The detoxification ability of sex-role reversed seahorses determines the sexual dimorphism in immune responses to benzo[a]pyrene exposure.

Sexual dimorphism in immune responses is an essential factor in environmental adaptation. However, the mechanisms involved remain obscure owing to the scarcity of data from sex-role-reversed species in stressed conditions. Benzo[a]pyrene (BaP) is one of the most pervasive and carcinogenic organic pollutants in coastal environments. In this study, we evaluated the potential effects on renal immunotoxicity of the sex-role-reversed lined seahorse (Hippocampus erectus) toward environmental concentrations BaP exposure. Our results discovered the presence of different energy-immunity trade-off strategies adopted by female and male seahorses during BaP exposure. BaP induced more severe renal damage in female seahorses in a concentration-dependent manner. BaP biotransformation and detoxification in seahorses resemble those in mammals. Benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BPDE) and 9-hydroxybenzo[a]pyrene (9-OH-BaP) formed DNA adducts and disrupted Ca2+ homeostasis may together attribute the renal immunotoxicity. Sexual dimorphisms in detoxification of both BPDE and 9-OH-BaP, and in regulation of Ca2+, autophagy and inflammation, mainly determined the extent of renal damage. Moreover, the mechanism of sex hormones regulated sexual dimorphism in immune responses needs to be further elucidated. Collectively, these findings contribute to the understanding of sexual dimorphism in the immunotoxicity induced by BaP exposure in seahorses, which may attribute to the dramatic decline in the biodiversity of the genus.

Behavioural responses of threespine stickleback with lateral line asymmetries to experimental mechanosensory stimuli.

Behavioural asymmetry, typically referred to as laterality, is widespread among bilaterians and is often associated with asymmetry in brain structure. However, the influence of sensory receptor asymmetry on laterality has undergone limited investigation. Here we used threespine stickleback (Gasterosteus aculeatus) to investigate the influence of lateral line asymmetry on laterality during lab simulations of three mechanosensation-dependent behaviours: predator evasion, prey localization and rheotaxis. We recorded the response of stickleback to impacts at the water surface and water flow in photic conditions and low-frequency oscillations in the dark, across four repeat trials. We then compared individuals' laterality with asymmetry in the number of neuromasts on either side of their body. Stickleback hovered with their right side against the arena wall 57% of the time (P<0.001) in illuminated surface impact trials and 56% of the time (P=0.085) in dark low-frequency stimulation trials. Light regime modulated the effect of neuromast count on laterality, as fish with more neuromasts were more likely to hover with the wall on their right during illumination (P=0.007) but were less likely to do so in darkness (P=0.025). Population-level laterality diminished in later trials across multiple behaviours and individuals did not show a consistent side bias in any behaviours. Our results demonstrate a complex relationship between sensory structure asymmetry and laterality, suggesting that laterality is modulated by multiple sensory modalities and is temporally dynamic.

Variation in the Sensory Space of Three-spined Stickleback Populations.

The peripheral sensory systems, whose morphological attributes help determine the acquisition of distinct types of information, provide a means to quantitatively compare multiple modalities of a species' sensory ecology. We used morphological metrics to characterize multiple sensory modalities-the visual, olfactory, and mechanosensory lateral line sensory systems-for Gasterosteus aculeatus, the three-spined stickleback, to compare how sensory systems vary in animals that evolve in different ecological conditions. We hypothesized that the dimensions of sensory organs and correlations among sensory systems vary in populations adapted to marine and freshwater environments, and have diverged further among freshwater lake-dwelling populations. Our results showed that among environments, fish differed in which senses are relatively elaborated or reduced. When controlling for body length, littoral fish had larger eyes, more neuromasts, and smaller olfactory tissue area than pelagic or marine populations. We also found differences in the direction and magnitude of correlations among sensory systems for populations even within the same habitat type. Our data suggest that populations take different trajectories in how visual, olfactory, and lateral line systems respond to their environment. For the populations we studied, sensory modalities do not conform in a predictable way to the ecological categories we assigned.

Immune defense parameters of wild fish as sensitive biomarkers for ecological risk assessment in shallow sea ecosystems: A case study with wild mullet (Liza haematocheila) in Liaodong Bay.

Environmental monitoring is important to the health management of an ecosystem. Biomarkers are particularly relevant because they are direct indicators of any toxic effects on organisms and are cheaper to use compared with chemical indicators, especially for extremely low-level organic contaminants. Fish can be significantly affected by pollutants, given their high trophic levels in aquatic food chains. Their immune function is closely related to their survival. The present study compared immune function-related parameters of wild mullet (Liza haematocheila) samples from low (Jinzhou) and high (Yingkou) polluted sites during the pre-winter (PW) and pre-breeding (PB) periods in Liaodong Bay, to evaluate the effect of water pollution on fish health and to explore potential biomarkers of coast water pollution. Compared with Jinzhou mullet, there was a significantly higher level of hematocrit in Yingkou mullet, but a significantly lower serum lysozyme level (P < 0.001), indicating that these fish were immunosuppressed. Significant differences occurred in the spleen between the two site populations. The abnormal: normal fish ratio in Yingkou L. haematochila was significantly higher than that of Jinzhou L. haematochila (2.5 times of that of Jinzhou during PB and nine times during PW). The splenic index of male Yingkou L. haematochila was 47.2% higher than that of Jinzhou L. haematochila in PW (P = 0.001). Moreover, histological observations showed that the spleen of the former was more congestive, with increased numbers (39.6% more) of melanomacrophage centers (MMCs) and changes in pigments (hemosiderin 8.3% higher and melanin 29.4% higher), compared with the latter. The splenic MMC area of Yingkou L. haematochila was significantly smaller than that of Jinzhou L. haematochila (P < 0.05) in PB, but showed no clear difference in PW (P > 0.05). Splenic MMC number was significantly higher in individual Yingkou L. haematochila with abnormal livers compared with normal Yingkou L. haematochila during both sampling periods. The splenic MMC area in abnormal livers was approximately four times those of normal individuals during PB in Yingkou L. haematochila. The number of splenic melanomacrophages (MM) in abnormal livers was approximately nine times those of the normal livers during PW. There were also differences in pigments in normal Yingkou individuals compared with normal Jinzhou samples during PW (melanin 29.4% higher and hemosiderin 8.3% higher). Based on these results, we suggest that serum lysozyme activity, splenic MM number and MMC (both number and area), and melanin of local fish have potential as sensitive biomarkers for the assessment of coastal water pollution.

Morphological and histopathological changes in seahorse (Hippocampus reidi) gills after exposure to the water-accommodated fraction of diesel oil.

Industrial activities and urbanization are the main sources of pollutants in estuarine environments. Diesel, which is widely used in urban and port activities, is an important source of hydrocarbons in the aquatic environment, and its water-accommodated fraction (WAF) is toxic to the local biota. This study was performed to analyze the effects of diesel oil WAF on fish. Specifically, we characterized the gill morphology of the seahorse Hippocampus reidi and analyzed the histopathological changes in the gills after exposure to 50% diesel oil WAF. Acute (12, 24, 48, and 96 h) and subchronic (168 and 336 h) toxicity tests were performed. Furthermore, a recovery protocol was conducted: after exposure to 50% WAF for 168 h, the fish were transferred and kept in seawater without contaminants for 336 h, for a total experimental period of 504 h. The seahorse branchial apparatus was found to be tufted with short filaments; the apical surfaces of the pavement cells in the filament and lamellar epithelia formed "crests" and had microridges. Mitochondria-rich cells were distributed exclusively in the lamellar epithelium, while mucous cells were distributed in the filament epithelium. All pathologies observed after acute and subchronic exposure featured progressive time-dependent alterations of lamellar structure that might disrupt gill physiological and metabolic functions. During the recovery period, the gill alterations were gradually repaired.

Repeatability of Adaptive Radiation Depends on Spatial Scale: Regional Versus Global Replicates of Stickleback in Lake Versus Stream Habitats.

The repeatability of adaptive radiation is expected to be scale-dependent, with determinism decreasing as greater spatial separation among "replicates" leads to their increased genetic and ecological independence. Threespine stickleback (Gasterosteus aculeatus) provide an opportunity to test whether this expectation holds for the early stages of adaptive radiation-their diversification in freshwater ecosystems has been replicated many times. To better understand the repeatability of that adaptive radiation, we examined the influence of geographic scale on levels of parallel evolution by quantifying phenotypic and genetic divergence between lake and stream stickleback pairs sampled at regional (Vancouver Island) and global (North America and Europe) scales. We measured phenotypes known to show lake-stream divergence and used reduced representation genome-wide sequencing to estimate genetic divergence. We assessed the scale dependence of parallel evolution by comparing effect sizes from multivariate models and also the direction and magnitude of lake-stream divergence vectors. At the phenotypic level, parallelism was greater at the regional than the global scale. At the genetic level, putative selected loci showed greater lake-stream parallelism at the regional than the global scale. Generally, the level of parallel evolution was low at both scales, except for some key univariate traits. Divergence vectors were often orthogonal, highlighting possible ecological and genetic constraints on parallel evolution at both scales. Overall, our results confirm that the repeatability of adaptive radiation decreases at increasing spatial scales. We suggest that greater environmental heterogeneity at larger scales imposes different selection regimes, thus generating lower repeatability of adaptive radiation at larger spatial scales.

High levels of docosahexaenoic acid are present in eight New World silversides (Pisces: Atherinopsidae)

Docosahexaenoic acid (DHA) is the most critical and least available omega-3 fatty acid in the Western human diet. Currently, the source of omega-3 long chain polyunsaturated fatty acids (LC-PUFA) is mainly dependent on wild fisheries, making this resource unsustainable in the foreseeable future. In recent years, a high rate of biosynthesis and accumulation of DHA has been discovered in a freshwater species (Chirostoma estor) belonging to the Atherinopsidae family. Interest in evaluating fatty acid composition in other members of the family has emerged, so this study compiles original data of flesh composition of eight atherinopsid species from freshwater and brackish environments, either wild or cultured. High levels of DHA (16 to 31%) were found in all analyzed members of the family, except in C. grandocule, independently of their habitat or origin. The analyzed species of the Jordani group (C. estor, C. promelas and C. humboldtianum) showed high DHA and low EPA levels (<0.5%) as previously reported for cultured C. estor. The low trophic niche of these atherinopsids and their fatty acid accumulation capabilities are factors that make these species noteworthy candidates for sustainable aquaculture.(AU)

O ácido docosahexaenóico (DHA) é o ácido graxo ômega-3 mais importante e menos disponível na dieta humana ocidental. Atualmente, a fonte de ácidos graxos poliinsaturados de cadeia longa ômega-3 (LC-PUFA) depende principalmente da pesca extrativista, tornando esse recurso insustentável em um futuro próximo. Nos últimos anos, uma alta taxa de biossíntese e acúmulo de DHA foi descoberta em uma espécie de água doce (Chirostoma estor) pertencente à família Atherinopsidae. Deste modo, surgiu o interesse em avaliar a composição de ácidos graxos em outros membros da família. Portanto, este estudo compila dados originais da composição de carne de oito espécies de aterinopsídeos de ambientes de água doce e salobra, selvagens ou cultivadas. Altos níveis de DHA (16 a 31%) foram encontrados em todos os membros da família analisados, exceto em C. grandocule, independentemente de seu habitat ou origem. As espécies analisadas do grupo Jordani (C. estor, C. promelas e C. humboldtianum) apresentaram altos níveis de DHA e EPA baixos (<0,5%), como relatado anteriormente para C. estor cultivado. O baixo nicho trófico desses aterinopsídeos e sua capacidade de acumulação de ácidos graxos são fatores que tornam essas espécies notáveis candidatas à aquicultura sustentável.(AU)

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.(AU)

El pez pipa (Microphis brachyurus) es un organismo dulceacuícola con alto potencial económico para la acuarofilia; sin embargo, es necesario implementar su cultivo a través de estudios de fisiología digestiva. Se evaluó el efecto del pH, temperatura e inhibidores sobre las actividades enzimáticas de proteasas ácidas y alcalinas. El pH óptimo de proteasas estomacales es de 2, mientras que el de proteases intestinales es de 10. La temperatura óptima de proteasas ácidas es de 35 ºC y las alcalinas de 45 ºC. La estabilidad térmica para proteasas ácidas y alcalinas es a los 35 ºC (más de 100% de actividad residual). La estabilidad a los diferentes pH de las proteasas ácidas es en 2 (50 % de la actividad residual), mientras que para las proteasas alcalinas es en 10 (90 % de la actividad residual). Las proteasas ácidas fueron inhibidas en 80% con pepstatina A y las proteasas alcalinas fueron altamente inhibidas con TLCK para tripsina (75%) y TPCK quimitripsina (80%). Finalmente, las metaloproteasas fueron inactivadas con EDTA en 70%. En conclusión, M. brachyurus tiene una buena capacidad digestiva al degradar una amplia variedad de proteinas debido a su alta actividad proteolítica.(AU)

A key metabolic gene for recurrent freshwater colonization and radiation in fishes.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

The role of variation and plasticity in parental care during the adaptive radiation of three-spine sticklebacks.

Phenotypic plasticity might influence evolutionary processes such as adaptive radiations. Plasticity in parental care might be especially effective in facilitating adaptive radiations if it allows populations to persist in novel environments. Here, we test the hypothesis that behavioral plasticity by parents in response to predation risk facilitated the adaptive radiation of three-spine sticklebacks. We compared the behavior of fathers across multiple ancestral (marine) and derived (freshwater) stickleback populations that differ in time since establishment. We measured behavioral plasticity in fathers in response to a predator found only in freshwater environments, simulating conditions marine males experience when colonizing freshwater. The antipredator behavior of males from newly established freshwater populations was intermediate between marine populations and well-established freshwater populations. In contrast to our predictions, on average, there was greater behavioral plasticity in derived freshwater populations than in ancestral marine populations. However, we found greater individual variation in behavioral reaction norms in marine populations compared to well-established freshwater populations, with newly established freshwater populations intermediate. This suggests that standing variation in behavioral reaction norms within ancestral populations might provide different evolutionary trajectories, and illustrates how plasticity can contribute to adaptive radiations.

Effects of a chronic exposure to different water temperatures and/or to an environmental cadmium concentration on the reproduction of the threespine stickleback (Gasterosteus aculeatus).

Knowledge about combined effects of chemicals and temperature on reproductive capacity of fish are rare in literature, especially when it comes to the effects of chronic low-dose chemical exposure combined to the thermal stress. The aim of the study was to evaluate the single and combined effects of temperature (16, 18, 21 °C) and an environmentally relevant concentration of waterborne cadmium (1 µg L-1, nominal concentration) on the reproductive outputs of threespine stickleback (Gasterosteus aculeatus), and their consequences on offspring survival parameters. The high temperature (21 °C) was the only factor that affected parental parameters (gonadosomatic index "GSI", and vitellogenin "VTG" particularly). On females, 21 °C had a stimulating effect on gonadal development evaluated by an early increase, followed by a sharp decrease of GSI, probably indicating gonadal atresia. Promoting effect of temperature was corroborated by an early production of VTG. In vitro fertilization assays showed interesting results, particularly cadmium effects. As it was supposed, high temperature had a negative impact on offspring parameters (significant decrease in survival and an increase of unhatched embryos). Parental exposure to the very low concentration of cadmium had also negative consequences on mortality rate (significant increase) and hatching rate (significant decrease). Our results indicate that in a global warming context, high temperature and its combination with contaminant may impact reproductive capacity of G. aculeatus, by decreasing parental investment (low eggs and/or sperm quality).

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Effects of chronic exposure to cadmium and temperature, alone or combined, on the threespine stickleback (Gasterosteus aculeatus): Interest of digestive enzymes as biomarkers.

The development of predictive, sensitive and reliable biomarkers is of crucial importance for aquatic biomonitoring to assess the effects of chemical substances on aquatic organisms, especially when it comes to combined effects with other stressors (e.g. temperature). The first purpose of the present study was to evaluate the single and combined effects of 90 days of exposure to an environmental cadmium concentration (0.5 µg L-1) and two water temperatures (16 and 21 °C) on different parameters. These parameters are involved in (i) the antioxidant system (superoxide dismutase activity -SOD- and total glutathione levels -GSH-), (ii) the energy metabolism, i.e. energy reserves (glycogen, lipids, proteins) and digestive enzymes (trypsin, amylase, intestinal alkaline phosphatase -IAP-), and (iii) biometric parameters (weight, length, Fulton's condition factor, and the gonadosomatic index -GSI-) of threespine stickleback (Gasterosteus aculeatus). The second purpose was to determine the interest of the three digestive enzymes as biomarkers in comparison with the other parameters. The higher temperature (21 °C) impacted the anti-oxidant and energy reserve parameters. In liver, GSH levels increased on day 60, while SOD decreased on days 15 and 90, with a significant decrease of protein and lipid energy reserves on day 90. In muscle, the higher temperature decreased SOD activity only on day 90. G. aculeatus biometric parameters were also impacted by the higher temperature, which limited stickleback growth after 90 days of exposure. In female sticklebacks, the GSI peaked on day 60 and decreased sharply on day 90, while the highest values were reached at day 90 in the control groups, suggesting impaired reproduction in sticklebacks raised at 21 °C. These results suggest that 21 °C is an upper-limit temperature for long-term physiological processes in sticklebacks. In contrast, very low-concentration cadmium exposure had no effect on classical biomarkers (energy reserves, antioxidant parameters, biometric parameters). However, digestive enzymes showed an interesting sensitivity to cadmium, which was emphasized by high temperature. The activity of the three digestive enzymes decreased significantly on day 90 when sticklebacks were exposed to cadmium alone, while the decrease was stronger and was recorded earlier (from day 15) when they were exposed to the cadmium-temperature combination. Compared to conventional measurements, digestive enzymes responded rapidly. This could be an important advantage for them to be used as early warning tools to reflect the health status of organisms, particularly for trypsin and IAP activities.

Interactive effects of salinity and temperature acclimation on gill morphology and gene expression in threespine stickleback.

Colonization of freshwater habitats from marine environments exposes organisms to novel combinations of temperature and salinity, but little is known about physiological responses to the interactive effects of these stressors. Here, we examined the effects of temperature (14 versus 4 °C) and salinity (11 versus 0.3 ppt) on gill gene expression in marine, anadromous, and freshwater populations of threespine stickleback (Gasterosteus aculeatus). Expression of the epithelial calcium channel was not affected by temperature or salinity, but had significantly higher expression in the freshwater ecotype. The combination of low temperature and low salinity had non-additive effects on the expression of the Na+/H+ exchanger. Fish exposed to the combination of low temperature and low salinity had expression levels similar to fish exposed to either factor in isolation. Expression of Na+,K+-ATPase α-subunit was greater in fish exposed to low temperature and low salinity than in fish exposed to the factors separately, and this effect was the most pronounced in the marine ecotype. We also examined the interactive effects of salinity and temperature on gill morphology in the marine ecotype, and observed non-additive effects. Low temperature increased the size of the interlamellar cell mass in fish held at 11 ppt, but not at 0.3 ppt, and the effect of low salinity was in the opposite direction in fish at high and low temperatures. These data demonstrate interactive effects of temperature and salinity and highlight that overwintering in cold freshwater was likely a physiological challenge for marine stickleback as they colonized freshwater following the last glaciation.

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

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-ß) and luteinizing hormone (lh-ß) 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-ß mRNA levels showed a peak at 48hps, while fsh-ß 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.

Morphology and hydro-sensory role of superficial neuromasts in schooling behaviour of yellow-eyed mullet (Aldrichetta forsteri).

The lateral line system is a mechanosensory organ found in all fish species and located on the skin or in subdermal canals. The basic functional units are superficial and canal neuromasts, which are involved in hydrodynamic sensing and cohesion in schooling fish. Yellow-eyed mullet (Aldrichetta forsteri) are an obligate schooling species found commonly in shallow coastal areas of New Zealand and Australia. Schooling is a fundamental part of their behavioural repertoire, yet little is known about the structure or functionality of the lateral line in this species. We used scanning electron microscopy to characterise the morphology of trunk superficial neuromasts. We then took a multi-sensory approach and conducted behavioural experiments comparing school structure in groups of fish with and without fully functioning lateral lines, under photopic and scotopic conditions. A highly developed hydro-sensing system exists on the trunk of yellow-eyed mullet consisting of superficial neuromasts containing hundreds of hair cells aligned, with respect to their most sensitive axis, in a rostrocaudal direction. Without functioning superficial neuromasts, schooling behaviour was disrupted under both photopic and scotopic conditions and the ability to detect stationary objects decreased. Results highlight the importance of this component of the lateral line system to schooling behaviour.

Convergent evolution of SWS2 opsin facilitates adaptive radiation of threespine stickleback into different light environments.

Repeated adaptation to a new environment often leads to convergent phenotypic changes whose underlying genetic mechanisms are rarely known. Here, we study adaptation of color vision in threespine stickleback during the repeated postglacial colonization of clearwater and blackwater lakes in the Haida Gwaii archipelago. We use whole genomes from 16 clearwater and 12 blackwater populations, and a selection experiment, in which stickleback were transplanted from a blackwater lake into an uninhabited clearwater pond and resampled after 19 y to test for selection on cone opsin genes. Patterns of haplotype homozygosity, genetic diversity, site frequency spectra, and allele-frequency change support a selective sweep centered on the adjacent blue- and red-light sensitive opsins SWS2 and LWS. The haplotype under selection carries seven amino acid changes in SWS2, including two changes known to cause a red-shift in light absorption, and is favored in blackwater lakes but disfavored in the clearwater habitat of the transplant population. Remarkably, the same red-shifting amino acid changes occurred after the duplication of SWS2 198 million years ago, in the ancestor of most spiny-rayed fish. Two distantly related fish species, bluefin killifish and black bream, express these old paralogs divergently in black- and clearwater habitats, while sticklebacks lost one paralog. Our study thus shows that convergent adaptation to the same environment can involve the same genetic changes on very different evolutionary time scales by reevolving lost mutations and reusing them repeatedly from standing genetic variation.

Plasma levels of immune factors and sex steroids in the male seahorse Hippocampus erectus during a breeding cycle.

To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study. The immune factors included monocytes/leucocytes (M/L), leucocyte phagocytic rate (LPR), immunoglobulin M (Ig M), interleukin-2 (IL-2), interferon-α (IFN-α), and lysozyme (LZM). The sex steroids included testosterone (T), 11-ketotestosterone (11-KT), 11ß-hydroxytestosterone (11ß-OHT), 17α-methyltestosterone (17α-MT), 17ß-estradiol (E2), and 17α-hydroxy-20ß-dihydroprogesterone (17α-20ß-P). Moreover, the immune metabolic activity of epithelium cells in the brood pouch at different breeding stages was also analyzed through ultrastructural observations of the abundance of cytoplasmic granules, mitochondria, endoplasmic reticulum, lysosomes, and exocytosis. The results show that a higher immune level was observed during pregnancy, particularly in the early and middle periods, and a lower immune level was noted during pre-pregnancy. Correspondingly, the epithelium cells in the brood pouch also showed a stronger immune metabolic activity during pregnancy and weaker activity during pre-pregnancy. Four sex steroids of T, 11ß-OHT, 17α-MT, and E2 were higher during pre-pregnancy and lower during post-pregnancy, whereas 11-KT and 17α-20ß-P, which were positively correlated with part immune factors, were higher during pregnancy. No negative correlations between sex steroids and immune factors were observed. In conclusion, the higher immune competence during pregnancy may indicate that parental care could improve immunity, which may be the major factor for no immunosuppressive effect of sex steroids during reproduction in the seahorse H. erectus, unlike noncaregiving fishes in which inhibitions of sex steroids on immunity are frequently observed. Moreover, higher 11-KT and 17α-20ß-P during pregnancy than during pre-pregnancy and post-pregnancy may suggest that these two steroids are also involved in parental care regulation.

Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure.

To get closer to the environmental reality, ecotoxicological studies should no longer consider the evaluation of a single pollutant, but rather combination of stress and their interaction. The aim of this study was to determine if responses of a fish to a sudden biological stress could be modified by a prior exposure to a chemical stress (a polymetallic contamination). For this purpose, in situ experiment was conducted in three ponds in the Haute-Vienne department (France). One pond was chosen for its high uranium concentration due to uranium mine tailings, and the two other ponds, which were not submitted to these tailings. Three-spined sticklebacks (Gasterosteus aculeatus) were caged in these ponds for 14 days. After this period, fish were submitted to a biological stress, exerted by lipopolysaccharides injection after anesthesia, and were sacrificed 4 days after these injections for multi-biomarkers analyses (leucocyte viability, phagocytic capacity and reactive oxygen species production, antioxidant peptide and enzymes, lipid peroxidation and DNA damage). The pond which received uranium mine tailings had higher metallic concentrations. Without biological stress, sticklebacks caged in this pond presented an oxidative stress, with increasing of reactive oxygen species levels, modification of some parts of the antioxidant system, and lipid peroxidation. Caging in the two most metal-contaminated ponds resulted in an increase of susceptibility of sticklebacks to the biological stress, preventing their phagocytic responses to lipopolysaccharides and modifying their glutathione contents and glutathione-S-transferase activity.

Rapid adaptive evolution of colour vision in the threespine stickleback radiation.

Vision is a sensory modality of fundamental importance for many animals, aiding in foraging, detection of predators and mate choice. Adaptation to local ambient light conditions is thought to be commonplace, and a match between spectral sensitivity and light spectrum is predicted. We use opsin gene expression to test for local adaptation and matching of spectral sensitivity in multiple independent lake populations of threespine stickleback populations derived since the last ice age from an ancestral marine form. We show that sensitivity across the visual spectrum is shifted repeatedly towards longer wavelengths in freshwater compared with the ancestral marine form. Laboratory rearing suggests that this shift is largely genetically based. Using a new metric, we found that the magnitude of shift in spectral sensitivity in each population corresponds strongly to the transition in the availability of different wavelengths of light between the marine and lake environments. We also found evidence of local adaptation by sympatric benthic and limnetic ecotypes to different light environments within lakes. Our findings indicate rapid parallel evolution of the visual system to altered light conditions. The changes have not, however, yielded a close matching of spectrum-wide sensitivity to wavelength availability, for reasons we discuss.