Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

ABSTRACT North America is a hotspot of freshwater mussel diversity. However, over the last century, many anthropogenic stressors have led to substantial declines in freshwater mussels of the families Unionidae and Margaritiferidae in North America. Conserving the remaining mussel fauna is a priority, as they play an integral role in freshwater ecosystems. The Little River in Arkansas is home to one of the last remaining populations of the federally endangered freshwater mussel, Arcidens wheeleri (Unionidae: Anodontini). Population dynamics information is lacking for A. wheeleri, and no population genetics study has been conducted on this species. A greater understanding of the genetic diversity within a population can serve as a benchmark for developing an effective management plan. We sampled A. wheeleri from three locations in the Little River. Genomic data were generated with a single-enzyme restriction-site-associated DNA sequencing approach to assess genetic diversity and structure of A. wheeleri in the Little River. Genetic structure analyses indicated one genetic population among the three locations, with limited, fine-scale subpopulation structure. Observed heterozygosity values were considerably lower than expected heterozygosity values, with Ho = 0.14 and He = 0.22, likely indicating a genetic bottleneck. Demographic analysis of the Little River population of A. wheeleri also suggests a historical bottleneck. Furthermore, a high inbreeding coefficient (FIS = 0.33) indicates A. wheeleri in the Little River is losing genetic diversity. Data generated indicate considerable risk of extirpation for A. wheeleri from the Little River and should serve as a baseline for future monitoring. Given its high risk of extinction, we recommend increased study of A. wheeleri across its range and on-the-ground conservation actions that include habitat protection and restoration, which are the only options until a successful host fish and protocols are identified for propagation.

Tularemia is a potentially fatal disease caused by the Category A bioterrorism agent Francisella tularensis. Aminoglycosides, fluoroquinolones, and tetracyclines can be used to treat tularemia; however, there is a high incidence of relapse and treatment failures when using these drugs. Furthermore, there is no tularemia vaccine licensed for use in the United States. Therefore, new antibiotics that target F. tularensis are being investigated. A novel family of resazurin-based antibiotics called resazomycins exhibit antimicrobial activity against F. tularensis and other Gram-negative pathogens including Neisseria gonorrhoeae. The mode of action of resazomycins has yet to be determined. To elucidate potential targets of resazurin (Rz), we screened for spontaneous Rz-resistant (Rzr) F. tularensis LVS mutants. Through the screen, 93% of all Rzr mutants sequenced contained mutations within the coding regions of FTL_0421, FTL_0895, and FTL_1504. In addition, 100% of all Rzr mutants sequenced contained a mutation approximately 50bp upstream of the gene FTL_0445. To understand the effect the mutation has on transcription of FTL_0445, RNA was isolated from wild-type LVS and an Rzr mutant (Rzr1). Quantitative reverse transcription PCR revealed FTL_0445 was upregulated in Rzr1 compared to wild-type LVS. To determine the role of FTL_0445 in Rz resistance, a FTL_0445 null deletion mutant is being generated in Rzr1 using standard molecular genetic techniques. Upon completion, the Rz sensitivity of the deletion mutant will be assessed using agar dilution assays. Understanding the role of FTL_0445 in Rz susceptibility would facilitate further development of these compounds as potential treatments for tularemia and gonorrhea.

Many species of bacteria, under conditions of stress or nutrient limitation, enter a state of dormancy referred to as viable but non-culturable (VBNC). VBNC bacteria persist in the environment, are difficult to detect and identify by many standard laboratory methods and can be altered in their susceptibility to antibiotics. Entry into the VBNC state is often accompanied by morphological changes; however, the mechanisms underlying this are poorly understood. Francisella transitions rapidly and spontaneously to a VBNC state, and therefore has the potential to be an excellent model organism for the study of this phenomenon. To investigate the transcriptome of VBNC F. tularensis LVS we extracted RNA from culturable and VBNC cells and carried out an RNA-Seq analysis using both Long-read Nanopore and Illumina Sequencing. Differentially expressed genes (DEGs) were identified using a DE-Seq pipeline. Over 300 genes were significantly upregulated and ~100 genes were down regulated in VBNC cells. Amongst the upregulated genes were some involved in the transport of metals or small molecules, and we have also identified putative transcriptional regulators that may be master controllers of the VBNC process. Our data represents the first transcriptomic analysis of F. tularensis LVS as it transitions into the VBNC state. Identifying genes that are involved in this transition is a critical first step in understanding the mechanisms that drive F. tularensis LVS into the VBNC state and will help us identify factors that allow the resuscitation of these bacteria. This will have important implications for understanding both environmental persistence and pathogenicity of Francisella species. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

The proliferation of genomic sequencing approaches has significantly impacted the field of phylogenetics. Target capture approaches provide a cost-effective, fast, and easily applied strategy for phylogenetic inference of non-model organisms. However, many existing pipelines used to create phylogenomic datasets from target capture data are incapable of incorporating whole genome sequencing data into their workflows. Here, we develop a highly efficient pipeline for capturing and de novo assembly of the targeted regions using whole genome re-sequencing reads. This new pipeline allows capturing targeted loci accurately and efficiently, and given its unbiased nature, can easily be expanded to be used with any other target capture probe set. We demonstrate the utility of our approach by incorporating whole genome sequencing data into a recently developed target capture probe set to reconstruct the evolutionary history of the freshwater mussel family Margaritiferidae, reconstructing supraspecific relationships outside the Unionidae family, providing the first comprehensive multi-loci phylogeny of the Margaritiferidae. We also provide a catalogue of well-curated functional annotations of the targeted regions for the target capture probe set, representing a complementary tool for scrutinizing phylogenetic inferences while expanding future applications of the probe set.

Phylogenomic studies with hundreds or thousands of loci are rare for most invertebrate groups, including freshwater gastropods. This can prevent understanding of phylogeny, which hinders many areas of research. Pleuroceridae is a family of freshwater snails that is highly imperiled and plays an essential role in the ecology of many freshwater systems of the eastern United States. However, the evolutionary history of the family is not understood, and the systematics of the family has not been revised in a modern framework. Pleurocerids display a variety of egg-deposition behaviors and shell shapes, making the family an ideal system for studying evolution of invertebrate life history and morphology. However, past mitochondrial-based phylogenetic analyses have failed to produce meaningful phylogenetic hypotheses, preventing conclusions about pleurocerid systematics and evolution. Here, we generated a novel anchored hybrid enrichment probe set with phylogenetic utility for Pleuroceridae. We sampled pleurocerids from across their range to test the probe set and generated a backbone phylogeny. Our analyses uncovered striking levels of polyphyly among currently accepted genera. Numerous species were also polyphyletic, indicative of unrecognized diversity. Phylogenetic patterns also revealed considerable convergence of shell morphologies. In contrast, anatomical and life history features appeared to be much less homoplastic. Despite generic paraphyly, high support for most major clades and phylogenetic cohesiveness of non-shell characters indicate utility of the AHE probe set for studying pleurocerid evolution.

Freshwater mussels are an important element of freshwater biodiversity and provide essential ecosystem services. However, mussels are among the most imperilled groups of organisms in the world. Although research has increased in recent years, information about range-wide genetic diversity and historical demography of most species is lacking. One such species is Cumberlandian combshell Epioblasma brevidens, which is listed as endangered under the US Endangered Species Act. Genetic diversity of E. brevidens was analysed using a high-resolution RADseq approach and included the previously overlooked Bear Creek population. Hypotheses were tested about population decline, comparative genetic diversity and population structure with model-based approaches enabled by a genome-scale dataset. Estimates of genetic differentiation among populations of E. brevidens were lower than past analyses, suggesting higher historical population connectivity than previously known. Demographic analyses indicate relatively recent splits among E. brevidens populations in the late Pleistocene to early Holocene, with clear founder effects in two populations. The Clinch River population has the highest genetic diversity and effective population size, despite demographic analyses revealing decline of this population since the Pleistocene. Analyses of both population structure and migration show evidence of past gene flow, but all populations are currently isolated by artificial barriers. Analyses indicate that populations began to decline before industrialization, but fragmentation and population extirpation has been exacerbated by modern habitat destruction. Relatively high genetic diversity in the Bear Creek population indicates that water quality improvements in the last 20 years have had a positive impact on population viability, offering promise for targeted management actions. In contrast, the Big South Fork population that has been presumed stable showed low genetic diversity and effective population size. Furthermore, genetic structure among sampled populations indicates that reintroduction efforts should use broodstock from as close to the reintroduction site as possible.

Resistance to antibiotic treatments coupled with the decline in antibiotic discovery has resulted in a steady increase in deaths caused by once “curable” bacterial infections. Developing new drugs is crucial to prevent more loss of life in the future. We discovered the compound resazurin exhibits antimicrobial activity against gram-negative bacteria including Francisella tularensis (Ft), however, certain strains of Ft have developed resistance to resazurin. Understanding how Ft develops resistance to resazurin will help with defining the mechanism by which resazurin elicits its antimicrobial effect. Whole genome sequencing of resazurin-resistant (Rzr) Ft LVS mutants revealed four mutations found in 93% of the isolates sequenced. Three mutations were within the coding regions of FTL_0421, FTL_0895, and FTL_1504 and the other mutation was 50 bp upstream of FTL_0445, likely disrupting expression of this gene. The focus of my project was to explore the role of FTL_0895 in resazurin susceptibility. To confirm this gene plays a role in the reduced susceptibility of the Rzr strains to resazurin, we cloned the wild-type copy of FTL_0895 into the Francisella vector pABST which contains the robust groE promotor of Ft. The resulting plasmid will be electroporated into one of the Rzr mutants and we will test the susceptibility of the complemented strain to resazurin, using time kill and agar dilution assays. If the susceptibility of the complemented strain to resazurin is restored, then it can be determined that FTL_0895 is a potential target of resazurin.

North American minnows of the Shiner Clade, within the family Leuciscidae, represent one of the most taxonomically complex clades of the order Cypriniformes due to the large number of taxa coupled with conserved morphologies. Species within this clade were moved between genera and subgenera until the community decided to lump many of the unclassified taxa with similar morphologies into one genus, Notropis, which has held up to 325 species. Despite phylogentic studies that began to re-elevate some genera merged into Notropis, such as Cyprinella, Luxilus, Lythrurus, and Pteronotropis, the large genus Notropis remained as a taxonomic repository for many shiners of uncertain placement. Recent molecular advances in sequencing technologies have provided the opportunity to re-examine the Shiner Clade using phylogenomic markers. Using a fish probe kit, we sequenced 90 specimens in 87 species representing 16 genera included in the Shiner Clade, with a resulting dataset of 1,004 loci and 286,455 base pairs. Despite the large dataset, only 32,349 bp (11.29%) were phylogenetically informative. In our maximum likelihood tree, 78% of nodes are 100% bootstrap supported demonstrating the utility of the phylogenomic markers at lower taxonomic levels. Unsurprisingly, species within Notropis as well as Hudsonius, Luxilus, and Alburnops are not resolved as monophyletic groups. Cyprinella is monophyletic if Cyprinella callistia is excluded, and Pteronotropis is monophyletic if it includes Hudsonius cummingsae. Taxonomic changes we propose are: restriction of species included in Alburnops and Notropis, elevation of the subgenus Hydrophlox, expansion of species included in Miniellus, movement of Hudsonius cummingsae to Pteronotropis, and resurrection of the genera Coccotis and Paranotropis. We additionally had two specimens of three species, Notropis atherinoides, Ericymba amplamala, and Pimephales vigilax and found signficant differences between the localities (1,086, 1,424, and 845 nucleotides respectively).

Non-indigenous species are a significant threat to freshwater biodiversity. In the Potomac Basin there are several known invasive crayfish species including F. virilis (virile crayfish) and F.rusticus (rusty crayfish). It is imperative to discover whether F. obscurus is in fact an invasive crayfish that needs a management plan or simply another native species that needs protection. F. obscurus is a wide-ranging crayfish species that is native to the northeastern United States. Populations found in the Atlantic Slope Drainage basins are considered invasive. Using CO1 and 16S primers, we will be creating a range-wide haplotype map to investigate these invasions.

Chelodina mccordi is a small freshwater turtle native to the islands of Roti and Timor in Indonesia. They are considered critically endangered according to the IUCN Red List due primarily to habitat loss and overcollection. Current conservation efforts aim to breed and release them into existing native populations to improve genetic pool quality and heterozygosity, although captive populations are extremely limited. Our study aims to determine the level of relatedness between six C. mccordi individuals held at the Audubon Zoo in New Orleans. Genetic markers indicate which individuals are siblings; this information will allow the Zoo to efficiently manage turtle breeding pairs to yield desired genetic diversity. DNA was extracted from each sample (n=6) and the nuclear marker R35x1 (RNA fingerprint protein 35, exon 1) was amplified. PCR product was sequenced using Sanger sequencing and 1,060 base pairs were recovered after assembly, multiple sequence alignment, and trimming. Across the entire alignment, individuals differed at only 4 sites with no clear pattern of relatedness. Based on these results, there is not enough variation in the exon region that was sequenced to confidently confirm the sibling status. Future directions of this project will involve sequencing a more variable intron region or using a Restriction Fragment Length Polymorphism (RFLP) method, if necessary. This research was carried out with financial support from the West Liberty University Biology and Zoo Science department.

Background Mygalomorph spiders represent a diverse, yet understudied lineage for which genomic level data has only recently become accessible through high-throughput genomic and transcriptomic sequencing methods. The Aptostichus atomarius species complex (family Euctenizidae) includes two coastal dune endemic members, each with inland sister species – affording exploration of dune adaptation associated patterns at the transcriptomic level. We apply an RNAseq approach to examine gene family conservation across the species complex and test for patterns of positive selection along branches leading to dune endemic species. Results An average of ~ 44,000 contigs were assembled for eight spiders representing dune ( n = 2), inland ( n = 4), and atomarius species complex outgroup taxa ( n = 2). Transcriptomes were estimated to be 64% complete on average with 77 spider reference orthologs missing from all taxa. Over 18,000 orthologous gene clusters were identified within the atomarius complex members, > 5000 were detected in all species, and ~ 4700 were shared between species complex members and outgroup Aptostichus species. Gene family analysis with the FUSTr pipeline identified 47 gene families appearing to be under selection in the atomarius ingroup; four of the five top clusters include sequences strongly resembling other arthropod venom peptides. The COATS pipeline identified six gene clusters under positive selection on branches leading to dune species, three of which reflected the preferred species tree. Genes under selection were identified as Cytochrome P450 2c15 (also recovered in the FUSTr analysis), Niemann 2 Pick C1-like, and Kainate 2 isoform X1. Conclusions We have generated eight draft transcriptomes for a closely related and ecologically diverse group of trapdoor spiders, identifying venom gene families potentially under selection across the Aptostichus atomarius complex and chemosensory-associated gene families under selection in dune endemic lineages.

Many freshwater gastropod species face extinction, including 79% of species in the family Pleuroceridae. The Oblong Rocksnail, Leptoxis compacta, is a narrow range endemic pleurocerid from the Cahaba River basin in central Alabama that has seen rapid range contraction in the last 100 years. Such a decline is expected to negatively affect genetic diversity in the species. However, precise patterns of genetic variation and gene flow across the restricted range of L. compacta are unknown. This lack of information limits our understanding of human impacts on the Cahaba River system and Pleuroceridae. Here, we show that L. compacta has likely seen a species-wide decline in genetic diversity, but remaining populations have relatively high genetic diversity. We also report a contemporary range extension compared to the last published survey. Our findings indicate that historical range contraction has resulted in the absence of common genetic patterns seen in many riverine taxa like isolation by distance as the small distribution of L. compacta allows for relatively unrestricted gene flow across its remaining range despite limited dispersal abilities. Two collection sites had higher genetic diversity than others, and broodstock sites for future captive propagation and reintroduction efforts should utilize sites identified here as having the highest genetic diversity. Broadly, our results support the hypothesis that range contraction will result in the reduction of species-wide genetic diversity, and common riverscape genetic patterns cannot be assumed to be present in species facing extinction risk.

The mygalomorph family Ctenizidae has a world-wide distribution and currently contains nine genera and 135 species. However, the monophyly of this group has long been questioned on both morphological and molecular grounds. Here, we use Anchored Hybrid Enrichment (AHE) to gather hundreds of loci from across the genome for reconstructing the phylogenetic relationships among the nine genera and test the monophyly of the family. We also reconstruct the possible ancestral ranges of the most inclusive clade recovered. Using AHE, we generate a supermatrix of 565 loci and 115,209 bp for 27 individuals. For the first time, analyses using all nine genera produce results definitively establishing the non-monophyly of Ctenizidae. A lineage formed exclusively by representatives of South African Stasimopus was placed as the sister group to the remaining taxa in the tree, and the Mediterranean Cteniza and Cyrtocarenum were recovered with high support as sister to exemplars of Euctenizidae, Migidae, and Idiopidae. All the remaining genera-Bothriocyrtum, Conothele, Cyclocosmia, Hebestatis, Latouchia, and Ummidia-share a common ancestor. Based on these results, we formally elevate this clade to the level of family. Our results definitively establish both the non-monophyly of the Ctenizidae and non-validity of the subfamilies Ummidiinae and Ctenizinae. In order to establish the placement of the remaining three ctenizid genera, Cteniza, Cyrtocarenum, and Stasimopus, thorough analyses within the context of a complete mygalomorph phylogenetic framework are needed. We formally describe the family Halonoproctidae Pocock 1901 and infer that the family's most recent common ancestor was likely distributed in western North America and Asia.

Phylogeographic studies frequently result in the elevation of subspecific taxa to species given monophyly, or the synonymy of subspecies that are not monophyletic. However, given limited or incongruent datasets, retention of subspecies can be useful to describe hypothesized incipient species or to illustrate interesting biological phenomena driving morphological diversity. Four subspecific taxa have been used to describe largely allopatric geographic variation within the species Pseudotriton ruber, a plethodontid salamander occupying stream and spring habitats across eastern North America: P. r. vioscai occurs in lowland Coastal Plain habitats, while P. r. ruber, P. r. nitidus, and P. r. schencki occupy upland regions in and around the Appalachian Mountains. Pseudotriton ruber co-occurs through its distribution with the aposematic newt Notophthalmus viridescens, and both species are hypothesized to be part of a Müllerian mimicry complex. In this study, we sequenced regions of two mitochondrial (cytochrome b, NADH dehydrogenase subunit 2) and one single copy nuclear protein-coding gene (pro-opiomelanocortin) from individuals sampled across much of the distribution of P. ruber and then used maximum-likelihood and Bayesian phylogenetic inference to test the monophyly of subspecies, reconstruct biogeographic history, and make inferences about morphological evolution. Phylogeographic hypotheses from mitochondrial and nuclear datasets described structure among populations of P. ruber which separated Coastal Plain and upland Appalachian populations, but subspecies were not monophyletic. Biogeographic reconstruction estimated the ancestor of all populations to have occupied and initially diverged in the Coastal Plain during the Pliocene (∼3.6mya), before one lineage subsequently invaded upland areas of Appalachia. Bold bright coloration of high elevation subspecies P. r. nitidus and P. r. schencki appears to have evolved twice. We hypothesize that the Müllerian mimicry complex with N. viridescens and P. ruber may provide a selective mechanism driving the co-evolution of striking bright and dull morphological variation among populations of both species. While P. ruber subspecies were not consistent with our criteria for diagnosing species (monophyly) and therefore could not be elevated to species, we advocate for the retention of subspecies because they describe hypotheses about an incipient species (P. r. vioscai) and how Müllerian mimicry may shape morphological diversity of species.

Spiders (Order Araneae) are massively abundant generalist arthropod predators that are found in nearly every ecosystem on the planet and have persisted for over 380 million years. Spiders have long served as evolutionary models for studying complex mating and web spinning behaviors, key innovation and adaptive radiation hypotheses, and have been inspiration for important theories like sexual selection by female choice. Unfortunately, past major attempts to reconstruct spider phylogeny typically employing the “usual suspect” genes have been unable to produce a well-supported phylogenetic framework for the entire order. To further resolve spider evolutionary relationships we have assembled a transcriptome-based data set comprising 70 ingroup spider taxa. Using maximum likelihood and shortcut coalescence-based approaches, we analyze eight data sets, the largest of which contains 3,398 gene regions and 696,652 amino acid sites forming the largest phylogenomic analysis of spider relationships produced to date. Contrary to long held beliefs that the orb web is the crowning achievement of spider evolution, ancestral state reconstructions of web type support a phylogenetically ancient origin of the orb web, and diversification analyses show that the mostly ground-dwelling, web-less RTA clade diversified faster than orb weavers. Consistent with molecular dating estimates we report herein, this may reflect a major increase in biomass of non-flying insects during the Cretaceous Terrestrial Revolution 125–90 million years ago favoring diversification of spiders that feed on cursorial rather than flying prey. Our results also have major implications for our understanding of spider systematics. Phylogenomic analyses corroborate several well-accepted high level groupings: Opisthothele, Mygalomorphae, Atypoidina, Avicularoidea, Theraphosoidina, Araneomorphae, Entelegynae, Araneoidea, the RTA clade, Dionycha and the Lycosoidea. Alternatively, our results challenge the monophyly of Eresoidea, Orbiculariae, and Deinopoidea. The composition of the major paleocribellate and neocribellate clades, the basal divisions of Araneomorphae, appear to be falsified. Traditional Haplogynae is in need of revision, as our findings appear to support the newly conceived concept of Synspermiata. The sister pairing of filistatids with hypochilids implies that some peculiar features of each family may in fact be synapomorphic for the pair. Leptonetids now are seen as a possible sister group to the Entelegynae, illustrating possible intermediates in the evolution of the more complex entelegyne genitalic condition, spinning organs and respiratory organs.

Spiders represent an ancient predatory lineage known for their extraordinary biomaterials, including venoms and silks. These adaptations make spiders key arthropod predators in most terrestrial ecosystems. Despite ecological, biomedical, and biomaterial importance, relationships among major spider lineages remain unresolved or poorly supported. Current working hypotheses for a spider "backbone" phylogeny are largely based on morphological evidence, as most molecular markers currently employed are generally inadequate for resolving deeper-level relationships. We present here a phylogenomic analysis of spiders including taxa representing all major spider lineages. Our robust phylogenetic hypothesis recovers some fundamental and uncontroversial spider clades, but rejects the prevailing paradigm of a monophyletic Orbiculariae, the most diverse lineage, containing orb-weaving spiders. Based on our results, the orb web either evolved much earlier than previously hypothesized and is ancestral for a majority of spiders or else it has multiple independent origins, as hypothesized by precladistic authors. Cribellate deinopoid orb weavers that use mechanically adhesive silk are more closely related to a diverse clade of mostly webless spiders than to the araneoid orb-weaving spiders that use adhesive droplet silks. The fundamental shift in our understanding of spider phylogeny proposed here has broad implications for interpreting the evolution of spiders, their remarkable biomaterials, and a key extended phenotype--the spider web.