Field-collected Sand Flies Research Articles

Sandfly Blood-Feeding Habits and Competence in Transmitting Ntepes Virus, a Recently Discovered Member of the Genus Phlebovirus.

Phleboviruses transmitted by sandflies are among emerging public health threats. A novel Phlebovirus named Ntepes virus (NTPV) was recently described and found to infect humans from a wide geographic area in Kenya. However, the entomologic risk factors of this virus such as the potential vectors and the transmission cycles remain poorly defined. This study assessed the ability of the colonized sandfly Phlebotomus duboscqi to transmit NTPV and determined the bloodmeal host sources of field-collected sandflies from the area where NTPV was found in Baringo County, Kenya. Five-day old laboratory-reared P. duboscqi were orally challenged with an infectious dose of NTPV (≈106.0 pfu/ml) and incubated for up to 15 days postinfection. Individual sandflies were dissected into abdomens, legs, and salivary glands and screened for the virus infection by cell culture. Of the 205 virus-exposed sandflies, 19.5% developed non-disseminated infections in the midgut, with no evidence of virus dissemination or transmission in legs and salivary glands, respectively. The midgut infection rates decreased with increasing extrinsic incubation period (Spearman's correlation, ρ = −0.71). Blood-fed specimens analyzed by polymerase chain reaction (PCR) and sequencing of a region of the mitochondrial 12S rRNA, revealed almost exclusive feeding on humans (98%) represented by the sandflies Sergentomyia schwetzi, S. clydei, S. antennata, S. squamipleuris, S. africana, and Phlebotomus martini. One specimen of S. clydei had fed on cattle (2%). These findings suggest P. duboscqi is an incompetent laboratory vector of NTPV. The high human-feeding rate by diverse sandfly species increases the likelihood of human exposure to pathogens associated with these sandflies. Assessment of the susceptibility of Sergentomyia species to NTPV is recommended given their high human-feeding tendency.

Detection of Leishmania donovani in Wild-Caught Phlebotomine Sand Flies in Endemic Focus of Leishmaniasis in Himachal Pradesh, India.

An endemic focus of cutaneous leishmaniasis (CL) is located in the Sutlej River Valley in Himachal Pradesh (India) in the north-western Himalaya where CL co-exists with visceral leishmaniasis (VL). In areas of the Indian subcontinent such as Rajasthan, cutaneous leishmaniasis is transmitted by Phlebotomus papatasi (Scopoli) and Phlebotomus salehi. In Himachal Pradesh, Phlebotomus longiductus (Parrot) is suspected to be the vector for CL. In the current study, sand flies were collected and tested for Leishmania infection and to confirm the vector species causing CL. Sand flies were collected during April and September of 2017–2019 from CL endemic villages of Rampur (Shimla), Nirmand (Kullu) and Nichar (Kinnaur) districts of Himachal Pradesh. The sand flies were identified as Phlebotomus (adlerius) longiductus (Parrot) and Phlebotomus (larrousisus) major (Annandale). The density of P. longiductus was found highest. The elevation of villages ranged from 947 m to 2,130 m and were far from the presence of subsoil water. Field collected sand flies tested positive (7.69%) for Leishmania donovani by PCR-RFLP. The L. donovani sequences detected from P. longiductus were 97% similar to L. donovani sequences reported from the cases of CL in Himachal Pradesh.The Leishmania positive sand flies were morphologically identified as Phlebotomus adlerious longiductus providing one step further evidence towards the vector status of CL in Himachal Pradesh. The findings of the study are of epidemiological significance for strategic planning of vector control for leishmaniasis in India.

Afrotropical sand fly-host plant relationships in a leishmaniasis endemic area, Kenya.

The bioecology of phlebotomine sand flies is intimately linked to the utilization of environmental resources including plant feeding. However, plant feeding behavior of sand flies remains largely understudied for Afrotropical species. Here, using a combination of biochemical, molecular, and chemical approaches, we decipher specific plant-feeding associations in field-collected sand flies from a dry ecology endemic for leishmaniasis in Kenya. Cold-anthrone test indicative of recent plant feeding showed that fructose positivity rates were similar in both sand fly sexes and between those sampled indoors and outdoors. Analysis of derived sequences of the ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL) from fructose-positive specimens implicated mainly Acacia plants in the family Fabaceae (73%) as those readily foraged on by both sexes of Phlebotomus and Sergentomyia. Chemical analysis by high performance liquid chromatography detected fructose as the most common sugar in sand flies and leaves of selected plant species in the Fabaceae family. Analysis of similarities (ANOSIM) of the headspace volatile profiles of selected Fabaceae plants identified benzyl alcohol, (Z)-linalool oxide, (E)-β-ocimene, p-cymene, p-cresol, and m-cresol, as discriminating compounds between the plant volatiles. These results indicate selective sand fly plant feeding and suggest that the discriminating volatile organic compounds could be exploited in attractive toxic sugar- and odor- bait technologies control strategies.

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10−3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Genetic Structure of Phlebotomus orientalis (Diptera: Psychodidae) in Leishmaniasis Endemic Foci of Sudan.

Visceral leishmaniasis (VL) remains a major concern in many parts of Sudan. The disease is transmitted by Phlebotomus orientalis. The objective of this study was to determine genetic structure of Phlebotomus orientalis population from 5 geographical regions in Sudan. A total of 194 individual sand flies were collected from 5 geographic regions in Sudan. The field collected sand flies were analyzed by Random Amplified Polymorphic DNA (RAPD) using 30 primers. Eight hundred and 65 bands from 4 RAPD primers were analyzed for genetic variation. A higher level of intrapopulational variability was detected in populations of P. orientalis from eastern Sudan compared to those populations from central and northern Sudan. Diagnostic bands were detected in populations of P. orientalis central Sudan. Hieratical clustering analysis showed clear clustering into 2 main populations with 1 population subdivided into 4 subpopulations. However, these populations did not show any correlation with their geographical origins. Furthermore, the low genetic differentiation among subpopulations was supported by fixation index (FST) estimated by analysis of molecular variance (AMOVA). It is concluded that the populations of P. orientalis from the selected areas in Sudan have a low genetic differentiation. However, assessment of genetic structure of P. orientalis populations is important for understanding the patterns of transmission of VL in different endemic areas.

Toxicity of Thiamethoxam on Field-Collected Sand Flies (Diptera: Psychodidae) From Different Regions of Turkey

Phlebotomine sand flies (Diptera: Psychodidae) are the vectors of human and canine leishmaniasis, sand fly fever, and other arboviruses and bacteria. The control of sand flies is recommended by World Health Organization throughout the world. In this study, we investigated the susceptibility of field-collected sand flies against thiamethoxam in Turkey. The adult sand flies collected were from five different locations in three provinces; Yeşilöz and Büyükpınar districts in Antalya province, Develi district in Kayseri province, and Üçpınar and Bayındırlık districts in Manisa province. They were investigated using two different doses (0.125 and 0.25 g ai/m2) of thiamethoxam under laboratory conditions. After 1-h exposure, all sand flies were found highly susceptible to thiamethoxam and both test doses caused 100% mortality after 24 h. Considering the KDT50 time, flies collected from Yeşilöz district in Antalya province were found to be most sensitive and flies collected from Üçpınar district in Manisa province were found as the most resistance to thiamethoxam. The results show that Turkish sand flies were highly susceptible to thiamethoxam and it can be used for the control of sand flies in the field.

Co-circulation of eight different phleboviruses in sand flies collected in the Northern Apennine Mountains (Italy)

The sand flies are the biological vectors of a variety of viruses belonging to the Phlebovirus genus. As several of these viruses, like Toscana virus, are important agents of diseases in humans, the definition the phleboviruses circulating in a particular area is an important health issue. This work presents results obtained between 2013 and 2016, by testing 25,853 field-collected sand flies, sampled at 50 sites in the Northern edge of the Apennine Mountains, in Emilia-Romagna and Lombardy regions (Italy). Isolation of the three reassortant Ponticelli I, II, and III viruses was successful from five pools of sand flies. These results, and phylogenetic analysis made by obtained sequences, suggest the presence of eight different viruses: Toscana virus, Fermo-like virus, Corfou virus, Ponticelli viruses, and two unknown putative viruses. The co-circulation of different phlebovirus reported in this study, indicate a very dynamic and complex situation, which deserves a more detailed investigation to characterize their possible pathogenicity to humans and animals.

Molecular detection of Wolbachia pipientis in natural populations of sandfly vectors of Leishmania infantum in endemic areas: first detection in Lutzomyia longipalpis.

A polymerase chain reaction-based method was used to screen sandflies for infection with Wolbachia (Rickettsiales: Rickettsiaceae), an intracellular bacterial endosymbiont found in many arthropods and filarial hosts. Positive results were obtained in five of 200 field-collected sandflies and were confirmed by sequencing. All sandflies were Lutzomyia longipalpis (Diptera: Psychodidae) captured in a region endemic for visceral leishmaniasis in Brazil. This is the first study to identify Wolbachia infection in this Lutzomyia species, which is the main vector of leishmaniasis in the study area. The low infection rate found in this study (2.5%), together with the lack of detection of Wolbachia in previous studies and the diversity found in the sequences analysed, suggests horizontal transmission to these sandflies.

Diversity of the Bacterial and Fungal Microflora from the Midgut and Cuticle of Phlebotomine Sand Flies Collected in North-Western Iran

BackgroundPhlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies.Methodology/Principal FindingsFive sand fly species: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates.Conclusion/SignificanceOur study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies.

Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper

The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.

Isolation of Leishmania mexicana (Kinetoplastida: Trypanosomatidae) from Lutzomyia anthophora (Diptera: Psychodidae) Collected in Texas

Three of 27 female Lutzomyia anthophora (Addis) collected in Texas from the nest of a southern plains woodrat, Neotoma micropus Baird, during October 1991 were infected with flagellate protozoans. Isolates were grown in Schneider's Drosophila medium supplemented with 20% fetal bovine serum, and isozyme analysis of two of the isolates determined the parasites to be Leishmania mexicana (Biagi). These are the first isolations of Leishmania from field-collected sand flies in North America north of Mexico. Possible reasons for the lack of human cases near the focus are presented.