Kristopher Silver

Extracellular vesicles (EVs) are nanoscale, lipid-bound structures released by cells across all domains of life. Once viewed as a means for discarding unwanted cellular components, they are now understood to be central mediators of intercellular communication. Much of what is known about EVs comes from mammalian systems, where extensive work has defined the major EV subtypes and the mechanisms that generate exosomes. These findings continue to serve as the primary reference for interpreting EV biology in other organisms. EVs have been isolated from diverse arthropods, including a few insect and tick species, yet the molecular pathways that produce them remain less characterized. Current evidence from Drosophila, mosquito cell lines, and tick systems shows that many of the core components driving exosome biogenesis in mammals, including ESCRT complexes, tetraspanins, lipid-modifying enzymes, Rab GTPases, and SNARE proteins, are present and, in several cases, experimentally validated in arthropods. These findings point to broad conservation of exosome biogenesis across taxa, while also highlighting key caveats, with most conclusions relying on only a small number of model systems. The definitions of EV subtypes remain unclear in many arthropods, and alternative EV biogenesis pathways have received little attention. Future studies that incorporate non-model species, apply rigorous EV characterization standards, and explore the roles of various EV subtypes will clarify how these pathways operate across arthropod lineages and how they differ from well-studied mammalian systems.

Background: Culicoides sonorensis biting midges transmit arboviruses that negatively affect animal welfare and production in ruminant livestock operations. However, little is known about the probing and ingestion (i.e., biting) behaviors that occur inside host tissues, even though these behaviors may directly affect pathogen acquisition and transmission. Electropenetrography (EPG) allows for indirect visualization and quantification of these behaviors by measuring the changes in electrical signals that arise during probing. Methods: Using an alternating current–direct current (AC–DC) electropenetrograph, a waveform library for C. sonorensis biting behaviors was constructed from recordings of 70 adult females fed to repletion on human hands. The waveforms were characterized using each combination of four Ri levels (10⁷, 10⁸, 10⁹, and 10¹⁰ ohms) and two electrical current types (AC, DC). Five response variables related to the count and duration of the waveforms were analyzed for each waveform family, applying likelihood ratio tests and Tukey’s procedure to detect significant differences among the means of the eight treatment groups. The probability of transitioning between the waveform families was assessed on the basis of a frequency table of transition events, and multiple exact binomial tests were used to identify nonrandom transition events. Videography and interruption experiments were used to correlate behaviors with waveforms. Results: Waveforms generated by C. sonorensis included waveform families J/K (stylet penetration through the skin); L (types 1, 2, 3, 4, and 5; preparation of an ingestion site); M (types 1, 2, 3, 4, and 5; ingestion); N (types 1 and 2; an unknown behavior that may be a resting phase); and W (withdrawal). The waveforms generally occurred in that order, sometimes with multiple transitions between L, M, and N. Significant differences in the number of waveform events by insect for J, K, and L were observed between some DC treatment groups. The optimal setting for EPG recordings of C. sonorensis probing on human hands was an Ri level of 10⁸ ohms using an applied DC signal of 75 millivolts (mV). Conclusions: EPG uniquely enhances our understanding of C. sonorensis probing and ingestion behaviors, which will facilitate further exploration and guide the development of EPG procedures for other biting midges and telmophagous insects. Graphical

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as "waveforms" that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

Mosquitoes transmit pathogens that negatively affect human and animal health. A greater understanding of their blood-feeding biology and interactions with hosts and pathogens could be exploited to develop new targets for controlling mosquito-borne diseases. Unfortunately, probing (i.e., biting) behaviors of mosquitoes are poorly understood because they occur inside host tissues. Here, a non-invasive procedure is described for using AC-DC electropenetrography (EPG) to indirectly visualize and quantify mosquito feeding behaviors by recording changes in electrical signals generated when probing and ingesting on human hands. Thin gold wires are attached to the mosquitoes using conductive silver glue and connected to the EPG instrument. The human host holds a substrate voltage probe in their hand. Probing of the host by the wired mosquito completes the electrical circuit, and electrical signals are recorded on a computer as "waveforms" that can then be measured and enumerated for analysis. This methodology has been used to characterize the probing and ingestion behaviors of Aedes aegypti and Culex tarsalis mosquitoes and can be applied to other mosquito species. EPG can be used to study the effects of pathogens, insecticides, and other factors on mosquito feeding behaviors.

BACKGROUND: Toxins of Bacillus thuringiensis subsp. israelensis ( Bti ) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi‐aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti ‐based commercial insecticide and its active ingredient in larval Culicoides sonorensis . A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti . RESULTS: The lethal concentration to kill 90% (LC ₉₀ ) of larvae of the commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. CONCLUSION: We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.

Culicoides are small biting midges with the capacity to transmit important livestock pathogens around much of the world, and their impacts on animal welfare are likely to expand. Hemorrhagic diseases resulting from Culicoides-vectored viruses, for example, can lead to millions of dollars in economic damages for producers. Chemical insecticides can reduce Culicoides abundance but may not suppress population numbers enough to prevent pathogen transmission. These insecticides can also cause negative effects on non-target organisms and ecosystems. RNA interference (RNAi) is a cellular regulatory mechanism that degrades mRNA and suppresses gene expression. Studies have examined the utility of this mechanism for insect pest control, and with it, have described the hurdles towards producing, optimizing, and applying these RNAi-based products. These methods hold promise for being highly specific and environmentally benign when compared to chemical insecticides and are more transient than engineering transgenic insects. Given the lack of available control options for Culicoides, RNAi-based products could be an option to treat large areas with minimal environmental impact. In this study, we describe the state of current Culicoides control methods, successes and hurdles towards using RNAi for pest control, and the necessary research required to bring an RNAi-based control method to fruition for Culicoides midges.

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.

Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The microbial and viral communities of ticks, including pathogenic microorganisms, are known to be highly diverse. However, the factors driving this diversity are not well understood. The tropical horse tick, Dermacentor nitens, is distributed throughout the Americas and it is recognized as a natural vector of Babesia caballi and Theileria equi, the causal agents of equine piroplasmosis. In this study, we characterized the bacterial and viral communities associated with partially fed Dermacentor nitens females collected using a passive survey on horses from field sites representing three distinct geographical areas in the country of Colombia (Bolivar, Antioquia, and Cordoba). RNA-seq and sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene were performed using the Illumina-Miseq platform (Illumina, San Diego, CA, USA). A total of 356 operational taxonomic units (OTUs) were identified, in which the presumed endosymbiont, Francisellaceae/Francisella spp., was predominantly found. Nine contigs corresponding to six different viruses were identified in three viral families: Chuviridae, Rhabdoviridae, and Flaviviridae. Differences in the relative abundance of the microbial composition among the geographical regions were found to be independent of the presence of Francisella-like endosymbiont (FLE). The most prevalent bacteria found in each region were Corynebacterium in Bolivar, Staphylococcus in Antioquia, and Pseudomonas in Cordoba. Rickettsia-like endosymbionts, mainly recognized as the etiological agent of rickettsioses in Colombia, were detected in the Cordoba samples. Metatranscriptomics revealed 13 contigs containing FLE genes, suggesting a trend of regional differences. These findings suggest regional distinctions among the ticks and their bacterial compositions.

Background: Vacuolar (H⁺)‐ATPase (V‐ATPase) is a multi‐subunit enzyme that hydrolyzes adenosine triphosphate (ATP) to transport protons across a cellular membrane, and it plays an important role in numerous biological processes, including in growth, development and immune responses. The c subunit of V‐ATPase is a highly conserved subunit of the rotatory proteolipid ring that is required for binding and transporting protons. To date, there are only a few published reports on V‐ATPase‐c functions in insects.Results: We identified and characterized the V‐ATPase‐c gene in Locusta migratoria, one of the most destructive agricultural insect pests in the world. LmV‐ATPase‐c was predominately expressed in Malpighian tubules of nymphs, followed by the hindgut and ovary, while the other tissues showed relatively low expression levels. Silencing of LmV‐ATPase‐c caused severe molting defects in nymphs and a high mortality rate of > 90%. Histological staining and microscopic examination of sections from the abdominal cuticle revealed the absence of newly formed cuticle in nymphs that were injected with dsLmV‐ATPase‐c. In addition, silencing of LmV‐ATPase‐c transcript levels significantly impaired RNA interference (RNAi) efficiency of a reporter gene. By quantifying double‐stranded RNA (dsRNA) amounts by quantitative polymerase chain reaction (PCR), we found that RNAi against LmV‐ATPase‐c provoked a dramatic accumulation of dsRNA in the endosomes of epidermal and midgut cells of Locusta migratoria.Conclusion: Our results indicate that LmV‐ATPase‐c is indispensable for the formation of new cuticle during the molting process and has pivotal functions in dsRNA escape from endosomes. LmV‐ATPase‐c might be a valuable target for developing new strategies for insect pest management. © 2022 Society of Chemical Industry.