Microbiome of Invasive Tick Species Haemaphysalis Longicornis in North Carolina, USA.

Ticks are one of the most important vectors of human and animal disease worldwide. In addition to pathogens, ticks carry a diverse microbiota of symbiotic and commensal microorganisms. In this study, we used next-generation sequencing (NGS) to survey the microbiomes of Haemaphysalis longicornis (Acari: Ixodidae) at different life stages collected from field populations in North Carolina (NC), USA. Sequence analyses were performed using QIIME2 with the DADA2 plugin and taxonomic assignments using the Greengenes database. Following quality filtering and rarefaction, the bacterial DNA sequences were assigned to 4795 amplicon sequence variants (ASVs) in 105 ticks. A core microbiome of H. longicornis was conserved across all ticks analyzed, and included bacterial taxa: Coxiella, Sphingomonas, Staphylococcus, Acinetobacter, Pseudomonas, Sphingomonadaceae, Actinomycetales, and Sphingobium. Less abundant bacterial taxa, including Rickettsia and Aeromonas, were also identified in some ticks. We discovered some ASVs that are associated with human and animal infections among the identified bacteria. Alpha diversity metrics revealed significant differences in bacterial diversity between life stages. Beta diversity metrics also revealed that bacterial communities across the three life stages were significantly different, suggesting dramatic changes in the microbiome as ticks mature. Based on these results, additional investigation is necessary to determine the significance of the Haemaphysalis longicornis microbiome for animal and human health.

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.

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.

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.

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.

Bacterial Isolates Derived from Nest Soil Affect the Attraction and Digging Behavior of Workers of the Red Imported Fire Ant, Solenopsis invicta Buren.

Populations of monogyne and polygyne red imported fire ants (RIFA), Solenopsis invicta Buren, are distributed throughout the southern United States. This ant species is hazardous to farm animals and workers, damages infrastructure, and depletes native arthropod populations. Colony expansion is affected by several biotic factors, but the effects of soil microbes on ant behavior related to soil excavation within nest sites have not been investigated. Consequently, we cultured bacteria from RIFA nest soils. The effects of individual bacterial isolates and bacterial cell densities on the choice of digging site as well as digging activity of monogyne and polygyne RIFA worker ants were evaluated in two-choice bioassays. Based on phylogenetic analysis, 17 isolates were selected and tested initially at 5 × 108 cells/mL and 20 workers per assay. Firmicutes (Bacillus, Paenibacillus, Brevibacillus) repelled the ants, but Arthrobacter woluwensis strongly attracted ants. Subsequently, the six isolates having the greatest positive or negative effects on ant behavior were evaluated at a lower bacterial cell and worker ant densities. Ant responses to these bacteria generally decreased as cell densities declined to 5 × 106 cells/mL. Observations of ant behavior during a three-hour, two-choice bioassay revealed that ants generally visited both control and bacteria-treated sand prior to making a digging site choice. Our research results indicate that soil bacteria may mediate ant nest expansion or relocation and foraging tunnel construction. Identification of bacterial metabolites that affect RIFA digging behavior merits additional research because these compounds may provide a basis for novel management strategies that repel RIFA away from sensitive infrastructure or attract fire ants to insecticidal baits.

Analyses of Bloodmeal Hosts and Prevalence of Rickettsia parkeri in the Gulf Coast Tick Amblyomma maculatum (Acari: Ixodidae) From a Reconstructed Piedmont Prairie Ecosystem, North Carolina.

Host feeding patterns and the prevalence of infection with Rickettsia parkeri were determined for the primary vector, Amblyomma maculatum Koch as well as sympatric tick species A. americanum (Linnaeus) and Dermacentor variabilis (Say) collected from a reconstructed prairie in the Piedmont region of North Carolina during 2011 and 2012. The occurrence of R. parkeri among A. maculatum adults and nymphs was 36.9% (45/122) and 33.3% (2/6), respectively. Rickettsia parkeri was detected in a single male A. americanum 2.3% (1/43). A PCR-reverse line blot hybridization assay of a 12S rDNA fragment amplified from remnant larval and nymphal bloodmeals of host-seeking ticks was used to identify bloodmeal hosts. Of the tick samples tested, bloodmeal host identification was successful for 29.3% (12/41) of adult A. americanum and 39.2% (20/51) of adult D. variabilis. For A. maculatum, bloodmeal host identification was successful for 50% (61/122) of adults collected from vegetation and 100% (4/4) of nymphs removed from cotton rats (Sigmodon hispidus Say and Ord). The cotton rat was the most common bloodmeal host with 59.0% (36/61) identified for adult A. maculatum. No statistically significant association was observed, however, between bloodmeal host and pathogen prevalence for any tick species. While the cotton rat was an important bloodmeal host for A. maculatum nymphs, this vertebrate did not appear to be the primary source of R. parkeri infection for A. maculatum.

Colonization by the Red Imported Fire Ant, Solenopsis invicta, Modifies Soil Bacterial Communities.

The long-standing association between insects and microorganisms has been especially crucial to the evolutionary and ecological success of social insect groups. Notably, research on the interaction of the two social forms (monogyne and polygyne) of the red imported fire ant (RIFA), Solenopsis invicta Buren, with microbes in its soil habitat is presently limited. In this study, we characterized bacterial microbiomes associated with RIFA nest soils and native (RIFA-negative) soils to better understand the effects of colonization of RIFA on soil microbial communities. Bacterial community fingerprints of 16S rRNA amplicons using denaturing gradient gel electrophoresis revealed significant differences in the structure of the bacterial communities between RIFA-positive and RIFA-negative soils at 0 and 10 cm depths. Illumina sequencing of 16S rRNA amplicons provided fine-scale analysis to test for effects of RIFA colonization, RIFA social form, and soil depth on the composition of the bacterial microbiomes of the soil and RIFA workers. Our results showed the bacterial community structure of RIFA-colonized soils to be significantly different from native soil communities and to evidence elevated abundances of several taxa, including Actinobacteria. Colony social form was not found to be a significant factor in nest or RIFA worker microbiome compositions. RIFA workers and nest soils were determined to have markedly different bacterial communities, with RIFA worker microbiomes being characterized by high abundances of a Bartonella-like endosymbiont and Entomoplasmataceae. Cloning and sequencing of the 16S rRNA gene revealed the Bartonella sp. to be a novel bacterium.

Tick Ecdysteroid Hormone, Global Microbiota/Rickettsia Signaling in the Ovary versus Carcass during Vitellogenesis in Part-Fed (Virgin) American Dog Ticks, Dermacentor variabilis.

The transovarial transmission of tick-borne bacterial pathogens is an important mechanism for their maintenance in natural populations and transmission, causing disease in humans and animals. The mechanism for this transmission and the possible role of tick hormones facilitating this process have never been studied. Injections of physiological levels of the tick hormone, 20-hydroxyecdysone (20E), into part-fed (virgin) adult females of the American dog tick, Dermacentor variabilis, attached to the host caused a reduction in density of Rickettsia montanensis in the carcass and an increase in the ovaries compared to buffer-injected controls. This injection initiates yolk protein synthesis and uptake by the eggs but has no effect on blood feeding. Francisella sp. and R. montanensis were the predominant bacteria based on the proportionality in the carcass and ovary. The total bacteria load increased in the carcass and ovaries, and bacteria in the genus Pseudomonas increased in the carcass after the 20E injection. The mechanism of how the Rickettsia species respond to changes in tick hormonal regulation needs further investigation. Multiple possible mechanisms for the proliferation of R. montanensis in the ovaries are proposed.

Sphingobacterium phlebotomi sp. nov., a new member of family Sphingobacteriaceae isolated from sand fly rearing substrate.

A Gram-stain-negative, rod-shaped, non-motile, non-spore-forming, aerobic bacterium, designated type strain SSI9T, was isolated from sand fly (Phlebotomus papatasi Scopoli; Diptera: Psychodidae) rearing substrate and subjected to polyphasic taxonomic analysis. Strain SSI9T contained phosphatidylethanolamine as a major polar lipid, MK-7 as the predominant quinone, and C16 : 1ω6c/C16 : 1ω7c, iso-C15 : 0, iso-C17 : 0 3-OH and C16 : 0 as the major cellular fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that SSI9T represents a member of the genus Sphingobacterium, of the family Sphingobacteriaceae sharing 96.5-88.0 % sequence similarity with other species of the genus Sphingobacterium. The results of multilocus sequence analysis using the concatenated sequences of the housekeeping genes recA, rplC and groL indicated that SSI9T formed a separate branch in the genus Sphingobacterium. The genome of SSI9T is 5 197 142 bp with a DNA G+C content of 41.8 mol% and encodes 4395 predicted coding sequences, 49 tRNAs, and three complete rRNAs and two partial rRNAs. SSI9T could be distinguished from other species of the genus Sphingobacterium with validly published names by several phenotypic, chemotaxonomic and genomic characteristics. On the basis of the results of this polyphasic taxonomic analysis, the bacterial isolate represents a novel species within the genus Sphingobacterium, for which the name Sphingobacterium phlebotomi sp. nov. is proposed. The type strain is SSI9T (=ATCC TSD-210T=LMG 31664T=NRRL B-65603T).

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.

Characterization of Long Non-Coding RNAs in the Bollworm, Helicoverpa zea, and Their Possible Role in Cry1Ac-Resistance.

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).

Oviposition-Site Selection of Phlebotomus papatasi (Diptera: Psychodidae) Sand Flies: Attraction to Bacterial Isolates From an Attractive Rearing Medium.

Phlebotomine sand flies are worldwide vectors of Leishmania parasites as well as other bacterial and viral pathogens. Due to the variable impact of traditional vector control practices, a more ecologically based approach is needed. The goal of this study was to isolate bacteria from the most attractive substrate to gravid Phlebotomus papatasi Scopoli sand flies and determine the role of bacterial volatiles in the oviposition attractancy of P. papatasi using behavioral assays. We hypothesized that gravid sand flies are attracted to bacterially derived semiochemical cues associated with breeding sites. Bacteria were isolated from a larvae-conditioned rearing medium, previously shown to be highly attractive to sand flies. The isolated bacteria were identified by amplifying and sequencing 16S rDNA gene fragments, and 12 distinct bacterial species were selected for two-choice olfactometer bioassays. The mix of 12 bacterial isolates elicited strong attraction at the lower concentration of 107 cells per ml and significant repellence at a high concentration of 109 cells per ml. Three individual isolates (SSI-2, SSI-9, and SSI-11) were particularly attractive at low doses. In general, we observed dose-related effects, with some bacterial isolates stimulating negative and some positive dose-response curves in sand fly attraction. Our study confirms the important role of saprophytic bacteria, gut bacteria, or both, in guiding the oviposition-site selection behavior of sand flies. Identifying the specific attractive semiochemical cues that they produce could lead to development of an attractive lure for surveillance and control of sand flies.

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.

The egg and larval pheromone dodecanoic acid mediates density-dependent oviposition of Phlebotomus papatasi.

BACKGROUND: Gravid females assess the conditions of oviposition sites to secure the growth and survival of their offspring. Conspecific-occupied sites may signal suitable oviposition sites but may also impose risk due to competition or cannibalism at high population density or heterogeneous larval stage structure, respectively. Chemicals in the habitat, including chemicals emitted from other organisms, serve as cues for females to assess habitat conditions. Here, we investigated the attraction and oviposition preference of the Old World cutaneous leishmaniasis vector, Phlebotomus papatasi, to young and old conspecific stages, including eggs and evaluated the effect of a semiochemical associated with eggs and neonate larvae. METHODS: Attraction and oviposition preference of Ph. papatasi to each of various life stages (eggs, first-, second-, third-, fourth-instar larvae, pupae and male and female adults) was investigated using cage and oviposition jar behavioral assays. Identification of organic chemical compounds extracted from eggs was performed using GC-MS and chemicals were tested in the same behavioral assays in a dose-response manner. Behavioral responses were statistically analyzed using logistic models. RESULTS: Gravid Ph. papatasi females were significantly attracted to and preferred to oviposit on medium containing young life stages (eggs and first instars). This preference decreased towards older life stages. Dose effect of eggs indicated a hump-shaped response with respect to attraction but a concave-up pattern with respect to oviposition. Chemical analysis of semiochemicals from eggs and first-instar larvae revealed the presence of dodecanoic acid (DA) and isovaleric acid. Sand flies were attracted to and laid more eggs at the lowest DA dose tested followed by a negative dose-response. CONCLUSIONS: Findings corroborated our hypothesis that gravid sand flies should prefer early colonized oviposition sites as indicators of site suitability but avoid sites containing older stages as indicators of potential competition. Findings also supported the predictions of our hump-shaped oviposition regulation (HSR) model, with attraction to conspecific eggs at low-medium densities and switching to repellence at high egg densities. This oviposition behavior is mediated by DA that was identified from surface extracts of both eggs and first-instar larvae. Isovaleric acid was also found in extracts of both stages.

Diel periodicity and visual cues guide oviposition behavior in Phlebotomus papatasi, vector of old-world cutaneous leishmaniasis.

BACKGROUND: Phlebotomine sand flies are vectors of human leishmaniases, important neglected tropical diseases. In this study, we investigated diel patterns of oviposition behavior, effects of visual cues on oviposition-site selection, and whether these affect the attraction of gravid Phlebotomus papatasi (Scopoli), the vector of old-world cutaneous leishmaniasis, to olfactory cues from oviposition sites. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate these questions, we conducted a series of experiments using attraction and oviposition assays within free-flight test chambers containing gravid females entrained under a 14:10 hrs light:dark photoperiod. By replacing sticky-screens or moist filter papers every three hours, we showed that oviposition site search occurs mainly in the latest part of the night whereas peak oviposition occurs during the early part of the night. Behavioral responses to olfactory oviposition cues are regulated by time-of-day and can be disrupted by transient exposure to a constant darkness photoperiod. Gravid females, but not any other stage, age, or sex, were attracted to dark, round oviposition jars, possibly resembling rodent burrow openings. This visual attraction disappeared in the absence of an illumination source. Egg deposition rate was not affected by jar color. Olfactory cues had the strongest effect when the visual cues were minimal. CONCLUSION AND SIGNIFICANCE: Our study showed, for the first time, that visual cues in the form of oviposition-site color, lighting level, and photoperiod are important in guiding the oviposition behavior of phlebotomine sand flies. Furthermore, such visual cues could modify the flies' sensitivity to olfactory oviposition cues. Our results suggest that chemosensory and visual cues are complementary, with visual cues used to orient gravid females towards oviposition sites, possibly at long- to medium-ranges during crepuscular periods, while olfactory cues are used to approach the burrow in darkness and assess its suitability at close-range. Implications to sand fly control are discussed.

A Diverse Microbial Community Supports Larval Development and Survivorship of the Asian Tiger Mosquito (Diptera: Culicidae).

Laboratory microcosm experiments were conducted to evaluate effects of bacteria isolated from senescent white oak leaves on the growth and survivorship of larval Aedes albopictus (Skuse). Larvae hatched from surface-sterilized eggs were reared in microcosms containing individual bacterial isolates, combined isolates (Porphyrobacter sp., Enterobacter asburiae, Acidiphilium rubrum, Pseudomonas syringae, and Azorhizobium caulinodans), a positive control containing a microbial community from an infusion of white oak leaves, and a negative control consisting of sterile culture media. Experiments were conducted for 21 d after which microcosms were deconstructed, larval survivorship was calculated, and bacteria contained in pupae, and adults that developed were quantified to determine rates of transstadial transmission. Positive control microcosms containing diverse microbial communities had an average (±SE) pupation rate of 89.3 (±5.8)% and average larval survivorship of 96.0 (± 2.3)%. Pupation in microcosms with bacterial isolates only occurred twice among all experimental replications; average larval survivorship ranged from 19 to 56%, depending on treatment. Larval growth was not found to be dependent on bacterial isolate density or isolate species, and larval survivorship was dependent on bacterial isolate density, not on isolate species. Potential mechanisms for failed development of larvae in microcosms with bacterial isolates are discussed. Bacterial isolates alone did not support larval development. High larval survivorship in positive control microcosms suggests that a diverse microbial community is required to complete larval development. Additional studies are needed to evaluate larval growth and survivorship on nonbacterial microbes, such as fungi and protozoa.