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.     

Diet of the killifish Rivulus marmoratus collected from land crab burrows,with further ecological notes

Synopsis One-hundred eleven specimens of the cyprinodont fish Rivulus marmoratus were trapped from burrows of the great land crab Cardisoma guanhumi in east central Florida and the entire alimentary tract removed for analysis of food items. Three categories of burrows (inactive, active and sealed) were sampled to examine possible relationships between burrow type and presence of R. marmoratus. Forty percent of the guts examined contained no food items, while the remaining 60% contained items of both terrestrial and aquatic origin. Capture rates among burrow categories did not vary significantly, and most collections consisted of 1 or 2 individuals per burrow. Mean standard length of fish within a burrow declined when more than 3 specimens were captured from a burrow. Supplemental observations on reproductive status and burrow water temperature are noted.     

Developmental morphology of the cottid fish Pseudoblennius marmoratus

Embryonic, larval, and juvenile development of a Japanese cottid fish Pseudoblennius marmoratus is described from laboratory-reared specimens. Eggs of P. marmoratus resembled those of two congeners, Pseudoblennius cottoides and Pseudoblennius percoides, but differed in their smaller diameter and yolk color. Although the larvae and juveniles of P. marmoratus were also similar to them in general morphology, the former differed in the lengths of newly hatched larvae, pigmentation patterns, number of pectoral fin rays, and dorsal fin spine elongation.     

Detection of aerolysin gene in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.     

Emersion in the mangrove forest fishRivulus marmoratus: a unique response to hydrogen sulfide

Synopsis The mangrove forest fishRivulus marmoratus (Cyprinodontidae) has frequently been observed out of water, a phenomenon generally attributed to habitat drying. We tested the hypothesis that hydrogen sulfide, a substance characteristically found in their environment, can serve as a stimulus for emersion. In the field we foundR. marmoratus in water with low to moderate levels (<250 ppb) of HS. In the laboratory,R. marmoratus leaped from water contaminated with H₂S at ecologically relevant concentrations (median response at 123 ppb). Aquatic hypoxia did not induce emersion, but prey capture did. Oxygen consumption by both juveniles and adults decreased significantly in air (27 and 25%, respectively). Our results suggest that avoidance of H₂S and the ability to survive terrestrial conditions enable this species to permanently occupy an area of the forest unavailable to other fishes. Furthermore, because a variety of stimuli lead to etriersion inR. marmoratus, terrestriality in this species is likely a generalized response to environmental stress as well as a means of exploiting terrestrial resources.     

The BAH domain, polybromo and the RSC chromatin remodelling complex

We isolated Rivulus marmoratus mitochondrial DNA by long-polymerase chain reaction with conserved primers, and sequenced it with 36 sets of internal conserved primers, which were designed from the extensive sequence similarities of mitochondrial DNA from several fish species. The R. marmoratus mitochondrial DNA has 17,329 bp with a conserved structural organization compared to those of other fish. Rivulus marmoratus mitochondrial DNA also has two nearly identical control regions. The basic characteristics of the R. marmoratus mitochondrial genome are discussed.     

Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

Homozygosity and Heterozygosity in three Populations of Rivulus marmoratus

Rivulus marmoratus is a self-fertilizing hermaphroditic fish found in the tropical Atlantic as populations of homozygous clones, with the exception of a single site in Belize where male fish are abundant and heterozygosity is the norm. The presence of male fish apparently leads to outcrossing and heterozygosity, but males have been found in limited numbers in other populations which are homozygous. DNA fingerprinting now reveals that the Belize population has remained heterozygous, with a high proportion of males (20–25%), for several years. In addition, two newly discovered populations with a lower percentage of males (1–2%) are reported from the Bahamas and Honduras. One of these populations (Bahamas) consists of homozygous clones, while the other (Honduras) displays a limited proportion of heterozygosity. The Honduras population is only the second outcrossing population known in this species, and the limited heterozygosity seen here may reflect the lower proportion of males.     

EVIDENCE THAT AN OUTCROSSING POPULATION IS A DERIVED LINEAGE IN A HERMAPHRODITIC FISH (RIVULUS MARMORATUS)

Rivulus marmoratus is the only known vertebrate with obligate, synchronous hermaphroditic fertilization. Males can be experimentally induced in the laboratory and are rare or absent in most populations, but at the isolated Twin Cays, Belize, locality, males are relatively abundant. At this locality, evidence of outcrossing has been documented in this otherwise automictic cloning species. Phylogenetic analysis of restriction sites and sequence characters revealed that all Florida and Belize western Caribbean populations (including Twin Cays) are phyletically indistinguishable yet divergent from eastern populations in Brazil and the Bahamas. Further, these western lineages shared a common ancestor more recently than all other populations. Therefore, the Twin Cays population is not a remnant ancestral outcrossing population. Outcrossing is suspected to have evolved as a phenotypically plastic character, and its expression in R. marmoratus may be dormant unless triggered by some ecological factor that is not well understood.     

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.     

AZFY-like sequence in fish,with comments on the evolution of theZFY family of genes in vertebrates

ZFY-like genes have been observed in a variety of vertebrate species. Although originally implicated as the primary testis-determining gene in humans and other placental mammals, more recent evidence indicates a role(s) outside that of testis determination. In this study, DNA from five species of fish,Carasius auratus, Rivulus marmoratus, Xiphophorus maculatus, X. milleri, andX. nigrensis was subjected to Southern blot analysis using a PCR-amplified fragment of mouseZFY-like sequence as a probe. Restriction fragment patterns were not polymorphic between sexes in any one species but showed a different pattern for each species. With one exception,Rivulus, a 3.1-kb band from theEcoRI digestion was common to all. Sequence and open reading frame analysis of this fragment showed a strong homology to other known vertebrateZFY-like genes. Of particular interest in this gene is a novel third finger domain similar to one human and one alligatorZFY-like gene. Our studies and others provide evidence for a family of vertebrateZFY genes, with those having this novel third finger being representative of the ancestral condition.     

Teleost Fish Genomes Contain a Diverse Array of L1 RetrotransposonLineages That Exhibit a Low Copy Number and High Rate of Turnover

Retrotransposable elements exhibit a wide range of variation in population dynamics, abundance, and lineage diversity among host genomes across taxa. This range of diversity is illustrated by a single well-defined constituent monophyletic clade of L1 non-LTR retrotransposons that is shared between mammalian and teleost fish genomes. Despite the clear phylogenetic relationships that exist between mammalian and teleost L1 sequences, these elements exhibit markedly different dynamics within their respective taxa. While mammalian genomes typically contain a single, abundant lineage of L1 elements that traces millions of years of evolution, the zebraflsh genome was recently shown to exhibit a high diversity of ancient lineages coexisting at a very low copy number and apparently exhibiting a high rate of turnover. In the present study, a combination of degenerate PCR, lineage-specific PCR, and genomic Southern blot analysis is utilized to demonstrate high L1 lineage diversity, low copy number, and a high proportion of polymorphic inserts in the genomes of the killifish species, Fundulus heteroclitus. Additional species surveyed by degenerate PCR include Cyprinodon variegatus, Rivulus marmoratus, and Menidia beryllina. These results further support the generality of the differences that exist in host–element dynamics between teleost fish and mammalian genomes with regard to L1 retrotransposons.Reviewing Editor: Dr. Axel Meyer     

Species‐specific PCR primers for the mitochondrial genome control region hypervariable region 1 of the reef fish Lutjanus sebae

Lutjanus sebae is an important commercial and recreational fish found throughout the Indo‐West Pacific region. Using universal primers for the mitochondrial genome control region hypervariable region 1 (HVR1), we isolated a 385‐bp fragment of HVR1 then designed specific primers near each end of this sequence in the conserved regions flanking the hypervariable region. These primers were shown to yield good quantity and quality of product and to be species specific. Of 20 L. sebae studied, there were eight segregating sites and eight haplotypes. These primers will provide a useful tool for population genetics studies, identification of larvae and eggs and for wildlife forensics.     

Specific DNA probes for the identification of the fish pathogen,Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile     

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.     

Isolation and differentiation of Rivulus hartii across Trinidad and neighboring islands

Diversification of freshwater fishes on islands is considered unlikely because the traits that enable successful colonization—specifically, broad salinity tolerances and the potential for oceanic dispersal—may also constrain post‐colonization genetic differentiation. Some secondary freshwater fish, however, exhibit pronounced genetic differentiation and geographic structure on islands, whereas others do not. It is unclear what conditions give rise to contrasting patterns of differentiation because few comparative reconstructions of population history have been carried out for insular freshwater fishes. In this study, we examined the phylogeography of Hart’s killifish (Rivulus hartii) across Trinidad, with reference to neighboring islands and northern South America, to test hypotheses of colonization and differentiation derived from comparable work on co‐occurring guppies (Poecilia reticulata). Geographic patterns of mitochondrial DNA haplotype variation and microsatellite genotype variation provide evidence of genetic differentiation of R. hartii among islands and across Trinidad. Our findings are largely consistent with patterns of geographically structured ancestry and admixture found in Trinidadian guppies, which suggests that both species share a history of colonization and differentiation and that post‐colonization diversification may be more common among members of insular freshwater fish assemblages than has been previously thought.     

Spawning in a cold bath: reproduction of polar and deep‐sea fish family Psychrolutidae

This study describes the spawning of the fatheads Psychrolutes marmoratus and Cottunculus granulosus, two psychrolutid species that inhabit the shelf edge and continental slope of the south‐west Atlantic. Females lay large eggs of c. 2·5 mm (P. marmoratus) and 4·5–5·0 mm (C. granulosus); fecundity is from the hundreds (C. granulosus) to a few thousand eggs (P. marmoratus). Egg maturation is synchronous in P. marmoratus with an autumn–winter peak of spawning and group‐synchronous in C. granulosus, which reproduces all year round. In the fishery, females predominate among adult fish in both species, possibly indicating male nest guarding on hard grounds inaccessible to fishing vessels. The reproductive strategy of representatives of the Psychrolutidae is similar to that of other sculpins of the superfamily Cottoidea.