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.

BALROG-ISO is a Nextflow pipeline for automated analysis of whole genome sequences of bacterial isolates to perform taxonomic classification, genomic annotation, annotation of antimicrobial resistance genes (ARGs), and prediction of ARG origin (e.g., plasmid, chromosomal). A final summary report additionally offers a comprehensive and user-friendly visualization of key quality metrics and annotation results. BALROG-ISO minimizes command inputs and streamlines modular processes, enabling the concurrent analysis of more genomic samples while also reducing manual job submission and analysis inconsistencies. Overall, BALROG-ISO is an adaptable workflow pipeline optimized for a One Health approach to the exploration of antimicrobial resistance in bacterial genomes.

Alternating current-direct current (AC-DC) electropenetrography (EPG) is a non-invasive approach for quantifying parasitic arthropods' probing and ingestion behaviors inside host tissues. Here, we present a procedure for using the Observer XT behavioral coding software to synchronize EPG waveforms with video recordings. We describe the steps for performing, importing, and analyzing simultaneous recordings. This protocol has potential applications for the initial correlation of waveforms with arthropod biological activities. For complete details on the use and execution of this protocol, please refer to Cooper et al.1.

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. 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. 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). 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.

BALROG-MON is a Nextflow pipeline for automated analysis of metagenomic long-read data to detect pathogens, annotate antimicrobial resistance genes (ARGs), link ARGs to specific pathogens, predict ARG origin (e.g., plasmid, chromosomal) and optionally perform steps like community analysis. With both assembly-based and assembly-free workflows, BALROG-MON is applicable to a wide range of sample types with low or high coverage, varying complexities and origins. Optional genome binning provides a comprehensive overview of ARGs within the dataset. BALROG-MON additionally presents results in summarized reports, overall serving as a flexible analysis tool for exploring diverse metagenomic samples for pathogens and antibiotic resistance.

This corrects the article DOI: 10.30802/AALAS-CM-23-000037When the above article was first published in the Vol 3 No 6 (December 2023) issue of Comparative Medicine, figure images were incorrectly associated with the figure legends. The correct version of this article has been reprinted in full in volume 74, issue 1 of the February issue of Comparative Medicine.The publisher apologizes for this error and any inconvenience caused.

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.

Insecticide formulations with safer environmental profiles and limited off-target effects are desirable to manage medical and veterinary pests. Silver nanoparticles are insecticidal against mosquitos, nonbiting midges, and other insects. The biting midge, Culicoides sonorensis Wirth and Jones, is a vector of agriculturally important pathogens in much of the United States, and this study aimed to examine the insecticidal properties of silver nanoparticles in larvae of this species. Mortality of third-instar larvae was assessed daily for 7 days after exposure to concentrations of silver nanoparticles, sorghum polymer particles, and hybrid silver-sorghum polymer particles. Both silver nanoparticles and silver-sorghum polymer particles were insecticidal, but sorghum polymer particles alone did not significantly contribute to larval mortality. Concentrations of 100 mg/liter of silver nanoparticles achieved >50% mortality at day 7, and 200 mg/liter treatments achieved >75% larval mortality within 24 h. The antimicrobial properties of silver nanoparticles were also examined, and culturable bacteria were recovered from larval-rearing media at 200 mg/liter but not at 400 mg/liter of silver nanoparticles. These data suggest that C. sonorensis larval mortality is primarily caused by silver nanoparticle toxicity and not by the reduction of bacteria (i.e., a larval food source). This work describes the first use of silver nanoparticles in C. sonorensis and shows the potential insecticide applications of these nanoparticles against this agricultural pest. The grain-polymer particles also successfully carried insecticidal silver nanoparticles, and their utility in loading diverse compounds could be a novel toxin delivery system for biting midges and similar pests.

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% (LC90) 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.

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.

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 .

A detailed understanding of mosquito probing and ingestion behaviors is crucial in developing novel interventions to interrupt the transmission of important human and veterinary pathogens, but these behaviors are difficult to observe as the mouthparts are inserted into the skin of the host. Electropenetrography (EPG) allows indirect observation, recording, and quantification of probing and ingestion behaviors of arthropods by visualizing the electrical waveform associated with these behaviors. The study of mosquito probing and ingestion behaviors has been limited to the use of human hands as host, which is not suitable for pathogen transmission studies. Mouse models of mosquito-borne diseases are a widely used and indispensable tool in this research, but previous attempts to use direct current EPG to study mosquito probing behaviors on mice have been unsuccessful. Accordingly, the present study used alternating or direct current (AC-DC) EPG to observe the ingestion behaviors of adult Aedes aegypti mosquitoes on a mouse host in real time under BSL-2 containment conditions with enhanced BSL-3 practices. Our results show that waveform families previously identified during Ae. aegypti probing and ingestion on human hands were observed using 100 mV of AC at an input resistance (Ri) of 107 Ohms (Ω) on CD1 mice. This work is a proof of concept for using mouse models for studying mosquito probing and ingestion behaviors with AC-DC EPG. In addition, these data show that the experimental setup used in these experiments is sufficient for conducting studies on mosquito probing and ingestion behaviors under BSL-2 containment conditions enhanced with BSL-3 practices. This work will serve as a foundation for using EPG to investigate the effects of pathogen infection on mosquito probing behaviors and to understand the real-time dynamics of pathogen transmission.

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.

Culex tarsalis Coquillett (Diptera: Culicidae) mosquitoes are capable of vectoring numerous pathogens affecting public and animal health. Unfortunately, the probing behaviors of mosquitoes are poorly understood because they occur in opaque tissues. Electropenetrography (EPG) has the potential to elucidate these behaviors by recording the electrical signals generated during probing. We used an AC-DC EPG with variable input resistors (Ri levels) to construct a waveform library for Cx. tarsalis feeding on human hands. Biological events associated with mosquito probing were used to characterize waveforms at four Ri levels and with two electrical current types. The optimal settings for EPG recordings of Cx. tarsalis probing on human hands was an Ri level of 107 Ohms using an applied signal of 150 millivolts alternating current. Waveforms for Cx. tarsalis included those previously observed and associated with probing behaviors in Aedes aegypti L. (Diptera: Culicidae): waveform families J (surface salivation), K (stylet penetration through the skin), L (types 1 and 2, search for a blood vessel/ingestion site), M (types 1 and 2, ingestion), N (type 1, an unknown behavior which may be a resting and digestion phase), and W (withdrawal). However, we also observed variations in the waveforms not described in Ae. aegypti, which we named types L3, M3, M4, and N2. This investigation enhances our understanding of mosquito probing behaviors. It also provides a new tool for the automated calculation of peak frequency. This work will facilitate future pathogen acquisition and transmission studies and help identify new pest and disease management targets.

The red flour beetle (Tribolium castaneum Herbst) and the confused flour beetle (Tribolium confusum Jacquelin du Val) are closely related species commonly infesting stored grains and milled grain products. Although the same insecticides are used to control both species, their susceptibilities to those insecticides often vary considerably. To explore possible contributions of cytochrome P450 (CYP) genes to the differential insecticide susceptibilities between T. castaneum and T. confusum, we selected six CYP orthologous genes to explore their possible contributions to differential insecticide susceptibilities to each of three commonly used insecticides (dichlorvos, pyrethrins, and esfenvalerate). After the expression of each CYP gene was suppressed by RNA interference (RNAi) in late larvae, we examined larval susceptibility to each insecticide. Our results showed that although T. castaneum were more susceptible to dichlorvos than T. confusum, none of these CYP genes appeared to play any significant role in differential dichlorvos susceptibility between the two species. In contrast, there were no significant differences in larval susceptibility to pyrethrins between the two species. However, suppression of CYP4BR3 expression increased larval susceptibility to pyrethrins in T. castaneum at 24 and 72 h after the insecticide exposure, but not in T. confusum. Furthermore, RNAi-mediated suppression of CYP4G7, CYP9Z5, CYP4BR3, and CYP345A1/6 significantly increased larval susceptibility to esfenvalerate in T. castaneum but not in T. confusum at 72 h. In addition, the suppression of CYP4G7 expression increased larval susceptibility to esfenvalerate at 96 h in both species. These results suggest that although CYP4G7 may play some role in esfenvalerate susceptibility in T. confusum, CYP4G7, CYP9Z5, CYP4BR3, and CYP345A1 significantly contribute to differential esfenvalerate susceptibilities between two Tribolium species. This study represents the first study to reveal molecular mechanisms causing differential insecticide susceptibilities between the two Tribolium species.

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.

Disruption of morphogenesis, an essential process in organismal development, can lead to disruption of biological processes, reduction in fitness, or even death of an organism. The roles of lethal giant larvae (Lgl) protein in maintaining tissue organization have been studied extensively in mammals, but little is known about this gene's roles in promoting correct tissue morphogenesis in insects. In this study, we identified an Lgl ortholog in Locusta migratoria. RT-qPCR results revealed that LmLgl was constitutively expressed during third, fourth, and fifth instar nymphs. Furthermore, LmLgl showed highest expression in the ovary followed by wing pads, midgut, hindgut, Malpighian tubules, and foregut of the third-instar nymphs. To examine the role of LmLgl in L. migratoria development, RNA interference was performed during nymphal stages. Silencing of LmLgl increased body size but decreased bodyweight by 9.0%. Histological sections of the midgut revealed abnormal large masses of disordered epithelial cells in dsLmLgl-injected nymphs. In addition, downregulation of LmLgl transcript levels significantly altered the morphological structure in midgut, resulting in the formation of tumor-like structures. Our results indicated that LmLgl may act as a tumor-suppressor gene, which plays an essential role in maintaining a normal morphological structure in the midgut of L. migratoria. Our results also suggest that LmLgl may be explored as a potential target for developing dsRNA-based biological pesticides for managing insect pests.

Rab proteins constitute the largest family of small GTPases, which play pivotal roles in intracellular membrane trafficking in all eukaryotes. A number of Rab genes have been identified in eukaryotes; however, very little information about these genes has been reported in insects. In the current study, for the first time we identified and characterized 27 Rab family genes from Locusta migratoria. Phylogenetic analysis and comparison of domain architecture indicated that Rab family genes are highly conserved among insect species. Tissue-dependent expression profiles indicated that expression of Rab genes was highest in the ovary, except for LmRab3, which was most highly expressed in hemolymph. The biological function of each Rab gene was investigated using RNA interference (RNAi). Double-stranded RNA targeting each Rab gene was injected into the hemocoel of nymphs and revealed that suppression of two Rab genes (LmRab5 and LmRab11A) caused 100% mortality. In addition, nymphs injected with dsLmRab5 exhibited severe phenotypic defects in the gastric caeca and midgut, while dsLmRab11A arrested the molting process. We then applied the RNAi of RNAi technique to test if silencing either of these two genes would affect the suppression of the lethal giant larvae (LmLgl) reporter gene and found that suppression of LmRab5 diminished the RNAi efficiency of LmLgl, whereas suppression of LmRab11A enhanced RNAi efficiency of LmLgl. These results indicate that Rab genes contribute differently to RNAi efficiency in different tissues. Our study provides a foundation for further functional investigations of Rab genes and their contributions to RNAi efficiency in L. migratoria.