Establishing an astaxanthin-rich live feed strain of Pseudodiaptomus annandalei.

This study aimed to establish an astaxanthin-rich strain of the calanoid copepod Pseudodiaptomus annandalei, through selective breeding based on RGB (red, green and blue) value, a parameter indicating color intensity. We evaluated the RGB value frequency distributions of the copepod populations, and selected individuals with the highest 10% and the lowest 10% RGB value over six generations. The RGB value, nauplii production, clutch interval and clutch number were assessed, and the genetic gain was calculated across generations (G0-G5). Two strains of copepods were selected and defined as dark body copepod strain (DBS) and light body copepod strain (LBS) at the end of experiment. Results revealed significantly lower RGB values (male: 121.5 ± 14.1; female: 108.8 ± 15) in the G5 DBS population compared to the G0 (male: 163.9 ± 13.1; female: 162.2 ± 14.6), with higher genetic gains of RGB values during G0 to G2. While DBS females exhibited longer clutch intervals in the G3 and G4, there was no significant difference in nauplii production between the two strains across all generations. Significantly higher astaxanthin content was found in the DBS copepods (0.04 µg/ ind.) compared to the LBS copepods (0.01 µg/ ind.) and the non-selective copepods (0.02 µg/ ind.) 20 months post selective breeding, validating the stability of the desired trait in the DBS strain. This study successfully established an astaxanthin-rich strain of P. annandalei, which provides implications for enhancing marine and brackish larviculture production.

Highly structured populations of copepods at risk to deep-sea mining: Integration of genomic data with demogenetic and biophysical modelling.

Copepoda is the most abundant taxon in deep-sea hydrothermal vents, where hard substrate is available. Despite the increasing interest in seafloor massive sulphides exploitation, there have been no population genomic studies conducted on vent meiofauna, which are known to contribute over 50% to metazoan biodiversity at vents. To bridge this knowledge gap, restriction-site-associated DNA sequencing, specifically 2b-RADseq, was used to retrieve thousands of genome-wide single-nucleotide polymorphisms (SNPs) from abundant populations of the vent-obligate copepod Stygiopontius lauensis from the Lau Basin. SNPs were used to investigate population structure, demographic histories and genotype-environment associations at a basin scale. Genetic analyses also helped to evaluate the suitability of tailored larval dispersal models and the parameterization of life-history traits that better fit the population patterns observed in the genomic dataset for the target organism. Highly structured populations were observed on both spatial and temporal scales, with divergence of populations between the north, mid, and south of the basin estimated to have occurred after the creation of the major transform fault dividing the Australian and the Niuafo'ou tectonic plate (350 kya), with relatively recent secondary contact events (<20 kya). Larval dispersal models were able to predict the high levels of structure and the highly asymmetric northward low-level gene flow observed in the genomic data. These results differ from most studies conducted on megafauna in the region, elucidating the need to incorporate smaller size when considering site prospecting for deep-sea exploitation of seafloor massive sulphides, and the creation of area-based management tools to protect areas at risk of local extinction, should mining occur.

Effects of ocean warming on the fatty acid and epigenetic profile of Acartia tonsa: A multigenerational approach.

The effects of climate change are becoming more prevalent, and it is important to know how copepods, the most abundant class in zooplankton, will react to changing temperatures as they are the main food source for secondary consumers. They act as key transferers of nutrients from primary producers to organisms higher up the food chain. Little is known about the effects of temperature changes on copepods on the long term, i.e., over several generations. Especially the epigenetic domain seems to be understudied and the question remains whether the nutritional value of copepods will permanently change with rising water temperatures. In this research, the effects of temperature on the fatty acid and epigenetic profiles of the abundant planktonic copepod Acartia tonsa were investigated, since we expect to see a link between these two. Indeed, changing methylation patterns helped copepods to deal with higher temperatures, which is in line with the relative abundance of the most important fatty acids, e.g., DHA. However, this pattern was only observed when temperature increased slowly. A sudden increase in temperature showed the opposite effect; Acartia tonsa did not show deviant methylation patterns and the relative abundance of DHA and other important fatty acids dropped significantly after several generations. These results suggest that local fluctuations in temperature have a greater effect on Acartia tonsa than an elevation of the global mean.

Comprehensive molecular characterisation of the complete mitogenome of Ergasilus tumidus and phylogenetic relationships of Copepoda inferred from mitogenomes.

Although parasitic copepods of the genus Ergasilus von Nordmann, 1832 are globally distributed parasites of fish, their phylogenetic relationships with other Copepoda are not clear, and the characteristics of their mitochondrial genomes (mitogenomes) are not thoroughly understood. The objective of this study was to address these knowledge gaps by sequencing the complete mitogenome of Ergasilus tumidus Markevich, 1940. The complete mitogenome (GenBank Acc. No. OQ596537) was 14,431 bp long and it comprised 13 protein-coding genes (PCGs), 22 tRNAs, two tRNAs, and two control regions (CRs). Phylogenetic analyses, conducted using concatenated nucleotide and amino acid sequences of 13 protein-coding genes, produced two partially incongruent topologies. While the order Calanoida was consistently resolved as the sister lineage to the other three orders, topological instability was observed in the relationships of the orders Cyclopoida, Siphonostomatoida and Harpacticoida. Siphonostomatoida clustered with Cyclopoida in the nucleotide-based phylogeny, but with Harpacticoida in the amino acid-based phylogeny. The latter topology conforms to the widely accepted relationships, but we speculate that the former topology is more likely to be the correct one. Our study provides a complete mitogenome sequence of E. tumidus, which helps us better understand the molecular evolution of the genus Ergasilus. Additionally, we suggest a different perspective on the controversial phylogenetic relationships among Siphonostomatoida, Cyclopoida and Harpacticoida, diverging from previously accepted views.

New species of Dermoergasilus Ho & Do, 1982 (Copepoda: Cyclopoida: Ergasilidae) parasitizing endemic cichlid Paretroplus polyactis (Bleeker) in Madagascar.

Dermoergasilus madagascarensis n. sp. is described from the gills of Paretroplus polyactis, an endemic cichlid fish in Madagascar, using a combined morphological (light microscopy and SEM) and molecular approach (partial 18S rDNA, 28S rDNA, and COI sequences). The new species is characterized mainly by possessing: (i) roughly pentagonal cephalosome; (ii) antennal endopodal segments covered with slightly inflated membrane; (iii) maxillule bearing 2 equally long outer setae and a minute inner seta; (iv) interpodal sternites of swimming legs ornamented with 3­4 rows of spinules; (v) genital segment and first abdominal somite both barrel-shaped; and (vi) a caudal ramus projecting into a digitiform process with inconspicuous terminal seta and bearing 3 terminal setae. The obtained DNA sequences of Malagasy species represent the first molecular data for species of Dermoergasilus. The 28S rDNA phylogeny showed the affiliation of D. madagascarensis n. sp. to Ergasilidae and its sister relationship with cosmopolitan Ergasilus sieboldi von Nordmann, 1832. The first checklist for all species of Dermoergasilus is provided.

Sex-Biased Transcription Expression of Vitellogenins Reveals Fusion Gene and MicroRNA Regulation in the Sea Louse Caligus rogercresseyi.

The caligid ectoparasite, Caligus rogercresseyi, is one of the main concerns in the Chilean salmon industry. The molecular mechanisms displayed by the parasite during the reproductive process represent an opportunity for developing novel control strategies. Vitellogenin is a multifunctional protein recognized as a critical player in several crustaceans' biological processes, including reproduction, embryonic development, and immune response. This study aimed to characterize the C. rogercresseyi vitellogenins, including discovering novel transcripts and regulatory mechanisms associated with microRNAs. Herein, vitellogenin genes were identified by homology analysis using the reference sea louse genome, transcriptome database, and arthropods vitellogenin-protein database. The validation of expression transcripts was conducted by RNA nanopore sequencing technology. Moreover, fusion gene profiling, miRNA target analysis, and functional validation were performed using luciferase assay. Six putative vitellogenin genes were identified in the C. rogercresseyi genome with high homology with other copepods vitellogenins. Furthermore, miR-996 showed a putative role in regulating the Cr_Vitellogenin1 gene, which is highly expressed in females. Moreover, vitellogenin-fusion genes were identified in adult stages and highly regulated in males, demonstrating sex-related expression patterns. In females, the identified fusion genes merged with several non-vitellogenin genes involved in biological processes of ribosome assembly, BMP signaling pathway, and biosynthetic processes. This study reports the genome array of vitellogenins in C. rogercresseyi for the first time, revealing the putative role of fusion genes and miRNA regulation in sea lice biology.

Diel metabolic patterns revealed by in situ transcriptome and proteome in a vertically migratory copepod.

Zooplankton undergo a diel vertical migration (DVM) which exposes them to gradients of light, temperature, oxygen, and food availability on a predictable daily schedule. Disentangling the co-varying and potentially synergistic interactions on metabolic rates has proven difficult, despite the importance of this migration for the delivery of metabolic waste products to the distinctly different daytime (deep) and nighttime (surface) habitats. This study examines the transcriptomic and proteomic profiles of the circumglobal migratory copepod, Pleuromamma xiphias, over the diel cycle. The transcriptome showed that 96% of differentially expressed genes were upregulated during the middle of the day - the period often considered to be of lowest zooplankton activity. The changes in protein abundance were more spread out over time, peaking (42% of comparisons) in the early evening. Between 9:00 and 15:00, both the transcriptome and proteome datasets showed increased expression related to chitin synthesis and degradation. Additionally, at 09:00 and 22:00, there were increases in myosin and vitellogenin proteins, potentially linked to the stress of migration and/or reproductive investment. Based on protein abundances detected, there is an inferred switch in broad metabolic processes, shifting from electron transport system in the day to glycolysis and glycogen mobilization in the afternoon/evening. These observations provide evidence of the diel impact of DVM on transcriptomic and proteomic pathways that likely influence metabolic processes and subsequent excretion products, and clarify how this behaviour results in the direct rapid transport of waste metabolites from the surface to the deep ocean.

Molecular quantification of parasitic sea louse larvae depends on species and life stage.

Sea lice cause substantial economic and environmental harm to Norway's aquaculture industry and wild salmonid populations. Rapid, accurate quantification of lice larval densities in coastal waters remains the greatest bottleneck for providing empirical data on infestation risk within wild salmon habitats and aquaculture production regions. We evaluated the capability of droplet digital PCR (ddPCR) as an absolute quantification method for the planktonic stages of two parasitic louse species, Lepeophtheirus salmonis (Krøyer) and Caligus elongatus (von Nordman). Results demonstrated linear relationships between the DNA quantity measured and the number of spiked larvae for both species and life stages. However, L. salmonis contained a significantly greater number of DNA copies than C. elongatus individuals and for C. elongatus, nauplii displayed a significantly higher number of DNA copies than copepodids. Our results suggest that ddPCR can effectively enumerate louse larvae, but interpreting ddPCR results differ between the two louse species. Obtaining larval abundance estimates from marine plankton samples will depend on the nauplii to copepodid ratio for C. elongatus, but not for L. salmonis.

Disproportionate role of nuclear-encoded proteins in organismal and mitochondrial thermal performance in a copepod.

Determining the mechanisms by which organisms evolve thermal tolerance is crucial to predicting how populations may respond to changes in local temperature regimes. Although evidence of relationships between mitochondrial background and thermal adaptation have been found, the presence of both nuclear-encoded and mitochondrial DNA (mtDNA)-encoded proteins warrants experiments aimed at parsing out the relative role of each genome in thermal adaptation. We investigated the relative role of mtDNA-encoded products in thermal tolerance between two divergent populations of Tigriopus californicus using first-generation (F1) hybrids that vary in maternally inherited mtDNA but are heterozygous for population-specific alleles across nuclear loci. We tested two measures of thermal tolerance, (1) survivorship to acute thermal stress and (2) thermal stability of mitochondrial performance in Complex I-fueled ATP synthesis, both across a range of increasing temperatures. We found that the southern population (San Diego, CA, USA) outperformed the northern population (Strawberry Hill, OR, USA) in survivorship, and that both reciprocal F1 hybrid crosses had intermediate survival. Mitochondria from the San Diego population displayed greater stability in ATP synthesis with increasing temperatures compared with those from Strawberry Hill. Interestingly, hybrids from both cross directions had synthesis profiles that were very similar to that of Strawberry Hill. Taken together, these results suggest that the relative role of the mtDNA in these phenotypes is negligible compared with that of elements encoded by nuclear DNA in this system.

Highly structured populations of deep-sea copepods associated with hydrothermal vents across the Southwest Pacific, despite contrasting life history traits.

Hydrothermal vents are extreme environments, where abundant communities of copepods with contrasting life history traits co-exist along hydrothermal gradients. Here, we discuss how these traits may contribute to the observed differences in molecular diversity and population genetic structure. Samples were collected from vent locations across the globe including active ridges and back-arc basins and compared to existing deep-sea hydrothermal vent and shallow water data, covering a total of 22 vents and 3 non-vent sites. A total of 806 sequences of mtDNA from the Cox1 gene were used to reconstruct the phylogeny, haplotypic relationship and demography within vent endemic copepods (Dirivultidae, Stygiopontius spp.) and non-vent-endemic copepods (Ameiridae, Miraciidae and Laophontidae). A species complex within Stygiopontius lauensis was studied across five pacific back-arc basins at eight hydrothermal vent fields, with cryptic species being restricted to the basins they were sampled from. Copepod populations from the Lau, North Fiji and Woodlark basins are undergoing demographic expansion, possibly linked to an increase in hydrothermal activity in the last 10 kya. Highly structured populations of Amphiascus aff. varians 2 were also observed from the Lau to the Woodlark basins with populations also undergoing expansion. Less abundant harpacticoids exhibit little to no population structure and stable populations. This study suggests that similarities in genetic structure and demography may arise in vent-associated copepods despite having different life history traits. As structured meta-populations may be at risk of local extinction should major anthropogenic impacts, such as deep-sea mining, occur, we highlight the importance of incorporating a trait-based approach to investigate patterns of genetic connectivity and demography, particularly regarding area-based management tools and environmental management plans.

Unmasking microsatellite deceptiveness and debunking hybridization with SNPs in four marine copepod species of Calanus.

Interspecific hybridization events are on the rise in natural systems due to climate change disrupting species barriers. Across taxa, microsatellites have long been the molecular markers of choice to identify admixed individuals. However, with the advent of high-throughput sequencing easing the generation of genome-wide datasets, incorrect reports of hybridization resulting from microsatellite technical artefacts have been uncovered in a growing number of taxa. In the marine zooplankton genus Calanus (Copepoda), whose species are used as climate change indicators, microsatellite markers have suggested hybridization between C. finmarchicus and C. glacialis, while other nuclear markers (InDels) never detected any admixed individuals, leaving the scientific community divided. Here, for the first time, we investigated the potential for hybridization among C. finmarchicus, C. glacialis, C. helgolandicus and C. hyperboreus using two large and independent SNP datasets. These were derived firstly from a protocol of target-capture applied to 179 individuals collected from 17 sites across the North Atlantic and Arctic Oceans, including sympatric areas, and second from published RNA sequences. All SNP-based analyses were congruent in showing that Calanus species are distinct and do not appear to hybridize. We then thoroughly re-assessed the microsatellites showing hybrids, with the support of published transcriptomes, and identified technical issues plaguing eight out of 10 microsatellites, including size homoplasy, paralogy, potential for null alleles and even two primer pairs targeting the same locus. Our study illustrates how deceptive microsatellites can be when applied to the investigation of hybridization.

Analysis of the transcriptional pathways associated with the induction of quiescent embryonic arrest in the calanoid copepod Acartia tonsa.

The copepod species Acartia tonsa (Dana)(Crustacea) have the unique ability to induce quiescent embryonic dormancy if adverse environmental conditions occur; a characteristic shared by 41 other species belonging to the superfamily Centropagoida in the Calanoida order. However, the transcriptional changes characterizing this process are not known. Here, we compare the transcriptome of embryos in arrested quiescence with the normal development to identify pathways and differentially regulated transcripts involved in quiescent embryogenesis. Quiescence was induced by incubating eggs at 4 °C with anoxia for 26 h(hr), while eggs undergoing normal immediate development were incubated at 16.9 °C in normoxia for 7 h (where gastrulation occurs) or 14 h (where organogenesis occurs) before collecting for RNA extraction and analysis by RNA-sequencing. Results indicate that the expression profile of the quiescent embryo is not as different from the normal embryonic gastrulation as initially expected: None of the mapped transcripts is uniquely expressed in quiescence. Moreover, in quiescence a large proportion of the annotated transcripts display expression values halfway in-between the normal, immediate developmental stages of gastrulation and organogenesis. In depth comparison between the organogenesis stage and quiescent samples, reveal a high degree of divergence, confirming that a developmental arrest has been induced through quiescence. Specifically: Stress response transcripts are prominent in the quiescent phase with a transcript like the mammalian autophagy gene Sequestosome-1/p62 (SQSTM) being upregulated. The present analysis provides a better understanding of the molecular mechanisms characterizing the quiescent embryonic state of A. tonsa.

Silencing of the Vasa gene by RNA Interference Affects Embryonic Development and Reproductive Output in the Sea Louse Caligus rogercresseyi.

The sea louse Caligus rogercresseyi is a major ectoparasitic copepod that causes significant economic losses in the salmon farming industry. Despite recent advancements, the mechanisms underlying germline and embryo development in this species remain poorly understood. The Vasa gene encodes a highly conserved DEAD box helicase that is required for germ cell formation and function in many species. In this study, the Vasa gene was characterized in C. rogercresseyi, and its expression and function were analyzed. Phylogenetic analysis showed that the Cr-Vasa gene product formed clusters in clades with Vasa proteins from closely related species of crustaceans. Cr-Vasa gene expression patterns were assessed by qPCR, and the results showed a significantly higher relative expression level in adult females compared to copepodid, chalimus, and adult male stages. Tissue-specific localization of Cr-Vasa mRNA in C. rogercresseyi was determined using chromogenic in situ hybridization, and strong positive signal was observed in male testes, but also in the intestine and cuticle, while in females, it was observed in the ovaries, oocytes, cuticle, intestine, and egg strings. RNAi-mediated gene silencing of Cr-Vasa impacted embryonic development and reproductive output in adult female lice. Females from the dsVasa-treated group displayed unusual phenotypes, including shorter egg strings with numerous extra-embryonic inclusions, irregularly shaped abnormal embryos, and aborted egg strings. This study provides insights into the role of the Vasa gene in C. rogercresseyi embryonic development and reproductive output, which may have implications for the control of this parasitic copepod in the salmon farming industry.

Transcriptomic analysis reveals responses to a polluted sediment in the Mediterranean copepod Acartia clausi.

Marine sediments are regarded as sinks for several classes of contaminants. Characterization and effects of sediments on marine biota now require a multidisciplinary approach, which includes chemical and ecotoxicological analyses and molecular biomarkers. Here, a gene expression study was performed to measure the response of adult females of the Mediterranean copepod Acartia clausi to elutriates of polluted sediments (containing high concentrations of polycyclic aromatic hydrocarbons, PAHs, and heavy metals) from an industrial area in the Southern Tyrrhenian Sea (Bagnoli-Coroglio). Functional annotation of the A. clausi transcriptome generated as reference here, showed a good quality of the assembly and great homology with other copepod and crustacean sequences in public databases. This is one of the few available transcriptomic resources for this widespread copepod species of great ecological relevance in temperate coastal areas. Differential expression analysis between females exposed to the elutriate and those in control seawater identified 1000 differentially expressed genes, of which 743 up- and 257 down-regulated. Within the up-regulated genes, the most represented functions were related to proteolysis (lysosomal protease, peptidase, cathepsin), response to stress and detoxification (heat-shock protein, superoxide dismutase, glutathione-S-transferase, cytochrome P450), and cytoskeleton structure (α- and ß-tubulin). Down-regulated genes were mostly involved with ribosome structure (ribosomal proteins) and DNA binding (histone proteins, transcription factors). Overall, these results suggest that processes such as transcription, translation, protein degradation, metabolism of biomolecules, reproduction, and xenobiotic detoxification were altered in the copepod in response to polluted elutriates. In conclusion, our results contribute to gaining information on the transcriptomic responses of copepods to polluted sediments. They will also prompt the selection of genes of interest to be used as biomarkers of exposure to PAHs and heavy metals in molecular toxicology studies on copepods, and in general, in comparative functional genomic studies on marine zooplankton.

Evolutionary Rates, Divergence Rates, and Performance of Individual Mitochondrial Genes Based on Phylogenetic Analysis of Copepoda.

Copepoda is a large and diverse group of crustaceans, which is widely distributed worldwide. It encompasses roughly 9 orders, whose phylogeny remains unresolved. We sequenced the complete mitochondrial genome (mitogenome) of Sinergasilus major (Markevich, 1940) and used it to explore the phylogeny and mitogenomic evolution of Copepoda. The mitogenome of S. major (14,588 bp) encodes the standard 37 genes as well as a putative control region, and molecular features are highly conserved compared to other Copepoda mitogenomes. Comparative analyses indicated that the nad2 gene has relatively high nucleotide diversity and evolutionary rate, as well as the largest amount of phylogenetic information. These results indicate that nad2 may be a better marker to investigate phylogenetic relationships among closely related species in Copepoda than the commonly used cox1 gene. The sister-group relationship of Siphonostomatoida and Cyclopoida was recovered with strong support in our study. The only topological ambiguity was found within Cyclopoida, which might be caused by the rapid evolution and sparse taxon sampling of this lineage. More taxa and genes should be used to reconstruct the Copepoda phylogeny in the future.

Genetic incompatibilities in reciprocal hybrids between populations of Tigriopus californicus with low to moderate mitochondrial sequence divergence.

All mitochondrial-encoded proteins and RNAs function through interactions with nuclear-encoded proteins, which are critical for mitochondrial performance and eukaryotic fitness. Coevolution maintains inter-genomic (i.e., mitonuclear) compatibility within a taxon, but hybridization can disrupt coevolved interactions, resulting in hybrid breakdown. Thus, mitonuclear incompatibilities may be important mechanisms underlying reproductive isolation and, potentially, speciation. Here we utilize Pool-seq to assess the effects of mitochondrial genotype on nuclear allele frequencies in fast- and slow-developing reciprocal inter-population F2 hybrids between relatively low-divergence populations of the intertidal copepod Tigriopus californicus. We show that mitonuclear interactions lead to elevated frequencies of coevolved (i.e., maternal) nuclear alleles on two chromosomes in crosses between populations with 1.5% or 9.6% fixed differences in mitochondrial DNA nucleotide sequence. However, we also find evidence of excess mismatched (i.e., noncoevolved) alleles on three or four chromosomes per cross, respectively, and of allele frequency differences consistent with effects involving only nuclear loci (i.e., unaffected by mitochondrial genotype). Thus, our results for low-divergence crosses suggest an underlying role for mitonuclear interactions in variation in hybrid developmental rate, but despite substantial effects of mitonuclear coevolution on individual chromosomes, no clear bias favoring coevolved interactions overall.

Without a pinch of salt: effect of low salinity on eggs and nauplii of the salmon louse (Lepeophtheirus salmonis).

The salmon louse is an economically important parasite on Atlantic salmon and poses a major threat to aquaculture. Several treatment methods have lost their effect due to resistance development in the lice. A rather new method for combatting sea lice is freshwater treatment where the various life stages of lice are differently affected by this treatment. In this study, we analyzed the effect of freshwater on the egg strings. A 3-h treatment with freshwater had a detrimental effect on the egg strings. First, the water penetrated the string, widening it, then entering the eggs and enlarging them. Finally, the ordered structure of the egg strings collapsed, and no alive animals hatched. Shorter treatments had a lower effectivity, and treatments with brackish water also showed milder effects. The egg strings were found to have a protective effect against low salinities, as hatched nauplii died rapidly under conditions that embryos survived. We also found that embryos react to low salinity on a molecular level by changing gene expression of several genes, when incubated in brackish water. Additionally, the hatching of embryos treated with brackish water was delayed in comparison to seawater controls.

Genomic Architecture of Hybrid Male Sterility in a Species Without Sex Chromosomes (Tigriopus californicus, Copepoda: Harpacticoida).

Sterility among hybrids is one of the most prevalent forms of reproductive isolation delineating species boundaries and is expressed disproportionately in heterogametic XY males. While hybrid male sterility (HMS) due to the "large X effect" is a well-recognized mechanism of reproductive isolation, it is less clear how HMS manifests in species that lack heteromorphic sex chromosomes. We evaluated differences in allele frequencies at approximately 460,000 SNPs between fertile and sterile F2 interpopulation male hybrids to characterize the genomic architecture of HMS in a species without sex chromosomes (Tigriopus californicus). We tested associations between HMS and mitochondrial-nuclear and/or nuclear-nuclear signatures of incompatibility. Genomic regions associated with HMS were concentrated on a single chromosome with the same primary 2-Mbp regions identified in one pair of reciprocal crosses. Gene Ontology analysis revealed that annotations associated with spermatogenesis were the most overrepresented within the implicated region, with nine protein-coding genes connected with this process found in the quantitative trait locus of chromosome 2. Our results indicate that a narrow genomic region was associated with the sterility of male hybrids in T. californicus and suggest that incompatibilities among select nuclear loci may replace the large X effect when sex chromosomes are absent.

Divergent RNA viruses infecting sea lice, major ectoparasites of fish.

Sea lice, the major ectoparasites of fish, have significant economic impacts on wild and farmed finfish, and have been implicated in the decline of wild salmon populations. As blood-feeding arthropods, sea lice may also be reservoirs for viruses infecting fish. However, except for two groups of negative-strand RNA viruses within the order Mononegavirales, nothing is known about viruses of sea lice. Here, we used transcriptomic data from three key species of sea lice (Lepeophtheirus salmonis, Caligus clemensi, and Caligus rogercresseyi) to identify 32 previously unknown RNA viruses. The viruses encompassed all the existing phyla of RNA viruses, with many placed in deeply branching lineages that likely represent new families and genera. Importantly, the presence of canonical virus-derived small interfering RNAs (viRNAs) indicates that most of these viruses infect sea lice, even though in some cases their closest classified relatives are only known to infect plants or fungi. We also identified both viRNAs and PIWI-interacting RNAs (piRNAs) from sequences of a bunya-like and two qin-like viruses in C. rogercresseyi. Our analyses showed that most of the viruses found in C. rogercresseyi occurred in multiple life stages, spanning from planktonic to parasitic stages. Phylogenetic analysis revealed that many of the viruses infecting sea lice were closely related to those that infect a wide array of eukaryotes with which arthropods associate, including fungi and parasitic tapeworms, implying that over evolutionary time there has been cross-phylum and cross-kingdom switching of viruses between arthropods and other eukaryotes. Overall, this study greatly expands our view of virus diversity in crustaceans, identifies viruses that infect and replicate in sea lice, and provides evidence that over evolutionary time, viruses have switched between arthropods and eukaryotic hosts in other phyla and kingdoms.

Developmental rate displays effects of inheritance but not of sex in interpopulation hybrids of Tigriopus californicus.

Coevolved genetic interactions within populations can be disrupted by hybridization resulting in loss of fitness in hybrid individuals (i.e., hybrid breakdown). However, the extent to which variation in fitness-related traits among hybrids is inherited across generations remains unclear, and variation in these traits may be sex-specific in hybrids due to differential effects of genetic incompatibilities in females and males. Here we present two experiments investigating variation in developmental rate among reciprocal interpopulation hybrids of the intertidal copepod Tigriopus californicus. Developmental rate is a fitness-related trait in this species that is affected by interactions between mitochondrial-encoded and nuclear-encoded genes in hybrids that result in variation in mitochondrial ATP synthesis capacities. First, we show that F2 -hybrid developmental rate is equivalent in two reciprocal crosses and is unaffected by sex, suggesting that breakdown of developmental rate is likely experienced equally by females and males. Second, we demonstrate that variation in developmental rate among F3 hybrids is heritable; times to copepodid metamorphosis of F4 offspring of fast-developing F3 parents (12.25 ± 0.05 days, µ ± SEM) were significantly faster than those of F4 offspring of slow-developing parents (14.58 ± 0.05 days). Third, we find that ATP synthesis rates in these F4 hybrids are unaffected by the developmental rates of their parents, but that mitochondria from females synthesize ATP at faster rates than mitochondria from males. Taken together, these results suggest that sex-specific effects vary among fitness-related traits in these hybrids, and that effects likely associated with hybrid breakdown display substantial inheritance across hybrid generations.