Search

Underwater acoustic tag telemetry was used to assess behavioural differences between juvenile wild-type (i.e. non-transgenic, NT) and growth hormone (GH) transgenic (T) coho salmon Oncorhynchus kisutch in a contained simulated ocean environment. T O. kisutch were found across days to maintain higher baseline swimming speeds than NT O. kisutch and differences in response to feeding were detected between T and NT genotypes. This is the first study to assess behaviour of GH transgenic salmonids in a marine environment and has relevance for assessing whether behavioural effects of GH overexpression seen in freshwater environments can be extrapolated to oceanic phases of the life cycle.

The linkage between the Y-chromosomal DNA marker GH-Y (growth hormone pseudogene) and the Y-associated sex-determining locus was examined in 31 populations of Coho Salmon Oncorhynchus kisutch, 29 populations of Chum Salmon O. keta, and 14 populations of Pink Salmon O. gorbuscha from North America (British Columbia [BC] and Yukon, Canada; Oregon and Washington, USA) and Japan to examine the stability of Y-chromosome sequences in salmonids. This marker is used for genetic sexing of multiple salmon species both for population studies and for aquaculture strain development. We found a strong association between GH-Y and sex in Coho Salmon (with rare exceptions seen in Washington and Oregon only), whereas the association of GH-Y and sex was weak in Chum Salmon and Pink Salmon. Up to 100% of males lacking GH-Y were found in some BC Chum Salmon populations, whereas there were no exceptional (GH-Y-negative) males in Japan, the Yukon, or Washington, indicating strong geographic clustering of GH-Y marker de...

Pre‐market/prospective environmental risk assessments (ERAs) contribute to risk analyses performed to facilitate decisions about the market introduction of regulated stressors. Robust ERAs begin with an explicit problem formulation, which involves among other steps: (1) formally devising plausible pathways to harm that describe how the deployment of a regulated stressor could be harmful; (2) formulating risk hypotheses about the likelihood and severity of such events; (3) identifying the information that will be useful to test the risk hypotheses; and (4) developing a plan to acquire new data for hypothesis testing should tests with existing information be insufficient for decision‐making. Here, we apply problem formulation to the assessment of possible adverse effects of RNA interference‐based insecticidal genetically modified (GM) plants, GM growth hormone coho salmon, gene drive‐modified mosquitoes and classical biological weed control agents on non‐target organisms in a prospective manner, and of neonicotinoid insecticides on bees in a retrospective manner. In addition, specific considerations for the problem formulation for the ERA of nanomaterials and for landscape‐scale population‐level ERAs are given. We argue that applying problem formulation to ERA maximises the usefulness of ERA studies for decision‐making, through an iterative process, because: (1) harm is defined explicitly from the start; (2) the construction of risk hypotheses is guided by policy rather than an exhaustive attempt to address any possible differences; (3) existing information is used effectively; (4) new data are collected with a clear purpose; (5) risk is characterised against well‐defined criteria of hypothesis corroboration or falsification; and (6) risk assessment conclusions can be communicated clearly. However, problem formulation is still often hindered by the absence of clear policy goals and decision‐making criteria (e.g. definition of protection goals and what constitutes harm) that are needed to guide the interpretation of scientific information. We therefore advocate further dialogue between risk assessors and risk managers to clarify how ERAs can address policy goals and decision‐making criteria. Ideally, this dialogue should take place for all classes of regulated stressors, as this can promote alignment and consistency on the desired level of protection and maximum tolerable impacts across regulated stressors.

Environmental conditions are known to affect phenotypic development in many organisms, making the characteristics of an animal reared under one set of conditions not always representative of animals reared under a different set of conditions. Previous results show that such plasticity can also affect the phenotypes and ecological interactions of different genotypes, including animals anthropogenically generated by genetic modification. To understand how plastic development can affect behavior in animals of different genotypes, we examined the feeding and risk-taking behavior in growth-enhanced transgenic coho salmon (with two- to threefold enhanced daily growth rates compared to wild type) under a range of conditions. When compared to wild-type siblings, we found clear effects of the rearing environment on feeding and risk-taking in transgenic animals and noted that in some cases, this environmental effect was stronger than the effects of the genetic modification. Generally, transgenic fish, regardless of rearing conditions, behaved similar to wild-type fish reared under natural-like conditions. Instead, the more unusual phenotype was associated with wild-type fish reared under hatchery conditions, which possessed an extreme risk averse phenotype compared to the same strain reared in naturalized conditions. Thus, the relative performance of genotypes from one environment (e.g., laboratory) may not always accurately reflect ecological interactions as would occur in a different environment (e.g., nature). Further, when assessing risks of genetically modified organisms, it is important to understand how the environment affects phenotypic development, which in turn may variably influence consequences to ecosystem components across different conditions found in the complexity of nature.

Most fish species do not have single-chromosome-based sex determination or display cytologically distinguishable sex chromosomes. The selective forces acting on homologous sequences in diploid autosomal versus haploid sex-chromosomal regions are expected to be distinct and thus to differentially influence genetic variation. In Chinook Salmon Oncorhynchus tshawytscha, the Y chromosome possesses a growth hormone pseudogene (ghp) that is linked to the sex-determination locus and is derived from the functional autosomal growth hormone 2 gene (gh2). Thus, examining these two paralogues provides a model with which to study the forces affecting the persistence of genetic variation between sex-linked and autosomal loci among individuals in Chinook Salmon populations. We characterized single-nucleotide polymorphisms in a 1.6-kb contiguous homologous region in gh2 and ghp in 315 individuals from 19 Chinook Salmon populations ranging from Russia to Alaska, British Columbia, and California. The ghp sequence w...

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, CTmax ) were observed among size-matched wild-type, domesticated, growth hormone (GH)-transgenic fish fed to satiation, and GH-transgenic fish on a ration-restricted diet. Instead, GH-transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, TAB ). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH-transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.

The first genetically modified (GM) fish intended for human consumption has recently stimulated significant scientific discussion and regulatory scrutiny regarding food safety and environmental ris ...

Summary 1. Although life-history theory predicts that juvenile growth rates should be high, there is substantial evidence that most juveniles grow below their physiological maximum. The endocrine system plays an important role in the determination of fundamental life-history traits, and hormones often serve as a link between an organism’s environment and the expression of a trait. Particularly, growth is a life-history trait, which is strongly associated with growth hormone (GH) in fish, as well as most vertebrates. 2. To elucidate trade-offs related to elevated GH in fish in a natural environment, we experimentally administrated GH exogenously to juvenile Atlantic salmon using sustained-release GH implants, at an earlier ontogenetic stage than previously achieved (1AE5 months). We assessed the effects on growth, dispersal and survival in contrasting environments. 3. Exogenous GH treatment increased the growth rate when fish were fed ad libitum in captivity. However, in a natural stream, GH treatment had a significant negative effect on growth and no apparent effect on survival or dispersal. This contrasts with previous studies conducted at later developmental stages, which show either a positive growth effect or no effect of elevated GH levels. 4. This study shows that environmental conditions strongly affect the response to GH and that under some natural conditions, it may also reduce growth. We suggest that the endogenous plasma GH levels may be maximizing growth during early, but not later, juvenile stages in nature.

We tested the fitness consequences of introgression of fast-growing domesticated fish into a wild population. Fry from wild and domesticated rainbow trout (Oncorhynchus mykiss) crosses, F1 hybrids, and first- and second-generation backcrosses were released into two natural lakes. Parentage analysis using microsatellite loci facilitated the identification of survivors, so fitness was estimated in nature from the first-feeding stage. Results indicated that under certain conditions, domesticated fish survived at least as well as wild fish within the same environment. Relative growth and survival of the crosses, however, were highly dependent on environment. During the first summer, fastest-growing crosses had the highest survival, but this trend was reversed after one winter and another summer. Although the F1 hybrids showed evidence of outbreeding depression because of the disruption of local adaptation, there was little evidence of outbreeding depression in the backcrosses, and the second-generation backcrosses exhibited a wild-type phenotype. This information is relevant for assessing the multigenerational risk of escaped or released domesticated fish should they successfully interbreed with wild populations and provides information on how to minimize detrimental impacts of a conservation breeding and/or management programme. These data also further understanding of the selection pressures in nature that maintain submaximal rates of growth.

Wild-genotype and growth hormone (GH) transgenic coho salmon (with dramatically enhanced growth potential) were used to examine the influence of genotype, age, body size, growth, and rearing conditions on the onset of seaward migration and to assess the potential consequences of the introduction of such transgenic fish on natural ecosystems and wild populations. When reared from the first feeding stage under naturalized stream conditions, there was no difference in survival or migratory timing between the two genotypes. However, larger fish migrated earlier in the season than smaller fish of both genotypes, and transgenic fish with higher specific spring growth rates migrated earlier in the season than slower-growing transgenic fish. Stream-reared fish of both genotypes also displayed increased migratory activity at dawn and dusk. Fish reared in the hatchery for 3 and 15 months before being released into the stream in August differed in onset of seaward migration due mainly to age (older fish migrated earlier in the season) and genotype (transgenic fish migrated before wild-type in younger fish). Further, hatchery-reared fish showed no diel pattern in activity during migration. In older fish, larger individuals migrated later in the season than small individuals, whereas there was no clear size effect in younger individuals. Thus, although small differences in spring migration timing were observed among groups, seaward migration in coho salmon was largely independent of major shifts in size and growth rate induced by GH transgenesis (i.e., transgenic fish migrated at approximately the normal time in the spring, rather than at the typical size). Further, early rearing conditions had a stronger effect on migratory behavior than did the growth-promoting transgene. Taking into account effects of migratory timing, growth, survival, and differential food conversion efficiencies, these data suggest that transgenic fish escaped from hatcheries would have a greater impact on stream ecosystems during early life compared to escaped wild-type fish. However, this difference may be reduced if rearing occurred in subsequent generations under wild conditions where growth rates of transgenic fish are reduced compared with hatchery conditions.

Summary 1. The potential risks of accidental or intentional introduction of transgenic organisms to nature are unknown. We have previously shown that, after being reared in the hatchery, growth hormone transgenic coho salmon Oncorhynchus kisutch can exert a stronger predation effect on natural prey under simulated natural conditions compared with hatchery-reared genetically wild conspecifics. However, when reared in a simulated natural environment, the difference between the two genotypes was greatly reduced. 2. Here, we tested if the difference in predation rate between the two genotypes after being reared in the hatchery diminishes with time spent in a simulated natural environment. Genetically wild coho salmon reared in the hatchery were size matched to younger satiation-fed (fast-growing) and same age restricted-fed (growth rates matched to wild-type) transgenic fish. These three types of predators were released into simulated natural habitats and their effects on prey survival and growth were monitored over a 2-month period. 3. Restricted-fed, but not satiation-fed, transgenic predators consumed significantly more prey (×2·7) relative to wild predators during the first month, with an increase to 3.8 times during the second period. Prey biomass decreased more in the presence of restricted-fed predators during the first period, but a faster growth of these prey during the second period compensated for the higher predation rate resulting in no significant difference in prey biomass among predators after 2 months. Transgenic predators grew more in length than wild type, but all three types converged in weight and condition over time. 4. Synthesis and applications. Behavioural phenotypes developed in the hatchery showed little plasticity after release. Hence, development of policy on the use of transgenic organisms must incorporate knowledge of the effect of environmental conditions, experienced by the organism prior to escape or release, on phenotype. The risk posed by culture-reared organisms may depend on the frequency of escapes, number of individuals escaping and age of escapees. Important to note is that wild-reared organisms may be biologically different from culture-reared and thus require separate evaluation. Our results will be important for science policy makers and regulators to consider when deciding whether to allow commercial application of transgenic species in aquaculture.

The purpose of this study was to examine, using a rainbow trout (Oncorhynchus mykiss) model system, the fitness consequences of three generations of introgression of genotypes adapted to two different environments (culture and nature). The experiments also isolated the influence of competitive interactions and risk of predation on the relative growth and survival of the wild and backcrossed lines. Line crosses representing fast-growing pure domestic (D), slow-growing pure wild (W), domestic x wild hybrids (F1), F1 x wild backcrosses (B1), and B1 x wild backcrosses (B2) were generated and reared under (1) culture conditions, (2) seminatural conditions with competition among genotypes, and (3) seminatural conditions under risk of predation. Survival of the fry in a seminatural environment with competition fit an additive model of gene action with the domestic fish having the highest survival and the wild fish the lowest, but under risk of predation outbreeding depression was suggested by low survival of the B2 lines. Evidence of a trade-off in growth and survival under risk of predation along with observations of genetically determined behavioral differences among the strains may provide some explanation for the observed differences in survival among the strains. This information is relevant to improving our evolutionary understanding of the interaction among genomes, and the influence of environment, during hybridization events. Results from this experiment indicate that alteration of phenotype likely played a prominent role in the reduced fitness experienced by progeny produced after three generations of introgression, supporting the theory that disruption of genotypes selected for adaptation to local conditions may be a primary cause of outbreeding depression in species such as salmon.

Potential ecological consequences on the natural environment of fast-growing transgenic fish with elevated intrinsic growth rates is an important question should such fish be allowed in commercial production systems. One trait that will strongly affect the spatial extent of consequences is the propensity of transgenic fish to disperse. We addressed this question in three experiments using different measures of spatial dispersal where we compared very young genotypically wild coho salmon with transgenic conspecifics in terms of: (i) group cohesion, (ii) exploratory behavior, and (iii) up- and downstream movements. Transgenic fish were more loosely aggregated, more likely to explore, and less likely to swim upstream, but equally likely to disperse downstream compared with genetically wild fish. These results show that dispersal behavior has been affected by transgenesis and that transgenic fish therefore may venture into habitats previously not used by wild fish. Given the importance of dispersal in ecological risk-assessment of transgenic fish, continued work within this area is warranted and experimental habitats should mimic the potential receiving natural habitats to which transgenic fish are likely to escape or be released to.

Selective breeding for enhanced growth in Pacific salmon Oncorhynchus spp. and other fish typically involves use of the largest mature individuals to breed for future generations of aquaculture broodstock. Owing to an altered selection regime, faster-growing fish may not be as adapted to the natural environment as wild fish. To increase understanding of the genetic changes underlying selection for enhanced growth that results in phenotypic differentiation of farmed from wild Pacific salmon, multiple generations of pure and hybrid families were generated for coho salmon O. kisutch, including pure farm (D), pure native (Ch; a natural strain propagated by wild and hatchery production), F1 and F2 hybrids, and F1 × wild backcross (BCh) genotypes. The family groups were reared in the laboratory under controlled conditions as (1) individual genotypic groups, (2) mixed groups under culture conditions, and (3) mixed groups under enriched (seminatural) conditions. The growth of the fish was tracked until s...

Substantial evidence from the animal kingdom shows that there is a trade-off between benefits and costs associated with rapid somatic growth. One would therefore expect growth rates under natural conditions to be close to an evolutionary optimum. Nevertheless, natural selection in many salmonid species appears to be toward larger size and earlier emergence from spawning redds, indicating a potential for increased growth rate to evolve. We tested how selection for genetic variants (growth hormone transgenic coho salmon, Oncorhynchus kisutch, with more than doubled daily growth rate potential relative to wild genotypes) depended on predator timing and food abundance during the early period of life (fry stage). In artificial redds, fry of the fast-growing genotypes showed a highly significant developmental shift, emerging from gravel nests approximately two weeks sooner, but with an 18.6% reduced survival, relative to wild-genotype fry. In seminatural streams, fry of the fast-growing genotypes suffered higher predation than those of wild genotypes when predators were present at the time of fry emergence, but this difference was less pronounced when food was scarce. In streams where predators were introduced after emergence, fry survived equally well regardless of food availability. Surviving fry grew faster in habitats provided with more food, and fast-growing genotypes also grew faster than wild genotypes when predators arrived late and food was abundant. Fewer fish migrated downstream past a waterfall when food availability was high and in the presence of predators, and wild-genotype fry were more likely to migrate than fry of the fast-growing genotypes. After being returned to the experimental streams after migration, fast-growing genotypes survived equally well as those of the same genotypes that did not migrate, whereas migrating wild genotypes experienced higher mortality relative to those of the same genotypes that did not migrate. Comparisons of growth rates between siblings retained under hatchery conditions and those from habitats with the fastest growth in the experimental stream revealed that growth rates were similar for wild genotypes in both environments, whereas the fast-growing genotypes in the streams only realized 90% of their growth potential. The present study has shown that a major shift in developmental timing can alter critical early stages affecting survival and can have a significant effect on fitness. Furthermore, ecological conditions such as food abundance and predation pressure can strongly influence the potential for fast-growing variants to survive under natural conditions. The large-scale removal of many predatory species around the world may augment the evolution of increased intrinsic growth rates in some taxa.

Growth hormone transgenic coho salmon Oncorhynchus kisutch fed at the same ration level as non-transgenic controls (Tc) had the same growth rate as non-transgenic controls (Nt). In contrast, growth hormone transgenic coho salmon (Tf) fed ad libitum ate about twice as much and had much higher growth rates than the other two groups. The most obvious result was the significantly larger caeca in the Tf group relative to both Nt or pair-fed Tc. The Tf fish had more caeca that were longer. The results suggested that the effect was indirect and the enlarged caeca required both the GHtransgene and hyperphagia to cause enlarged caecal capacity. A small part of the results, however, also suggested that there was a direct effect of the GHtransgene on some gut tissues, particularly the intestine.

Coho salmon Oncorhynchus kisutch were genetically altered to produce growth hormone without regulation, causing them grow 11 times larger (on average) than control fish after 14 months. This technology has potential benefits for the aquaculture industry, but the environmental risk associated with the escape of transgenic fish into the wild is not known. To partially address this issue, we experimentally investigated how well transgenic salmon survive when given a choice to consume food in a predator's presence. If transgenic salmon are to retain their growth advantage, we predict that they must also be more effective at competing for food than wild salmon and be willing to suffer higher mortality rates when foraging. The relative competitive abilities of transgenic and control salmon at two different ages were tested with an unequal competitors ideal free distribution. A larger proportion of transgenic salmon fed within the system, although they were not overrepresented at a higher-quantity food ...

The reproductive performances of growth-enhanced transgenic, hatchery, and cultured nontransgenic coho salmon Oncorhynchus kisutch were examined to investigate the consequences of reproductive interaction between growth hormone (GH)–transgenic fish and wild fish that may occur if transgenic salmon escaped into the natural environment. We examined adult morphological phenotypes, gamete quantity and quality, in vitro offspring production, courtship and spawning behavior, male competitive behavior, and transgene transmission to offspring. Transgenic, hatchery, and cultured nontransgenic fish required 2, 3, and 3 or 4 years, respectively, to reach sexual maturation. No differences in male gamete quantity or in vitro offspring production were observed. Transgenic females were more fecund than hatchery females but had smaller eggs. Fewer transgenic females spawned than hatchery females under experimental conditions, and transgenic females displayed consistently low levels of courtship behavior. In nonc...

We compared the performance of normal and growth hormone-transgenic coho salmon feeding on surface drifting edible and inedible novel prey items in various social environments. With an inherently higher appetite, we predicted that transgenic fish would be more willing to feed on novel prey, and that visual company with another fish would enhance this difference further. Transgenic and normal fish, of similar size and age, were equally willing to attack both the edible (live insects) and inedible (artificial angling lure flies) prey, but transgenic fish did so faster and were more likely to make repeated attacks. Transgenic fish managed to seize and consume the edible prey after fewer attacks than did normal fish. However, swallowing of prey took longer than for normal fish. More transgenic individuals interacted with the inedible prey compared with normal salmon, and initially, transgenic fish in visual company with another fish also interacted more with the prey than single transgenic or any constellation of normal focal fish. With repeated exposures, the number of individuals attacking and the number of interactions with the prey decreased. These responses were stronger in transgenic fish, partly explained by the initially low response in normal fish. The observed differences are most likely the consequences of elevated levels of growth hormone in transgenic fish generating enhanced feeding motivation and reinforcement capacity. In a natural environment, the performance of a growth hormone-transgenic fish may therefore depend on the relative abundance of profitable vs. unprofitable prey, as well as the presence of other transgenic individuals.

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non-transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non-transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non-transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth-accelerated coho salmon.

Growth hormone (GH) gene transgenesis has allowed the production of salmon with an inherently increased growth potential, on average two to threefold higher compared with daily specific growth rates observed in normal, non-transgenic fish. This difference quickly results in animals of very different sizes at age, and is associated with specific morphological effects and enhanced appetites in transgenic animals. However, less is known of the feeding and antipredator behaviour of GH-transgenic fish, information that can help with predictions of potential ecological consequences of release or escape of transgenic fish into the wild. In a series of experiments, transgenic (T) and normal (N) coho salmon of varying age and size (from 0.5 to 40 g, 3.5-21 mo) were studied singly, in pairs, and in groups during feeding and simulated predation threat. Vertical position generally did not differ between T and N fry, but at larger size (> 4 g) T fish remained closer to the surface than N fish both during feeding and predatory attacks, probably as a consequence of inherent differences in feeding motivation and later reinforcement by associative learning. This difference in vertical position was not the result of competition as it remained even after either fish in the pair had been removed. In nature, where predators may attack from above (birds) or below (fish), this kind of behaviour may translate into higher risk of predation, which could increase mortality and lower the fitness of transgenic fish, unless their increased growth rate can compensate for the increased risk-taking.

Oxygen uptake of growth hormone transgenic coho salmon Oncorhynchus kisutch was measured in individual fish with a closed-system respirometer and was compared with that of similar-sized non-transgenic control coho salmon during starvation and when fed a fixed ration or to satiation. Transgenic and control fish did not differ in their standard oxygen uptake after 4 days of starvation, although control fish had a higher routine oxygen uptake, scope for spontaneous activity and initial acclimation oxygen uptake. During feeding, transgenic fish ate significantly more than control fish, and had an overall oxygen uptake that was 1.7 times greater than control fish. When fish that had eaten the same per cent body mass were compared, transgenic fish had an oxygen uptake that was 1.4 times greater than control fish. Differences in oxygen uptake in growth hormone transgenic coho salmon and non-transgenic fish appear to be due to the effects of feeding, acclimation and activity level, and not to a difference in basal metabolism.

Routine oxygen consumption [Mo 2 ) was 35% higher in 1 day starved and 21% higher in 4 day starved adult transgenic coho salmon Oncrhynchus kisutch relative to end of migration ocean-ranched coho salmon. Critical swimming speed (U crit ) and Mo 2 at U crit (Mo 2max ) were significantly lower in 4 day starved transgenic coho salmon (1.25BL s -1 ;8.79 mg O 2 kg -1 min -1 ) compared to ocean-ranched coho salmon (1.60BL s -1 ; 9.87mgO 2 kg -1 min -1 ). Transgenic fish swam energetically less efficiently than ocean-ranched fish, as indicated by a poorer swimming economy at U crit (Mo 2max U -1 crit ). Although Mo 2max was lower in transgenic coho salmon, the excess post-exercise oxygen consumption (EPOC) measured during the first 20min of recovery was significantly larger in transgenic coho salmon (44.1 mgO 2 kg -1 ) compared with ocean-ranched coho salmon (34.2mg O 2 kg -1 ). which had a faster rate of recovery.

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.

The taxonomy of the PKX myxosporean, cause of proliferative kidney disease (PKD) of salmonids, has long been an enigma because only immature spores are consistently associated with the infection. In the early 1990s, one of us (M.L.K.) proposed that PKX may be the extrasporogonic form of Sphaerospora oncorhynchi, which has been observed in salmonids where PKD is enzootic. Recently, the sequence of the small subunit ribosomal DNA (SSU rDNA) of PKX was deposited in GenBank (U70623). We designed PKX-specific primers by aligning this sequence with that of coho salmon Oncorhynchus kisutch and with those of other myxosporeans. With these primers, a portion of the SSU rDNA of PKX was amplified and sequenced from the kidneys of infected salmonids from British Columbia, Washington, California, Newfoundland, and England. The maximum variation in sequence across 764 base pairs (bp) among all PKX isolates, including the original sequence (GenBank U70623), was 0.8%. Results indicated that PKX isolates from dif...

Rules of zoological nomenclature, morphological data, and ribosomal DNA sequence data support the validity of the genus Nucleospora, and its placement in the family Enterocytozoonidae. Although Nucleospora exhibits most of the distinguishing morphological characteristics of the family Enterocytozoonidae Cali and Owen, 1990, the distinctively different hosts (fish and humans, respectively) and sites of development (the nuclei of immature blood cells and the cytoplasm of enterocytes) support the placement of Nucleospora and Enterocytozoon into separate genera. Ribosomal DNA sequence comparisons between Nucleospora salmonis and Enterocytozoon bieneusi showed 19.8% genetic divergence in the large and small subunit regions. Although more inter- and intrageneric comparisons are needed before percent homology of ribosomal DNA can be used as a criterion for the separation of genera, the genetic divergence between the two species is sufficiently large to deter suppression of the genus Nucleospora as a junior synonym of Enterocytozoon. A polymerase chain reaction test for the detection of N. salmonis in chinook salmon (Oncorhynchus tshawytscha), based on N. salmonis-specific ribosomal DNA sequence, is described.

Increases in hepatic CYP1A1 messenger RNA (mRNA) levels were quantitated, using a reverse-transcriptase–competitive-polymerase-chain-reaction assay, from juvenile chinook salmon following exposure to treated bleached-kraft mill effluent (BKME). Two tissue preservation techniques, alcohol fixation and freezing, were compared and both found to be valid, although absolute values obtained differed between the methods. The results of this study show that juvenile chinook respond with a dose-dependent induction of CYP1A1 mRNA to levels of treated BKME found under winter, low-flow conditions downstream of pulp mills in the Fraser River, British Columbia, Canada. The validation of alcohol fixation as a sampling technique for studies on mRNA levels improves the ease of field sampling and increases the feasibility of using CYP1A1 mRNA level as a bioindicator of exposure of fish to specific chemical contaminants.

Although many studies implicate sex steroids in the process of sexual differentiation, the exact role played by these substances in lower vertebrates, especially teleost fish, is not clear, since it is not conclusively known whether sex steroids are the cause or an early consequence of sex differentiation. By hormonal manipulation and specific crossings, it is possible to produce all‐female stocks of the otherwise gonochoristic chinook salmon (Oncorhynchus tshawytscha). These all‐female stocks are an excellent model to study the effects of steroids on sexual differentiation since the genetic sex is known before the actual differentiation of the embryonic gonads takes place. In the present study, we show that treatment with a nonsteroidal inhibitor of aromatase, the enzyme that catalyzes the conversion from androgens to estrogens, for only 2 hours when the gonads were bipotent, caused genetic females to develop into normal males. Furthermore, these males had testes which were indistinguishable both in size and in structure from those of genetic males, and completed all the stages of spermatogenesis. At 2 years of age, these males produced viable sperm capable of inducing normal embryonic development when used to fertilize eggs, with a resulting all‐female progeny. These results provide strong support for Yamamoto's theory (Yamamoto, '69) that androgens and estrogens are natural sex inducers in gonochoristic fish, and suggest that aromatase plays a pivotal role in the sex differentiation of salmon. Thus, by brief treatment with an aromatase inhibitor at a specific time in development, an organism was induced to develop a funtional, phenotypic sex different from its genetic sex. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc., A Wiley Company

Production of trisomic-3L Drosophila melanogaster has allowed further investigation of compensated levels of gene expression in autosomal trisomies. We find that four enzyme loci on this arm produce diploid levels of gene product in trisomic-3L larvae. For one of these genes, we show that all three alleles are expressed at similar levels. Two genes on 3L display dose-dependent levels of gene product, and their location, relative to the four compensating loci, indicates that these two classes of genes are not regionally separated. In trisomic-2R larvae, the level of enzyme produced from on 2R-linked gene was dose dependent. In contrast, measurements of five loci on the X chromosome in metafemales (X trisomies) suggest that most genes are compensated in these individuals. Heat-shock gene expression in trisomic-3L salivary glands was qualitatively similar to diploids. The quantities of the small hsps (from the 67B cluster on 3L) suggest that these four genes respond independently to the trisomic condition; two produce compensated levels of protein, whereas the other two produce dose-dependent levels of protein. The amount of hsp 83 produced in trisomies was similar to diploids (compensated). However, quantification of hsp 83 RNA showed that a dose-dependent level of transcript was produced. This implies that hsp 83 compensation is controlled post-transcriptionally.