Early detection and recovery of river herring spawning habitat use in response to a mainstem dam removal.

Historical loss of river and stream habitats due to impassable dams has contributed to the severe decline of many fish species. Anadromous fishes that migrate from the sea to freshwater streams to spawn have been especially impacted as dams restrict these fish from accessing ancestral spawning grounds. In 2018, Bloede Dam was removed from the Patapsco River near Baltimore, Maryland, restoring approximately 100 km of potential habitat for migratory fish. We assessed the response of anadromous river herring, alewife (Alosa pseudoharengus) and blueback herring (Alosa aestivalis), to this dam removal by monitoring environmental DNA (eDNA) and eggs from 2015 to 2021 at locations upstream and downstream of the dam site during their spawning migrations. We additionally assessed the presence of fish by collecting electrofishing samples and tracked the movements of individual adult fish within the river using passive integrated transponder (PIT) tags. No adult river herring, eDNA, or eggs were detected upstream of Bloede Dam in the four years prior to its removal despite the presence of a fish ladder. Our results suggest initial habitat use recovery by spawning river herring in the first year post-removal, although a relatively small proportion of the population in the river used the newly accessible habitat. In the three years post-removal, the likelihood of detecting river herring eDNA upstream of the former dam site increased to 5% for alewife and 13% for blueback herring. Two adult fish were also collected in electrofishing samples upstream of the dam site in 2021. We found no evidence of changes in egg abundance and no tagged fish were detected upstream of the dam site post-removal. While long term monitoring is needed to assess population changes, this study highlights the value of integrating methods for comprehensive understanding of habitat use following dam removal.

Cross-Hemispheric Genetic Diversity and Spatial Genetic Structure of Callinectes sapidus Reovirus 1 (CsRV1).

The movement of viruses in aquatic systems is rarely studied over large geographic scales. Oceanic currents, host migration, latitude-based variation in climate, and resulting changes in host life history are all potential drivers of virus connectivity, adaptation, and genetic structure. To expand our understanding of the genetic diversity of Callinectes sapidus reovirus 1 (CsRV1) across a broad spatial and host life history range of its blue crab host (Callinectes sapidus), we obtained 22 complete and 96 partial genomic sequences for CsRV1 strains from the US Atlantic coast, Gulf of Mexico, Caribbean Sea, and the Atlantic coast of South America. Phylogenetic analyses of CsRV1 genomes revealed that virus genotypes were divided into four major genogroups consistent with their host geographic origins. However, some CsRV1 sequences from the US mid-Atlantic shared high genetic similarity with the Gulf of Mexico genotypes, suggesting potential human-mediated movement of CsRV1 between the US mid-Atlantic and Gulf coasts. This study advances our understanding of how climate, coastal geography, host life history, and human activity drive patterns of genetic structure and diversity of viruses in marine animals and contributes to the capacity to infer broadscale host population connectivity in marine ecosystems from virus population genetic data.

Genome-wide analysis of natural and restored eastern oyster populations reveals local adaptation and positive impacts of planting frequency and broodstock number.

The release of captive-bred plants and animals has increased worldwide to augment declining species. However, insufficient attention has been given to understanding how neutral and adaptive genetic variation are partitioned within and among proximal natural populations, and the patterns and drivers of gene flow over small spatial scales, which can be important for restoration success. A seascape genomics approach was used to investigate population structure, local adaptation, and the extent to which environmental gradients influence genetic variation among natural and restored populations of Chesapeake Bay eastern oysters Crassostrea virginica. We also investigated the impact of hatchery practices on neutral genetic diversity of restored reefs and quantified the broader genetic impacts of large-scale hatchery-based bivalve restoration. Restored reefs showed similar levels of diversity as natural reefs, and striking relationships were found between planting frequency and broodstock numbers and genetic diversity metrics (effective population size and relatedness), suggesting that hatchery practices can have a major impact on diversity. Despite long-term restoration activities, haphazard historical translocations, and high dispersal potential of larvae that could homogenize allele frequencies among populations, moderate neutral population genetic structure was uncovered. Moreover, environmental factors, namely salinity, pH, and temperature, play a major role in the distribution of neutral and adaptive genetic variation. For marine invertebrates in heterogeneous seascapes, collecting broodstock from large populations experiencing similar environments to candidate sites may provide the most appropriate sources for restoration and ensure population resilience in the face of rapid environmental change. This is one of a few studies to demonstrate empirically that hatchery practices have a major impact on the retention of genetic diversity. Overall, these results contribute to the growing body of evidence for fine-scale genetic structure and local adaptation in broadcast-spawning marine species and provide novel information for the management of an important fisheries resource.

Genome-wide analysis of acute low salinity tolerance in the eastern oyster Crassostrea virginica and potential of genomic selection for trait improvement.

As the global demand for seafood increases, research into the genetic basis of traits that can increase aquaculture production is critical. The eastern oyster (Crassostrea virginica) is an important aquaculture species along the Atlantic and Gulf Coasts of the United States, but increases in heavy rainfall events expose oysters to acute low salinity conditions, which negatively impact production. Low salinity survival is known to be a moderately heritable trait, but the genetic architecture underlying this trait is still poorly understood. In this study, we used ddRAD sequencing to generate genome-wide single-nucleotide polymorphism (SNP) data for four F2 families to investigate the genomic regions associated with survival in extreme low salinity (<3). SNP data were also used to assess the feasibility of genomic selection (GS) for improving this trait. Quantitative trait locus (QTL) mapping and combined linkage disequilibrium analysis revealed significant QTL on eastern oyster chromosomes 1 and 7 underlying both survival and day to death in a 36-day experimental challenge. Significant QTL were located in genes related to DNA/RNA function and repair, ion binding and membrane transport, and general response to stress. GS was investigated using Bayesian linear regression models and prediction accuracies ranged from 0.48 to 0.57. Genomic prediction accuracies were largest using the BayesB prior and prediction accuracies did not substantially decrease when SNPs located within the QTL region on Chr1 were removed, suggesting that this trait is controlled by many genes of small effect. Our results suggest that GS will likely be a viable option for improvement of survival in extreme low salinity.

Chromosome-level genome assembly of the blue crab, Callinectes sapidus.

The blue crab, Callinectes sapidus (Rathbun, 1896) is an economically, culturally, and ecologically important species found across the temperate and tropical North and South American Atlantic coast. A reference genome will enable research for this high-value species. Initial assembly combined 200× coverage Illumina paired-end reads, a 60× 8 kb mate-paired library, and 50× PacBio data using the MaSuRCA assembler resulting in a 985 Mb assembly with a scaffold N50 of 153 kb. Dovetail Chicago and HiC sequencing with the 3d DNA assembler and Juicebox assembly tools were then used for chromosome scaffolding. The 50 largest scaffolds span 810 Mb are 1.5-37 Mb long and have a repeat content of 36%. The 190 Mb unplaced sequence is in 3921 sequences over 10 kb with a repeat content of 68%. The final assembly N50 is 18.9 Mb for scaffolds and 9317 bases for contigs. Of arthropod BUSCO, ∼88% (888/1013) were complete and single copies. Using 309 million RNAseq read pairs from 12 different tissues and developmental stages, 25,249 protein-coding genes were predicted. Between C. sapidus and Portunus trituberculatus genomes, 41 of 50 large scaffolds had high nucleotide identity and protein-coding synteny, but 9 scaffolds in both assemblies were not clear matches. The protein-coding genes included 9423 one-to-one putative orthologs, of which 7165 were syntenic between the two crab species. Overall, the two crab genome assemblies show strong similarities at the nucleotide, protein, and chromosome level and verify the blue crab genome as an excellent reference for this important seafood species.

Are bivalves susceptible to domestication selection? Using starvation tolerance to test for potential trait changes in eastern oyster larvae.

Conservation efforts are increasingly being challenged by a rapidly changing environment, and for some aquatic species the use of captive rearing or selective breeding is an attractive option. However, captivity itself can impose unintended artificial selection known as domestication selection (adaptation to culture conditions) and is relatively understudied for most marine species. To test for domestication selection in marine bivalves, we focused on a fitness-related trait (larval starvation resistance) that could be altered under artificial selection. Using larvae produced from a wild population of Crassostrea virginica and a selectively bred, disease-resistant line we measured growth and survival during starvation versus standard algal diet conditions. Larvae from both lineages showed a remarkable resilience to food limitation, possibly mediated by an ability to utilize dissolved organic matter for somatic maintenance. Water chemistry analysis showed dissolved organic carbon in filtered tank water to be at concentrations similar to natural river water. We observed that survival in larvae produced from the aquaculture line was significantly lower compared to larvae produced from wild broodstock (8 ± 3% and 21 ± 2%, respectively) near the end of a 10-day period with no food (phytoplankton). All larval cohorts had arrested growth and depressed respiration during the starvation period and took at least two days to recover once food was reintroduced before resuming growth. Respiration rate recovered rapidly and final shell length was similar between the two treatments Phenotypic differences between the wild and aquaculture lines suggest potential differences in the capacity to sustain extended food limitation, but this work requires replication with multiple selection lines and wild populations to make more general inferences about domestication selection. With this contribution we explore the potential for domestication selection in bivalves, discuss the physiological and fitness implications of reduced starvation tolerance, and aim to inspire further research on the topic.

Tracking genetic diversity in a large-scale oyster restoration program: effects of hatchery propagation and initial characterization of diversity on restored vs. wild reefs.

The release of hatchery-propagated fish and shellfish is occurring on a global scale, but the genetic impacts of these practices are often not fully understood and rarely monitored. Slow recovery of depleted eastern oyster populations in the Chesapeake Bay, USA has prompted a hatchery-based restoration program focused in the Choptank River, Maryland consisting of the mass release of hatchery-produced juveniles from local, wild broodstock. To evaluate potential genetic effects of this program, we (1) examined changes in genetic diversity (allelic richness, heterozygosity) and the effective number of breeders (Nb) over the hatchery production cycle with microsatellite-based parentage of natural, mass- and controlled-spawned cohorts, and (2) compared genetic diversity and effective population size (Ne) of a restored reef to wild source populations. Mass-spawned cohorts showed high variance in reproductive contribution, particularly among males, leading to a 45% average reduction in Nb from spawning adult numbers and higher relatedness-lower magnitude reductions in heterozygosity and significant reductions in allelic richness were also observed. While controlled-spawns (single-male fertilizations of pooled eggs) reduced male variance, overall reproductive variance (Vk) remained high. Finally, oysters sampled from a restored reef displayed comparable Ne, genetic diversity, and relatedness to samples from wild populations, with no significant genetic differentiation among them. Overall, the hatchery-based results and initial field-based population genetic analyses suggest that despite reductions in diversity from parents to offspring owing to high Vk, enhancement with rotated, wild broodstock appears to have maintained genetic diversity in a restored reef population compared to proximal wild populations.

Environmental DNA analysis of river herring in Chesapeake Bay: A powerful tool for monitoring threatened keystone species.

Environmental DNA (eDNA) sampling has emerged as a powerful tool to detect and quantify species abundance in aquatic environments. However, relatively few studies have compared the performance of eDNA-based abundance estimates to traditional catch or survey approaches in the field. Here, we have developed and field-tested a qPCR assay to detect eDNA from alewife and blueback herring (collectively known as 'river herring'), comparing eDNA-based presence and abundance data to traditional methods of quantification (ichthyoplankton sampling and adult observations). Overall, the qPCR assay showed very high target specificity in lab trials, and was successful in detecting river herring for 11/12 Chesapeake Bay tributaries in spring 2015 and 2016, with 106 out of 445 samples exhibiting positive eDNA hits. We found a strong correlation between eDNA abundance and ichthyoplankton count data (Spearman's Rho = 0.52), and Phi-tests (correlation of presence/absence data) showed higher correlation between eDNA and ichthyoplankton data (Phi = 0.45) than adult data (Phi = 0.35). Detection probability was significantly lower on western vs. eastern shore tributaries of Chesapeake Bay, and blueback herring and alewife were more likely detected on the western and eastern shores, respectively. Temporal patterns of eDNA abundance over the spring spawning season revealed that alewife were present in high abundances weeks ahead of blueback herring, which aligns with known differences in spawning behavior of the species. In summary, the eDNA abundance data corresponded well to other field methods and has great potential to assist future monitoring efforts of river herring abundance and habitat use.

Genetic load in marine animals: a review.

Marine invertebrates and fish are well known for their remarkable genetic diversity, which is commonly explained by large population size and the characteristic dispersive nature of their early, planktonic life history. Other potential sources of diversity in marine animals, such as a higher mutation rate, have been much less considered, though evidence for a high genetic load in marine bivalves has been accumulating for nearly half a century. In this review, I examine evidence for a higher genetic load in marine animals from studies of molecular marker segregation and linkage over the last 40 years, and survey recent work examining mutational load with molecular evolution approaches. Overall, marine animals appear to have higher genetic load than terrestrial animals (higher dn/ds ratios, inbreeding load, and segregation dis`tortion), though results are mixed for marine fish and data are lacking for many marine animal groups. Bivalves (oysters) have the highest loads observed among marine animals, comparable only to long-lived plants; however, more data is needed from other bivalves and more marine invertebrate taxa generally. For oysters, a higher load may be related to a chronically lower effective population size that, in concert with a higher mutational rate, elevate the number of deleterious mutations observed. I suggest that future studies use high-throughput sequencing approaches to examine (1) polymorphism in genome-scale datasets across a wider range of marine animals at the population level and (2) intergenerational mutational changes between parents and offspring in crosses of aquaculture species to quantify mutation rates.

Density drives polyandry and relatedness influences paternal success in the Pacific gooseneck barnacle, Pollicipes elegans.

BACKGROUND: Polyandry is a common mating strategy in animals, increasing female fitness through direct (material) and indirect (genetic) benefits. Most theories about the benefits of polyandry come from studies of terrestrial animals, which have relatively complex mating systems and behaviors; less is known about the potential benefits of polyandry in sessile marine animals, for which potential mates may be scarce and females have less control over pre-copulatory mate choice. Here, we used microsatellite markers to examine multiple paternity in natural aggregations of the Pacific gooseneck barnacle Pollicipes elegans, testing the effect of density on paternity and mate relatedness on male reproductive success. RESULTS: We found that multiple paternity was very common (79% of broods), with up to five fathers contributing to a brood, though power was relatively low to detect more than four fathers. Density had a significant and positive linear effect on the number of fathers siring a brood, though this relationship leveled off at high numbers of fathers, which may reflect a lack of power and/or an upper limit to polyandry in this species. Significant skew in male reproductive contribution in multiply-sired broods was observed and we found a positive and significant relationship between the proportion of offspring sired and the genetic similarity between mates, suggesting that genetic compatibility may influence reproductive success in this species. CONCLUSIONS: To our knowledge, this is the first study to show high levels of multiple paternity in a barnacle, and overall, patterns of paternity in P. elegans appear to be driven primarily by mate availability. Evidence of paternity bias for males with higher relatedness suggests some form of post-copulatory sexual selection is taking place, but more work is needed to determine whether it operates during or post-fertilization. Overall, our results suggest that while polyandry in P. elegans is driven by mate availability, it may also provide a mechanism for females to ensure fertilization by compatible gametes and increase reproductive success in this sessile species.

Characterization of microsatellite loci and repeat density in the gooseneck barnacle, Pollicipes elegans, using next generation sequencing.

Pollicipes elegans is a commercially important and biogeographically significant rocky-shore gooseneck barnacle found along the eastern Pacific coasts of Peru, El Salvador, and Mexico. Little is known about its reproductive biology, and no genetic resources exist despite its growing importance as a fisheries species in the region. Next generation sequencing methods can provide rapid and cost-effective development of molecular markers such as microsatellites, which can be applied to studies of paternity, parentage, and population structure in this understudied species. Here, we used Roche 454 pyrosequencing to develop microsatellite markers in P. elegans and made genomic comparisons of repeat density and repeat class frequency with other arthropods and more distantly related taxa. We identified 13 809 repeats of 1-6 bp, or a density of 9744 bp of repeat per megabase queried, which was intermediate in the range of taxonomic groups compared. Comparison of repeat class frequency distributions revealed that P. elegans was most similar to Drosophila melanogaster rather than the more closely related crustacean Daphnia pulex. We successfully isolated 15 polymorphic markers with an average of 9.4 alleles per locus and average observed and expected heterozygosities of 0.501 and 0.597, respectively. Four loci were found to be out of Hardy-Weinberg equilibrium, likely due to the presence of null alleles. A preliminary population genetic analysis revealed low but significant differentiation between a Peruvian (n = 47) and Mexican (n = 48) population (F(ST) = 0.039) and markedly reduced genetic diversity in Peru. These markers should facilitate future studies of paternity, parentage, and population structure in this species.

Environmental stress increases selection against and dominance of deleterious mutations in inbred families of the Pacific oyster Crassostrea gigas.

The deleterious effects of inbreeding are well documented and of major concern in conservation biology. Stressful environments have generally been shown to increase inbreeding depression; however, little is known about the underlying genetic mechanisms of the inbreeding-by-stress interaction and to what extent the fitness of individual deleterious mutations is altered under stress. Using microsatellite marker segregation data and quantitative trait locus (QTL) mapping methods, I performed a genome scan for deleterious mutations affecting viability (viability or vQTL) in two inbred families of the Pacific oyster Crassostrea gigas, reared in a stressful, nutrient-poor diet and a favourable, nutrient-rich diet, which had significant effects on growth and survival. Twice as many vQTL were detected in the stressful diet compared with the favourable diet, resulting primarily from substantially greater mortality of homozygous genotypes. At vQTL, estimates of selection (s) and dominance (h) were greater in the stressful environment (= 0.86 vs. 0.54 and = 0.35 vs. 0.18, in stressful and nonstressful diets, respectively). There was no evidence of interaction between vQTL. Individual vQTL differed across diets in selection only, or in both selection and dominance, and some vQTL were not affected by diet. These results suggest that stress-associated increases in selection against individual deleterious alleles underlie greater inbreeding depression with stress. Furthermore, the finding that inbreeding-by-environment interaction appears, to some extent, to be locus specific, helps to explain previous observations of lineage-specific expression of inbreeding depression and environment-specific purging, which have important implications for conservation and evolutionary biology.

Quantitative trait locus analysis of stage-specific inbreeding depression in the Pacific oyster Crassostrea gigas.

Inbreeding depression and genetic load have been widely observed, but their genetic basis and effects on fitness during the life cycle remain poorly understood, especially for marine animals with high fecundity and high, early mortality (type-III survivorship). A high load of recessive mutations was previously inferred for the Pacific oyster Crassostrea gigas, from massive distortions of zygotic, marker segregation ratios in F(2) families. However, the number, genomic location, and stage-specific onset of mutations affecting viability have not been thoroughly investigated. Here, we again report massive distortions of microsatellite-marker segregation ratios in two F(2) hybrid families, but we now locate the causative deleterious mutations, using a quantitative trait locus (QTL) interval-mapping model, and we characterize their mode of gene action. We find 14-15 viability QTL (vQTL) in the two families. Genotypic frequencies at vQTL generally suggest selection against recessive or partially recessive alleles, supporting the dominance theory of inbreeding depression. No epistasis was detected among vQTL, so unlinked vQTL presumably have independent effects on survival. For the first time, we track segregation ratios of vQTL-linked markers through the life cycle, to determine their stage-specific expression. Almost all vQTL are absent in the earliest life stages examined, confirming zygotic viability selection; vQTL are predominantly expressed before the juvenile stage (90%), mostly at metamorphosis (50%). We estimate that, altogether, selection on vQTL caused 96% mortality in these families, accounting for nearly all of the actual mortality. Thus, genetic load causes substantial mortality in inbred Pacific oysters, particularly during metamorphosis, a critical developmental transition warranting further investigation.