Oxytocin influences parental care in male threespine stickleback across multiple time scales.

Oxytocin (OXT) and its homologs are known to regulate parental care in vertebrates, but it is unknown what role these neuropeptides may play in the evolutionary loss of care. Here, we compared two recently diverged ecotypes of threespine stickleback (Gasterosteus aculeatus) that differ in parental care. Males of the common ecotype provide obligate, uniparental care to their offspring, whereas males of the white ecotype abandon their offspring after fertilization. To test if OXT plays a role in the loss of care, we manipulated OXT in males of both ecotypes via intraperitoneal injection of a vehicle control, OXT single- or double-dose, or an OXT antagonist. We observed the behavioral response to injection at two time points for commons (0 and 4 days post-fertilization (dpf)) and one for whites (0 dpf). Our results suggest that, in commons, OXT promotes the onset of care but not its maintenance. Notably, commons that ultimately terminated their clutches did not respond to OXT at 0 dpf, which may have contributed to their failure to transition to a state of care. Whites responded to OXT manipulation in a different manner than commons, suggesting that the loss of care in whites is not due to a loss of sensitivity to OXT, or insufficient levels of OXT ligand, but rather an evolutionary change to the underlying parental circuit that OXT is acting on. These results provide evidence that ancient hormonal systems like OXT can contribute to losses of care over multiple timescales.

Tapeworm infection affects sleep-like behavior in three-spined sticklebacks.

Sleep is a complex and conserved biological process that affects several body functions and behaviors. Evidence suggests that there is a reciprocal interaction between sleep and immunity. For instance, fragmented sleep can increase the probability of parasitic infections and reduce the ability to fight infections. Moreover, viral and bacterial infections alter the sleep patterns of infected individuals. However, the effects of macro-parasitic infections on sleep remain largely unknown, and measuring sleep in non-model organisms remains challenging. In this study, we investigated whether macro-parasite infections could alter sleep-like behavior of their hosts. We experimentally infected three-spined sticklebacks (Gasterosteus aculeatus), a freshwater fish, with the tapeworm Schistocephalus solidus and used a hidden Markov model to characterize sleep-like behavior in sticklebacks. One to four days after parasite exposure, infected fish showed no difference in sleep-like behavior compared with non-exposed fish, and fish that were exposed-but-not-infected only showed a slight reduction in sleep-like behavior during daytime. Twenty-nine to 32 days after exposure, infected fish showed more sleep-like behavior than control fish, while exposed-but-not-infected fish showed overall less sleep-like behavior. Using brain transcriptomics, we identified immune- and sleep-associated genes that potentially underlie the observed behavioral changes. These results provide insights into the complex association between macro-parasite infection, immunity, and sleep in fish and may thus contribute to a better understanding of reciprocal interactions between sleep and immunity.

Associations Between Gut Microbiota Diversity and a Host Fitness Proxy in a Naturalistic Experiment Using Threespine Stickleback Fish.

The vertebrate gut microbiota is a critical determinant of organismal function, yet whether and how gut microbial communities affect host fitness under natural conditions remains largely unclear. We characterised associations between a fitness proxy-individual growth rate-and bacterial gut microbiota diversity and composition in threespine stickleback fish introduced to large semi-natural ponds. We detected a 63% higher richness of bacterial taxa (α-diversity) in the guts of high-fitness fish compared to low-fitness fish, which might be driven by stronger bacterial dispersal among high-fitness fish according to the fit of a neutral community model. Further, microbial communities of high-fitness fish were more similar to one another (i.e., exhibited lower ß-diversity) than those of low-fitness fish. The lower ß-diversity found to be associated with higher host fitness is consistent with the Anna Karenina principle-that there are fewer ways to have a functional microbiota than a dysfunctional microbiota. Our study links differences in α- and ß-diversity to a fitness-related trait in a vertebrate species reared under naturalistic conditions and our findings provide a basis for functional tests of the fitness consequences of host-microbiota interactions.

Host ecotype and rearing environment are the main drivers of threespine stickleback gut microbiota diversity in a naturalistic experiment.

Host-microbiota interactions play a critical role in the hosts' biology, and thus, it is crucial to elucidate the mechanisms that shape gut microbial communities. We leveraged threespine stickleback fish (Gasterosteus aculeatus) as a model system to investigate the contribution of host and environmental factors to gut microbiota variation. These fish offer a unique opportunity for experiments in naturalistic conditions; we reared benthic and limnetic ecotypes from three different lakes in experimental ponds, allowing us to assess the relative effects of shared environment (pond), geographic origin (lake-of-origin), trophic ecology and genetics (ecotype) and biological sex on gut microbiota α- and ß-diversity. Host ecotype had the strongest influence on α-diversity, with benthic fish exhibiting higher diversity than limnetic fish, followed by the rearing environment. ß-diversity was primarily shaped by rearing environment, followed by host ecotype, indicating that environmental factors play a crucial role in determining gut microbiota composition. Furthermore, numerous bacterial orders were differentially abundant across ponds, underlining the substantial contribution of environmental factors to gut microbiota variation. Our study illustrates the complex interplay between environmental and host ecological or genetic factors in shaping the stickleback gut microbiota and highlights the value of experiments conducted under naturalistic conditions for understanding gut microbiota dynamics.

The transgenerational consequences of paternal social isolation and predation exposure in threespined sticklebacks.

Parents routinely encounter stress in the ecological environment that can affect offspring development (transgenerational plasticity: TGP); however, parents' interactions with conspecifics may alter how parents respond to ecological stressors. During social buffering, the presence of conspecifics can reduce the response to or increase the speed of recovery from a stressor. This may have cascading effects on offspring if conspecifics can mitigate parental responses to ecological stress in ways that blunt the transmission of stress-induced transgenerational effects. Here, we simultaneously manipulated both paternal social isolation and experience with predation risk prior to fertilisation in threespined stickleback (Gasterosteus aculeatus). We generated offspring via in-vitro fertilisation to allow us to isolate paternal effects mediated via sperm alone (i.e. in the absence of paternal care). If social buffering mitigates TGP induced by paternal exposure to predation risk, then we expect the transgenerational effects of predation exposure to be weaker when a conspecific is present compared to when the father is isolated. Offspring of predator-exposed fathers showed reduced anxiety-like behaviour and tended to be captured faster by the predator. Fathers who were socially isolated also had offspring that were captured faster by a live predator, suggesting that paternal social isolation may have maladaptive effects on how offspring respond to ecological stressors. Despite additive effects of paternal social isolation and paternal predation risk, we found no evidence of an interaction between these paternal treatments, suggesting that the presence of a conspecific did not buffer fathers and/or offspring from the effects of predation risk. Our results suggest that socially induced stress is an important, yet underappreciated, mediator of TGP and can elicit transgenerational effects even in species that do not form permanent social groups. Future studies should therefore consider how the parental social environment can affect both within and trans-generational responses to ecological stressors.

Alternative splicing in parallel evolution and the evolutionary potential in sticklebacks.

Repeatability of adaptation to similar environments provides opportunity to evaluate the predictability of natural selection. While many studies have investigated gene expression differences between populations adapted to contrasting environments, the role of post-transcriptional processes such as alternative splicing has rarely been evaluated in the context of parallel adaptation. To address the aforementioned knowledge gap, we reanalysed transcriptomic data from three pairs of threespine stickleback (Gasterosteus aculeatus) ecotypes adapted to marine or freshwater environment. First, we identified genes with repeated expression or splicing divergence across ecotype pairs, and compared the genetic architecture and biological processes between parallelly expressed and parallelly spliced loci. Second, we analysed the extent to which parallel adaptation was reflected at gene expression and alternative splicing levels. Finally, we tested how the two axes of transcriptional variation differed in their potential for evolutionary change. Although both repeated differential splicing and differential expression across ecotype pairs showed tendency for parallel divergence, the degree of parallelism was lower for splicing than expression. Furthermore, parallel divergences in splicing and expression were likely to be associated with distinct cis-regulatory genetic variants and functionally unique set of genes. Finally, we found that parallelly spliced genes showed higher nucleotide diversity than parallelly expressed genes, indicating splicing is less susceptible to genetic variation erosion during parallel adaptation. Our results provide novel insight into the role of splicing in parallel adaptation, and underscore the contribution of splicing to the evolutionary potential of wild populations under environmental change.

Effects of maternal age and environmental enrichment on learning ability and brain size.

It is well known that maternal age at reproduction affects offspring lifespan and some other fitness-related traits, but it remains understudied whether maternal senescence affects how offspring respond to their environments. Early environment often plays a significant role in the development of an animal's behavioral phenotype. For example, complex environments can promote changes in cognitive ability and brain morphology in young animals. Here, we study whether and how maternal effect senescence influences offspring plasticity in cognition, group behavior, and brain morphology in response to environmental complexity. For this, juvenile 3-spined sticklebacks from young and old mothers (i.e. 1-yr and 2-yr-old) were exposed to different levels of environmental enrichment and complexity (i.e. none, simple, and complex), and their behavior, cognitive ability, and brain size were measured. Exposing fish to enriched conditions improved individual learning ability assessed by a repeated detour-reaching task, increased the size of the whole brain, and decreased aggressive interactions in the shoal. Maternal age did not influence the inhibitory control, learning ability, and group behavioral responses of offspring to the experimental environmental change. However, maternal age affected how some brain regions of offspring changed in response to environmental complexity. In offspring from old mothers, those exposed to the complex environment had larger telencephalons and cerebellums than those who experienced simpler environments. Our results suggest that maternal effect senescence may influence how offspring invest in brain functions related to cognition in response to environmental complexity.

Improved assembly of the Pungitius pungitius reference genome.

The nine-spined stickleback (Pungitius pungitius) has been increasingly used as a model system in studies of local adaptation and sex chromosome evolution but its current reference genome assembly is far from perfect, lacking distinct sex chromosomes. We generated an improved assembly of the nine-spined stickleback reference genome (98.3% BUSCO completeness) with the aid of linked-read mapping. While the new assembly (v8) was of similar size as the earlier version (v7), we were able to assign 4.4 times more contigs to the linkage groups and improve the contiguity of the genome. Moreover, the new assembly contains a ∼22.8 Mb Y-linked scaffold (LG22) consisting mainly of previously assigned X-contigs, putative Y-contigs, putative centromere contigs, and highly repetitive elements. The male individual showed an even mapping depth on LG12 (pseudo X chromosome) and LG22 (Y-linked scaffold) in the segregating sites, suggesting near-pure X and Y representation in the v8 assembly. A total of 26,803 genes were annotated, and about 33% of the assembly was found to consist of repetitive elements. The high proportion of repetitive elements in LG22 (53.10%) suggests it can be difficult to assemble the complete sequence of the species' Y chromosome. Nevertheless, the new assembly is a significant improvement over the previous version and should provide a valuable resource for genomic studies of stickleback fishes.

Predictable and Divergent Change in the Multivariate P Matrix during Parallel Adaptation.

AbstractAdaptation to replicated environmental conditions can be remarkably predictable, suggesting that parallel evolution may be a common feature of adaptive radiation. An open question, however, is how phenotypic variation itself evolves during repeated adaptation. Here, we use a dataset of morphological measurements from 35 populations of threespine stickleback, consisting of 16 parapatric lake-stream pairs and three marine populations, to understand how phenotypic variation has evolved during transitions from marine to freshwater environments and during subsequent diversification across the lake-stream boundary. We find statistical support for divergent phenotypic covariance (P) across populations, with most diversification of P occurring among freshwater populations. Despite a close correspondence between within-population phenotypic variation and among-population divergence, we find that variation in P is unrelated to total variation in population means across the set of populations. For lake-stream pairs, we find that theoretical predictions for microevolutionary change can explain more than 30% of divergence in P matrices across the habitat boundary. Together, our results indicate that divergence in variance structure occurs primarily in dimensions of trait space with low phenotypic integration, correlated with disparate lake and stream environments. Our findings illustrate how conserved and divergent features of multivariate variation can underlie adaptive radiation.

Inferring the evolution of reproductive isolation in a lineage of fossil threespine stickleback, Gasterosteus doryssus.

Darwin attributed the absence of species transitions in the fossil record to his hypothesis that speciation occurs within isolated habitat patches too geographically restricted to be captured by fossil sequences. Mayr's peripatric speciation model added that such speciation would be rapid, further explaining missing evidence of diversification. Indeed, Eldredge and Gould's original punctuated equilibrium model combined Darwin's conjecture, Mayr's model and 124 years of unsuccessfully sampling the fossil record for transitions. Observing such divergence, however, could illustrate the tempo and mode of evolution during early speciation. Here, we investigate peripatric divergence in a Miocene stickleback fish, Gasterosteus doryssus. This lineage appeared and, over approximately 8000 generations, evolved significant reduction of 12 of 16 traits related to armour, swimming and diet, relative to its ancestral population. This was greater morphological divergence than we observed between reproductively isolated, benthic-limnetic ecotypes of extant Gasterosteus aculeatus. Therefore, we infer that reproductive isolation was evolving. However, local extinction of G. doryssus lineages shows how young, isolated, speciating populations often disappear, supporting Darwin's explanation for missing evidence and revealing a mechanism behind morphological stasis. Extinction may also account for limited sustained divergence within the stickleback species complex and help reconcile speciation rate variation observed across time scales.

Parallel evolution despite low genetic diversity in three-spined sticklebacks.

When populations repeatedly adapt to similar environments they can evolve similar phenotypes based on shared genetic mechanisms (parallel evolution). The likelihood of parallel evolution is affected by demographic history, as it depends on the standing genetic variation of the source population. The three-spined stickleback (Gasterosteus aculeatus) repeatedly colonized and adapted to brackish and freshwater. Most parallel evolution studies in G. aculeatus were conducted at high latitudes, where freshwater populations maintain connectivity to the source marine populations. Here, we analysed southern and northern European marine and freshwater populations to test two hypotheses. First, that southern European freshwater populations (which currently lack connection to marine populations) lost genetic diversity due to bottlenecks and inbreeding compared to their northern counterparts. Second, that the degree of genetic parallelism is higher among northern than southern European freshwater populations, as the latter have been subjected to strong drift due to isolation. The results show that southern populations exhibit lower genetic diversity but a higher degree of genetic parallelism than northern populations. Hence, they confirm the hypothesis that southern populations have lost genetic diversity, but this loss probably happened after they had already adapted to freshwater conditions, explaining the high degree of genetic parallelism in the south.

Experimental evidence for adaptive divergence in response to a warmed habitat reveals roles for morphology, allometry and parasite resistance.

Ectotherms are expected to be particularly vulnerable to climate change-driven increases in temperature. Understanding how populations adapt to novel thermal environments will be key for informing mitigation plans. We took advantage of threespine stickleback (Gasterosteus aculeatus) populations inhabiting adjacent geothermal (warm) and ambient (cold) habitats to test for adaptive evolutionary divergence using a field reciprocal transplant experiment. We found evidence for adaptive morphological divergence, as growth (length change) in non-native habitats related to head, posterior and total body shape. Higher growth in fish transplanted to a non-native habitat was associated with morphological shape closer to native fish. The consequences of transplantation were asymmetric with cold sourced fish transplanted to the warm habitat suffering from lower survival rates and greater parasite prevalence than warm sourced fish transplanted to the cold habitat. We also found divergent shape allometries that related to growth. Our findings suggest that wild populations can adapt quickly to thermal conditions, but immediate transitions to warmer conditions may be particularly difficult.

Late Pleistocene stickleback environmental genomes reveal the chronology of freshwater adaptation.

Directly observing the chronology and tempo of adaptation in response to ecological change is rarely possible in natural ecosystems. Sedimentary ancient DNA (sedaDNA) has been shown to be a tractable source of genome-scale data of long-dead organisms1,2,3 and to thereby potentially provide an understanding of the evolutionary histories of past populations.4,5 To date, time series of ecosystem biodiversity have been reconstructed from sedaDNA, typically using DNA metabarcoding or shotgun sequence data generated from less than 1 g of sediment.6,7 Here, we maximize sequence coverage by extracting DNA from ∼50× more sediment per sample than the majority of previous studies1,2,3 to achieve genotype resolution. From a time series of Late Pleistocene sediments spanning from a marine to freshwater ecosystem, we compare adaptive genotypes reconstructed from the environmental genomes of three-spined stickleback at key time points of this transition. We find a staggered temporal dynamic in which freshwater alleles at known loci of large effect in marine-freshwater divergence of three-spined stickleback (e.g., EDA)8 were already established during the brackish phase of the formation of the isolation basin. However, marine alleles were still detected across the majority of marine-freshwater divergence-associated loci, even after the complete isolation of the lake from marine ingression. Our retrospective approach to studying adaptation from environmental genomes of three-spined sticklebacks at the end of the last glacial period complements contemporary experimental approaches9,10,11 and highlights the untapped potential for retrospective "evolve and resequence" natural experiments using sedaDNA.

Predator-induced transgenerational plasticity of parental care behaviour in male three-spined stickleback fish across two generations.

Parental care is a critical determinant of offspring fitness, and parents adjust their care in response to ecological challenges, including predation risk. The experiences of both mothers and fathers can influence phenotypes of future generations (transgenerational plasticity). If it is adaptive for parents to alter parental care in response to predation risk, then we expect F1 and F2 offspring who receive transgenerational cues of predation risk to shift their parental care behaviour if these ancestral cues reliably predict a similarly risky environment as their F0 parents. Here, we used three-spined sticklebacks (Gasterosteus aculeatus) to understand how paternal exposure to predation risk prior to mating alters reproductive traits and parental care behaviour in unexposed F1 sons and F2 grandsons. Sons of predator-exposed fathers took more attempts to mate than sons of control fathers. F1 sons and F2 grandsons with two (maternal and paternal) predator-exposed grandfathers shifted their paternal care (fanning) behaviour in strikingly similar ways: they fanned less initially, but fanned more near egg hatching. This shift in fanning behaviour matches shifts observed in response to direct exposure to predation risk, suggesting a highly conserved response to pre-fertilization predator exposure that persists from the F0 to the F1 and F2 generations.

Elemental content of a host-parasite relationship in the threespine stickleback.

Parasite infections are ubiquitous and their effects on hosts could play a role in ecosystem processes. Ecological stoichiometry provides a framework to study linkages between consumers and their resource, such as parasites and their host, and ecosystem process; however, the stoichiometric traits of host-parasite associations are rarely quantified. Specifically, it is unclear whether parasites' elemental ratios closely resemble those of their host or if infection is related to host stoichiometry, especially in vertebrate hosts. To answer such questions, we measured the elemental content (%C, %N, and %P) and molar ratios (C:N, C:P, and N:P) of parasitized and unparasitized Gasterosteus aculeatus (three-spined stickleback) and their cestode parasite, Schistocephalus solidus. Host and parasite elemental content were distinct from each other, and parasites were generally higher in %C and lower in %N and %P. Parasite infections were related to host C:N, with infected hosts being lower in C:N. Parasite elemental content was independent of their host, but parasite body mass and parasite density were important drivers of parasite stoichiometry. Overall, these potential effects of parasite infections on host stoichiometry along with parasites' distinct elemental compositions suggest parasites may further contribute to differences in how individual hosts store and recycle nutrients.

Opening a can of worms: a test of the co-infection facilitation hypothesis.

Parasitic infections are a global occurrence and impact the health of many species. Coinfections, where two or more species of parasite are present in a host, are a common phenomenon across species. Coinfecting parasites can interact directly or indirectly via their manipulation of (and susceptibility to) the immune system of their shared host. Helminths, such as the cestode Schistocephalus solidus, are well known to suppress immunity of their host (threespine stickleback, Gasterosteus aculeatus), potentially facilitating other parasite species. Yet, hosts can evolve a more robust immune response (as seen in some stickleback populations), potentially turning facilitation into inhibition. Using wild-caught stickleback from 20 populations with non-zero S. solidus prevalence, we tested an a priori hypothesis that S. solidus infection facilitates infection by other parasites. Consistent with this hypothesis, individuals with S. solidus infections have 18.6% higher richness of other parasites compared to S. solidus-uninfected individuals from the same lakes. This facilitation-like trend is stronger in lakes where S. solidus is particularly successful but is reversed in lakes with sparse and smaller cestodes (indicative of stronger host immunity). These results suggest that a geographic mosaic of host-parasite co-evolution might lead to a mosaic of between-parasite facilitation/inhibition effects.

Ultrastructural analysis of throat dermal tissue and chromatophore components in the threespine stickleback (Gasterosteus aculeatus).

The threespine stickleback (Gasterosteus aculeatus) is an important model for studying the evolution of nuptial coloration, but histological analyses of color are largely lacking. Previous analyses of one nuptial coloration trait, orange-red coloration along the body, have indicated carotenoids are the main pigment producing this color. In addition, recent gene expression studies found variation in the correlates of throat coloration between the sexes and between populations, raising the possibility of variation in the mechanisms underlying superficially similar coloration. We used transmission electron microscopy (TEM) to investigate the histological correlates of color in the throat dermal tissue of threespine stickleback from Western North America, within and between sexes, populations, and ecotypes. Ultrastructural analysis revealed carotenoid-containing erythrophores to be the main chromatophore component associated with orange-red coloration in both males and females across populations. In individuals where some darkening of the throat tissue was present, with no obvious orange-red coloration, erythrophores were not detected. Melanophore presence was more population-specific in expression, including being the only chromatophore component detected in a population of darker fish. We found no dermal chromatophore units within colorless throat tissue. This work confirms the importance of carotenoids and the erythrophore in producing orange-red coloration across sexes, as well as melanin within the melanophore in producing darkened coloration, but does not reveal broad histological differences among populations with similar coloration.

Estimating the magnitude and sensitivity of energy fluxes for stickleback hosts and Schistocephalus solidus parasites using the metabolic theory of ecology.

Parasites are ubiquitous, yet their effects on hosts are difficult to quantify and generalize across ecosystems. One promising metric of parasitic impact uses the metabolic theory of ecology (MTE) to calculate energy flux, an estimate of energy lost to parasites. We investigated the feasibility of using metabolic scaling rules to compare the energetic burden of parasitism among individuals. Specifically, we found substantial sensitivity of energy flux estimates to input parameters used in the MTE equation when using available data from a model host-parasite system (Gasterosteus aculeatus and Schistocephalus solidus). Using literature values, size data from parasitized wild fish, and a respirometry experiment, we estimate that a single S. solidus tapeworm may extract up to 32% of its stickleback host's baseline metabolic energy requirement, and that parasites in multiple infections may collectively extract up to 46%. The amount of energy siphoned from stickleback to tapeworms is large but did not instigate an increase in respiration rate in the current study. This emphasizes the importance of future work focusing on how parasites influence ecosystem energetics. The approach of using the MTE to calculate energy flux provides great promise as a quantitative foundation for such estimates and provides a more concrete metric of parasite impact on hosts than parasite abundance alone.

Shoaling behaviour in response to turbidity in three-spined sticklebacks.

Many fresh and coastal waters are becoming increasingly turbid because of human activities, which may disrupt the visually mediated behaviours of aquatic organisms. Shoaling fish typically depend on vision to maintain collective behaviour, which has a range of benefits including protection from predators, enhanced foraging efficiency and access to mates. Previous studies of the effects of turbidity on shoaling behaviour have focussed on changes to nearest neighbour distance and average group-level behaviours. Here, we investigated whether and how experimental shoals of three-spined sticklebacks (Gasterosteus aculeatus) in clear (<10 Nephelometric Turbidity Units [NTU]) and turbid (~35 NTU) conditions differed in five local-level behaviours of individuals (nearest and furthest neighbour distance, heading difference with nearest neighbour, bearing angle to nearest neighbour and swimming speed). These variables are important for the emergent group-level properties of shoaling behaviour. We found an indirect effect of turbidity on nearest neighbour distances driven by a reduction in swimming speed, and a direct effect of turbidity which increased variability in furthest neighbour distances. In contrast, the alignment and relative position of individuals was not significantly altered in turbid compared to clear conditions. Overall, our results suggest that the shoals were usually robust to adverse effects of turbidity on collective behaviour, but group cohesion was occasionally lost during periods of instability.

FIRST REPORT IN CANADA OF CERATONOVA GASTEROSTEA (CNIDARIA: MYXOZOA) IN THREESPINE STICKLEBACK GASTEROSTEUS ACULEATUS FROM THE FRASER RIVER, BRITISH COLUMBIA.

A survey of threespine stickleback (Gasterosteus aculeatus L.) and juvenile Chinook salmon (Oncorhynchus tshawytscha (Walbaum)) from the lower Fraser River, British Columbia, Canada and adjacent marine waters was conducted for the presence of myxozoan parasites of the genus Ceratonova Atkinson, Foott, and Bartholomew, 2014 (Bivalvulida, Ceratomyxidae). Quantitative polymerase chain reaction (PCR; small subunit ribosomal RNA gene) combined with conventional PCR and sequencing (ribosomal internal transcribed spacer region 1) detected Ceratonova gasterostea Atkinson, Foott, and Bartholomew, 2014 in 89 of 269 (33.1%) sticklebacks from the Fraser River and in 1 of 51 (2%) from Howe Sound. In contrast, Ceratonova shasta (Atkinson, Foott, and Bartholomew, 2014) was detected in 2 of 26 (7.7%) Chinook salmon from the Fraser River. Reciprocal infections were not detected. Light microscopic examination of the stickleback intestine revealed the presence of an intense infection with large numbers of pseudoplasmodia which interacted with enterocytes through pseudopodia-like projections. Myxospores with characteristic elongate valve cells were visible within mature pseudoplasmodia and free in the intestinal lumen. This is the first report of C. gasterostea in Canada.