Independent Innexin Radiation Shaped Signaling in Ctenophores.

Innexins facilitate cell-cell communication by forming gap junctions or nonjunctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here, we identify and phylogenetically characterize the complete set of innexins of four ctenophores: Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, and Beroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of the M. leidyi innexin cluster suggests that this cluster has been maintained due to constraints related to gene regulation. We describe the basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

Integrating Embryonic Development and Evolutionary History to Characterize Tentacle-Specific Cell Types in a Ctenophore.

The origin of novel traits can promote expansion into new niches and drive speciation. Ctenophores (comb jellies) are unified by their possession of a novel cell type: the colloblast, an adhesive cell found only in the tentacles. Although colloblast-laden tentacles are fundamental for prey capture among ctenophores, some species have tentacles lacking colloblasts and others have lost their tentacles completely. We used transcriptomes from 36 ctenophore species to identify gene losses that occurred specifically in lineages lacking colloblasts and tentacles. We cross-referenced these colloblast- and tentacle-specific candidate genes with temporal RNA-Seq during embryogenesis in Mnemiopsis leidyi and found that both sets of candidates are preferentially expressed during tentacle morphogenesis. We also demonstrate significant upregulation of candidates from both data sets in the tentacle bulb of adults. Both sets of candidates were enriched for an N-terminal signal peptide and protein domains associated with secretion; among tentacle candidates we also identified orthologs of cnidarian toxin proteins, presenting tantalizing evidence that ctenophore tentacles may secrete toxins along with their adhesive. Finally, using cell lineage tracing, we demonstrate that colloblasts and neurons share a common progenitor, suggesting the evolution of colloblasts involved co-option of a neurosecretory gene regulatory network. Together these data offer an initial glimpse into the genetic architecture underlying ctenophore cell-type diversity.

Malignant canine mammary epithelial cells shed exosomes containing differentially expressed microRNA that regulate oncogenic networks.

BACKGROUND: Breast (mammary) cancers in human (BC) and canine (CMT) patients share clinical, pathological, and molecular similarities that suggest dogs may be a useful translational model. Many cancers, including BC, shed exosomes that contain microRNAs (miRs) into the microenvironment and circulation, and these may represent biomarkers of metastasis and tumor phenotype. METHODS: Three normal canine mammary epithelial cell (CMEC) cultures and 5 CMT cell lines were grown in serum-free media. Exosomes were isolated from culture media by ultracentrifugation then profiled by transmission electron microscopy, dynamic light scattering, and Western blot. Exosomal small RNA was deep-sequenced on an Illumina HiSeq2500 sequencer and validated by qRT-PCR. In silico bioinformatic analysis was carried out to determine microRNA gene and pathway targets. RESULTS: CMEC and CMT cell lines shed round, "cup-shaped" exosomes approximately 150-200 nm, and were immunopositive for exosomal marker CD9. Deep-sequencing averaged ~ 15 million reads/sample. Three hundred thirty-eight unique miRs were detected, with 145 having > ±1.5-fold difference between one or more CMT and CMEC samples. Gene ontology analysis revealed that the upregulated miRs in this exosomal population regulate a number of relevant oncogenic networks. Several miRNAs including miR-18a, miR-19a and miR-181a were predicted in silico to target the canine estrogen receptor (ESR1α). CONCLUSIONS: CMEC and CMT cells shed exosomes in vitro that contain differentially expressed miRs. CMT exosomal RNA expresses a limited number of miRs that are up-regulated relative to CMEC, and these are predicted to target biologically relevant hormone receptors and oncogenic pathways. These results may inform future studies of circulating exosomes and the utility of miRs as biomarkers of breast cancer in women and dogs.

Metabarcoding reveals environmental factors influencing spatio-temporal variation in pelagic micro-eukaryotes.

Marine environments harbour a vast diversity of micro-eukaryotic organisms (protists and other small eukaryotes) that play important roles in structuring marine ecosystems. However, micro-eukaryote diversity is not well understood. Likewise, knowledge is limited regarding micro-eukaryote spatial and seasonal distribution, especially over long temporal scales. Given the importance of this group for mobilizing energy from lower trophic levels near the base of the food chain to larger organisms, assessing community stability, diversity and resilience is important to understand ecosystem health. Herein, we use a metabarcoding approach to examine pelagic micro-eukaryote communities over a 2.5-year time series. Bimonthly surface sampling (July 2009 to December 2011) was conducted at four locations within Mobile Bay (Bay) and along the Alabama continental shelf (Shelf). Alpha-diversity only showed significant differences in Shelf sites, with the greatest differences observed between summer and winter. Beta-diversity showed significant differences in community composition in relation to season and the Bay was dominated by diatoms, while the Shelf was characterized by dinoflagellates and copepods. The northern Gulf of Mexico is heavily influenced by the Mobile River Basin, which brings low-salinity nutrient-rich water mostly during winter and spring. Community composition was correlated with salinity, temperature and dissolved silicate. However, species interactions (e.g. predation and parasitism) may also contribute to the observed variation, especially on the Shelf, which warrants further exploration. Metabarcoding revealed clear patterns in surface pelagic micro-eukaryote communities that were consistent over multiple years, demonstrating how these techniques could be greatly beneficial to ecological monitoring and management over temporal scales.

Bacterial associates of two Caribbean coral species reveal species-specific distribution and geographic variability.

Scleractinian corals harbor microorganisms that form dynamic associations with the coral host and exhibit substantial genetic and ecological diversity. Microbial associates may provide defense against pathogens and serve as bioindicators of changing environmental conditions. Here we describe the bacterial assemblages associated with two of the most common and phylogenetically divergent reef-building corals in the Caribbean, Montastraea faveolata and Porites astreoides. Contrasting life history strategies and disease susceptibilities indicate potential differences in their microbiota and immune function that may in part drive changes in the composition of coral reef communities. The ribotype structure and diversity of coral-associated bacteria within the surface mucosal layer (SML) of healthy corals were assessed using denaturing gradient gel electrophoresis (DGGE) fingerprinting and 454 bar-coded pyrosequencing. Corals were sampled at disparate Caribbean locations representing various levels of anthropogenic impact. We demonstrate here that M. faveolata and P. astreoides harbor distinct, host-specific bacteria but that specificity varies by species and site. P. astreoides generally hosts a bacterial assemblage of low diversity that is largely dominated by one bacterial genus, Endozoicomonas, within the order Oceanospirillales. The bacterial assemblages associated with M. faveolata are significantly more diverse and exhibit higher specificity at the family level than P. astreoides assemblages. Both corals have more bacterial diversity and higher abundances of disease-related bacteria at sites closer to the mainland than at those furthest away. The most diverse bacterial taxa and highest relative abundance of disease-associated bacteria were seen for corals near St. Thomas, U.S. Virgin Islands (USVI) (2.5 km from shore), and the least diverse taxa and lowest relative abundance were seen for corals near our most pristine site in Belize (20 km from shore). We conclude that the two coral species studied harbor distinct bacterial assemblages within the SML, but the degree to which each species maintains specific microbial associations varies both within each site and across large spatial scales. The taxonomic scale (i.e., phylum versus genus) at which scientists examine coral-microbe associations, in addition to host-elicited factors and environmental fluctuations, must be considered carefully in future studies of the coral holobiont.

Comparative Genomic and Transcriptomic Analyses Revealed Twenty-Six Candidate Genes Involved in the Air-Breathing Development and Function of the Bighead Catfish Clarias macrocephalus.

The bighead catfish (Clarias macrocephalus) and channel catfish (Ictalurus punctatus) are freshwater species in the Siluriformes order. C. macrocephalus has both gills and modified gill structures serving as an air-breathing organ (ABO), while I. punctatus does not possess such an organ, and cannot breathe in air, providing an excellent model for studying the molecular basis of ABO development in teleost fish. To investigate the critical time window for the development of air-breathing function, seven development stages were selected based on hypoxia challenge results, and RNA-seq was performed upon C. macrocephalus to compare with the non-air-breathing I. punctatus. Five-hundred million reads were generated and 25,239 expressed genes were annotated in C. macrocephalus. Among those, 8675 genes were differentially expressed across developmental stages. Comparative genomic analysis identified 1458 C. macrocephalus specific genes, which were absent in I. punctatus. Gene network and protein-protein interaction analyses identified 26 key hub genes involved in the air-breathing function. Three top candidate genes, mb, ngb, hbae, are mainly associated with oxygen carrying, oxygen binding, and heme binding activities. Our study provides a rich data set for exploring the genomic basis of air-breathing function in C. macrocephalus and offers insights into the adaption to hypoxic environments.

Microsatellites reveal origin and genetic diversity of Eurasian invasions by one of the world's most notorious marine invader, Mnemiopsis leidyi (Ctenophora).

Marine invasions are taking place at an increasing rate. When occurring in blooms, zooplanktivorous comb jellies of the genus Mnemiopsis are able to cause pelagic regime shifts in coastal areas and may cause the collapse of commercially important fish populations. Using microsatellites, developed for the first time in the phylum Ctenophora, we show that Mnemiopsis leidyi has colonized Eurasia from two source regions. Our preliminary data set included four sites within the putative source region (US East Coast and Gulf of Mexico) and 10 invaded locations in Eurasian waters. Bayesian clustering and phylogeographic approaches revealed the origin of earlier invasions of the Black and Caspian Sea in the 1980s/1990s within or close to the Gulf of Mexico, while the 2006 invasion of the North and Baltic Seas can be directly traced to New England (pairwise F(ST) = 0). We found no evidence for mixing among both gene pools in the invaded areas. While the genetic diversity (allelic richness) remained similar in the Baltic Sea compared to the source region New England, it was reduced in the North Sea, supporting the view of an initial invasion of Northern Europe to a Baltic Sea port. In Black and Caspian Sea samples, we found a gradual decline in allelic richness compared to the Gulf of Mexico region, supporting a stepping-stone model of colonization with two sequential genetic founder events. Our data also suggest that current practices of ballast water treatment are insufficient to prevent repeated invasions of gelatinous zooplankton.

Comparative Transcriptome Analysis During the Seven Developmental Stages of Channel Catfish (Ictalurus punctatus) and Tra Catfish (Pangasianodon hypophthalmus) Provides Novel Insights for Terrestrial Adaptation.

Tra catfish (Pangasianodon hypophthalmus), also known as striped catfish, is a facultative air-breather that uses its swim bladder as an air-breathing organ (ABO). A related species in the same order (Siluriformes), channel catfish (Ictalurus punctatus), does not possess an ABO and thus cannot breathe in the air. Tra and channel catfish serve as great comparative models for investigating possible genetic underpinnings of aquatic to land transitions, as well as for understanding genes that are crucial for the development of the swim bladder and the function of air-breathing in tra catfish. In this study, hypoxia challenge and microtomy experiments collectively revealed critical time points for the development of the air-breathing function and swim bladder in tra catfish. Seven developmental stages in tra catfish were selected for RNA-seq analysis based on their transition to a stage that could live at 0 ppm oxygen. More than 587 million sequencing clean reads were generated, and a total of 21,448 unique genes were detected. A comparative genomic analysis between channel catfish and tra catfish revealed 76 genes that were present in tra catfish, but absent from channel catfish. In order to further narrow down the list of these candidate genes, gene expression analysis was performed for these tra catfish-specific genes. Fourteen genes were inferred to be important for air-breathing. Of these, HRG, GRP, and CX3CL1 were identified to be the most likely genes related to air-breathing ability in tra catfish. This study provides a foundational data resource for functional genomic studies in air-breathing function in tra catfish and sheds light on the adaptation of aquatic organisms to the terrestrial environment.

Neonatal porcine endometrial development and epithelial proliferation affected by age and exposure to estrogen and relaxin.

In the pig, temporospatially regulated proliferation of uterine luminal (LE) and glandular (GE) epithelium between birth (postnatal day=PND 0) and PND 15 is essential for success of endometrial development. Exposure of gilts to estrogen (E) or relaxin (RLX) during this period disrupts uterine development, and neonatal E exposure can compromise adult uterine function. Neonatal uterotrophic effects of E and RLX, administered for 2 days beginning on PND 12, can be inhibited with the antiestrogen ICI 182,780 (ICI) indicating crosstalk between RLX and E signaling systems. Here, objectives were to determine effects of: (study 1) neonatal age and (study 2) exposure to E, RLX, and ICI on porcine neonatal uterine histoarchitecture and patterns of epithelial cell proliferation as reflected by proliferating cell nuclear antigen labeling index. In study 1, uteri were obtained on PND 0, 3, 6, 9, 12 and 15. Glandular epithelium, absent at birth, was observed by PND 3. Overall, epithelial labeling index increased from birth to PND 3, declined from PND 6-9 in LE and GE, and increased to PND 15 in GE. In study 2, uteri were collected on PND 14 after administration of vehicle, E, or RLX for 2 days, or following pretreatment with ICI. Alone, E was uterotrophic and adenogenic and increased labeling index in both LE and GE. Both RLX and ICI increased proliferation in GE. Effects of E and RLX were attenuated by ICI, providing further support for crosstalk between these signaling systems in the developing neonatal porcine endometrium.

Complete Genome Sequence of Streptomyces sp. Strain CCM_MD2014, Isolated from Topsoil in Woods Hole, Massachusetts.

Here, we present the complete genome sequence of Streptomyces sp. strain CCM_MD2014 (phylum Actinobacteria), isolated from surface soil in Woods Hole, MA. Its single linear chromosome of 8,274,043 bp in length has a 72.13% G+C content and contains 6,948 coding sequences.

Complete Genome Sequence of Curtobacterium sp. Strain MR_MD2014, Isolated from Topsoil in Woods Hole, Massachusetts.

Here, we present the 3,443,800-bp complete genome sequence of Curtobacterium sp. strain MR_MD2014 (phylum Actinobacteria). This strain was isolated from soil in Woods Hole, MA, as part of the 2014 Microbial Diversity Summer Program at the Marine Biological Laboratory in Woods Hole, MA.

Dispersed oil disrupts microbial pathways in pelagic food webs.

Most of the studies of microbial processes in response to the Deepwater Horizon oil spill focused on the deep water plume, and not on the surface communities. The effects of the crude oil and the application of dispersants on the coastal microbial food web in the northern Gulf of Mexico have not been well characterized even though these regions support much of the fisheries production in the Gulf. A mesocosm experiment was carried out to determine how the microbial community off the coast of Alabama may have responded to the influx of surface oil and dispersants. While the addition of glucose or oil alone resulted in an increase in the biomass of ciliates, suggesting transfer of carbon to higher trophic levels was likely; a different effect was seen in the presence of dispersant. The addition of dispersant or dispersed oil resulted in an increase in the biomass of heterotrophic prokaryotes, but a significant inhibition of ciliates, suggesting a reduction in grazing and decrease in transfer of carbon to higher trophic levels. Similar patterns were observed in two separate experiments with different starting nutrient regimes and microbial communities suggesting that the addition of dispersant and dispersed oil to the northern Gulf of Mexico waters in 2010 may have reduced the flow of carbon to higher trophic levels, leading to a decrease in the production of zooplankton and fish on the Alabama shelf.