Report of the Second European Zebrafish Principal Investigator Meeting in Karlsruhe, Germany, March 21-24, 2012.

The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.

Population genomic tests of models of adaptive radiation in Lake Victoria Region cichlid fish

Adaptive radiation is usually thought to be associated with speciation, but the evolution of intraspecific polymorphisms without speciation is also possible. The radiation of cichlid fish in Lake Victoria (LV) is perhaps the most impressive example of a recent rapid adaptive radiation, with 600+ very young species. Key questions about its origin remain poorly characterized, such as the importance of speciation versus polymorphism, whether species persist on evolutionary time scales, and if speciation happens more commonly in small isolated or in large connected populations. We used 320 individuals from 105 putative species from Lakes Victoria, Edward, Kivu, Albert, Nabugabo and Saka, in a radiation-wide amplified fragment length polymorphism (AFLP) genome scan to address some of these questions. We demonstrate pervasive signatures of speciation supporting the classical model of adaptive radiation associated with speciation. A positive relationship between the age of lakes and the average genomic differentiation of their species, and a significant fraction of molecular variance explained by above-species level taxonomy suggest the persistence of species on evolutionary time scales, with radiation through sequential speciation rather than a single starburst. Finally the large gene diversity retained from colonization to individual species in every radiation suggests large effective population sizes and makes speciation in small geographical isolates unlikely.

Hotshots, hot spots, and female preference: exploring lek formation models with a bower-building cichlid fish

In many animals, males congregate in leks that females visit for the sole purpose of mating. We observed male and female behavior on 3 different-sized leks of the bower-building cichlid fish Nyassachromis cf. microcephalus to test predictions of 3 prominent lek models: the "hotshot," "hot spot," and "female preference" models. In this system, we were able to refine these predictions by distinguishing between indirect mate choice, by which females restrict their set of potential mates in the absence of individual male assessment, and direct mate choice, by which females assess males and their territories through dyadic behavioral interactions. On no lek were males holding central territories favored by indirect or direct mate choice, contrary to the prediction of the hotshot model that leks form because inferior males establish territories surrounding hotshot males preferred by females. Average female encounter rate of males increased with lek size, a pattern typically interpreted as evidence that leks form through female preference for lekking males, rather than because males congregate in hot spots of high female density. Female propensity to engage in premating behavior once courted did not increase with lek size, suggesting female preference for males on larger leks operated through indirect choice rather than direct choice based on individual assessment. The frequency of male-male competitive interactions increased with lek size, whereas their foraging rate decreased, implying a cost to males maintaining territories on larger leks. Together these data most strongly support the female preference model, where females may benefit through indirect mate choice for males able to meet the competitive cost of occupying larger leks.

Genetics of male nuptial colour divergence between sympatric sister species of a Lake Victoria cichlid fish

The hypothesis of sympatric speciation by sexual selection has been contentious. Several recent theoretical models of sympatric speciation by disruptive sexual selection were tailored to apply to African cichlids. Most of this work concludes that the genetic architecture of female preference and male trait is a key determinant of the likelihood of disruptive sexual selection to result in speciation. We investigated the genetic architecture controlling male nuptial colouration in a sympatric sibling species pair of cichlid fish from Lake Victoria, which differ conspicuously in male colouration and female mating preferences for these. We estimated that the difference between the species in male nuptial red colouration is controlled by a minimum number of two to four genes with significant epistasis and dominance effects. Yellow colouration appears to be controlled by one gene with complete dominance. The two colours appear to be epistatically linked. Knowledge on how male colouration segregates in hybrid generations and on the number of genes controlling differences between species can help us assess whether assumptions made in simulation models of sympatric speciation by sexual selection are realistic. In the particular case of the two sister species that we studied a small number of genes causing major differences in male colouration may have facilitated the divergence in male colouration associated with speciation.

Assortative mating among Lake Malawi cichlid fish populations is not simply predictable from male nuptial colour

Background: Research on the evolution of reproductive isolation in African cichlid fishes has largely focussed on the role of male colours and female mate choice. Here, we tested predictions from the hypothesis that allopatric divergence in male colour is associated with corresponding divergence in preference. Methods: We studied four populations of the Lake Malawi Pseudotropheus zebra complex. We predicted that more distantly-related populations that independently evolved similar colours would interbreed freely while more closely-related populations with different colours mate assortatively. We used microsatellite genotypes or mesh false-floors to assign paternity. Fisher's exact tests as well as Binomial and Wilcoxon tests were used to detect if mating departed from random expectations. Results: Surprisingly, laboratory mate choice experiments revealed significant assortative mating not only between population pairs with differently coloured males, but between population pairs with similarly-coloured males too. This suggested that assortative mating could be based on nonvisual cues, so we further examined the sensory basis of assortative mating between two populations with different male colour. Conducting trials under monochromatic (orange) light, intended to mask the distinctive male dorsal fin hues (blue v orange) of these populations, did not significantly affect the assortative mating by female P. emmiltos observed under control conditions. By contrast, assortative mating broke down when direct contact between female and male was prevented. Conclusion: We suggest that non-visual cues, such as olfactory signals, may play an important role in mate choice and behavioural isolation in these and perhaps other African cichlid fish. Future speciation models aimed at explaining African cichlid radiations may therefore consider incorporating such mating cues in mate choice scenarios.

The antioxidant glutathione in the fish cell lines EPC and BCF-2: response to model pro-oxidants as measured by three different fluorescent dyes

Reduced glutathione (GSH) protects cells against injury by oxidative stress and maintains a range of vital functions. In vitro cell cultures have been used as experimental models to study the role of GSH in chemical toxicity in mammals; however, this approach has been rarely used with fish cells to date. The present study aimed to evaluate sensitivity and specificity of three fluorescent dyes for measuring pro-oxidant-induced changes of GSH contents in fish cell lines: monochlorobimane (mBCl), 5-chloromethylfluorescein diacetate (CMFDA) and 7-amino-4-chloromethylcoumarin (CMAC-blue). Two cell lines were studied, the EPC line established from a skin tumour of carp Cyprinus carpio, and BF-2 cells established from fins of bluegill sunfish Lepomis macrochirus. The cells were exposed for 6 and 24 h to low cytotoxic concentrations of pro-oxidants including hydrogen peroxide, paraquat (PQ), copper and the GSH synthesis inhibitor, L-buthionine-SR-sulfoximine (BSO). The results indicate moderate differences in the GSH response between EPC and BF-2 cells, but distinct differences in the magnitude of the GSH response for the four pro-oxidants. Further, the choice of GSH dye can critically affect the results, with CMFDA appearing to be less specific for GSH than mBCl and CMAC-blue.

The state of in vitro science for use in bioaccumulation assessments for fish

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Program's Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

Mechanisms of rapid sympatric speciation by sex reversal and sexual selection in cichlid fish

Mechanisms of speciation in cichlid fish were investigated by analyzing population genetic models of sexual selection on sex-determining genes associated with color polymorphisms. The models are based on a combination of laboratory experiments and field observations on the ecology, male and female mating behavior, and inheritance of sex-determination and color polymorphisms. The models explain why sex-reversal genes that change males into females tend to be X-linked and associated with novel colors, using the hypothesis of restricted recombination on the sex chromosomes, as suggested by previous theory on the evolution of recombination. The models reveal multiple pathways for rapid sympatric speciation through the origin of novel color morphs with strong assortative mating that incorporate both sex-reversal and suppressor genes. Despite the lack of geographic isolation or ecological differentiation, the new species coexists with the ancestral species either temporarily or indefinitely. These results may help to explain different patterns and rates of speciation among groups of cichlids, in particular the explosive diversification of rock-dwelling haplochromine cichlids.