Metazoan endoparasites of snakes from Argentina: Review and checklist with distributional notes and remarks.

This article presents a checklist of metazoan parasites of snakes from Argentina, along with a comprehensive review of the relevant literature published between 1922 and June 2023, covering various aspects of interest. We compiled 34 species of metazoan endoparasites from 28 studies. The subclass Digenea showed the highest number of species (n = 22 species), followed by the phylum Nematoda (n = 8 species), and the subclass Pentastomida (n = 3 species and 1 taxa inquirenda). Dipsadidae was the family of snakes with the most species examined for metazoan endoparasites (n = 20 species). In contrast, Viperidae had the largest number of specimens surveyed (n = 343). Of 23 provinces, 15 (65.2%) presented at least one report of metazoan endoparasites in snakes. The northeastern provinces showed the highest richness of metazoan endoparasites and host diversity. Many articles focused on taxonomy, but studies on parasite ecology were not found. Although taxonomic accuracy was high in most reports, some records were correctly deposited in zoological collections or geo-referenced. This is the first attempt to include all groups of metazoan endoparasites of snakes from Argentina in a single checklist in the last century.

Irreducible Complexity of Hox Gene: Path to the Canonical Function of the Hox Cluster.

The evolution of major taxa is often associated with the emergence of new gene families. In all multicellular animals except sponges and comb jellies, the genomes contain Hox genes, which are crucial regulators of development. The canonical function of Hox genes involves colinear patterning of body parts in bilateral animals. This general function is implemented through complex, precisely coordinated mechanisms, not all of which are evolutionarily conserved and fully understood. We suggest that the emergence of this regulatory complexity was preceded by a stage of cooperation between more ancient morphogenetic programs or their individual elements. Footprints of these programs may be present in modern animals to execute non-canonical Hox functions. Non-canonical functions of Hox genes are involved in maintaining terminal nerve cell specificity, autophagy, oogenesis, pre-gastrulation embryogenesis, vertical signaling, and a number of general biological processes. These functions are realized by the basic properties of homeodomain protein and could have triggered the evolution of ParaHoxozoa and Nephrozoa subsequently. Some of these non-canonical Hox functions are discussed in our review.

Selection by differential survival among marine animals in the Phanerozoic.

The Gaia hypothesis posits that the Earth and its biosphere function as a single self-stabilizing system, but a key challenge is explaining how this could have arisen through Darwinian evolution. One theory is that of "selection by differential survival," in which a clade's extinction probability decreases with age as it accumulates adaptations resisting environmental disturbances. While this is hard to assess during early Earth history, we can assess whether this process operated among marine animal genera throughout the Phanerozoic. To that end, we analyzed time ranges of 36,117 extinct animal genera using fossil occurrence data from the Paleobiology Database in order to calculate marine metazoan extinction age selectivity, extinction rates, and speciation rates over the Phanerozoic. We identify four signatures of selection by differential survival: lower extinction rates among older lineages, heritability and taxonomically nested propagation of extinction resistance, reduced age selectivity during rare environmental perturbations, and differential extinction rather than speciation as the primary driver of the phenomenon. Evidence for this process at lower taxonomic levels also implies its possibility for life as a whole - indeed, the possibility of Gaia.

The Evolution and Characterization of the RNA Interference Pathways in Lophotrochozoa.

In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs, and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is typically loaded by a specific Argonaute homolog. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Within Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries suggested that in all Trochozoa, the proteins related to the endo-siRNA pathway have been lost, a part of them in some phyla (i.e. Nemertea, Bryozoa, Entoprocta), while all of them in all the others. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

The alternative enzymes-bearing tunicates lack multiple widely distributed genes coding for peripheral OXPHOS subunits.

The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.

Brief History of Placozoa.

Placozoans are morphologically the simplest free-living animals. They represent a unique window of opportunities to understand both the origin of the animal organization and the rules of life for the system and synthetic biology of the future. However, despite more than 100 years of their investigations, we know little about their organization, natural habitats, and life strategies. Here, we introduce this unique animal phylum and highlight some directions vital to broadening the frontiers of the biomedical sciences. In particular, understanding the genomic bases of placozoan biodiversity, cell identity, connectivity, reproduction, and cellular bases of behavior are critical hot spots for future studies.

Long-Term Culturing of Placozoans (Trichoplax and Hoilungia).

The phylum Placozoa remains one of the least explored among early-branching metazoan lineages. For over 130 years, this phylum had been represented by the single species Trichoplax adhaerens-an animal with the simplest known body plan (three cell layers without any organs) but complex behaviors. Recently, extensive sampling of placozoans across the globe and their subsequent genetic analysis have revealed incredible biodiversity with numerous cryptic species worldwide. However, only a few culture protocols are available to date, and all are for one species only. Here, we describe the breeding of four different species representing two placozoan genera: Trichoplax adhaerens, Trichoplax sp. H2, Hoilungia sp. H4, and Hoilungia hongkongensis originating from diverse biotopes. Our protocols allow to culture all species under comparable conditions. Next, we outlined various food sources and optimized strain-specific parameters enabling long-term culturing. These protocols can facilitate comparative analyses of placozoan biology and behaviors, which together will contribute to deciphering general principles of animal organization.

The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Anti-biofouling performance and microbial communities of an integrated fixed-film activated sludge membrane bioreactor with a fibrous carrier material: Pilot-scale demonstration.

Widespread use of membrane bioreactors for high-performance wastewater treatment depends on the prevention of biofouling during membrane filtration, which can reduce operating costs. Biofouling is usually prevented using mechanical and chemical membrane treatment methods, which can be time-consuming and expensive. In this study, we developed bio-capsules as a fluidizing carrier material in an integrated fixed-film activated sludge membrane bioreactor (IFAS-MBR). The bio-capsules were prepared from moniliform polyvinylidene chloride fibrous balls enclosed in a spherical plastic basket, and could harbor protozoa and metazoa. A pilot-scale anoxic-oxic IFAS-MBR system with a total volume of 132 m3 was operated to remove organic carbon and nitrogen from municipal wastewater at a high permeate flux (0.84 m3/m2/day). The efficacy of the bio-capsules and the prokaryotic/eukaryotic community structures in the system were investigated. After operation for 1 year, the system demonstrated stable removal of organic carbon (76.0 % ± 15.5 % as total organic carbon, 93.1 ± 5.3 % as BOD, and 88.5 ± 5.2 % as CODMn) and nitrogen (71.3 % ± 9.3 %) despite fluctuations in the influent concentrations. Increases in transmembrane pressure (TMP) were retarded from its increase rates from 0.56 kPa/day to 0.149-0.224 kPa/day by the bio-capsules, and the TMP was kept constant at around 20 kPa throughout the operational period. High-throughput sequencing of 16S rRNA gene amplicons showed that the prokaryotic family Pirellulaceae was metabolically active and correlated with the TMP. According to the 18S rRNA gene sequencing, the eukaryotic metazoan Bdelloidea was more abundant in the bio-capsules than in activated sludge, which was supported by microscopic observations. These results suggest that the application of bio-capsules prevents increases in the TMP by harboring the procaryotes and eukaryotes responsible for biofouling mitigation in the IFAS-MBR system.

Scientific history, biogeography, and biological traits predict presence of cryptic or overlooked species.

Genetic data show that many nominal species are composed of more than one biological species, and thus contain cryptic species in the broad sense (including overlooked species). When ignored, cryptic species generate confusion which, beyond biodiversity or vulnerability underestimation, blurs our understanding of ecological and evolutionary processes and may impact the soundness of decisions in conservation or medicine. However, very few hypotheses have been tested about factors that predispose a taxon to contain cryptic or overlooked species. To fill this gap, we surveyed the literature on free-living marine metazoans and built two data sets, one of 187,603 nominal species and another of 83 classes or phyla, to test several hypotheses, correcting for sequence data availability, taxon size and phylogenetic relatedness. We found a strong effect of scientific history: the probability of a taxon containing cryptic species was highest for the earliest described species and varied among time periods potentially consistently with an influence of prevailing scientific theories. The probability of cryptic species being present was also increased for species with large distribution ranges. They were more frequent in the north polar and south polar zones, contradicting previous predictions of more cryptic species in the tropics, and supporting the hypothesis that many cryptic species diverged recently. The number of cryptic species varied among classes, with an excess in hydrozoans and polychaetes, and a deficit in actinopterygians, for example, but precise class ranking was relatively sensitive to the statistical model used. For all models, biological traits, rather than phylum, appeared responsible for the variation among classes: there were fewer cryptic species than expected in classes with hard skeletons (perhaps because they provide good characters for taxonomy) and image-forming vision (in which selection against heterospecific mating may enhance morphological divergence), and more in classes with internal fertilisation. We estimate that among marine free-living metazoans, several thousand additional cryptic species complexes could be identified as more sequence data become available. The factors identified as important for marine animal cryptic species are likely important for other biomes and taxa and should aid many areas in biology that rely on accurate species identification.

Metazoa-level USCOs as markers in species delimitation and classification.

Metazoa-level universal single-copy orthologs (mzl-USCOs) are universally applicable markers for DNA taxonomy in animals that can replace or supplement single-gene barcodes. Previously, mzl-USCOs from target enrichment data were shown to reliably distinguish species. Here, we tested whether USCOs are an evenly distributed, representative sample of a given metazoan genome and therefore able to cope with past hybridization events and incomplete lineage sorting. This is relevant for coalescent-based species delimitation approaches, which critically depend on the assumption that the investigated loci do not exhibit autocorrelation due to physical linkage. Based on 239 chromosome-level assembled genomes, we confirmed that mzl-USCOs are genetically unlinked for practical purposes and a representative sample of a genome in terms of reciprocal distances between USCOs on a chromosome and of distribution across chromosomes. We tested the suitability of mzl-USCOs extracted from genomes for species delimitation and phylogeny in four case studies: Anopheles mosquitos, Drosophila fruit flies, Heliconius butterflies and Darwin's finches. In almost all instances, USCOs allowed delineating species and yielded phylogenies that corresponded to those generated from whole genome data. Our phylogenetic analyses demonstrate that USCOs may complement single-gene DNA barcodes and provide more accurate taxonomic inferences. Combining USCOs from sources that used different versions of ortholog reference libraries to infer marker orthology may be challenging and, at times, impact taxonomic conclusions. However, we expect this problem to become less severe as the rapidly growing number of reference genomes provides a better representation of the number and diversity of organismal lineages.

Nonribosomal Peptide Synthetases in Animals.

Nonribosomal peptide synthetases (NRPSs) are a class of cytosolic enzymes that synthesize a range of bio-active secondary metabolites including antibiotics and siderophores. They are widespread among both prokaryotes and eukaryotes but are considered rare among animals. Recently, several novel NRPS genes have been described in nematodes, schistosomes, and arthropods, which led us to investigate how prevalent NRPS genes are in the animal kingdom. We screened 1059 sequenced animal genomes and showed that NRPSs were present in 7 out of the 19 phyla analyzed. A phylogenetic analysis showed that the identified NRPSs form clades distinct from other adenylate-forming enzymes that contain similar domains such as fatty acid synthases. NRPSs show a remarkably scattered distribution over the animal kingdom. They are especially abundant in rotifers and nematodes. In rotifers, we found a large variety of domain architectures and predicted substrates. In the nematode Plectus sambesii, we identified the beta-lactam biosynthesis genes L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase, and deacetoxycephalosporin C synthase that catalyze the formation of beta-lactam antibiotics in fungi and bacteria. These genes are also present in several species of Collembola, but not in other hexapods analyzed so far. In conclusion, our survey showed that NRPS genes are more abundant and widespread in animals than previously known.

Fully automated annotation of mitochondrial genomes using a cluster-based approach with de Bruijn graphs.

A wide range of scientific fields, such as forensics, anthropology, medicine, and molecular evolution, benefits from the analysis of mitogenomic data. With the development of new sequencing technologies, the amount of mitochondrial sequence data to be analyzed has increased exponentially over the last few years. The accurate annotation of mitochondrial DNA is a prerequisite for any mitogenomic comparative analysis. To sustain with the growth of the available mitochondrial sequence data, highly efficient automatic computational methods are, hence, needed. Automatic annotation methods are typically based on databases that contain information about already annotated (and often pre-curated) mitogenomes of different species. However, the existing approaches have several shortcomings: 1) they do not scale well with the size of the database; 2) they do not allow for a fast (and easy) update of the database; and 3) they can only be applied to a relatively small taxonomic subset of all species. Here, we present a novel approach that does not have any of these aforementioned shortcomings, (1), (2), and (3). The reference database of mitogenomes is represented as a richly annotated de Bruijn graph. To generate gene predictions for a new user-supplied mitogenome, the method utilizes a clustering routine that uses the mapping information of the provided sequence to this graph. The method is implemented in a software package called DeGeCI (De Bruijn graph Gene Cluster Identification). For a large set of mitogenomes, for which expert-curated annotations are available, DeGeCI generates gene predictions of high conformity. In a comparative evaluation with MITOS2, a state-of-the-art annotation tool for mitochondrial genomes, DeGeCI shows better database scalability while still matching MITOS2 in terms of result quality and providing a fully automated means to update the underlying database. Moreover, unlike MITOS2, DeGeCI can be run in parallel on several processors to make use of modern multi-processor systems.

Regeneration among animals: An evolutionary hypothesis related to aquatic versus terrestrial environment.

The present hypothesis tries to explain animal regeneration in relation to the life cycles and environment of different animals. Regeneration is a basic phenomenon present since the origin of life in the sea, as testimonial in lower or more complex extant marine animals. Aquatic animals that evolved an indirect development, forming larvae and transiting into the adult stage through metamorphosis, use gene networks present in their genome for these transformations. In case of injury or organ loss as adults, they can re-utilize most or part of the gene networks previously activated during larval growth and metamorphosis. In contrast, terrestrial animals that evolved life cycles with the elimination of larvae and metamorphosis for the adaptation to land conditions lost some of the genes implicated in these post-developmental processes and consequently also the ability to regenerate. Few arthropods and lizards are capable to form hydrated regenerative blastemas with a similar consistence of embryonic tissues. The present hypothesis submits that regeneration cannot be activated in the dry land environment and consequently was largely or completely abolished in terrestrial animals. After injury or organ loss, nematodes, most arthropods and terrestrial vertebrates can only form scars or a limited healing or regengrow in juveniles. This is a process where somatic growth is superimposed to wound healing so that the apparent regeneration derives from the combination from both processes. When full growth is terminated these terrestrial animals can only heal by scarring.

How many metazoan species live in the world's largest mineral exploration region?

The global surge in demand for metals such as cobalt and nickel has created unprecedented interest in deep-sea habitats with mineral resources. The largest area of activity is a 6 million km2 region known as the Clarion-Clipperton Zone (CCZ) in the central and eastern Pacific, regulated by the International Seabed Authority (ISA). Baseline biodiversity knowledge of the region is crucial to effective management of environmental impact from potential deep-sea mining activities, but until recently this has been almost completely lacking. The rapid growth in taxonomic outputs and data availability for the region over the last decade has allowed us to conduct the first comprehensive synthesis of CCZ benthic metazoan biodiversity for all faunal size classes. Here we present the CCZ Checklist, a biodiversity inventory of benthic metazoa vital to future assessments of environmental impacts. An estimated 92% of species identified from the CCZ are new to science (436 named species from a total of 5,578 recorded). This is likely to be an overestimate owing to synonyms in the data but is supported by analysis of recent taxonomic studies suggesting that 88% of species sampled in the region are undescribed. Species richness estimators place total CCZ metazoan benthic diversity at 6,233 (+/-82 SE) species for Chao1, and 7,620 (+/-132 SE) species for Chao2, most likely representing lower bounds of diversity in the region. Although uncertainty in estimates is high, regional syntheses become increasingly possible as comparable datasets accumulate. These will be vital to understanding ecological processes and risks of biodiversity loss.

On the Unknown Proteins of Eukaryotic Proteomes.

To study unknown proteins on a large scale, a reference system has been set up for the three better studied eukaryotic kingdoms, built with 36 proteomes as taxonomically diverse as possible. Proteins from 362 other eukaryotic proteomes with no known homologue in this set were then analyzed, focusing noteworthy on singletons, that is, on such proteins with no known homologue in their own proteome. Consistently, for a given species, no more than 12% of the singletons thus found are known at the protein level, according to Uniprot. In addition, since they rely on the information found in the alignment of homologous sequences, predictions of AlphaFold2 for their tridimensional structure are poor. In the case of metazoan species, the number of singletons rarely exceeds 1000 for the species the closest to the reference system (divergence times below 75 Myr). Interestingly, in the cases of viridiplantae and fungi, larger amounts of singletons are found for such species, as if the timescale on which singletons are added to proteomes were different in metazoa and in other eukaryotic kingdoms. In order to confirm this phenomenon, further studies of proteomes closer to those of the reference system are, however, needed.

The removal of dissolved organic matter by marine sponges is a function of its composition and concentration: An in situ seasonal study of four Mediterranean species.

Sponges are unique among metazoans in their ability to use dissolved organic matter (DOM), the largest pool of organic matter in the ocean, as a major food source. The effect of variations in DOM abundance and composition on its uptake by sponges has rarely been studied. We examined, in situ, the seasonal uptake of DOM by four sponges [2 species with high microbial abundance (HMA) and 2 with low microbial abundance (LMA)] in the northwestern Mediterranean. Dissolved organic carbon (DOC) showed a strong seasonality with 3-fold higher concentrations in fall-winter (256 ± 16 µmol L-1, mean ± SE) than in spring-summer (88 ± 3 µmol L-1). Dissolved organic nitrogen (DON) showed the opposite trend, with higher summer concentrations (8.9 ± 0.4 µmol L-1) and mean concentrations of 2.5-3.4 µmol L-1 in the other seasons. DOC removal by all sponge species increased linearly with its ambient concentration, but only above a DOC removal threshold that was threefold higher in fall-winter (198 µmol L-1) than in spring-summer (74 µmol L-1). All species showed a concentration-dependent DON removal, but LMA sponges removed more DON than HMA sponges. The DOC removal rate (normalized to sponge volume) was 2-3 times higher in fall-winter, when ambient DOC levels were high, than in spring-summer. Sponges efficiently removed clusters of the fluorescent DOM (FDOM) associated with protein-rich DOM, but not those associated with humic material. The clear threshold for DOC removal and the protein-like FDOM uptake pattern suggest that the quality and quantity of DOM control its removal and transformation by marine sponges. Our results indicate that marine sponges transform the composition of the coastal DOM pool, thereby affecting its fate. It is postulated that the DOM excreted by the sponges is more recalcitrant; consequently, sponge activity enhances carbon sequestration in benthic habitats in a similar fashion to that of the oceanic 'microbial pump'.

Habitat type and host grazing regimen influence the soil microbial diversity and communities within potential biting midge larval habitats.

BACKGROUND: Biting midges (Culicoides spp.) are important vectors of diverse microbes such as viruses, protozoa, and nematodes that cause diseases in wild and domestic animals. However, little is known about the role of microbial communities in midge larval habitat utilization in the wild. In this study, we characterized microbial communities (bacterial, protistan, fungal and metazoan) in soils from disturbed (bison and cattle grazed) and undisturbed (non-grazed) pond and spring potential midge larval habitats. We evaluated the influence of habitat and grazing disturbance and their interaction on microbial communities, diversity, presence of midges, and soil properties. RESULTS: Bacterial, protistan, fungal and metazoan community compositions were significantly influenced by habitat and grazing type. Irrespective of habitat and grazing type, soil communities were dominated by phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria (Bacteria); Apicomplexa, Cercozoa, Ciliophora, Ochrophyta (Protists); Chytridiomycota, Cryptomycota (Fungi) and Nematoda, Arthropoda (Metazoa). The relative abundance of Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, Verrucomicrobia (Bacteria); Apicomplexa, Lobosa (Protists); Ascomycota, Blastomycotina, Cryptomycota (Fungi); and Platyhelminthes (Metazoa) were significantly affected by grazing type. Of note, midge prevalence was higher in grazed sites (67-100%) than non-grazed (25%). Presence of midges in the soil was negatively correlated with bacterial, protistan, fungal and metazoan beta diversities and metazoan species richness but positively correlated with protistan and fungal species richness. Moreover, total carbon (TC), nitrogen (TN) and organic matter (OM) were negatively correlated with the presence of midges and relative abundances of unclassified Solirubrobacterales (Bacteria) and Chlamydomonadales (Protists) but positively with Proteobacteria and unclassified Burkholderiales (Bacteria). CONCLUSIONS: Habitat and grazing type shaped the soil bacterial, protistan, fungal and metazoan communities, their compositions and diversities, as well as presence of midges. Soil properties (TN, TC, OM) also influenced soil microbial communities, diversities and the presence of midges. Prevalence of midges mainly in grazed sites indicates that midges prefer to breed and shelter in a habitat with abundant hosts, probably due to greater accessibility of food (blood meals). These results provide a first glimpse into the microbial communities, soil properties and prevalence of midges in suspected midge larval habitats at a protected natural prairie site.

Transcriptome mining extends the host range of the Flaviviridae to non-bilaterians.

The flavivirids (family Flaviviridae) are a group of positive-sense RNA viruses that include well-documented agents of human disease. Despite their importance and ubiquity, the timescale of flavivirid evolution is uncertain. An ancient origin, spanning millions of years, is supported by their presence in both vertebrates and invertebrates and by the identification of a flavivirus-derived endogenous viral element in the peach blossom jellyfish genome (Craspedacusta sowerbii, phylum Cnidaria), implying that the flaviviruses arose early in the evolution of the Metazoa. To date, however, no exogenous flavivirid sequences have been identified in these hosts. To help resolve the antiquity of the Flaviviridae, we mined publicly available transcriptome data across the Metazoa. From this, we expanded the diversity within the family through the identification of 32 novel viral sequences and extended the host range of the pestiviruses to include amphibians, reptiles, and ray-finned fish. Through co-phylogenetic analysis we found cross-species transmission to be the predominate macroevolutionary event across the non-vectored flavivirid genera (median, 68 per cent), including a cross-species transmission event between bats and rodents, although long-term virus-host co-divergence was still a regular occurrence (median, 23 per cent). Notably, we discovered flavivirus-like sequences in basal metazoan species, including the first associated with Cnidaria. This sequence formed a basal lineage to the genus Flavivirus and was closer to arthropod and crustacean flaviviruses than those in the tamanavirus group, which includes a variety of invertebrate and vertebrate viruses. Combined, these data attest to an ancient origin of the flaviviruses, likely close to the emergence of the metazoans 750-800 million years ago.