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.     

Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Experimental infection of Phlebotomus perniciosus and Phlebotomus tobbi with different Leishmania tropica strains

Cutaneous leishmaniasis due to Leishmania tropica is increasingly documented in Europe and the Middle East. Besides its specific vector, Phlebotomus sergenti, permissive Phlebotomus sand flies are suspected as potential vectors of L. tropica. We investigated the susceptibility of two widely distributed species, Phlebotomus perniciosus and Phlebotomus tobbi. Laboratory-reared sand flies were infected experimentally with L. tropica strains differing in lipophosphoglycan epitopes, geographical distribution and epidemiology. High infection rates, heavy parasite loads and fully developed late-stage infections including colonization of the stomodeal valve were observed in all parasite-vector combinations. Our findings demonstrate that P. perniciosus and P. tobbi are susceptible to different L. tropica strains and may play a role in their circulation in endemic foci of Europe, the Middle East and North Africa.     

Genetic polymorphism in sympatric species of the genus Phlebotomus, with special reference to Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera, Phlebotomidae)

The Random Amplified Polymorphic DNA assay was used to study genetic variation within and between five Phlebotomus species belonging to three subgenera: P. (Larroussius) ariasi, P. (L.) longicuspis, P. (L.) perniciosus, P.(Paraphlebotomus) sergenti and P. (Phlebotomus) papatasi sympatric in southern Spain and proven vector of leishmaniasis. Two cluster analysis were proposed: one according to sandfly species and populations, the second according individual specimens of Phlebotomus perniciosus, Phlebotomus longicuspis s.l. and intermediate morphological specimens between these species. The results obtained are closely correlated with the taxonomy classically accepted for the subgenera and with the automatic classifications made by other authors which use morphological and isoenzymatic data. The validity of the species Phlebotomus longicuspis is also discussed.     

Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as vectors of Leishmania infantum in Tbilisi, Georgia

A survey of potential vector sand flies was conducted in the neighboring suburban communities of Vake and Mtatsminda districts in an active focus of visceral Leishmaniasis (VL) in Tbilisi, Georgia. Using light and sticky-paper traps, 1,266 male and 1,179 female sand flies were collected during 2006-2008. Five Phlebotomus species of three subgenera were collected: Phlebotomus balcanicus Theodor and Phlebotomus halepensis Theodor of the subgenus Adlerius; Phlebotomus kandelakii Shchurenkova and Phlebotomus wenyoni Adler and Theodor of the subgenus Larroussius; Phlebotomus sergenti Perfil'ev of the subgenus Paraphlebotomus. Phlebotomus sergenti (35.1%) predominated in Vake, followed by P. kandelakii (33.5%), P. balcanicus (18.9%), P. halepensis (12.2%), and P. wenyoni (0.3%). In Mtatsminda, P. kandelakii (76.8%) comprised over three fourths of collected sand flies, followed by P. sergenti (12.6%), P. balcanicus (5.8%), P. halepensis (3.7%), and P. wenyoni (1.1%). The sand fly season in Georgia is exceptionally short beginning in early June, peaking in July and August, then declining to zero in early September. Of 659 female sand flies examined for Leishmania, 12 (1.8%) specimens without traces of blood were infected including 10 of 535 P. kandelakii (1.9%) and two of 40 P. balcanicus (5.0%). Six isolates were successfully cultured and characterized as Leishmania by PCR. Three isolates from P. kandelakii (2) and P. balcanicus (1) were further identified as L. infantum using sequence alignment of the 70 kDa heat-shock protein gene. Importantly, the sand fly isolates showed a high percent identity (99.8%-99.9%) to human and dog isolates from the same focus, incriminating the two sand fly species as vectors. Blood meal analysis showed that P. kandelakii preferentially feeds on dogs (76%) but also feeds on humans. The abundance, infection rate and feeding behavior of P. kandelakii and the infection rate in P. balcanicus establish these species as vectors in the Tbilisi VL focus.     

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.     

Base-line susceptibility of Phlebotomus papatasi to insecticides

Susceptibility of the leishmaniasis vector sandfly Phlebotomus papatasi (Scopoli) to three insecticides was assessed on a colony originating in the Jordan Valley, Israel. Results are considered to show susceptibility to DDT and permethrin. The results for methoxychlor indicated the possibility of heterogeneity in the population and tolerance towards this compound.     

Postglacial dispersal of Phlebotomus perniciosus into France

Phlebotomus perniciosus was identified morphologically in samples from France and northeast Spain, and individuals were then characterized at three polymorphic isoenzyme loci (by isoelectrofocusing) and at the mitochondrial DNA locus (by comparative DNA sequence analysis of a fragment of the Cytochrome b gene). The four polymorphic loci gave conflicting patterns of population relationships, which can be explained by hypothesizing different amounts of gene introgression at each locus when two distinctive lineages met in southern France or northeast Spain after isolation in southern Italy and Spain during the Pleistocene Ice Ages. P. perniciosus is an important vector of leishmania infantum and so these population differentiation studies are relevant for predicting the emergence and spread of leishmaniasis in relation to environmental changes, including climate.     

Phlebotomus from Madagascar (Diptera: Psychodidae). III--Description of Phlebotomus (Anaphlebotomus) fontenillei n. sp

The male of Phlebotomus (Anaphlebotomus) fontenillei n. sp. is described from Namoroka area (Madagascar). Its belongs to the subgenus Anaphlebotomus: style with four spines, coxite without basal process and paramere with two branches. It shares with P. berentiensis an original and exclusive antennal formula: 2/III-XII which distinguishes them from P. fertei. P. fontenillei n. sp. differs mainly from P. berentiensis by about 40 setae in tuft on the ventral face of the coxite, the length of the genital ducts and the position of the spines on the style. Sequence of the second internal transcribed spacer (ITS2) of the ribosomal DNA (rDNA) is very informative: the male of P. fontenillei n. sp. cannot be linked to the female of P. huberti (male unknown) regarding the size of amplified DNA fragment (459 bp versus 600 respectively) and the high degree of variability. There are few differences (10 mutations) between the sequences of P. fontenillei n. sp. and P. berentiensis which are closely related species.