Biomolecular Minerals and Volcanic Glass Bio-Mimics to Control Adult Sand Flies, the Vector of Human Leishmania Protozoan Parasites.

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.

Acaricidal Biominerals and Mode-of-Action Studies against Adult Blacklegged Ticks, Ixodes scapularis.

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.

Detection of Orientia spp. Bacteria in Field-Collected Free-Living Eutrombicula Chigger Mites, United States.

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 Blood Feeding Prevention Using an Extra-Low DC Voltage Charged Cloth.

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.

Rickettsia amblyommatis in Ticks: A Review of Distribution, Pathogenicity, and Diversity.

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.

Internal and external microbiota of home-caught Anopheles coluzzii (Diptera: Culicidae) from Côte d'Ivoire, Africa: Mosquitoes are filthy.

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.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility.

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.

Biology, Systematics, Microbiome, Pathogen Transmission and Control of Chiggers (Acari: Trombiculidae, Leeuwenhoekiidae) with Emphasis on the United States.

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.

Rickettsia felis and Other Rickettsia Species in Chigger Mites Collected from Wild Rodents in North Carolina, USA.

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.

Mechanical Acaricides Active against the Blacklegged Tick, Ixodes scapularis.

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.

Mosquito-Textile Physics: A Mathematical Roadmap to Insecticide-Free, Bite-Proof Clothing for Everyday Life.

Garments treated with chemical insecticides are commonly used to prevent mosquito bites. Resistance to insecticides, however, is threatening the efficacy of this technology, and people are increasingly concerned about the potential health impacts of wearing insecticide-treated clothing. Here, we report a mathematical model for fabric barriers that resist bites from Aedes aegypti mosquitoes based on textile physical structure and no insecticides. The model was derived from mosquito morphometrics and analysis of mosquito biting behavior. Woven filter fabrics, precision polypropylene plates, and knitted fabrics were used for model validation. Then, based on the model predictions, prototype knitted textiles and garments were developed that prevented mosquito biting, and comfort testing showed the garments to possess superior thermophysiological properties. Our fabrics provided a three-times greater bite resistance than the insecticide-treated cloth. Our predictive model can be used to develop additional textiles in the future for garments that are highly bite resistant to mosquitoes.

Bacterial Microbiota of Field-Collected Helicoverpa zea (Lepidoptera: Noctuidae) from Transgenic Bt and Non-Bt Cotton.

The bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is an important agricultural pest in U.S. cotton and is managed using transgenic hybrids that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). The reduced efficacy against H. zea caterpillars of Bt plants expressing Cry toxins is increasing in the field. In a first step towards understanding Bt cotton-bollworm-microbiota interactions, we investigated the internal bacterial microbiota of second-third stadium H. zea collected in the field from non-Bt versus Bt (WideStrike) cotton in close proximity (in North Carolina, USA). The bacterial populations were analyzed using culture-dependent and -independent molecular approaches. We found that WideStrike samples had a higher bacterial density and diversity per larva than insects collected from non-Bt cotton over two field seasons: 8.42 ± 0.23 and 5.36 ± 0.75 (log10 colony forming units per insect) for WideStrike compared to 6.82 ± 0.20 and 4.30 ± 0.56 for non-Bt cotton for seasons 1 and 2, respectively. Fifteen phyla, 103 families, and 229 genera were identified after performing Illumina sequencing of the 16S rRNA. At the family level, Enterobacteriaceae and Enterococcaceae were the most abundant taxa. The Enterococcaceae family was comprised mostly of Enterococcus species (E. casseliflavus and another Enterococcus sp.). Members of the Enterococcus genus can acidify their environment and can potentially reduce the alkaline activation of some Bt toxins. These findings argue for more research to better understand the role of cotton-bollworm-bacteria interactions and the impact on Bt toxin caterpillar susceptibility.

New Mosquitocide Derived From Volcanic Rock.

Malaria, dengue, yellow fever, and the Zika and West Nile Viruses are major vector-borne diseases of humans transmitted by mosquitoes. According to the World Health Organization, over 80% of the world's population is at risk of contacting these diseases. Insecticides are critical for mosquito control and disease prevention, and insect insecticide resistance is on the increase; new alternatives with potentially different modes of action from current chemistry are needed. During laboratory screening of industrial minerals for insecticide activity against Anopheles gambiae (Giles) (Diptera: Culicidae) we discovered a novel mechanical insecticide derived from volcanic rock (MIVR) with potential use as a residual spray. In modified WHO cone tests, the time to 50% mortality was 5 h under high-humidity conditions. MIVR treated surfaces demonstrated no mosquito repellency. In field studies where the mechanical insecticide was applied to wood using standard spray equipment and then placed under stilt homes in New Orleans, LA, the residual activity was >80% after 9 wk against Aedes aegypti (L.) (Diptera: Culicidae), Aedes albopictus (Skuse) (Diptera: Culicidae) and Culex quinquefasciatus (Say) (Diptera: Culicidae) (with similar efficacy to a positive chemical insecticide control). In scanning electron microcopy studies, the MIVR was transferred as particles mostly to the legs of the mosquito. This wettable powder made from volcanic rock is a mechanical insecticide representing a potential new mode of action different from current chemistry for mosquito control and is in commercial development under the trade name Imergard™WP as an indoor and outdoor residual spray.

Development of an Insecticide-Free Trapping Bednet to Control Mosquitoes and Manage Resistance in Malaria Vector Control: A New Way of Thinking.

Mosquito-borne malaria kills 429,000 people each year with the problem being acute in sub-Saharan Africa. The successes gained with long-lasting pyrethroid-treated bednets are now in jeopardy because of wide-spread, pyrethroid resistance in mosquitoes. Using crowd modeling theory normalized for standard bednet architecture, we were able to design an attract-trap-kill technology for mosquitoes that does not require insecticides. Using three-dimensional polyester knitting and heat fixation, trap funnels were developed with high capture efficacy with no egression under worst-case laboratory conditions. Field testing in Africa in WHO huts with Gen1-3 T (trap)-Nets validated our model, and as predicted, Gen3 had the highest efficacy with a 4.3-fold greater trap-kill rate with no deterrence or repellency compared to Permanet 2.0, the most common bednet in Africa. A T-Net population model was developed based on field data to predict community-level mosquito control compared to a pyrethroid bednet. This model showed the Gen3 non-insecticidal T-Net under field conditions in Africa against pyrethroid resistant mosquitoes was 12.7-fold more efficacious than single chemical, pyrethroid-treated nets.

Multiple Known Mechanisms and a Possible Role of an Enhanced Immune System in Bt-Resistance in a Field Population of the Bollworm, Helicoverpa zea: Differences in Gene Expression with RNAseq.

Several different agricultural insect pests have developed field resistance to Bt (Bacillus thuringiensis) proteins (ex. Cry1Ac, Cry1F, etc.) expressed in crops, including corn and cotton. In the bollworm, Helicoverpa zea, resistance levels are increasing; recent reports in 2019 show up to 1000-fold levels of resistance to Cry1Ac, a major insecticidal protein in Bt-crops. A common method to analyze global differences in gene expression is RNA-seq. This technique was used to measure differences in global gene expression between a Bt-susceptible and Bt-resistant strain of the bollworm, where the differences in susceptibility to Cry1Ac insecticidal proteins were 100-fold. We found expected gene expression differences based on our current understanding of the Bt mode of action, including increased expression of proteases (trypsins and serine proteases) and reduced expression of Bt-interacting receptors (aminopeptidases and cadherins) in resistant bollworms. We also found additional expression differences for transcripts that were not previously investigated, i.e., transcripts from three immune pathways-Jak/STAT, Toll, and IMD. Immune pathway receptors (ex. PGRPs) and the IMD pathway demonstrated the highest differences in expression. Our analysis suggested that multiple mechanisms are involved in the development of Bt-resistance, including potentially unrecognized pathways.

Role of long non-coding RNA in DEET- and fipronil-mediated alteration of transcripts associated with Phase I and Phase II xenobiotic metabolism in human primary hepatocytes.

Human exposure to environmental chemicals both individually and in combination occurs frequently world-wide most often with unknown consequences. Use of molecular approaches to aide in the assessment of risk involved in chemical exposure is a growing field in toxicology. In this study, we examined the impact of two environmental chemicals used in and around homes, the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels of enzymes potentially involved in xenobiotic metabolism and on long non-coding RNAs (lncRNAs). Primary human hepatocytes were treated with these two chemicals both individually and in combination. Using RNA-Seq, we found that 10 major enzyme categories involved in phase 1 and phase 2 xenobiotic metabolism were significantly (α = 0.05) up- and down-regulated (i.e., 100 µM DEET-19 transcripts, 89% up and 11% down; 10 µM fipronil-52 transcripts, 53% up and 47% down; and 100 µM DEET +10 µM fipronil-69 transcripts, 43% up and 57% down). The altered genes were then mapped to the human genome and their proximity (within 1,000,000 bp) to lncRNAs examined. Unique proximities were discovered between altered lncRNA and altered P450s (CYP) and other enzymes (DEET, 2 CYP; Fipronil, 6 CYP and 15 other; and DEET + fipronil, 7 CYP and 21 other). Many of the altered P450 transcripts were in multiple clusters in the genome with proximal altered lncRNAs, suggesting a regulator function for the lncRNA. At the gene level there was high percent identity for lncRNAs near P450 clusters, but this relationship was not found at the transcript level. The role of these altered lncRNAs associated with xenobiotic induction, human diseases and chemical mixtures is discussed.

ImergardTMWP: A Non-Chemical Alternative for an Indoor Residual Spray, Effective against Pyrethroid-Resistant Anopheles gambiae (s.l.) in Africa.

Malaria is the deadliest mosquito-borne disease and kills predominantly people in sub-Saharan Africa (SSA). The now widespread mosquito resistance to pyrethroids, with rapidly growing resistance to other insecticide classes recommended by the World Health Organization (WHO), may overturn the successes gained in mosquito control in recent years. It is of utmost importance to search for new, inexpensive, and safe alternatives, with new modes of action, that might improve the efficacy of current insecticides. The efficacy of a novel mechanical insecticidal mineral derived from volcanic rock, ImergardTMWP, was investigated to determine its efficacy as a stand-alone residual wall spray and as a mixture with deltamethrin (K-Othrine® Polyzone) in experimental huts in Cove, Benin. The evaluation was conducted with susceptible (Kisumu) and wild-type Anopheles gambiae (s.l.). Deltamethrin applied alone demonstrated 40-45% mortality (at 72 h post-exposure) during the first four months, which declined to 25% at six months for wild An. gambiae from Cove. ImergardTMWP alone and mixed with deltamethrin, under the same assay conditions, produced 79-82% and 73-81% mortality, respectively, during the same six-month period. ImergardTMWP met the 80% WHO bio-efficacy threshold for residual activity for the first five months with 78% residual activity at six months. ImergardTMWP can be used as a mixture with chemical insecticides or as a stand-alone pesticide for mosquito control in Africa.

Control of Filth Flies, Cochliomyia macellaria (Diptera: Calliphoridae), Musca domestica (Diptera: Muscidae), and Sarcophaga bullata (Diptera: Sarcophagidae), Using Novel Plant-Derived Methyl Ketones.

Filth flies live in close proximity to humans and livestock and transmit pathogens. Current control relies on chemical insecticides, and flies can develop resistance to these insecticides. The public is also interested in natural and safer insecticides. Therefore, alternative pesticides compatible with the synanthropic nature of flies are needed. Four plant aliphatic methyl ketones were evaluated for control of adult house flies, Musca domestica L., blow flies, Cochliomyia macellaria (F.), and gray flesh flies, Sarcophaga bullata (Parker). In sealed petri dish assays, 2-heptanone, 2-octanone, 2-nonanone, and 2-undecanone exhibited fumigant activity against house flies with 24-h LC50s of 6.9, 7.5, 8.0, and 9.2 µg/cm3, respectively. Further research focused on undecanone (a U.S. EPA-registered biopesticide). When tested in larger enclosures at 1.7, 2.3, and 2.8 µg/cm3, undecanone provided 60.4, 82.2, and 94.4% house fly mortality; 56.9, 75.6, and 92.5% flesh fly mortality; and 62.1, 84.5, and 97.9% blow fly mortality, respectively, after a 2-h exposure. In a two-choice behavioral assay with 194.6 µg/cm2 of the test compound on the treatment versus an untreated surface of the same area, the overall mean repellencies for blow flies were 84.7% for undecanone versus 87.6% for N,N-diethyl-meta-toluamide (DEET). For house flies, mean repellencies were 80.7% for undecanone and 84.9% for DEET. The house fly topical LD50 for undecanone was 58.1 µg per fly. Undecanone was far less expensive for filth fly control than the gold standard for insect fumigation, methyl bromide.

Bacterial microbiome of the chigger mite Leptotrombidium imphalum varies by life stage and infection with the scrub typhus pathogen Orientia tsutsugamushi.

Scrub typhus is a mites-borne rickettsiosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. The disease is potentially life threatening and is prevalent in tropical Asia, islands of the western Pacific Ocean and northern Australia where an estimated one million cases occur annually. Orientia tsutsugamushi is transmitted by the bite of larval mites in the genus Leptotrombidium. In the present study, the composition of the microbiome in larvae, deutonymphs and adult males and females from laboratory colonies of L. imphalum that were infected as well as uninfected with O. tsutsugamushi were investigated by high-throughput sequencing of the bacterial 16S rRNA gene. Notably, the bacterial microbiomes of infected adult females were dominated by sequences of O. tsutsugamushi and an unidentified species of Amoebophilaceae, which together comprised 98.2% of bacterial sequences. To improve the taxonomic resolution of the Amoebophilaceae OTU a nearly full length sequence of the 16S rRNA gene was amplified, cloned, and Sanger sequenced. Infected female mites had 89 to 92% nucleotide identity with the Amoebophilaceae family, indicating that the bacterium was likely to be a species of a novel genus. The species composition of bacterial communities varied between mite life stages regardless of their infection status. Uninfected adults exhibited greater species diversity than adults infected with O. tsutsugamushi. In the infected colony, the rate of filial infection with Orientia was less than 100%. Larval and male mites that were PCR-negative for Orientia contained low numbers of sequences of Amoebophilaceae (0.01 and 0.06%, respectively) in their taxonomic profiles, suggesting that a mutualistic relationship exists between the novel species of Amoebophilaceae and O. tsutsugamushi. Our study findings provide the basis for further research to determine the influence of the novel Amoebophilaceae species on the bacterial microbiome and on vector susceptibility to and transovarial transmission of O. tsutsugamushi.

Differential Expression Profile of lncRNAs from Primary Human Hepatocytes Following DEET and Fipronil Exposure.

While the synthesis and use of new chemical compounds is at an all-time high, the study of their potential impact on human health is quickly falling behind, and new methods are needed to assess their impact. We chose to examine the effects of two common environmental chemicals, the insect repellent N,N-diethyl-m-toluamide (DEET) and the insecticide fluocyanobenpyrazole (fipronil), on transcript levels of long non-protein coding RNAs (lncRNAs) in primary human hepatocytes using a global RNA-Seq approach. While lncRNAs are believed to play a critical role in numerous important biological processes, many still remain uncharacterized, and their functions and modes of action remain largely unclear, especially in relation to environmental chemicals. RNA-Seq showed that 100 µM DEET significantly increased transcript levels for 2 lncRNAs and lowered transcript levels for 18 lncRNAs, while fipronil at 10 µM increased transcript levels for 76 lncRNAs and decreased levels for 193 lncRNAs. A mixture of 100 µM DEET and 10 µM fipronil increased transcript levels for 75 lncRNAs and lowered transcript levels for 258 lncRNAs. This indicates a more-than-additive effect on lncRNA transcript expression when the two chemicals were presented in combination versus each chemical alone. Differentially expressed lncRNA genes were mapped to chromosomes, analyzed by proximity to neighboring protein-coding genes, and functionally characterized via gene ontology and molecular mapping algorithms. While further testing is required to assess the organismal impact of changes in transcript levels, this initial analysis links several of the dysregulated lncRNAs to processes and pathways critical to proper cellular function, such as the innate and adaptive immune response and the p53 signaling pathway.