Mar Huertas

Non-invasive measurement of steroids in fish-holding water: important considerations when applying the procedure to behaviour studies

Fish behaviourists are increasingly turning to non-invasive measurement of steroid hormones in holding water, as opposed to blood plasma. When some of us met at a workshop in Faro, Portugal, in September, 2007, we realised that there were still many issues concerning the application of this procedure that needed resolution, including: Why do we measure release rates rather than just concentrations of steroids in the water? How does one interpret steroid release rates when dealing with fish of different sizes? What are the merits of measuring conjugated as well as free steroids in water? In the ‘static’ sampling procedure, where fish are placed in a separate container for a short period of time, does this affect steroid release—and, if so, how can it be minimised? After exposing a fish to a behavioural stimulus, when is the optimal time to sample? What is the minimum amount of validation when applying the procedure to a new species? The purpose of this review is to attempt to answer these questions and, in doing so, to emphasize that application of the non-invasive procedure requires more planning and validation than conventional plasma sampling. However, we consider that the rewards justify the extra effort.

Stocking Density at Early Developmental Stages Affects Growth and Sex Ratio in the European Eel (Anguilla anguilla)

To investigate the effect of stocking density on growth and sex ratio in European eel, four constant density conditions were tested during the transition from the glass to the elver stage for 90 days (Period 1). The test conditions combined the weight of fish per unit surface or volume (surface density or volume density) resulting in four experimental groups: low surface density (0.5 kg/m2) and low volume density (5 kg/m3) (group S0.5V5); low surface density (0.5 kg/m2) and high volume density (10 kg/m3) (group S0.5V10); high surface density (2 kg/m2) and low volume density (5 kg/m3) (group S2V5); and high surface density (2 kg/m2) and high volume density (10 Kg/m3) (group S2V10). Subsequently, fish from the S0.5V5, S2V5, and S2V10 groups were transferred to low density conditions (0.1–0.4 kg/m2 or 0.1–0.3 kg/m3) for another 21 months (630 days; Period 2). After Period 1, fish maintained at high surface density, regardless of the volume density, showed higher standard growth rates (SGRs) and RNA/DNA ratio in muscle than those cultured at low surface density. The percentage of mortality was similar in three of the groups (34.2%–41.8%), but not in the S2V10 group (83.3%). At the end of Period 2, most fish (about 95%) exhibited fully differentiated gonads, but different sex ratios were observed in each group. Thus, the S2V5 group showed a higher proportion of females (36.1%) and a lower proportion of males (56.8%) than the S0.5V5 group (11.4% and 72.5%, respectively), while all survivor fish from the S2V10 group developed into females. The gonadosomatic index and SGR were higher in females than in males. These results suggest that glass eels maintained at high surface density during the first months of growth tend to develop into females. The data also indicate that growth and sex ratio are linked processes during eel development, with growth seeming to be sex dependant rather than being influenced by the density conditions in which glass eels are maintained.

Deep sequencing of the olfactory epithelium reveals specific chemosensory receptors are expressed at sexual maturity in the European eel Anguilla anguilla.

Vertebrate genomes encode a diversity of G protein‐coupled receptor (GPCR) that belong to large gene families and are used by olfactory systems to detect chemical cues found in the environment. It is not clear however, if individual receptors from these large gene families have evolved roles that are specific to certain life stages. Here, we used deep sequencing to identify differentially expressed receptor transcripts in the olfactory epithelia (OE) of freshwater, seawater and sexually mature male eels (Anguilla anguilla). This species is particularly intriguing because of its complex life cycle, extreme long‐distance migrations and early‐branching position within the teleost phylogeny. In the A. anguillaOE, we identified full‐length transcripts for 13, 112, 6 and 38 trace amine‐associated receptors, odorant receptors (OR) and type I and type II vomeronasal receptors (V1R and V2R). Most of these receptors were expressed at similar levels at different life stages and a subset of OR and V2R‐like transcripts was more abundant in sexually mature males suggesting that ORs and V2R‐like genes are important for reproduction. We also identified a set of GPCR signal transduction genes that were differentially expressed indicating that eels make use of different GPCR signal transduction genes at different life stages. The finding that a diversity of chemosensory receptors is expressed in the olfactory epithelium and that a subset is differentially expressed suggests that most receptors belonging to large chemosensory gene families have functions that are important at multiple life stages, while a subset has evolved specific functions at different life stages.

Chemical communication in the Genus Anguilla: a minireview

Eels of the Genus Anguilla are important fish in both scientific and economic terms. Unfortunately, current stocks of the European eel in particular ( Anguilla anguilla L.) are becoming increasingly endangered. Chemical communication plays important roles in several key aspects of fish biology. Due to its complex life-cycle, the eel offers a unique opportunity to study various aspects of olfactory biology and chemical communication almost in isolation, feeding (during freshwater ‘yellow’ stage), adaptation to seawater, migration and, finally, reproduction. The aim of this brief review is to summarise what is known about chemical communication in the eel (during reproduction in particular) and, more importantly, stimulate further research. To date, eels have been shown to have high olfactory sensitivity to substances released by conspecifics. Both the bile fluid and skin mucus have been implicated as the routes of release of these odorants but other routes, such as the urine, cannot be excluded. Furthermore, the nature of these odorants depends on both the sex and reproductive status of the donor; exposure to water conditioned by mature con-specifics causes a stimulation of sexual maturation in immature eels. These results are consistent with a role for chemical communication in eel reproduction. The European eel is, therefore, proposed as a model species for studies on olfaction and chemical communication in fish because of its ancestral status, its high olfactory sensitivity and its physiological plasticity. However, more research is necessary; specifically, identification of the key odorants involved, their behavioural and physiological effects and how the olfactory system may change at various stages of its life-cycle.

(+)- and (−)-Petromyroxols: Antipodal Tetrahydrofurandiols from Larval Sea Lamprey (Petromyzon marinus L.) That Elicit Enantioselective Olfactory Responses

(+)- and (-)-petromyroxol [(+)-1 and (-)-1, respectively], two novel tetrahydrofuran (THF)-diol fatty acid enantiomers, were isolated from water conditioned with larval sea lamprey. We herein describe their isolation and subsequent resolution using chiral chromatography. The absolute configuration of each enantiomer was determined by a combination of Mosher ester analysis and comparison with related natural and synthetic products. Electro-olfactogram (EOG) assays indicated that (+)-petromyroxol (1) possesses potent olfactory activity for sea lamprey.

Tilapia male urinary pheromone stimulates female reproductive axis

Mozambique tilapia males congregate in leks where they establish dominance hierarchies and attract females to spawn in sandy pits. Dominant males store more urine than subordinates and the pattern of urination and the high sensitivity of females to male urine suggest chemical signalling via the urine. Here we show that pre-ovulated and post-spawn females when exposed to dominant male urine increased significantly, in less than 1h, the release rate of the maturation-inducing steroid 17,20β-dihydroxypregn-4-en-3-one which is maintained elevated for at least 6h. This indicates a pheromonal role for male urine in the synchronisation of spawning. Furthermore, we show that the lack of affinity of 17,20βP to sex steroid binding globulin explains, at least partly, its rapid release and lack of detection in the blood. Thus tilapia urine involvement in several communication processes confirms that cichlids have evolved a sophisticated chemical signalling system together with their complex visual, acoustic and behavioural displays.

Petromyzonin, a hexahydrophenanthrene sulfate isolated from the larval sea lamprey (Petromyzon marinus L.).

A new hexahydrophenanthrene sulfate was identified from water conditioned with sea lamprey larvae ( Petromyzon marinus ) and named petromyzonin. Its structure was unequivocally elucidated on the basis of spectroscopic analyses including comparison with spectra of known compounds. The absolute configuration was determined by electronic circular dichroism. Petromyzonin may function as a chemical signal, as it elicited responses in electro-olfactogram recording with a dynamic concentration-response relationship and a detection threshold of 10(-11) M.

A Review of Research in Fish Pheromones

This review provides selected examples of several types of chemical signals and cues important for the social behavior of fish. Alarm substances evoke antipredator behaviors, typified by increased shoaling, refuging, freezing, dashing, area avoidance, and reduced foraging. Migratory pheromones are employed by some fish species that migrate long distances to locate home streams or spawning grounds. Many fishes employ sex pheromones to attract members of the opposite sex or to elicit spawning behavior. Steroids, prostaglandins, bile acids and amino acids have all been shown to serve as sex pheromones in fishes. Pheromones can also be used to recognize kin and establish hierarchies. Nonspecific diet metabolites as well as specific pheromones are important in chemical mediation of social behavior in fish. The use of pheromones in fisheries started long before scientific proof of their existence. Sea lamprey (Petromyzon marinus) pheromone is the first vertebrate pheromone that has been tested in the field as a pest control agent. Other potential applications of pheromone usage include conservation of endangered species or aquaculture. Similar approaches could also be useful for other aquatic organisms, including crustaceans.

Evidence for partial overlap of male olfactory cues in lampreys.

ABSTRACT Animals rely on a mosaic of complex information to find and evaluate mates. Pheromones, often consisting of multiple components, are considered to be particularly important for species-recognition in many species. Although the evolution of species-specific pheromone blends is well described in many insects, very few vertebrate pheromones have been studied in a macro-evolutionary context. Here, we report a phylogenetic comparison of multi-component male odours that guide reproduction in lampreys. Chemical profiling of sexually mature males from eleven species of lamprey, representing six of ten genera and two of three families, indicated that the chemical profiles of sexually mature male odours are partially shared among species. Behavioural assays conducted with four species sympatric in the Laurentian Great Lakes indicated asymmetric female responses to heterospecific odours, where Petromyzon marinus were attracted to male odour collected from all species tested, but other species generally preferred only the odour of conspecifics. Electro-olfactogram recordings from P. marinus indicated that although P. marinus exhibited behavioural responses to odours from males of all species, at least some of the compounds that elicited olfactory responses were different in conspecific male odours compared with heterospecific male odours. We conclude that some of the compounds released by sexually mature males are shared among species and elicit olfactory and behavioural responses in P. marinus, and suggest that our results provide evidence for partial overlap of male olfactory cues among lampreys. Further characterization of the chemical identities of odour components is needed to confirm shared pheromones among species. Summary: Olfactory cues, critical to the behaviour and physiology of sea lamprey (Petromyzon marinus), exhibit partial overlap among lamprey species.

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation

BackgroundThe vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males.ResultsUsing a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity.ConclusionsThis study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.