About the presence of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae) in Eastern Sicily, Italy

The note reports the data of a three-year sand fly investigation (1997-99) carried out in Eastern Sicily (Italy) with the aim to study the distribution of Phlebotomus sergenti. The survey involved a densely inhabited area at the foot of Mount Etna and the area of Iblei mounts. A total of 9,095 sand flies, of which 63.4% males, were captured. Five species belonging to the genus Phlebotomus (P. perniciosus, P. perfiliewi, P. neglectus, P. sergenti and P. papatasi) and one to the genus Sergentomyia (S. minuta) were identified. Both the prevalence and distribution of the species were different within the two areas studied. In Mount Etna area, P. perniciosus (77.7%) was the prevalent species followed by S. minuta (19.8%), P. sergenti (2.0%), P. neglectus (0.3%) and P. papatasi (0.2%). While in Iblei mounts region S. minuta (84.5%) showed the highest prevalence, followed by P. perniciosus (14.4%), P. perfiliewi (0.9%) and P. neglectus (0.1%). Here, P. sergenti was a very rare species (< 0.02). P. sergenti was mostly associated to domestic habitats of peri-urban and urban zones located between two and 750 m a.s.l. The density values of P. sergenti, expressed as number of specimens/m2 of sticky trap, were between 0.3 and 5.5 with the highest value in the hilly collecting sites. The low observed abundance of P. sergenti does not allow to draw any prediction on the role that the species could play in the transmission of leishmaniasis in Sicily.     

A comparison of the intraspecific variability of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae).

Phlebotomus sergenti populations from different areas of the Mediterranean basin are known to exhibit high intraspecific variability. Previous studies of ITS2 revealed the presence of two branches that may represent sibling species. To corroborate this finding by other tools, two colonies of P. sergenti originating from Turkey and Israel, each belonging to a different ITS2 branch, were compared by three different methods: geometric morphometric analysis of wing shape, RAPD (random amplified polymorphic DNA), and cross-mating study. For geometric morphometric analysis, two-dimensional Cartesian coordinates of 16 landmarks from the wings were digitized and analyzed. Significant shape differences were found between colonies but not between sexes within each colony. RAPD results formed two distinctive clades corresponding to the origin of the colony but also showed heterogenity among members of both colonies. In cross-mating studies, viable hybrid F1 and F2 progeny were obtained when both Turkish males/Israeli females and Israeli males/Turkish females were crossed. F1 progeny was included in RAPD analysis and these hybrids formed a distinctive clade with an intermediate position between the two parental clades. No significant differences were found in egg production of crossed sand flies. The cross-mating study showed that there is no reproductive barrier between P. sergenti from different geographical areas. On the other hand, RAPD and geometric morphometric analysis revealed a significant difference between colonies and confirmed the suitability of previous ITS2 analysis for discrimination among sand fly populations. Further development of molecular markers should resolve a possible existence of sibling species within Phlebotomus sergenti.     

Molecular Characterization of Gregarines from Sand Flies (Diptera: Psychodidae) and Description of Psychodiella n. g. (Apicomplexa: Gregarinida)

ABSTRACT. Sand fly and mosquito gregarines have been lumped for a long time in the single genus Ascogregarina and on the basis of their morphological characters and the lack of merogony been placed into the eugregarine family Lecudinidae. Phylogenetic analyses performed in this study clearly demonstrated paraphyly of the current genus Ascogregarina and revealed disparate phylogenetic positions of gregarines parasitizing mosquitoes and gregarines retrieved from sand flies. Therefore, we reclassified the genus Ascogregarina and created a new genus Psychodiella to accommodate gregarines from sand flies. The genus Psychodiella is distinguished from all other related gregarine genera by the characteristic localization of oocysts in accessory glands of female hosts, distinctive nucleotide sequences of the small subunit rDNA, and host specificity to flies belonging to the subfamily Phlebotominae. The genus comprises three described species: the type species for the new genus— Psychodiella chagasi ( Adler and Mayrink 1961 ) n. comb., Psychodiella mackiei ( Shortt and Swaminath 1927 ) n. comb., and Psychodiella saraviae ( Ostrovska, Warburg, and Montoya-Lerma 1990 ) n. comb. Its creation is additionally supported by sequencing data from other gregarine species originating from the sand fly Phlebotomus sergenti . In the evolutionary context, both genera of gregarines from mosquitoes ( Ascogregarina ) and sand flies ( Psychodiella ) have a close relationship to neogregarines; the genera represent clades distinct from the other previously sequenced gregarines.     

ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera,Psychodidae): possible consequences in their ability to transmit Leishmania tropica

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.     

Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae)

Phlebotomus papatasi ( Scopoli, 1786 ) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps.     

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.     

Host-parasite relationships of Ascogregarina chagasi (Eugregarinorida, Aseptatorina, Lecudinidae) in Lutzomyia longipalpis (Diptera: Psychodidae)

The life cycle of Ascogregarina chagasi in larvae and in adult female Lutzomyia longipalpis was studied by light and electron microscopy. Sporozoites and young gamonts were attached to the epithelial lining of the larval midgut via an osmiophilic contact zone. The mucron of young gamonts was bordered by an invaginated pellicular fold, and an electron-opaque vesicular structure was observed adjacent to it. Sporozoites possessed an apical complex and were bound by a double membrane with underlying subpellicular microtubules. The gamont pellicle was uniformly corrugated and consisted of two cortical membranes and a plasma membrane. Mature gamonts and gametocysts were found in the posterior ectoperitrophic space of third and fourth instar larval midguts and in the haemocoel of adult flies. Gametocysts in adult females adhered to the genital accessory glands, where they were encased in electron-dense capsules secreted by the fly through haemocyte-mediated humoral immune reactions. Oocytes were spindle-shaped and bound by a double-layered wall with a discernible polar plug at each end. Sporulation was endogenous, occurring within gametocysts in the midguts of larvae or the accessory glands of adult females. FITC-phalloidine staining of all stages of A. chagasi except mature gametocysts produced bright fluorescence, indicating the presence of a diffuse, actin-like protein in the cytoplasm.     

Ascogregarina saraviae n. sp. (Apicomplexa: Lecudinidae) in Lutzomyia lichyi (Diptera: Psychodidae)

Ascogregarina saraviae n. sp. (Apicomplexa: Lecudinidae) is described from wild-caught Lutzomyia lichyi (Diptera: Psychodidae) females. Gametocysts adhered to the hemocoel side of the genital accessory gland walls and oocysts were injected into their lumina. Sporulated oocysts were ellipsoidal, 12.4 x 5.8 (11.6-13.1 x 5.6-5.9) micrometers, contained eight sporozoites and a refractile residuum. The elongate form of A. saraviae n. sp. oocysts, and their more delicate walls, clearly distinguish them from oocysts of A. chagasi (Adler & Mayrink, 1961).     

Ultrastructure of the seminal vesicle of Phlebotomus perniciosus Newstead (Diptera, Psychodidae)

The seminal vesicles of Phlebotomus perniciosus were investigated by light microscopy, confocal scanning laser microscopy and by scanning and transmission electron microscopy. They have a complex structure, and three different morphological compartments called A, B and C are distinguished on the basis of their position and fine structure. Compartment A is continuous with the vasa deferentia and consists of a cylindrical wall limiting a lumen in which the spermatozoa are stored. Compartment B is hemispherical and surrounds compartment A like a muff. Compartment C constitutes an external coat surrounding A and B. The epithelial cells of each compartment are characterized by morphologically different secretory granules. The ultrastructural features of these cells are described and their role in sandfly reproductive biology is discussed.     

Ascogregarina saraviae N. Sp. (Apicomplexa: Lecudinidae) in Lutzomyia lichyi (Diptera: Psychodidae)

Ascogregarina saraviae n. sp. (Apicomplexa: Lecudinidae) is described from wild-caught Lutzomyia lichyi (Diptera: Psychodidae) females. Gametocysts adhered to the hemocoel side of the genital accessory gland walls and oocysts were injected into their lumina. Sporulated oocysts were ellipsoidal, 12.4 × 5.8 (11.6–13.1 × 5.6–5.9) micrometers, contained eight sporozoites and a refractile residuum. The elongate form of A. saraviae n. sp. oocysts, and their more delicate walls, clearly distinguish them from oocysts of A. chagasi (Adler & Mayrink, 1961).     

Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae)

Phlebotomus papatasi is a proven vector of Leishmania major which is one of the causative agents of cutaneous leishmaniasis in the Old World. Although it has a wide geographical range, its population structure is not yet well understood. In an effort to better understand the population dynamics of this vector, we developed a panel of di‐ and trinucleotide microsatellite markers, using a magnetic bead hybridization enrichment protocol. These microsatellite loci showed three to seven alleles with an expected heterozygosity range between 0.702 and 0.876. The level of polymorphisms found in this study suggests that these microsatellite loci can be used for population analysis of P. papatasi.     

Comparative demography of the sand fly Phlebotomus papatasi (Diptera: Psychodidae) at constant temperatures.

We measured reproductive and population parameters of adult sand flies, Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae), in environmental chambers maintained at temperatures of 15, 18, 20, 25, 28, and 32 degrees C. Based on cohorts of adults at each temperature regime, horizontal life tables were constructed using established laboratory colonies initiated from specimens collected in Sanliurfa Province, southeastern Anatolia, Turkey. The fecundity and longevity of the insects were both highly variable, depending on the temperature. At 15 degrees C, all of the cohort females died before laying eggs, so the construction of a life table for this temperature regime was not possible. Within a range of 18 to 32 degrees C, the longevity of adult P. papatasi increased as the temperature decreased; at 15 degrees C, the mean survival times of females and males were 19.04 +/- 6.94 days (9-35) and 17.84 +/- 7.11 days (9-33), respectively. While the highest number of eggs was found in the cohort at 28 degrees C (44.08 +/- 7.79), this was only 3.60 +/- 1.55 in the cohort at 32 degrees C and 2.8 +/- 0.9 in the cohort at 18 degrees C. This result showed that extreme temperatures negatively affect the fecundity of this species. The cohort reared at 28 degrees C exhibited the highest intrinsic rates of population increase (r(m)) for P. papatasi. The r(m) ranged from 0.098 at 28 degrees C to 0.007 at 18 degrees C. The cohort placed at 28 degrees C was found to be significantly different (P < 0.01) from the other cohorts producing the fewest progeny in terms of net reproductive rate, R(0), (15.87). The values for mean generation time (T) were estimated to vary from 36 days to 271 days depending on temperature. Principal Component Analysis (PCA) confirmed results from the previous studies that the cohort at 28 degrees C orientated and clustered as a distinct group along the first two PCs.     

First description of the male of Phlebotomus betisi Lewis and Wharton, 1963 (Diptera: Psychodidae)

The male of Phlebotomus (Larroussius) betisi is described from Malayan caves. Several males have been caught in association with P. betisi females. Males and females have been associated by ecology, biogeography, morphology and molecular biology (homology of the ND4 mtDNA sequences).     

How to increase the population of a Phlebotomus perniciosus (Diptera: Psychodidae) colony: a new method

The sandfly Phlebotomus perniciosus is the most widespread vector of Leishmania infantum in Spain. Laboratory colonisation represents the most feasible source of information on the biology of these insects, but in conducting any study, the density of individuals in the colony may drop to such an extent that it is sometimes difficult to recover the initial population levels. A new technique was tested for the recovery of sandfly eggs in three different colonies; the recovery rate was studied by comparing the standard method of mass rearing with this new method of colony management. The results demonstrate a mean increase of 18.4% in adult production, a growth in colony productivity that justifies the inclusion of this process in the routine maintenance of any colony of sandflies.     

Free amino acids in wild-caught Phlebotomus perniciosus and P. perfiliewi (Diptera: Psychodidae).

Free amino acids occurring in wild Phlebotomus perniciosus and P. perfiliewi were analyzed using high performance liquid chromatography. For both species the total concentration of amino acids, per unit weight, is over three times greater in females than in males. Qualitative inter-sex differences in amino acid compositions were also discovered: the most significant involving higher proportions of taurine and gamma-aminobutyric acid in females and higher proportions of arginine and glutamic acid in males. Although amino acid composition is consistent between females of both species, there are significant differences between males. The most important difference between males is an increase in tyrosine levels, at the expense of taurine, in P. perniciosus (as compared with P. perfiliewi). It is suggested that the intra- and inter-specific variation in amino acid composition identified in these studies could be due to ecological factors, such as differences in the sandflies natural amino acid sources (honeydew and/or plants), although a physiological explanation is not ruled out.     

Leishmania major and L. donovani: effects on proteolytic enzymes of Phlebotomus papatasi (Diptera, Psychodidae)

Phlebotomus papatasi is susceptible to Leishmania major which it transmits in nature, but is resistant to L. donovani. The present study compares the effect of L. major and L. donovani on the proteolytic activity of P. papatasi gut enzymes. The experiments measured digestion of C14-labeled globin by gut homogenates of flies. Homogenates were prepared from flies fed on serum only (controls) or from flies fed serum containing promastigotes or their dried culture overlayer. In other experiments, the promastigotes or dried culture overlayers were added in vitro to the gut homogenate of control flies. Proteolytic activity of gut homogenate from flies infected with L. major was about one-third less than that of controls, while that from flies infected with L. donovani was one-third greater. Ingestion of L. major dried culture overlayer had an effect on flies similar to that of the promastigotes, while L. donovani dried culture overlayer produced no significant effect. When added to gut homogenate in vitro, promastigotes of both species promoted proteolysis as did dried culture overlayer of L. major. Dried culture overlayer of L. donovani, however, had an opposite effect. It is suggested that the observed reduction in proteolytic activity caused by L. major infection may result from inhibition of enzyme production.     

Bionomics of Phlebotomus papatasi (Diptera: Psychodidae) in an endemic focus of zoonotic cutaneous leishmaniasis in central Iran.

Following an epidemiological survey of zoonotic cutaneous leishmaniasis (ZCL) in several villages of Badrood, a rural district north of the city of Natanz, central Iran, Phlebotomus (Phlebotomus) papatasi Scopoli were found to be naturally infected with Leishmania (Leishmania) major zymodeme MON-26. Sand flies were collected and dissected biweekly from rodent burrows from May to October 2001. Leptomonad infection rates varied between 6.7% and 22.0%, being greatest in September, coinciding with peak activity of P. papatasi, two-three months before the highest incidence of ZCL human cases in November-December. The leptomonad infection rate was 1.1% of the 94 P. papatasi captured indoors. In ELISA testing of 520 P. papatasi blood meals during Sept. 2001 and Aug. 2002, the proportion giving positive reactions for human, sheep, cow, goat, rodent, and bird were 31.2%, 69.6%, 63%, 38.8%, 24.7%, and 21.8%, respectively. This report thus incriminates P. papatasi as the vector of zoonotic cutaneous leishmaniasis in this part of Iran.