Phenotypic plasticity in the growth of the self-fertilizing hermaphroditic fish Rivulus marmoratus

Temperature, salinity, food ration and interactions among them had significant effects on overall growth of hatchlings of the self-fertilizing hermaphrodite Rivulus marmoratus . Low temperature (19° C) suppressed growth. At 26° C, the treatment groups at 12 and 40‰ salinity had higher final wet masses than the group at 1‰. When provided with high food ration, individuals whose parents were provided with low food had a significantly higher specific growth rate in the first month than those whose parents were provided with high food ration. This effect was also found in the final wet masses. It appears that genetic variation in this species occurs only between strains, yet within a strain there is considerable scope for phenotypic plasticity. The combination of a fixed genotype and plastic expression, along with a suite of specialized physiological characteristics, may explain how a self-fertilizing hermaphrodite can thrive in such an abiotically stressful and variable habitat as an estuary.     

How geitonogamous selfing affects sex allocation in hermaphrodite plants

Does the mode of self-pollination affect the evolutionarily stable allocation to male vs. female function? We distinguish the following scenarios. (1) An ‘autogamous’ species, in which selfing occurs within the flower prior to opening. The pollen used in selfing is a constant fraction of all pollen grains produced. (2) A species with ‘abiotic pollination’, in which selfing occurs when pollen dispersed in one flower lands on the stigma of a nearby flower on the same plant (geitonogamy). The selfing rate increases with male allocation but a higher selfing rate does not mean a reduced export of pollen. (3) An ‘animal-pollinated’ species with geitonogamous selfing. Here the selfing rate also increases with male allocation, but pollen export to other plants in the population is a decelerating function of the number of simultaneously open flowers. In all three models selfing selects for increased female allocation. For model 3 this contradicts the general opinion that geitonogamous selfing does not affect evolutionarily stable allocations. In all models, the parent benefits more from a female-biased allocation than any other individual in the population. In addition, in models 2 and 3, greater male allocation results in more local mate competition. In model 3 and in model 2 with low levels of inbreeding depression, hermaphroditism is evolutionarily stable. In model 2 with high inbreeding depression, the population converges to a fitness minimum for the relative allocation to male function. In this case the fitness set is bowed inwards, corresponding with accelerating fitness gain curves. If the selfing rate increases with plant size, this is a sufficient condition for size-dependent sex allocation (more allocation towards seeds in large plants) to evolve. We discuss our results in relation to size-dependent sex allocation in plants and in relation to the evolution of dioecy.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Methyltestosterone efficiently induces male development in the self-fertilizing hermaphrodite fish, Kryptolebias marmoratus

A hermaphrodite fish, Kryptolebias marmoratus, is the only known vertebrate that reproduces by self-fertilization. In nature, males have been rarely observed. Low-temperature treatment during late embryonic stages is known to induce males but its efficacy is variable. Here we report that 17alpha-methyltestosterone (MT) treatment of the embryos converted most of the fish to males. We examined a time course of this male induction with histological and marker gene expression analyses. Oogenesis started in the gonads of the control embryo at hatching; spermatogenesis did not start until two months after hatching. In the MT-treated fish, oogenesis started initially as in the control but stopped completely within one month after hatching. Instead, spermatogonial proliferation started earlier than in the control fish and progressed to full spermatogenesis. Expression profiles of the sex-specific marker genes corresponded well with histological observations. From one month after hatching, expression of an oocyte-specific marker, figalpha, and a testicular somatic cell marker, dmrt1, started to increase in the control and in the MT-treated fish, respectively.     

Age, Growth, and Sexual Development in the Self-fertilizing Hermaphroditic Fish Rivulus marmoratus

We studied age, growth, and sexual development in the early life intervals of the self-fertilizing mangrove killi-fish, Rivulus marmoratus. Newly hatched (day 0) individuals had sagittal otoliths of 60m radius, with about 30 increments. Sequential sampling until about day 60 after hatching yielded otoliths with the number of increments outside the 60m radius equal to the daily age of the fish. Alizarin complexone marking of otoliths also confirmed the increments were daily, and demonstrated the applicability of this technique to field studies for capture-mark-recapture, or age and growth estimates. Individuals fed a restricted amount of food formed fewer daily otolith growth increments than fish fed to satiation each day. Using histological analysis for identifying gonad morphogenesis, we found no correlation between gonadal development and external appearance (caudal ocellus, orange fin colouration) in young fish of known ages. The caudal ocellus was not present until 9mm total length, and developed thereafter. Of 136 individuals examined, fish less than 17.2mm total length (TL, n = 124) were females. Testicular tissue first appeared among individuals 17–18mm TL (n = 3), while some individuals greater than 18mm TL (n = 8) were functional hermaphrodites. The single male in our study was relatively of small body size (9.6mm, day 37) with a distinct caudal ocellus, indicating that it is presumably a primary male.     

Extreme homogeneity and low genetic diversity in Kryptolebias ocellatus from south-eastern Brazil suggest a recent foundation for this androdioecious fish population

This study documents unexpectedly low levels of intra and interpopulation genetic diversity in Kryptolebias ocellatus, an androdioecious and predominantly self-fertilizing killifish from south-eastern Brazil. This finding generally is inconsistent with the established opinion that the K. ocellatus and K. marmoratus clade originated in this geographic region and later dispersed northward into the Caribbean.     

Presence of primary and secondary males in a population of the protogynous Synbranchus marmoratus

Synbranchus marmoratus , the 'swamp eel', is a protogynous diandric fish. The primary and secondary males can be distinguished from each other easily by differences in gonadal morphology. Primary males have lobular, unrestricted testes with central efferent ducts. The secondary male has a 'lamellar' testis, efferent ducts are present in the ventral region (new formation), lateral supports and it is covered by the former ovarian capsule. The length of primary male varies from 13 to 88 cm while secondary males range from 56 to 91 cm. Transitional individuals vary between 45 and 60 cm in length. The swamp eel population studied is composed of 80% primary males and 20% secondary males. Although gonadosomatic indices are always higher in primary males, they increase in secondary ones as the newly acquired stage progresses. The lamellar organization of the gonad of secondary males, the absence of this kind of testis in individuals smaller than 56 cm, and the presence of hermaphroditic fish, is evidence for protogyny in this species.     

Evolutionary rescue in randomly mating,selfing, and clonal populations

Severe environmental change can drive a population extinct unless the population adapts in time to the new conditions (“evolutionary rescue”). How does biparental sexual reproduction influence the chances of population persistence compared to clonal reproduction or selfing? In this article, we set up a one‐locus two‐allele model for adaptation in diploid species, where rescue is contingent on the establishment of the mutant homozygote. Reproduction can occur by random mating, selfing, or clonally. Random mating generates and destroys the rescue mutant; selfing is efficient at generating it but at the same time depletes the heterozygote, which can lead to a low mutant frequency in the standing genetic variation. Due to these (and other) antagonistic effects, we find a nontrivial dependence of population survival on the rate of sex/selfing, which is strongly influenced by the dominance coefficient of the mutation before and after the environmental change. Importantly, since mating with the wild‐type breaks the mutant homozygote up, a slow decay of the wild‐type population size can impede rescue in randomly mating populations.     

Plasma Sex Steroid Levels and Steroidogenesis in the Gonad of the Self-fertilizing Fish Rivulus marmoratus

Synopsis The mangrove killifish, Rivulus marmoratus, is the only known self-fertilizing vertebrate. This species is sexually dimorphic; sexually mature individuals are either hermaphrodite or primary and secondary males. Although the mangrove killifish has a unique reproductive strategy, there has been no study on the reproductive endocrinology of this species. Thus we investigated plasma sex steroid hormone levels and steroidogenesis in the gonads of R. marmoratus by enzyme linked immunosorbent assay (ELISA). Plasma 17β-estradiol (E2) and 11-ketotestosterone (11-KT) were detected both in hermaphrodite and in primary male. Ovarian follicles (follicle-enclosed oocytes) from hermaphrodites, which were categorized into early yolk stage and late yolk stage, and testis tissue of primary males were cultured with different concentrations of 17α-hydroxyprogesterone (OHP) or testosterone (T) for 24 h. Production of T, E2, 11-KT and 17α-20 β-dihydroxy-4-pregnen-3-one (17α,20β-P) in the medium from tissue culture were measured by ELISA. Early and late ovarian follicles of hermaphrodites and testis pieces of primary males synchronously secreted E2, 11-KT, and 17α,20β-P following incubation with OHP or T. We conclude that both hermaphrodite and primary male of the mangrove killifish secrete estrogen, androgen, and progestin synchronously.     

Strong population structure despite evidence of recent migration in a selfing hermaphroditic vertebrate, the mangrove killifish (Kryptolebias marmoratus)

We employ a battery of 33 polymorphic microsatellite loci to describe geographical population structure of the mangrove killifish (Kryptolebias marmoratus), the only vertebrate species known to have a mixed-mating system of selfing and outcrossing. Significant population genetic structure was detected at spatial scales ranging from tens to hundreds of kilometres in Florida, Belize, and the Bahamas. The wealth of genotypic information, coupled with the highly inbred nature of most killifish lineages due to predominant selfing, also permitted treatments of individual fish as units of analysis. Genetic clustering algorithms, neighbour-joining trees, factorial correspondence, and related methods all earmarked particular killifish specimens as products of recent outcross events that could often be provisionally linked to specific migration events. Although mutation is the ultimate source of genetic diversity in K. marmoratus, our data indicate that interlocality dispersal and outcross-mediated genetic recombination (and probably genetic drift also) play key proximate roles in the local 'clonal' dynamics of this species.     

Size‐dependent sex allocation in a simultaneous hermaphrodite parasite

Most models of sex allocation distinguish between sequential and simultaneous hermaphrodites, although an intermediate sexual pattern, size‐dependent sex allocation, is widespread in plants. Here we investigated sex allocation in a simultaneous hermaphrodite animal, the tapeworm Schistocephalus solidus, in which adult size is highly variable. Sex allocation was determined using stereological techniques, which allow measuring somatic and reproductive tissues in a common currency, namely volume. We investigated the relationships between individual volume and allocation to different reproductive tissues using an allometric model. One measure of female allocation, yolk gland volume, increased more than proportionally with individual volume. This is in contrast to the measure of male allocation, testis volume, which showed a strong tendency to increase less than proportionally with individual volume. Together these patterns led to sex allocation being strongly related to individual volume, with large individuals being more biased towards female allocation. We discuss these findings in the light of current ideas about size‐dependent sex allocation in, primarily, plants and try to extend them to simultaneous hermaphrodite animals.     

Inbreeding depression and selfing rates in a self-compatible,hermaphroditic species,Schiedea membranacea (Caryophyllaceae)

Inbreeding depression and selfing rates were investigated in Schiedea membranacea (Caryophyllaceae), a hermaphroditic species endemic to the Hawaiian Islands. Most theoretical models predict high inbreeding depression in outcrossing hermaphroditic species and low inbreeding depression in inbreeding species. Although high outcrossing rates and high levels of inbreeding depression are characteristic of many species of Schiedea, self- fertilization is common among relatives of hermaphroditic S. membranacea, and high selfing rates and low levels of inbreeding depression were predicted in this species. Sixteen individuals grown in the greenhouse were used to produce selfed and outcrossed progeny. Inbreeding depression, which was evident throughout the stages measured (percentage viable seeds per capsule, mean seed mass, percentage seed germination, percentage seedling survival, and biomass after 8 mo), averaged 0.70. Inbreeding depression among maternal families varied significantly for all measured traits and ranged from −0.12 to 0.97. Using isozyme analysis, the multilocus selfing rate varied from 0.13 to 0.38 over 4 yr. Contrary to the initial prediction of high selfing and low inbreeding depression based on phylogenetic relationships within Schiedea, low selfing rates and high levels of inbreeding depression were found in S. membranacea. These results indicate that outcrossing is stable in this species and maintained by high levels of inbreeding depression.     

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.     

Association behaviour of the self-fertilizing Kryptolebias marmoratus (Poey): the influence of microhabitat use on the potential for a complex mating system

Preliminary observations were conducted to identify conspicuous body postures and movements of males and hermaphrodites in the mangrove killifish Kryptolebias marmoratus . These behaviours were used quantitatively to examine the social interactions for experimental pairings of K. marmoratus of different sexual states ( i.e. simultaneous hermaphrodite and male) in an aquarium with an open-water area and simulated crab burrows. This allowed observation of behaviours that could not be observed in the field. Kryptolebias marmoratus , regardless of sexual state and experimental treatment, spent 40% of the time in the burrow. Hermaphrodites exhibited a preference for associating with males rather than other hermaphrodites. The observed complexity of displayed behaviours and interactions between paired conspecifics indicate that K. marmoratus has a rich repertoire of social behaviour not predicted for a strictly selfing species. Also, land crab burrows play an important role in their social interactions.     

A hermaphroditic Arctic charr from Loch Rannoch, Scotland

An individual Arctic charr from Loch Rannoch was found to contain a gonad the anterior portion of which consisted of ovarian tissue. This is an extremely rare condition in salmonid fish and in this instance is possibly the result of environmental contamination.     

Early development of the self-fertilizing mangrove killifish <Emphasis Type="Italic">Rivulus marmoratus</Emphasis> reared in the laboratory

The mangrove killifish Rivulus marmoratus was reared at 25°±1°C and 17ppt salinity from 0 to 100 days after hatching (DAH), and its early development was described by examining growth and morphometric parameters, meristic characters (vertebral and fin-ray counts), bone-cartilage development, and pigmentation. Growth was isometric for preanal length, head length, snout length, body depth, pectoral-fin length, dorsal-fin length, anal-fin length, and caudal-peduncle depth. Negative allometric growth was observed in eye diameter and gape size. Meristic counts (mean±SD) for vertebrae (34.2±0.4) and dorsal- (8.6±0.5), anal- (11.4±0.5), and caudal-fin rays (30.2±0.8) were complete at 0 DAH (n=5), whereas pectoral-fin rays and pelvic-fin rays were complete by 30 DAH (14.5±0.4, n=5) and 60 DAH (4.2±0.8, n=5). Full ossification of meristic elements proceeded in the following sequence: vertebrae (by 30 DAH), caudal-, dorsal-, and anal-fin rays (by 60 DAH), pectoral-fin rays (between 60 DAH and 100 DAH), and pelvic-fin rays (by 100 DAH). Both morphological characters and meristic counts indicate that this species can be considered to be a juvenile after 9.8mm in standard length (20 DAH).     

Differences in life-history traits in two clonal strains of the self-fertilizing fish, <Emphasis Type="BoldItalic">Rivulus marmoratus</Emphasis>

Synopsis We compared life-history traits such as fecundity, sex ratio, reproductive cycle, age at sexual maturity, embryonic period, egg size, early growth and morphology in two clonal strains (PAN-RS and DAN) of the mangrove killifish, Rivulus marmoratus, under constant rearing conditions. We found a positive relationship between growth and reproductive effort. Fecundity was significantly higher in the PAN-RS strain than in the DAN strain. The sex ratio was significantly different, with DAN producing more primary males than PAN-RS. Spawning and ovulation cycle did not clearly differ between the strains. PAN-RS showed a significantly higher growth rate than DAN from 0 to 100 days after hatching, however, age at sexual maturity, embryonic period, egg size, and morphometric and meristic characteristics (vertebral and fin-ray counts) did not differ between the two strains. The high fecundity of PAN-RS may provide an increased chance of offspring survival, while the attainment of sexual maturity at a smaller size in DAN may allow them to invest earlier in reproduction to increase breeding success. Variations in the life-history traits of PAN-RS and DAN may be adaptive strategies for life in their natural habitat, which consists of mangrove estuaries with a highly variable environment.     

Molecular cloning and characterization of theta-class glutathione S-transferase (GST-T) from the hermaphroditic fish Rivulus marmoratus and biochemical comparisons with alpha-class glutathione S-transferase (GST-A)

We cloned and sequenced full-length cDNA of a theta-class-like glutathione S-transferase (GST-T) from liver tissue of the self-fertilizing fish Rivulus marmoratus. The full-length cDNA of rm-GST-T was 907 bp in length containing an open reading frame of 666 bp that encoded a 221-amino acid putative protein. Its derived amino acid sequence was clustered with other vertebrate theta-class GSTs in a phylogenetic tree. The deduced amino acid sequence of theta-like rm-GST (rm-GST-T) was compared with both classes (alpha and theta) of GST and alpha-class rm-GST (rm-GST-A). Tissue-specific expression of two rm-GST mRNAs was investigated using real-time RT-PCR. To further characterize the catalytic properties of this enzyme along with rm-GST-A, we constructed the recombinant theta-like rm-GST plasmid with a 6 x His-Tag at the N-terminal of rm-GST-T cDNA. Recombinant rm-GST-T was highly expressed in transformed Escherichia coli, and its soluble fraction was purified by His-Tag affinity column chromatography. The kinetic properties and effects of pH and temperature on rm-GST-T were further studied, along with enzyme activity and inhibition effects, and compared with recombinant rm-GST-A. These results suggest that recombinant rm-GSTs such as rm-GST-A and rm-GST-T play a conserved functional role in R. marmoratus.     

Lifetime reproductive output in a hermaphrodite cestode when reproducing alone or in pairs: a time cost of pairing

The cestode Schistocephalus solidus is a facultatively self-fertilising simultaneous hermaphrodite. Here we test for differences in the starting point, the rate, and the magnitude of egg production between individuals allowed to reproduce alone (only self-fertilisation possible) or in pairs (both self- and cross-fertilisation possible). Specifically, we want to distinguish between alternative processes responsible for the lower egg production in paired individuals observed in an earlier study (Wedekind et al., 1998). We designed an improved in vitro system, replacing the bird final host that allows us to measure, with high temporal resolution, the timing and magnitude of lifetime egg production of worms in these two social situations. We found that the experimental groups did not differ significantly in the starting point of egg production. However, the temporal pattern in egg production differed between them, in that paired individuals had a lower rate of egg production. This, however, did not lead to a significant reduction in lifetime egg production, as pairs compensated for the lower rate by producing eggs longer than single individuals. We argue that the lower rate of egg production may nevertheless lead to a time cost of pairing in the study species, and that this cost is likely to represent a cost of outcrossing due to sexual selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

The cyprinodont fish Rivulus (Aplocheiloidei: Rivulidae) in Trinidad and Tobago: molecular evidence for marine dispersal, genetic isolation and local differentiation

Mitochondrial DNA sequences (756 bp) were obtained from the cytochrome b gene of 36 Rivulus individuals collected from 10 sites in Trinidad and one site in Tobago. Eight haplotypes were identified. Low genetic divergence (0.5%) between one western Trinidad (Blue Basin) haplotype and Rivulus hartii from north-western Venezuela (Paria peninsula) and high genetic divergence ( c. 11%) between these and the remaining other Trinidad and Tobago haplotypes suggests that the islands were colonized by two lineages. The commoner haplotype is distributed throughout lowland Trinidad, possibly a reflection of flooding of the Orinoco River leading to high dispersal between watersheds. Rivulus from higher altitude (Northern Range) localities that would not have been affected by such flooding show high genetic divergence between sites. The genetic differentiation between northern and southern watersheds suggests isolation between some of these drainages.