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Abstract Background Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two human neglected tropical diseases that cause major disabilities. Mass administration of drugs targeting the microfilarial stage has reduced transmission and eliminated these diseases in several countries but a macrofilaricidal drug that kills or sterilizes the adult worms is critically needed to eradicate the diseases. The causative agents of onchocerciasis and lymphatic filariasis are filarial worms that harbor the endosymbiotic bacterium Wolbachia. Because filarial worms depend on Wolbachia for reproduction and survival, drugs targeting Wolbachia hold great promise as a means to eliminate these diseases. Methods To better understand the relationship between Wolbachia and its worm host, adult Brugia pahangi were exposed to varying concentrations of doxycycline, minocycline, tetracycline and rifampicin in vitro and assessed for Wolbachia numbers and worm motility. Worm motility was monitored using the Worminator system, and Wolbachia titers were assessed by qPCR of the single copy gene wsp from Wolbachia and gst from Brugia to calculate IC50s and in time course experiments. Confocal microscopy was also used to quantify Wolbachia located at the distal tip region of worm ovaries to assess the effects of antibiotic treatment in this region of the worm where Wolbachia are transmitted vertically to the microfilarial stage. Results Worms treated with higher concentrations of antibiotics had higher Wolbachia titers, i.e. as antibiotic concentrations increased there was a corresponding increase in Wolbachia titers. As the concentration of antibiotic increased, worms stopped moving and never recovered despite maintaining Wolbachia titers comparable to controls. Thus, worms were rendered moribund by the higher concentrations of antibiotics but Wolbachia persisted suggesting that these antibiotics may act directly on the worms at high concentration. Surprisingly, in contrast to these results, antibiotics given at low concentrations reduced Wolbachia titers. Conclusion Wolbachia in B. pahangi display a counterintuitive dose response known as the “Eagle effect.” This effect in Wolbachia suggests a common underlying mechanism that allows diverse bacterial and fungal species to persist despite exposure to high concentrations of antimicrobial compounds. To our knowledge this is the first report of this phenomenon occurring in an intracellular endosymbiont, Wolbachia, in its filarial host.

Wolbachia is a genus containing obligate, intracellular endosymbionts with arthropod and nematode hosts. Numerous studies have identified differentially expressed transcripts in Wolbachia endosymbionts that potentially inform the biological interplay between these endosymbionts and their hosts, albeit with discordant results. Here, we re-analyze previously published Wolbachia RNA-Seq transcriptomics data sets using a single workflow consisting of the most up-to-date algorithms and techniques, with the aim of identifying trends or patterns in the pan-Wolbachia transcriptional response. We find that data from one of the early studies in filarial nematodes did not allow for robust conclusions about Wolbachia differential expression with these methods, suggesting the original interpretations should be reconsidered. Across datasets analyzed with this unified workflow, there is a general lack of global gene regulation with the exception of a weak transcriptional response resulting in the upregulation of ribosomal proteins in early larval stages. This weak response is observed across diverse Wolbachia strains from both nematode and insect hosts suggesting a potential pan-Wolbachia transcriptional response during host development that diverged more than 700 million years ago.

Abstract Background Wolbachia are the most widely spread endosymbiotic bacteria, present in a wide variety of insects and two families of nematodes. As of now, however, relatively little genomic data has been available. The Wolbachia symbiont can be parasitic, as described for many arthropod systems, an obligate mutualist, as in filarial nematodes or a combination of both in some organisms. They are currently classified into 16 monophyletic lineage groups (“supergroups”). Although the nature of these symbioses remains largely unknown, expanded Wolbachia genomic data will contribute to understanding their diverse symbiotic mechanisms and evolution. Results This report focuses on Wolbachia infections in three pseudoscorpion species infected by two distinct groups of Wolbachia strains, based upon multi-locus phylogenies. Geogarypus minor harbours wGmin and Chthonius ischnocheles harbours wCisc, both closely related to supergroup H, while Atemnus politus harbours wApol, a member of a novel supergroup S along with Wolbachia from the pseudoscorpion Cordylochernes scorpioides (wCsco). Wolbachia supergroup S is most closely related to Wolbachia supergroups C and F. Using target enrichment by hybridization with Wolbachia-specific biotinylated probes to capture large fragments of Wolbachia DNA, we produced two draft genomes of wApol. Annotation of wApol highlights presence of a biotin operon, which is incomplete in many sequenced Wolbachia genomes. Conclusions The present study highlights at least two symbiont acquisition events among pseudoscorpion species. Phylogenomic analysis indicates that the Wolbachia from Atemnus politus (wApol), forms a separate supergroup (“S”) with the Wolbachia from Cordylochernes scorpioides (wCsco). Interestingly, the biotin operon, present in wApol, appears to have been horizontally transferred multiple times along Wolbachia evolutionary history.

ABSTRACT Endosymbiotic Wolbachia bacteria are known to influence the host physiology, microbiota composition, and dissemination of pathogens. We surveyed a population of Tabanus nigrovittatus, commonly referred to as “greenheads,” from Crane Beach (Ipswich, MA, USA) for the presence of the alphaproteobacterial symbiont Wolbachia. We studied the COI (mitochondrial cytochrome oxidase) marker gene to evaluate the phylogenetic diversity of the studied specimens. The DNA sequences show strong similarity (between 99.9 and 98%) among the collected specimens but lower similarity to closely related entries in the NCBI database (only between 96.3 and 94.7%), suggesting a more distant relatedness. Low levels of Wolbachia presence necessitated a nested PCR approach, and using 5 markers (ftsZ, fbpA, dnaA, coxA, and gatB), we determined that two recognized “supergroups” of Wolbachia species were represented in the studied specimens, members of clades A and B. Using next-generation sequencing, we also surveyed the insect gut microbiomes of a subset of flies, using Illumina and PacBio 16S rRNA gene sequencing with barcoded primers. The composition of Proteobacteria also varied from fly to fly, with components belonging to Gammaproteobacteria making up the largest percentage of organisms (30 to 70%) among the microbiome samples. Most of the samples showed the presence of Spiroplasma, a member of the phylum Mollicutes, although the frequency of its presence was variable, ranging from 2 to 57%. Another noteworthy bacterial phylum consistently identified was Firmicutes, though the read abundances were typically below 10%. Of interest is an association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes, suggesting that the presence of Wolbachia affects the host microbiome. IMPORTANCE Tabanus nigrovittatus greenhead populations contain two supergroups of Wolbachia endosymbionts, members of supergroups A and B. Analysis of the greenhead microbiome using next-generation sequencing revealed that the majority of bacterial species detected belonged to Gammaproteobacteria, with most of the samples also showing the presence of Spiroplasma, a member of the Mollicutes phylum also known to infect insects. An association between Wolbachia presence and higher Alphaproteobacteria representation in the microbiomes suggests that Wolbachia presence affects the host microbiome composition.

Parasitic nematodes causing onchocerciasis and lymphatic filariasis rely on a bacterial endosymbiont, Wolbachia, which is a validated therapeutic target. Here, Clare et al. perform a high-throughput screen of 1.3 million compounds and identify 5 chemotypes with faster kill rates than existing anti-Wolbachia drugs.

Abstract Lateral gene transfer (LGT) from microbial symbionts to invertebrate animals is described at an increasing rate, particularly between Wolbachia endosymbionts and their diverse invertebrate hosts. We sought to assess the use of a capture system to cost-effectively sequence such LGT from the host genome. The sequencing depth of Illumina paired end data obtained with a Wolbachia capture system correlated well with that for an Illumina paired end data set used to detect LGT in Wolbachia-depleted B. malayi (p-value:

ABSTRACT Endosymbiotic Wolbachia bacteria and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, life span, stem cell activity, and resistance to human pathogens, in insect vectors. This work shows that Wolbachia bacteria, which reside mainly in the fly germline, affect the microbial species present in the fly gut in a lab-reared strain. Drosophila melanogaster hosts two main genera of commensal bacteria—Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Sampling of the microbiome of axenic flies fed with equal proportions of both bacteria shows that the presence of Wolbachia bacteria is a significant determinant of the composition of the microbiome throughout fly development. However, this effect is host genotype dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia bacteria on Imd and reactive oxygen species pathways, the main regulators of immune response in the fly gut, was measured. The presence of Wolbachia bacteria does not induce significant changes in the expression of the genes for the effector molecules in either pathway. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia bacteria and gut microbes. Confocal analysis shows that Wolbachia bacteria are absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of composition of the microbiome by Wolbachia bacteria is more complex than a direct bacterial interaction or the effect of Wolbachia bacteria on fly immunity. The findings reported here highlight the importance of considering the composition of the gut microbiome and host genetic background during Wolbachia-induced phenotypic studies and when formulating microbe-based disease vector control strategies. IMPORTANCE Wolbachia bacteria are intracellular bacteria present in the microbiome of a large fraction of insects and parasitic nematodes. They can block mosquitos’ ability to transmit several infectious disease-causing pathogens, including Zika, dengue, chikungunya, and West Nile viruses and malaria parasites. Certain extracellular bacteria present in the gut lumen of these insects can also block pathogen transmission. However, our understanding of interactions between Wolbachia and gut bacteria and how they influence each other is limited. Here we show that the presence of Wolbachia strain wMel changes the composition of gut commensal bacteria in the fruit fly. Our findings implicate interactions between bacterial species as a key factor in determining the overall composition of the microbiome and thus reveal new paradigms to consider in the development of disease control strategies.

Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.

Abstract Background Wolbachia endosymbionts are a proven target for control of human disease caused by filarial nematodes. However, little is known about the occurrence of Wolbachia in taxa closely related to the superfamily Filarioidea. Our study addressed the status of Wolbachia presence in members of the superfamily Dracunculoidea by screening the human parasite Dracunculus medinensis and related species from wildlife for Wolbachia. Findings D. medinensis, D. lutrae and D. insignis specimens were all negative for Wolbachia colonization by PCR screening for the Wolbachia ftsZ, 16S rRNA and Wolbachia surface protein (wsp) sequences. The quality and purity of the DNA preparations was confirmed by amplification of nematode 18S rRNA and cytochrome c oxidase subunit I sequences. Furthermore, Wolbachia endobacteria were not detected by whole mount fluorescence staining, or by immunohistochemistry using a Wolbachia-specific antiserum. In contrast, positive control Brugia malayi worms were shown to harbour Wolbachia by PCR, fluorescence staining and immunohistochemistry. Conclusions Three examined species of Dracunculus showed no evidence of Wolbachia endobacteria. This supports that members of the superfamily Dracunculoidea are free of Wolbachia. Within the order Spirurida, these endosymbionts appear restricted to the Filarioidea.

An innovation is one of those things that society looks at and says, if we make this part of the way we live and work, it will change the way we live and work. —Dean Kamen One of my primary objects is to form the tools so the tools themselves shall fashion the work and give to every part its just proportion. —Eli Whitney It's what you learn after you know it all that counts. —Harry S. Truman Since their initial development nearly 20 years ago, molecular biology kits have evolved from simple protocols and reagents for cloning of DNA to the more recent complex reagent sets that enable whole genomic sequencing. Initially met with resistance by some who felt that using them deprived researchers of the basic knowledge of how to create reagents, molecular biology kits have taken on an important role in the biological sciences. In this article we describe kit development, why kits have succeeded in molecular biology, and how they have paved the way for the more recent widespread use of core facilities.

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.

Wolbachia are among the most abundant symbiotic microbes on earth; they are present in about 66% of all insect species, some spiders, mites and crustaceans, and most filarial nematode species. Infected filarial nematodes, including many pathogens of medical and veterinary importance, depend on Wolbachia for proper development and survival. The mechanisms behind this interdependence are not understood. Interestingly, a minority of filarial species examined to date are naturally Wolbachia-free.We used 454 pyrosequencing to survey the genomes of two distantly related Wolbachia-free filarial species, Acanthocheilonema viteae and Onchocerca flexuosa. This screen identified 49 Wolbachia-like DNA sequences in A. viteae and 114 in O. flexuosa. qRT-PCR reactions detected expression of 30 Wolbachia-like sequences in A. viteae and 56 in O. flexuosa. Approximately half of these appear to be transcribed from pseudogenes. In situ hybridization showed that two of these pseudogene transcripts were specifically expressed in developing embryos and testes of both species.These results strongly suggest that the last common ancestor of extant filarial nematodes was infected with Wolbachia and that this former endosymbiont contributed to their genome evolution. Horizontally transferred Wolbachia DNA may explain the ability of some filarial species to live and reproduce without the endosymbiont while other species cannot.

Context: "Tensegrity" is a structural-organization model initially described in the architecture and design fields. By applying tensegrity design principles to biological structures, scientists have developed "biotensegrity" to explain a complex systems-on-systems structural-organization philosophy for integrated human movements. Objective: To provide a brief historical overview of tensegrity and biotensegrity principles, including recommendations and benefits for integrating these structural models into athletic training education. Background: Tensegrity and biotensegrity structures require constant interaction between continuous tension and discontinuous compression elements that connect through focal adhesion points. During the 1970s and 1980s, scientists applied tensegrity concepts to biological organisms to create an integrated model of human structure and interaction. Since then, biotensegrity has grown as an accepted biological structural model capable of explaining complex and integrated human movements. Synthesis: By teaching tensegrity and biotensegrity principles, athletic training educators can provide athletic training students with a basic and consistent human body structural model. With this knowledge, students can better comprehend the integrated kinetic chain, including current and future prevention, examination, and rehabilitation paradigms. Results: Although absent from the Practice Analysis, seventh edition, and the 2020 Commission on Accreditation of Athletic Training Education curricular content standards, tensegrity and biotensegrity relate to many injury prevention, examination, treatment, and rehabilitation concepts regularly taught in professional athletic training programs. Recommendation(s): Athletic training educators should consider ways to incorporate biotensegrity models into professional athletic training programs to improve critical thinking and whole-person health care principles of athletic training students. Conclusion(s): Integrating tensegrity and biotensegrity principles into professional athletic training programs provides a structural hierarchy of human body organization that athletic training students can apply to a multitude of current and future methodical approaches.

Context: Advanced clinical practice is inherent in contemporary athletic training education, such as residency programs and Doctor of Athletic Training programs; however, as a concept, advanced clinical practice in athletic training has been poorly studied to date. Objective: To explore athletic trainers' perceptions of advanced clinical practice. Design: Cross-sectional. Setting: Online survey with open-ended questions. Patients or Other Participants: Three hundred fifty of 1992 athletic trainers accessed the survey (17.6% access rate); 321 respondents completed at least 1 open-ended question; and 196 completed the survey in its entirety (61.1% completion rate). Intervention(s): We used a 13-item survey including demographic items (9 items) and open-ended response questions (4 items). Main Outcome Measure(s): Guided by the consensual qualitative research approach, a 3-person data analysis team coded the open-ended responses. Each member coded 50 responses and a consensus codebook was developed. Two members of the team coded the remaining responses, which were confirmed by the third member. Emergent data were organized into themes and categories, and frequency counts were determined for each category. Results: Athletic trainers' definitions of advanced clinical practice were categorized into 4 emergent categories: (1) formal training and education; (2) informal training and education; (3) knowledge, skills, and behaviors; and (4) experience and uncertainty. Conclusions: The categories of formal and informal training and education focused on athletic trainers acquiring additional knowledge and skills through mechanisms such as postprofessional degree programs, residency programs, or other areas of study. The knowledge, skills, and behaviors category included areas related to specialized skills and the core competencies. These 3 categories aligned with one another to provide both the types of knowledge, skills, and behaviors that define advanced clinical practice, and the specific mechanisms through which an athletic trainer can achieve advanced clinical practice.

Context: The ''Strategic Priorities for Athletic Training Education'' promote the development of clinical specialists and advanced practice leaders; however, little is known about the characteristics of advanced practice athletic trainers. Peer health professions have formal education and training programs and specialized credentials to denote advanced practice clinicians; however, such mechanisms are only now emerging in athletic training. As training and credentialing programs advance, it is important to understand the perceived characteristics of clinicians engaged in advanced clinical practice. Objective: To explore how athletic trainers perceive the characteristics of clinicians advanced practice clinicians. Design: Cross-sectional. Setting: Self-reported online survey. Participants: Of 1992 athletic trainers invited to participate, 350 accessed the survey (17.6% access rate); 321 respondents completed at least 1 open-ended question, and 196 completed the entire survey (61.1% completion rate). Main Outcome Measures: Consensual qualitative research approach consisting of a 3-person team was used to analyze the open-ended responses. Members individually coded the initial set of responses and then met to develop a consensus codebook. Remaining responses were coded by 2 team members and were confirmed by the third member. Data were organized into themes and categories, and frequency counts were used for each category. Results: Four categories of characteristics associated with advanced practice clinicians emerged: (1) intrapersonal skills, (2) interpersonal skills, (3) discipline specific knowledge and skills, and (4) experience. Conclusions: Intrapersonal skills including lifelong learning, critical thinking, and willingness to mentor others were identified, in addition to strong communication and leadership skills. Discipline-specific knowledge and skills were also associated with advanced clinical practice, including specialized skills and advanced integration of the core competencies. Accredited Athletic Training Residency programs and Doctor of Athletic Training programs should strive to foster these characteristics within their students to facilitate the development of advanced practice athletic trainers.

Context: The use of point-of-care ultrasound (POCUS) is increasingly becoming a standard of care for sports medicine and orthopedic physicians. As such, there will be increased demand for athletic trainers to have knowledge and skills in diagnostic ultrasound. Objective: To provide key considerations for integration of diagnostic ultrasound into athletic training education and address challenges and barriers to such integration. Background: Advancements in designing portable, user-friendly ultrasound units have enabled clinicians to perform POCUS as an adjunct to the physical exam, resulting in improved patient care. Increased demands for POCUS across medicine have prompted medical schools to integrate diagnostic ultrasound throughout the curriculum, providing athletic training education programs a framework to consider within their curriculum. Synthesis: Integrating diagnostic ultrasound throughout the curriculum provides focused experiences to prepare, conduct, and link imaging to clinical and physical exam skills. Diagnostic ultrasound has a role in visualizing structures in an anatomy course, as an adjunct to physical exam in medical conditions, orthopedic exam, and diagnosis courses, and as a visual feedback tool in rehabilitation courses, thus enabling it to be integrated throughout existing curriculum. Barriers to diagnostic ultrasound include cost, time, and faculty expertise. Results: Cost sharing or use of ultrasound simulators can overcome cost barriers. Use of online educational modules to deliver content virtually is effective and allows face-to-face time to be spent in hands-on experiential learning. Finally, developing interprofessional partnerships to ''teach the teacher'' is an effective model in teaching faculty about diagnostic ultrasound. Recommendation(s): Educational programs should consider ways to overcome barriers and integrate diagnostic ultrasound into curriculum equipping future athletic trainers with knowledge and skills for POCUS, thus increasing their effectiveness on a health care team. Conclusion(s): Proactive integration of diagnostic ultrasound into athletic training education will equip graduates to add value to a health care team.

Context: The use of point-of-care diagnostic ultrasound is increasing in health care, specifically among sports medicine clinicians as an adjunct to the physical exam. Given the role of athletic trainers in interdisciplinary sports medicine teams, athletic training educational programs should consider integrating this noninvasive imaging modality into curricula. Objective: To provide a framework for integrating diagnostic ultrasound imaging content into existing athletic training curricula. Background: A phased approach to incorporating ultrasound imaging into existing courses with minimal disruption is important for adoption. Foundational knowledge for skill performance begins with early exposure to ultrasound concepts and is followed by phased integration of hands-on ultrasound imaging into athletic training courses. Description: Content delivery considerations, such as online modules and technology needs, to enhance hands-on learning is discussed. Examples of integrating diagnostic ultrasound imaging throughout the curriculum, including anatomy, clinical assessment, and manual therapy courses, are provided. Clinical Advantage(s): Integrating ultrasound throughout curricula teaches students how to use and interpret ultrasound images as an adjunct to physical exam, enhancing the athletic trainer's value on a health care team and improving clinical practice. Ultrasound imaging can also be used as a valuable feedback mechanism during the performance of hands-on athletic training skills, including special tissue tests and manual therapy techniques. Conclusion(s): Following initial exposure to ultrasound imaging, the inclusion of diagnostic ultrasound instruction in athletic training curricula can expose students to ultrasound imaging, basic concepts, transducer characteristics, and image interpretation, which is a valuable adjunct to clinical practice.