Sand Fly larvae are capable of positive chemotaxis: a proof of concept study using Phlebotomus papatasi (Diptera: Psychodidae) as a model species.

Phlebotomine sand flies are important vectors of medical and veterinary importance, transmitting pathogens, such as the Leishmania parasites, responsible for 700,000 to 1 million new cases of leishmaniasis every year. The vast majority of the current sand fly surveillance and control tools are tailored against the adult stages, due to the limited knowledge on the ecology of the larval stages. Since vector control is primarily an ecological problem, an in-depth understanding of the behavior of the target insect pests across all the different life stages of their development is required prior to the development of effective control strategies. It is well known that chemical cues play an important role in insect behavior. While there are numerous studies investigating the behavior of adult sand flies in response to chemical sources, there is currently no information available on the response of their larval stages. In this study, novel bioassays were constructed to investigate the effect of chemical cues (gustatory and olfactory) on the behavior of Phlebotomus papatasi (Scopoli) sand fly larvae. The larvae exhibited a clear food preference within a few hours of exposure in a 2-choice bioassay, while, also, demonstrated positive chemotaxis in response to volatile stimuli emitted from their preferred food source. Identification of the specific chemical compounds (or the combination thereof) eliciting attractance response to sand fly immature stages could lead to the development of innovative, and targeted (larval-specific) tools for the surveillance, and management of these important public health pests.

Experimental Colonization of Sand Flies (Lutzomyia longipalpis; Diptera: Psychodidae) by Bartonella ancashensis.

Background: Bartonella ancashensis is a recently described Bartonella species endemic to Peru, where it causes verruga peruana in humans. While the arthropod vector of B. ancashensis transmission is unknown, human coinfections with Bartonella bacilliformis suggest that phlebotomine sand flies are a vector. Materials and Methods: To address the hypothesis that sand flies are involved in the bacterium's transmission, Lutzomyia longipalpis sand flies were used as an infection model, together with green fluorescent protein-expressing B. ancashensis. Results: Results showed that bacterial infections were clearly established, limited to the anterior midgut of the female fly, and maintained for roughly 7 days. At 3-7 days postinfection, a prominent microcolony of aggregated bacteria was observed in the anterior midgut, immediately distal to the stomodeal valve of the esophagus. In contrast, eggs, diuretic fluid, feces, and other tissues were not infected. Conclusion: These results suggest that certain sand fly species within the endemic zone for B. ancashensis may play a role in the bacterium's maintenance and possibly in its transmission to humans.

A Composite Recombinant Salivary Proteins Biomarker for Phlebotomus argentipes Provides a Surveillance Tool Postelimination of Visceral Leishmaniasis in India.

Incidence of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) has declined by more than 95% since initiation of the elimination program in 2005. As the ISC transitions to the postelimination surveillance phase, an accurate measurement of human-vector contact is needed to assure long-term success. To develop this tool, we identified PagSP02 and PagSP06 from saliva of Phlebotomus argentipes, the vector of Leishmania donovani in the ISC, as immunodominant proteins in humans. We also established the absence of cross-reactivity with Phlebotomus papatasi saliva, the only other human-biting sand fly in the ISC. Importantly, by combining recombinant rPagSP02 and rPagSP06 we achieved greater antibody recognition and specificity than single salivary proteins. The receiver operating characteristics curve for rPagSP02 + rPagSP06 predicts exposure to Ph. argentipes bites with 90% specificity and 87% sensitivity compared to negative control sera (P >.0001). Overall, rPagSP02 + rPagSP06 provides an effective surveillance tool for monitoring vector control efforts after VL elimination.

No Evidence of rVSV-Ebola Virus Vaccine Replication or Dissemination in the Sand Fly Phlebotomus papatasi.

Following vaccination with the live attenuated, recombinant vesicular stomatitis virus Indiana serotype Ebola virus (rVSV-EBOV) vaccine, persons may exhibit a transient vaccine-associated viremia. To investigate the potential for Old World sand flies to transmit this vaccine following feeding on a viremic person, we fed laboratory-reared Phlebotomus papatasi an artificial blood meal containing 7.2 log10 plaque-forming units of rVSV-EBOV. Replication or dissemination was not detected in the body or legs of any P. papatasi collected at seven (n = 75) or 15 (n = 75) days post-feed. These results indicate a low potential for rVSV-EBOV to replicate and disseminate in P. papatasi, a species whose geographic distribution ranges from Morocco to southwest Asia and as far north as southern Europe.

Use of In Vivo Imaging System Technology in Leishmania major BALB/c Mouse Ear Infection Studies.

Novel treatments for cutaneous leishmaniasis (CL) are needed, due to current lack of effective universal treatments, increasing resistance among the parasite, and toxic effects or impracticality of the current therapeutics. Models of direct infection with high number of Leishmania parasites in the current research of CL involving the BALB/c mouse or Golden Syrian Hamster are considered not suitable for the assessment of antileishmanial drug efficacy because of the lack of disease similarities with humans. The saliva of the sand fly vector is known to affect the host response to infection by the Leishmania parasite. Here, we build upon a previous BALB/c model infected with luciferase-expressing Leishmania major parasites. In the present study, we infect the ear dermis instead of the foot pad or base of the tail, and compare multiple methods of infection, using parasites alone or mixed with either bites from uninfected sand flies (Phlebotomus duboscqi Diptera Psychodidae:Neveu-Lemaire) or salivary gland sonicate from sand flies. Our data show a dose-response of bioluminescent signal (which represents the parasite load at the infection site), dermal lesion development, and Leishmania Donovan Units in liver and spleen. This in vivo L. major ear infection model, once optimized, can be used for assessing the efficacy of drug compounds that have been determined as very effective in the other, high inoculum CL models.

Laboratory colonization and mass rearing of phlebotomine sand flies (Diptera, Psychodidae).

Laboratory colonies of phlebotomine sand flies are necessary for experimental study of their biology, behaviour and mutual relations with disease agents and for testing new methods of vector control. They are indispensable in genetic studies and controlled observations on the physiology and behaviour of sand flies, neglected subjects of high priority. Colonies are of particular value for screening insecticides. Colonized sand flies are used as live vector models in a diverse array of research projects, including xenodiagnosis, that are directed toward control of leishmaniasis and other sand fly-associated diseases. Historically, labour-intensive maintenance and low productivity have limited their usefulness for research, especially for species that do not adapt well to laboratory conditions. However, with growing interest in leishmaniasis research, rearing techniques have been developed and refined, and sand fly colonies have become more common, enabling many significant breakthroughs. Today, there are at least 90 colonies representing 21 distinct phlebotomine sand fly species in 35 laboratories in 18 countries worldwide. The materials and methods used by various sand fly workers differ, dictated by the availability of resources, cost or manpower constraints rather than choice. This paper is not intended as a comprehensive review but rather a discussion of methods and techniques most commonly used by researchers to initiate, establish and maintain sand fly colonies, with emphasis on the methods proven to be most effective for the species the authors have colonized. Topics discussed include collecting sand flies for colony stock, colony initiation, maintenance and mass-rearing procedures, and control of sand fly pathogens in colonies.

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.

Efficacy of Permethrin Treated Bed Nets Against Leishmania major Infected Sand Flies.

Insecticide treated nets (ITNs) are a potential tool to help control sand flies and prevent Leishmaniasis. However, little is currently known about the response of Leishmania infected sand flies to ITNs. In this study, Phlebotomus duboscqi sand flies were infected with the parasite Leishmania major. Infected and noninfected sand flies were then evaluated against permethrin treated and untreated bed nets in a laboratory assay that required sand flies to pass through suspended netting material to feed on a mouse serving as an attractive host. The number of sand flies passing through the nets and blood feeding was recorded. There was not a significant difference in the ability of infected or noninfected sand flies to move through treated or untreated nets. Fewer sand flies entered the permethrin treated nets compared to the untreated nets, indicating that permethrin creates an effective barrier. The results show that in addition to reducing the nuisance bites of noninfected sand flies, ITNs also protect against Leishmania infected sand flies and therefore can play in key role in reducing the rates of Leishmaniasis. This study is important to the Department of Defense as it continues to develop and field new bed nets to protect service members.

Blood-Feeding Behaviors of Anopheles stephensi but not Phlebotomus papatasi are Influenced by Actively Warming Guinea Pigs (Cavia porcellus) Under General Anesthesia.

Animal models are often used to study hematophagous insect feeding behavior and evaluate products such as topical repellents. However, when these models are used the study animals often experience significant drops in core body temperature because of the effects of anesthesia. This study used a guinea pig model to investigate whether maintaining a normothermic core body temperature during anesthesia influenced the rate of Anopheles stephensi and Phlebotomus papatasi blood feeding. Experiments were conducted with anesthetized animals that had their body temperatures either maintained with a warming device or were allowed to drop naturally. Results showed that when guinea pigs were actively warmed by a heating device, An. stephensi feeding behavior was similar at the beginning and end of anesthesia. However, when a warming device was not used, fewer An. stephensi took a blood meal after the animals' temperatures had dropped. Phlebotomus papatasi were not as sensitive to changes in temperature and feeding rates were similar whether a warming device was used or not. These results are discussed and it is recommended that warming devices are used when conducting feeding experiments with insects sensitive to changes in host body temperature, such as An. stephensi.

A sand fly salivary protein vaccine shows efficacy against vector-transmitted cutaneous leishmaniasis in nonhuman primates.

Currently, there are no commercially available human vaccines against leishmaniasis. In rodents, cellular immunity to salivary proteins of sand fly vectors is associated to protection against leishmaniasis, making them worthy targets for further exploration as vaccines. We demonstrate that nonhuman primates (NHP) exposed to Phlebotomus duboscqi uninfected sand fly bites or immunized with salivary protein PdSP15 are protected against cutaneous leishmaniasis initiated by infected bites. Uninfected sand fly-exposed and 7 of 10 PdSP15-immunized rhesus macaques displayed a significant reduction in disease and parasite burden compared to controls. Protection correlated to the early appearance of Leishmania-specific CD4(+)IFN-γ(+) lymphocytes, suggesting that immunity to saliva or PdSP15 augments the host immune response to the parasites while maintaining minimal pathology. Notably, the 30% unprotected PdSP15-immunized NHP developed neither immunity to PdSP15 nor an accelerated Leishmania-specific immunity. Sera and peripheral blood mononuclear cells from individuals naturally exposed to P. duboscqi bites recognized PdSP15, demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homology to mammalian proteins, further demonstrating its potential as a component of a vaccine for human leishmaniasis.

Immuno-chromatographic wicking assay for the rapid detection of dengue viral antigens in mosquitoes (Diptera: Culicidae).

There is a threat for dengue virus (DENV) reemergence in many regions of the world, particularly in areas where the DENV vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), are readily available. However, there are currently no accurate and reliable diagnostic methods to provide critical, real-time information for early detection of DENV within the vector populations to implement appropriate vector control and personal protective measures. In this article, we report the ability of an immuno-chromatographic assay developed by VecTOR Test Systems Inc. to detect DENV in a pool of female Aedes mosquitoes infected with any of the four viral serotypes. The DENV dipstick assay was simple to use, did not require a cold chain, and provided clear results within 30 min. It was highly specific and did not cross-react with samples spiked with West Nile, yellow fever, Japanese encephalitis, Rift Valley fever, chikungunya, Venezuelan equine encephalomyelitis, Ross River, LaCrosse, or Caraparu viruses. The DENV assay can provide real-time critical information on the presence of DENV in mosquitoes to public health personnel. Results from this assay will allow a rapid threat assessment and the focusing of vector control measures in high-risk areas.

Field evaluation of a wicking assay for the rapid detection of Rift Valley fever viral antigens in mosquitoes.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in domestic ungulates as well as humans in Africa. There is a concern that outbreaks of Rift Valley fever may continue and that this virus may spread into regions where it had not previously been detected. Surveillance and rapid detection are critical to the initiation of an effective disease control program. Here we report on the field evaluation in Kenya of the VectorTest RVFV antigen assay, modeled on the VecTest assay for West Nile virus. The dipsticks provided results in <20 min, were easy to use, and did not require a laboratory with containment facilities. Although none of the field-collected mosquitoes were infected with RVFV, the dipstick provided a clear positive result with pools of field-collected mosquitoes spiked with a single positive, irradiated (to inactivate any infectious virus) mosquito. Similarly, the dipstick was able to detect virus from pools of mosquitoes captured during the RVFV outbreak in 2007. The RVFV dipstick assay was highly specific with only a single weak false positive out of 266 pools tested (specificity > 99.6%). The RVFV assay can provide a rapid, safe, easy-to-use preliminary test to alert public health personnel to the presence of RVFV in mosquitoes in a given area. Results from this assay will allow for more rapid medical threat assessments and the focusing of vector control measures on high-risk areas.