Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback (Gasterosteus aculeatus)

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.     

Spiggin levels are reduced in male sticklebacks infected with Schistocephalus solidus

Using an enzyme-linked immunosorbent assay (ELISA) based technique, Schistocephalus solidus infection was shown to considerably reduce levels of spiggin in the kidney of male three-spined sticklebacks Gasterosteus aculeatus from an oligotrophic upland lake. These results suggested a graduated effect of the infection on the reproductive physiology of male three-spined sticklebacks.     

Molecular cloning and characterization of a nuclear androgen receptor activated by 11-ketotestosterone

Although 11-ketotestosterone is a potent androgen and induces male secondary sex characteristics in many teleosts, androgen receptors with high binding affinity for 11-ketotestosterone or preferential activation by 11-ketotestosterone have not been identified. So, the mechanism by which 11-ketotestosterone exhibits such high potency remains unclear. Recently we cloned the cDNA of an 11-ketotestosterone regulated protein, spiggin, from three-spined stickleback renal tissue. As spiggin is the only identified gene product regulated by 11-ketotestosterone, the stickleback kidney is ideal for determination of the mechanism of 11-ketotestosterone gene regulation. A single androgen receptor gene with two splicing variants, belonging to the androgen receptor-β subfamily was cloned from stickleback kidney. A high affinity, saturable, single class of androgen specific binding sites, with the characteristics of an androgen receptor, was identified in renal cytosolic and nuclear fractions. Measurement of ligand binding moieties in the cytosolic and nuclear fractions as well as to the recombinant receptor revealed lower affinity for 11-ketotestosterone than for dihydrotestosterone. Treatment with different androgens did not up-regulate androgen receptor mRNA level or increase receptor abundance, suggesting that auto-regulation is not involved in differential ligand activation. However, comparison of the trans-activation potential of the stickleback androgen receptor with the human androgen receptor, in both human HepG2 cells and zebrafish ZFL cells, revealed preferential activation by 11-ketotestosterone of the stickleback receptor, but not of the human receptor. These findings demonstrate the presence of a receptor preferentially activated by 11-ketotestosterone in the three-spined stickleback, so far the only one known in any animal.     

Molecular cloning and characterization of spiggin. An androgen-regulated extraorganismal adhesive with structural similarities to von Willebrand Factor-related proteins

One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (alpha, beta, and gamma). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.     

Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.     

Variation in the reproductive potential of Schistocephalus infected male sticklebacks is associated with 11-ketotestosterone titre

Parasites can impact host reproduction by interfering with host endocrine systems, but the adaptive nature of such effects is disputed. Schistocephalus solidus plerocercoids are parasites of three-spined sticklebacks Gasterosteus aculeatus that are often associated with impaired host reproduction. Here, we relate reproductive behavior and physiology to levels of the androgen 11-ketotestosterone (11KT) in naturally infected and non-infected male sticklebacks from two UK populations. In one population infected males harbored heavy infections and showed uniformly reduced 11KT titres and kidney spiggin (nesting glue protein) content compared to non-infected fish. However in a second population infection levels were more variable and males with smaller infections recorded 11KT and spiggin titres that overlapped those of non-infected fish; among infected males from this population 11KT and kidney spiggin also both correlated negatively with infection severity. Male reproductive behavior correlated closely with 11KT titre in both populations, and infected males with high 11KT levels exhibited normal reproductive behavior. Our results suggest that Schistocephalus infection per se does not block reproductive development in male sticklebacks, and that some male fish may have the ability to breed whilst infected. Our results are not consistent with the hypothesis that Schistocephalus adaptively castrates male hosts via endocrine disruption; rather they support the hypothesis that reproductive disruption is a side effect of the energetic costs of infection.     

Nesting, courtship and kidney hypertrophy in Schistocephalus-infected male three-spined stickleback from an upland lake

The effect of Schistocephalus solidus infection on reproductive development and behaviour of male three-spined stickleback Gasterosteus aculeatus from an upland lake (Llyn Frongoch, Wales, U.K.) was investigated. Uninfected and infected males were collected on two separate occasions during the 2005 breeding season and encouraged to build nests under favourable laboratory conditions. Male nuptial colouration, courtship levels and nesting activity were recorded daily over two separate 21 day studies. Completed nests were removed, encouraging males to build multiple nests. On termination of each study, males were dissected and the kidney and any S. solidus plerocercoids were removed and weighed. In contrast to uninfected males, which readily built multiple nests, no infected males completed a nest during either study. Infected males also exhibited significantly reduced courtship levels, nesting activity, nuptial colouration, kidney development and body condition, compared with uninfected fish. Among infected three-spined stickleback, courtship and nesting behaviours tended to be more severely impacted in fish harbouring heavier plerocercoid burdens. In contrast to other populations, male three-spined stickleback from Llyn Frongoch appear to be unable to overcome the negative impacts of infections on reproductive development and behaviour when provided with short-term favourable conditions. Discrepancies between populations might have been related to the trophic status at the site of capture, emphasizing the importance of studying the biological effects of infections under different environmental conditions.     

Genetic evidence for male-biased dispersal in the three-spined stickleback (Gasterosteus aculeatus)

Sex-biased dispersal is capable of generating population structure in nonisolated populations and may affect adaptation processes when selective conditions differ among populations. Intrasexual competition for local resources and/or mating opportunities predicts a male-biased dispersal in polygynous species and a female bias in monogamous species. The patterns of sex-biased dispersal in birds and mammals are well explained by their respective mating systems, but the picture emerging from fish studies is still mixed. Using neutral genetic markers, we investigated whether there is any evidence for sex-biased dispersal among Baltic Sea populations of the three-spined stickleback ( Gasterosteus aculeatus ). The null hypothesis of non sex-biased dispersal was rejected in favour of male-biased dispersal in this species. As the three-spined stickleback has a polygynous mating system, the observed male bias in dispersal is consistent with the hypothesis that local mate competition might drive the observed pattern. Although more research both on the proximate and ultimate causes behind the observed pattern is needed, our results serve as a first step towards understanding patterns of sex-biased dispersal in this species.     

Changes in the diet of the coastal cod <Emphasis Type="Italic">Gadus morhua marisalbi</Emphasis> in the Kandalaksha Gulf of the White Sea under conditions of increased abundance of three-spined stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>

The data on feeding of coastal cod Gadus morhua marisalbi in the area of the Chupa Bay (Kandalaksha Gulf of the White Sea) are presented. In the food spectrum of cod, over 25 cm long fish and their eggs dominated (77.5% by frequency of occurrence and 91.7% by weight). Other groups of food organisms occupied a secondary place. Among fish, three-spined stickleback Gasterosteus aculeatus was the most prominent food item in the diet of cod (48.3% by weight). The published data on long-term changes of the diet of cod are considered, which are mostly related to considerable fluctuations of the abundance of three-spined stickleback in the White Sea. It is shown that, at present, three-spined stickleback again plays an important role in the diet of the White Sea cod     

Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer

Cytochrome P450 1 (CYP1) mRNA induction patterns in three-spined stickleback (Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3',4,4',5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A, CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.     

Fecundity of the three-spined stickleback, Gasterosteus aculeatus (L.)

The number of eggs spawned by female three-spined sticklebacks Gasterosteus aculeatus (L.) was highly correlated with the size of the fish expressed either as total length or as weight after spawning. There were no significant correlations between the size of the eggs measured either as wet weight per egg or as dry weight per egg, and the size of the fish. Nor were there significant correlations between the calorific value of the eggs and the length or weight of the fish. Regressions relating egg production to the length and the weight of the fish are given. These results and an analysis of previous studies on the fecundity of the stickleback suggest that variations in fecundity are primarily a reflection of variation in the size of the fish at maturity, and that this size is related to the race of the stickleback and to environmental conditions such as food supply which influence the growth of sticklebacks.     

Behind closed doors: use of visual cover by courting male three-spined stickleback, Gasterosteus aculeatus

Species that form breeding aggregations, such as the three-spined stickleback, have ample opportunity to observe and interact with others in their respective populations. This exposes courting pairs to distraction or disruption from onlooking individuals. Thus, courting inconspicuously could be beneficial to males. We tested whether males would court inconspicuously by housing them in individual tanks with visual access to a neighbouring male. A dummy of a gravid female three-spined stickleback was presented to a male in the front quadrant of the tank while screens concealed either the front quarter or front half of the tank from the neighbour. When a portion of the tank was concealed from a neighbouring rival, males courted more in the concealed areas than those that were exposed to a rival. Hence, in the presence of rivals, males can adjust their courtship behaviour in ways that could increase their mating success. Whether this reflects an active ‘strategy’ or a passive result of the male being less distracted by its neighbour is presently unknown.     

Effects of an antiandrogen,cyproterone acetate,on the kidney of the three-spined stickleback (Gasterosteus aculeatus L.)

The fine structure of the kidney is studied in the sexually mature male three-spined stickleback after administration of an antiandrogen, cyproterone acetate. Under these conditions, the dedifferentiation of renal tubules is characterized by the same involutive processes as those induced by castration, with the difference that cyproterone acetate only begins to act after 14 days whereas after castration the first signs of involution are visible after 7 days. The ultrastructural modifications affect the nucleoli, the rough endoplasmic reticulum and the Golgi apparatus. They reflect an inhibition of the secretory process. The results obtained demonstrate that administration of cyproterone acetate to male sticklebacks has an inhibitory effect on renal target cells, apparently indistinguishable from the changes induced by lack of male sex hormone, and that this drug may be a valid substitute for castration in fish.     

Comparison of taste preferences in the three-spined Gasterosteus aculeatus and nine-spined Pungitius pungitius sticklebacks from the White Sea Basin

Similarity between the taste preferences of classical taste substances and free L-amino acids in adult three-spined stickleback Gasterosteus aculeatus (forma leiurus) and nine-spined stickleback Pungitius pungitius (forma laevis) was found. The strongest and the most significant responses in both species were evoked by citric acid and cysteine, asparatic, and glutamic acids. Sodium chloride, calcium chloride, sucrose, and the remaining 18 amino acids do not elicit a statistically significant effect on the consumption of agar-agar pellets by the fish or have weak taste attractiveness. Similarity of taste preferences in the three-spined and nine-spined sticklebacks are supported by correlation analysis. Absolute values of the consumption of pellets of the same types are also similar in the two species. There were, however, differences in the behavioral taste response, repeated snaps, and the duration of processing of the pellets. The taste response of the nine-spined stickleback is more similar to taste responses in fish of the limnophilic complex than to the response in the three-spined stickleback. It is hypothesized that taste spectra may be very similar in fish with similar ecology and feeding patterns not only in sticklebacks, but also in other related species.     

Empirical evidence of a trade-off between reproductive effort and expectation of future reproduction in female three-spined sticklebacks

Optimal life-history models generally predict that the reproductive effort of iteroparous organisms may increase with age, as their expectation of future reproduction decreases. The population of three-spined sticklebacks (Gasterosteus aculeatus) in the Camargue (Rhone River Delta, France) is annual, all adults dying after their first breeding season. As the three-spined stickleback is a multiple spawner, we tested the hypothesis that reproductive effort may increase during the breeding season on field data. From 1987 to 1998, 653 female sticklebacks were collected in the field during the breeding seasons. The body size, body weight and weights of the liver, gonads and carcass were measured for these individuals. Only gravid females with mature eggs (176 fish) were included in the analysis. Considering the female three-spined stickleback as a capital breeder, the energetic resources available for allocation between soma and gonads were estimated by its body weight. Somatic condition decreased during the breeding season and reproductive effort (gonad weight relative to body weight) increased. These patterns did not vary significantly between years. These observed variations in reproductive effort during the breeding season can be interpreted as empirical evidence of a trade-off between reproductive effort and expectation of future reproduction.     

Role of histamine as a putative inhibitory transmitter in the honeybee antennal lobe

Background

Nests are built in various animal taxa including fish. In systems with exclusive male parental care, the choice of a nest site may be an important component of male fitness. The nest site may influence male attractiveness as a mate, and male, embryo, and juvenile survival probabilities. Reproductively active three-spined stickleback males establish and defend a territory in which they build a nest. Territories can differ remarkably in qualities that influence male and female reproductive success like predation risk or abiotic factors such as dissolved oxygen concentration or lighting conditions. The latter may be important because in sticklebacks the extended visual capability into the ultraviolet (UV) wave range plays a role in female mate choice. Males are thus expected to be choosy about the habitat in which they will build their nest.

Results

We tested nest-site choice in male three-spined sticklebacks with respect to different UV lighting conditions. Reproductively active males were given the simultaneous choice to build their nest either in an UV-rich (UV+) or an UV-lacking (UV-) environment. Males exhibited no significant nest-site preferences with respect to UV+ or UV-. However, larger males and also heavier ones completed their nests earlier.

Conclusion

We found that UV radiation as well as differences in luminance had no influence on nest-site choice in three-spined sticklebacks. Males that built in the UV-rich environment were not different in any trait (body traits and UV reflection traits) from males that built in the UV-poor environment. There was a significant effect of standard length and body mass on the time elapsed until nest completion in the UV experiment. The larger and heavier a male, the faster he completed his nest. In the brightness control experiment there was a significant effect only of body mass on the duration of nest completion. Whether nest building preferences with respect to UV lighting conditions are context dependent needs to be tested for instance by nest-site choice experiment under increased predation risk.     

Endocrine disrupting effects on the nesting behaviour of male three‐spined stickleback Gasterosteus aculeatus L.

The analysis of patterns of temporal variability in the nesting behaviour of male three-spined stickleback Gasterosteus aculeatus exposed to the synthetic oestrogen, 17β‐ethinylestradiol, revealed immediate, but transient, treatment‐related effects. Glueing frequency and time spent near nest were significantly reduced in exposed fish at the beginning of the experiment. The expression of these behaviours subsequently recovered and there was no effect of treatment on nest building success.     

Feeding and Refuge Use by Small Fish in the Presence of Cyanobacteria Blooms

We studied the effect of cyanobacteria on foraging and refuge use in small fish. We measured pike larval feeding in the presence of cyanobacteria by counting leftover prey. Our results showed that feeding by pike larvae on zooplankton prey decreased significantly in the presence of non-toxic cyanobacteria. The behaviour can be due to lowered vision caused by turbidity or clogging of the gills. Further, we tested whether the three-spined stickleback use toxic cyanobacteria as a refuge against predators in a choice experiment. The choice experiment was performed in a Y-maze fluviarum, where the fish could select between two different environments. Our results support the refuge use hypothesis because the three-spined stickleback clearly preferred toxic cyanobacteria to the chemical predator signal. To conclude, cyanobacteria decrease feeding rates in fish larvae, but may function as important refuge for e.g. sticklebacks, during predation pressure in pelagic algal blooms.