Control of phlebotomine sandflies

Phlebotomine sandflies (Diptera: Psychodidae) transmit many zoonotic diseases (arboviruses, bartonelloses and especially leishmaniases) of importance to human health in at least 80 countries. Measures used to control adult sandflies (Lutzomyia and Phlebotomus) include the use of insecticides (mostly pyrethroids) for residual spraying of dwellings and animal shelters, space-spraying, insecticide-treated nets, impregnated dog-collars and personal protection through application of repellents/insecticides to skin or fabrics. Because the breeding-sites of sandflies are generally unknown, control measures that act specifically against immatures are not feasible, although the effectiveness of a few biological and chemical agents has been demonstrated in laboratory evaluations. Reports of insecticide-resistance refer to only three sandfly species (P. papatasi, P. argentipes and S. shorttii) against DDT in one country (India), although there are reports of DDT-tolerance in several countries. Current knowledge of sandfly susceptibility to various insecticides is summarized. Constraints and advantages of different compounds, formulations and delivery methods for sandfly control under different environmental conditions are discussed.     

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.     

The phlebotomine sandflies of Venezuelan Amazonia

Phlebotomine sandflies were surveyed in two ecologically contrasted areas, the northern moist and southern wet tropical forests, of the Territorio Federal Amazonas, Venezuela. Three new taxa and twenty-one new records were added to the previously known species list for Venezuelan sandflies, which now totals eighty species. Both sexes of Lutzomyia (Psychodopygus) killicki sp.n., L. (Trichophoryomyia) bettinii sp.n., L. (Nyssomyia) olmeca reducta subsp.n. and and the females of L. bernalei Osorno et al., Brumptomyia pintoi Costa Lima and L. begonae (Ortiz & Torres) are described and illustrated.     

Natural breeding places of phlebotomine sandflies

Methods of finding larvae and pupae of phlebotomine sandflies (Diptera: Psychodidae) are described and the known types of breeding sites used by sandflies are listed. Three ways of detecting sandfly breeding places are the use of emergence traps placed over potential sources to catch newly emerged adult sandflies; flotation of larvae and pupae from soil, etc., and desiccation of media to drive out the larvae. Even so, remarkably little information is available on the ecology of the developmental stages of sandflies, despite their importance as vectors of Leishmania, Bartonella and phleboviruses affecting humans and other vertebrates in warmers parts of the world. Regarding the proven or suspected vectors of leishmaniases, information on breeding sites is available for only 15 out of 29 species of sandflies involved in the Old World and 12 out of 44 species of sandflies involved in the Americas, representing approximately 3% of the known species of Phlebotominae. Ecotopes occupied by immature phlebotomines are usually organically rich moist soils, such as the rain forest floor (Lutzomyia intermedia, Lu. umbratilis, Lu. whitmani in the Amazon; Lu. gomezi, Lu. panamensis, Lu. trapidoi in Panama), or contaminated soil of animal shelters (Lu. longipalpis s.l. in South America, Phlebotomus argentipes in India; P. chinensis in China; P. ariasi, P. perfiliewi, P. perniciosus in Europe). Developmental stages of some species (P. langeroni and P. martini in Africa; P. papatasi in Eurasia; Lu. longipalpis s.l. in South America), have been found in a wide range of ecotopes, and many species of sandflies employ rodent burrows as breeding sites, although the importance of this niche is unclear. Larvae of some phlebotomines have been found in what appear to be specialized niches such as Lu. ovallesi on buttress roots of trees in Panama; P. celiae in termite hills in Kenya; P. longipes and P. pedifer in caves and among rocks in East Africa. Old World species found as immatures in the earthen floor of human habitations include P. argentipes, P. chinensis, P. martini and P. papatasi. Much more information on sandfly breeding sites is required to facilitate their control by source reduction.     

Faunistic study of sandflies in Greece

A faunistic study of phlebotomine sandflies was carried out on the mainland and on four islands in Greece between 1999 and 2004. Sandflies were collected in 18 areas, and the population structure was observed and recorded. A total of 10 species were identified; their distribution is presented here. Of these, the species Phlebotomus neglectus Tonnoir and P. perfiliewi Parrot (Diptera: Psychodidae), epidemiologically the most important vectors of leishmaniases and sandfly fever in Greece, were shown to be present in the main endemic foci of the country.     

Ootaxonomy and eggshell ultrastructure of Phlebotomus sandflies

The eggshell structure of four sandfly species: Phlebotomus perniciosus Newstead, P.perfiliewi Parrot, P.papatasi Scopoli and P.duboscqi Neveu-Lemaire, was examined by scanning and transmission electron microscopy (SEM and TEM). At the TEM level, the eggshell appears to have a homogeneous vitelline envelope and a thick chorion. At SEM level, the eggshell of all species is characterized by the outer chorion forming a series of fifteen to twenty longitudinal sinuous ridges, cross-linked in places to form a pattern of polygons, each line of the chorion consisting of columns arranged in a palisade. The aeropyle region of the egg is described for the first time in phlebotomine sandflies. Specific characters of the eggshell topography are described for distinguishing between these and other species of Phlebotomus.     

Interspecific hybridization and genetic variability of Phlebotomus sandflies

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est-3, Idh-1, Mdh-2, Mpi, Tre-1 and Tre-3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est-3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est-3, Idh-1, Me, Mpi, Pgd, Tre-1 and Tre-3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh-2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5-8 polymorphic loci (10-12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.     

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.     

Morphometric and molecular differentiation of Phlebotomus (Phlebotomus) sandflies

The closely related sandfly species of the subgenus Phlebotomus namely, Phlebotomus papatasi (Scopoli, 1786), Phlebotomus duboscqi Neveu‐Lemair, 1906 and Phlebotomus bergeroti Parrot, 1934 (Diptera: Psychodidae), are major vectors of Leishmania major (Kinetoplastida: Trypanosomatidae), the causative agent of cutaneous leishmaniasis in the Old World. Although allopatric in most of their distribution, the three species exist sympatrically in many places in central and eastern Sudan. Males of the three species can be distinguished using morphological characters; however, females are much harder to identify, thus complicating epidemiological studies. We carried out a morphometric and a molecular study to determine reliable morphological features and develop a polymerase chain reaction (PCR) assay for distinguishing females of these species. Males and females from each species were collected from sites in Sudan, East Africa and from one site in Mali, West Africa. Males were analysed morphologically and 20 characters and 10 character ratios were used in a stepwise discriminant analysis. This led to the identification of four characters with high discriminant loading scores sufficient for accurate male species identification. Male DNA was then used for the development of a PCR‐based species diagnostic based on the second internal transcribed spacer (ITS2) of the ribosomal DNA. A set of four primers was developed to generate fragment sizes that are specific to each species and can reliably identify females as well as hybrid DNA. Both the morphometric and the molecular findings of this study have important applications for studies of the epidemiology of cutaneous leishmaniasis.     

Comparison of three methods for age-grading of female Neotropical phlebotomine sandflies

Abstract. Changes in appearance of the accessory glands (AG), ovaries (OV) and the absence or presence of crease marks at the edges of the genital atrium armature (GA) were compared as methods to age-grade female laboratory-bred (Lutzomyia migonei) and wild-caught (Lu.youngi, Lu.spinicrassa, Lu.ovallesi) Neotropical sandflies.
Dissections at different physiological stages revealed that all parous and high proportions of nulliparous (4/38 unmated, 12/12 mated) females showed similar residual AG secretions. Hence AG appearance (nullipars with AG translucent, versus yellowish opaque granulated AG previously regarded as signs of parousness) is unreliable for age-grading.
Readily recognizable changes in both GA (98% creased) and the OV (100% yellowish with loose ovarioles) indicated that these organs could be employed to discriminate between nulliparous and parous females. Despite finding one misdiagnosed female (a nullipar having creased GA indicative of parity), the GA method of age-grading is recommended because females may be examined satisfactorily at any stage of the gonotrophic cycle. Moreover, the GA characteristics of pickled or dry preserved sandflies as well as fresh slide-mounted specimens can be determined easily.
Among sixty-five wild-caught Lutzomyia females dissected (mostly Lu.spinicrassa and Lu.youngi) , the proportions of specimens yielding preparations suitable for examination were 97% for GA, 89% for OV, but only 58% for AG, which reinforces GA as the preferred character for age-grading.     

Chorion patterns on eggs of Lutzomyia sandflies from the Peruvian Andes

Abstract. Scanning electron microscopy (SEM) was used to depict and describe the eggs of five species of phlebotomine sandfly from the Andean region of Peru: Lutzomyia caballeroi, Lu.noguchii, Lu.peruensis, Lu.tejadai and Lu.verrucarum. Two new types of chorionic sculpturing of sandfly eggs are reported: these were named disperse (Lu.tejadai) and verrucose (Lu.noguchii). The aeropylar area of the eggs is also described for the first time for neotropical sandflies. These character states appear to vary for ecological rather than phylogenetic reasons and could be used for species identification.