Publications by authors named "Marion Sebire"

15 Publications

  • Page 1 of 1

Skin swabbing is a refined technique to collect DNA from model fish species.

Sci Rep 2020 10 23;10(1):18212. Epub 2020 Oct 23.

Department of Neuroscience, Psychology and Behaviour, College of Life Sciences, University of Leicester, Leicester, LE1 7RH, UK.

Model fish species such as sticklebacks and zebrafish are frequently used in studies that require DNA to be collected from live animals. This is typically achieved by fin clipping, a procedure that is simple and reliable to perform but that can harm fish. An alternative procedure to sample DNA involves swabbing the skin to collect mucus and epithelial cells. Although swabbing appears to be less invasive than fin clipping, it still requires fish to be netted, held in air and handled-procedures that can cause stress. In this study we combine behavioural and physiological analyses to investigate changes in gene expression, behaviour and welfare after fin clipping and swabbing. Swabbing led to a smaller change in cortisol release and behaviour on the first day of analysis compared to fin clipping. It also led to less variability in data suggesting that fewer animals need to be measured after using this technique. However, swabbing triggered some longer term changes in zebrafish behaviour suggesting a delayed response to sample collection. Skin swabbing does not require the use of anaesthetics and triggers fewer changes in behaviour and physiology than fin clipping. It is therefore a more refined technique for DNA collection with the potential to improve fish health and welfare.
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http://dx.doi.org/10.1038/s41598-020-75304-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584585PMC
October 2020

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries.

Chemosphere 2020 Oct 6;256:126946. Epub 2020 May 6.

Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset DT4 8UB, United Kingdom; The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, EH25 9RG, United Kingdom.

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6-77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 μg ml), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.
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http://dx.doi.org/10.1016/j.chemosphere.2020.126946DOI Listing
October 2020

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Gen Comp Endocrinol 2018 02 2;257:97-105. Epub 2017 Aug 2.

Department of Zoology, Stockholm University, Stockholm, Sweden.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-β) and luteinizing hormone (lh-β) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-β mRNA levels showed a peak at 48hps, while fsh-β did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.
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http://dx.doi.org/10.1016/j.ygcen.2017.07.030DOI Listing
February 2018

Application of Passive Sampling to Characterise the Fish Exometabolome.

Metabolites 2017 Feb 14;7(1). Epub 2017 Feb 14.

Centre for Environment, Fisheries and Aquaculture Science, Cefas Weymouth Laboratory, Weymouth, Dorset DT4 8UB, UK.

The endogenous metabolites excreted by organisms into their surrounding environment, termed the exometabolome, are important for many processes including chemical communication. In fish biology, such metabolites are also known to be informative markers of physiological status. While metabolomics is increasingly used to investigate the endogenous biochemistry of organisms, no non-targeted studies of the metabolic complexity of fish exometabolomes have been reported to date. In environmental chemistry, Chemcatcher (Portsmouth, UK) passive samplers have been developed to sample for micro-pollutants in water. Given the importance of the fish exometabolome, we sought to evaluate the capability of Chemcatcher samplers to capture a broad spectrum of endogenous metabolites excreted by fish and to measure these using non-targeted direct infusion mass spectrometry metabolomics. The capabilities of C18 and styrene divinylbenzene reversed-phase sulfonated (SDB-RPS) Empore™ disks for capturing non-polar and polar metabolites, respectively, were compared. Furthermore, we investigated real, complex metabolite mixtures excreted from two model fish species, rainbow trout () and three-spined stickleback (). In total, 344 biological samples and 28 QC samples were analysed, revealing 646 and 215 / peaks from trout and stickleback, respectively. The measured exometabolomes were principally affected by the type of Empore™ (Hemel Hempstead, UK) disk and also by the sampling time. Many peaks were putatively annotated, including several bile acids (e.g., chenodeoxycholate, taurocholate, glycocholate, glycolithocholate, glycochenodeoxycholate, glycodeoxycholate). Collectively these observations show the ability of Chemcatcher passive samplers to capture endogenous metabolites excreted from fish.
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http://dx.doi.org/10.3390/metabo7010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372211PMC
February 2017

Microarray analysis of di-n-butyl phthalate and 17α ethinyl-oestradiol responses in three-spined stickleback testes reveals novel candidate genes for endocrine disruption.

Ecotoxicol Environ Saf 2016 Feb 19;124:96-104. Epub 2015 Oct 19.

Department of Biology, University of Turku, 20014 Turku, Finland; Natural History Museum, University of Oslo, Oslo NO-0318, Norway.

Phthalate esters are plasticizers frequently found in wastewater effluents. Previous studies on phthalates have reported anti-androgenic activity in mammals, causing concerns of their potential effects on the reproduction of aquatic organisms. Another group of environmental endocrine disrupters, steroidal estrogens, are known to inhibit steroid biosynthesis in the gonads, but the effects related to spermatogenesis are not well understood in fish. In this study, three-spined sticklebacks were exposed to di-n-butyl phthalate (DBP) and 17α ethinyl-oestradiol (EE2) at nominal concentrations 35μg/L and 40ng/L, respectively, for four days. The aim of the study was to obtain insight into the acute transcriptional responses putatively associated with endocrine disruption. RNA samples from eight individual male fish per treatment (including controls) were used in microarray analysis, covering the expression of approximately 21,000 genes. In the EE2 treatment the results show transcriptional downregulation of genes associated with steroid biosynthesis pathway and up-regulation of genes involved in pathways related to epidermal growth factor signaling and xenobiotic metabolism. The transcriptional response to DBP was in general weaker than to EE2, but based on enrichment analysis, we suggest adverse effects on retinoid metabolism, creatine kinase activity and cell adhesion. Among the genes showing highest fold changes after DBP treatment compared to control was the teleost fish -specific cytochrome P450 17A2. Overall, this study promotes our understanding on molecular responses to anti-androgens and estrogens in fish testes.
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http://dx.doi.org/10.1016/j.ecoenv.2015.09.039DOI Listing
February 2016

Prozac affects stickleback nest quality without altering androgen, spiggin or aggression levels during a 21-day breeding test.

Aquat Toxicol 2015 Nov 25;168:78-89. Epub 2015 Sep 25.

Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom.

Pharmaceuticals are increasingly being used in human and veterinary medicine, and their presence in the aquatic environment may present a threat to non-target aquatic organisms. The selective serotonin reuptake inhibitor fluoxetine (Prozac) has been reported to affect diverse behaviours (feeding, aggression, and reproduction) and also the endocrine system (steroid biosynthesis pathway) in fish. To investigate these claims further, and in particular effects on androgen synthesis, male three-spined sticklebacks (Gasterosteus aculeatus) were exposed to fluoxetine at 0, 3.2, 10 and 32μg/L in a flow-through system for 21 days. Their sex was determined prior to exposure using a non-invasive method to collect DNA for determining the genetic sex, reported here for the first time. This was necessary as the exposure required males of a non-breeding status which had not developed secondary characteristics. Post exposure a number of biochemical (serotonin, steroid and spiggin levels) and apical (aggressive behaviour) endpoints were measured. No effects were detected on morphometric parameters, spiggin or androgen (11-ketotestosterone) levels. However, all fluoxetine-exposed male fish had higher cortisol levels in comparison to the control fish, although this effect only persisted throughout the whole exposure duration at the highest concentration (32μg/L). In addition, the ratio of 5-HIAA/5-HT (serotonin metabolite/serotonin) was significantly lower in the brains of males exposed to fluoxetine at all concentrations tested. Although we found no differences in the number of nests built by the males, the quality of the nests produced by the fluoxetine-exposed males was generally inferior consisting only of a basic, rudimentary structure. Males exposed to 32μg/L of fluoxetine displayed a delayed response to a simulated threat (rival male via own mirror image) and were less aggressive (number of bites and attacks) toward their mirror image, but these differences were not statistically significant. In summary, fluoxetine exposure resulted in reduced serotonergic activity in the male three-spined stickleback brain suggesting that the mechanism of action between humans and fish is at least partially conserved. Furthermore, this study provided additional evidence of cross-talk between the serotonergic and stress axes as demonstrated by the perturbations in cortisol levels. This potentially complex interaction at brain level may be responsible for the effects observed on nest quality, an endpoint with serious ecological consequences for this species. Finally, despite our hypothesis (an effect on steroid biosynthesis, based on limited literature evidence), we observed no effects of fluoxetine exposure (at the concentrations and duration employed) on male stickleback androgen levels.
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http://dx.doi.org/10.1016/j.aquatox.2015.09.009DOI Listing
November 2015

Environmental chemicals active as human antiandrogens do not activate a stickleback androgen receptor but enhance a feminising effect of oestrogen in roach.

Aquat Toxicol 2015 Nov 26;168:48-59. Epub 2015 Sep 26.

University of Exeter, Biosciences, College of Life & Environmental Sciences, Exeter EX4 4QD, United Kingdom. Electronic address:

Sexual disruption is reported in wild fish populations living in freshwaters receiving discharges of wastewater treatment works (WwTW) effluents and is associated primarily with the feminisation of males by exposure to oestrogenic chemicals. Antiandrogens could also contribute to the feminisation of male fish, but there are far less data supporting this hypothesis and almost nothing is known for the effects of oestrogens in combination with antiandrogens in fish. We conducted a series of in vivo exposures in two fish species to investigate the potency on reproductive-relevant endpoints of the antiandrogenic antimicrobials triclosan (TCS), chlorophene (CP) and dichlorophene (DCP) and the resin, abietic acid (AbA), all found widely in WwTW effluents. We also undertook exposures with a mixture of antiandrogens and a mixture of antiandrogens in combination with the oestrogen 17α-ethinyloestradiol (EE2). In stickleback (Gasterosteus aculeatus), DCP showed a tendency to reduce spiggin induction in females androgenised by dihydrotestosterone (DHT), but these findings were not conclusive. In roach (Rutilus rutilus), exposures to DCP (178 days), or a mixture of TCS, CP and AbA (185 days), or to the model antiandrogen flutamide (FL, 178 days) had no effect on gonadal sex ratio or on the development of the reproductive ducts. Exposure to EE2 (1.5ng/L, 185 days) induced feminisation of the ducts in 17% of the males and in the mixture of antiandrogens (TCS, CP, AbA) in combination with EE2, almost all (96%) of the males had a feminised reproductive ducts. In stickleback androgen receptor (ARα and ARβ) transactivation assays, the model antiandrogens, FL and procymidone inhibited 11-ketotestosterone (11-KT) induced receptor activation, but none of the human antiandrogens, TCS, CP, DCP and AbA had an effect. These data indicate that antimicrobial antiandrogens in combination can contribute to the feminisation process in exposed males, but they do not appear to act through the androgen receptor in fish.
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http://dx.doi.org/10.1016/j.aquatox.2015.09.014DOI Listing
November 2015

In vivo endocrine effects of naphthenic acids in fish.

Chemosphere 2013 Nov 12;93(10):2356-64. Epub 2013 Sep 12.

Department of Biology, University of Bergen, Bergen, Norway. Electronic address:

Oil pollution from various sources, including exploration, production and transportation, is a growing global concern. The highest toxicity of hydrocarbon pollutants is associated with the water-soluble phase compounds, including naphthenic acids, a known component found in all hydrocarbon deposits. Recently, naphthenic acids (NAs) have shown estrogenic and anti-androgenic effects in vitro. For this reason we investigated the potential effects of two commercial mixtures of naphthenic acids on fish in vivo, using the three-spined stickleback (Gasterosteus aculeatus) as a model species. Anti-androgenic and estrogenic properties of tested compounds were evaluated using the androgenized female stickleback screen (AFSS) and a variant of the 21-d fish screen (TG230) respectively. One-dimensional gas chromatography-mass spectrometry (GC-MS) showed that the complex commercial NAs mixtures were dominated by acyclic carboxylic acids. In one experiment (freshwater) we found a clear effect of NA exposure on spiggin levels; this was contrary to our hypothesis since NAs enhanced the androgenic potency of DHT (when co-administered) without inducing spiggin when tested in the absence of DHT. Exposure to NAs did not have a statistically significant effect on vitellogenin (Vtg) production in male stickleback, although the Vtg responses were increasing with increasing exposure concentrations. This study shows that in contrast to previous in vitro data, NAs did not exhibit either estrogenic or anti-androgenic properties in vivo, at the concentrations tested. On the contrary, at least in freshwater, NAs appear to have an overall androgenic effect that is not mediated via the androgen receptor involved in spiggin synthesis. Possible reasons for this discrepancy between in vitro and in vivo results as well as between our studies are discussed.
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http://dx.doi.org/10.1016/j.chemosphere.2013.08.033DOI Listing
November 2013

Short-term exposure to a treated sewage effluent alters reproductive behaviour in the three-spined stickleback (Gasterosteus aculeatus).

Aquat Toxicol 2011 Sep 27;105(1-2):78-88. Epub 2011 May 27.

Cefas Weymouth Laboratory, The Nothe, Weymouth, Dorset DT4 8UB, UK.

Some UK sewage treatment work (STW) effluents have been found to contain high levels of anti-androgenic activity, but the biological significance of this activity to fish has not been determined. The aim of this study was to investigate the effects of exposure to a STW effluent with anti-androgenic activity on the reproductive physiology and behaviour of three-spined sticklebacks (Gasterosteus aculeatus). Fish were exposed to a STW effluent (50 and 100%, v/v) with a strong anti-androgenic activity (328.56±36.83 μgl(-1) flutamide equivalent, as quantified in a recombinant yeast assay containing the human androgen receptor) and a low level of oestrogenic activity (3.32±0.66 ngl(-1) oestradiol equivalent, quantified in a recombinant yeast assay containing the human oestrogen receptor) for a period of 21 days in a flow-through system in the laboratory. Levels of spiggin, an androgen-regulated protein, were not affected by the STW effluent exposure, nor were levels of vitellogenin (a biomarker of oestrogen exposure), but the reproductive behaviour of the males was impacted. Males exposed to full strength STW effluent built fewer nests and there was a significant reduction in male courtship behaviour for exposures to both the 50 and 100% STW effluent treatments compared with controls. The effect seen on the reproduction of male sticklebacks may not necessarily have been as a consequence of the endocrine active chemicals present in the STW effluent alone, but could relate to other features of the effluent, such as turbidity that can impair visual signalling important for courtship interactions. Regardless the specific causation, the data presented show that effluents from STW have an impact on reproductive behaviour in male sticklebacks which in turn affects reproductive performance/outcome. The study further highlights the use of fish behaviour as a sensitive endpoint for assessing potential effects of contaminated water bodies on fish reproduction.
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http://dx.doi.org/10.1016/j.aquatox.2011.05.014DOI Listing
September 2011

The model anti-androgen flutamide suppresses the expression of typical male stickleback reproductive behaviour.

Aquat Toxicol 2008 Oct 13;90(1):37-47. Epub 2008 Aug 13.

Cefas Weymouth laboratory, Environment and Animal Health, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK.

Over the past 15 years considerable attention has been given to the presence in the environment of endocrine disrupting chemicals (EDCs) that may have harmful effects on organisms. Specific test guidelines for the detection of EDCs used for short-term fish screening assays have been developed by the Organisation for Economic Cooperation and Development (OECD). Compared to the core species used in the OECD guidelines, the three-spined stickleback (Gasterosteus aculeatus) has an additional and unique endpoint for (anti-)androgenic substances through the androgen-dependent glue protein (spiggin) used in the nest building. Here we describe a specific behavioural assay that was developed in parallel to the OECD protocol, utilising unique behavioural features of sticklebacks. In the assay, a photoperiod of 16L:8D (light:dark) and a temperature of 17+/-1 degrees C was used to induce breeding in quiescent male sticklebacks that were simultaneously exposed for a 21-day period to the mammalian anti-androgen flutamide (FL) at 100, 500 and 1000 microg/l (plus a water control). Spiggin production and the reproductive behaviour (nest building and courtship) of male sticklebacks were the main measured endpoints. The control fish entered an active breeding cycle including nest building and courtship behaviours as expected due to the stimulating temperature and photoperiodic conditions. The FL-exposed males showed significantly lower spiggin levels at 500 and 1000 microg/l. In addition, there was a significant decrease in the number of nests built by the FL-treated males at 100 microg/l with no nest built at 500 and 1000 microg/l. Finally, FL affected the courtship behaviour of the males with a significant reduction of the number of zigzags towards the female. When the breeding status of the stickleback males is controlled, the behavioural assay developed here is a suitable tool for the detection of androgen antagonists.
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http://dx.doi.org/10.1016/j.aquatox.2008.07.016DOI Listing
October 2008

The organophosphorous pesticide, fenitrothion, acts as an anti-androgen and alters reproductive behavior of the male three-spined stickleback, Gasterosteus aculeatus.

Ecotoxicology 2009 Jan 21;18(1):122-33. Epub 2008 Sep 21.

Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK.

Fenitrothion (FN) is a widely used organophosphorous pesticide that has structural similarities with the clinical anti-androgen flutamide. The potential for FN to act as an anti-androgen (at exposures of 1, 50, and 200 microg FN/l over a 26-day period) was assessed in male three-spined sticklebacks, Gasterosteus aculeatus, by measuring kidney spiggin concentration, nest-building, and courtship behavior. Spiggin is the glue protein that male sticklebacks use to build their nests and is directly controlled by androgens. FN exposure significantly reduced spiggin production as well as nest-building activity. It also adversely affected courtship--especially the 'zigzag dance' and biting behavior of the males. FN thus appears to have anti-androgenic effects on both the physiology and behavior of the male stickleback.
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http://dx.doi.org/10.1007/s10646-008-0265-2DOI Listing
January 2009

Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Mar Environ Res 2008 Jul 26;66(1):161-3. Epub 2008 Feb 26.

Cefas Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, UK.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.
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http://dx.doi.org/10.1016/j.marenvres.2008.02.049DOI Listing
July 2008

Three-spined stickleback: an emerging model in environmental endocrine disruption.

Environ Sci 2007 ;14(5):263-83

Cefas Weymouth Laboratory, Barrack Road, Weymouth, Dorset DT4 8UB, UK.

The three-spined stickleback, a small teleost species with habitats that range from full marine to fresh water bodies across the whole Northern hemisphere, has a number of advantages for endocrine disruption research. It is the only teleost species with an unambiguous biomarker for androgens, the presence of the glue protein spiggin in the male kidney, which can be measured by enzyme-linked immunosorbent assay (ELISA). The androgen assay has been adopted to detect antiandrogens in two different ways and an homologous ELISA for stickleback vitellogenin is also available. DNA markers for molecular sex determination are available; thus, sex ratios can also be used for in situ biomonitoring. In addition, the critical period of sexual differentiation has been determined and the occurrence of intersex fish has been reported several times. The species full genome sequence is almost complete. All aspects of stickleback biology (ecology, evolution, behavior, physiology, endocrinology) are well documented. In European waters, the stickleback is the only fish that can bring laboratory and field studies together and allow the true impact of endocrine disruptors on fish populations to be evaluated.
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June 2008

Non-invasive measurement of 11-ketotestosterone, cortisol and androstenedione in male three-spined stickleback (Gasterosteus aculeatus).

Gen Comp Endocrinol 2007 May 20;152(1):30-8. Epub 2007 Feb 20.

The Centre for Environment, Fisheries and Aquaculture Science, Weymouth, Dorset DT4 8UB, UK.

The androgen 11-ketotestosterone (11-KT) plays an important role in reproductive physiology and behaviour in male teleosts. In the three-spined stickleback, Gasterosteus aculeatus, the plasma concentrations of 11-KT are related to the breeding status of the fish. Sticklebacks are relatively small (generally less than 1g) and in order to obtain sufficient plasma for assay of 11-KT, it has been necessary in the past to sacrifice the fish. In this paper, we report on the development of a non-invasive procedure for measuring 11-KT, cortisol and androstenedione (Ad) in the three-spined stickleback. Validation of the procedure included the demonstration that the rate of release of steroids into the water was correlated to their plasma concentrations. Ten males that were kept at a low temperature and short photoperiod were moved to high temperature and long photoperiod to initiate spermatogenesis and breeding. Every two to four days, for a total of 53 days, males were removed and placed in a beaker containing 50-ml water for 30 min. The water was then processed by solid phase extraction for radioimmunoassay. Males were presented with females on days 13/14, 18/19 and 44/45. 11-KT was originally undetectable but built up gradually to reach an average release rate of between 1 and 2.5 ng/g/h between days 16 and 45 and then started to decline (but non-significantly). Ad release reached a plateau of 1 ng/g/h about day 20. However, from days 44/45 to 51, there was a highly significant rise in the rate of release of Ad to 5 ng/g/h. On days 44/45, five of the males mated successfully and five did not. However, there were no significant differences in 11-KT or Ad release rates between the two groups. Cortisol release rates fluctuated with no pattern throughout the study. The results show that it is possible to make measurements on sex and stress steroid production in sticklebacks without recourse to anaesthesia, bleeding or sacrificing the fish. The procedure is potentially a powerful tool for the study of the link between steroids and behaviour in this useful sentinel species.
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http://dx.doi.org/10.1016/j.ygcen.2007.02.009DOI Listing
May 2007

Settlement behaviour of marine invertebrate larvae measured by EthoVision 3.0.

Biofouling 2004 Aug-Oct;20(4-5):211-7

School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.

Submerged marine surfaces are rapidly colonized by fouling organisms. Current research is aimed at finding new, non-toxic, or at least environmentally benign, solutions to this problem. Barnacles are a major target organism for such control as they constitute a key component of the hard fouling community. A range of standard settlement assays is available for screening test compounds against barnacle cypris larvae, but they generally provide little information on mechanism(s) of action. Towards this end, a quick and reliable video-tracking protocol has been developed to study the behaviour of the cypris larvae of the barnacle, Balanus amphitrite, at settlement. EthoVision 3.0 was used to track individual cyprids in 30-mm Petri dishes. Experiments were run to determine the optimal conditions vis-a-vis acclimation time, tracking duration, number of replicates, temperature and lighting. A protocol was arrived at involving a two Petri dish system with backlighting, and tracking over a 5-min period after first acclimating the cyprids to test conditions for 2 min. A minimum of twenty replicates was required to account for individual variability in cyprid behaviour from the same batch of larvae. This methodology should be widely applicable to both fundamental and applied studies of larval settlement and with further refinements, to that of smaller fouling organisms such as microalgae and bacteria.
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http://dx.doi.org/10.1080/08927010400011674DOI Listing
May 2005