TaqMan assays for the genetic identification of delta smelt (Hypomesus transpacificus) and wakasagi smelt (Hypomesus nipponensis)

We have developed species-specific TaqMan assays for two California fish species, the threatened delta smelt (Hypomesus transpacificus) and the introduced wakasagi smelt (Hypomesus nipponensis). The assays are capable of correctly identifying each species with 100% accuracy, with no cross-species amplification. We anticipate these assays will prove useful for future scientific studies requiring genetic species identification (e.g. predation of smelt) or monitoring (e.g. detection of delta smelt near water diversions).     

Resident spawning of Japanese smelt,Hypomesus nipponensis,along gravel shorelines in Lake Nojiri,Central Japan

In Lake Nojiri, the Japanese smelt, Hypomesus nipponensis McAllister, 1963, population has been thought to be maintained by artificial spawning, and stocking efforts as natural spawning in in-flowing streams is unlikely due to the lack of inflowing stream habitat. In this study, novel resident Japanese smelt spawning was observed along the lakeshore in shallow areas with clean gravel and flow. Spawning occurred at night during early March to mid-April and eggs, confirmed on gravel substrate, progressed to the eyed state in 5–6 weeks. Although natural spawning was observed, the ratio of eggs that progressed to the eyed stage was minimal, possibly due to thick algae cover in areas with low flow velocity. Therefore, resident Japanese smelt spawning along the lake shoreline is physically possible and potentially contributes to the overall population in Lake Nojiri, but in-lake spawning alone presumably is not a biologically viable method for population sustainability due to low survival rates and egg density at present. Environmental improvements such as construction of fish ladders to suitable in-flowing spawning habitat, introduction of clean gravel to shorelines, and water level management adjusted to the smelt spawning run would contribute to higher recruitment by natural smelt reproduction, and consequently enhance the smelt production in Lake Nojiri.

     

Comparative environmental tolerances of threatened delta smelt (Hypomesus transpacificus) and introduced wakasagi (H. nipponensis) in an altered California estuary

In California’s Sacramento-San Joaquin estuary, environmental protection and habitat restoration efforts directed at a threatened native osmerid, the delta smelt (Hypomesus transpacificus), are complicated by the presence of a morphologically similar non-native congener, the wakasagi (H. nipponensis), transported to the estuary from upstream reservoirs. In order to better define delta smelt critical habitat and to evaluate the potential for habitat overlap by these two species, we compared the tolerances of the two species to temperature, salinity, and water velocity, environmental factors that vary spatially and temporally within the estuary. For fishes acclimated to 17°C and fresh water (0 ppt), we measured critical thermal maxima and minima, chronic upper salinity tolerance limits, and critical swimming velocities. Wakasagi had higher critical thermal maxima (29.1°C vs. 25.4°C for delta smelt), lower critical thermal minima (2.3°C vs. 7.5°C for delta smelt), higher upper salinity tolerances (26.8 ppt vs. 19.1 ppt for delta smelt), and swam faster (for 6–6.9 cm SL fish, 43.3 cm s^–1 vs. 28.2 cm s^–1 for delta smelt) than delta smelt. This suggests that the wide seasonal and year-to-year fluctuations in temperature, salinity, and flow typical in the estuary would not exclude wakasagi, although their eggs and larvae may be less tolerant. With respect to these factors, the native delta smelt may be at a physiological disadvantage, particularly in habitats with suboptimal environmental conditions, and may be excluded from shallow-water habitat restoration sites, which are characterized by poor circulation, low flows, and more environmentally extreme conditions. The low abundance of wakasagi in the estuary recorded to date may indicate that factors other than temperature, salinity, and flow determine wakasagi distribution. Received: 25 August 1999 / Accepted: 22 November 1999     

Spawning ecology of Japanese surf smelt, Hypomesus pretiosus japonicus (Osmeridae), in Otsuchi Bay, northeastern Japan

The spawning habits of Japanese surf smelt, Hypomesus pretiosus japonicus (Japanese name chika), were studied from 1992 to 1995 at Akaiso Beach in Otsuchi Bay off the Pacific coast of northern Honshu. Investigations were conducted in relation to time, tidal rhythm, sand grain size on the spawning ground, and sex and age of spawners. Spawning occurred from late March to early May with its peak at the spring tide period during full moon in April. Before dark, fish gathered to school from 1–3 m depth, and 10–20 m off the shoreline of the spawning beach. Just before dark, they repeatedly approached the shoreline and stayed near the shoreline to spawn just after dark. In the spawning process, three intervals, i.e. aggregation, approach, and spawning, were recognized and these intervals are thought to be functionally connected. Mating occurred in a unit or group formed by one female and several males. During 1992–1995, except for 1994, spawning took place in a restricted region of the beach where backwash was stronger than other areas, and most sand grains were more than 1 mm in diameter and coarser than those of other areas. Although spawning was observed near the shoreline (34 to 120 cm tide level), most of the eggs were dispersed by backwash and each became attached to a few sand grains close to a step formed at 1–2 m depth off the spawning site. Spawners were composed mainly of age 0+ fish and some 1+ fish. Males usually outnumbered females on the spawning ground. Their spawning styles are discussed in relation to their ecology and habitat.     

Structural and functional characterization of a C-type lectin-like antifreeze protein from rainbow smelt (Osmerus mordax).

Antifreeze proteins (AFPs) are produced by several cold-water fish species. They depress physiological freezing temperatures by inhibiting growth of ice crystals and, in so doing, permit the survival of these fish in seawater cooler than their normal freezing temperatures. The type II AFP from rainbow smelt (Osmerus mordax), which is a member of the C-type lectin superfamily, was characterized in terms of its Ca2+-binding quaternary structure and the role of its single N-linked oligosaccharide. The protein core of the smelt AFP, shown through sequence homology to be a C-type lectin carbohydrate-recognition domain, was found to be protease resistant. Smelt AFP was also shown to bind Ca2+, as determined by ruthenium red staining and a conformational change on Ca2+ binding detected by intrinsic fluorescence. The N-linked oligosaccharide was found to have no effect on protease resistance, dimerization, or antifreeze activity. Thus its role, if any, in the antifreeze function of this protein remains unknown. Smelt AFP was also shown to be a true intermolecular dimer composed of two separate subunits. This dimerization did not require the presence of N-linked oligosaccharide or bound Ca2+. Smelt AFP dimerization has implications for the effective solution concentration and measurement of its activity. This finding may also lead to new interpretation of the mechanism of ice-growth inhibition by this AFP.     

Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance

Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfidebonded 14 kDa protein was targeted for accumulation in its pro and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciensmediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plantproduced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wildtype AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for largescale production: but no difference in frost resistance was observed between transgenic and wildtype plants during the onset of early fall frosts.     

Analyses of growth processes of pond smelt, Hypomesus nipponensis, in Lake Ogawara, Japan, through the use of daily otolith increments

It has been reported that pond smelt, Hypomesus nipponensis, in Lake Ogawara, Japan, appear in small and large size groups during spawning despite being an annual fish. It is hypothesized that pond smelt have a bimodal life history, anadromous for large size groups and resident in the lake for small size groups. We calculated the body length and growth rate of the small and large size groups through the use of daily otolith increments and compared growth parameters between groups. In addition, the growth processes of resident fish in Lake Ogawara and anadromous fish in the adjacent sea were examined and compared with small and large size groups, respectively. We found that the two size groups diverged after 40–50 days from hatching with significant size groups present after 50 days. Through otolith increment analysis the growth processes of the small and large size groups were correlated with resident and anadromous forms respectively. These results revealed the utility of otolith increment analyses to clarify population structure of this species.     

Analysis of the mitochondrial DNA variation in pond smelt Hypomesus olidus (Osmeridae)

Pond smelt Hypomesus olidus (Pallas, 1814), one of the five species of the genus Hypomesus, family Osmeridae, was examined for intraspecific variation of the mitochondrial DNA cytb (1062 bp) and COI (567 bp) genes. Among the ten single substitutions discovered, only one, leading to the substitution of isoleucine by valine, was nonsynonymous, while the remaining substitutions were synonymous. The degree of genetic divergence among pooled nucleotide sequences in H. olidus populations examined constituted 0.4% on average, ranging from 0.2 to 0.6%. These values were not higher than the levels of divergence between the individuals within the populations. Phylogenetic analysis of the populations examined did not reveal their subdivision depending of their geographic location, and pointed to the absence of intraspecific differentiation of the species.     

Hyperactive antifreeze protein from fish contains multiple ice-binding sites

Antifreeze proteins (AFPs) are produced to prevent freezing in many fish species that are exposed to icy seawater. There are a number of nonhomologous types of AFPs, diverse in both sequence and structure, which share the function of binding to ice and inhibiting its growth. We recently discovered a hyperactive AFP in the winter flounder and related species that is many-fold more active than other fish AFPs. Like the 3-4-kDa type I AFPs, it is alanine-rich and highly helical, but this 17-kDa protein is considerably larger and forms a dimer. We have sequenced the cDNA encoding this new AFP to gain insight into its structure and evolutionary relationship to the type I AFP family. The gene is clearly homologous to the righteye flounder type I AFP genes. Thus we have designated this protein "hyperactive type I AFP" (hyp-type I). The sequence of hyp-type I AFP supports a structural model in which two extended 195-amino acid alpha-helices form an amphipathic homodimer with a series of linked Ala- and Thr-rich patches on the surface of the dimer, each of which resembles ice-binding sites of type I AFPs. The superior activity of hyp-type I AFP may derive from the large combined surface area of the ice-binding sites, recognition of multiple planes of ice, and protection of the basal plane from ice growth.     

Analysis of ice-binding sites in fish type II antifreeze protein by quantum mechanics

Many organisms living in cold environments can survive subzero temperatures by producing antifreeze proteins (AFPs) or antifreeze glycoproteins. In this paper we investigate the ice-binding surface of type II AFP by quantum mechanical methods, which, to the best of our knowledge, represents the first time that molecular orbital computational approaches have been applied to AFPs. Molecular mechanical approaches, including molecular docking, energy minimization, and molecular dynamics simulation, were used to obtain optimal systems for subsequent quantum mechanical analysis. We selected 17 surface patches covering the entire surface of the type II AFP and evaluated the interaction energy between each of these patches and two different ice planes using semi-empirical quantum mechanical methods. We have demonstrated the weak orbital overlay phenomenon and the change of bond orders in ice. These results consistently indicate that a surface patch containing 19 residues (K37, L38, Y20, E22, Y21, I19, L57, T56, F53, M127, T128, F129, R17, C7, N6, P5, G10, Q1, and W11) is the most favorable ice-binding site for both a regular ice plane and an ice plane where water O atoms are randomly positioned. Furthermore, for the first time the computation results provide new insights into the weakening of the ice lattice upon AFP binding, which may well be a primary factor leading to AFP-induced ice growth inhibition.     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

Freezing of a fish antifreeze protein results in amyloid fibril formation

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.     

Morphological variability of pond smelt <Emphasis Type="Italic">Hypomesus olidus</Emphasis> (Osmeridae) from some water bodies of the Far East

Morphological variability of four characters in 13 samples of pond smelt Hypomesus olidus is investigated using three parameters: fluctuating asymmetry, geographic variability, and variability of characters. The differences in the level of fluctuating asymmetry in H. olidus from various water bodies of the Far East and, thus, in the stability of development depend on the differences in conditions of embryonic and early postembryonic development. Only one character of four (mean number of soft rays in pectoral fins) shows presence of geographic variability in smelt: the mean value of this character increase from north to south. Estimation of variability of characters demonstrates that the maximum fluctuations of the variation coefficient are characteristic of the number of infraorbital bones and the minimal fluctuations are characteristic of the number of soft rays in pectoral fins.     

Microsatellites from the burbot (Lota lota), a freshwater gadoid fish (Teleostei)

We developed 21 polymorphic dinucleotide microsatellite loci, (CA)_n and (CT)_n, for the Holarctic freshwater fish, Lota lota, using an enriched genomic library protocol. The species has an interesting life history because winter‐spawning adults migrate over long distances to form spawning aggregations, a behaviour which should maintain genetic homogeneity across large spatial scales. Availability of the reported microsatellites will facilitate the investigation of population genetic structure with regard to postglacial colonization history and conservation strategies. The primers were screened on 30 individuals from a natural population (Lake Constance, southern Germany), revealing three to 24 alleles per locus with expected heterozygosities ranging from 0.48 to 0.93.     

Glochidia of the freshwater mussel,Anodonta anatina, affecting the anadromous European smelt (Osmerus eperlanus) from the Eider estuary, Germany

During a 2-year survey on external disease conditions of fish between January 1988 and December 1989, 0.2% of 40011 smelt, Osmerus eperlanus , from 30 stations in the German Wadden Sea and estuaries of tributary rivers, were infected with glochidia of the freshwater mussel Anodonta anatina . Seasonality was marked, with glochidia only being observed in March and April 1989, when prevalences were 37% and 15% respectively, in fish 10–20cm long at the most heavily infected site. A marked increase in prevalence in fish 15cm in length and longer, suggested that only temporarily resident spawning fish (as opposed to resident juveniles) were infected. Infection was almost exclusively restricted to the Eider estuary, where prevalence decreased with increasing salinity. It is concluded that the glochidia carried by smelt returning from their freshwater spawning sites in the Eider River to the sea would have died, thus representing a loss to the 1989 cohort of A. anatina from the Eider. The potential importance of the smelt– A. anatina relationship as indicator for the detection of (a) spawning mussel populations and of (b) possible ecological effects of climate changes is noted.     

Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.     

Gnathostoma binucleatum (Spirurida: Gnathostomatidae) from the freshwater fish in Tabasco, Mexico

Human gnathostomiasis is a food-born parasitic disease of relative importance in many countries in Southeast Asia. It is caused by several species of nematodes of the genus Gnathostoma. In Mexico is an emerging public health problem since 1970, when first cases were reported. Until today, larval morphometric characters that have been proposed to differentiate between the three species of Gnathostoma present in this country, are not satisfactory. Recently, the presence of advanced third-stage larvae AdvL3 (infective form for humans) in freshwater fishes from Pantanos de Centla, Tabasco. was recorded but their specific identity was not clarified . Examination of four species of freshwater fishes from the same locality revealed that three of them: Petenia splendida (n=58), Cichlasoma managuense (n=35) and Gobiomorus dormitor (n=9) were infected by 15 AdvL3 of Gnathostoma binucleatum. Specific identity was obtained comparing the internal transcribed spacer 2 (ITS2) of the ribosomal DNA with sequences reported in Genbank. This is the first record of G. binucleatum in P. splendida and G. dormitor from Tabasco and the first specific determination of the parasite in the locality.     

The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne)

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.     

Chlorophycean parasite on a marine fish,Sillago japonica (Japanese sillago)

A green spotted Japanese sillago (Sillago japonica) was caught by a fisherman and brought to the laboratory for pathological inspection. The green spots were abundant on the lateral line and more extensively so within the mouth cavity. In both sites, green spots were embedded within the fish flesh and formed 2–3 mm dome-shaped colonies. SEM revealed these colonies to harbor numerous unknown cells with small, surface warts (ornamentations). Molecular analysis showed the cells were Desmodesmus (D. komarekii), a common freshwater coccoid green alga found in ponds and rivers worldwide. It is uncertain how the host fish came to be infected with the alga which was not merely attached externally but embedded within the flesh and inside the mouth cavity. This is the first case of parasitic form of coccoid green algae in marine fish and provides new insights into the variable nature of green algae.