Single Fish Research Articles

Development of multiple robotic fish cooperation platform

This article presents the development of a multiple robotic fish cooperation platform, which is established by employing a group of radio-controlled, multi-link fish-like robots. This work is inspired by the observation from nature that the capability of one single fish is limited, as in order to survive the atrocious circumstances in the sea, fish often swim in schools. The analogical situations occur in the robotic fish case. In engineering applications, most missions are so complex that they must be accomplished by effective cooperation of multiple fish robots. The platform presented in this article, as a novel test bed for multiple robotic fish cooperation, can be applied to different types of complex tasks. More importantly, it provides a good platform to test and verify all kinds of algorithms and strategies for cooperation of multiple underwater mobile robots. We use two cooperative tasks as examples to heuristically demonstrate the performance of this platform.

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, Notemigonus crysoleucas

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R 2: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R 2: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.

A stochastic modelling approach to describing the dynamics of an experimental furunculosis epidemic in Chinook salmon, Oncorhynchus tshawytscha (Walbaum)

A susceptible-infected-removed (SIR) stochastic model was compared to a susceptible-latent-infectious-removed (SLIR) stochastic model in terms of describing and capturing the variation observed in replicated experimental furunculosis epidemics, caused by Aeromonas salmonicida. The epidemics had been created by releasing a single infectious fish into a group of susceptible fish (n = 43) and progress of the epidemic was observed for 10 days. This process was replicated in 70 independent groups. The two stochastic models were run 5000 times and after every run and every 100 runs, daily mean values of each compartment were compared to the observed data. Both models, the SIR model (R(2) = 0.91), and the SLIR model (R(2) = 0.90) were successful in predicting the number of fish in each category at each time point in the experimental data. Moreover, between-replicate variability in the stochastic model output was similar to between-replicate variability in the experimental data. Generally, there was little change in the goodness of fit (R(2)) after 200 runs in the SIR model whereas 500 runs were necessary to have stable predictions with the SLIR model. In the SIR model, on an individual replicate basis, approximately 80% of 5000 simulated replicates had R(2) = 0.7 and above, whereas this ratio was slightly higher (82%) with the SLIR model. In brief, both models were equally effective in predicting the observed data and its variance but the SLIR model was advantageous because it differentiated the latent, i.e. infected but not having the ability to discharge pathogen, from the infectious fish.

Measurement of fish steroids in water—a review

Measurement of fish steroids in water provides a non-invasive alternative to measurement in blood samples, offering the following advantages: zero or minimal intervention (i.e. no anaesthetic, bleeding or handling stress); results not being biased by sampling stress; repeat measurements on the same fish; the possibility of making non-lethal measurements on small and/or rare fish; integrating the response of many (or of single) fish; and allowing concurrent monitoring of behaviour or physiology. The procedure is relatively new and, although applications are still fairly limited, there are several themes and potential problem areas that are worthy of review.

Ontogenetic changes of learning capability under reward conditioning in striped knifejaw Oplegnathus fasciatus juveniles

To elucidate the ontogenetic changes in learning capability of the striped knifejaw Oplegnathus fasciatus, which is a common rocky-shore member in Japan, 28 individuals (2–10 cm standard length [SL]) of laboratory-reared fish under reward conditioning were trained. A single fish was first conditioned to go to one end of two arms of a Y-maze by being given a reward (food pellets), and once the fish has cleared the original problem, the rewarded side was reversed. All fish cleared at least the original problem, although fish at 2–4 cm SL showed lower scores because they learned more slowly or did not clear the reversal problem. Fish showed better learning capability as they grew. Fish of ca. 7 cm SL showed the highest score with quick learning of both the original and reversal problems. Their learning curve (relationship between SL and individual score) was approximated by a quadratic curve with the maximum score at approximately 7 cm SL. It was speculated that fish at its recruitment stage requires a high learning capability to adapt to the new environment of the rocky shore, which is composed of more complex microhabitats compared to their previous pelagic habitat.

Numerical study on propulsive performance of fish-like swimming foils

Two-dimensional numerical simulations are performed to study the propulsive performance of fish-like swimming foils using the immersed-boundary method. A single fish as well as two fishes in tandem arrangement are studied. First, the effect of the phase speed on the propulsive performance of a single fish is analyzed. The wake structures and pressure distribution near the wavy fish are also examined. The results show good correlation with those by previous researchers. Second, two tandem fishes with the same phase speed and amplitude are studied. The results show that the fish situated directly behind another one endure a higher thrust than that of a single one.

Behavioural responses in wild cod ( Gadus morhua L.) exposed to fish holding water

Local fishermen in several areas of Norway assert that salmon farms have caused the wild migrating cod to change their migratory behaviour, so that they no longer enter their natural spawning grounds in the fjords. If the asserted changes in behaviour of wild cod populations can be linked to establishment of salmon farms, water-soluble odorants are then possible candidates to explain such a connection. Chemical stimulants are important to the individual fish's conception of the surrounding environment. High density stocks of fish in a farm are expected to release large amounts of waterborne information. The present laboratory experiments were conducted to test behavioural responses in Atlantic cod exposed to water containing metabolites and waste from farmed salmon. The trials were conducted on single fish in a multiple chamber preference system. The results show that migrating wild Atlantic cod chose to spend more time in chambers without addition of water from the salmon tank, regardless of their maturation status, and even at very low concentrations (0.2%). The avoidance is probably due to presence of chemical compounds with olfactory properties from salmon, since anosmic cod did not elicit such response. Farmed cod, on the other hand, does not avoid water from the salmon tank, and stationary wild cod caught nearby a fish farm had a less pronounced response as compared to wild migrating cod. The response seen is not species specific, as wild migrating cod responded similar to water from a tank holding farmed cod as to water from salmon. These results do not preclude fishermen's observations that cod change their behaviour in areas with fish farming activity. Such a change in behaviour could be a response to water-soluble odorants, but needs to be validated and detailed in further laboratory experiments as well as in nature before any conclusions can be made.

Development of a quantitative scanning sonar system and sea trial for a fish school of herring

A quantitative sonar system enabling data recording, calibration, and numerical analysis of fish schools was developed based on the commercial scanning sonar. This system is equipped with a newly developed algorithm for estimation of the abundance of a fish school using three-dimensional echo integration. Estimation was made on the condition that the target strength of all fish is identical and known and the weight of a single fish is known. The field recording was performed to evaluate the algorithm. The school of spring spawning herring were scanned three-dimensionally and recorded using two different scanning methods. Two methods were applied in different distances between the school and vessel. The volume and three-dimensional echo integration for these two recorded dates were calculated and compared to each other. It was found that the variance of three-dimensional echo integration is smaller compared with the variance of volume. A compensation of abundance estimation using average target strength was tried and its effectivity was examined.

The influence of habitat characteristics and conspecifics on attraction and survival of coral reef fish juveniles

In marine species with a pelagic larval stage, search behavior and selection of a suitable reef habitat can maximize the settlement success of recently settled juveniles and their subsequent performance (growth and survival of juveniles). Our objective was to test this hypothesis for a single target coral reef fish species ( Chromis viridis) at Moorea Island. C. viridis settle on living coral colonies of Porites rus already populated with conspecifics. In the present study (conducted in experimental cages), we found that: 1) mortality rate of recently settled juveniles of C. viridis was lower in the settlement habitat (living coral colonies of P. rus) than in other habitats having physical structure different from those of P. rus colonies; 2) C. viridis juveniles preferentially colonized coral heads of P. rus with conspecifics present rather than uninhabited coral heads and they also preferentially colonized uninhabited coral heads rather than coral heads with heterospecifics; 3) mortality rate of C. viridis juveniles did not vary with the presence or absence of conspecifics or heterospecifics on P. rus colonies. Overall, the study allows us to highlight that site selection by juveniles for habitat containing conspecifics does not benefit their short term mortality rates, suggesting that in the short term at least, site selection has little importance.

Hierarchical status and colour preference in Nile tilapia (Oreochromis niloticus)

We studied the colour preference of isolated Nile tilapia (Oreochromis niloticus) and whether previous residence or body size can affect environmental colour choice. In the first phase, a cylindrical tank was divided into five differently coloured compartments (yellow, blue, green, white and red), a single fish was introduced into the tank and the frequency at which this fish visited each compartment was recorded over a 2-day study period. An increasingly larger fish (approx +2 cm in length each time) was then added into the tank on each of days 3, 5 and 7 (=four fish in the tank by day 7), and the frequency at which each fish visited the different compartments of the tank was observed twice a day to obtain visit frequency data on the differently sized fishes. This experiment was replicated six times. In the first phase, the solitary fish established residence inside the yellow compartment on the first and second days. Following the introduction of a larger fish, the smaller fish was displaced from the occupied compartment. Nile tilapia possibly shows this preference for yellow as a function of its visual spectral sensitivity and/or the spectral characteristics of its natural environment. Moreover, body size is an important factor in determining hierarchical dominance and territorial defence, and dominant fish chose the preferred environmental colour compartment as their territory.

Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae).

Coral reefs are known for their high level of biodiversity, but parasite biodiversity has not been evaluated. Cases such as Epinephelus maculatus, described here, show that the numerical estimation of parasite biodiversity in coral reefs could reach more than ten times the number of fish species; consequently, the extinction of certain fish species from endangered coral reefs would result in the co-extinction of at least ten times the number of parasite species. E. maculatus is a grouper of intermediate size (1-2 kg) and common in the coral reefs of New Caledonia, South Pacific. Based on the examination of more than 800 monogenean specimens, 12 species of monogeneans (ten diplectanids and two ancyrocephalids) were differentiated on the gills. These species of diplectanids have not been found in other epinephelines in the same area and thus are considered as specific to this host. In addition, three species of copepods, and isopod larvae, are present on the gills; E. maculatus thus has a total of 16 species of gill ectoparasites, which can be found together on a single individual fish. Diplectanids include Laticola dae Journo & Justine, 2006, which is the most abundant species representing about 50% of the specimens, and nine species which are rare, each representing 2-7% of the specimens: Diplectanum uitoe n. sp. and eight species of Pseudorhabdosynochus Yamaguti, 1958. D. uitoe, provisionally attributed to Diplectanum Diesing, 1858, is characterised by a small conical penis with internal walls. Pseudorhabdosynochus auitoe n. sp., P. buitoe n. sp., P. cuitoe n. sp., P. duitoe n. sp., P. euitoe n. sp. and P. fuitoe n. sp. are differentiated on the basis of the morphology of the sclerotised vagina, but are very similar in other characteristics; P. guitoe n. sp. is characterised by a quadriloculate organ with very thick walls and a very small sclerotised vagina; and P. huitoe n. sp. is characterised by its sclerotised vagina and by very long ventral and dorsal haptoral bars. Two rare (2-3% of specimens) ancyrocephalids, Haliotrema epinepheli Young, 1969 and Haliotrema sp., are briefly described in relation to the male copulatory organs and haptoral bars; H. epinepheli is apparently a generalist species found in various epinephelines and other fish species. A table of the 50 species of diplectanids (Pseudorhabdosynochus, Laticola Yang et al., 2006, Echinoplectanum Justine & Euzet, 2006 and Diplectanum) from serranids is provided.

P-930: Ploidy of day 5 and 6 embryos reaching blastocyst and classified as chromosomally abnormal after single cell biopsy and PGD on day 3

To assess D5 and 6 ploidy and cell numbers in some embryos donated to research that reached post-compaction and were diagnosed as abnormal by Day 3 FISH results. The purpose of this study was not to determine the error rate of PGD, which can only be accomplished by reanalysis of all abnormal embryos, and not only those reaching blastocyst, but to determine if morphology can help detect those errors. Cross-sectional analysis. Some embryos reported as abnormal by Day 3 single blastomere FISH, which reached various post-compaction developmental stages by days 5-6, were frozen. Those which survived freeze-thaw and underwent uneventful nuclear fixation were analysed (N=23). FISH analyses involved chromosomes X,Y,13,15,16,17,18,21,22 and were performed at Reprogenetics, N.J. Day 5 or 6 embryos with <28% abnormal cells were classified as minimal mosaics; mean cell number/embryo was 79.5±44.6 (range:10-230 cells). Of the embryos reported by Day 3 PGD as abnormal, 23 reached day 5 or 6 and survived thawing. Of those, 8 developed into blastocysts with ≥90cells. For embryos classified by PGD as monosomies other than X and 21, the proportion forming blastocysts with ≥90 cells was 2/5. Of those two, one was a minimal mosaic and the other an extensive mosaic with most cells abnormal. Average number of cells for Day 5/6 Extensive Mosaic, Minimal Mosaic, Monosomy X or 21, other monosomies and Trisomy embryos was 78, 87.3, 75, 10, and 64.5, respectively. The frequency of chromosome anomalies and number of Day 5/6 embryos reaching ≥90cells as compared to Day 3 FISH findings is shown in the Table.Tabled 1 This approach does not provide the error rate of PGD since only blastocysts were reanalysed and not arrested embryos. It is well known (Sandalinas et al. 2001) that some abnormalities survive less often to blastocyst than normal embryos, thus the population here has been enriched with normal embryos.These findings indicate that the majority of embryos reaching blastocyst stage previously classified by Day 3 PGD as complex, trisomy or monosomy X or 21 were abnormal. Their average numbers of cells did not differ from normal embryos. In contrast, the few monosomies other than X and 21 reaching blastocyst were not monosomies but extensive mosaics or minimal mosaics. We suggest that for those embryos, a blastocyst biopsy could be considered and if confirmed normal, replaced.

Seabream ghrelin: cDNA cloning, genomic organization and promoter studies

Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from sea-bream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5'-flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5'-flanking region. However, a number of putative transcription factor-binding sites different from the human counterpart were found in the 5'-flanking region of the seabream ghrelin gene, suggesting that different cis- and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5'-flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5'-flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.

On the species-specific biotransformation of dibenzo[ a, l]pyrene

We were aimed at investigating the activation of the carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[ a, l]pyrene (DB[ a, l]P) in Chinese hamster V79 cells that express single human, rat or fish cytochrome P450 (CYP) enzymes. DB[ a, l]P is detectable in environmental samples and has been characterized as the most potent carcinogenic species among all PAHs as yet tested in rodent bioassays. Metabolite profiles and metabolite-dependent cytotoxic and clastogenic activities were monitored. The total turnover of CYP-mediated transformation of DB[ a, l]P was as follows: human CYP1B1 > fish CYP1A1 ≈ human CYP1A1 ≫ rat CYP1A2 > rat CYP1A1. By contrast, enzyme forms that are not classified as being members of family CYP1, such as CYP2A6, 2E1, 2B1, and 3A4, failed to catalyze any detectable conversion of this substrate. All CYP1A1 enzymes tested formed both the K-region trans-8,9- and the trans-11,12-dihydrodiol, whereas human CYP1B1 failed to catalyze K-region activation. In cells expressing human or fish CYP1A1, human CYP1B1, and rat CYP1A2, the (−)- trans-11,12-dihydrodiol was formed enantiospecifically. DB[ a, l]P-dependent cytotoxicities (EC 50) were found in the following order: human CYP1A1 (12 nM) > fish CYP1A1 (30 nM) > human CYP1B1 (45 nM) ≫ other forms. In addition, an appreciable micronuclei formation was detected in human CYP1A1- and 1B1-expressing cells during exposure to DB[ a, l]P. Our study demonstrates that human CYP1A1, 1B1 and fish CYP1A1 are able to transform DB[ a, l]P into genotoxic derivatives in appreciable amounts. In contrast, CYP enzymes from rat predominantly target the K-region of DB[ a, l]P and thus are serving more a rather protective route of biotransformation. Together our data suggest that humans might be more susceptible to DB[ a, l]P-induced carcinogenicity than rats.

Bacterial diversity in a marine hatchery: Balance between pathogenic and potentially probiotic bacterial strains

Aquatic hatcheries contain diverse microbial communities that include pathogenic, innocuous and beneficial bacteria, and the ability to maintain a proper balance of this microflora may be the key to a successful culture environment. Herein, we undertook to identify the bacterial diversity present in a marine hatchery in British Columbia that cultures both fish and shellfish species. Bacterial strains were cultured from numerous microalgae and shellfish species as well as a single marine fish (sablefish) species grown in the facility. In addition, several bacterial isolates were taken from wild bivalve shellfish used as broodstock (geoducks and oysters), surrounding marine waters and sediments, and from macroalgae collected in the field. Characterizations were limited to culturable bacteria, as the ultimate aim of the project was to identify strains that were beneficial and could be used as probionts in the future. Of the 598 bacterial isolates cultured, 172 unique phylotypes were identified through 16S ribosomal DGGE genotyping. Sixty percent of the unique phylotypes were sequenced, yielding 112 different strains/species of bacteria. Twenty-two percent of the bacterial strains were found to be ubiquitous in the hatchery and marine environment, having been identified in three or more species. Potential pathogens were identified by their strain ID, ability to lyse red blood cells, or prevalence in moribund individuals. Sixteen of the strains identified were known fish or shellfish pathogens, and six strains were reported human pathogens. Fifty-seven percent of the bacterial isolates tested were haemolytic positive and 23% were identified from moribund or dead larvae. In addition, Vibrio logei, a luminous bacterial species that is symbiotic in many shellfish species, was identified as a potential pathogen of sablefish larvae.

Reducing sea turtle by‐catch in pelagic longline fisheries

AbstractReducing by‐catch of sea turtles in pelagic longline fisheries, in concert with activities to reduce other anthropogenic sources of mortality, may contribute to the recovery of marine turtle populations. Here, we review research on strategies to reduce sea turtle by‐catch. Due to the state of management regimes in most longline fisheries, strategies to reduce turtle interactions must not only be effective but also must be commercially viable. Because most research has been initiated only recently, many results are not yet peer‐reviewed, published or readily accessible. Moreover, most experiments have small sample sizes and have been conducted over only a few seasons in a small number of fisheries; many study designs preclude drawing conclusions about the independent effect of single factors on turtle by‐catch and target catch rates; and few studies consider effects on other by‐catch species. In the US North Atlantic longline swordfish fishery, 4.9‐cm wide circle hooks with fish bait significantly reduced sea turtle by‐catch rates and the proportion of hard‐shell turtles that swallowed hooks vs. being hooked in the mouth compared to 4.0‐cm wide J hooks with squid bait without compromising commercial viability for some target species. But these large circle hooks might not be effective or economically viable in other longline fisheries. The effectiveness and commercial viability of a turtle avoidance strategy may be fishery‐specific, depending on the size and species of turtles and target fish and other differences between fleets. Testing of turtle avoidance methods in individual fleets may therefore be necessary. It is a priority to conduct trials in longline fleets that set gear shallow, those overlapping the most threatened turtle populations and fleets overlapping high densities of turtles such as those fishing near breeding colonies. In addition to trials using large 4.9‐cm wide circle hooks in place of smaller J and Japan tuna hooks, other fishing strategies are under assessment. These include: (i) using small circle hooks (≤ 4.6‐cm narrowest width) in place of smaller J and Japan tuna hooks; (ii) setting gear below turtle‐abundant depths; (iii) single hooking fish bait vs. multiple hook threading; (iv) reducing gear soak time and retrieval during daytime; and (v) avoiding by‐catch hotspots through fleet communication programmes and area and seasonal closures.

Single Fish Egg DNA Extraction for PCR Amplification

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and connective tissue of eggs. From various sizes of a single egg of walleye pollack (Theragra chalcogramma), the amounts of total nucleic acids were reproducibly obtained to be 18.25 ± 1.92 μg per egg. Using DNA templates diluted ranging 1/100–1/105, PCR amplification for the mitochondrial cytochrome b (Cytb) gene was successfully performed, and the 1/102 diluted template yielded the best result in PCR amplification for three different DNA marker genes. This method is quite simple and economical, and enables to provide the high throughput often demanded by the stock identification of marine biomass, in which large numbers of specimens of single fish eggs must be analyzed.

Multiple natriuretic peptides coexist in the most primitive extant ray-finned fish, bichir Polypterus endlicheri

Natriuretic peptides (NPs) have diversified from a single NP in cyclostomes and elasmobranchs to multiple NPs in ray-finned fishes where ANP, BNP, VNP, and/or up to four CNPs (CNP-1, 2, 3, and/or 4) have been identified. To trace the evolutionary diversification of NPs in fishes, we analyzed the bichir ( Polypterus endlicheri), believed to be the most primitive extant ray-finned fish, for the presence of any NPs by a PCR-based method using primers that amplify all NP cDNAs identified to date. We have cloned cDNAs encoding ANP, BNP, VNP from the heart and three CNPs (CNP-1, 3, and 4) from the brain. An extensive search for CNP-2 from the brain was not successful. The C-terminus of bichir ANP presented an amidation signal as in ray-finned fish ANP. The bichir BNP mRNA had AUUUA repeats in the 3′-untranslated region as observed in all BNP cDNAs of vertebrates. The bichir VNP had a long C-terminal ‘tail’ sequence extending from the intramolecular ring as does teleost VNP. The three bichir CNPs are structurally similar to each teleost counterpart and are grouped after molecular phylogenetic analyses. ANP was most abundantly expressed in the atrium, BNP in the ventricle, and VNP was expressed in both atrium and ventricle. The three CNPs are most abundantly expressed in the brain, and CNP-4 transcripts were found in small amounts in the ventricle and kidney. Taken together, it is clear that all major NPs exist prior to the whole genome duplication that occurred in the teleost lineage. Furthermore, this is the first observation that CNP-3, ANP, BNP, and VNP, whose genes are colocalized in the same chromosome, coexist in a single fish species including teleosts, thereby confirming that CNP-3 is not an ortholog of VNP, and that ANP, BNP, and VNP genes were generated by tandem duplication from the CNP-3 gene.

Extreme levels of intra-specific divergence among Cape Peninsula populations of the Cape galaxias, Galaxias zebratus Castelnau 1861, reveals a possible species complex

The Cape galaxias, Galaxias zebratus, is part of the paleao-endemic fauna characteristic of the south-western Cape, South Africa, and is the only galaxiid found in continental Africa. A 284-bp fragment of the cytochrome b region of the mtDNA was sequenced from 48 individual galaxiids, representing 10 populations from the Cape Peninsula. Five sequences, for four additional populations sampled at the extremes of the species range, were obtained from the literature. Analysis of cyt b mtDNA from these 14 populations of G. zebratus revealed five distinct and highly divergent lineages with low levels of intra-population mtDNA haplotype diversity. A new and distinct genetic lineage is described from the southern part of the Cape Peninsula. Estimates of genetic divergence between populations ranged from <1% to >17%. The observed level of sequence divergence represents the largest yet reported for any single fish species. The distribution of these lineages and their degree of sequence divergence refutes a model of isolation by distance. Results suggest that periods of low sea level may have been important in creating opportunities and alternative routes for dispersal and migration for Cape Peninsula populations.

Frequency of Functional Sex Change in Two Populations ofDascyllus MelanurusConforms to a Prediction from Sex Allocation Theory

The present study examined gonadal development and sexual pattern in two populations of the black-tailed dascyllus Dascyllus melanurus in Madang, Papua New Guinea (5°10′S, 145°48′E) from 31 March to 26 June 1996. Two populations differed in their social organizations. In the Guzem population, small fish occurred in groups of 10–20 around single coral colonies and large fish occurred in groups of 50–100 over a continuous cover of corals. In the Kranket population, fish occurred in groups of a few to 20 around single or clusters of two to three corals. A total of 81 and 119 fish, respectively, were examined histologically. After an initially undifferentiated state, gonads of D. melanurus developed oocytes in the primary growth stage, followed by an ovarian lumen. From this ovarian state or from more developed ovaries, some gonads developed into testes through degeneration of oocytes and development of spermatogenic tissue. No fish had gonads that simultaneously contained both degenerating vitellogenic oocytes and developing spermatogenic tissue, but some fish had gonads that contained both degenerating oocytes in the primary growth stage and developing spermatogenic tissue (termed as mixed-stage gonads). The size distributions of fish with mixed-stage gonads differed between the two populations in a manner that was predicted from the theories of sex allocation and mating systems. In the Guzem population, most fish with mixed-stage gonads were smaller than the smallest mature female, which indicated that functional sex change was infrequent. In the Kranket population, the majority of fish with mixed-stage gonads were within the size range of mature females, which indicated that functional sex change was present and was more frequent than in the former population. Presence of fish with mixed-stage gonads in the size range of mature females indicated that D. melanurus was a protogynous hermaphrodite, but two populations differed in the frequency of functional sex change.