Differential gene expression in normal and intersex Crustacea: New horizons for investigating impacts of pollution, parasitism and climate change.

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences


Intersexuality is the abnormal condition when an organism displays characteristics of being both male and female. The study of intersexuality in wildlife provides excellent means for studying comparative biology of abnormal conditions in reproductive determination and/or differentiation. Intersex in crustaceans can occur through genetic abberations, parasitism, disrupted environmental sex determination (ESD) and through chemical exposure. The genetic knowledge of crustacean groups is currently very limited making the assessment of mechanisms behind environmental sex determination (ESD); feminsing parasites; and pollution very limited in this ecologically important group: thus hot-topics in environmental sciences such as endocrine disruption and climate change are difficult to assess without a clear understanding of the biology of ecologically relevant species. Considerable information exists at the physiological, individual and population level impacts of temperature, photoperiod, and parasitism on the sex determination in amphipods. In addition, several studies have suggested sexual disruption might also be occuring in this group following exposure to environmental contaminents. Following on from a NERC 454 pilot study into gene expression in normal and intersex gonadal tissues, this project would utilise high-throughput sequencing technologies to complete a suite of archived biological tissues for specific gene expression in male and female amphipods and two intersex phenotypes (intersex males and females); increasing substantially the genetic knowledge for this and crustacean groups in general. Intra and inter specific differences between organs of males, females, intersex males and intersex females would be compared by digital transcriptmics. Following some evidence that suggests that male crustaceans maybe impacted by pollution in a way that effects their male hormones and cause demasculinisation (a more female-like appearance). Further studies would experimently induce de-masculinisation in an intertidal amphipod using three methods; firstly, physically dissecting the male (androgenic) glands; secondly, through infection with feminising parasites; and thirdly, through chemical exposure to some known endocrine disrupting chemicals. These experiments would be used to examine comparisons in the genes being up/down regulated following each experimental manipulation. Key genes would be selected and utilised to develop assays to assess whether demasculinisation is occuring in field collected specimens from clean and polluted sites. All genetic data acquired during these studies would be made publically available through a specially designed database entitled AmphiBASE.


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Short S (2012) A widespread and distinctive form of amphipod intersexuality not induced by known feminising parasites. in Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation

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Short S (2014) Vitellogenin is not an appropriate biomarker of feminisation in a crustacean. in Aquatic toxicology (Amsterdam, Netherlands)

Description This grant was part of a complemented of an award to Dr A. Ford Portsmouth University grant number NE/G004587/1. The key discoveries related to this project are reported under the main entry.
Exploitation Route This project generated resources which can be used for a range of marine sciences and profound implication in relation to chemical and parasite endocrine disruption in marine crustacean sp.
Sectors Chemicals,Education,Environment

Description Molecular underpinning to similar behavioural manipulation in two convergent parasite-host models
Amount £38,000 (GBP)
Organisation University of Surrey 
Department Daphne Jackson Trust
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 03/2021
Title Assembly of transcriptome for the intertidal amphipod Echinogammarus marinus constructed using various tissues and developmental stages 
Description Assembly of transcriptome for the intertidal amphipod Echinogammarus marinus. This constructed from sequenced reads made using RNA isolated from Echinogammarus marinus muscle, head, hepatopancreas and gonadal tissues dissected from parasitised, unparasitised, male, female, and juveniles (n=14 adults and 10 juveniles at various stages of development). Assembly name: E marinus normalised Flx Assembly, Newbler/Mira Merged assembly Assembly description: Contig count 46248 Base count 36699305 Min contig length 300 Mean contig length 793 Max contig length 8468 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact This database has directly facilitated the generation of multiple published studies on the subject of intersexuality: For example: Short, S., Yang, G., Guler, Y., Kille, P., and Ford, A.T. Crustacean intersexuality is feminisation without de-masculinisation: implications for environmental toxicology. (2014) Environmental Science and Technology 48:13520-13529. Short, S., Yang, G., Kille, P., and Ford, A. Vitellogenin is not an appropriate biomarker of feminisation in a crustacean. Aquatic Toxicology (2013): 153:89-97. Short, S., Yang, G., Kille, P., and Ford, A. A widespread and distinctive form of amphipod intersexuality not induced by known feminising parasites. Sexual Development (2012): 6:320-324. Short, S., Guler, Y., Yang, G., Kille, P., and Ford, A. Paramyxean-microsporidian co-infection in amphipods: Is the consensus that Microsporidia can feminise their hosts presumptive? International Journal for Parasitology (2012): 42:683-691. It has also made possible investigations into the molecular basis of amphipod responses to antidepressant exposure and parasite infection: For example: Bossus, M., Guler, Y., Short, S., Morrison, E., and Ford A. Behavioural and transcriptional changes in the amphipod Echinogammarus marinus exposed to two antidepressants, Fluoxetine and Sertraline. Aquatic Toxicology (2014): 151:46-56 Guler, Y., Short, S., Green Etxabe A, Kille, P., and Ford, A. (2015) Impacts of a newly identified behaviour-altering trematode on its host amphipod: from the level of gene expression to population. Parasitology (2015): 142 (12):1469-1480. 
URL http://afterparty.bio.ed.ac.uk
Description Dr. Grant Stentiford, Dr. Kelly Bateman and Dr. Stephen Feist: Characterising the ultrastructure of Paramyxid intracellular parasites associated with feminisation of host crustaceans. 
Organisation Centre For Environment, Fisheries And Aquaculture Science
Department Weymouth Laboratory
Country United Kingdom 
Sector Public 
PI Contribution We researched Paramyxid-microsporidian co-infection in amphipods and found striking levels of co-infection between paramyxid and microsporidian parasites. Short, S., Guler, Y., Yang, G., Kille, P., and Ford, A. Paramyxean-microsporidian co-infection in amphipods: Is the consensus that Microsporidia can feminise their hosts presumptive? International Journal for Parasitology (2012): 42:683-691 This raised the possibility that the two intracellular parasites were physically associated within their host cells, specially, that this association took the form of the microsporidian hyper-parasitising the larger paramyxid parasite. This kind of hyperparasitism had been suggested following ultrastructural analysis of other paramyxid species that infect shellfish. We provided infected material to allow ultrastructural analysis using TEM.
Collaborator Contribution The team at Cefas Weymouth prepared the tissues and performed TEM analysis of the infected tissues to reveal the ultrastructure of host cells co-infected with both paramyxid and microsporidian parasite.
Impact The TEM analysis of the infected tissues revealed paramyxid and microsporidian spores sharing the same cells but not physically associated. The work suggests that microsporidian and paramyxid parasites have a range relationships in marine invertebrates: accidental co-infections (observed in amphipods infected with some microsporidian-paramxyid species combinations) - through to almost absolute co-infection but no physical association (the case revealed by this collaboration) - through to hyperparasitism, reported in a later publication from the Cefas team: Hyperspora aquatica n. gn., n. sp.(Microsporidia), hyperparasitic in Marteilia cochillia (Paramyxida), is closely related to crustacean-infecting microspordian taxa. GD Stentiford, A Ramilo, E Apollo et al Parasitology (2017) 144 (2):186-199 This spectrum of relationships in economically important invertebrates will be an future area of collaboration between the our team and Cefas. Specially, the transcriptomics and genomics of co-infecting and hyperparasitic intracellular parasites.
Start Year 2014
Description International Environmental and Omits Synthesis (EOS) Conference 2013 (Cardiff University) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The talk was entitled: Parasite and environmentally induced intersexuality in amphipods: transcriptomic insights into feminisation, immune response and crustacean biomarkers of sexual dysfunction. It concentrated on the pitfalls and challenges of applying 'omic' technology to organisms previously unstudied at the molecular level.

The conference brought together researchers and organisations from a range of disciplines with shared interests in the development of new approaches for data handling, generation and analysis in environmental comics. The areas of interest included bioinformatics, DNA-barcoding, genomics, metagenomics, metabarcoding, transcriptomics, proteomics, metabolomics, epigenetics, evolutionary and ecological omics, phylogenetics, study of ancient DNA and anthropology, new tools, resources and training, and beyond as applied to the study of the natural environmental and environmentally relevant organisms and systems. The resulting interaction and exchange of ideas has helped develop novel approaches, new collaborations and the establishment of a wider integrated 'Omic community'.
Year(s) Of Engagement Activity 2013
URL http://environmentalomics.org/ieos2013/
Description NERC Planet Earth Podcast (January 2015) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact NERC Planet Earth Podcast - A look at the effects of drugs on marine life. This podcast conducted and recorded by Richard Hollingham dealt with much of the work associated with this award. It covered our findings on parasitic feminisation and when into more recent research studying the influence of antidepressants on amphipod biology. It was published on the Natural Environment Research Council's research news website, Planet Earth Online (PEO). The Planet Earth Podcast attracts around 4,000 people/month on Planet Earth Online. It's also hosted on iTunes and has 20,000 followers.
Year(s) Of Engagement Activity 2015
URL http://www.nerc.ac.uk/planetearth/podcasts/
Description Press release to publicise the findings associated with this award: Male crustaceans win battle against being feminised 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press realise (see below) to publicise the findings of the transcriptomic analysis of crustaceans feminised by parasitism and pollution. The findings were reported in many media outlets, e.g. Science Daily (https://www.sciencedaily.com/releases/2014/11/141110083402.htm).

Male crustaceans can 'lock down' their maleness to avoid being completely feminised by seawater contaminated by feminising pollutants, according to scientists.

New research by scientists at the University of Portsmouth has shown that crustaceans turned partially into females retain a core of masculinity, and they may have learned how to do it after evolutionary battles with parasites.

The researchers have also published the entire genetic code for the amphipod crustacean they studied, which they hope will lead to even better understanding of their biology.

The research is published in Environmental Science and Technology.

One of the researchers Dr Alex Ford said: "We've known for some time that fish change sex if they're subjected to even small amounts of oestrogen in the water, but until now we didn't know what was happening to crustaceans.

"What we found is that once a crustacean has decided to be male, it can lock down its maleness. It will still become feminised in many respects, but at its core, it will remain male. This has important implications for how we study the effects of potential feminising pollutants on these creatures."

Fish and some other aquatic creatures are increasingly changing sex because the rivers and oceans are receiving a steady stream of feminising pollutants in sewage and industrial effluent.

Dr Ford and his co-author Dr Stephen Short are marine biologists at the Institute of Marine Sciences at Portsmouth and have been studying the effect of different chemicals on a range of organisms for several years.

Dr Short said: "We don't know why crustaceans, but not vertebrates, have this ability to hold on to their male-ness, but we know crustaceans have been engaged in long evolutionary battles with feminising parasites which turn males into females in order to transmit to the next generation via the eggs of their hosts.

"It could be that this history has given crustaceans strategies to cope with feminisation and this is now proving useful in the face of human pollution."

Some crustaceans decide whether to be male or female soon after hatching. It is, Dr Ford says, "a little window of plasticity".

"In fish and in humans and other mammals if a creature becomes more female, they automatically will become less male as a result. It's like a seesaw. Crustaceans are wired differently and by becoming female, they don't necessary lose their maleness," he said.

The research was part of a four-year project that looked at the effects of parasites and pollution on crustaceans and was funded by the Natural Environment Research Council (NERC).

Dr Ford has previously found that a miniscule amount of drugs - equivalent to a drop in an Olympic sized swimming pool -commonly found in human waste can have a dramatic effect on aquatic life, including changing the speed at which some creatures can swim, to their ability to reproduce.

He said: "Crustaceans are the most diverse creatures in our oceans and, until now, we didn't know a lot about their molecular biology.

"By publishing the entire set of genes we are giving biologists a whole suite of tools to further study many aspects of their biology including their endocrine, nervous and immune systems."
Year(s) Of Engagement Activity 2014
URL http://uopnews.port.ac.uk/2014/11/10/shellfish-win-battle-against-being-feminised/
Description Publicise the discovery of new feminising parasites in UK crustacean populations 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact The press release (see below) was used to write stories in many publications read by the general public, including the independent.

Scientists have discovered a new parasite, previously unknown to science, which may be responsible for turning shrimps and other crustaceans from male to female. The research, which reverses 40 years of scientific thinking, means scientists can start addressing the problem of gender imbalance in crustaceans caused by feminising parasites. Marine biologists at the University of Portsmouth have discovered a new species of paramyxean, a type of parasite, which they believe feminises its host. They found that paramyxean almost always exists alongside another well-known parasite known as microsporidians, which scientists previously believed to be the feminising force. Their research led them to conclude that the existing parasite simply 'hitchhikes' a ride with the new parasite which carries out the work of sex reversal. The research is funded by the Natural Environment Research Council and published in the International Journal for Parasitology.

The newly discovered paramyxean parasite is related to a group of organisms known to cause catastrophic sexual dysfunction in molluscs. The issue can be disastrous for commercial mussel and oyster bed operations and can have major consequences further up the food chain. In amphipod crustaceans such as shrimps (Echinogammarus marinus), both parasites infect their hosts by transmitting vertically from 'mother' to offspring and converting the host in its early, most vulnerable stages of life. Vertically transmitted parasites depend on the reproductive success of their hosts to survive and by turning them female, all parties have the best chance of survival.

ReserachNewsAlex Ford Dr Alex Ford from the University's Institute of Marine Science (IMS) said that the discovery could re-write the textbooks. He said: "It means that we can start looking at a whole different genus causing the feminisation of crustaceans. And this is vital research because we're seeing a gender imbalance which is a serious ecological problem affecting species further up the food chain. Marine creatures such as shrimps and molluscs are food for fish and seabirds which mean the consequences could be profound.

Dr Stephen Short from the IMS said that these species are additionally vulnerable because industrial pollutants such as plastics, oils and toxic PCBs in the water makes them less able to fight infection or attack. "Newborn crustaceans are sexless and are very open to being directed a particular way. What we're currently seeing is worrying for our marine habitats because the problem is chronic and widespread."
Year(s) Of Engagement Activity 2012
URL http://www.port.ac.uk/research/news/title,161889,en.html
Description SETAC Barcelona (2015): The development and application of crustacean biomarkers to monitor reproductive endocrine disruption. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk given on the subject of developing and applying crustacean biomarkers to monitor reproductive endocrine disruption. This was given as part of the SETC 2015 meeting in Barcelona entitled 'Environmental protection in a multi-stressed world: challenges for science, industry and regulators'. Sparked discussion on the use of current biomarkers developed for vertebrate species and created a plan for the further validation of these putative biomarkers tailored to crustacean endocrinology.
Year(s) Of Engagement Activity 2015
URL http://barcelona.setac.eu/general_info/welcome!/?contentid=790&pr_id=766&last=769&sub=790
Description SETAC/iEOS - Joint Focused Topic Meeting on Environmental and (eco)toxicological Omics and Epigenetics (Ghent, Belgium 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The Society of Environmental Toxicology and Chemistry (SETAC) and the UK Environmental Omics Synthesis Centre (iEOS) are organised a joint focused topic meeting on environmental and (eco)toxicological omics and epigenetics. The application of 'omics in environmental and (eco)toxicological science is a fast moving area of research, with new understanding of the epigenome bringing an important new perspective to understanding of species response to environmental perturbations. Thus the jointly organised meeting focused on bringing together the best possible scientists and practitioners in the field of Environmental and (Eco)toxicological Omics and Epigenetics to address key questions within the field and to identify areas that require immediate attention, such as the nature of dose response relationships for epigenetic effects and how to incorporate this information in chemical risk assessment. The meeting showcased new scientific approaches and insights, as well as their practical and regulatory applications.

The symposium consisted of platform and poster presentations from selected abstracts as well as opening lectures from world-renowned invited speakers. Invited speakers provided an overview of key advances that have been made within the field and also highlighted current technologies used for studying epigenetic processes (e.g. companies involved in biochemical test method or sequencing). This enabled discussions on the available technologies, costs, and development of suitable epigenetic assays for chemical risk assessment. Plenary discussions led by the invited speakers and session chairs from academia, government and industry as well as an expert panel debate provided vibrant environment to discuss and identify the key areas that require further investigation and immediate attention, and generated recommendations for developing or validating epigenetic tests and methods.

The talk given was titled: Using transcriptomics to understand crustacean intersexuality: untangling feminisation, demasculinisa6on, parasitic and environmental influences.
Year(s) Of Engagement Activity 2016
URL http://epigenetics.setac.eu/?contentid=1068
Description Seminar at Cefas (Weymouth) in 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A invited talk at Cefas Weymouth on the subject of Microsporidian/paramyxid co-infection transcriptomics. The audience included experts on invertebrate pathology, parasitology and bioinformatics. The talk lead to a discussion about how best to analyse parasitic gene patterns from samples consisting of a mixture of of both host tissue and parasite spores. It also encourages further collaboration to sequence the genome of avirulent paramyxid and microsporidian parasites.
Year(s) Of Engagement Activity 2017