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.     

Reproductive and developmental responses in the self-fertilizing fish,Rivulus marmoratus,induced by the plasticizer,di-n-butylphthalate

Synopsis Specialized life-history attributes of the cyprinodontiform fish, Rivulus marmoratus, allow continuous life cycle testing to reveal effects of chemicals or environmental stresses upon fertilization, fecundity, egg viability, embryonic development, sex ratios, frequency of growth or skeletal anomalies, as well as other biological markers. This study reports responses in fecundity, viability of embryos, and skeletal anomalies during and after exposure of parental fish to the plasticizer, di-n-butylphthalate (DBP). Skeletal anomalies among offspring were classified as mild, moderate, or severe compared with non-deformed normal offspring. The frequency of skeletal anomalies increased from 4% (all categories combined) in controls, to 10% and 19% of the offspring from adults exposed to 1 and 2 mg I^-1 DBP, respectively. DBP treatment was conducted over a 21 week interval, followed by a 9 week post-treatment observation interval. During post-treatment, frequency of skeletal anomalies decreased to less than 5% in all groups. Contributions No. 610, Environmental Research Laboratory, Gulf Breeze, Florida 32561 U.S.A. The information in this document has been funded wholly by the U.S. Environmental Protection Agency, and has been subjected to the Agency's peer and administrative review. Mention of commercial products or trade names does not constitute the Agency's endorsement or recommendation for use.     

Abundant males in populations of a selfing hermaphrodite fish, Rivulus marmoratus, from some Belize cays

The killifish Rivulus marmoratus is the only known selfing hermaphroditic vertebrate, and males of the species are usually exceedingly rare or non-existent in nature. Collections on several Belize cays in 1988 and 1989 yielded 13.5–24% males. Factors responsible for this unusually high proportion of males are currently not understood. Likewise, the biological significance of males in populations which otherwise consist of selfing hermaphrodites (with internal fertilization) is problematic and awaits further study.     

Adaptive advantages of myrmecochory: the predator-avoidance hypothesis tested over a wide geographic range

The predator‐avoidance hypothesis states that once released from the parent plant, myrmecochorous seeds are rapidly taken by ants to their nests, where they are protected from predators. Previous studies conducted to test this hypothesis have frequently neglected two major aspects necessary for its verification: 1) the influence of processes acting after the seed release and 2) the spatial evenness of such processes. Thus, large‐scale variations in the mechanisms acting beyond seed release, and possibly influencing seed escape from predators, remain poorly documented. Here, we present the results of a post‐dispersal seed‐removal experiment on the myrmecochorous herb Helleborus foetidus, aimed at verifing the predator‐avoidance hypothesis by considering two key post‐release aspects of seed fate: seed destination (dispersed or nondispersed) and seed burial (buried or not buried). Experiments were performed in four different regions in the Iberian Peninsula. After three days of exposure of seeds to the main predator (fieldmice Apodemus sylvaticus), ca 30% of the seeds were removed. Seed destination affected the proportion of seeds escaping predation, but the sign, magnitude and statistical significance of the effect varied among the geographical regions. In the southern region (Cazorla), seeds dispersed in ant nests or intermediate destinations suffered scarcely any predation, but seeds under reproductive‐age plants experienced losses ca 50%. Conversely, in the northern region (Caurel), seeds in nests suffered significantly greater losses than seeds under plants or intermediate destinations, suggesting that nests were especially unsafe destinations. Seed burial had a strong impact on seed escape from predators, and its effect was highly consistent among geographical regions. In view of the consistency of its effect at different spatial scales, seed burial was a more general mechanism for predation avoidance than seed relocation to ant nests, which was habitat‐ and/or ant‐species‐dependent. Our results thus only partially support the predator‐avoidance hypothesis for the evolution of myrmecochory.     

Persistence of Mhc heterozygosity in homozygous clonal killifish,Rivulus marmoratus: implications for the origin of hermaphroditism

The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced. Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose approximately 20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci, as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by mutations, each of which spread through the entire population and was fixed independently in the emerging clones.     

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.     

Genomic cloning of a heat shock cognate 71-1 gene (HSC71-1) from the hermaphroditic fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae).

The self-fertilizing fish Rivulus marmoratus (R. marmoratus) heat shock cognate 71 (HSC71) gene was cloned and characterized recently (Park et al., 2001). Here, we report the isolation of a homologue of the R. marmoratus HSC71 gene via screening of an R. marmoratus genomic DNA library. A 12,591 bp genomic fragment was sequenced and found to contain a 2844 bp open reading frame that consisted of 8 exons and showed high similarity to the previously reported R. marmoratus HSC71 gene. The two genes differed slightly at exons 5 and 8, and intron 3. On a deduced amino acid sequence level, the two R. marmoratus HSC71 genes were highly similar (89.3% in amino acid residues). In this paper, the author presented a homologous gene (R. marmoratus HSC71-1) similar to R. marmoratus HSC71 gene.     

Cytogenetic analysis of chromosomal aberrations of peripheral lymphocytes in workers occupationally exposed to nickel.

The authors carried out a cytogenetic examination of chromosomal aberrations of peripheral lymphocytes (100 cells evaluated in each sample) with simultaneous monitoring of the level of exposure by means of determination of nickel in the urine, serum and hair. The series included 21 workers occupationally exposed to nickel at two workshops producing NiO (6 persons) and NiSO4 (15 persons) in a chemical plant. At the same time a comparable control group, i.e., 19 workers of the same chemical plant but without any direct occupational nickel exposure (clerks, service men, etc.), were examined in the same way. In the exposed group chromosomal aberrations of peripheral lymphocytes were detected with an average value of 6.41 +/- 1.9% (range 2-14%); in the group producing NiO it was, on the average, 9.5 +/- 3.2% (range 7-14%) whereas in the NiSO4 production workers it was only 5.2 +/- 1.9% (range 2-10%). There was a dependence of chromosomal aberrations of peripheral lymphocytes on the exposure time and on the nickel content of the biological material. Significantly increased values (in contrast to the normal value of chromosomal aberrations of peripheral lymphocytes, up to 2%) were detected in the control group as well (average value of 4.05 +/- 2.27%, range 1-10%). The authors explain this fact by the nickel-polluted environment of the whole observed chemical plant.     

Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: identification of a novel muscle-preferred regulatory element

To further our understanding of the role of stress proteins in development as well as in adaptation of fish to adverse environmental conditions, we undertook molecular analyses of stress protein encoding genes from the hermaphroditic teleost Rivulus marmoratus. We isolated a genomic clone containing the Hsc71 gene (rm-hsc71m) and its upstream sequences. rm-Hsc71m is not induced by external stress, but is enriched in a tissue-specific manner during early development. In adult, the strongest expression appeared in skeletal muscle, whereas lower expression was seen in the gill, eye and brain. To understand the regulatory basis of high muscle expression of rm-hsc71m, transfection of R.marmoratus muscle tissue was performed using 5′ deletion fragments containing the rm-hsc71m promoter driving EGFP expression. An upstream region from –2.7 to –1.9 kb was identified as a muscle-specific regulatory region. Within this region, we identified at least three sites with the novel sequence TGTnACA interacting with a fish muscle factor having an M_r of 32 000. Our data indicate that rm-hsc71m expression in skeletal muscle is controlled by a muscle-specific regulatory element containing this novel motif.     

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.     

Cytogenetic analysis of a Mediterranean Gobiid fish Gobius paganellus L., 1758 from Turkey

In this study, cytogenetic analysis of Gobius paganellus, a Mediterranean Gobiid fish from the southern coast of Turkey, was carried out by modified air-dried technique. Phytohemaglutinine pretreatment was performed before the conventional air-dried technique to increase the mitotic index. Chromosomes were obtained using gill tissue. As a result, it was determined that the diploid chromosome number of Gobius paganellus is 2n = 44 (NF = 45), comprising one large metacentric and 43 acrocentric chromosomes.     

Cytogenetic analysis of the noxious bloodsucker Boophthora erythrocephala (Diptera: Simuliidae) from different geographic zones

Boophthora erythrocephala is a widely spread species inhabiting both small polluted water-bodies and large rivers. Three populations from Samara region in Russia and from Donetsk and Chernigov regions in the Ukraine have been studied. Five populations of this species in Russia were described earlier (Polyanskaya, Tsapygina, 1968; Petrukhina, 1972). For identification of this species the chromosome map of Adler and Werner (personal communication) was used. Comparison of 8 populations studied was performed. All of them belong to the species B. erythrocephala but everyone has its own individual characteristics. Inversion polymorphism was observed in each population comprising from 88 to 100 %. Mean number of heterozygous inversions per individual is low fluctuating around 3.6. There is one inversion in IIS observed in all populations that proves panmixy among them. Other inversions occur in a part of the individuals.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.     

Environmental marginality and geographic range limits: a case study with Arabidopsis lyrata ssp. lyrata

Understanding the factors that govern the distribution of species is a central goal of evolutionary ecology. It is commonly assumed that geographic range limits reflect ecological niche limits and that species experience increasingly marginal conditions towards the edge of their ranges. Using spatial data and ecological niche models we tested these hypotheses in Arabidopsis lyrata. Specifically, we asked whether range limits coincide with predicted niche limits in this system and whether the suitability of sites declines towards the edge of the species’ range in North America. We further explored patterns of environmental change towards the edge of the range and asked whether genome‐wide patterns of genetic diversity decline with increasing peripherality and environmental marginality. Our results suggest that latitudinal range limits coincide with niche limits. Populations experienced increasingly marginal environments towards these limits – though patterns of environmental change were more complex than most theoretical models for range limits assume. Genomic diversity declined towards the edge of the species’ range and with increasing distance from the estimated centre of the species’ niche in environmental space, but not with the suitability of sites based on niche model predictions. Thus while latitudinal range limits in this system are broadly associated with niche limits, the link between environmental conditions and genetic diversity (and thus the adaptive potential of populations) is less clear.     

Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.     

Cytogenetic analysis of chorionic villi: a technical assessment

Summary Eighty-five samples of chorionic villi from women undergoing prenatal diagnosis at 8 to 12 weeks' gestation were subjected to cytogenetic analysis. Samples were prepared by a direct technique that permits limited analysis within two hours and by a short-term culture technique that permits detailed structural analysis within one week. An adequate number of cell divisions for cytogenetic analysis was obtained from 96% of living fetuses. Using both the direct technique and short-term culture, satisfactory banded chromosomal preparations were made in 93% of cases. Eleven of 12 pregnancies (92%) shown by ultrasound to be dead shortly before sampling, had cytogenetic abnormalities. Further studies are needed to develop banding definition equivalent to that available on cultured amniocytes.