Ten species specific microsatellite loci for Lahontan cutthroat trout,Oncorhynchus clarki henshawi

Ten polymorphic microsatellite loci (containing tri and tetra‐nucleotide repeats) were developed for the Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a subspecies of cutthroat trout listed as threatened under the United States Endangered Species Act. Polymorphism was assessed for 445 individuals from 12 populations representing eight watersheds spread throughout the three Distinct Population Segments defined for this subspecies. All loci were polymorphic (X? = 19, range 7–26 alleles). All loci were in Hardy–Weinberg equilibrium (HWE) except for one locus (OCH 9) in a single population (P < 0.00014 after Bonferroni correction for multiple tests).     

Landscape topography and the distribution of Lahontan cutthroat trout (Oncorhynchus clarki henshawi) in a high desert stream

Lahontan cutthroat trout, Oncorhynchus clarki henshawi, are currently limited in their distribution to a patchwork of small isolated populations, the result of habitat degradation and natural variation in landscape and in-stream conditions. The objectives of this study were to determine if landscape topography influences trout distribution, and if water temperatures control this response. The work was carried out in a sub-basin of the Quinn River system, McDermitt Creek, which drains the sagebrush desert of southeastern Oregon and northern Nevada. Headwater tributaries of this creek consist of alternating canyon-confined and valley bounded reaches. Trout within these systems are challenged by low discharge and high temperatures during the summer, and anchor ice during the winter. Contiguous whole stream surveys were used to look at trout distribution during the summer of 2003 and spring and fall of 2004. Our results suggested that topography can affect trout distribution. Trout numbers were highest in areas with greater numbers of nick-points (the transition zones between less confined and more confined valley segments) and greater valley confinement. Additionally, in the downstream portion of our headwater reaches, more trout were found in nick-points than expected based on the availability of this habitat type. Our data suggest that hyporheic inputs may be high in such areas, thus providing trout with shelter from warm water in the summer, anchor ice in the winter, and shallow stream depths during all seasons. Spatial occurrence of these areas of refugia can be taken into consideration when planning land use activities and restoration efforts. Further research is required to confirm that topography can affect the distribution of Lahontan cutthroat trout in other systems, and to better understand the mechanisms behind these patterns.     

Multiple hemoglobins of the cutthroat trout, Salmo clarki

Nine hemoglobins were purified from blood of Salmo clarki by ion-exchange chromatography and preparative isoelectric focusing. The subunit structures of eight of the purified hemoglobins were studied by electrophoresis of globins in the presence of urea. Six are alpha 2 beta 2 tetramers while two appear to be heterotetramers of the type alpha alpha' beta 2 and alpha alpha' beta beta'. The effects of pH, nucleotides, and temperature on the oxygen equilibria of the purified hemoglobins were studied. Five hemoglobins with isoelectric points from 9.1 to 7.1 and one minor hemoglobin with an isoelectric point of 5.9 appear to have essentially identical oxygen binding properties. All have similar oxygen equilibria which are independent of pH and temperature and not affected by saturating amounts of ATP. Another minor hemoglobin with an isoelectric point below 5.9 has similar oxygen equilibria except for a possible pH dependence. Two hemoglobins, with isoelectric points of 6.5 and 6.4, have oxygen binding properties which are strongly pH and temperature dependent. Addition of ATP or GTP causes a large decrease in the oxygen affinity without affecting the cooperativity of oxygen binding. The effect of GTP is slightly greater than that of ATP. No significant differences were observed in the oxygen equilibria of these two hemoglobins. The red blood cells of S. clarki were found to contain large amounts of both ATP and GTP, with an ATP:GTP ratio of 3:1. Both nucleotides may be important modulators of hemoglobin oxygen affinity in S. clarki, in contrast to the situation in S. gairdneri, in which red blood cell GTP concentrations are considerably lower. The presence of six or possibly seven hemoglobins with identical oxygen binding properties in S. clarki suggests that, to a large extent, the physiological role of multiple hemoglobins in this species involves phenomena not directly related to the oxygen binding properties of the hemoglobins.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.     

Electrophoretic variation in muscle lactate dehydrogenase in Snake Valley cutthroat trout, Salmo clarki subsp.

1. Electrophoretic variation observed in muscle A group lactate dehydrogenase in Snake Valley cutthroat trout (Salmo clarki subsp.) suggested the presence of two variant alleles at the A1 locus and a null allele at the A2 locus. 2. The taxonomic status of the Snake Valley cutthroat trout was reviewed.     

Discovery and characterization of novel genetic markers for use in the management of Lahontan cutthroat trout (Oncorhynchus clarkii henshawi)

The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) is threatened by habitat destruction, over‐harvest and hybridization with nonnative trout. Currently, three Geographic Management Units (GMUs) are recognized within the taxon. Here, we describe a suite of 68 single‐nucleotide polymorphism (SNP) genetic markers for use in the study and management of Lahontan cutthroat trout and a closely related subspecies, the Paiute cutthroat trout (O. c. seleneris). These include markers variable within the two subspecies (n = 35), diagnostic for the two subspecies (n = 23) and diagnostic for Yellowstone cutthroat trout (O. c. bouvieri) and other closely related subspecies (n = 10). Sixty‐three markers were discovered by Sanger sequencing of 171 EST loci in an ascertainment panel including Lahontan cutthroat trout from four populations representing all GMUs. Five markers were identified in a secondary sequencing effort with a single population of Lahontan cutthroat trout. TaqMan assays were validated on six Lahontan cutthroat trout populations and a diverse panel of other trout. Over 90% of the markers variable in Lahontan cutthroat trout were polymorphic in at least two populations, and 66% were variable within all three GMUs. All Lahontan diagnostic markers were also fixed for the Lahontan allele in Paiute cutthroat trout. Most of the Yellowstone diagnostic markers can also be used for this purpose in other cutthroat trout subspecies. This is the first set of SNP markers to be developed for Lahontan cutthroat trout, and will be an important tool for conservation and management.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.     

The development of gill arches and gill blood vessels of the rainbow trout,Salmo gairdneri

Gill development begins on the sixth day of incubation at 10^°C and is complete by 31 days (hatching). Gill arches are formed by fusion and perforation of ectoderm and endoderm across the pharyngeal wall. A primary branchial artery forms within each arch and a second branchial artery forms as a branch from its ventral end. A series of filament loop vessels forms connecting the two arteries and when several are patent a unidirectional blood flow is established via afferent (second) branchial artery, filament loop vessels to efferent (primary) branchial artery. Part of the efferent branchial artery just above its junction with the afferent branchial artery constricts and occludes. It is suggested that this change in the pattern of blood flow is dependent on differences in resistance of the two branchial arteries. A later extension of the gill ventrally is thought not to be homologous with similar regions in elasmobranchs and Acipenser.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Glucose transport across the gill of the rainbow trout, Salmo gairdneri

Glucose fluxes across the gills were measured in freshwater-adapted trout (Salmo gairdneri) using an in vitro, perfused-head preparation. A large asymmetry was observed for the primary lamellar pathway, glucose permeability in the serosa-to-mucosa direction being up to 24 times greater than the permeability in the mucosa-to-serosa direction. Chloride cells appeared to possess a maximal rate of transport, or TMG, of 79 mumol/hr per 100 g. Phlorizin, phloretin and, to a lesser extent, harmaline caused an increase in the rate of glucose efflux. The results suggest that the tubulo-vesicular reticulum, into which plasma is introduced under low pressure, may be regarded as a reabsorption site for glucose in a way similar to the nephron proximal tubule. Thus, essential molecules such as glucose are removed while excess or non-essential substances are excreted into the external medium.     

Rapid changes in the phospholipid composition of gill membranes during thermal acclimation of the rainbow trout,Salmo gairdneri

Summary The phospholipid composition of gill tissue was determined in rainbow trout (Salmo gairdneri) undergoing thermal acclimation between 5°C and 20°C for a period of up to 28 days. Proportions of phosphatidylethanolamine (PE) and cardiolipin (CL) increased during cold acclimation and decreased during warm acclimation; proportions of phosphatidylcholine (PC) changed in the opposite direction (i.e., decreased during cold acclimation). In contrast, levels of phosphatidylserine,-inositol, and sphingomyelin did not vary significantly. Thermal modulation of headgroup composition occurred rapidly as reflected by changes in the ratio of PC-to-PE, which rose significantly from 2.40±0.09 to 2.92±0.09 within 72 h of transfer from 5 to 20°C; adaptation to 5°C was equally rapid. Proportions of PE changed more rapidly than those of PC during cold adaptation, whereas the opposite was true during warm acclimation. Both the time course and the direction of the observed changes in phospholipid composition suggest that such adjustments may contribute to the homeoviscous regulation of membrane properties, particularly during the initial stages of thermal adaptation.     

Environmental salinity selectively modifies the outer-ring deiodinating activity of liver, kidney and gill in the rainbow trout

We here analyzed the effect of a mild hyperosmotic challenge on the activities of deiodinases type I (D1) and II (D2) in the trout liver, and D1 in kidney and gill, two organs involved in osmoregulation. FW-adapted immature rainbow trout were transferred to 5 per thousand SW and killed 0.5, 1, 2, 4, 8 12, 24 and 48 h post-transfer (PT). Fish maintained in FW served as controls. Hepatic, renal and branchial D1 and hepatic D2 activities were assessed as well as circulating levels of T(3), T(4) and cortisol. Hyperosmotic challenge elicited significant and sustained decreases in kidney D1 and liver D2 activities at 8 h PT, which returned to control values at 48 h PT. In contrast, liver and gill D1 activities exhibited no significant change throughout the study. Also, significant increases in circulating T(4) at 2-4 and 48 h PT were observed. Circulating T(3) remained unmodified until 24-48 h PT, when it rose sharply. Simultaneously, cortisol showed a trend towards increase during the initial 4 h PT, which attained significance at 48 h PT. The present findings demonstrate that a mild hypertonic challenge is sufficient to elicit responses in the trout thyroidal axis. Hormonal changes in the circulatory compartment are in accordance with those previously described for migratory salmonids. A novel aspect of our findings is the organ-specific differential response exhibited by ORD-enzymes when trout are exposed to a mildly different osmotic environment. Our findings further establish the uniqueness of fish thyroid physiology, and can be of value in further understanding the evolutionary aspects of this ORD family of deiodinases.     

The structure of the gill of the trout,Salmo gairdneri (Richardson)

Summary A light and electron microscopic study was made of the structure of the gill arch, filament and secondary lamella of Salmo gairdneri R. Blood pathways through the gill were traced from serial histological sections, and from the examination of ink perfused tissue and perspex casts formed following resin injection of the circulatory system.The epithelium covering the gill consists of unspecialized, dark, chloride and mucous cells. The distribution of specialized cells appears to be related to gill function. The basement membrane underlying the epithelium consists of three layers, the inner collagen layer being continuous with the connective tissue core of the gills.Blood supply to the secondary lamellar respiratory surface is via branchial, filament and secondary lamellar arteries. Blood spaces of the secondary lamellae are delimited by pillar cells containing what appears to be contractile material. The marginal channel of each lamella is bounded distally by cells of endothelial origin. A network of lymph spaces within the filaments connects with efferent branchial arteries. Nutritionary capillaries within the filaments connect with afferent branchial arteries. No shunts between afferent and efferent filament arteries were found.Data from this study and previous physiological and histopathological studies suggest a mechanism for the control of blood flow to suit the respiratory requirements of the fish. This mechanism involves a system of recruitment of additional respiratory units and changes in overall blood flow patterns.This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1971 and for which M. M. was in receipt of a studentship from the Natural Environmental Research Council. The authors are grateful for the support given by research grants from the M.R.C (P.T.) and the N.E.R.C. (M.M.), and to Prof. G. M. Hughes in whose department the work was carried out.     

Interaction for food and space between experimental populations of juvenile coho salmon (Oncorhynchus kisutch) and coastal cutthroat trout (Salmo clarki) in a laboratory stream

Populations of juvenile coho salmon and coastal cutthroat trout frequently cohabit small coastal streams in western North America. The pattern and mechanism of interactions for food and space between experimental populations of underyearlings of these two salmonids were examined at 3, 5 and 13 °C in a laboratory riffe/pool environment simulating winter and summer conditions. When tested separately at 13 °C, their habitat demands were similar and approximately 60–75% of either species occurred in pools. When tested together they segregated, with approximately 75% of coho in pools and up to 63% of cutthroat trout in riffles. In winter, at 3 °C, both species preferred pools and overhead cover, whether tested separately or together. At 5 °C, they partially segregated in a pattern similar to, but far less pronounced than, that in summer. Both species used similar forms of aggressive behaviour, although aggressive displays were more frequently used by coho, while nipping was more frequently used by cutthroat trout. Both salmonids were most aggressive when food was presented, irrespective of season, although coho responded with greater rapidity and intensity to feeding than did cutthroat trout. When tested together in summer, aggressiveness was high for both species, and agonistic interference by coho in pools and cutthroat trout in riffles appeared to largely account for their segregation. At 3 °C, aggression was low and both species weakly defended pools. At 5 °C, their aggression rose considerably and they partially segregated in a pattern resembling that at 13 °C. The mechanism of segregation between these two salmonids is clearly that of Nilsson's interactive type, which presumably functions to attenuate competition when streams are most likely to be resource limiting; typically, that is the late summer period of low flows and relatively high fish population densities.Based on a dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Institute of Animal Resource Ecology, University of British Columbia, Vancouver, B.C.Ministry of Agriculture and Fisheries, Fisheries Research Division, P.O. Box 8324, Riccarton Christchurch, New Zealand     

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.     

The effects of aluminum and acid on the gill morphology in rainbow trout,Salmo gairdneii

Synopsis The objective was to determine the effects of acid and aluminum in acidified hard and soft water on the histology and morphometry of rainbow trout gills, and to determine relevant toxicity indicators within the gill tissue. Acid and aluminum promoted measurable primary epithelial hyperplasia which proved to be a reliable biological indicator of acid and aluminum contamination and possibly of some predictive value. Low levels of aluminum and acid resulted in hypertrophied chloride cells, suggesting a role in adapting to the contaminants. High concentrations of aluminum (>10 molI^-1) caused chloride cell necrosis and consequently a decline in cell numbers over time. Aluminum precipitates accumulating within the chloride cell cytoplasm probably lead to impaired function prior to cell degeneration. The morphological alterations resulted in a decrease in water space between secondary lamellae (up to 40% within 14 d) which may reduce the efficiency of gas exchange. Twice the aluminum was required in hard water to elicit a similar soft water tissue response. Pathological changes were more severe with aluminum at pH 5.2 than at pH 4.7; results of aluminum speciation suggest that both labile and non-labile fractions are responsible for the induction of gill lesions. Low levels of aluminum may protect fish from the effects of high hydrogen ion concentration.     

Cadmium-induced changes in gill morphology of zebrafish, Brachydanio rerio (Hamilton–Buchanan), and rainbow trout, Salmo gairdneri Richardson

Freshwater zebrafish and brackish water rainbow trout were exposed to different concentrations of cadmium for up to 6 weeks. The gill morphology was examined by light and electron microscopy, and a morphometric analysis was performed. The morphometric study of the secondary lamellae revealed an increase in the portion exterior to the basal lamina, resulting in an increased diffusion distance, after exposure to cadmium concentrations of 10μg l⁻¹ and above. In both species this was due to an increase in volume of the non-tissue spaces of the secondary lamellar epithelium. Furthermore, the water space between neighbouring secondary lamellae was clearly reduced. Morphological examination revealed some gross alterations compared with control fish. These initially consisted in curling of the secondary lamellae and finally resulted in local teleangiectasia. Partial lifting of the secondary lamellar epithelium from the pillar cells resulted in large non-tissue spaces which were invaded by leucocytes. The first sign of degeneration was observed in the chloride cells, which were characterized by a dispersed cytoplasm and a smooth apical plasma membrane.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.