Abstract Background Visceral leishmaniasis, caused by Leishmania donovani and transmitted by Phlebotomus argentipes, remains a major public health challenge in the Indian subcontinent. Sand fly populations are controlled by using different insecticides, particularly dichlorodiphenyltrichloroethane (DDT) and pyrethroids such as alpha-cypermethrin. Prolonged and irregular use of these insecticides raises concern about the development of resistance in sand fly populations. Methods This study aimed to compare metabolic detoxification and knockdown resistance (kdr) mutations in Vgsc and ace-1 between nine sprayed (indoor residual spray, IRS) villages and one unsprayed (non-IRS) village in Muzaffarpur, Bihar, India. A total of 10 Ph. argentipes from each village were used for metabolic detoxification and 10–25 Ph. argentipes were used for target site insensitivity mutations. Homogenized Ph. argentipes aliquots were used for different enzymes activity and DNA was used for the sequence analysis of Vgsc and ace-1 genes to access the presence of kdr mutations. Results Biochemical assays revealed that levels of detoxification enzymes, including glutathione S-transferase (GST), esterase (PNPA), and cytochrome P450 monooxygenase, were elevated in IRS villages, suggesting localized metabolic resistance. Molecular screening of the voltage-gated sodium channel (Vgsc) gene revealed high frequencies of knockdown resistance (kdr) mutations at codon 1014, with serine (L1014S) (48.5%) being the most prevalent, followed by wild type leucine (L1014) (39.5%). No mutations were detected at codon 119 of the ace-1 gene, indicating the sensitivity to organophosphates in the sand fly population. Conclusions The results suggested that continuous and repeated exposure to the synthetic pyrethroid may exert selective pressure, leading to early signs of resistance in Ph. argentipes, mediated through metabolic detoxification mechanisms and mutation in the kdr gene. These findings underscore the importance of ongoing resistance monitoring and the implementation of rotational insecticide strategies to support sustained efforts toward the elimination of visceral leishmaniasis. Graphical Abstract

Visceral leishmaniasis derived from Leishmania donovani is transmitted by sand flies (Phlebotomus argentipes) throughout the Indian subcontinent. Although considered anthroponotic, L. donovani infects other mammals susceptible to sand fly bites, including dogs. Aggressive strategies to reduce sand fly populations in India have led to flies seeking nonhuman hosts, so understanding the role of dogs in L. donovani transmission has become critical. Our study investigated L. donovani infection in dogs and the potential for such infections to be transmitted back to sand flies. We performed xenodiagnosis by using P. argentipes on dogs (n = 73) with quantitative PCR-detectible parasitemia in both endemic and outbreak villages. We found that 12% (9/73) of dogs were infectious to sand flies during winter and rainy seasons. Patients with visceral leishmaniasis remain primary sources of L. donovani transmission, but our findings suggest a possible link between canine infection and human exposure.

Abstract Background The Centers for Disease Control and Prevention (CDC) bottle bioassay is a commonly used susceptibility test for measuring insect response to insecticide exposure. However, inconsistencies and high variability in insect response when conducting CDC bottle bioassays have been reported in previous publications. We hypothesized that the CDC bottle bioassay results may be compromised when expected and actual insecticide concentrations in the bottles are not equivalent and that inadequate bottle cleaning and/or loss during insecticide introduction and bottle storage steps could be responsible. We explored this hypothesis by quantifying insecticides using gas chromatography tandem mass spectrometry (GC-MS/MS) in bottles that had been cleaned, prepared, and stored according to the CDC guidelines. Methods We investigated the bottle cleaning, preparation, and storage methods outlined in the CDC bottle bioassay procedure to identify sources of irreproducibility. We also investigated the effectiveness of cleaning bottles by autoclaving because this method is commonly used in insecticide assessment laboratories. The two insecticides used in this study were chlorpyrifos and lambda-cyhalothrin (λ-cyhalothrin). Insecticides were removed from glass bioassay bottles by rinsing with ethyl-acetate and n-hexane and then quantified using GC-MS/MS. Results The CDC bottle bioassay cleaning methods did not sufficiently remove both insecticides from the glass bottles. The cleaning methods removed chlorpyrifos, which has higher water solubility, more effectively than λ-cyhalothrin. Chlorpyrifos experienced significant loss during the bottle-coating process whereas λ-cyhalothrin did not. As for bottle storage, no significant decreases in insecticide concentrations were observed for 6 h following the initial drying period for either insecticide. Conclusions The CDC bottle bioassay protocol is susceptible to producing inaccurate results since its recommended bottle cleaning method is not sufficient and semi-volatile insecticides can volatilize from the bottle during the coating process. This can lead to the CDC bottle bioassay producing erroneous LC50 values. High levels of random variation were also observed in our experiments, as others have previously reported. We have outlined several steps that CDC bottle bioassay users could consider that would lead to improved accuracy and reproducibility when acquiring toxicity data. Graphical Abstract

Abstract Background Visceral leishmaniasis (VL), known as Kala-azar on the Indian subcontinent, is a parasitic disease caused by the flagellated protozoa Leishmania donovani and can be fatal if left untreated. The sand fly Phlebotomus argentipes is the only proven vector of VL in the Southeast Asia region, and VL control in this region has relied on the use of synthetic insecticides for indoor residual spraying (IRS). The use of DDT in VL control programmes has led to the development of resistance to this insecticide in sand flies, resulting in DDT being replaced with the insecticide alpha-cypermethrin. However, alpha-cypermethrin has a similar mode of action as DDT and, therefore, the risk of resistance development in sand flies increases under the pressure of regular exposure to this insecticide. In the present study we assessed the susceptibility status of wild-caught sand flies and F1 progeny using the CDC bottle bioassay. Methods Sand flies were collected from 10 villages in Muzaffarpur District, Bihar, India. Eight of these villages are receiving continuous IRS with alpha-cypermethrin, one village had discontinued IRS with alpha-cypermethrin and one village had never received IRS with alpha-cypermethrin. The collected sand flies were exposed to a pre-determined diagnostic dose for a specific time duration (3 µg/ml for 40 min), and knockdown and mortality at 24 h post-exposure were recorded. Results Knockdown ranged from 91.19% to 99.47% for wild-caught sand flies and from 91.70% to 98.89% for their F1 progeny. At 24 h post-exposure, mortality ranged from 89.34% to 98.93% for wild-caught sand flies and from 90.16% to 98.33% for F1 progeny. Conclusions The results of this study showed that P. argentipes is potentially developing resistance, signalling the need for continuous monitoring and vigilance to sustain the validation of elimination once achieved. Graphical Abstract

Visceral leishmaniasis (VL) is a vector-borne protozoan disease, which can be fatal if left untreated. Synthetic chemical insecticides are very effective tools for controlling of insect vectors, including the sand fly Phlebotomus argentipes, the vector of VL in the Indian subcontinent. However, repeated use of the same insecticide with increasing doses potentially can create high selection pressure and lead to tolerance and resistance development. The objective of this study was to determine the lethal concentrations and assess levels of susceptibility, diagnostic doses and times to death of laboratory-reared P. argentipes to five insecticides that are used worldwide to control vectors. Using the Center for Disease Control and Prevention (CDC) bottle bioassay, 20–30 sand flies were exposed in insecticide- coated 500-ml glass bottles. Flies were then observed for 24 hours and mortality was recorded. Dose-response survival curves were generated for each insecticide using QCal software and lethal concentrations causing 50%, 90% and 95% mortality were determined. A bioassay was also conducted to determine diagnostic doses and diagnostic times by exposing 20–30 flies in each bottle containing set concentrations of insecticide. Mortality was recorded at 10-minute intervals for 120 minutes to generate the survival curve. Phlebotomus argentipes are highly susceptible to alpha-cypermethrin, followed by deltamethrin, malathion, chlorpyrifos, and least susceptible to DDT. Also, the lowest diagnostic doses and diagnostic times were established for alpha-cypermethrin (3μg/ml for 40 minutes) to kill 100% of the flies. The susceptibility data, diagnostic doses and diagnostic times presented here will be useful as baseline reference points for future studies to assess insecticide susceptibility and resistance monitoring of field caught sand flies and to assist in surveillance as VL elimination is achieved in the region.

Leishmaniasis on the Indian subcontinent is thought to have an anthroponotic transmission cycle. There is no direct evidence that a mammalian host other than humans can be infected with Leishmania donovani and transmit infection to the sand fly vector. The aim of the present study was to evaluate the impact of sand fly feeding on other domestic species and provide clinical evidence regarding possible non-human reservoirs through experimental sand fly feeding on cows, water buffalo goats and rodents. We performed xenodiagnosis using colonized Phlebotomus argentipes sand flies to feed on animals residing in villages with active Leishmania transmission based on current human cases. Xenodiagnoses on mammals within the endemic area were performed and blood-fed flies were analyzed for the presence of Leishmania via qPCR 48hrs after feeding. Blood samples were also collected from these mammals for qPCR and serology. Although we found evidence of Leishmania infection within some domestic mammals, they were not infectious to vector sand flies. Monitoring infection in sand flies and non-human blood meal sources in endemic villages leads to scientific proof of exposure and parasitemia in resident mammals. Lack of infectiousness of these domestic mammals to vector sand flies indicates that they likely play no role, or a very limited role in Leishmania donovani transmission to people in Bihar. Therefore, a surveillance system in the peri-/post-elimination phase of visceral leishmaniasis (VL) must monitor absence of transmission. Continued surveillance of domestic mammals in outbreak villages is necessary to ensure that a non-human reservoir is not established, including domestic mammals not present in this study, specifically dogs.

Abstract Insecticides can exert strong selection on insect pest species, including those that vector diseases, and have led to rapid evolution of resistance. Despite such rapid evolution, relatively little is known about standing genetic variation for resistance in insecticide‐susceptible populations of many species. To help fill this knowledge gap, we generated genotyping‐by‐sequencing data from insecticide‐susceptible Phlebotomus papatasi and Lutzomyia longipalpis sand flies that survived or died from a sub‐diagnostic exposure to either permethrin or malathion using a modified version of the Centers for Disease Control and Prevention bottle bioassay. Multi‐locus genome‐wide association mapping methods were used to quantify standing genetic variation for insecticide resistance in these populations and to identify specific alleles associated with insecticide survival. For each insecticide treatment, we estimated the proportion of the variation in survival explained by the genetic data (i.e., “chip” heritability) and the number and contribution of individual loci with measurable effects. For all treatments, survival to an insecticide exposure was heritable with a polygenic architecture. Both P. papatasi and L. longipalpis had alleles for survival that resided within many genes throughout their genomes. The implications for resistance conferred by many alleles, as well as inferences made about the utility of laboratory insecticide resistance association studies compared to field observations, are discussed.

During the course of a plague epizootic, decimation of rodent host populations may result in the transfer of fleas to alternate or phoretic hosts, including to sympatric raptors that prey on rodents. We studied flea abundance and flea species assemblages on burrowing owls (Athene cunicularia hypugaea) in southwestern Idaho before (2012 - 2014), during (2015 - 2016), and after (2017) an epizootic of plague in Piute ground squirrels (Urocitellis mollis). We examined (1) if a larger proportion of burrowing owl nests contained fleas, (2) the likelihood that owls within a high flea abundance class increased, and (3) if owls harbored ground squirrel fleas during the epizootic. Using a flea abundance index assigned to 1,184 owls from 236 nests, the proportion of nests and the likelihood that owls had high flea abundance decreased rather than increased during epizootic years. Moreover, of 3,538 collected fleas from owls at 143 nests, no fleas were species that Piute ground squirrels typically harbor. Instead, Pulex irritans was the predominant flea collected in all study years (> 99%). Thus, although raptors may play a role in plague, there was no evidence that the die-off of ground squirrels resulted in higher flea intensity in burrowing owls or that they served as frequent accidental or phoretic hosts for ground squirrel fleas that could potentially be infectious with Yersinia pestis.

In Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population.

Phlebotomus papatasi Scopoli is a medically important insect that has been successfully colonized in the laboratory, and blood feeding is critical for colony propagation. There has been much interest in developing established protocols for in vitro blood-feeding systems. The objective of this study was to determine if a Parafilm membrane and a hog's gut membrane could be successfully used with in vitro feeding systems. We evaluated percentages of P. papatasi females that blood fed on different blood-feeding systems (a mouse, a Hemotek feeder, or a glass feeder) used with either a Parafilm or a hog's gut membrane, with cohorts of 250 and 500 P. papatasi females, and with or without external exhalations. For all feeding system combinations, female P. papatasi blood fed in higher percentages when in cohorts of 500 individuals and in the presence of exhalations. Higher percentages of P. papatasi fed on a mouse, but this study also demonstrates that P. papatasi will readily feed with in vitro feeding systems using a Parafilm membrane or a hog's gut membrane. This study suggests that female P. papatasi may use an invitation effect to blood feed and are attracted to blood sources via chemical olfaction cues, both of which have been characterized in other blood-feeding arthropods. Our study demonstrates that a Parafilm membrane or a hog's gut membrane, in conjunction with the Hemotek or glass feeder system, is potentially a viable alternative to live rodents to blood feed a colony of P. papatasi.

Insecticide resistance to synthetic chemical insecticides is a worldwide concern in phlebotomine sand flies (Diptera: Psychodidae), the vectors of Leishmania spp. parasites. The CDC bottle bioassay assesses resistance by testing populations against verified diagnostic doses and diagnostic times for an insecticide, but the assay has been used limitedly with sand flies. The objective of this study was to determine diagnostic doses and diagnostic times for laboratory Lutzomyia longipalpis (Lutz & Nieva) and Phlebotomus papatasi (Scopoli) to ten insecticides, including pyrethroids, organophosphates, carbamates, and DDT, that are used worldwide to control vectors.

Knowledge about the distribution and abundance of the western black-legged tick, Ixodes pacificus Cooley and Kohls, in Utah is limited. Recent concerns over tick-borne diseases in Utah, primarily Lyme disease, have reinvigorated the need to understand the distribution and habitats favored by this tick species. We surveyed 157 sites throughout Utah to examine the distribution, abundance, and habitat of I. pacificus. In total, 343 adult ticks were collected from 2011 to 2013. Specifically, 119 I. pacificus, 217 Dermacentor andersoni Stiles, six D. albipictus Packard, and one D. hunteri Bishopp were collected. Overall, tick abundance was relatively low in the areas evaluated in Utah. I. pacificus collections were limited to sites above 1700m. Ninety-two percent of I. pacificus were captured in the Sheeprock Mountains in Tooele County. I. pacificus positive collection sites were characterized by Gambel oak (Quercus gambelii Nuttall), juniper (Juniperus spp. L.), big sagebrush (Artemisia tridentata Nuttall) and black sagebrush (A. nova Nelson), and mixed grass habitat. All I. pacificus ticks were tested for the presence of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner, sensu stricto) using real-time PCR. All ticks tested negative for B. burgdorferi. The likelihood of encountering I. pacificus and acquiring Lyme disease in the areas evaluated in Utah is considerably low due to low tick abundance and limited distribution, as well as low prevalence (or absence) of B. burgdorferi in Utah.

Western Burrowing Owls (Athene cunicularia hypugaea) are small, ground-dwelling owls of western North America that frequent prairie dog (Cynomys spp.) towns and other grasslands. Because they rely on rodent prey and occupy burrows once or concurrently inhabited by fossorial mammals, the owls often harbor fleas. We examined the potential role of fleas found on burrowing owls in plague dynamics by evaluating prevalence of Yersinia pestis in fleas collected from burrowing owls and in owl blood. During 2012-2013, fleas and blood were collected from burrowing owls in portions of five states with endemic plague-Idaho, Oregon, Washington, Colorado, and South Dakota. Fleas were enumerated, taxonomically identified, pooled by nest, and assayed for Y. pestis using culturing and molecular (PCR) approaches. Owl blood underwent serological analysis for plague antibodies and nested PCR for detection of Y. pestis. Of more than 4750 fleas collected from owls, Pulex irritans, a known plague vector in portions of its range, comprised more than 99.4%. However, diagnostic tests for Y. pestis of flea pools (culturing and PCR) and owl blood (PCR and serology) were negative. Thus, even though fleas were prevalent on burrowing owls and the potential for a relationship with burrowing owls as a phoretic host of infected fleas exists, we found no evidence of Y. pestis in sampled fleas or in owls that harbored them. We suggest that studies similar to those reported here during plague epizootics will be especially useful for confirming these results.

Knowledge about the distribution and abundance of the western black-legged tick, Ixodes pacificus Cooley and Kohls, in Utah is limited. Recent concerns over tick-borne diseases in Utah, primarily Lyme disease, have reinvigorated the need to understand the distribution and habitats favored by this tick species. We surveyed 157 sites throughout Utah to examine the distribution, abundance, and habitat of I. pacificus. In total, 343 adult ticks were collected from 2011 to 2013. Specifically, 119 I. pacificus, 217 Dermacentor andersoni Stiles, six D. albipictus Packard, and one D. hunteri Bishopp were collected. Overall, tick abundance was relatively low in the areas evaluated in Utah. I. pacificus collections were limited to sites above 1700 m. Ninety-two percent of I. pacificus were captured in the Sheeprock Mountains in Tooele County. I. pacificus positive collection sites were characterized by Gambel oak (Quercus gambelii Nuttall), juniper (Juniperus spp. L.), big sagebrush (Artemisia tridentata Nuttall) and black sagebrush (A. nova Nelson), and mixed grass habitat. All I. pacificus ticks were tested for the presence of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt, and Brenner, sensu stricto) using real-time PCR. All ticks tested negative for B. burgdorferi. The likelihood of encountering I. pacificus and acquiring Lyme disease in the areas evaluated in Utah is considerably low due to low tick abundance and limited distribution, as well as low prevalence (or absence) of B. burgdorferi in Utah.

Chemical insecticides are effective for controlling Lutzomyia and Phlebotomus sand fly (Diptera: Psychodidae) vectors of Leishmania parasites. However, repeated use of certain insecticides has led to tolerance and resistance. The objective of this study was to determine lethal concentrations (LCs) and lethal exposure times (LTs) to assess levels of susceptibility of laboratory Lutzomyia longipalpis (Lutz and Nieva) and Phlebotomus papatasi (Scopoli) to 10 insecticides using a modified version of the World Health Organization (WHO) exposure kit assay and Centers for Disease Control and Prevention (CDC) bottle bioassay. Sand flies were exposed to insecticides coated on the interior of 0.5-gallon and 1,000-ml glass bottles. Following exposure, the flies were allowed to recover for 24 h, after which mortality was recorded. From dose-response survival curves for L. longipalpis and P. papatasi generated with the QCal software, LCs causing 50, 90, and 95% mortality were determined for each insecticide. The LCs and LTs from this study will be useful as baseline reference points for future studies using the CDC bottle bioassays to assess insecticide susceptibility of sand fly populations in the field. There is a need for a larger repository of sand fly insecticide susceptibility data from the CDC bottle bioassays, including a range of LCs and LTs for more sand fly species with more insecticides. Such a repository would be a valuable tool for vector management.

Residential temperature and humidity are associated with multiple health effects. Studies commonly use single-point measures to estimate indoor temperature and humidity exposures, but there is little evidence to support this sampling strategy. This study evaluated the relationship between single-point and continuous monitoring of air temperature, apparent temperature, relative humidity, and absolute humidity over four exposure intervals (5-min, 30-min, 24-hr, and 12-days) in 9 northern Utah homes, from March-June 2012. Three homes were sampled twice, for a total of 12 observation periods. Continuous data-logged sampling was conducted in homes for 2–3 wks, and simultaneous single-point measures (n = 114) were collected using handheld thermo-hygrometers. Time-centered single-point measures were moderately correlated with short-term (30-min) data logger mean air temperature (r = 0.76, β = 0.74), apparent temperature (r = 0.79, β = 0.79), relative humidity (r = 0.70, β = 0.63), and absolute humidity (r = 0.80, β = 0.80). Data logger 12-day means were also moderately correlated with single-point air temperature (r = 0.64, β = 0.43) and apparent temperature (r = 0.64, β = 0.44), but were weakly correlated with single-point relative humidity (r = 0.53, β = 0.35) and absolute humidity (r = 0.52, β = 0.39). Of the single-point RH measures, 59 (51.8%) deviated more than ±5%, 21 (18.4%) deviated more than ±10%, and 6 (5.3%) deviated more than ±15% from data logger 12-day means. Where continuous indoor monitoring is not feasible, single-point sampling strategies should include multiple measures collected at prescribed time points based on local conditions.