Swim bladder as a primary site of mycobacterial infection in Nothobranchius 'belly sliders'.

The swim bladder inflates early after fish hatching via its interconnection with the digestive tract (ductus pneumaticus). This interconnection may serve as a portal to foreign particles, including bacteria, causing deficiencies in primary swim bladder inflation. We histologically examined 134 African annual killifish (genus Nothobranchius) with secondary loss of swim bladder function ('belly sliders'). We demonstrate that these fish lost the ability of air regulation in their swim bladders likely due to Mycobacterium spp. infection at an individual-specific age. Nearly all examined belly sliders had thickened swim bladder walls, and their swim bladder was filled with material containing mycobacteria, cell debris, young monocytic cells and phagocyting macrophages. Mycobacterial infection was restricted to the swim bladder in juveniles, where mycobacteria likely enter the host through the ductus pneumaticus. Infection in adults was systemic and mycobacteria were present in all examined organs. Presence of mycobacteria in the epithelial lining and submucosal layers of the digestive tract of adults suggests that it may also serve as the entrance site of infection. We suspect 2 sources of Mycobacterium contamination: dietary (with bloodworms) and/or contaminated hatching substrate. These sources of contamination may be eliminated by use of laboratory dry feed and egg disinfection prior to hatching.

Parasitic cuckoo catfish exploit parental responses to stray offspring.

Interspecific brood parasitism occurs in several independent lineages of birds and social insects, putatively evolving from intraspecific brood parasitism. The cuckoo catfish, Synodontis multipunctatus, the only known obligatory non-avian brood parasite, exploits mouthbrooding cichlid fishes in Lake Tanganyika, despite the absence of parental care in its evolutionary lineage (family Mochokidae). Cuckoo catfish participate in host spawning events, with their eggs subsequently collected and brooded by parental cichlids, though they can later be selectively rejected by the host. One scenario for the origin of brood parasitism in cuckoo catfish is through predation of cichlid eggs during spawning, eventually resulting in a spatial and temporal match in oviposition by host and parasite. Here we demonstrate experimentally that, uniquely among all known brood parasites, cuckoo catfish have the capacity to re-infect their hosts at a late developmental stage following egg rejection. We show that cuckoo catfish offspring can survive outside the host buccal cavity and re-infect parental hosts at a later incubation phase by exploiting the strong parental instinct of hosts to collect stray offspring. This finding implies an alternative evolutionary origin for cuckoo catfish brood parasitism, with the parental response of host cichlids facilitating its evolution. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Local variation in embryo development rate in annual fish.

Extreme asynchrony in embryo development, a typical feature of annual killifish living in temporary pools, represents a bet-hedging strategy to cope with unpredictable rainfall. African annual killifish are distributed across a large precipitation gradient, raising the potential for local adaptation in the degree of developmental asynchrony (e.g. higher in arid areas, lower in humid areas). Eight populations of two sister species, Nothobranchius furzeri and Nothobranchius kadleci, from sites along the rainfall gradient were tested and compared for asynchrony and duration of embryo development. Degree of asynchrony and mean duration of embryo development did not differ across the examined range. Despite generally high developmental variability, fish from more arid regions (where rain is more erratic) produced a significantly higher proportion of short-developing embryos. Comparable developmental asynchrony, regardless of precipitation level, suggests that all populations tested need to cope with some level of rainfall stochasticity. By producing more short-developing embryos, however, fish in more arid areas with relatively more erratic rains are better adapted to very short pool durations and are more likely to produce multiple offspring generations within a single rainy season.

The occurrence of Trichinella species in the cougar Puma concolor couguar from the state of Colorado and other regions of North and South America.

Trichinella species are zoonotic nematodes that infect wild carnivores and omnivores throughout the world. We examined the prevalence and species of Trichinella infections in cougars (Puma concolor couguar) from Colorado, United States. Tongues from cougars were examined by pepsin-HCl artificial digestion to detect Trichinella spp. larvae. The species or genotype of individual worms was identified by multiplex polymerase chain reaction (PCR). Trichinella spp. larvae were detected in 17 of 39 cougars (43.6% (28.7-59.5%)). Five of the cougars (12.8%) were infected with T. murrelli, 3 (7.7%) were infected with T. pseudospiralis, and 1 (2.6%) had Trichinella genotype T6. Trichinella spp. larvae from eight cougars were not identified at the species level, due to degraded DNA. The high prevalence of Trichinella spp. in cougars from Colorado and reports of the parasite in other populations of Puma spp. suggest that this large predator is a key mammalian reservoir.

Maternal source of variability in the embryo development of an annual killifish.

Organisms inhabiting unpredictable environments often evolve diversified reproductive bet-hedging strategies, expressed as production of multiple offspring phenotypes, thereby avoiding complete reproductive failure. To cope with unpredictable rainfall, African annual killifish from temporary savannah pools lay drought-resistant eggs that vary widely in the duration of embryo development. We examined the sources of variability in the duration of individual embryo development, egg production and fertilization rate in Nothobranchius furzeri. Using a quantitative genetics approach (North Carolina type II design), we found support for maternal effects rather than polyandrous mating as the primary source of the variability in the duration of embryo development. The number of previously laid eggs appeared to serve as an internal physiological cue initiating a shift from rapid-to-slow embryo developmental mode. In annual killifish, extensive phenotypic variability in progeny traits is adaptive, as the conditions experienced by parents have limited relevance to the offspring generation. In contrast to genetic control, with high phenotypic expression and heritability, maternal control of traits under natural selection prevents standing genetic diversity from potentially detrimental effects of selection in fluctuating environments.

Seasonal dynamics in community structure, abundance, body size and sex ratio in two species of Neotropical annual fishes.

Seven ephemeral pools on the coastal plain of southern Brazil were found to be inhabited by three annual and 22 non-annual fish species. Two common annual species (Austrolebias minuano and Cynopoecilus fulgens) exhibited clear seasonal dynamics, with the appearance of young fishes in the austral autumn (May to June) and a decline in abundance over the seasonal cycle. The third annual species, Austrolebias wolterstorffii, was rare. No seasonal dynamics were observed in non-annual fishes. The relative abundance of non-annual fishes compared with annual fishes increased over the seasonal cycle, but they coexisted widely. The size structure of annual fishes suggested the presence of a single age cohort in most pools though a second age cohort was registered in one pool in August, coinciding with a large flooding. Strong sexual dimorphism in body size was found in C. fulgens throughout the seasonal cycle, while no sexual dimorphism in body size was found in A. minuano. Female-biased sex ratios were recorded in both common annual fish species in the last three sampling dates (in spring), but not during the first two sampling dates (in winter). The natural lifespan of annual fishes was <8 months. Annual fishes disappeared before habitat desiccation in half of the pools, while non-annual fishes were still present.

Strategic exploitation of fluctuating asymmetry in male Endler's guppy courtship displays is modulated by social environment.

Lateral asymmetry in signalling traits enables males to strategically exploit their best side. In many animals, both body colouration and fluctuating asymmetry are signals of male attractiveness. We demonstrated experimentally that even sexually naïve male Poecilia wingei were able to identify their most attractive side (i.e. that with a higher proportion of carotenoid pigmentation) and use it preferentially during courtship. Notably, males retained their strategic signalling in a male-biased social environment, whereas they ceased to signal strategically in a female-biased environment. The degree of asymmetry in colouration did not affect overall courtship activity. Strategic lateralization in courtship displays was strongest and most repeatable in the male-biased social environment where males competed with rivals for matings. Individual asymmetry in colouration changed considerably over a period of 3 months. This suggests that colouration is a dynamic feature during adulthood and that males are capable of tracking and strategically exploiting their lateral asymmetry in accordance with their social environment.

The function of multiple ejaculations in bitterling.

In some taxa, males perform multiple ejaculations, which may function in sperm competition or in maintaining a baseline density of spermatozoa in the female reproductive tract to ensure fertilization, a process that has been termed 'topping up'. We investigated the function of multiple ejaculations in two species of bitterling, the European bitterling (Rhodeus amarus) and Chinese rose bitterling (Rhodeus ocellatus). Bitterling oviposit in living freshwater mussels, with fertilization taking place within the mussel gill cavity. Thus, although fertilization is external, the mussel is analogous to the female reproductive tract in an internally fertilizing species. We measured the frequency of ejaculations and mussel inspections by individual males of two bitterling species in 28 replicated mesocosms and examined focal male responses to rival ejaculations and the presence of females in spawning condition. We used a model of ejaculatory behaviour to simulate the temporal abundance of spermatozoa in mussels. Male R. amarus exhibited high rates of ejaculation and inspection of the siphons of mussels and increased their ejaculation rate in response to the presence of females in spawning condition. Rhodeus ocellatus showed lower overall rates of ejaculation, but significantly elevated ejaculation rate in response to rival ejaculations. The ejaculatory strategy of R. amarus is one that maintains a minimum level of spermatozoa in mussels, which is elevated when the probability of oviposition increases. In contrast, R. ocellatus engages more directly in sperm competition with rivals. We discuss these results in the context of the function of multiple ejaculations and male mating tactics.

Alternative intrapopulation life-history strategies and their trade-offs in an African annual fish.

In ephemeral habitats, the same genotypes cope with unpredictable environmental conditions, favouring the evolution of developmental plasticity and alternative life-history strategies (ALHS). We tested the existence of intrapopulation ALHS in an annual killifish, Nothobranchius furzeri, inhabiting temporary pools. The pools are either primary (persisting throughout the whole rainy season) or secondary (refilled after desiccation of the initial pool), representing alternative niches. The unpredictable conditions led to the evolution of reproductive bet-hedging with asynchronous embryonic development. We used a common garden experiment to test whether the duration of embryonic period is associated with post-embryonic life-history traits. Fish with rapid embryonic development (secondary pool strategy, high risk of desiccation) produced phenotypes with more rapid life-history traits than fish with slow embryonic development (primary pool strategy). The fast fish were smaller at hatching but had larger yolk sac reserves. Their post-hatching growth was more rapid, and they matured earlier. Further, fast fish grew to a smaller body size and died earlier than slow fish. No differences in fecundity, propensity to mate or physiological ageing were found, demonstrating a combination of plastic responses and constraints. Such developmentally related within-population plasticity in life history is exceptional among vertebrates.

No evidence for host specialization or host-race formation in the European bitterling (Rhodeus amarus), a fish that parasitizes freshwater mussels.

Coevolutionary relationships between parasites and hosts can elevate the rate of evolutionary changes owing to reciprocal adaptations between coevolving partners. Such relationships can result in the evolution of host specificity. Recent methodological advances have permitted the recognition of cryptic lineages, with important consequences for our understanding of biological diversity. We used the European bitterling (Rhodeus amarus), a freshwater fish that parasitizes unionid mussels, to investigate host specialization across regions of recent and ancient sympatry between coevolving partners. We combined genetic data (12 microsatellite and 2 mitochondrial markers) from five populations with experimental data for possible mechanisms of host species recognition (imprinting and conditioning). We found no strong evidence for the existence of cryptic lineages in R. amarus, though a small proportion of variation among individuals in an area of recent bitterling-mussel association was statistically significant in explaining host specificity. No other measures supported the existence of host-specific lineages. Behavioural data revealed a weak effect of conditioning that biased behavioural preferences towards specific host species. Host imprinting had no effect on oviposition behaviour. Overall, we established that populations of R. amarus show limited potential for specialization, manifested as weak effects of host conditioning and genetic within-population structure. Rhodeus amarus is the only species of mussel-parasitizing fish in Europe, which contrasts with the species-rich communities of bitterling in eastern Asia where several host-specific bitterling occur. We discuss costs and constraints on the evolution of host-specific lineages in our study system and more generally.

Phylogeny, genetic variability and colour polymorphism of an emerging animal model: the short-lived annual Nothobranchius fishes from southern Mozambique.

Nothobranchius are a group of small, extremely short-lived killifishes living in temporary savannah pools in Eastern Africa and that survive annual desiccation of their habitat as dormant eggs encased in dry mud. One mitochondrial (COI) and three nuclear (CX32.2, GHITM, PNP) loci were used to investigate the phylogenetic relationship of Nothobranchius species from southern and central Mozambique. This group shows marked variation in captive lifespan at both the inter- and intraspecific levels; lifespan varies from a few months to over a year. As their distribution encompasses a steep gradient between semi-arid and humid habitats, resulting in contrasting selection pressures on evolution of lifespan and associated life history traits, Mozambican Nothobranchius spp. have recently become a model group in studies of ageing, age-related disorders and life history evolution. Consequently, intraspecific genetic variation and male colour morph distribution was also examined in the recovered clades. Using Bayesian species tree reconstruction and single loci analyses, three large clades were apparent and their phylogenetic substructure was revealed at the inter- and intra-specific levels within those clades. The Nothobranchius furzeri and Nothobranchius orthonotus clades were strongly geographically structured. Further, it was demonstrated that male colour has no phylogenetic signal in N. furzeri, where colour morphs are sympatric, but is associated with two reciprocally monophyletic groups in Nothobranchius rachovii clade, where colour morphs are parapatric. Finally, our analysis showed that a polymorphism in the Melanocortin1 receptor gene (which controls pigmentation in many vertebrates and was a candidate gene of male colouration in N. furzeri) is unrelated to colour phenotypes of the study species. Our results raise significant implications for future comparative studies of the species and populations analysed in the present work.

Age structure of annual Nothobranchius fishes in Mozambique: is there a hatching synchrony?

The age structures of populations of African annual Nothobranchius spp. were examined for the first time. Daily increments in sagittal otoliths of Nothobranchius furzeri, Nothobranchius kadleci, Nothobranchius orthonotus and Nothobranchius rachovii from southern and central Mozambique were used for age determination. Four hypotheses were tested: (1) timing of hatching is consistent with the calendar onset of the rainy season, (2) hatching is synchronized within a population in a pool, (3) there is a difference in hatching date between geographical regions differing in mean total annual precipitation and (4) sympatric Nothobranchius spp. hatch at the same time. The results show that daily increment analysis represents an applicable method for age determination in Nothobranchius spp. Despite a significant positive relationship between age and size of fishes, a pronounced variation in fish size at an age precluded the use of fish size as a valid age marker. Timing of hatching was not consistent with the calendar onset of the rainy season. Interpopulation variability was observed in the degree of hatching date synchronization within a population. Hatching dates were relatively uniform in some populations, while there was considerable variability in others. Differences in timing of hatching date were found in only 1 of 2 years within the three regions investigated (Chefu, lower Limpopo and Sofala regions), each of which differed in mean total annual rainfall. The hatching dates of sympatric Nothobranchius spp. were marginally different, but further testing on a larger sample is needed for conclusive results.

Seasonal dynamics in population characteristics of European bitterling Rhodeus amarus in a small lowland river.

The seasonal dynamics of biological traits in a population of European bitterling Rhodeus amarus from a small river in central Europe (River Kyjovka, Czech Republic) were investigated from April 2007 to March 2008. The reproductive season lasted from early April to mid-June, with a peak in late April when > 50% of females possessed ripe eggs in their ovaries. The sex ratio was female-biased, with a higher bias following the peak in reproduction, suggesting higher mortality of males than females over the reproductive period. The population was effectively annual, with a major decrease in adult fish abundance at the end of the reproductive period. Highest relative lipid content and condition factor were observed in February, both decreasing with an increase in investment into gonads. Seasonal dynamics in somatic condition, relative lipid content and gonad mass, and their comparison with other R. amarus populations, suggest that the study population allocated a disproportionally high level of resources to reproduction.

Range-wide population genetic structure of the European bitterling (Rhodeus amarus) based on microsatellite and mitochondrial DNA analysis.

An understanding of recent evolutionary processes is essential for the successful conservation and management of contemporary populations, especially where they concern the introduction or invasion of species outside their natural range. However, the potentially negative implications of intraspecific introductions and invasions have attracted less attention, although they also represent a potential threat to biodiversity, and are commonly facilitated through human activities. The European bitterling (Rhodeus amarus) is a small cyprinid fish that decreased greatly in its distribution during the 1970s and 1980s and was subsequently included on many European conservation lists. This decline appears to have reversed, and the extent of its distribution now exceeds its former range. We used a combination of 12 microsatellite markers and cytochrome b sequences on a large data set (693 individuals) across the current range of the European bitterling to investigate possible scenarios for its colonization of Europe. We show that the inferred history of colonization of Europe was largely congruent between mitochondrial and nuclear markers. The most divergent mtDNA lineages occur in the Aegean region but probably are not reproductively isolated as the Aegean populations also displayed mtDNA haplotypes from other lineages and nuclear data indicated their close relationship to Danubian populations. Much of Europe is currently populated by descendants of two main lineages that came to natural secondary contact in western Europe. An approximate Bayesian computation analysis indicates different dates for admixture events among western and central European populations ranging from the last deglaciation (natural) to the last few centuries (human-assisted translocations).

Diet overlap among three sympatric African annual killifish species Nothobranchius spp. from Mozambique.

The diet patterns of three Nothobranchius species (N. furzeri, N. orthonotus and N. rachovii), small, short-lived annual killifish from temporary pools in African savannah were investigated. Four sites with contrasting fish density and water surface area were sampled in 2008 and 2009 in southern Mozambique. Stomach content analysis showed that all the species examined were generalists, with diets largely based on aquatic invertebrates. The same invertebrate prey categories were consumed by all three species, but their relative proportions varied across species. The largest species, N. orthonotus, showed the most distinct diet and consumed vertebrates (juvenile lungfish Protopterus annectens and larval Amphibia) and a relatively high proportion of Odonata, Coleoptera and Ephemeroptera larvae. The diet of the other two species (N. furzeri and N. rachovii) showed a stronger overlap, did not include vertebrates, but was rich in small crustaceans (Cladocera, Copepoda, Ostracoda and Conchostraca). Mosquito (Diptera) larvae formed only a negligible part of the diet of all the three species.

Distribution, colour polymorphism and habitat use of the African killifish Nothobranchius furzeri, the vertebrate with the shortest life span.

Intensive collection in southern Mozambique across and outside the potential range of Nothobranchius furzeri, the species with the shortest recorded life span among vertebrates used as a model in ageing research, revealed that, contrary to previous data, it is a widespread species. It occurs in small freshwater pools south of the Save River and north of the Incomati River, including basins of the Limpopo, Changane, Chefu, Mazimechopes and Vaneteze Rivers. During collection in February 2008 (the second part of the rainy season), populations were strongly female biased (mean, 28% of males across 19 populations), and there was a spatial pattern in female bias among metapopulations. Populations varied in the proportion of male colour morphs. Fourteen populations were composed exclusively of the red male phenotype, three populations of the yellow male phenotype and 12 populations were mixed. Overall, the red phenotype was more common, but there was strong geographical variation in morph proportion, with yellow males more abundant at the periphery and red male dominance in the centre of the range of N. furzeri in the Limpopo basin. Nothobranchius furzeri was sympatric with Nothobranchius orthonotus (35% of investigated pools) and Nothobranchius rachovii (27% of sites). Analysis of habitat use of N. furzeri is presented; N. furzeri was associated with pools containing a soft muddy substratum and turbid water.

River discharge drives recruitment success of the European bitterling Rhodeus amarus in a regulated river in central Europe.

A 15 year data set revealed that the abundance of 0+ year Rhodeus amarus in a regulated river was negatively associated with mean river discharge, while mean standard length was positively related to the cumulative number of degree-days > or =10 degrees C. Results are discussed in the context of the recent invasion success of R. amarus.

Seasonal change in the opportunity for sexual selection.

Environmental and population parameters that influence the strength of sexual selection may vary considerably over the course of the reproductive season. However, the potential for sexual selection frequently fails to translate into variation in reproductive success among individuals. We investigated seasonal changes in variation in reproductive success, measured as the opportunity for sexual selection, using parentage analysis in 20 experimental populations of the European bitterling (Rhodeus amarus, Cyprinidae), a small freshwater fish with a promiscuous, resource-based mating system. We showed that although the largest males sired most offspring over the entire reproductive season, variation in reproductive success and hence the opportunity for sexual selection was low at the start of the season but increased significantly at its end. This seasonal difference probably arose from the superior competitive endurance of large males and from a higher temporal clustering of reproductively active females at the start of the breeding season than later in the season. The spatial distribution of oviposition sites had a negligible effect on the variation in reproductive success. We discuss the potential implications of our results for the importance and strength of sexual selection in natural populations.

Experimental infection of domestic cats (Felis domesticus) with Cytauxzoon manul from Pallas' cats (Otocolobus manul).

In a random, blind study, six domestic cats were assigned to two treatment groups that received either sterile water or dexamethasone by subcutaneous injection prior to intravenous inoculation with Pallas' cat (Otocolobus manul) blood infected with Cytauxzoon manul. A seventh domestic cat served as a control and was inoculated only with sterile water. Cats were monitored for clinical signs consistent with cytauxzoonosis, and periodically screened for hemoparasitemia. All domestic cats (6/6) that received Pallas' cat blood infected with C. manul developed a low but detectible parasitemia by 9 days post-inoculation, yet remained clinically healthy. All domestic cats (7/7) were subsequently challenged with Cytauxzoon felis and developed clinical signs typical of cytauxzoonosis within 5 days post-challenge. Affected animals were euthanized and cytauxzoonosis was confirmed by histopathology. While inoculation of domestic cats with Pallas' cat blood infected with C. manul induced a parasitemia, it did not cause disease or provide protection against challenge with C. felis. Further studies are warranted to determine the potential for interspecies transmission and disease with C. manul.

The costs and benefits in an unusual symbiosis: experimental evidence that bitterling fish (Rhodeus sericeus) are parasites of unionid mussels in Europe.

Interspecific symbiotic relationships involve a complex network of interactions, and understanding their outcome requires quantification of the costs and benefits to both partners. We experimentally investigated the costs and benefits in the relationship between European bitterling fish (Rhodeus sericeus) and freshwater mussels that are used by R. sericeus for oviposition. This relationship has hitherto been thought mutualistic, on the premise that R. sericeus use mussels as foster parents of their embryos while mussels use R. sericeus as hosts for their larvae. We demonstrate that R. sericeus is a parasite of European mussels, because it (i) avoids the cost of infection by mussel larvae and (ii) imposes a direct cost on mussels. Our experiments also indicate a potential coevolutionary arms race between bitterling fishes and their mussel hosts; the outcome of this relationship may differ between Asia, the centre of distribution of bitterling fishes, and Europe where they have recently invaded.