Logu Ponnusamy

In recent decades, vector-borne diseases have re-emerged and prolonged at alarming rates, triggering significant morbidity and mortality worldwide. Particularly, larvae of trombiculid mites, commonly known as chiggers, are widespread ectoparasites of vertebrates and known to bite humans. They transmit a human pathogen, including bacteria, such as Orientia spp. (Scrub typhus), Bartonella spp. and Rickettsia spp. has been reported. Scrub typhus has an expanding known distribution, with most diseases occurring across South and East Asia, Middle East, South America, and in sub-Saharan Africa. Importantly, however, the microbiomes of chigger species remain unexplored in the USA. Our long-term goals are to identify novel microbial organisms as potential pathogens, symbionts, and commensals associated with chigger, to understand the wildlife reservoirs for these microbes and, to develop new diagnostic tests.

Our areas of research in 2023 are the following: (1) Determine proof of concept for using wing beat patterns and machine learning for remote identification of bollworm versus budworm moths in cotton; (2) Determine whether the microbiome associated with bollworm larval resistance to Bt cotton can transfer to adults and the next generation; and (3) Conduct preliminary egg staining assessments of bollworm versus budworm eggs for egg ID.

Larvae of trombiculid mites (Acari: Trombiculidae), commonly known as chiggers, bite humans outdoors, transmit pathogens causing human disease that on a worldwide basis sickens millions of people and causes death. This pilot project will use next-generation sequencing to provide the first characterization of the trombiculid bacterial microbiome in the United States. The research will investigate chigger species that acquired bacteria from feeding on wild animal hosts and their potential role in the epidemiology of tick-associated rickettsiosis, illness related to scrub typhus that might produce adverse human health conditions.

This project will describe the microbiome from the invasive Asian Long-horned Tick.

Phlebotomine sand flies transmit protozoan parasites (Leishmania spp.), bacterial (Bartonella bacilliformis), and viral pathogens . Because no vaccines are available against these pathogens, reduction of exposure to sand fly bites is the most effective disease prevention measure. An alternative approach to the traditional delivery of an insecticide to the vector is to bring the vector to the insecticide using an attractant. In the context of controlling vector-borne disease, oviposition-site attractants are expected to be highly effective because they target gravid females that are responsible for transmission of the pathogen and amplifying vector populations. Decomposing organic matter is the main food source for sand fly larvae. Central to our ongoing and proposed research is the proposition that natural selection has tuned the olfactory system of gravid females to odorants emanating from optimal oviposition substrates that indicate suitable conditions and nutrients for larval development. We therefore hypothesize that gravid sand flies are differentially attracted in a dose-dependent manner to a blend of fecal- and microbially-derived chemical cues associated with the decomposition of fecal material, as well as to signals from eggs and larvae which indicate suitable oviposition sites. Results of previous field and lab studies indicate that gravid females of both old- and new-world sand fly species are attracted to odors emanating from conspecific eggs as well as to host feces conditioned by feeding larvae. In a preliminary study, we showed that rearing medium of 2nd/3rd instars was highly attractive for Phlebotomus papatasi. We cultured 12 bacterial isolates from this medium, and initiated dose-response studies of their effect on the oviposition behavior of gravid females. Our overall goal is to develop and optimize an attractive blend of semiochemicals that would function as a lure for oviposition-site seeking females of Ph. papatasi sand flies. In the future, we will also test the efficacy of this lure on new-world sand flies (Lutzomyia longipalpis) and use our methodologies to develop new lures for this and related species. We will apply an integrated interdisciplinary approach including behavioral, electrophysiological, and microbiological studies to systematically address the following specific aims: Aim 1. Identify the most attractive and oviposition stimulating conspecific stages, rearing medium, and saprophytic microbes. Rationale & Hypotheses: (a) Conspecifics: Previous research suggests that gravid females are attracted to conspecific eggs, larvae and pupae. Because sand fly larvae exhibit density-dependent cannibalism, we hypothesize a dose-dependent reversal of oviposition response (i.e., upside-down parabola): females should be attracted to conspecifics at low density, and repelled at high density; (b) Rearing medium and microbes: We hypothesize that host feces and bacteria and fungi associated with host feces are attractive to gravid female flies. Approach: Task 1. Conduct behavioral assays to identify the most attractive life-stage, the optimal density of this life stage, and the concentration of extracts of this life stage that elicits maximal attraction. Task 2. Assess the interaction between rearing medium decomposition and larval stage using olfactometer and oviposition preference assays (preliminary results indicate that 2nd/3rd instar rearing medium is most attractive, but the independent contribution of larvae and rearing medium needs to be evaluated). Task 3. Isolate and identify culturable microbial strains from the most attractive rearing medium and conduct dose-response studies to identify the most attractive strains. Aim 2. Isolate and identify oviposition attractants and stimulants from the most attractive conspecific stage, rearing medium, and microbial isolates. Rationale & Hypothesis: Gravid females are expected to use at least two major classes of chemosensory cues: Volatile cues that guide orientation to oviposition sites and contact-based cues that stimulate oviposition. We hypothesize that the olfactory and gustatory cues that guide orientation to oviposition sites and egg laying can be extracted with organic solvents, assayed behaviorally and electrophysiologically, and chemically characterized. Approach: Task 1. Using bioassay-guided extraction and fractionation , isolate chemical fractions from the most attractive sources characterized in Aim 1. Task 2. Identify the most attractive odorants from the active fractions and from the isolated microbial strains through SPME, headspace collections, GC-EAD, and GC-MS. Aim 3: Develop an optimal blend of oviposition attractants and stimulants and evaluate it at the micro- and meso-scales. Rationale & Hypothesis: Development of a sand fly oviposition lure will require a blend of chemicals that represent to gravid females an optimal oviposition site. We hypothesize that a blend of odorants from conspecifics, host feces and microbial isolates can be optimized as an effective sand fly lure. Olfactometer bioassays occur at the micro-scale of a few decimeters. But an attractive lure needs to be effective in the field and its efficacy should be shown over a larger distance that requires sustained oriented flight. Approach: Task 1. Bioassay newly identified compounds and previously identified attractants from conspecifics and host feces. Task 2. Using additive assays, test various blends of the most attractive odorants derived from conspecifics, host feces and microbial isolates using olfactometers. Task 3. Evaluate the most attractive blend in a wind tunnel.

The US Department of Labor statistics report that agriculture is one of the most dangerous occupations in the country. If chronic health conditions are included in this analysis, the threat is even greater. It is well documented that agricultural workers and families including children that live in or near the agricultural workplace suffer from poor health compared to the general US population. Although there has been research on many risk factors associated with the farm, forestry activities, and the home environment associated with these occupations (especially the farm), the impact of filth files on worker and family health has not been investigated. Filth flies cause diarrheal diseases and spread multiple-antibiotic resistant bacteria from animals to adults and children. The military estimates that 10% or more of their deployed service personnel are sickened by fly-borne diarrheal diseases at any given time. The extent of the risk to the farm and forestry community is unknown but based on military estimates is expected to be high. The objective of this proposal is to examine the microbiome of filth flies in the farm environment. Our focus will be the blow fly, Phormia regina, the major fly in the US found infesting and involved in the decompensation of dead animals and vegetation. These flies are also attracted to the Ag workplace, homes and your next meal. Our research in the future will focus on other filth flies, e.g., house flies and flesh flies. Blow fly adults will be trapped on a university farm where there is both plant and animal production in process and where this farm is located in proximity to an urban neighborhood and a farmers market. The external and internal DNA from replicate flies will be extracted, the bacteria 16S ribosomal RNA (rRNA) gene amplified and sequenced, and the sequence data used to identify the bacteria species and potential presence of antibiotic-resistant genes. We will examine both alpha and beta microbial diversity between fly samples and bacteria origins and identify flies to the lowest possible taxonomic unit (potentially down to species). Statistical and phylogenic analyses will be conducted using QIIME II, phylogenetics, and cloning and additional DNA sequencing of bacterial genomic DNA if needed. Antibiotic resistance will be assessed by targeted PCR using know (specific) primers that would amplify antibiotic resistant microbial genes. The role of the bacteria in human disease will be limited to what is known from the scientific literature for the particular bacteria identified from the flies collected. The data generated in this proposal will be used to develop proposals for USDA, NIOSH, NIH, NIEHS, EPA, DOE, and DOD.

The mosquito Aedes aegypti is the primary vector of human viruses causing dengue and yellow fever, diseases of global public health importance. Aedes aegypti is a peri-domestic species that lays desiccation-resistant eggs in water-filled human-made containers. In the proposed research, we will conduct a comprehensive investigation of the interaction of bacteria and Ae. aegypti eggs. The main goal of the proposed project is to identify two groups of bacteria-produced chemical cues, one that stimulates and another that inhibits egg hatching. Our hypothesis, based on extensive preliminary results, that bacteria-associated chemical cues regulate hatching is a departure from the generally accepted paradigm that egg hatching is stimulated by low dissolved oxygen (DO) concentration resulting from rapid bacterial growth. Our preliminary data show that a 1-day old mixed culture of 14 bacterial species, originally isolated from an experimental mosquito habitat, stimulates egg hatching under both high and low DO conditions. However, under supposedly stimulatory low DO conditions, eggs fail to hatch when submerged in an 8-day old culture of the same mix of bacterial species, suggesting that the same bacteria species can produce stimulatory and inhibitory cues, depending on the age of culture. In the proposed research, we will screen 14 bacterial species isolates from experimental mosquito habitats for bioactivity in stimulating and inhibiting egg hatch. Bacterial species exhibiting highly stimulatory or inhibitory bioactivity will be selected for further research. Chemical compounds associated with bacterial species will be identified by using bioassay-guided solvent extraction and fractionation coupled with chemical analytical techniques, including high-performance liquid chromatography and gas chromatography, and mass spectrometry. The activity of putative bioactive compounds will be verified in egg hatching bioassays. Once identified, the cue(s) that control hatching may be used to disrupt hatching of the pharate larva from the mosquito egg and offer innovative tools for control of a virus vector of global importance. The proposed research will provide a more complete understanding of the strategies that Ae. aegypti eggs employ to maximize survival under changing environmental conditions. Moreover, the project will provide tools and reagents for future research on the physiological events during the transition from embryo to neonate, including gene expression patterns and studies of sensory physiology

Tick-borne diseases are of growing importance in the US. Within the US there are geographic variations in tick vectors as well as the types of tick-borne diseases. Despite the public health importance of tick-borne diseases such as Lyme disease, Rocky Mountain Spotted Fever (RMSF), Babesiosis and Human Monocytic Ehrlichiosis (HME), there are huge gaps in our understanding of their natural history and epidemiology. The aim of this pilot project is to use Massively Parallel Pyrosequencing to describe the microbial flora of various stages and species of ticks from North Carolina, New York and Connecticut. We will use these pilot data as the basis for future grant proposals. Our long term goals are to characterize and understand the pathogens, symbionts and commensals in the tick microbiome. This information can be used to better characterize modes of transmission and wildlife reservoirs and to develop new diagnostic tests.