Oral administration of Escherichia coli lipopolysaccharide enhances the immune system of striped catfish,Pangasianodon hypophthalmus (Sauvage)

Lipopolysaccharide (LPS), a component of Gram negative bacteria, was reported as important immunostimulant for fish. In this study, striped catfish were fed diets containing different Escherichia coli LPS concentrations (0%, 0.01% and 0.05%) for 2 weeks and then fed control feed (0% LPS) for 4 weeks. Plasma cortisol and glucose were rather low and did not differ significantly among treatments (P > 0.05). The respiratory burst activity, lysozyme, complement, total of antibody as well as mortality in fish challenged with Edwardsiella ictaluri were recorded every 2 weeks (W2, W4 and W6). The lysozyme activity significantly increased in fish treated with LPS (P < 0.05) in W2, W4 and W6. The highest values of respiratory burst activity were observed at week 4 in fish fed 0.01% LPS. There were significant differences in total of antibody between fish fed LPS (0.01%) and control in W2, W4. The challenge test with Edwardsiella ictaluri showed that fish fed 0.01% LPS had lower cumulative mortality (40%, 33% and 42%) compared with the fish fed 0.05% LPS (50%, 40% and 47%) and control fish (40%, 57% and 53%) in the three difference sampling times respectively. These results suggest that feed supplemented with 0.01% LPS could enhance immunity of striped catfish after 2 weeks of oral administration and fish could be protected against bacterial infection during the following 4 weeks.     

Weaning methods using formulated feeds for snakehead (Channa striata and Channa micropeltes) larvae

The culture of snakehead fish (Channa striata and Channa micropeltes) in Vietnam is limited, and snakehead culture has been banned in Cambodia, because traditional practices include capture of fingerlings from the wild as seed, as well as capture of small‐size (also known as trash fish or low‐value) fish. As hatchery breeding technology has improved, we investigated the optimal weaning practices for these two species. Both laboratory experiments and farm trials were conducted. For C. striata, the optimal weaning procedure is to begin at 17 days after hatch (dah) and wean the fish at 10% replacement of live feed with formulated feed per day. However, for C. micropeltes, the optimal procedure is to wait until 40 dah to begin weaning and then to wean the fish with a 10% replacement of live feed with formulated feed every 3 days. These results should enable farmers to domesticate snakehead culture in Vietnam and Cambodia and eliminate reliance on fish captured from the wild as both seed and feed.     

Stress response of Salmo salar (Linnaeus 1758) facing low abundance infestation of Caligus rogercresseyi (Boxshall & Bravo 2000), an object in the tank,and handling

This study looks at how low infestation loads of adult Caligus rogercresseyi and other stressors affect the physiology of Salmo salar. Experimental fish groups were with (infested) or without (control) exposure to the parasite. The parasite cohort was followed for 78 days post‐infestation (dpi), and only adult lice were observed. Additional stressors were applied at 60 and 75 dpi. The analysis included measurements of fish physiology and weight. Low‐level infestations by adult C. rogercresseyi for more than 50 dpi induced moderate stress in S. salar as well as a high energy demand and increased small skin mucous cells. Threshold lice loads were identified, and above those loads, a high stress response was observed. Additional stressors altered fish physiology, inducing downregulation of the cortisol response after the first stressor and upregulation after the second stressor, but infested fish responded more strongly. Parasitism by C. rogercresseyi is energetically demanding, affecting the primary and secondary responses (e.g. cortisol and glucose levels), as well as the tertiary response (fish weight).     

Understanding imperfect detection in a San Francisco Estuary long‐term larval and juvenile fish monitoring programme

Imperfect detection can present a significant challenge when monitoring for a rare and imperilled species. Here, a long‐term larval and early‐juvenile fish monitoring programme in the upper San Francisco Estuary was examined to evaluate its overall reliability in detecting various fish species, including the imperilled delta smelt, Hypomesus transpacificus McAllister, for which the programme was designed. Using occupancy modelling, detection probability of species with pelagic larval or juvenile life stages was found to be generally high (≥.95) based on the current sampling effort of three larval net tows per site. However, detection probability can vary considerably from year to year depending on the species’ level of larval production. Water temperature and turbidity were identified as important predictors of occurrence for young‐of‐year delta smelt, longfin smelt Spirinchus thaleichthys (Ayres) and striped bass Morone saxatilis (Walbaum), and there was evidence for fish size selectivity by the sampling gear in all three species. These results highlight the need to consider adaptively managing detection probability by increasing sampling effort in years when young‐of‐year delta smelt abundance is expected to be low, especially when information on the species’ occurrence at a particular region is critical.     

Effect of guava Psidium guajava (L.) aqueous extract diet on growth performance,intestinal morphology,immune response and survival of Oreochromis niloticus challenged with Aeromonas hydrophila

An 84‐day feeding trial was carried out to evaluate the effects of aqueous Psidium guajava leaf extract (PGE) on growth, intestinal morphology, physiology, immune response and susceptibility of Oreochromis niloticus to Aeromonas hydrophila. Diets containing 0% (P0), 0.25% (P1), 0.50% (P2), 0.75% (P3) and 1.00% (P4) PGE were fed to triplicate groups of fish (mean weight; 1.32 ± 0.04 g) for 84 days. After the 84‐day feeding trial, test fish were injected with pathogenic A. hydrophila and then fed for 14 days. More feed were consumed in groups of fish fed PGE‐treated diets and resulted in significantly higher weight gain and feed intake. Incidentally, there was an increase in the calculated area of absorption of fish fed PGE diets, as accounted for by marginally higher villi length and width. Antioxidant and immune response were improved with PGE inclusion in diets as total protein, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase significantly increased (p < 0.05) in fish fed PGE diets. Results of the challenge test with A. hydrophila revealed that the highest mortality (100%) was recorded in P0. This study revealed that inclusion of P. guajava extract in the diet of O. niloticus improved growth, nutrient utilization, immune system and survival of O. niloticus fingerlings.     

Mycobacteria in aquarium fish: results of a 3‐year survey indicate caution required in handling pet‐shop fish

Fish are commonly infected with non‐tuberculous mycobacteria (NTM), which should be regarded as potential pathogens when handling aquarium fish and equipment. This study examined 107 aquarium fish from pet shops. Cultivation of the fish samples using different selective media was conducted for identification of NTM. Isolates were identified using the GenoType Mycobacterium common mycobacteria and additional species assays, sequencing of the 16S rRNA and rpoB genes, and real‐time PCR assay for identification of Mycobacterium (M.) marinum. Among the investigated fish, 79.4% (85/107) were positive for mycobacteria, with 8.2% (7 of 85) having two mycobacterial species present. Among the positive fish, the common pathogens M. marinum, Mycobacterium fortuitum (M. fortuitum group) and Mycobacterium chelonae were identified in approx. 90% of fish and other NTM species in 10%, including Mycobacterium peregrinum/septicum, Mycobacterium gordonae, Mycobacterium arupense, Mycobacterium kansasii, Mycobacterium ulcerans and Mycobacterium setense. The well‐known human pathogen M. marinum was present in 10.6% of the positive fish (9 of 85). The species of mycobacteria identified in the study are not only recognized as aquarium fish pathogens, but can also cause pathology in humans. Microbiological and clinical communities should therefore be sensitized to the role of NTM in infections associated with exposure to aquarium fish.     

Influence of dietary administration of a probiotic strain Shewanella putrefaciens on senegalese sole (Solea senegalensis,Kaup 1858) growth,body composition and resistance to Photobacterium damselae subsp piscicida

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 10⁹ cells g^?1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.     

Effects of dietary fish oil replacement by various vegetable oils on growth performance,body composition and fatty acid profile of juvenile Malaysian mahseer,Tor tambroides

The current study was carried out to investigate the effects of replacing dietary fish oil with different vegetable oil sources and ratios on the growth performance and tissue fatty acid profiles of juvenile Tor tambroides. Three different vegetable oils (sunflower oil, linseed oil and palm oil) were used in two different substitution ratios (50% and 100%). A diet without replacement (100% fish oil) was used as a control. Triplicate groups of T. tambroides juveniles (5.0 ± 0.4 g) were fed the test diets for 9 weeks. The highest weight gain was observed in fish fed palm oil diets that, of course, were reared in aquaria. There was a significant effect (P < 0.05) of the percentage of fish oil replacement on the liver weight after controlling for the effect of fish weight (P < 0.05). The viscera weight significantly increased (P < 0.05) in fish on vegetable oil diets, and its highest amount observed among fish fed palm oil diets. Vegetable oil inclusion significantly reduced eicosapentaenoic acid and docosahexaenoic acid (DHA) concentrations in both muscle and liver of fish except for fish on 50% palm oil diet that had similar liver DHA content with those on control diet.     

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)₃] and Group IV [with Al(OH)₃ and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.     

Evaluation of raw and hydrothermically processed leguminous seeds as supplementary feed for the growth of two Indian major carp species

To study the effect of four different raw and hydrothermically processed leguminous seeds, namely Glycine max (soybean), Vigna radiata (moong), Vigna ungiculata (cowpea) and Cyamopsis tetragonaloba (guar) on growth in the fingerlings of Cirrhinus mrigala (mrigal) and Labeo rohita (rohu), two experiments were conducted. The first experiment was conducted under laboratory conditions (LD 12:12 at 25 °C) and the mrigal fingerlings were fed @2% BW d^?1 for 30 days. Before using the seeds as feeds, they were hydrothermically processed (15 lbs at 121 °C for 15 min) for the elimination of antinutrient factors (ANF). The results indicate that 15 minute autoclaving significantly reduces the trypsin inhibitor activity. Phytase‐phosphorous and tannins were also analysed from raw and treated beans. Studies on mrigal have revealed that weight gain was significantly low (P < 0.05) in the fish fed on raw beans compared with the fingerlings fed on processed bean proteins. Among the various raw diets used, significantly highest growth (P < 0.05) was observed in fish fingerlings fed on raw moong, followed by raw soybean, cowpea and guar. On the other hand, when beans were processed, the highest growth was observed in fingerlings fed on processed soybean. Studies have further revealed that hydrothermal processing also affected the values of the feed conversion ratio, gross conversion efficiency, protein efficiency ratio, gross protein retention, gross energy retention and ammonia excretion. Proximate composition of the carcass revealed high accumulation of protein, fat and energy in fingerlings fed on processed soybean in comparison with other diets. Keeping in view the higher weight gain in fish fed on hydrothermically processed bean proteins, only processed bean diets were fed to the fingerlings of mrigal and rohu stocked in fish ponds (experiment 2). A significant (P < 0.05) increase in mean fish weight, and specific growth rate was observed in fingerlings fed on hydrothermically processed soybean, followed by moong and cowpea. Weight gain for mrigal fingerlings remained significantly low in comparison to the rohu fingerlings fed on similar diets. An investigation on the effects of feeds on water quality parameters indicate that the trophic status of the ponds where soybean was used as fish feed remained high in comparison to the other treatments. A significant positive correlation of nutrients, NPP and plankton population with fish growth was also observed.     

Occurrence and prevalence of infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss) cultured in cages in the Black Sea

Rainbow trout (Oncorhynchus mykiss) cultured in cage systems in the South Eastern Black Sea were surveyed for the type, occurrence and prevalence of infectious pancreatic necrosis virus (IPNV). Two nearby farms (designated as Farm A and Farm B) were visited monthly in 2007 and 2008. At each farm, 385 fish were selected randomly from five cages. Another farm with infected trout from a hatchery also was monitored for IPNV from the transfer to harvest. IPNV was found to be prevalent in both farms surveyed. In Farm A, IPNV was present throughout the growing period, from January to May, and all five randomly sampled cages tested positive for IPNV in March and April of 2007. In Farm B, IPNV was present only in February and March in 2007, and in 2008, IPNV was observed in January (two cages) and February (one cages) at low levels. Interestingly, IPNV was absent 2 weeks after transfer to the sea at 17.5°C. The same strain of IPNV, genotype III that was isolated from the same stock of fish at the hatchery, reoccurred when water temperatures dropped to 12°C in December in the Black Sea. Transferring fish to the sea at high water temperatures could lessen the negative impacts of IPNV on growth of rainbow trout in brackish water.     

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.     

Effect of different culture conditions on the structural diversity of prokaryote communities in the sediment of earth ponds stocked with gilthead seabream Sparus aurata (Linnaeus, 1758)

In this study, we evaluate the use of polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) for monitoring the effect of different aquaculture practices on sediment prokaryote (Archaea and Bacteria) communities. The effect of initial fish (gilthead seabream Sparus aurata) stocking density on the structural diversity of prokaryote communities of earth ponds bottom sediments was evaluated using PCR‐DGGE after a 5 month grow‐out period. An identical approach was used to monitor the effect of supplying different fish feeds [commercial feed (CD) versus an ecofeed (ECO)]. One additional variable was the use of copper sulphate (CuSO₄) as an algicide in some of the experimental rearing tanks. The statistical analyses of prokaryote community profiles showed that the presence of fish in earth ponds significantly influenced the structure of sediment prokaryote communities, when compared with earth ponds without fish, independently of the stocking density. Our results also indicated that the structure of the prokaryote communities of earth ponds supplied with the ECO feed shared a strong similarity with that fed CD. Curiously, the use of CuSO₄ in ponds receiving the ECO feed promoted significant differences on the structural composition of the bacterial community, but not on the archaeal community. DGGE molecular fingerprints are suitable for fast evaluation of new management practices in food‐fish production on earth ponds by monitoring shifts on microbial communities in bottom sediments.     

The effect of foraging and ontogeny on the prevalence and intensity of the invasive parasite Anguillicola crassus in the European eel Anguilla anguilla

Infection patterns of the invasive Anguillicola crassus nematode were investigated in a population of the European eel Anguilla anguilla where parasite invasion is very recent, Loch Lomond, Scotland. Intensity levels of the parasite were associated with differences in fish ontogeny and trophic ecology. Although eels foraged on both fish and invertebrates, individuals which were smaller and fed on invertebrates (>70% contribution to diet) were found to contain a greater number of swim bladder parasites compared to larger eel with a predominance of fish (>60% contribution) in their diet. Within affected fish, a significant negative relationship was found between fish length and parasite intensity, with smaller individuals having higher parasite intensity than larger individuals. This study indicates that food intake and infection risk are linked in this recently infected host–parasite system. From a management perspective increasing our understanding of how infection intensity and repeated exposure is linked to resource use in an ecosystem is important for the future management of this endangered species in Europe.     

Evaluation of growth performance and intestinal barrier function in Arctic Charr (Salvelinus alpinus) fed yeast (Saccharomyces cerevisiae), fungi (Rhizopus oryzae) and blue mussel (Mytilus edulis)

Arctic charr (Salvelinus alpinus) were fed for 99 days on experimental diets with 40% of fish meal replaced, on a crude protein basis, with intact yeast (Saccharomyces cerevisiae) (ISC), extracted yeast (ESC), Rhizopus oryzae fungus (RHO) or de‐shelled blue mussels (Mytilus edulis) (MYE). The fish were evaluated for growth performance, nutrient digestibility and fish intestinal function. Growth performance, retention of crude protein and sum of amino acids were not affected in fish fed diets ISC or MYE compared with those fed the reference (REF) diet. However, fish fed diet ISC displayed decreased digestibility of crude protein and indispensable amino acids and decreased intestinal barrier function compared with fish fed the REF diet. Fish fed diet ESC exhibited decreased growth performance and protein retention, but had comparable digestibility to fish fed the REF diet. Fish fed diets MYE and RHO showed similar performance in terms of growth, nutrient digestibility and intestinal barrier function. Overall, the results indicated that blue mussel and intact S. cerevisiae yeast are promising protein sources for Arctic charr.     

Improving low‐temperature stress tolerance of tilapia,Oreochromis niloticus: A functional analysis of Astragalus membranaceus

A 54‐day experiment was conducted to investigate the effects of dietary supplementation of Astragalus membranaceus extract powder (AMEP) on the low temperature tolerance of genetically improved farmed tilapia, Oreochromis niloticus. The base diet was supplemented with 0.0% (NC), 0.1% (NP0.1), 0.2% (NP0.2), and 0.4% (NP0.4) AMEP. Tilapia with an initial body weight of 21.35 ± 0.17 g were fed three times a day for 14 days at 30°C and then fed for 40 days while the water temperature decreased at a rate of 0.5°C/day. The results showed that survival, final weight, and the rate of weight gain in fish in the NP0.1, NP0.2, and NP0.4 groups were higher than those of fish in the NC group. The contents of glucose, triglyceride, cholesterol, and nitric oxide in the serum of fish fed diets supplemented with AMEP were all significantly lower than those of fish in the NC group. In contrast, fish fed with AMEP‐supplemented diets had higher liver superoxide dismutase, glutathione peroxidase and catalase activities, and lower malonaldehyde content than those of fish in the NC group at 10°C. The expression of the growth‐arrest‐specific 2 gene in the liver was significantly lower in the NP0.2 and NP0.4 groups than in the NC group. The results of this study demonstrated that the optimal level of AMEP supplementation was 0.2% under the conditions used in the current study. Optimum dietary AMEP can promote growth performance and facilitate the survival of tilapia under low‐temperature stress.     

Outbreak of hirame rhabdovirus infection in cultured spotted sea bass Lateolabrax maculatus on the western coast of Korea

In this study, we determined the cause of a disease outbreak in spotted sea bass, Lateolabrax maculatus reared in culture cages on the western coast of Korea in 2013. The major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. No external morphological signs of infection were visible, except for a darkening in colour. No parasites or pathological bacteria were isolated from the diseased fish; however, epithelioma papulosum cyprini (EPC) cells inoculated with tissue homogenates from the diseased fish showed cytopathic effects (CPEs). Virus particles in the EPC cells were bullet‐shaped, 185–225 nm long and 70–80 nm wide, characteristic of Rhabdoviridae. Polymerase chain reaction analyses of homogenized tissues from the diseased fish and supernatants of cell cultures with CPEs indicated specific, 553‐bp‐long fragments corresponding to the matrix protein gene of the hirame rhabdovirus (HIRRV). Phylogenetically, the HIRRV phosphoprotein gene of spotted sea bass was more closely related to phosphoproteins from Chinese and Polish HIRRV strains than from other Korean strains. To our knowledge, this is the first report of HIRRV infection in cultured spotted sea bass.     

Long‐chain polyunsaturated fatty acid metabolism in carnivorous marine teleosts: Insight into the profile of endogenous biosynthesis in golden pompano Trachinotus ovatus

Golden pompano Trachinotus ovatus is an important farmed carnivorous marine teleost. Although some enzymes for long‐chain polyunsaturated fatty acid (LC‐PUFA) biosynthesis have been identified, the ability of T. ovatus for endogenous biosynthesis is unknown. Here, we evaluated in vivo LC‐PUFA synthesis in a 56‐day culture experiment using six diets (D1–D6) formulated with linseed and soybean oils to produce dietary linolenic/linoleic acid (ALA/LA) ratios ranging from 0.14 to 2.20. The control diet (D0) used fish oil as lipid source. The results showed that, compared with the corresponding indices of fish fed D0, the weight gain rate and specific growth rate as well as the contents of eicosapentaenoic (EPA) and docosahexaenoic acids in tissues (liver, muscle, brain and eye) of D1–D6 groups were significantly lower (p < .05). These data suggested that T. ovatus could not synthesize LC‐PUFA from C₁₈ PUFA or such ability was very low. However, tissue levels of 20:4n‐3 in fish fed diets D1–D6 were higher than that of D0 fish (p < .05), and positively correlated with dietary ALA/LA ratio, while levels of EPA showed no difference among the D1–D6 groups. These results indicated that Δ5 desaturation, required for the conversion of 20:4n‐3 to EPA, may be lacking or very low, suggesting incomplete LC‐PUFA biosynthesis ability in T. ovatus.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.