Building consensus around the assessment and interpretation of Symbiodiniaceae diversity.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Expressing the Pro-Apoptotic Reaper Protein via Insertion into the Structural Open Reading Frame of Sindbis Virus Reduces the Ability to Infect Aedes aegypti Mosquitoes.

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the Drosophila pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. In order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti, we constructed MRE/rprORF, a version of SINV containing reaper inserted into the structural open reading frame (ORF) as an in-frame fusion. MRE/rprORF successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, induced apoptosis in cultured cells, and caused increased effector caspase activity in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed significantly less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in the minority of mosquitoes that became infected with MRE/rprORF was not significantly different from control virus. Deep sequencing of virus populations from ten mosquitoes infected with MRE/rprORF indicated that the reaper insert was stable, with only a small number of point mutations and no deletions being observed at frequencies greater than 1%. Our results indicate that expression of Reaper by this method significantly reduces infection prevalence, but if infection is established then Reaper expression has limited ability to continue to suppress replication.

Primary Microbial Succession in the Anchialine Ecosystem.

When new land is created, initial microbial colonization lays the foundation for further ecological succession of plant and animal communities. Primary microbial succession of new aquatic habitats formed during volcanic activity has received little attention. The anchialine ecosystem, which includes coastal ponds in young lava flows, offers an opportunity to examine this process. Here, we characterized microbial communities of anchialine habitats in Hawaii that were created during volcanic eruptions in 2018. Benthic samples from three habitats were collected ∼2 years after their formation and at later time points spanning ∼1 year. Sequence profiling (16S and 18S) of prokaryotic and eukaryotic communities was used to test whether communities were similar to those from older, established anchialine habitats, and if community structure changed over time. Results show that microbial communities from the new habitats were unlike any from established anchialine microbial communities, having higher proportions of Planctomycetota and Chloroflexi but lower proportions of green algae. Each new habitat also harbored its own unique community relative to other habitats. While community composition in each habitat underwent statistically significant changes over time, they remained distinctive from established anchialine habitats. New habitats also had highly elevated temperatures compared to other habitats. These results suggest that idiosyncratic microbial consortia form during early succession of Hawaiian anchialine habitats. Future monitoring will reveal whether the early communities described here remain stable after temperatures decline and macro-organisms become more abundant, or if microbial communities will continue to change and eventually resemble those of established habitats. This work is a key first step in examining primary volcanic succession in aquatic habitats and suggests young anchialine habitats may warrant special conservation status.

Infection of Aedes aegypti Mosquitoes with Midgut-Attenuated Sindbis Virus Reduces, but Does Not Eliminate, Disseminated Infection.

Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.

Discovery of Novel Biosynthetic Gene Cluster Diversity From a Soil Metagenomic Library.

Soil microorganisms historically have been a rich resource for natural product discovery, yet the majority of these microbes remain uncultivated and their biosynthetic capacity is left underexplored. To identify the biosynthetic potential of soil microorganisms using a culture-independent approach, we constructed a large-insert metagenomic library in Escherichia coli from a topsoil sampled from the Cullars Rotation (Auburn, AL, United States), a long-term crop rotation experiment. Library clones were screened for biosynthetic gene clusters (BGCs) using either PCR or a NGS (next generation sequencing) multiplexed pooling strategy, coupled with bioinformatic analysis to identify contigs associated with each metagenomic clone. A total of 1,015 BGCs were detected from 19,200 clones, identifying 223 clones (1.2%) that carry a polyketide synthase (PKS) and/or a non-ribosomal peptide synthetase (NRPS) cluster, a dramatically improved hit rate compared to PCR screening that targeted type I polyketide ketosynthase (KS) domains. The NRPS and PKS clusters identified by NGS were distinct from known BGCs in the MIBiG database or those PKS clusters identified by PCR. Likewise, 16S rRNA gene sequences obtained by NGS of the library included many representatives that were not recovered by PCR, in concordance with the same bias observed in KS amplicon screening. This study provides novel resources for natural product discovery and circumvents amplification bias to allow annotation of a soil metagenomic library for a more complete picture of its functional and phylogenetic diversity.

Red Coloration in an Anchialine Shrimp: Carotenoids, Genetic Variation, and Candidate Genes.

Red coloration is a widely distributed phenotype among animals, yet the pigmentary and genetic bases for this phenotype have been described in relatively few taxa. Here we show that the Hawaiian endemic anchialine shrimp Halocaridina rubra is red because of the accumulation of astaxanthin. Laboratory colonies of phylogenetically distinct lineages of H. rubra have colony-specific amounts of astaxanthin that are developmentally, and likely genetically, fixed. Carotenoid supplementation and restriction experiments failed to change astaxanthin content from the within-colony baseline levels, suggesting that dietary limitation is not a major factor driving coloration differences. A possible candidate gene product predicted to be responsible for the production of astaxanthin in H. rubra and other crustaceans is closely related to the bifunctional cytochrome P450 family 3 enzyme CrtS found in fungi. However, homologs to the enzyme thought to catalyze ketolation reactions in birds and turtles, CYP2J19, were not found. This work is one of the first steps in linking phenotypic variation in red coloration of H. rubra to genotypic variation. Future work should focus on (1) pinpointing the genes that function in the bioconversion of dietary carotenoids to astaxanthin, (2) examining what genomic variants might drive variation in coloration among discrete lineages, and (3) testing more explicitly for condition-dependent carotenoid coloration in crustaceans.

Phenotypic Comparability from Genotypic Variability among Physically Structured Microbial Consortia.

Microbiomes represent the collective bacteria, archaea, protist, fungi, and virus communities living in or on individual organisms that are typically multicellular eukaryotes. Such consortia have become recognized as having significant impacts on the development, health, and disease status of their hosts. Since understanding the mechanistic connections between an individual's genetic makeup and their complete set of traits (i.e., genome to phenome) requires consideration at different levels of biological organization, this should include interactions with, and the organization of, microbial consortia. To understand microbial consortia organization, we elucidated the genetic constituents among phenotypically similar (and hypothesized functionally-analogous) layers (i.e., top orange, second orange, pink, and green layers) in the unique laminated orange cyanobacterial-bacterial crusts endemic to Hawaii's anchialine ecosystem. High-throughput amplicon sequencing of ribosomal RNA hypervariable regions (i.e., Bacteria-specific V6 and Eukarya-biased V9) revealed microbial richness increasing by crust layer depth, with samples of a given layer more similar to different layers from the same geographic site than to their phenotypically-analogous layer from different sites. Furthermore, samples from sites on the same island were more similar to each other, regardless of which layer they originated from, than to analogous layers from another island. However, cyanobacterial and algal taxa were abundant in all surface and bottom layers, with anaerobic and chemoautotrophic taxa concentrated in the middle two layers, suggesting crust oxygenation from both above and below. Thus, the arrangement of oxygenated vs. anoxygenated niches in these orange crusts is functionally distinct relative to other laminated cyanobacterial-bacterial communities examined to date, with convergent evolution due to similar environmental conditions a likely driver for these phenotypically comparable but genetically distinct microbial consortia.

Disparate responses to salinity across species and organizational levels in anchialine shrimps.

Environmentally induced plasticity in gene expression is one of the underlying mechanisms of adaptation to habitats with variable environments. For example, euryhaline crustaceans show predictable changes in the expression of ion-transporter genes during salinity transfers, although studies have typically been limited to specific genes, taxa and ecosystems of interest. Here, we investigated responses to salinity change at multiple organizational levels in five species of shrimp representing at least three independent invasions of the anchialine ecosystem, defined as habitats with marine and freshwater influences with spatial and temporal fluctuations in salinity. Although all five species were generally strong osmoregulators, salinity-induced changes in gill physiology and gene expression were highly species specific. While some species exhibited patterns similar to those of previously studied euryhaline crustaceans, instances of distinct and atypical patterns were recovered from closely related species. Species-specific patterns were found when examining: (1) numbers and identities of differentially expressed genes, (2) salinity-induced expression of genes predicted a priori to play a role in osmoregulation, and (3) salinity-induced expression of orthologs shared among all species. Notably, ion transport genes were unchanged in the atyid Halocaridina rubra while genes normally associated with vision and light perception were among those most highly upregulated. Potential reasons for species-specific patterns are discussed, including variation among anchialine habitats in salinity regimes and divergent evolution in anchialine taxa. Underexplored mechanisms of osmoregulation in crustaceans revealed here by the application of transcriptomic approaches to ecologically and taxonomically understudied systems are also explored.

Na+ /K+ -ATPase gene duplications in clitellate annelids are associated with freshwater colonization.

Major habitat transitions, such as those from marine to freshwater habitats or from aquatic to terrestrial habitats, have occurred infrequently in animal evolution and may represent a barrier to diversification. Identifying genomic events associated with these transitions can help us better understand mechanisms that allow animals to cross these barriers and diversify in new habitats. Study of the Capitella telata and Helobdella robusta genomes allows examination of one such habitat transition (marine to freshwater) in Annelida. Initial examination of these genomes indicated that the freshwater leech H. robusta contains many more copies (12) of the sodium-potassium pump alpha-subunit (Na+ /K+ -ATPase) gene than does the marine polychaete C. telata (2). The sodium-potassium pump plays a key role in maintenance of cellular ionic balance and osmoregulation, and Na+ /K+ -ATPase duplications may have helped annelids invade and diversify in freshwater habitats. To assess whether the timing of Na+ /K+ -ATPase duplications coincided with the marine-to-freshwater transition in Clitellata, we used transcriptomic data from 18 annelid taxa, along with the two genomes, to infer a species phylogeny and identified Na+ /K+ -ATPase gene transcripts in order to infer the timing of gene duplication events using tree-based methods. The inferred timing of Na+ /K+ -ATPase duplication events is consistent with the timing of the initial marine-to-freshwater transition early in the history of clitellate annelids, supporting the hypothesis that gene duplications may have played a role in the annelid diversification into freshwater habitats.

Systematic Revision of Symbiodiniaceae Highlights the Antiquity and Diversity of Coral Endosymbionts.

The advent of molecular data has transformed the science of organizing and studying life on Earth. Genetics-based evidence provides fundamental insights into the diversity, ecology, and origins of many biological systems, including the mutualisms between metazoan hosts and their micro-algal partners. A well-known example is the dinoflagellate endosymbionts ("zooxanthellae") that power the growth of stony corals and coral reef ecosystems. Once assumed to encompass a single panmictic species, genetic evidence has revealed a divergent and rich diversity within the zooxanthella genus Symbiodinium. Despite decades of reporting on the significance of this diversity, the formal systematics of these eukaryotic microbes have not kept pace, and a major revision is long overdue. With the consideration of molecular, morphological, physiological, and ecological data, we propose that evolutionarily divergent Symbiodinium "clades" are equivalent to genera in the family Symbiodiniaceae, and we provide formal descriptions for seven of them. Additionally, we recalibrate the molecular clock for the group and amend the date for the earliest diversification of this family to the middle of the Mesozoic Era (∼160 mya). This timing corresponds with the adaptive radiation of analogs to modern shallow-water stony corals during the Jurassic Period and connects the rise of these symbiotic dinoflagellates with the emergence and evolutionary success of reef-building corals. This improved framework acknowledges the Symbiodiniaceae's long evolutionary history while filling a pronounced taxonomic gap. Its adoption will facilitate scientific dialog and future research on the physiology, ecology, and evolution of these important micro-algae.

Crossing the Divide: Admixture Across the Antarctic Polar Front Revealed by the Brittle Star Astrotoma agassizii.

The Antarctic Polar Front (APF) is one of the most well-defined and persistent oceanographic features on the planet and serves as a barrier to dispersal between the Southern Ocean and lower latitudes. High levels of endemism in the Southern Ocean have been attributed to this barrier, whereas the accompanying Antarctic Circumpolar Current (ACC) likely promotes west-to-east dispersal. Previous phylogeographic work on the brittle star Astrotoma agassizii Lyman, 1875 based on mitochondrial genes suggested isolation across the APF, even though populations in both South American waters and the Southern Ocean are morphologically indistinguishable. Here, we revisit this finding using a high-resolution 2b-RAD (restriction-site-associated DNA) single-nucleotide polymorphism (SNP)-based approach, in addition to enlarged mitochondrial DNA data sets (16S rDNA, COI, and COII), for comparison to previous work. In total, 955 biallelic SNP loci confirmed the existence of strongly divergent populations on either side of the Drake Passage. Interestingly, genetic admixture was detected between South America and the Southern Ocean in five individuals on both sides of the APF, revealing evidence of recent or ongoing genetic contact. We also identified two differentiated populations on the Patagonian Shelf with six admixed individuals from these two populations. These findings suggest that the APF is a strong but imperfect barrier. Fluctuations in location and strength of the APF and ACC due to climate shifts may have profound consequences for levels of admixture or endemism in this region of the world.

Phylogenomic analyses of Crassiclitellata support major Northern and Southern Hemisphere clades and a Pangaean origin for earthworms.

BACKGROUND: Earthworms (Crassiclitellata) are a diverse group of annelids of substantial ecological and economic importance. Earthworms are primarily terrestrial infaunal animals, and as such are probably rather poor natural dispersers. Therefore, the near global distribution of earthworms reflects an old and likely complex evolutionary history. Despite a long-standing interest in Crassiclitellata, relationships among and within major clades remain unresolved. METHODS: In this study, we evaluate crassiclitellate phylogenetic relationships using 38 new transcriptomes in combination with publicly available transcriptome data. Our data include representatives of nearly all extant earthworm families and a representative of Moniligastridae, another terrestrial annelid group thought to be closely related to Crassiclitellata. We use a series of differentially filtered data matrices and analyses to examine the effects of data partitioning, missing data, compositional and branch-length heterogeneity, and outgroup inclusion. RESULTS AND DISCUSSION: We recover a consistent, strongly supported ingroup topology irrespective of differences in methodology. The topology supports two major earthworm clades, each of which consists of a Northern Hemisphere subclade and a Southern Hemisphere subclade. Divergence time analysis results are concordant with the hypothesis that these north-south splits are the result of the breakup of the supercontinent Pangaea. CONCLUSIONS: These results support several recently proposed revisions to the classical understanding of earthworm phylogeny, reveal two major clades that seem to reflect Pangaean distributions, and raise new questions about earthworm evolutionary relationships.

Discovery and evolution of novel hemerythrin genes in annelid worms.

BACKGROUND: Despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin (Hr) evolutionary history. Four subgroups of Hrs have been documented, including: circulating Hr (cHr), myohemerythrin (myoHr), ovohemerythrin (ovoHr), and neurohemerythrin (nHr). Annelids have the greatest diversity of oxygen carrying proteins among animals and are the only phylum in which all Hr subgroups have been documented. To examine Hr diversity in annelids and to further understand evolution of Hrs, we employed approaches to survey annelid transcriptomes in silico. RESULTS: Sequences of 214 putative Hr genes were identified from 44 annelid species in 40 different families and Bayesian inference revealed two major clades with strong statistical support. Notably, the topology of the Hr gene tree did not mirror the phylogeny of Annelida as presently understood, and we found evidence of extensive Hr gene duplication and loss in annelids. Gene tree topology supported monophyly of cHrs and a myoHr clade that included nHrs sequences, indicating these designations are functional rather than evolutionary. CONCLUSIONS: The presence of several cHrs in early branching taxa suggests that a variety of Hrs were present in the common ancestor of extant annelids. Although our analysis was limited to expressed-coding regions, our findings demonstrate a greater diversity of Hrs among annelids than previously reported.

Geographic structure in the Southern Ocean circumpolar brittle star Ophionotus victoriae (Ophiuridae) revealed from mtDNA and single-nucleotide polymorphism data.

Marine systems have traditionally been thought of as "open" with few barriers to gene flow. In particular, many marine organisms in the Southern Ocean purportedly possess circumpolar distributions that have rarely been well verified. Here, we use the highly abundant and endemic Southern Ocean brittle star Ophionotus victoriae to examine genetic structure and determine whether barriers to gene flow have existed around the Antarctic continent. Ophionotus victoriae possesses feeding planktotrophic larvae with presumed high dispersal capability, but a previous study revealed genetic structure along the Antarctic Peninsula. To test the extent of genetic differentiation within O. victoriae, we sampled from the Ross Sea through the eastern Weddell Sea. Whereas two mitochondrial DNA markers (16S rDNA and COI) were employed to allow comparison to earlier work, a 2b-RAD single-nucleotide polymorphism (SNP) approach allowed sampling of loci across the genome. Mitochondrial data from 414 individuals suggested three major lineages, but 2b-RAD data generated 1,999 biallelic loci that identified four geographically distinct groups from 89 samples. Given the greater resolution by SNP data, O. victoriae can be divided into geographically distinct populations likely representing multiple species. Specific historical scenarios that explain current population structure were examined with approximate Bayesian computation (ABC) analyses. Although the Bransfield Strait region shows high diversity possibly due to mixing, our results suggest that within the recent past, dispersal processes due to strong currents such as the Antarctic Circumpolar Current have not overcome genetic subdivision presumably due to historical isolation, questioning the idea of large open circumpolar populations in the Southern Ocean.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Here We Are, But Where Do We Go? A Systematic Review of Crustacean Transcriptomic Studies from 2014-2015.

Despite their economic, ecological, and experimental importance, genomic resources remain scarce for crustaceans. In lieu of genomes, many researchers have taken advantage of technological advancements to instead sequence and assemble crustacean transcriptomes de novo However, there is little consensus on what standard operating procedures are, or should be, for the field. Here, we systematically reviewed 53 studies published during 2014-2015 that utilized transcriptomic resources from this taxonomic group in an effort to identify commonalities as well as potential weaknesses that have applicability beyond just crustaceans. In general, these studies utilized RNA-Seq data, both novel and publicly available, to characterize transcriptomes and/or identify differentially expressed genes (DEGs) between treatments. Although the software suite Trinity was popular in assembly pipelines and other programs were also commonly employed, many studies failed to report crucial details regarding bioinformatic methodologies, including read mappers and the utilized parameters in identifying and characterizing DEGs. Annotation percentages for assembled transcriptomic contigs were low, averaging 32% overall. While other metrics, such as numbers of contigs and DEGs reported, correlated with the number of sequence reads utilized per sample, these did reach apparent saturation with increasing sequencing depth. Most disturbingly, a number of studies (55%) reported DEGs based on non-replicated experimental designs and single biological replicates for each treatment. Given this, we suggest future RNA-Seq experiments targeting transcriptome characterization conduct deeper (i.e., 50-100 M reads) sequencing while those examining differential expression instead focus more on increased biological replicates at shallower (i.e., ∼10-20 M reads/sample) sequencing depths. Moreover, the community must avoid submitting for review, or accepting for publication, non-replicated differential expression studies. Finally, mining the ever growing publicly available transcriptomic data from crustaceans will allow future studies to focus on hypothesis-driven research instead of continuing to simply characterize transcriptomes. As an example of this, we utilized neurotoxin sequences from the recently described remipede venom gland transcriptome in conjunction with publicly available crustacean transcriptomic data to derive preliminary results and hypotheses regarding the evolution of venom in crustaceans.

Developmental Transcriptomics of the Hawaiian Anchialine Shrimp Halocaridina rubra Holthuis, 1963 (Crustacea: Atyidae).

Many crustacean species progress through a series of metamorphoses during the developmental transition from embryo to adult. The molecular genetic basis of this transition, however, is not well characterized for a large number of crustaceans. Here, we employ multiple RNA-Seq methodologies to identify differentially expressed genes (DEGs) between "early" (i.e., Z1 - Z2) as well as "late" (i.e., Z3 - Z4) larval and adult developmental stages of Halocaridina rubra Holthuis (1963), an atyid shrimp endemic to the environmentally variable anchialine ecosystem of the Hawaiian Islands. Given the differences in salinity tolerance (narrow vs. wide range), energy acquisition (maternal yolk-bearing vs. microphagous grazing), and behavior (positively phototactic vs. not) between larvae and adults, respectively, of this species, we hypothesized the recovery of numerous DEGs belonging to functional categories relating to these characteristics. Consistent with this and regardless of methodology, hundreds of DEGs were identified, including upregulation of opsins and other light/stimulus detection genes and downregulation of genes related to ion transport, digestion, and reproduction in larvae relative to adults. Furthermore, isoform-switching, which has been largely unexplored in crustacean development, appears to be pervasive between H. rubra larvae and adults, especially among structural and oxygen-transport genes. Finally, by comparing RNA-Seq methodologies, we provide recommendations for future crustacean transcriptomic studies, including a demonstration of the pitfalls associated with identifying DEGs from single replicate samples as well as the utility of leveraging "prepackaged" bioinformatics pipelines.

Salinity-induced changes in gene expression from anterior and posterior gills of Callinectes sapidus (Crustacea: Portunidae) with implications for crustacean ecological genomics.

Decapods represent one of the most ecologically diverse taxonomic groups within crustaceans, making them ideal to study physiological processes like osmoregulation. However, prior studies have failed to consider the entire transcriptomic response of the gill - the primary organ responsible for ion transport - to changing salinity. Moreover, the molecular genetic differences between non-osmoregulatory and osmoregulatory gill types, as well as the hormonal basis of osmoregulation, remain underexplored. Here, we identified and characterized differentially expressed genes (DEGs) via RNA-Seq in anterior (non-osmoregulatory) and posterior (osmoregulatory) gills during high to low salinity transfer in the blue crab Callinectes sapidus, a well-studied model for crustacean osmoregulation. Overall, we confirmed previous expression patterns for individual ion transport genes and identified novel ones with salinity-mediated expression. Notable, novel DEGs among salinities and gill types for C. sapidus included anterior gills having higher expression of structural genes such as actin and cuticle proteins while posterior gills exhibit elevated expression of ion transport and energy-related genes, with the latter likely linked to ion transport. Potential targets among recovered DEGs for hormonal regulation of ion transport between salinities and gill types included neuropeptide Y and a KCTD16-like protein. Using publically available sequence data, constituents for a "core" gill transcriptome among decapods are presented, comprising genes involved in ion transport and energy conversion and consistent with salinity transfer experiments. Lastly, rarefication analyses lead us to recommend a modest number of sequence reads (~10-15M), but with increased biological replication, be utilized in future DEG analyses of crustaceans.