Population structure and geographical subdivision of the Leishmania major vector Phlebotomus papatasi as revealed by microsatellite variation

Abstract Multi‐locus microsatellite typing (MLMT) has been employed to infer the population structure of Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) sandflies and assign individuals to populations. Phlebotomus papatasi sandflies were collected from 35 sites in 15 countries. A total of 188 P. papatasi individuals were typed using five microsatellite loci, resulting in 113 different genotypes. Unique microsatellite signatures were observed for some of the populations analysed. Comparable results were obtained when the data were analysed with Bayesian model and distance‐based methods. Bayesian statistic‐based analyses split the dataset into two distinct genetic clusters, A and B, with further substructuring within each. Population A consisted of five subpopulations representing large numbers of alleles that were correlated with the geographical origins of the sandflies. Cluster B comprised individuals collected in the Middle East and the northern Mediterranean area. The subpopulations B1 and B2 did not, however, show any further correlation to geographical origin. The genetic differentiation between subpopulations was supported by F statistics showing statistically significant (Bonferroni‐corrected P < 0.005) values of 0.221 between B2 and B1 and 0.816 between A5 and A4. Identification of the genetic structure of P. papatasi populations is important for understanding the patterns of dispersal of this species and to developing strategies for sandfly control.     

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.     

Cutaneous leishmaniasis caused by Leishmania infantum transmitted by Phlebotomus tobbi

Transmission of cutaneous leishmaniasis (CL) caused by Leishmania infantum was studied in South Anatolia, Turkey. Small, non-ulcerating lesions prevailed and patients were negative in rK39 tests for antibody detection for human visceral leishmaniasis (VL). The most abundant sand fly species, Phlebotomus tobbi, was found positive for Leishmania promastigotes with a prevalence of 1.4% (13 out of 898 dissected females). The isolated strains were identical with those obtained from patients with CL and were typed as L. infantum. Phylogenetic analysis revealed similarity to MON-188 and a clear difference from the MON-1 clade. Blood-meal identification showed that P. tobbi feeds preferentially on cattle and humans. This finding, the high number of CL patients and relative scarcity of dogs in the focus, suggests that the transmission cycle could be anthroponotic.     

Isolation (with enrichment) and characterization of trinucleotide microsatellites from Phlebotomus perniciosus,a vector of Leishmania infantum

Mitochondrial DNA characterization of the sandfly Phlebotomus perniciosus has not resolved the population structure of its Iberian lineage. For this purpose, four AGC‐ and seven AGG‐class microsatellite loci were characterized, after their isolation using Biotin‐Avidin enrichment and the screening of plasmid libraries by polymerase chain reaction. Of the five polymorphic loci analysed in four Spanish populations, four showed patterns of allele diversity consistent with migration from a southern Ice Age refuge. Estimates of the historical migration rates of P. perniciosus will help to predict the effects of global warming on its range and that of Leishmania infantum, the parasitic protozoan it transmits.     

Sandflies of the Phlebotomus perniciosus complex: mitochondrial introgression and a new sibling species of P. longicuspis in the Moroccan Rif

The bloodsucking adult females of Phlebotomus perniciosus Newstead and P. longicuspis Nitzulescu (Diptera: Psychodidae) are important vectors of the protozoan Leishmania infantum Nicolle (Kinetoplastida: Trypanosomatidae) in western Mediterranean countries. The species status of the two phlebotomine sandflies was assessed, along with the epidemiological implications. Individual sandflies from three Moroccan Rif populations were characterized morphologically, isoenzymatically (by the isoelectrofocusing of alleles at the polymorphic enzyme loci of HK, GPI and PGM), and by comparative DNA sequence analysis of a fragment of mitochondrial Cytochrome b (mtDNA). By reference to the character profiles of specimens from other locations, including southern Spain and the type-locality countries, the Moroccan flies were placed in three lineages: first, the lineage of P. perniciosus, which contained two mtDNA sublineages, one (pnt) widely distributed and associated with the morphology of the male types from Malta, and the other (pna) associated with a P. longicuspis-like male morphology; second, the lineage of P. longicuspis sensu stricto, including typical forms from Tunisia; and third, a new sibling species of P. longicuspis. The mtDNA sublineage (pnt) of typical P. perniciosus was also found in some P. longicuspis from Morocco, indicating interspecific hybridization. The typical race of P. perniciosus occurs in Italy as well as in Malta, Tunisia and Morocco. It is replaced in southern Spain by the Iberian race (with the pni mtDNA sublineage). The discovery of interspecific gene introgression and a new sibling species mean that previous records of the two morphospecies do not necessarily reflect their true vectorial roles or geographical and ecological distributions.     

Structure and function of the spermathecal complex in the phlebotomine sandflyPhlebotomus papatasi Scopoli (Diptera: Psychodidae): II. Post-copulatory histophysiological changes during the gonotrophic cycle

The spermathecal complex ofPhlebotomus papatasi Scopoli (Diptera: Psychodidae) undergoes histological and physiological changes during its gonotropic cycle. The present histochemical study revealed a mucopolysaccharide secretory mass in the spermathecae of the newly emerged sandfly. Sperm competition occurs when two or more males compete to fertilize an ovum in the female reproductive tract. In this study, spermatophores of two or more competing males were deposited at the base of the spermathecal ducts, which originate from the female bursa copulatrix. This suggests that females play a role in sperm displacement, which is defined as any situation in which the last male to mate with a female fertilizes maximum number her eggs. A blood meal ingested by the female for ovary development and egg laying stimulates the release of sperm from the spermatophore. The spermatozoa then migrate to the lumen of the spermatheca. The ultrastructure of spermatozoa comprises a head with double-layered acrosomal perforatorium, an elongate nucleus, and the axoneme with a 9 + 9 + 0 flagellar pattern. This axomene differs from the aflagellate axoneme of other Psychodinae. Morphological changes, such as the casting off of the acrosomal membrane, and histological changes in the spermatophore are also described. Mating plugs that have been described previously in sandflies appear to be artefacts. Females ofP. papatasi may be inseminated more than once during each gonotrophic cycle, and additional inseminations may be necessary for each cycle. The relationships between the volumes of the sperm and the spermatheca were calculated to determine sperm utilization and fecundity ofP. papatasi. As the females ofP. papatasi mate polyandrously, the anatomical and physiological complexity of the spermathecal complex may be related to post-copulatory sexual selection.     

Salivary gland hyaluronidase in various species of phlebotomine sand flies (Diptera: psychodidae)

Hyaluronidase activity was detected and partially characterized in salivary gland extracts of females of six sand fly species. In Phlebotomus papatasi and Lutzomyia longipalpis the enzyme was active over a broad pH range; the pH optimum was 5.0. Besides high cleaving activity towards hyaluronic acid, it hydrolyzed chondroitin sulfates A and C. Hyaluronidases of various sand fly species differed in structure and sensitivity to reducing conditions. In the subgenera Phlebotomus (P. papatasi and P. duboscqi) and Adlerius (P. halepensis) the predominant active form of the enzyme was monomeric with the same apparent molecular weight under nonreducing and reducing conditions (around 65 kDa for P. papatasi and P. duboscqi and 110 kDa for P. halepensis). In P. sergenti the enzyme occurred as a putative homodimer but remained active under reducing conditions when separated into 60 kDa subunits. In L. longipalpis and P. perniciosus the activity was detectable under non-reducing conditions only. In P. duboscqi, low enzyme activity was found also in males. Salivary gland hyaluronidases of sand flies share characteristics with endo-N-acetyl-hexosaminidases of mammalian sperm cells and corresponding venom enzymes of Hymenoptera. Hypothetically, they facilitate blood meal acquisition but also may modulate immune reactions of the host and promote pathogen transmission.     

Cellulase Activity of Leishmania major in the Sandfly Vector and in Culture

ABSTRACT. The secretion of cellulose-degrading enzymes by Leishmania promastigotes in culture and in the sandfly vector was demonstrated. Two types of activity of cellulase enzyme-complexes were measured: endoglucanases, which randomly cleave cellulose chains and cellobioydrolases, which remove cellobiose from the nonreducing end of the molecule. The assays demonstrated that enzymes with these activities were secreted into the culture medium by Leishmania major, L. donovani , and L. braziliensis . These activities were also found in cultures of Sauroleishmania agamae, Leptomonas seymouri, Herpetomonas muscarum, Crithidia fasciculata and Trypanosoma brucei brucei that had a relatively low endoglucanase activity. Both endoglucanase and cellobiohydrolase activities were found in the gut of L. major-infected Phlebotomus papatasi , while gut preparations of uninfected sandflies had only cellobiohydrolase activity. The similar growth of L. major parasites in medium supplemented with either cellulose or glucose suggests these parasites can utilize cellulose.     

Effect of trapping method on species identification of phlebotomine sandflies by MALDI‐TOF MS protein profiling

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood‐feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI‐TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in‐house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI‐TOF MS protein profiling for species identification.     

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar,India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.