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.

Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle.

Systemic control uses the vertebrate hosts of zoonotic pathogens as "Trojan horses," killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.

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.

Microbial ecology of sand fly breeding sites: aging and larval conditioning alter the bacterial community composition of rearing substrates.

BACKGROUND: Sand flies vector several human pathogens, including Leishmania species, which cause leishmaniases. A leishmaniasis vaccine does not yet exist, so the most common prevention strategies involve personal protection and insecticide spraying. However, insecticides can impact non-target organisms and are becoming less effective because of the evolution of resistance. An alternative control strategy is the attract-and-kill approach, where the vector is lured to a lethal trap, ideally located in oviposition sites that will attract gravid females. Oviposition traps containing attractive microbes have proven successful for the control of some mosquito populations but have not been developed for sand flies. Gravid female sand flies lay their eggs in decomposing organic matter on which the larvae feed and develop. Studies have demonstrated that gravid females are particularly attracted to larval conditioned (containing eggs and larvae) and aged rearing substrates. An isolate-based study has provided some evidence that bacteria play a role in the attraction of sand flies to conditioned substrates. However, the overall bacterial community structure of conditioned and aged substrates and how they change over time has not been investigated. METHODS: The goal of this study was to characterize the bacterial communities of rearing and oviposition substrates that have been shown to vary in attractiveness to gravid sand flies in previous behavioral studies. Using 16S rRNA amplicon sequencing we determined the bacterial composition in fresh, aged, and larval-conditioned substrates at four time points representing the main life-cycle stages of developing sand flies. We compared the diversity, presence, and abundance of taxa across substrate types and time points in order to identify how aging and larval-conditioning impact bacterial community structure. RESULTS: We found that the bacterial communities significantly change within and between substrates over time. We also identified bacteria that might be responsible for attraction to conditioned and aged substrates, which could be potential candidates for the development of attract-and-kill strategies for sand flies. CONCLUSION: This study demonstrated that both aging and larval conditioning induce shifts in the bacterial communities of sand fly oviposition and rearing substrates, which may explain the previously observed preference of gravid female sand flies to substrates containing second/third-instar larvae (conditioned) and substrates aged the same amount of time without larvae (aged).

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).

Spatial Bet Hedging in Sand Fly Oviposition: Factors Affecting Skip Oviposition in Phlebotomus papatasi Sand Flies.

Skip oviposition is considered as an example of spatial bet hedging and involves a tradeoff between the benefit of reducing the risk of complete reproductive failure due to stochastic loss of a breeding site and the energetic and mortality costs associated with dispersal across several oviposition sites. Skip oviposition has been studied extensively for container-breeding mosquitoes but has never been studied with sand flies. By conducting a series of bioassays using solitary gravid females exposed to varying numbers of oviposition sites and to oviposition sites of variable quality at small (oviposition jar) and medium (free-flight cage) scales, we showed that sand flies exhibited skip oviposit at both scales. Specifically, with low-quality oviposition sites, females spread their eggs across several oviposition sites with total egg clutch size remaining constant and number of eggs per oviposition site decreasing with increasing number of oviposition sites. With variable quality sites, sand flies biased their oviposition to sites containing increasing levels of organic matter (OM), but also laid eggs in poor-quality sites; a behavior consistent with spatial bet hedging. We also demonstrated that the presence of OM stimulated larger egg clutch size and increased percent skip oviposition. Skip oviposition was less frequent at the free flight cage scale. But, at this scale, females were shown to be stimulated to lay more eggs when in the presence of other females than when alone.

Larval Conditioning and Aging of Sand Fly Rearing Medium Affect Oviposition Site Selection in Phlebotomus papatasi (Diptera: Psychodidae) Sand Flies.

Sand fly larvae develop in sheltered humid habitats containing decaying organic matter on which they feed. Previously, we showed that gravid females of Phlebotomus papatasi Scopoli (Diptera: Psychodidae) are attracted to and stimulated to lay eggs on larval rearing medium containing larvae. That study, however, did not control for the possible effect of medium aging. Our goal in this study was to evaluate the effect of larval substrate conditioning on attraction and oviposition responses of Ph. papatasi sand flies while controlling for the effect of substrate aging. Initially, we confirmed that the pretreatment fresh larval food sources (to be used as larval conditioned and unconditioned media) did not differ with respect to their effect on attraction and oviposition responses. The larval conditioned medium was produced by rearing larvae to the second/third-instar stage over 3 wk using the same larval food source. To produce larval unconditioned medium, the same amount of fresh larval food was added to a control rearing cup that did not contain larvae but was aged under identical time and conditions. Two-choice bioassays were conducted to evaluate gravid female's attraction and oviposition response to larval conditioned and unconditioned media. We found that gravid females were significantly attracted (P < 0.05) to larval conditioned medium when compared with unconditioned medium under the same amount of time and conditions. However, no such difference was found with respect to oviposition response. Both attraction and oviposition responses were significantly increased for larval conditioned and unconditioned media in comparison to the initial fresh larval food source.

Temporal Bet-Hedging in Sand Fly Oviposition: Pharate Phlebotomus papatasi Sand Fly Neonates Regulate Hatching Time in Response to Organic Matter and Proximity to Conspecific Eggs.

In phlebotomine sand fly, the topic of the factors stimulating or inhibiting egg hatching has been largely ignored. In this study, we evaluated the hypothesis that pharate neonate sand flies are able to regulate their hatching time adaptively in response to cues indicative of the presence of food or potential competitors. In this study, we evaluated the independent and combined effects of organic matter (OM) extract and proximity to conspecific eggs on the hatching proportion of Phlebotomus papatasi eggs. In one set of larval rearing jars, we introduced 16 eggs into a small hole in the center of a plaster base of the jar. In another set, we introduced a single egg into each small hole of a 4-by-4 symmetrical array. To one set, we added an aqueous OM extract, and to the other, we added deionized water (DI). OM stimulated egg hatching while egg clustering slightly inhibited egg hatching. Results of this experiment are biologically important because they show, for the first time, that pharate sand fly neonates are able to adaptively regulate their hatching time in response to external cues.

Peridomestic conditions affect La Crosse virus entomological risk by modifying the habitat use patterns of its mosquito vectors.

Anthropogenic land-use change may affect the transmission risk for endemic vector-borne diseases such as La Crosse encephalitis. In this study, we applied a comparative ecological approach to evaluate differences in vector species abundance, gonotrophic status, and environmental variables among six residential habitats (historical case houses) and six paired adjacent forest patches in a La Crosse virus endemic area of North Carolina. A total of 93,158 container Aedes spp. eggs were obtained by ovistrips and 1,040 resting mosquito adults were collected by large-bore aspiration from 10 June through 8 October, 2010. At sites characterized by high densities of artificial containers, the totals of eggs and adult mosquitoes were higher in the peridomestic plots. At sites characterized by lower densities of artificial containers, the totals of eggs and adult mosquitoes were higher in the forested plots. Although Aedes triseriatus, the primary La Crosse virus vector, was more commonly found in forested habitat overall, in sites characterized by high density of artificial containers, it was found in higher abundance in the peridomestic habitat. Similarly, the proportion of gonotrophically active (parous and gravid) mosquitoes was higher in the peridomestic habitat for sites with highartificial container density. Our study suggests that La Crosse virus transmission risk may be higher at peridomestic habitats with higher densities of artificial containers and thus reinforces the importance of public health measures to improve source reduction efforts.

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.

The Potential of Systemic Control of Sand Flies Using Fipronil in the Novel Leishmania major (Kinetoplastida: Trypanosomatidae) Reservoirs Meriones tristrami (Rodentia: Muridea) and Meriones crassus (Rodentia: Muridea).

Leishmania major (Yakimoff & Schokhor, 1914), an important causative agent of Old World Cutaneous Leishmaniasis (CL), is transmitted by sand flies among a limited number of gerbilline reservoir-species. We can take advantage of this strong dependency to break the pathogen transmission cycle by using systemic insecticides that render the host toxic to the blood-feeding vector. We evaluated the potential of this approach with two novel reservoir species, incriminated for CL expansion in several sites in the Middle East. Specifically, we evaluated: 1) the residuality of the systemic insecticide fipronil in Meriones tristrami (Thomas, 1892) fed on fipronil-treated baits and 2) the treatments' adulticide effect on sand flies that blood fed on treated and untreated M. tristrami and M. crassus (Sundevall, 1842). We fed M. tristrami with food pellets containing 0.1 g/kg fipronil and used gas chromatograph-mass spectrometery analysis and bioassays to examine its residual toxicity to blood-feeding female sand flies. In M. tristrami, fipronil was rapidly metabolized to fipronil sulfone, found in the blood, urine, and feces for ≥31 d after fipronil admission. The survival of sand flies that blood fed on fipronil-treated M. tristrami and M. crassus was significantly reduced for at least 15 and 9 d respectively, after fipronil admission. These results hold promise for the potential contribution of systemic control approaches to CL integrated management strategies against novel CL (due to L. major) outbreaks in Israel and elsewhere.

Systemic Control of Cutaneous Leishmaniasis Sand-Fly Vectors: Fipronil-Treated Rodent Bait Is Effective in Reducing Phlebotomus papatasi (Diptera: Psychodidae) Female Emergence Rate From Rodent Burrows.

The strong dependency of some vectors on their host as a source of habitat can be viewed as a weak link in pathogen's transmission cycles using the vertebrate host as a 'Trojan horse' to deliver insecticides directly to the vector-host point of contact (hereafter 'systemic control'). This could, simultaneously, affect the survival of blood-feeding females and coprophagic larvae. Sand-flies, vectors of leishmaniasis worldwide, are often dependent on their bloodmeal host as a source of habitat and may therefore be good candidates for systemic control. In the present study, we field-tested this methodology by baiting Meriones crassus (Sundevall, 1842) (Rodentia:Muridea) with Fipronil-treated food pellets and evaluated its effect on reducing sand-fly emergence rate, in general, and of that of blood-fed females, in particular. We demonstrated 86% reduction in the abundance of female sand-flies that exit burrows of Fipronil-treated jirds, whereas male abundance was unaffected. Furthermore, whereas in control burrows 20% of the females were blood-fed, in treatment burrows no blood-fed females were detected. Sand-fly abundance outside the burrows was not affected by burrow treatment. This highlights the focal specificity of this method: affecting female sand-flies that feed on the reservoir host. This should result in the reduction of the pathogen transmission rate in the vicinity of the treated area by reducing the prevalence of leishmania-infected sand-fly females. These results hold promise for the potential of the systemic control approach in this system. Our next-step goal is to test this methodology at a large-scale cutaneous leishmaniasis control program.

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.

Horizontal distribution affects the vertical distribution of native and invasive container-inhabiting Aedes mosquitoes within an urban landscape.

The vertical dimension constitutes an important niche axis along which mosquitoes may adjust their distribution. Here, we evaluated whether the vertical distribution of container-inhabiting Aedes mosquitoes differs along a gradient of anthropogenic land-use intensity within an urban landscape. Using a pulley system, we hung oviposition cups at three heights (ground level, 4.5, and 9 m) and in three habitats: forest, park, and a built environment. We hypothesized that mosquito abundance and diversity would be highest in the least disturbed forest habitat, decrease in the park, and be lowest at the UNC-Greensboro campus. We also expected Aedes albopictus (Skuse) and Ae. triseriatus (Say) to mainly oviposit at ground level and Ae. hendersoni (Cockerell) at canopy height. Aedes albopictus was the most common species (68.8%) collected in all three habitat types and was the only species found in the built environment. In that habitat, Ae. albopictus exhibited a bimodal distribution with the lowest activity at the intermediate height (4.5 m). Aedes triseriatus (28.9%) did not differ in egg abundance between the forest and park habitats but did exhibit diverse vertical habitat use while avoiding the canopy in the park habitat. Aedes hendersoni (2.3%) was the most sylvatic species and oviposited only at ground level. Our results indicate that the vertical distribution of mosquitoes is affected by the type of habitat in which they occur, and that this variation could be driven via local-scale modification of microclimatic factors.

Quantitative and Qualitative Costs of Autogeny in Phlebotomus papatasi (Diptera: Psychodidae) Sand Flies.

Most sand flies and mosquitoes require a bloodmeal for egg production, but when blood-sources are scarce, some of them can reproduce without it, so called facultative autogeny. The evolution of autogenous reproduction is thought to involve a trade-off between the benefit of reproducing in the absence of bloodmeal hosts versus the quantitative cost of reduced fecundity and/or or qualitative effect on reduced offspring development and survivorship. We blood-fed (BF) some Phlebotomous papatasi (Scopoli) sand fly females on mice while keeping others (from the same cohort) not BF. We then compared the fecundity of BF and non-blood-fed (NBF) females and also evaluated their egg mass and hatching rate, larval development rate and survivorship, pupa mass and eclosion rates, and progeny fecundity. Among NBF females, only 55% became gravid and produced three times less mature oocytes than BF ones. Autogenous females laid 3.5 and 5.7 times fewer eggs in individual and multi-female bioassays, respectively. Egg mass and hatching rate were not affected by blood-feeding. Individual-larvae bioassays suggested reduced survival during larval stages in the autogenous group. In multi-larvae bioassays, overall and especially pupae survival was significantly reduced in the autogenous group. Development rate was slower and pupal mass was reduced in progeny from autogenous mothers. These effects were particularly apparent at high larval density. Mothers' blood-feeding history did not affect daughter's fecundity. Studies on the costs of autogeny provides insights on the evolution of blood feeding. Moreover, it also provides insights regarding potential implications of autogeny to the emergence of vector-borne diseases.

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.

Attraction and oviposition preferences of Phlebotomus papatasi (Diptera: Psychodidae), vector of Old-World cutaneous leishmaniasis, to larval rearing media.

BACKGROUND: As part of a project aimed at developing oviposition attractants for the control and surveillance of Phlebotomus papatasi (a vector of Old-World cutaneous leishmaniasis), we tested the hypothesis that gravid sand flies are attracted to chemical cues emanating from the growth medium of conspecific larvae - predominantly larvae-conditioned host feces that represents a suitable oviposition site. We report the results of a systematic assessment of media from various developmental stages of the sand fly using oviposition and olfactometer behavioral assays. METHODS: We conducted multiple-choice oviposition assays in 500 mL Nalgene jars. Six treatments were placed on separate filter paper discs at the bottom of the jar: 2(nd)/3(rd) larval instar medium, 4(th) larval instar/pupae medium, frass from expired colonies, larval food (aged rabbit chow and rabbit feces mix), rabbit feces, and a solvent (water) control. Fifty gravid females were introduced into each jar. Cumulative number of eggs laid on each filter paper per jar was counted at different time intervals from digital images. Attraction of gravid sand flies to these six treatments was assayed with a 3-chamber linear olfactometer. Twenty gravid females were transferred to the middle chamber of the olfactometer and their distribution in treatment and control chambers was recorded after 3 h. RESULTS: Almost no eggs were oviposited during the first 72 h following a blood-meal. Cumulative egg deposition increased drastically in the next 24 h (hours 73-96), with a slight non-significant increasing trend thereafter. Comparing mean cumulative egg deposition among the six treatments, we found that significantly more eggs were oviposited on 2(nd)/3(rd) larval rearing medium followed by 4(th) instar/pupae rearing medium. Oviposition preference did not vary over time. The olfactometer results were consistent with the oviposition assays, with 2(nd)/3(rd) larval rearing medium being the most attractive, followed by 4(th) instar/pupae rearing medium. CONCLUSION: The key finding of this study is that gravid, laboratory reared, Ph. papatasi sand flies are significantly more attracted to rearing medium of the most biologically active larval stages (2(nd)/3(rd) instar and 4(th) instar/pupae). This finding indicates that sand fly-digested host food and feces is attractive to gravid females and suggests that the larvae and larval gut microbiome may be involved in conditioning the oviposition substrate and possibly the production of oviposition attractants and stimulants.

Molecular identification of Aedes triseriatus and Aedes hendersoni by a novel duplex polymerase chain reaction assay.

Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

Zoonotic disease in a peripheral population: persistence and transmission of Leishmania major in a putative sink-source system in the Negev Highlands, Israel.

Populations at the edge of their geographic distributions are referred to as peripheral populations. Very little attention has been given to this topic in the context of persistence of infectious disease in natural populations. In this study, we examined this question using zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major in the Negev Desert of Israel as a model system. Here, we suggest that the regional persistence of Phlebotomus papatasi populations and L. major transmission in the Sede Boqer region could be explained through processes akin to sink-source and/or mainland-island metapopulation dynamics. Given its potentially enzootically superior ecological conditions, we hypothesize that the Zin Valley ecotope constitutes the "mainland" or the "source" patch for the Sede Boqer area where L. major transmission is persistent and resistant to local extinctions (die-outs) whereas the local sand fly populations on the Zin Plateau ("island patch" or "sink patch") are more prone to local extinctions. Between 2007 and 2008, we trapped sand flies and sand rats in the two areas and compared sand fly abundance and L. major infection prevalence in both. In both 2007 and 2008, sand fly abundance was high and continuous in the Zin Wadi but low and discontinuous in the Zin Plateau. Infection prevalence of sand rats was significantly higher in the Wadi (13%) compared with the Zin Plateau (3%). Minimum infection rate in sand flies did not differ significantly between the two areas. Overall, our results are consistent with the premise that the Zin Valley population is relatively robust in terms of L. major transmission, whereas transmission is potentially more tenuous in the plateau. Understanding the biotic and abiotic processes enabling the persistence of L. major and other vector-borne diseases in peripheral disease foci is important for predicting the effect of anthropogenic land use and climate change.