helping to enhance the beetle's chances of survival in new habitats

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Gut microbiome of invasive insects across the Pacific

Molecular techniques are being used to investigate how symbiotic microbial communities may be influencing insect invasiveness

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eveloping a rapid field test D for kauri dieback

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Researchers are developing an effective, inexpensive field test for Phytophthora agathidicida, the causal agent for kauri dieback disease

16 Our neglected biodiversity 20 Using DNA to improve

Unitec scientists and students are using molecular methods to uncover our hidden and often neglected biodiversity

chicken welfare

High-throughput DNA sequencing is being used to

investigate if there are underlying genetic causes of

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chicken pecking behaviour

The power of pathogens

Unitec scientists and students have been researching natural herbicides to control an invasive weed that threatens New Zealand’s native ecosystem

Editor Dr Dan Blanchon Writers Dr Stephane Boyer, Dr Marie-Caroline Lefort, Dr Richard Winkworth, Dr Peter de Lange, Dr Jessica Walker, Dr Nick Waipara, Associate Professor Marcus Williams, Dr Diane Fraser Communications Claire McCarthy Design Tineswari Maruthamuthu

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Printing Unitec Copy Centre Published by Unitec Institute of Technology Private Bag 92025, Victoria Street West Auckland 1142, New Zealand ISSN 1176-7391 Phone 0800 10 95 10 www.unitec.ac.nz

Old problems can benefit from new solutions

From its origins in the first half of the 20th Century, molecular biology has rapidly developed to revolutionise biology.

With a greater understanding of DNA and genetics, scientists have been able to investigate gene regulation, behaviour and evolution in a range of organisms, including humans.

This has created great possibilities for fields such as medicine and agriculture, but has also raised some ethical concerns.

The vast amounts of genomic data now available to us unlock opportunities for investigating and providing new insights into problems that we have wrestled with for decades. We can create rapid diagnostic tests for diseases that affect humans, crops or livestock; inventory communities of organisms; and we can describe the physiology of an animal or understand how genes function in a given ecological situation.

Research done at Unitec as part of the Applied Molecular Solutions Research Focus area is taking existing technologies and developing new approaches to address problems and questions generated by our community and industry stakeholders.

Some exciting projects are already underway at Unitec, ranging from studying the diet of Westland Petrels, investigating the effects of differences in insect gut microbiomes on their invasiveness, cataloguing the biodiversity of lichens, fungi, beetles, ferns and other organisms, to looking at the underlying genetic causes of behaviour in chickens. Funding for these projects has been received from a range of sources including Auckland Council, Department of Conservation, Australia Pacific Science Foundation, Bio-Protection Research Centre and Unitec.

Our research is focusing on four main areas: biodiversity assessment; detection of pest species and pathogens and the diagnosis of disease; studying the underlying genetic causes of diseases and their spread; and the assessment of animal welfare.

If you have any questions about the research articles in this issue of Advance, please contact the Unitec Research and Enterprise Office.

We’d love to hear from you.

INTRODUCTION

This winter 2016 edition is themed to focus on Applied Molecular Solutions (molecular biology), which is one of Unitec’s Strategic Research Foci.

The challenge now for the Applied Molecular Solutions Research Focus area is to broaden the range of industries and disciplines we’re engaging with in the use of molecular tools, and be a catalyst for cutting-edge research at Unitec and beyond.

Dr Dan Blanchon Associate Professor & Curator Unitec Herbarium Head of Environmental and Animal Sciences

research@unitec.ac.nz 09 8154321 Extn 8574

Winter 2016

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ADVANCE SHORTS SHORTS

New research leadership team Tūāpapa Rangahau (our new name for the Research and Enterprise Office) welcomed three new ‘Research Partners’ earlier this year. Along with the Dean of Research and Enterprise, Marcus Williams, the team comprises two ‘win’ positions, which find new partnership and funding opportunities and two ‘sustain’ positions, which ensure research contracts and operations are delivered to a high standard.

Irene Kereama-Royal, Research Partner, Rangahau Māori & Development holds a ‘win’ position that will specialise in Māori research and public service relationships/ funding. Irene is a lawyer with extensive experience in innovation, funding and advisory roles within organisations as diverse as FoRST, HortResearch and Technology NZ. Working closely with Irene is Gregor Steinhorn, Research

Partner Enterprise. This ‘win’ position has a focus on the private sector and industry partnerships. Gregor has both science and business Masters degrees and experience in the New Zealand innovation ecology stemming from his varied roles at Comvita.

Zoe McKechnie, Research Partner Performance will ‘sustain’ research business, cultivating key external relationships and managing all funded contracts, which average around 70 per annum – and are growing. With a degree in Biomedical Science, Zoe has extensive experience in research management, including eight years combined with Auckland Cancer Society Research Centre and UniServices, both at the University of Auckland.

Marcus Williams, Dean of Research and Enterprise also holds a ‘sustain’ position with oversight of Unitec’s research and enterprise strategy,

including the development of research productivity and research impact.

The specialist expertise, networks and experience that Irene, Gregor and Zoe bring to Unitec will be accessible to and benefit all research and enterprise activity at Unitec, and not be specific to any division or centre.

They will, however, liaise with, and maintain communications into, one or more of Unitec’s new networks in the organisation’s new academic model.

Research Partners team, Auckland, 2016. Image © Marcus Williams

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ePress update

That's why researchers Dr Dan Blanchon and Mel Galbraith have created a series with Unitec's digital publishing house ePress that brings together the disciplines that underpin biosecurity into one place. Four papers have been published so far, bringing staff, students and associates into an ongoing conversation.

The first paper sees the editors discuss the interdisciplinary nature of biology, while the second homes in on science.

Dr Glenn Aguilar employs species distribution modelling to predict the suitability of New Zealand to the infamous Queensland Fruit Fly. The third paper takes a different approach. Author Jack Craw argues in an opinion piece that while the vast majority of media attention given to biosecurity is on national border security, it is regional and local council pest management strategies that need public scrutiny.

Meanwhile, Graham Jones and Dr Diane Fraser have been investigating the honey-robbing Argentinian ant. Their pilot study exploring beekeepers’ awareness of this invasive species marks the fourth and latest paper in the series.

At ePress, we value innovative approaches to presenting research and that makes Perspectives in

Biosecurity a perfect fit for our digital publishing house. Just as you won't find the series entrenched within a purely scientific narrative, you won't find its presentation entrenched within a purely academic layout. As well as pdfs free for you to download, the multi-format series includes an interactive map accompanied with snack-size data and other dynamic features. It's just one of our multimedia publications; visit ePress.unitec.ac.nz to see them all.

SHORTS

It's more than just the science community who should be paying ongoing attention to that pesky fruit fly found in Grey Lynn, argue two Unitec researchers. From our social interactions to cultural nuances and the development of 'cyberinfrastructure', effectively managing biosecurity in New Zealand is a topic that embraces multiple disciplines.

Tachnid Fly, Auckland, 2015. Image © Mel Galbraith

Epilobium research voucher It might look like a weed and aptly has the common name of ‘hairy willowherb’, but Epilobium hirtigerum is a threatened indigenous New Zealand herb of ‘Nationally Critical’ conservation status. This plant species is now extremely limited in distribution but surprisingly has been found in an area of land under housing development at Hobsonville Point in Auckland.

Unitec Environmental and Animal Sciences lecturer Dr Diane Fraser and applied for a Metro Scheme Research Voucher to support research into the plant biology and spatial characteristics of its distribution. The Metro Voucher Scheme allows not-for-profit groups and businesses access to $5000 worth of research support.

Chris Ferkins (GeckoTrustNZ/Auckland Council) and Mary Stewart (Auckland Council) have been beavering away to establish a relatively small reserve in the Hobsonville development for protection of this rare plant that has also been dubbed the ‘Hobsonville kakapo’.

The voucher has been of valuable help with the site conservation of Epilobium hirtigerum by providing funding for Andrew Marshall (a recent graduate of Unitec’s Bachelor of Applied Science degree) to complete a baseline biodiversity survey of the reserve and provided summer studentship funding for two current degree students (Kelly Hayhurst and Sinead Spedding) to complete small research projects for their self-directed study course.

Unfortunately, there is very little known about the ecology of this species and, hence, management of the area is difficult. The GeckoTrustNZ, on behalf of the project, approached

Kelly and Sinead’s projects are to assess the growth of Epilobium in relation to managed and unmanaged sites, water table and competition from other plant species. To do this, they are supervised by Dr Glenn Aguilar in mapping the presence and growth characteristics of the plant.

Outcomes are keenly awaited to add to the knowledge of management and preservation of this threatened species.

Epilobium hirtigerum, Hobsonville, Auckland, 2015. Image © Andrew Marshall

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Future-proofing the Petrel Molecular analysis of the Westland Black Petrel's diet is aiding in the conservation of the small population of rare birds ECOLOGY

Unitec senior lecturer Dr Stephane Boyer is intent on ensuring the survival of the Westland Black Petrel/TÄ iko (Procellaria westlandica). For two years, he has been utilising molecular analysis to detail the bird's diet during the critically important breeding season.

By collecting and analysing non-invasive DNA samples, with help from undergraduate research students, Boyer has been aiming to determine whether the species is still heavily reliant on waste from the fishing industry, as was discovered in a 1998 Lincoln University study by Amanda N. D. Freeman.

The rare bird, which is native to the West Coast of New Zealand's South Island, is classified as vulnerable by the International Union for Conservation of Nature. If care is not taken to manage its fragile habitat, food supply and the threat of predators, it will risk extinction.

There is an estimated annual breeding population of just 3000-5000 pairs of Petrels.

They fly as far as South America and Australia in the summer and, in winter, return to nest in an area of forest in the Paparoa National Park, south of the tourist hotspot of Punakaiki.

It is the only place in the world where they nest, therefore conserving the area and its birdlife is crucial. The birds bring visitors to the area, with one tour company offering sunrise and sunset tours for those who have travelled from around the world to witness the 'return of the Petrel '.

Boyer's research began when he was working at Lincoln University, helping the group Conservation Volunteers to restore and maintain the Petrel's habitat. This involved replanting vegetation and ensuring that no major development, mining or building would occur in the area.

By finding out the exact contents of the bird's diet, their food supply could also be monitored.

Boyer's prediction was that he would find different species in the bird's diet than

Westland Black Petrel / TÄ iko (Procellaria westlandica) 4

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– Paparoa National Park, Punakaiki

ECOLOGY

"If care is not taken to manage its fragile habitat, food supply and the threat of predators, it will risk extinction"

those found in 1998, due to changes in both the environment and the fishing industry over the last 18 years.

“I'm interested to see what has changed, whether [the birds] are shifting to something different, whether they are still very much dependent on this fishery waste,” he explains.

“Due to uncertainty in future sea-surface temperature, fish stocks may vary greatly from year to year, which could decrease fishery waste, thereby impacting adult survival during the breeding season.

“In a recent study by Te Papa staff, Susan Waugh found that the main driver of Westland Petrel population growth was adult survival during the breeding season, with hoki fishery catch being a good predictor of adult survival.”

While a four-fold increase in the amount of hoki caught in the area may have increased the bird's dependence on fishery waste, improvement in fishing practices and

regulatory changes may have lowered the quantity of waste, and future regulatory changes could lower this even further. Since the 1998 study, more advanced collection and analysis methods have enabled a more detailed picture to be taken.

“The diet analysis method used in 1998 required forced regurgitation and could only apply to relatively intact pieces of flesh or skeleton,” Boyer says. “The results therefore represent an only partial picture of the Petrels’ diet.

We now have much better analysis tools and protocols, so we don't even need to approach the birds. It is possible to draw a much more accurate and detailed picture of the birds’ diet, including detecting all prey species predated by individual birds.”

Because the birds are at sea during the day, Boyer was able to collect faecal samples from the entrance of their burrows, without disturbing the nests or the birds themselves.

Westland Black Petrel in flight, Kaikoura, 2013. Image © Philip Griffin

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ECOLOGY

"The bird's diet is still largely made up of fishery waste, with 44 species of fish and octopus present”

Samples were taken in April and September, the start and end of the nesting season. Boyer hopes to determine what is being fed to the chicks, and what the adults are eating during the incubation period.

“That will give us a much bigger picture of what they eat as a species,” he says.

The DNA samples have undergone molecular analysis in the Unitec Applied Molecular Solutions laboratory, followed by next-generation sequencing analysis at New Zealand Genomics Ltd. Preliminary findings, based on an initial pilot study, show that while the bird's diet has changed since 1998, it is still largely made up of fishery waste, with 44 species of fish and octopus present. The second, larger set of samples is currently being analysed, with final results expected in June.

contact Dr Stephane Boyer sboyer@unitec.ac.nz

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Westland Black Petrel, Kaikoura, 2013. Image © Philip Griffin

MOLECULAR BIOLOGY

Molecular analysis assists restoration project

A study of the native carabid's diet is helping to enhance the beetle's chances of survival in new habitats For three years, Unitec's Dr Stephane Boyer has been analysing the diet of native carabids (Megadromus guerinii) found on Christchurch's Banks Peninsula. The science lecturer's findings have provided essential knowledge for the restoration of Ōtamahua/Quail Island, and will aid in the understanding of native beetles, including their role in food webs and function in New Zealand native ecosystems.

Native carabid beetle (Megadromus guerinii) – Banks Peninsula, Christchurch

Carabid beetles play an important role. The predatory insects help to contribute to soil fertility, vegetation renewal and weed control, and provide a food source for other animals.

Boyer's diet analysis, funded by the Brian Mason Trust, aimed to determine why 44 of the black flightless beetles, native to Banks Peninsula, did not survive translocation to Quail Island.

Megadromus guerinii, Banks Peninsula, 2015. Image © Mike Bowie

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After molecular analysis of the carabid’s faeces, Boyer has determined that the hypothesised lack of food on the island was most likely not responsible for the beetle's inability to survive.

“We found that the native ground beetle feeds on a wide variety of insect larvae including flies, other beetles and moths, many of which are present on Quail Island,” he says.

“Therefore, reasons for the disappearance of the translocated individuals are unlikely to relate to inappropriate food.”

University ecologist Mike Bowie explained in his 2008 paper, Ecological restoration of the invertebrate fauna on Quail Island (Ōtamahua).

With help from a number of volunteers, the Trust has attempted to restore the island to its pre-human state, through the replanting of over 100 species of native plants and controlling invasive predators, such as deer, possums and rats.

MOLECULAR BIOLOGY

“The predatory insects help to contribute to soil fertility, vegetation renewal and weed control, and provide a food source for other animals”

His study was part of an ongoing restoration project on Quail Island. The small island, inside Lyttleton Harbour on Banks Peninsula, was home to the now extinct New Zealand Quail and a number of other species, before the land was cleared by Maori and European settlers.

The 85ha recreational reserve, administered by the Department of Conservation (DoC), has been the focus of restoration efforts for the past 20 years by the Ōtamahua/Quail Island Ecological Restoration Trust, in partnership with DoC and Te Hapu o Ngāti Wheke of Rāpaki.

“The main aim of the Trust was to facilitate the restoration of indigenous vegetation and fauna on Ōtamahua/Quail Island and provide refuge for locally extinct or rare and endangered species of the Banks Peninsula region,” trustee and Lincoln

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Although the island is now largely predator-free (apart from mice, which are difficult to eradicate), it has been hard for native species to recolonise the reserve, due to it being an island.

As Boyer explains, “although the new conditions have prompted the return of a number of birds, such as bellbirds and kereru, and penguins are now nesting on the island, Quail Island remains out of reach for many other native animals that would have likely lived on the island prior to human arrival.”

Bowie, Boyer's former Lincoln University colleague, has been on the Quail Island Trust for over 15 years, and has been instrumental in reintroducing native species to the island. “On Banks Peninsula there are roughly 50 [species of] carabid beetle, whereas on Quail Island there are probably only about five,” Bowie explains.

“The ones we are particularly interested in are the Banks Peninsula endemic species.”

“After discovering that the prey species of the carabid were largely available on Quail Island, diet was ruled out as an issue in their survival”

“These artificial shelters were also used to monitor the survival of the animals after release,” says Boyer. “Although most translocations were successful, there is one particular case where 22 male and 22 female native ground beetles were reintroduced, but none could be recovered after a year.”

Bowie enlisted Boyer's help with this issue, knowing he was well qualified for such research, his PhD having focused on the ecology of invasive and native cockroaches on two French islands in the Indian Ocean. Much of Boyer's other work has focused on invertebrates, and the conservation of native species.

With predators largely eradicated, Boyer thought a possible explanation for the carabids’ death was the lack of appropriate food on the island.

To ascertain the exact contents of their diet, with help from students, carabids were collected from Banks Peninsula. These were placed in petri dishes lined with paper towels.

The beetle's faecal matter was then collected and analysed, and the beetles returned to their original location. Initial samples provided insufficient data, so experiments were repeated the next year. Former post-doctoral student Dr Richard Winkworth helped with the more complex sequence analysis.

“This [data] was analysed using next-generation DNA sequencing methods,” explains Boyer.

“This allowed us to identify which prey were

eaten, from the DNA traces that remain in the faeces of predators after digestion.”

Boyer says he was not entirely surprised at the range found in the carabid's diet.

After discovering that the prey species of the carabid were largely available on Quail Island, diet was ruled out as an issue in their survival.

MOLECULAR BIOLOGY

The Trust reintroduced a number of these carabids, along with weta and leaf vein slugs.

These were collected from Banks Peninsula and transported to Quail Island in weta motels – hollow wooden containers which the weta crawls into – and wooden discs, which carabids naturally inhabit.

The next hypothesis is that the lack of habitat available on the island was responsible for their inability to thrive.

Carabids nest under old pieces of wood or rotting tree trunks, and there are not a large number of these on the island.

While Boyer is not involved in this phase of the project, including the next attempt at translocating carabids, he plans to make recommendations relating to their habitat, such as leaving more wooden discs to rot on the island. These will mimic dead tree trunks and provide safe havens from mice, possible predators of the beetles. Boyer also recommends using the same diet analysis techniques in future restoration projects.

“The molecular protocol developed for this study is applicable to other carabid beetles and other invertebrate predators in general,” he explains.

“It is advisable to conduct this sort of analysis prior to the translocation of endangered species in areas outside of their current distribution.”

contact Dr Stephane Boyer sboyer@unitec.ac.nz

Megadromus guerinii, Banks Peninsula, 2015. Image © Mike Bowie

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Gut microbiome of invasive insects across the Pacific Molecular techniques are being used to investigate how symbiotic microbial communities may be influencing insect invasiveness COMMUNITY CONNECTION

"Some species are pests due to the microbes that live in symbiosis in their gut"

Isolated islands are threatened by arrival of invasive species For millions of years, New Zealand and the Pacific Islands have been extremely isolated, which has led to the development of native ecosystems with unique biodiversity. But with the increase in human travels and exchanges of goods in past centuries, this isolation is not quite what it used to be.

New species have been arriving en masse, some of which can have devastating effects on native biodiversity and also on agriculture.

We call them vermin, pests or invasive species. The ones attacking crops are mainly insects and they can cause millions of dollars of damage to the New Zealand agriculture industry.

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Their impact can also be very detrimental to communities whose livelihoods rely primarily on local agriculture and in areas where biosecurity resources are limited, which is the case for a number of Pacific Islands.

Traditionally, insect pest species are controlled with pesticides. But pesticides may also affect species that are beneficial for crops, such as those that improve the nutrients found in soil or that feed on the pests themselves. It is therefore essential to develop efficient tools to identify these detrimental species and targeted measures to control them before they reach damaging population sizes, but without affecting the non-pest species.

COMMUNITY CONNECTION

How can we control insect pest species without affecting other beneficial insects?

What makes a species beneficial or detrimental has been the subject of much debate. Here at Unitec, a group of scientists thinks that some species are pests due to the microbes that live in symbiosis in their gut.

Most plants produce toxins and other chemicals to deter insects, but pest species still manage to eat and digest the plants. Just like gut microbes help humans digest food, some microbes are

helping insects to digest plants. Recent advances in microbiology have highlighted the crucial role of gut microbiome in insect growth, development and environmental adaptation to their hosts.

In the case of invasive insects, it is possible that the contribution of the microbes goes beyond this, and also helps the insect to 'overcome' the toxic chemicals that plants produce to protect themselves.

Dr Marie-Caroline Lefort with Costelytra zealandica grub, Auckland, 2016. Image Š Dr Stephane Boyer

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The MADII project COMMUNITY COMMUNITY CONNECTION COMPUTING CONNECTION

Using modern molecular analyses, it is possible to compare the types of microbes that live in the guts of pest and non-pest insect species.

The aim of the MADII project (Microbiome As Driver of Insect Invasiveness) is to use molecular tools to determine the role that gut microbes play in digesting food and handling plant defence chemicals.

What researchers are looking for is a particular set of microbes that would only be present in the guts of pest species and not in beneficial species.

If they could identify this 'invasiveness signature', new biological control solutions that target those microbes, rather than the insect itself, could be developed. Without their special gut microbes, pest species are likely to be much less of a problem, and this would help reduce the need for pesticides. Such solutions would also ensure

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the preservation of the biodiversity of beneficial insects occurring in the ecosystem in question.

Because Unitec is primarily a tertiary institution, the researchers working on this project wanted it to also be an educational science project. MADII is facilitated through a newly established UNESCO UNITWIN network (University Twinning and Networking Programme) that links New Zealand tertiary institutions with others from the Pacific Small Island Developing States (PSIDS), and aims at enhancing these institutions’ capacities through resources, knowledge sharing and collaborative work in education and natural sciences.

Costelytra zealandica grub, Auckland, 2016. Image Š Kaytee Shum

What has been done so far The project started in January this year with support from Unitec through the Strategic Research Fund. The team comprises eight Unitec staff and students as well as collaborators from three other institutions across New Zealand (Bio-Protection Research Centre, Massey University and Waiariki Institute of Technology) and from the University of the South Pacific in Fiji.

To test the research hypothesis, two model species have been identified and chosen to carry out the study in New Zealand, while collaborators in Fiji are currently investigating the best species model to use in their country.

The invasive Scarab Grass Grub Costelytra zealandica and its close congener but non-invasive species C. brunneum have already been sampled in the North and South Island. The second model comprises the introduced and invasive Black Field Cricket (sampling completed) and the New Zealand

COMMUNITY CONNECTION

"Without their special gut microbes, pest species are likely to be much less of a problem, and this would help reduce the need for pesticides" native Small Field Cricket for which the team is working at identifying collection sites.

The guts of over 100 of these insects have been dissected and stored at extremely low temperatures to preserve their DNA, while waiting to be prepared for molecular analyses.

The team is hoping to get the results from the molecular analyses of the New Zealand species in early September, and those from the Fijian species by the end of the year.

A new website/research platform has also been launched to facilitate the collaboration between New Zealand and Fiji: http://lefortmc.wix.com/madii. Progress on the project can be monitored on the website, and tutorials about molecular techniques and insect dissections will be made available for the general public and to support the learning of the tertiary students involved in the project.

contact Dr Marie-Caroline Lefort mlefort@unitec.ac.nz

Black Field Cricket, Auckland, 2016. Image Š Dr Marie-Caroline Lefort

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Developing a rapid field test for kauri dieback Researchers are developing an effective, inexpensive field test for Phytophthora agathidicida, the causal agent for kauri dieback disease BIOLOGY

Kauri (Agathis australis) is a New Zealand endemic tree with significant cultural and ecological importance. The once-extensive kauri forests of northern New Zealand were reduced to small fragments by logging and gum extraction during the late 19th and early 20th centuries. Now what remains of the kauri forests is threatened by kauri dieback, a devastating disease characterised by leaf yellowing, canopy thinning, trunk lesions and, ultimately, tree death.

First recognised in the 1970s the disease is now widespread and affects many of the remaining kauri stands.

The disease is caused by Phytophthora agathidicida, a fungal-like organism found in the soil. Literally translated the name Phytophthora means 'the plant destroyer' and this group of organisms – there are about 100 currently recognised species – is capable of causing enormous damage to both agricultural and natural ecosystems. As an example, a relative of P. agathidicida was responsible for the 1845 Irish potato famine.

Currently, detection of P. agathidicida in environmental samples is time consuming, requiring specialist laboratory equipment and highly trained staff. It takes several weeks to first isolate the organism from a sample and then to grow it on a specialised medium until it can be identified morphologically. This method has

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certainly proved useful, but the time needed to obtain a result limits its usefulness in the context of day-to-day monitoring.

To date, interest has mostly been in determining the extent of the problem, but if we want to move towards controlling its spread, then rapid field tests could have significant impact on our ability to address kauri dieback. For example, such a test would allow nurseries to ensure that their stock was free from P. agathidicida.

The Bio-Protection Research Centre and Unitec have funded the project, which is led

by Professor Peter Lockhart and Dr Richard Winkworth. It uses an approach that pairs high throughput sequencing with an emerging DNA amplification technology. The first step has been to generate whole mitochondrial genome sequences for a range of Phytophthora species found here in New Zealand and overseas.

Mitochondrial genomes are good targets for the development of molecular diagnostics as they differ significantly between species and they are present in high copy number.

"Literally translated the name Phytophthora means 'the plant destroyer' "

The team is also using a novel methodology that allows them to generate these genome resources faster and more cost-effectively than elsewhere. The test itself is based on the Loop-mediated AMPlification (LAMP) technology. The genome resources are used to identify genetic signatures that distinguish P. agathidicida from other Phytophthora species.

The researchers then engineer the test so that a positive result is returned only if these regions – and hence the organism – are present in the sample. The result is inexpensive, rapid and highly sensitive on-site genetic diagnostics; that is, tests that take less than an hour, cost $1-2 per test and that can be carried out on site.

Currently, the team has generated genomic resources for more than 40 Phytophthora pathogens including P. agathidicida from several locations. This database is allowing the researchers to develop tests for P. agathidicida as well as other Phytophthora species from agricultural and native ecosystems in New Zealand.

Rapid DNA diagnostics have the potential to be used for the detection of pathogens and diseases in a wide range of situations.

On-site diagnostics could be applied wherever

BIOLOGY

Genetic tests targeting them are therefore both highly selective and sensitive.

"Kauri dieback is a devastating disease characterised by leaf yellowing, canopy thinning, trunk lesions and, ultimately, tree death"

timely identifications or cost-effective monitoring of a biological agent is needed. Applications include human health, environmental quality, conservation, forestry, agriculture and border biosecurity. Such tools have the potential to positively impact our capacity to protect the New Zealand environment as well as the economic sustainability of our primary industries.

“Our goal is to develop reliable, inexpensive and field-deployable diagnostics for pest and disease monitoring in a range of contexts,” says Winkworth, now a Senior Lecturer at Massey University. “For some, this means determining not only whether a pest is present, but also how much of it is there.

This technology allows us to do that in a single test.

“With such tools we can better assess disease risk and therefore target our control measures more effectively – saving money and reducing the environmental impact,” says Winkworth.

In the long term, more targeted and effective use of pesticides is also likely to reduce the appearance of resistance.

contact Dr Richard Winkworth R.C.Winkworth@massey.ac.nz

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BIOLOGY

Our neglected biodiversity Conservation of the 'unfashionable': the use of DNA sequence data to identify our neglected biodiversity Our knowledge of the biodiversity of New Zealand is unevenly spread. Charismatic or larger organisms such as birds, reptiles, fish and higher plants are generally well known and well-studied. We do however have more neglected corners of our biodiversity.

For a range of reasons some taxonomic groups have not been as extensively studied as the better-known groups of organisms.

There may be few or no experts in that field in New Zealand; the organisms may be microscopic, have cryptic distinguishing characteristics, or may live in inaccessible places and environments.

These neglected taxonomic groups, such as fungi and many groups of invertebrates, are thought to represent a significant part of our native diversity. Some of them may be threatened with extinction, but without knowing anything about them, it is rarely possible to even attempt to manage them.

Dr Peter de Lange is a Principal Science Advisor at the Department of Conservation (DoC) and

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a research fellow at Unitec. He collaborates with Dr Dan Blanchon from Unitec’s Applied Molecular Solutions Research Focus on a range of projects studying lichenised fungi, or 'lichens'.

“Lichens are a significant part of New Zealand's biodiversity, but they are poorly studied, poorly known. We are beginning to understand that they are very important bioindicators of climate change and changing forest structure and health.

Because we often don’t know what species we are dealing with, we can’t effectively manage them – we need to know what we are dealing with. DoC is increasingly broadening its approach to include all forms of life in New Zealand, including more cryptic things like lichens,” says de Lange.

The last full review of the lichen species in New Zealand, started in 2009 and published in 2012 by a DoC threat classification panel, recognised 1799 different lichens and associated fungi as being found in New Zealand. The purpose of this review was to determine if

Lichen, Auckland, 2016. Images © Dr Dan Blanchon

BIOLOGY

“Lichens are very important bioindicators of climate change and changing forest structure and health”

any lichen species were threatened with extinction. It turned out that more than half of the species were so poorly known that they were listed as ‘Data Deficient', and therefore could not be assigned to a threat category.

In 2016, a new threat classification panel for lichens (led by de Lange and including Unitec’s Dr Dan Blanchon) will meet again to determine the threat status for New Zealand’s lichens.

The task is a daunting one, as conservative estimates now suggest we may have as many as 2500 different lichens and associated fungi in New Zealand.

“A large proportion of New Zealand lichens are considered to be ‘Data Deficient' – in some cases we are not confident that they are actually real species,” says de Lange.

Part of the solution lies in the judicious use of molecular technologies such as DNA sequencing.

While there are few active lichenologists in Australasia, worldwide there are several laboratories working on different lichen genera Lichen, Auckland, 2016. Images © Dr Dan Blanchon

and families, many of which are represented by species in New Zealand. Their work, and research carried out in the Applied Molecular Solutions laboratory at Unitec by Blanchon, de Lange and Dr Sofia Chambers involves extracting DNA from lichen specimens, generating DNA sequence data and comparing this with other published sequences available in online databases. A vast amount of genetic data is available for comparison on international databases such as Genbank.

“Lichens are a little odd to work with, as they are actually communities of organisms, made up of a fungus, which 'farms' captured algae and/or cyanobacteria. In practice, the scientific name of the lichen refers to the fungal partner, and it is the DNA of the fungus which is usually compared,” says Blanchon.

Comparison of DNA sequences can uncover or identify species new to science, confirm or dispose of species names and help us to understand the relationships between or within Winter 2016

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species worldwide. “DNA is the only reliable way of getting to grips with problematic groups – getting unknown taxa down to at least genus level. When studying lichens, without getting DNA sequence data you are dreaming if you think you are going to do a good job,” says de Lange.

Blanchon and de Lange are collaborating on a range of lichen projects, most involving the use of DNA sequence data. One interesting project involved a hunt for a mysterious 'missing' lichen called Ramalodium dumosum. This lichen had only been collected once (in 1981), by a collector called John Bartlett, from coastal cliffs at Huia near Auckland and was never seen again.

A thorough survey of the Manukau Harbour coastline (including an accidental discovery of a nudist beach) uncovered one possible candidate for the elusive R. dumosum. This unappealing microscopic gelatinous black lichen was taken back to the lab for DNA analysis. Comparison of the ITS DNA sequence data with Genbank sequences indicated that this specimen was actually a new species of Enchylium rather than Ramalodium, replacing one mystery with another.

BIOLOGY COMPUTING

Another example of the value of molecular data is from the Chatham Islands (Rekohu).

Rangatira (South-East) Island is the site of an attempt to restore population numbers of the critically threatened Chatham Island Black Robin.

The succession of the native plant pohuehue (Muehlenbeckia australis) had been identified as a possible barrier to the Black Robin recovery programme on the island.

However, before removing the pohuehue, it was important to determine if there would be negative effects on other species. A lichen was collected from the pohuehue by de Lange,

and an examination back in the lab at Unitec suggested it could be a new species. DNA data has subsequently shown that it is indeed a species previously unknown to science.

The use of molecular data can also cause surprises with common lichen species. Blanchon hosted a Royal Society of New Zealand Teacher Fellow, Glenys Hayward. Hayward and Blanchon studied the common and widespread lichen Ramalina celastri, a species found in virtually every backyard in Auckland. In Australia, the species was separated into two subspecies, but in New Zealand we only recognised one species.

Comparison of DNA sequence data from specimens collected from New Zealand and Australia showed that we should recognise two separate species, R. celastri and R. ovalis (an old name for specimens mainly found in the South Island of New Zealand and eastern Australia), adding one species to the total known lichens for New Zealand.

In addition, collaboration in worldwide studies can show that in some cases where we previously thought we had species with a worldwide distribution, we are wrong.

For example, at Unitec we are part of the PARSYS consortium, investigating members of the family Parmeliaceae all over the world.

We are currently revising the species of the genus Parmotrema for New Zealand. Initial results are indicating that a common species around Auckland that we have been calling Parmotrema perlatum is not in fact that species.

The species with this name has been listed as a culinary herb in places such as India – and based on our data, what we have here is not that species, which appears to be restricted to the Northern Hemisphere.

“A common species around Auckland that we have been calling Parmotrema perlatum is not in fact that species"

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Lichen, Auckland, 2016. Images © Dr Dan Blanchon

BIOLOGY

“Voucher specimens substantiate claims of occurrence and can provide useful information on the geographical distribution and ecology of species"

One essential step in the use of molecular tools such as DNA sequencing for studying lichen species is in the creation of voucher specimens – the preserved sample of the lichen the DNA was taken from, lodged and protected for posterity in a collection such as a herbarium.

The DNA sequence data is linked to the collection number of the specimen and this specimen can be checked by other scientists – such peer review is an essential part of the scientific process. Voucher specimens substantiate claims of occurrence and can provide useful information on the geographical distribution and ecology of species.

“Unitec is the only lichen-specialist herbarium in New Zealand,” notes de Lange. “Some other herbaria maintain lichen collections, but they may not have specialists working there or may not be actively adding to or curating their collections. Unitec provides a specific type of service to the New Zealand people that none of the other New Zealand herbaria do.”

With between 1800 and 2500 lichens to choose from, the list of projects for the Unitec and DoC researchers is nearly endless, but every puzzle they solve is a win for our understanding of New Zealand’s biodiversity.

Lichen, Auckland, 2016. Images © Dr Dan Blanchon

contact Dr Dan Blanchon dblanchon@unitec.ac.nz Dr Peter de Lange pdelange@doc.govt.nz

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Using DNA to improve chicken welfare High-throughput DNA sequencing is being used to investigate if there are underlying genetic causes of chicken pecking behaviour GENETICS

Feather pecking, where aggressive chooks target others with hard jabs and pulls to feathers, is one of the most significant animal welfare issues. In severe cases it can cause skin trauma, cannibalism and death. New research suggests it could also be genetically linked to depression.

In a typical brood of chickens there are three types of birds – those that peck, those that get pecked and 'control' birds that do neither.

Figuring out what causes this behaviour and finding some potential solutions has been the focus of Unitec animal welfare lecturer and researcher Dr Jessica Walker, who has been working with Massey University and adjunct evolutionary biologist Professor Peter Lockhart.

Together they have used the increasingly important high-throughput DNA sequencing technology to figure out if the problem lies in the DNA. While DNA has been used often in animal welfare science, high-throughput sequencing allows a researcher to rapidly and relatively cheaply generate huge amounts of DNA sequence data.

The main aim of the project was to look at the new technology as a research tool to improve animal welfare in intensively farmed chickens.

Rather than just looking at standard behaviour or stress levels, Walker says, high-throughput

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sequencing has offered a whole new world of possibilities. “The data you get out is amazing and it’s just a matter of trying to figure out which parts you can isolate and use,” she says.

The second aim of the project was to determine if there were any genomic differences between chickens that display feather pecking behaviour, those that are recipients of feather pecking, and that third group of those that neither peck nor are pecked. It turns out there is.

The researchers are finding that there are significant differences between genes that are switched on and switched off within the three groups of chickens. One gene – TPH2, which is related to serotonin – is found in much higher levels in peckers than in other birds.

"Feather pecking could also be genetically linked to depression"

In humans, serotonin is the chemical associated with feelings of wellbeing and happiness. When a person suffers depression, a lack of serotonin is seen as one cause. It is possible this big henhouse problem is linked to the same issue.

GENETICS

"There are three types of birds – those that peck, those that get pecked and 'control' birds that do neither" “Often the ones being pecked have a lower level of serotonin, and we associate them with having a lower quality of life or welfare. What we are seeing with chickens that are pecking is they seem to have higher levels of serotonin,” says Walker. Working out what that means and how to use the information are the next steps.

The researchers plan to go through all the genes that are turned on, look at where the significant differences are among the three groups, then look at what these genes relate to and what that could mean in terms of welfare. They’ll also look at what other researchers have published around feather

pecking, which is one of the most complex, as well as pressing, issues facing the industry.

A solution could be as simple as separating the differently disposed chickens, although there are many other factors in chicken farming that could contribute to behaviour.

"The problem is it’s complicated by environmental issues as well, they’re so cramped and close together," says Walker. "There are a whole lot of other variables that come together with feather pecking as well. All we’re really trying to do is get right back to that brain chemistry and take away the influence of the environment, although that plays a massive role on chicken welfare and how they behave with one another.”

contact Dr Jessica Walker jwalker@unitec.ac.nz

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The power of pathogens Unitec scientists and students have been researching natural herbicides to control an invasive weed that threatens New Zealand’s native ecosystem

BIOSECURITY

Research on natural enemies of the invasive African club moss (Selaginella kraussiana) is helping to protect New Zealand's native ecosystems, while enabling Unitec students and graduates to undertake industry-generated research. Led by Dr Dan Blanchon, Associate Professor and Head of Environmental and Animal Sciences at Unitec, and Dr Nick Waipara, Auckland Council's principal advisor of biosecurity, students and graduates have been investigating possible natural pathogens of the invasive introduced plant. The hope is to find a fungus which could be used to create a mycoherbicide (fungal herbicide) that would help to control the plant, which originates from Madagascar and other wet areas of Africa.

Through DNA testing of approximately 100 fungi found on discoloured or wilted leaves of the club moss, two promising fungal isolates have emerged as natural enemies of the club moss. There are a number of fungal isolates still to be tested, with the hopes that an even stronger candidate will be found. “In an ideal world, we're looking for something that really kills or knocks back the club moss but doesn't attack anything else,” explains Blanchon.

African club moss is common in Auckland and Northland, but the Department of Conservation is now seeing it spread further, including into parts of the West Coast. The plant establishes itself in shady, damp environments, spreading along tracks and into the bush on people’s boots, through rainwater and through its own spores.

“It is going to be a pest of national significance,” Waipara says.

The research project was initially commissioned

by Auckland Council in 2011, with the aim of finding a biological control for the invasive plant, as per their current pest management strategy.

If one can be found locally, it will save having to look overseas to the plant’s original environment.

This would be an expensive exercise and potential risks are involved if another species was to be introduced to New Zealand.

Waipara asked Unitec to look at the impacts of the African club moss plant on the native environment and to explore possible solutions to these impacts. Though the weed and its negative effects were known, biological control research had not been

African club moss (Selaginella kraussiana) – Auckland and Northland

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undertaken in New Zealand or overseas, causing the weed to be overlooked in many pest management programmes. “This weed is an issue for northern New Zealand and hasn't had a lot of research around it,” he explains.

The plant is hard to control, as it creates thick layers of ground cover, which smother native plants and seedlings. Using general herbicides would kill these seedlings, mosses, ferns and lichens as well as the African club moss, which would return at a faster rate than the native species and inhibit their growth. “What you really want is to target this [club moss] specifically,” explains Blanchon.

The tools that the Council can use against the plant are limited, and being able to combine biocontrol with chemicals and hand removal would help, says Waipara.

“The more armoury you have against some of these pests the better.”

With a background in scientific research himself, Waipara has been collaborating with Blanchon and students on the research. The Council runs a biosecurity studentship summer programme with Unitec, and initially funded summer student Hayley Nessia to undertake research, followed

Culture plate, Auckland, 2016. Image © Dr Dan Blanchon

BIOSECURITY

"African club moss is common in Auckland and Northland, but is now spreading further, including into parts of the West Coast" by other students and graduates, including Matt McClymont, Christy Reynolds, Sarah Killick and Orhan Er. Impact studies conducted showed that the plant had an invasive impact in the native ecosystem, including suppressing seedling growth, and causing a significant decrease in the abundance of native plants.

The next stage involved looking for pathogenic fungal isolates to reduce these impacts.

Samples of unhealthy-looking plants were collected from areas of Auckland and Northland, including parts of the Waitakere and Hunua Ranges, Whangarei and Waiheke Island.

These were screened in the Unitec laboratory for fungi.

A sample of each fungal isolate was taken and identified through a number of processes, including DNA sequencing through Massey University. These fungal isolates were reapplied to damaged and undamaged samples of the club moss, to check for pathogenicity.

Two promising pathogens emerged, including Phoma selaginellicola, which attacks African club moss significantly but does not kill it.

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"If none of the fungal isolates are suitable as a biocontrol, the next phase may involve the Council going offshore"

BIOSECURITY

However, as it seems to be specific to African club moss, the bonus is that it would not kill other plants if used as a mycoherbicide.

The other candidate is Pestalotiopsis clavispora, though it is more of a 'generalist', says Blanchon, so other plants could be at risk if it was used as a biocontrol agent.

Whether or not the pathogenic fungi would be successful as biocontrol agents in the field has not been determined yet. The project is nearing completion, and soon, Blanchon and his team will submit recommendations to the Council.

If they have found an isolate that shows enough promise as a biological control, the Council may look to develop this as a mycoherbicide. A number of trials would need to be conducted to ensure it is safe, and to gauge any potential adverse effects on native club mosses and other native species.

If none of the fungal isolates are suitable as a biocontrol, the next phase may involve the Council going offshore. Other options include taking an integrated approach, says Blanchon, such as spraying an initial low-dose herbicide followed by a mycoherbicide that would hopefully kill the weakened African club moss. “The low-dose herbicide would weaken everything, but the native stuff should recover,” he explains.

Whatever the outcome, Waipara says Unitec's research has been critical in helping the Council to determine their next step. It has been beneficial for Unitec as well, says Blanchon, with students and graduates getting to work on a real-life research project which combines biodiversity with biosecurity. They have also had the opportunity to be hired as researchers and to contribute to a number of published studies.

contact Dr Dan Blanchon dblanchon@unitec.ac.nz Dr Nick Waipara Nick.Waipara@aucklandcouncil.govt.nz

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Selaginella kraussiana and culture plates, Auckland, 2016. Images © Dr Dan Blanchon

"The challenge now for the Applied Molecular Solutions Research Focus area is to broaden the range of industries and disciplines we’re engaging with in the use of molecular tools, and be a catalyst for cutting-edge research at Unitec and beyond" – Dr Dan Blanchon

phone 0800 10 95 10 fax +64 9 815 2905 web www.unitec.ac.nz

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