Intraspecific differentiation of sandflies specimens by optical spectroscopy and multivariate analysis

Lutzomyia longipalpis and Lutzomyia cruzi are the main sandflies species involved in the transmission of Leishmania infantum protozoan in Brazil. The morphological characteristics can be used for species identification of males specimens, while females are indistinguishable. Although, sandflies identification is essential to understand vectorial capacity, and susceptibility to infectious agents or insecticides, there is a lack of new strategies for specimen identification. In this study, Fourier transform infrared photoacoustic spectroscopy combined with multivariate analysis identified intraspecific differences between Lutzomyia populations. Successfully group clustering was achieved by principal component analysis. The main differences observed can be related to the protein content of the specimens. A classification with 100% accuracy was obtained using machine learning approach, allowing the identification of sandflies specimens.     

Leishmaniasis in Sardinia. III. Soil analysis of a breeding site of three species of sandflies

Soil analysis was carried out on samples obtained from a breeding-site of the sandflies Phlebotomus perniciosus Newstead, P. perfiliewi perfiliewi Parrot and Sergentomyia minuta (Rondani) in Sardinia. Soil texture, pH, CaCo3, organic matter and water content showed no correlation with the number of sandflies that emerged from the places where soil samples were taken. It appears that pre-imaginal stages of these phlebotomine sandflies are associated with a relatively stable, cool and humid environment protected from sunshine and rain, rich in clay and in organic nitrogen.     

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.     

Further evidence that deltamethrin-impregnated collars protect domestic dogs from sandfly bites

In many foci of zoonotic visceral leishmaniasis (ZVL), domestic dogs are important reservoir hosts of the causative Leishmania parasites transmitted by phlebotomine sandflies (Diptera: Psychodidae). We tested the protective value of impregnated dog collars (20 g plastic containing deltamethrin 800 mg ai) against Phlebotomus papatasi (Scopoli) sandflies in Iran. For each assay, the dog was sedated and caged in a net with 70-100 wild-caught sandflies overnight (23.30-06.30 hours). Dogs wearing the collars were bitten by approximately 80% fewer sandflies than before collars were fitted, i.e. 51% vs. 11% of hungry female flies exposed. Sandfly mortality rates following 20 h exposure to dogs with collars (18%) or without collars (17%) were not significantly different. Effects of collars were tested when dogs had been wearing them for 8 days. A previous trial against the sandfly P. perniciosus Newstead in France, using smaller dogs, showed that effects of such collars were not fully realized until they had been worn for 2 weeks or more; they remained effective for at least 8 months and killed significant proportions of the sandflies exposed. Present results with P. papatasi, confirming that this simple device provides effective protection against sandflies, are considered sufficiently encouraging to justify a community-wide field trial of deltamethrin-impregnated dog collars against ZVL vector sandflies in Iran.     

The effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment in preventing Phlebotomus perniciosus from feeding on dogs

A controlled clinical trial was performed to assess the effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment (recommended dosage) in preventing adult female sandflies (Phlebotomus perniciosus) from feeding on dogs. Sandfly mortality was also assessed. Twelve beagle dogs were used in the study. Prior to treatment they were checked for their attractiveness to sandflies, ranked accordingly to generate partner triplets of equivalent sensitivity to sandflies: four control dogs, four treated with the pyriprole and four with the metaflumizone spot-on. The dogs were challenged with 50 unfed adult female sandflies (8-10 days old), in cages for one hour on Day 1 and Day 7. The sandflies were checked for blood feeding and mortality at one hour, 24 hours and 48 hours after exposure to the dogs. A very poor anti-feeding effect (near 7%) was seen on sandflies with the metaflumizone combined with amitraz and no antifeeding effect was seen with pyriprole. The sandfly mortality effect as a result of exposure to treated dogs was under 20% for the two spot-on. The two formulations could not be proposed in a leishmaniosis prevention program.     

Sugar meal sources for the phlebotomine sandfly Lutzomyia longipalpis in Ceará State, Brazil

Abstract. In the Brazilian village of BoqueiraTo do Renato Parente, Ceará State, where visceral leishmaniasis is endemic, significant variation in the abundance of phlebotomine sandflies between different types of vegetation was demonstrated by castor oil sticky trap collections. Population densities of Lutzomyia longipalpis (Lutz & Neiva) sandflies were higher in beans and maize crops than in the surrounding open and tree scrub. Significant relationships were detected between the abundances of sandflies and aphids, suggesting aphid honey dew as a potential food source. Sugar meal analysis, using high-performance anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) and the cold anthrone test, was used to demonstrate that significantly more sandflies fed on bean plants contaminated with aphids and honeydew than on uncontaminated beans. Furthermore, higher concentrations of sugars were detected in flies which fed on aphid/honeydew-contaminated beans, suggesting that sugar was more easily available and/or that honeydew/aphids act as phagostimulants for sandflies. Amongst wild sandflies collected from a bean field, significantly more female sandflies were 'sugar-positive' than males, though the sugar concentrations in positive sandflies were similar for both sexes. The concentrations of di- and trisaccharides in sandfly homogenates, honeydew extracts and phloem exudates were very low.     

Molecular evolution of the period gene in sandflies

The molecular evolution of the clock gene period was studied in Phlebotomine sandflies (Diptera: Psychodidae). Comparison of the synonymous and nonsynonymous substitution rates between sandflies and Drosophila revealed a significantly higher evolutionary rate in the latter in three of the four regions analyzed. The differences in rate were higher in the sequences flanking the Thr–Gly repetitive domain, a region that has expanded in Drosophila but remained stable and short in sandflies, a result consistent with the coevolutionary scenario proposed for this region of the gene. An initial phylogenetic analysis including eight neotropical sandfly species and one from the Old World was also carried out. The results showed that only the subgenus Nyssomyia is well supported by distance (neighbor-joining) and maximum parsimony analysis. The grouping of the other species from the subgenus Lutzomyia and Migonei group shows very low bootstrap values and is not entirely consistent with classical morphological systematics of the genus Lutzomyia.     

Sampling methods for phlebotomine sandflies

A review is presented of methods for sampling phlebotomine sandflies (Diptera: Psychodidae). Among approximately 500 species of Phlebotominae so far described, mostly in the New World genus Lutzomyia and the Old World genus Phlebotomus, about 10% are known vectors of Leishmania parasites or other pathogens. Despite being small and fragile, sandflies have a wide geographical range with species occupying a considerable diversity of ecotopes and habitats, from deserts to humid forests, so that suitable methods for collecting them are influenced by environmental conditions where they are sought. Because immature phlebotomines occupy obscure terrestrial habitats, it is difficult to find their breeding sites. Therefore, most trapping methods and sampling procedures focus on sandfly adults, whether resting or active. The diurnal resting sites of adult sandflies include tree holes, buttress roots, rock crevices, houses, animal shelters and burrows, from which they may be aspirated directly or trapped after being disturbed. Sandflies can be collected during their periods of activity by interception traps, or by using attractants such as bait animals, CO2 or light. The method of trapping used should: (a) be suited to the habitat and area to be surveyed, (b) take into account the segment of the sandfly population to be sampled (species, sex and reproduction condition) and (c) yield specimens of appropriate condition for the study objectives (e.g. identification of species present, population genetics or vector implication). Methods for preservation and transportation of sandflies to the laboratory also depend on the objectives of a particular study and are described accordingly.     

Vectors of the filaria Thamugadia ivaschkini (Splendidofilariidae) in the southern Turkmen SSR

1903 specimens of sandflies and mosquitoes were examined for their spontaneous infection with larvae of Thamugadia ivaschkini. The experimental infection of sandflies and mosquitoes was conducted on infected lizards. It has been established that the vectors of this filaria in the south of Turkmenia are sandflies of the genus Phlebotomus and Sergentomyia arpaclensis. The development of the helminth carries out in thoracic muscles of sandflies, the first infective larvae appear in 7 to 17 days after the infection of vectors.     

Hourly Activity and Natural Infection of Sandflies (Diptera: Psychodidae) Captured from the Aphotic Zone of a Cave,Minas Gerais State,Brazil

Sandflies are holometabolous insects that are of great epidemiological importance in the neotropical region as vectors of leishmaniases. Caves are ecotopes that significantly differ from external environments and, among the insects that live or visit their internal area and adjacent environment, sandflies are commonly found. Based on this context, the objective of this work was to examine the period of activity of sandflies in the cave environment in the aphotic zone. Thus, four sandfly captures were conducted, one in each season of the year, in a cave where studies on the bioecological aspects of sandfly fauna have been conducted since 2008. In this same study, we have also noticed the presence of flagellates in some captured females. Catches were carried out for 24 hours using a Shannon trap, light bait, and cave walls were actively searched. We collected a total of 638 sandflies, representing 11 species. The most abundant species and with more intense period of activity were, in descending order: Lu. cavernicola (62%), Ev. spelunca (16%) and Ev. sallesi (14%). A total of 69 females were dissected to check for natural infection, and in five specimens we found living flagellated forms: two Ev. spelunca, two Ev. sallesi and one Sc. sordellii. This study shows that the activity of some species caught in the aphotic zone of the cave, especially Lu. cavernicola, differs from what has already been reported in previous sandfly captures, which are almost always conducted at night and during twilight. The existence of sandflies that were naturally infected with flagellates and the lack of awareness regarding the behaviour of sandflies in cave environments are strong indicators of the need for further study on this group of insects in this ecotope, as a safety measure to protect the visitors of such environment.     

Attraction of Lutzomyia longipalpis to human skin odours

Abstract. Male and female Lutzomyia longipalpis sandflies showed attraction to human skin emanations placed on warmed glass Petri dishes. Unfed virgin females were more strongly attracted than males, which also showed attraction. Four human subjects were tested and significant variation was found between the numbers of sandflies attracted to their skin emanations. This suggests that some individuals were more attractive than others. There was a significant difference between the response shown by sandflies from the Jacobina and Lapinha regions of Brazil, suggesting that sandflies from the Jacobina region were more anthropophilic. In addition, sandflies from Jacobina had a significantly higher level of activity than those from Lapinha. The role of sandfly attraction to humans as a risk factor in Leishmania transmission is considered.     

Molecular crosstalks in Leishmania-sandfly-host relationships

Sandflies (Diptera: Phlebotominael are vectors of Leishmania parasites, causative agents of important human and animal diseases with diverse manifestations. This review summarizes present knowledge about the vectorial part of Leishmania life cycle and parasite transmission to the vertebrate host. Particularly, it focuses on molecules that determine the establishment of parasite infection in sandfly midgut. It describes the concept of specific versus permissive sandfly vectors, explains the epidemiological consequences of broad susceptibility of permissive sandflies and demonstrates that genetic exchange may positively affect Leishmania fitness in the vector. Last but not least, the review describes recent knowledge about circulating antibodies produced by hosts in response to sandfly bites. Studies on specificity and kinetics of antibody response revealed that anti-saliva IgG could be used as a marker of host exposure to sandflies, i.e. as a useful tool for evaluation of vector control.     

Phlebotomine sandfly ecology on the Indian subcontinent: does village vegetation play a role in sandfly distribution in Bihar,India?

Visceral leishmaniasis (VL) is a disease that results in approximately 50 000 human deaths annually. It is transmitted through the bites of phlebotomine sandflies and around two‐thirds of cases occur on the Indian subcontinent. Indoor residual spraying (IRS), the efficacy of which depends upon sandfly adults resting indoors, is the only sandfly control method used in India. Recently, in Bihar, India, considerable sandfly numbers have been recorded outdoors in village vegetation, which suggests that IRS may control only a portion of the population. The purpose of this study was to revisit previously published results that suggested some sandflies to be arboreal and to rest on outlying plants by using Centers for Disease Control light traps to capture sandflies in vegetation, including banana plants and palmyra palm trees, in two previously sampled VL‐endemic Bihari villages. Over 3500 sandflies were trapped in vegetation over 12 weeks. The results showed the mean number of sandflies collected per trap night were significantly higher in banana trees than in other vegetation (P = 0.0141) and in female rather than male palmyra palm trees (P = 0.0002). The results raise questions regarding sandfly dispersal, oviposition and feeding behaviours, and suggest a need to refine current control practices in India and to take into account an evolving understanding of sandfly ecology.     

Surveillance of arthropod vector-borne infectious diseases using remote sensing techniques: a review

Epidemiologists are adopting new remote sensing techniques to study a variety of vector-borne diseases. Associations between satellite-derived environmental variables such as temperature, humidity, and land cover type and vector density are used to identify and characterize vector habitats. The convergence of factors such as the availability of multi-temporal satellite data and georeferenced epidemiological data, collaboration between remote sensing scientists and biologists, and the availability of sophisticated, statistical geographic information system and image processing algorithms in a desktop environment creates a fertile research environment. The use of remote sensing techniques to map vector-borne diseases has evolved significantly over the past 25 years. In this paper, we review the status of remote sensing studies of arthropod vector-borne diseases due to mosquitoes, ticks, blackflies, tsetse flies, and sandflies, which are responsible for the majority of vector-borne diseases in the world. Examples of simple image classification techniques that associate land use and land cover types with vector habitats, as well as complex statistical models that link satellite-derived multi-temporal meteorological observations with vector biology and abundance, are discussed here. Future improvements in remote sensing applications in epidemiology are also discussed.     

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge

Abstract The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E.coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M. luteus infection since anti-Zs. coli activity was similar in haemolymph from both E.coli oxM.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.     

Studies on the behaviour and control of phlebotomine sandflies using experimental houses

Programmes for the control of phlebotomine sandflies (Diptera: Psychodidae), the vectors of leishmaniases, mainly target adults because larval breeding sites are generally unknown or inaccessible. To determine how blood‐questing sandfly females enter homes and to develop means for their control, an experimental house (EH) was constructed in a village endemic for cutaneous leishmaniasis. Initially, carbon dioxide (CO₂)‐baited suction traps were installed inside the EH to attract and capture sandflies. For other experiments, the windows of the EH were fitted with CO₂‐baited window entrance traps (WETs) that allow each window to be considered as a separate unit. The majority of captures inside the EH and in WETs consisted of Phlebotomus sergenti, a species that enters inhabited houses relatively infrequently. Analyses of collections in WETs and in sticky traps on external walls showed that sandflies entered windows having landed previously on the wall below or either side of the window. Shelves constructed below windows significantly reduced the numbers of sandflies that entered both the EH and inhabited houses. The lining of internal walls with insecticide‐impregnated fabric significantly increased mortality rates of sandflies captured inside the EH. To reduce the biting burden imposed by phlebotomine sandflies, several control measures must be integrated and sustained.     

Honeydew sugars in wild-caught Phlebotomus ariasi detected by high performance liquid chromatography (HPLC) and gas chromatography (GC)

Phlebotomus ariasi Tonnoir sandflies were caught in light traps hung in oak trees and in a house in the Cévennes focus of leishmaniasis in the South of France. The flies were cryopreserved either immediately on removal from the traps, or after starvation for 6-7 days, or after 6-7 days starvation followed by exposure to oak infested with the aphid genera Lachnus or Thelaxes. After transportation to the laboratory, the sandflies were thawed and aqueous extracts of the crushed flies were analysed for their carbohydrate content using high performance liquid chromatography (HPLC) and gas chromatography (GC). Starved female sandflies lacked significant amounts of any saccharides. Four types of sugar, melezitose and its hydrolysis products turanose, glucose and fructose, were observed in flies which had been starved previously and then placed with Lachnus infested oak. The results also indicate the presence of hydrolysis products of melezitose: (a) in flies previously starved and placed with Thelaxes infested oak, (b) in P.ariasi cryopreserved direct from the light traps hung in oak trees infested with Lachnus and Thelaxes, and (c) flies caught in a house. Unidentifiably small quantities of a trisaccharide were also detected in the latter groups of flies. In previous tests, sugars were detected in P.ariasi after their exposure to aphid-infested oak (Quercus ilex L.), but not when P.ariasi females were exposed to washed oak leaves without aphids. The results indicate that P.ariasi feed on melezitose and/or turanose, the main local source of which is aphid honeydew. A better understanding of sugar meal sources of sandflies using HPLC and GC techniques will assist in our understanding of sandfly/Leishmania relationships, parasite transmission and epidemiology.     

Sandfly distribution and abundance in a tropical rain forest

1. The distribution patterns of sandflies (Diptera: Psychodidae) upon tree buttresses were studied in tropical rain forest at Finca la Selva in the Caribbean lowlands of Costa Rica. 2. Four species of sandfly, Lutzomyia shannoni Dyar, L. trapidoi F. & H., L. ylephiletor F. & H. and L. vespertilionis F. & H. comprising 97% of those caught, used tree buttresses as diurnal resting sites. Their distribution on the buttresses was aggregated. 3. During the dry season tree species had no significant effect upon the distribution of the sandflies. However, during the wet season the distributions of two of the species, L. trapidoi and L. ylephiletor, were significantly affected by the species of tree; it is suggested that some species of tree may provide greater protection from rainfall than others. 4. L. vespertilionis was restricted to a single buttress on each positive tree. Distribution of this species is evidently determined by the distribution of its host animal, the bat (Emballonuridae). Female flies feed upon the bat's blood and male flies may be attracted to the bat as it provides a source of female sandflies. It is suggested, therefore, that tree buttresses serve as sandfly swarming sites. 5. Within a large buttress the sandflies are not randomly distributed but are aggregated in particular areas. Within these aggregations, the sandflies are vertically zoned upon the buttress with a shift in species composition with height. Two hypothesis were suggested to account for this distribution pattern: a response to an environmental gradient or an interaction between the four species of fly.     

The relationship between CDC light-trap and human-bait catches of endophagic sandflies (Diptera: Psychodidae) in the Peruvian Andes

Abstract. A study was carried out in the Peruvian Andes to test the suitability of CDC light traps for monitoring changes in the human-landing rate of endophagic phlebotomine sandflies, following house-spraying with pyrethroid insecticide. On four pairs of consecutive nights, sandflies were caught inside eight sprayed and eight unsprayed houses, either by human bait or by CDC light traps. The sandflies collected were Lutzomyia verrucarum (97%) and Lu. peruensis (3%), both probable vectors of Leishmania peruviana , and the species composition was unaffected by house-spraying. A non-linear relationship was detected between light-trap and human-bait catches, but the relationship did not diverge significantly from linearity within the range of sandfly abundance found in most houses in the endemic area (i.e. between 3 and 200 sandflies/house-night), and did not differ significantly between sprayed and unsprayed houses. However, light trap catches had a significantly lower proportion of blood-fed females in sprayed than in unsprayed houses, probably due to an insecticidal effect on post-blood-feeding behaviour. The proportion of Lu. verrucarum was significantly higher in light trap than in human bait catches, indicating that Lu. peruensis is either more anthropophilic or less phototropic than Lu. verrucarum.