Expanding non-invasive approaches for fish-health monitoring: A survey of the epidermal mucous metabolomes of phylogenetically diverse freshwater fish species.

There is a pressing need for more-holistic approaches to fisheries assessments along with growing demand to reduce the health impacts of sample collections. Metabolomic tools enable the use of sample matrices that can be collected with minimal impact on the organism (e.g., blood, urine, and mucus) and provide high-throughput, untargeted biochemical information without the requirement of a sequenced genome. These qualities make metabolomics ideal for monitoring a wide range of fish species, particularly those under protected status. In the current study, we surveyed the relative abundances of 120 endogenous metabolites in epidermal mucus across eight freshwater fish species belonging to seven phylogenetic orders. Principal component analysis was used to provide an overview of the data set, revealing strong interspecies relationships in the epidermal mucous metabolome. Normalized relative abundances of individual endogenous metabolites were then used to identify commonalities across multiple species, as well as those metabolites that showed notable species specificity. For example, taurine was measured in high relative abundance in the epidermal mucus of common carp (Cyprinus carpio), northern pike (Esox lucius), golden shiner (Notemigonus crysoleucas), rainbow trout (Oncorhynchus mykiss), and rainbow smelt (Osmerus mordax), whereas γ-amino butyric acid (GABA) exhibited a uniquely high relative abundance in flathead catfish (Pylodictis olivaris). Finally, hierarchical cluster analysis was used to evaluate species relatedness as characterized by both the epidermal mucous metabolome (phenotype) and genetic phylogeny (genotype). This comparison revealed species for which relatedness in the epidermal mucous metabolome composition closely aligns with phylogenetic relatedness (e.g., N. crysoleucas and C. carpio), as well as species for which these two measures are not well aligned (e.g., P. olivaris and Polyodon spathula). These, and other findings reported here, highlight novel areas for future research with fish, including development of epidermal mucous-based markers for non-invasive health monitoring, sex determination, and hypoxia tolerance.

No observed developmental effects in early life stages of capelin (Mallotus villosus) exposed to a water-soluble fraction of crude oil during embryonic development.

The rise in offshore oil and gas operations, maritime shipping, and tourism in northern latitudes enhances the risk of oil spills to sub-Arctic and Arctic coastal environments. Therefore, there is a need to understand the potential adverse effects of petroleum on key species in these areas. Here, we investigated the effects of oil exposure on the early life stages of capelin (Mallotus villosus), an ecologically and commercially important Barents Sea forage fish species that spawns along the coast of Northern Norway. Capelin embryos were exposed to five different concentrations (corresponding to 0.5-19 µg/L total PAHs) of water-soluble fraction (WSF) of crude oil from 6 days post fertilization (dpf) until hatch (25 dpf), and development of larvae in clean seawater was monitored until 52 dpf. None of the investigated endpoints (embryo development, larval length, heart rate, arrhythmia, and larval mortality) showed any effects. Our results suggest that the early life stages of capelin may be more robust to crude oil exposure than similar life stages of other fish species.

Visual, spectral, and microchemical quantification of crystalline anomalies in otoliths of wild and cultured delta smelt.

Developmental abnormalities in otoliths can impact growth and survival in teleost fishes. Here, we quantified the frequency and severity of developmental anomalies in otoliths of delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish that is endemic to the San Francisco Estuary. Left-right asymmetry and anomalous crystalline polymorphs (i.e., vaterite) were quantified and compared between wild and cultured populations using digital image analysis. Visual estimates of vaterite were validated using X-ray diffraction, Raman spectroscopy, laser ablation ICPMS, and electron probe microanalysis. Results indicated that cultured delta smelt were 80 times more likely to contain a vateritic otolith and 18 times more likely to contain relatively large (≥ 15%) amounts of vaterite. Similarly, cultured fish exhibited 30% greater asymmetry than wild fish. These results indicate that cultured delta smelt exhibit a significantly higher frequency of vestibular abnormalities which are known to reduce fitness and survival. Such hatchery effects on otolith development could have important implications for captive culture practices and the supplementation of wild fish populations with cultured individuals.

Gross morphology, histology, and ultrastructure of the olfactory rosette of a critically endangered indicator species, the Delta Smelt, Hypomesus transpacificus.

The Delta Smelt (Hypomesus transpacificus) is a small, semi-anadromous fish native to the San Francisco Bay-Delta Estuary and has been declared as critically endangered. Their olfactory biology, in particular, is poorly understood and a basic description of their sensory anatomy is needed to advance our understanding of the sensory ecology of species to inform conservation efforts to manage and protect them. We provide a description of the gross morphology, histological, immunohistochemical, and ultrastructural features of the olfactory rosette in this fish and discuss some of the functional implications in relation to olfactory ability. We show that Delta Smelt have a multilamellar olfactory rosette with allometric growth. Calretinin immunohistochemistry revealed a diffuse distribution of olfactory receptor neurons within the epithelium. Ciliated, microvillous and crypt neurons were clearly identified using morphological and immunohistochemical features. The olfactory neurons were supported by robust ciliated and secretory sustentacular cells. Although the sense of smell has been overlooked in Delta Smelt, we conclude that the olfactory epithelium has many characteristics of macrosmatic fish. With this study, we provide a foundation for future research into the sensory ecology of this imperiled fish.

Effects of water accommodated fraction of physically and chemically dispersed heavy fuel oil on beach spawning capelin (Mallotus villosus).

Due to a northward shift in off-shore activities, including increased shipping traffic and oil and gas exploration there is a growing focus on the potential effects of oil pollution on Arctic marine ecosystems. Capelin (Mallotus villosus) is a small fish and a member of the smelt family, and is a key species in the marine food chain. Capelin are seasonally abundant in the Northern Atlantic and in coastal Arctic waters, e.g. in western Greenland and in the Barents Sea, where it undertakes aggregated spawning in the intertidal and subtidal zone. To study the possible effects of oil pollution on the physiology and development of early life stages in capelin, freshly fertilised capelin eggs were exposed to a water accommodated fraction of physically (WAF) and chemically (CEWAF) dispersed heavy fuel oil (IFO30) for 72 h. Subsequent mortality, hatching success, larvae malformations, growth and CYP1A/EROD activity was measured over a 4-week period. The nominal exposure concentrations of WAF and CEWAF were between 0.02 and 14.5 mg total hydrocarbon content (THC) L-1 and 0.5-304 mg THC L-1, respectively. Egg mortality correlated significantly with WAF exposure concentration. The proportions of hatched eggs decreased with increasing CEWAF exposure concentration. Further, the percentage of malformed larvae with craniofacial abnormalities, body axis defects, generally under developed larvae, reduced total body length (dwarfs), correlated significantly with exposure concentrations in both CEWAF and WAF treatments. The four types of the predominant malformations were distributed differently in two parallel experiments. At the biochemical level, we observed a significant relationship between CEWAF exposure concentration and CYP1A/EROD activity in newly hatched larvae and this effect persisted for 3 weeks after the 72 h exposure. We conclude that even short-term exposure to both heavy fuel oil WAF and CEWAF, at environmentally relevant THC concentrations following an oil spill, may induce adverse developmental effects on the vulnerable early life stages of capelin. The mechanisms responsible for the observed effects on mortality, growth and embryo development in capelin eggs and embryos following WAF and CEWAF exposure require further studies.

Characterization of P2X7R and its function in the macrophages of ayu, Plecoglossus altivelis.

P2X purinoceptor 7 (P2X7R), an ATP-gated ion channel, plays an important role during the innate immune response in mammals. However, relatively little is known about the role of P2X7R in the fish immune system. Here, we cloned a cDNA sequence encoding ayu (Plecoglossus altivelis) P2X7R (aP2X7R). The predicted protein was composed of 574 amino acid residues with a P2X family signature, two transmembrane domains, and a long C-terminal. aP2X7R transcripts were mainly distributed in ayu immune tissues and significantly increased in all tested tissues and in macrophages after Listonella anguillarum infection. The aP2X7R protein was upregulated significantly in macrophages upon bacterial challenge. An antibody against the ectodomain of aP2X7R (aEPAb) and an antagonist (oATP) were used to block aP2X7R. aP2X7R siRNA was also used to knockdown the receptor expression in ayu macrophages. Cell death induced by ATP was significantly inhibited in ayu macrophages after aEPAb, oATP, or siRNA treatment. Moreover, aP2X7R ablation also resulted in suppression of phagocytic activity and ATP-induced bacterial killing in ayu macrophages. Our results indicated that aP2X7R was upregulated after infection and mediated cell death, phagocytosis, and bacterial killing of ayu macrophages.

Toxicity of crude oil and pyrene to the embryos of beach spawning capelin (Mallotus villosus).

Due to a northward shift in oil and gas activities, there is an increasing need to understand the potential anthropogenic impacts of oil-related compounds on sub-Arctic and Arctic organisms, particularly those in coastal habitats. Capelin (Mallotus villosus), a key fish species in the Barents Sea ecosystem, undertakes aggregated spawning at both intertidal and subtidal coastal localities in northern Norway. To investigate the sensitivity of capelin embryos to oil compounds, newly fertilized capelin eggs were collected from a spawning beach and exposed until hatch (32 days) to either the water soluble fraction of crude oil or the single PAH compound, pyrene. Threshold levels for egg mortality, development and hatching success were determined. Concentrations of 40 µg/L crude oil (∑26 PAHs) and 55 µg/L pyrene significantly increased embryonic mortality rates and decreased hatching success, compared with controls, indicating that a potential oil spill in the vicinity of capelin spawning grounds may cause significant impacts. No significant incidence of adverse effects such as yolk sac oedema, pericardia oedema, haemorrhages, craniofacial abnormalities, premature hatch or inhibited growth was observed. Histological studies of hatched larvae did not reveal specific sublethal effects in tissues and organs. Developmental delays and subsequent embryo death were noticed at the period of eye pigmentation in affected groups. Early life-history stages of capelin are sensitive indicators of PAH impacts, but the mechanisms responsible for the toxic effects require further investigation.

[Molecular cloning, sequence analysis and expression pattern of hepcidin gene in ayu (Plecoglossus altivelis)].

Hepcidin, a member of cysteine-rich antimicrobial peptides, plays an important role in both fish adaptive immunity and the regulation of iron metabolism. In this paper, the nucleotide sequence of a full-length cDNA clone for ayu (Plecoglossus altivelis) hepcidin gene, 763 nucleotides in length, was determined. Ayu hepcidin gene contained a complete open reading frame (ORF) encoding an 85-amino-acid peptide with a molecular weight of 9.7 k. A signal peptide of 24 residues existed in hepcidin N-terminus. The ayu hepcidin mature peptide sequence contained 25 amino acids with eight cysteines that formed four disulfide bonds. Sequence comparison and phylogenetic analysis showed that ayu hepcidin was most similar to Atlantic salmon (Salmo salar), and the relationships of the different hepcidin coincided well with the evolutionary relationships of their organisms. In healthy ayu, hepcidin mRNA was mainly expressed in the liver, spleen, kidney, heart, and muscle. After Listonella anguillarum infection, liver hepcidin mRNA expression change was determined by quantitative real-time PCR (qRT-PCR) method. Hepcidin transcripts of ayu liver were significantly up-regulated and peaked at 12 h. These results suggest that hepcidin may be involved in the immune response of ayu.

Molecular cloning of liver Wap65 cDNA in ayu (Plecoglossus altivelis) and mRNA expression changes following Listonella anguillarum infection.

The teleost warm temperature acclimation related 65 kDa protein (Wap65) is a plasma glycoprotein with the potential roles in heat adaptation, heme recycling, immune response and copper metabolism. It is most homologous to the mammalian hemopexin, which is the plasma transporter of heme. A full-length cDNA clone of the Wap65 gene, 1,534 bp in size, was isolated from the fish ayu (Plecoglossus altivelis). Its deduced amino acid sequence of 439 residues had 60.4-65.4% and 38.3-47.3% identical to fish Wap65-2-type and Wap65-1-type sequences, respectively. In phylogenetic analysis, aWap65 grouped tightly with those fish Wap65-2-type sequences. In healthy control fish, the highest mRNA signal for aWap65 was from the liver, moderately high in brain and gill, and but weaker in spleen, kidney, muscle, heart and intestine. In Listonella anguillarum-infected fish, aWap65 transcripts were significantly increased in liver, while no obvious changes in other tissues at 12 hpi. However, aWap65 transcripts were significantly increased in various tissues at 24 hpi when hemolysis developing, suggesting that aWap65 might be involved in the immune response of ayu.

Purification, characterization, cDNA cloning, and expression of asialofetuin-binding C-type lectin from eggs of shishamo smelt (Osmerus [Spirinchus] lanceolatus).

A novel C-type lectin (OLABL) was isolated from the eggs of shishamo smelt [Osmerus (Spirinchus) lanceolatus] by affinity chromatography on asialofetuin-Sepharose. OLABL had a molecular mass of 29 kDa on SDS-PAGE under nonreducing conditions and two subunits with masses of 15 kDa (OLABL-H) and 14 kDa (OLABL-L) under reducing conditions. Thus, OLABL is a heterodimeric protein. cDNA sequence analysis revealed that the H- and L-subunits of OLABL were composed of 137 and 136 amino acid residues, respectively, and showed almost identical (95%) sequences, with slight differences in the N-terminal and C-terminal regions. Since each subunit contained only the characteristic motif of C-type lectin-like domain (CTLD), EPN-E-WND, OLABL is a member of group VII of the CTLD-containing protein family. Although OLABL had an EPN sequence that is known as a mannose-specific motif found in the collectin family, OLABL agglutinated rabbit erythrocytes without the addition of Ca(2+) ion, and this activity was inhibited by l-rhamnose and d-galactose derivatives, but not by d-mannose and d-glucose. These results indicate that OLABL has similar characteristics to AJL-2, a calcium-independent lactose specific lectin isolated from Japanese eel skin mucus. Recombinant OLABLs (rHisOLABLs), His-tagged homodimers of the H- and L-subunits, were refolded from inclusion bodies expressed by Escherichia coli. rHisOLABL-L was recovered as a soluble form, but rHisOLABL-H was hardly dissolved in a renaturing buffer. The specific activities of rHisOLABL-L, rHisOLABL-H, and native OLABL were 500, 36, and 20, respectively. These findings suggest that the combination of subunits may affect the solubility and activity of these dimeric form lectins.

Present status and changes of the phytoplankton community after invasion of Neosalanx taihuensis since 1982 in a deep oligotrophic plateau lake, Lake Fuxian in the subtropical China.

Phytoplankton assemblages in the subtrophical oligotrophic Lake Fuxian, the second deepest lake in China, were investigated monthly from September 2002 to August 2003. A total of 113 species belonging to seven phyla were identified, among them, a filamentous green alga, Mougeotia sp., dominated almost throughout the study period and comprised most of the total phytoplankton biomass. Mougeotia sp. has made a substantial development during the past decades: it was absent in 1957, only occasionally present in 1983, increased substantially in 1993, and became predominant in 2002-2003. It is likely that natural invasion of the Taihu Lake noodlefish (Neosalanx taihuensis) has led to a change of dominant herbivorous zooplankton from small to large calanoid, which has increased grazing pressure on small edible algae, and thus has indirectly favored the development of the inedible filamentous Mougeotia sp.

Influence of a dispersant on the bioaccumulation of phenanthrene by topsmelt (Atherinops affinis).

Chemical dispersants enhance oil spill dispersion by forming water-accommodated micelles with oil droplets. However, how dispersants alter bioavailability and subsequent bioaccumulation of hydrocarbons is not well understood. Thus, the goal was to investigate the influence of a chemical dispersant on the disposition (uptake, biotransformation, and depuration) of a model hydrocarbon, [14C]-phenanthrene ([14C]PHN), by larval topsmelt (Atherinops affinis). Exposure was via aqueous-only or combined dietary and aqueous routes from a water-accommodated fraction (WAF) of Prudhoe Bay Crude Oil (PBCO) or a WAF of Corexit 9527-dispersed PBCO (DO). Trophic transfer was measured by incorporating into exposure media both a rotifer (Brachionus plicatilis) as food for the fish and a phytoplankton (Isochrysis galbana) as food for the rotifers. Short-term (4 h) bioconcentration of PHN was significantly decreased in topsmelt when oil was treated with dispersant (P < 0.05), but differences diminished after 12 h. When trophic transfer was incorporated, PHN accumulation was initially delayed but after 12 h attained similar levels. Dispersant use also significantly decreased the proportion of biotransformed PHN (as 9-phenanthrylsulfate) produced by topsmelt (P < 0.05). However, overall PHN depuration was not affected by dispersant use. Thus, chemical dispersant use in oil spill response may reduce short-term uptake but not long-term accumulation of hydrocarbons such as PHN in pelagic fish.

Type II antifreeze protein from a mid-latitude freshwater fish, Japanese smelt (Hypomesus nipponensis).

A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.