Filth flies pose a health risk because of the microbes they carry outside and inside of their bodies to humans and animals. Mostly synthetic chemical insecticides are used for fly control. Alternative approaches with a different mode of action are needed because of increasing fly resistance to pesticides. We used a modified World Health Organization cone test to determine the efficacy of the minerals produced by diatoms (diatomaceous earth) as a mechanical insecticide against adults of the house fly, Musca domestica, the secondary screwworm, Cochliomyia macellaria, and the grey flesh fly, Sarcophaga bullata, under low and high humidity. The use of mechanical insecticides as an alternative to kill filth flies has received minimal consideration. However, recent research showed that expanded perlite, a new mechanical insecticide made from volcanic rock, was highly efficacious against mosquitoes and flies. Mortality for diatomaceous earth in this paper at 30°C and 50% relative humidity was observed as early as 2 h after exposure with 50% and 90% mortality observed at 3.5 and 4.6 h (the LT50 and LT90, respectively) in M. domestica. The LT50 and LT90 increased as the size of the fly species increased (M. domestica to C. macellaria to S. bullata) and increased for all three species when the relative humidity increased from 50 to 70%. These results suggest dehydration was the mode of action. Scanning electron micrographs of C. macellaria adults 2 h after diatomaceous earth exposure, showed the flies were evenly self-covered with the mineral with no obvious damage to the cuticle. Proof of concept was demonstrated that diatomaceous earth as a residual surface treatment could be used as an alternative for filth fly control.

Chiggers (larval Trombiculid mites) serve as vectors for Orientia species that cause scrub typhus, a potentially serious illness in humans with a broadening global distribution. To date, there is limited research on the chigger microbiome in the United States (US) compared to some other parts of the world. Investigating chigger bacterial communities is essential for understanding the potential role they play in pathogen transmission dynamics within these arthropods. This study investigated the bacterial communities of free-living chiggers collected from sites across the three ecoregions in North Carolina using 16S rDNA gene targeted next-generation sequencing. Molecular identification of the chigger revealed three species: Eutrombicula splendens, Eutrombicula tinami, and Pseudoschoengastia sp. All three trombiculid mite species occurred at least once in the Mountains and Piedmont, except for E. tinami, which was absent from the Coastal Plain ecoregion. Microbiome analysis revealed significant differences in alpha and beta diversity among the collection sites for E. splendens. No significant differences in overall microbiome diversity were observed between E. splendens and Pseudoschoengastia sp., the two dominant chigger species. However, the microbiome of E. splendens alone exhibited significant differences in both Shannon diversity and beta diversity between Orientia-infected and uninfected individuals. Within E. splendens, genera like Brevibacillus and Telluria were more abundant in Orientia-positive chiggers, while Methylobacterium was more abundant in Orientia-negative chiggers. We also found potentially pathogenic bacterial genera, including Rickettsia, Listeria, Legionella, Staphylococcus, and Streptococcus sequences. These findings suggest that geography and Orientia infection influence chigger-associated bacterial communities, potentially affecting their vector competence.

Deployed armed forces and the public engaged in outdoor activities are at high risk for mosquito bites and the diseases they transmit. Current mosquito bite-resistant garments prevent blood-feeding with slow-release insecticide formulations. Many people today want to avoid contact with pesticides, especially in their clothing. Insecticide treated clothing also is costly and requires regulatory agency approvals. Using mosquito bite-resistant mathematical textile models and a WholeGarment® knitting technique, a seamless garment was constructed with military-compliant, no-melt, no-drip flame retardant yarns using an AiryPique knit architecture. The garment was 99.5% bite proof in walk-in cage bioassays with 200 Aedes aegypti host-seeking mosquitoes where the human subjects did not move for 20 min. A standard flame test and a PyroManTM flammability study validated the garment’s fire protection, a requirement for military uniforms. The thermal physiological comfort tests (air permeability, wetting time/radius, thermal resistance, evaporative resistance, and sweating thermal manikin test) were similar to current army combat uniforms and appropriate for use in everyday clothing. Bite prevention occurred by physically blocking the insect mouth parts from obtaining a blood meal. The knitting technique is well-suited for mass production of bite-resistant clothing through automation, significantly reducing labor, time, and cost by optimizing “fit on demand” for different body types compared to traditional manufacturing methods. This innovation provides a non-insecticidal, safe, scalable, and efficient solution for protecting individuals against mosquito bites.

Caterpillar pests and their microbiota are of growing interest in relation to pest control, including any future use of living wild-type or genetically modified microbes for pest suppression and risk assessment. This study investigated whether bacteria associated with field-collected larval bollworms, Helicoverpa zea, persist through metamorphosis to the adult, potentially enabling the insects to transport microbes over long distances as the moths disperse. Fifth stadium, fully fed larvae were collected from non-Bt cotton in the field and allowed to pupate without additional feeding and emerge as adults in the laboratory. Bacterial community composition and diversity were assessed at each stage using Illumina sequencing of the 16S rRNA gene. Analysis of the internal bacterial communities revealed that alpha and beta diversity differed significantly between larvae, pupae, and adults. Despite this difference, 79 amplicon sequence variants were retained from the larval to the adult stage. The 5 most abundant taxa shared between larvae, pupae, and adults were Enterobacter, Klebsiella, an undefined Enterobacteriaceae species, Enterococcus casseliflavus, and Klebsiella oxytoca. Some of these bacteria are typical gut microbes. Further research is needed to characterize the composition and functional roles of these bacteria in H. zea, to evaluate their influence across generations, and to determine whether bacteria can be acquired by the larvae from their environment and persist into the adult stage. The role of the caterpillar microbiome in the context of modern cotton agroecosystems remains a largely understudied area of insect-microbe ecology.

The insect molting process is critical to growth and development and is regulated in part by the neuropeptides corazonin, eclosion hormone, and α and β bursicon. We found messages in a synganglion transcriptome from adult, female American dog ticks, Dermacentor variabilis (that do not molt), with a high similarity to the larval insect neuropeptides that control molting. The phylogenetic analysis of the tick putative neuropeptides compared to other arthropods is discussed in detail. The relative gene expression of these peptides was determined by quantitative PCR during the following adult developmental stages: (i) virgin, unfed 0–24 h after entering the adult stage (non-host-seeking), (ii) host-seeking, unfed, and not mated (3 d after emergence), (iii) part-fed (unmated, attached to host; 1st and 3rd day after emergence), (iv) mated (females are part-fed; allowed to mate for ≤1 day, 7th day after emergence), (v) mated repletes (completion of blood feeding but still attached to host), and (vi) post-drop-off (from host) with egg laying starting within 1 d of detachment. Eclosion hormone transcript levels peaked at mating and at drop-off. Bursicon α levels were highest just after molting into adults, with a second smaller peak in replete females. Bursicon β levels were highest (32-fold) post-drop-off. Corazonin message levels peaked in part-feds and were much higher (40-fold) in repletes compared to 0–24 h after emergence. RNAi suppression of the corazonin message by injection in newly molted ticks reduced oviposition and the number of vitellogenic eggs in the ovaries at drop-off but had no apparent effect on host-seeking, partial feeding, mating, feeding to repletion, and drop-off. The possible roles of these transcripts in adult, female tick development are discussed.

The longhorned tick (LHT), Haemaphysalis longicornis Neumann (Acari: Ixodidae), is a serious invasive pest in North America where its geographical range is expanding with high densities associated with commercial animal production. There are only a few chemical pesticides available for LHT control, which can lead to the evolution of resistant strains. Diatomaceous earth (DE) was shown to be effective in killing some important tick species but was not examined for LHTs. When LHT nymphs were dipped for about 2–4 s into DE, transferred to Petri dishes (one tick/dish), and incubated at 30 °C and 70% relative humidity, the median survival time was 4.5 h. A locomotor activity assay showed that there was no difference in the overall distance traveled between the DE-treated and control ticks except during the first 2 h after exposure. In a field-simulated study in which a dose of 5.0 g DE/m2 was applied to pine needle litter infested with LHT, all the LHTs were dead at 24 h with no control mortality. Scanning electron micrographs showed the mineral adhering to all surfaces of the tick. The results indicated that DE is effective in killing nymphal LHTs and could be an alternative to the use of chemical acaricides with the advantage of managing pesticide resistance through the killing by a different mode of action and could be used for organically certified animal husbandry.

Chiggers are larval mites that pose a significant health risk globally via the spread of scrub typhus. However, fundamental studies into the bacterial microbiome in North America have never been considered. In this investigation, chiggers were collected in the wild from two locally common rodent host species (i.e., Sigmodon hispidus and Peromyscus leucopus) in three different ecoregions of North Carolina (NC), United States to investigate the composition of their bacterial communities, including potential pathogens. DNA was extracted from the chiggers, and the V3-V4 regions of the bacterial 16S rRNA gene were sequenced using next-generation sequencing (NGS). Alpha diversity metrics revealed significant differences in bacterial diversity among different collection counties. Beta diversity metrics also revealed that bacterial communities across counties were significantly different, suggesting changes in the microbiome as the environment changed. Specifically, we saw that the two western NC collection counties had similar bacterial composition as did the two eastern collection counties. In addition, we found that the chigger microbiome bacterial diversity and composition differed between rodent host species. The 16S rRNA sequence reads were assigned to 64 phyla, 106 orders, 199 families, and 359 genera. The major bacterial phylum was Actinobacteria. The most abundant species were in the genera Corynebacterium, Propionibacterium, class ZB2, and Methylobacterium. Sequences derived from potential pathogens within the genera Orientia and Rickettsia were also detected. Our findings provide the first insights into the ecology of chigger microbiomes in the US. Further research is required to determine if the potential pathogens found detected in chiggers are a threat to humans and wildlife.

Diatomaceous earth (DE) recently was shown to rapidly kill ticks. Proof of concept for use of DE to kill the American dog tick, Dermacentor variabilis, the most widely distributed native three-host tick in North America, has not been investigated. Unfed D. variabilis nymphs dipped into DE and incubated at 30 °C and 50 versus 70

Sand flies (Diptera: Psychodidae) serve as vectors for transmitting protozoan parasites, Leishmania spp., that cause the disease called leishmaniasis. The main approach to controlling sand flies is the use of chemical insecticides. The discovery of alternative methods for their control is needed because of potential health risks of chemical insecticides and development of sand fly resistance to these pesticides. The biomineral produced by diatoms (diatomaceous earth, DE; Celite) and a volcanic glass bio-mimic (Imergard) have been shown by our group to be efficacious against mosquitoes, filth flies, and ticks but never studied for the control of sand flies. In a modified World Health Organization cone test, 50% of adult Phlebotomus papatasi sand flies at 29 ± 1 °C, 55 ± 5% RH, and 12:12 LD, when exposed to Imergard and Celite, were dead in 13.08 and 7.57 h, respectively. Proof of concept was established for the use of these biominerals for sand fly and leishmaniasis disease control. Using a light source as an attractant to the minerals had no significant effect on the LT50, the time to 50% mortality. The LT50 at a higher relative humidity of 70 ± 5% increased to 20.91 and 20.56 h for Imergard and Celite, respectively, suggesting their mode of action was dehydration. Scanning electron microscopy of dead sand flies showed high coating levels of Celite only on the sides of the thorax and on the tarsi, suggesting an alternative mode of action for mechanical insecticides.

Rickettsia amblyommatis is a potentially pathogenic species of Rickettsia within the spotted fever group vectored by ticks. While many studies have been published on this species, there is debate over its pathogenicity and the inhibitory role it plays in diagnosing illnesses caused by other spotted fever group Rickettsia species. Many publications have recorded the high infection prevalence of R. amblyommatis in tick populations at a global scale. While this species is rather ubiquitous, questions remain over the epidemiological importance of this possible human pathogen. With tick-borne diseases on the rise, understanding the exact role that R. amblyommatis plays as a pathogen and inhibitor of infection relative to other tick-borne pathogens will help public health efforts. The goal of this review was to compile the known literature on R. amblyommatis, review what we know about its geographic distribution, tick vectors, and pathogenicity, assess relatedness between various international strains from ticks by phylogenetic analysis and draw conclusions regarding future research needed.

Scrub typhus, a rickettsial disease caused by Orientia spp., is transmitted by infected larval trombiculid mites (chiggers). We report the molecular detection of Orientia species in free-living Eutrombicula chiggers collected in an area in North Carolina, USA, to which spotted fever group rickettsiae infections are endemic.

Mosquito vector-borne diseases such as malaria and dengue pose a major threat to human health. Personal protection from mosquito blood feeding is mostly by treating clothing with insecticides and the use of repellents on clothing and skin. Here, we developed a low-voltage, mosquito-resistant cloth (MRC) that blocked all blood feeding across the textile and was flexible and breathable. The design was based on mosquito head and proboscis morphometrics, the development of a novel 3-D textile with the outer conductive layers insulated from each other with an inner, non-conductive woven mesh, and the use of a DC (direct current; extra-low-voltage) resistor-capacitor. Blockage of blood feeding was measured using host-seeking Aedes aegypti adult female mosquitoes and whether they could blood feed across the MRC and an artificial membrane. Mosquito blood feeding decreased as voltage increased from 0 to 15 volts. Blood feeding inhibition was 97.8% at 10 volts and 100% inhibition at 15 volts, demonstrating proof of concept. Current flow is minimal since conductance only occurs when the mosquito proboscis simultaneously touches the outside layers of the MRC and is then quickly repelled. Our results demonstrated for the first time the use of a biomimetic, mosquito-repelling technology to prevent blood feeding using extra-low energy consumption.

Ticks in the USA are the most important arthropod vector of microbes that cause human and animal disease. The blacklegged tick, Ixodes scapularis, the focus of this study, is able to transmit the bacteria that causes Lyme disease in humans in the USA. The main approach to tick control is the use of chemical acaricides and repellents, but known and potential tick resistance to these chemicals requires the discovery of new methods of control. Volcanic glass, Imergard, was recently developed to mimic the insecticide mode of action of the minerals from diatoms (diatomaceous earth, DE) for the control of malaria mosquitoes in Africa. However, studies on the use of these minerals for tick control are minimal. In a dipping assay, which was put into DE (Celite), the times of 50 and 90% death of adult female I. scapularis were 7.3 and 10.5 h, respectively. Our mimic of DE, Imergard, killed ticks in 6.7 and 11.2 h, respectively. In a choice-mortality assay, ticks moved onto a treated surface of Imergard and died at 11.2 and 15.8 h, respectively. Ticks had greater locomotor activity before death when treated by dipping for both Imergard and Celite versus the no-mineral control. The ticks after making contact with Imergard had the mineral covering most of their body surface shown by scanning electron microscopy with evidence of Imergard inside their respiratory system. Although the assumed mode of action of Imergard and Celite is dehydration, the minerals are not hygroscopic, there was no evidence of cuticle damage, and death occurred in as little as 2 h, suggesting minimal abrasive action of the cuticle. Semi-field and field studies are needed in the future to examine the practical use of Imergard and Celite for tick control, and studies need to examine their effect on tick breathing and respiratory retention of water.

Chiggers are vectors of rickettsial pathogenic bacteria, Orientia spp., that cause the human disease, scrub typhus, in the Asian–Pacific area and northern Australia (known as the Tsutsugamushi Triangle). More recently, reports of scrub typhus in Africa, southern Chile, and the Middle East have reshaped our understanding of the epidemiology of this disease, indicating it has a broad geographical distribution. Despite the growing number of studies and discoveries of chigger-borne human disease outside of the Tsutsugamushi Triangle, rickettsial pathogens in chigger mites in the US are still undetermined. The aim of our study was to investigate possible Rickettsia DNA in chiggers collected from rodents in North Carolina, USA. Of 46 chiggers tested, 47.8% tested positive for amplicons of the 23S-5S gene, 36.9% tested positive for 17 kDa, and 15.2% tested positive for gltA. Nucleotide sequence analyses of the Rickettsia-specific 23S-5S intergenic spacer (IGS), 17 kDa, and gltA gene fragments indicated that the amplicons from these chiggers were closely related to those in R. felis, R. conorii, R. typhi, and unidentified Rickettsia species. In this study, we provide the first evidence of Rickettsia infection in chiggers collected from rodents within the continental USA. In North Carolina, a US state with the highest annual cases of spotted fever rickettsioses, these results suggest chigger bites could pose a risk to public health, warranting further study.

Cases of Lyme disease in humans are on the rise in the United States and Canada. The vector of the bacteria that causes this disease is the blacklegged tick, Ixodes scapularis. Current control methods for I. scapularis mainly involve chemical acaricides. Unfortunately, ticks are developing resistance to these chemicals, and more and more, the public prefers non-toxic alternatives to chemical pesticides. We discovered that volcanic glass, ImergardTM WP, and other industrial minerals such as Celite 610 were efficacious mechanical insecticides against mosquitoes, filth flies, and agricultural pests. In this report, when 6–10- and 50–70-day old unfed I. scapularis nymphs were dipped for 1–2 s into Celite, the time to 50% mortality (LT50) was 66.8 and 81.7 min, respectively, at 30 °C and 50% relative humidity (RH). The LT50 was actually shorter at a higher 70% RH, 43.8 min. Scanning electron microscopy showed that the ticks were coated over most of their body surface, including partial to almost total coverage of the opening to their respiratory system. The other mechanical insecticide, Imergard, had similar efficacy against blacklegged unfed nymphs with an LT50 at 30 °C and 50% RH of 70.4 min. Although more research is needed, this study suggests that industrial minerals could be used as an alternative to chemical pesticides to control ticks and Lyme disease.

Over the past 10 years, studies using high-throughput 16S rRNA gene sequencing have shown that mosquitoes harbor diverse bacterial communities in their digestive system. However, no previous research has examined the total bacteria community inside versus outside of mosquitoes and whether bacteria found on the outside could represent a potential health threat through mechanical transfer. We examined the bacterial community of the external surface and internal body of female Anopheles coluzzii adults collected from homes in Côte d’Ivoire, Africa, by Illumina sequencing of the V3 to V4 region of 16S rRNA gene. Anopheles coluzzii is in the Anopheles gambiae sensu lato (s.l.) species complex and important in the transmission of malaria. The total 16S rRNA reads were assigned to 34 phyla, 73 orders, 325 families, and 700 genera. At the genus level, the most abundant genera inside and outside combined were Bacillus, Staphylococcus, Enterobacter, Corynebacterium, Kocuria, Providencia, and Sphingomonas. Mosquitoes had a greater diversity of bacterial taxa internally compared to the outside. The internal bacterial communities were similar between homes, while the external body samples were significantly different between homes. The bacteria on the external body were associated with plants, human and animal skin, and human and animal infections. Internally, Rickettsia bellii and Rickettsia typhi were found, potentially of importance, since this genus is associated with human diseases. Based on these findings, further research is warranted to assess the potential mechanical transmission of bacteria by mosquitoes moving into homes and the importance of the internal mosquito microbiota in human health.

Multiple insect pest species have developed field resistance to Bt-transgenic crops. There has been a significant amount of research on protein-coding genes that contribute to resistance, such as the up-regulation of protease activity or altered receptors. However, our understanding of the role of non-protein-coding mechanisms in Bt-resistance is minimal, as is also the case for resistance to chemical pesticides. To address this problem relative to Bt, RNA-seq was used to examine statistically significant, differential gene expression between a Cry1Ac-resistant (~100-fold resistant) and Cry1Ac-susceptible strain of Helicoverpa zea, a prevalent caterpillar pest in the USA. Significant differential expression of putative long non-coding RNAs (lncRNAs) was found in the Cry1Ac-resistant strain (58 up- and 24 down-regulated gene transcripts with an additional 10 found only in resistant and four only in susceptible caterpillars). These lncRNAs were examined as potential pseudogenes and for their genomic proximity to coding genes, both of which can be indicative of regulatory relationships between a lncRNA and coding gene expression. A possible pseudogenic lncRNA was found with similarities to a cadherin. In addition, putative lncRNAs were found significantly proximal to a serine protease, ABC transporter, and CYP coding genes, potentially involved in the mechanism of Bt and/or chemical insecticide resistance. Characterization of non-coding genetic mechanisms in Helicoverpa zea will improve the understanding of the genomic evolution of insect resistance, improve the identification of specific regulators of coding genes in general (some of which could be important in resistance), and is the first step for potentially targeting these regulators for pest control and resistance management (using molecular approaches, such as RNAi and others).

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

Chiggers are the larval stage of Trombiculidae and Leeuwenhoekiidae mites of medical and veterinary importance. Some species in the genus Leptotrombidium and Herpetacarus vector Orientia species, the bacteria that causes scrub typhus disease in humans. Scrub typhus is a life-threatening, febrile disease. Chigger bites can also cause dermatitis. There were 248 chigger species reported from the US from almost every state. However, there are large gaps in our knowledge of the life history of other stages of development. North American wide morphological keys are needed for better species identification, and molecular sequence data for identification are minimal and not clearly matched with morphological data. The role of chiggers in disease transmission in the US is especially understudied, and the role of endosymbionts in Orientia infection are suggested in the scientific literature but not confirmed. The most common chiggers in the eastern United States were identified as Eutrombicula alfreddugesi but were likely misidentified and should be replaced with Eutrombicula cinnabaris. Scrub typhus was originally believed to be limited to the Tsutsugamushi Triangle and the chigger genus, Leptotrombidium, but there is increasing evidence this is not the case. The potential of Orientia species establishing in the US is high. In addition, several other recognized pathogens to infect humans, namely Hantavirus, Bartonella, Borrelia, and Rickettsia, were also detected in chiggers. The role that chiggers play in these disease transmissions in the US needs further investigation. It is possible some of the tick-borne diseases and red meat allergies are caused by chiggers.

Pesticide-free, 3-D, spacer fabrics (Plant Armor Generation (PA Gen) 1 and 2) were investigated for proof-of-concept as an insect barrier to protect plants and improve plant agronomics for organic farming. The time to 50% penetration (TP50) for tobacco thrips, Frankliniella fusca (Hinds) adults in laboratory Petri dish bioassays was 30 and 175 min for PA Gen 1 and 2, respectively, and 12 min for the control (a commercially available, single layer-crop cover, Proteknet). PA Gen 2 was ≥90% resistant to penetration of unfed caterpillar neonates, Helicoverpa zea (Boddie), while the TP50‘s for Gen 1 and Proteknet were 3.1 and 2.35 h, respectively. In small cage studies, PA Gen 2 covered potted cabbage plants were 100% resistant to penetration by these insects through 10 d after which the study was ended. In small field plot studies for 3 summer months, cabbage plants grew approximately twice as fast when covered versus not covered with Gen 1 and Gen 2 without the need for insecticides or herbicides. This was not observed for the control crop cover. Martindale abrasion tests demonstrated Gen 1 and 2 were at least 6- and 1.8-fold more durable than the control crop cover used. Data are also presented on percentage light, water, air, and water vapor penetration across each textile and operational temperatures and humidity for cabbage plants covered and uncovered in small field plots.