Sand Fly Phlebotomus Research Articles

Transfection of Leishmania tropica with green fluorescent protein (gfp) gene and investigation of the in vitro drug effect

Cutaneous leishmaniasis (CL) is a parasitic disease transmitted by vector sand flies Phlebotomus and Lutzomyia. This disease is characterized by long time non-healing skin lesions, and caused by Leishmania species. CL is the most common infection in Eastern and Southeastern Anatolia in Turkey and L.tropica is known as the main agent of the disease. Number of cases is increasing in our country in time because of malnutrition, migration, travel, low socioeconomic level and ecological changes. For the treatment, the pentavalent antimonials are often used as intralesionally for many years, and it was reported that resistant cases have increased in recent years. New treatment methods and anti-Leishmanial activity of new agents have been investigated because of side effects, resistance development and toxic reactions of the present drugs. These studies are first carried out in vitro and afterwards with in vivo experimental animal models. Reporter gene technology has been used to investigate a variety of purposes like biological events in microorganisms and the efficacy and resistance of drugs in recent years. The major areas that green fluorescent protein (gfp) used are that they can be incorporated into different genes to determine the amount of expression of these genes in different organisms and can be used as markers in living cells. Especially gfp gene, which encodes the green fluorescent protein, is widely used nowadays. Gene-based assays have several advantages like being easy to follow-up, inexpensive and have improved biosecurity. The aim of the present study was to perform the transfection of L.tropica with "enhanced gfp (egfp)" and in vitro usefulness of gfp-transfectants as a drug screening model in comparison to the conventional methods. Promastigotes of L.tropica were transfected with p6.5/egfp by electroporation and selected for tunicamycin-resistance as previously described. L.tropica promastigotes transfected with gfp and in vitro effect of meglumine animoniate was assessed using different methods such as fluorescence microscopy, fluorometer and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide) assay. The use of gfp-transfected Leishmania strains was found more rapid and more sensitive by fluorescent microscopy and fluorometry than conventional assays for the evaluation of potential anti-leishmanial agents. Consequently, stable gfp-transfected Leishmania species will be used in vitro and in vivo for screening of anti-leishmanial drugs and vaccine development as well as for understanding the biology of the host-parasite interactions at the cellular level. As a result ot this study, gfp transfected model using a Turkish L.tropica isolate was established to be used in further studies.

Laboratory transmission of an Asian strain of Leishmania tropica by the bite of the southern European sand fly Phlebotomus perniciosus

Imported cases of anthroponotic cutaneous leishmaniasis due to Leishmania tropica are increasingly documented in Europe. We investigated the ability of Phlebotomus perniciosus, a competent vector of Leishmania infantum widespread in southwestern Europe, to support the growth and transmissibility of an Asian strain of L. tropica recently isolated from a refugee. Parasite growth behavior was investigated in laboratory-reared sand flies fed artificially with promastigotes as well as in sand flies infected after biting on footpad lesions induced in hamsters by promastigote inoculation. The evolution of infection was checked by gut microscopy and quantitative real-time PCR, and it was found to be similar between promastigote- and amastigote-initiated infections. In 80% of infected sand flies, despite survival and flourishing growth of promastigotes after blood digestion and defecation, either the parasites died, or failed to migrate to the foregut and/or to mature into infective forms. However, in the remaining 20% L. tropica developed into abundant metacyclic promastigotes. The quantitative real-time PCR assay detected variable loads of gut promastigotes irrespective of morphological evidence of viability or progressive/final death. Parasite transmissibility was investigated by exposing naive hamsters to P. perniciosus previously infected on chronic lesions induced in hamsters which survived to take a second blood meal. Two months post exposure, lesions developed in skin sites bitten by sand flies confirmed to harbor metacyclic promastigotes; in the following months, the presence of viable and transmissible L. tropica parasites in lesions was demonstrated by xenodiagnosis assays. Our findings support the hypothesis that, in particular epidemiological situations, P. perniciosus may play the role of an occasional L. tropica vector.

Leishmaniosis in dogs and cats

Leishmania are vector-borne protozoan parasites. Infection with these parasites can result in a range of clinical diseases, dependent upon the infecting species. Leishmania infantum is the main species causing leishmaniosis in dogs and cats in Europe, as well as visceral and cutaneous forms of leishmaniosis in humans. Dogs are the main reservoir, but cats and other potential vertebrate reservoirs have been also reported. Sand flies (Phlebotomus spp.) are the main vectors, but non-vectorial transmission (e.g. venereal, transplacental) is possible. Lacking a gold-standard diagnostic test, diagnosis of leishmaniosis is achieved mainly based on clinical signs, skin histopathology, serological detection of specific immune responses against Leishmania spp., and molecular detection of the parasite DNA in tissues using polymerase chain reaction. Correct and early diagnosis is essential for timely institution of treatment and for minimising the transmission of Leishmania from infected animals to vectors. Meglumine antimoniate and allopurinol are the most widely used anti-leishmanial drugs, although miltefosine is emerging as a useful alternative to meglumine, especially in the renally-compromised patient. Vaccination to prevent disease is also available, but only for dogs. The advent of effective insecticide-impregnated collars and topical (‘spot-on’) formulations, and insights into the appropriate management of leishmaniosis, lends a hopeful outlook for the future. This article discusses biology, epidemiology, diagnosis, and management of leishmaniosis in dogs and cats, and explains the importance of connecting clinical and research communities in a ‘One Health’ approach for effective surveillance and control of this disease.

Morphometric discrimination between females of two isomorphic sand fly species, Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Phlebotominae) in endemic and non-endemic foci of zoonotic cutaneous leishmaniasis in Iran

Objective: To delineate reliable morphological characteristics for identifying and separating female Phlebotomus caucasicus and Phlebotomus mongolensis which exist sympatrically in the main foci of zoonotic cutaneous leishmaniasis in Iran. Methods: Sand flies were collected using sticky trap papers from active colonies of rodent burrows installed from 16 catching sites. Morphometric measurements were analyzed of 87 Phlebotomus caucasicus and 156 Phlebotomus mongolensis. Univariate and multivariate analysis were carried out to determine significant morphometric variables for discrimination of the two species. Finally, seven morphological characteristics of 65 female Phlebotomus caucasicus and 124 female Phlebotomus mongolensis were described. Results: Univariate and multivariate analyses of 10 morphometric variables via Discriminant Function Analysis (DFA) and Principal Component Analysis (PCA) showed that five morphometric variables had an accuracy of 100% for discriminating female Phlebotomus caucasicus and Phlebotomus mongolensis. Moreover, PCA revealed that the five morphometric variables with the highest loadings separated these two species. Morphological studies on antennal flagellum (and its associated structures) and mouth-parts of female specimens demonstrated significant differences in several structures. Conclusions: The results show that morphological and morphometrical features can be used to discriminate two female isomorphic species, Phlebotomus caucasicus and Phlebotomus mongolensis accurately.

Real-time PCR using high-resolution melting analysis technology for diagnosis of Leishmania and determination of types of clinical samples

Leishmaniasis is a disease group carried by Phlebotomus and Lutzomyia sand flies infected with Leishmania and is mostly observed in rural areas. In this study, using high-resolution melting analysis (HRMA), we aim to identify the active types of leishmaniasis, which are inadequately identified by classical methods. Samples of 85 patients were examined in the study. Six of the patients were suspected of having visceral leishmaniasis (VL) and 79 cutaneous leishmaniasis (CL). The slides prepared from the samples were stained with Giemsa stain and examined under a light microscope. The results were compared with those determined in real-time PCR. When the real-time PCR result was positive for Leishmania , we determined the type by HRMA. Among 85 Leishmania amastigote samples, 28 (32.9%) of them were detected and accepted as positive by microscopic examination. On the other hand, 25 (29.4%) of the 85 samples were found as positive using real-time PCR. In addition, when 25 Leishmania-positive samples were examined by HRMA, the results showed that 21 (84%) were L. tropica, 3 (12%) were L. major, and 1 (4%) was L. infantum. Based on our findings, we conclude that using real-time PCR and HRMA is useful in determining the cause of the disease in leishmaniasis.

Genomic characterisation of Cuiaba and Charleville viruses: arboviruses (family Rhabdoviridae, genus Sripuvirus) infecting reptiles and amphibians.

Viruses in the family Rhabdoviridae are ecologically very diverse, infecting mammals, birds, reptiles, fish, plants and a wide range of other terrestrial and aquatic invertebrates. The genus Sripuvirus currently comprises five viruses that appear to circulate in transmission cycles involving reptiles and sandflies. Here, we report an analysis of the complete coding sequences of Cuiaba virus (CUIV), isolated from an amphibian (Bufo marinus) collected in Brazil, and Charleville virus (CHVV), isolated from sandflies (Phlebotomus sp.) and lizards (Gehyra australis), collected in Australia. CUIV and CHVV cluster phylogenetically with the sripuviruses in maximum likelihood trees generated from complete L protein (RdRp) sequences. They also share with sripuviruses unique features in genome organisation, including an additional gene (U1) between the matrix protein (M) gene and glycoprotein (G) gene, and an alternative long open reading frame near the start of the G ORF that encodes a predicted transmembrane protein. In view of their phylogenetic relationships, similar genome organisations and similar ecological characteristics, we propose the assignment of CUIV and CHVV as novel members of the genus Sripuvirus.

Broadening the tools for studying sand fly breeding habitats: A novel molecular approach for the detection of phlebotomine larval DNA in soil substrates

Phlebotomine sand flies constitute a group of haematophagous insects of great medical and veterinary importance. Despite the significant knowledge on the biology and behavior of adult sand flies in the wild, there is little information available on the ecology of their larval stages due to difficulties of detecting them in the soil. So far, investigations on sand fly breeding sites have been based on methods to catch emerging adults or on microscopic examination of the soil for the presence of immatures, neither of which is very precise or efficient for studying immatures in the field. Thus, there is a need for a better method to detect, identify and quantify immatures in soil samples. To address this issue we developed a novel molecular genetic approach for the detection of sand fly larval DNA within soil samples. Different numbers of live larvae of Phlebotomus papatasi and P. tobbi (alone or in combination), were mixed with soil and analyzed to identify and quantify the sand flies. By modifying already existing soil DNA extraction protocols in combination with a real-time PCR using species-specific primers, we successfully detected as little as one larva/40 ml of soil. We could also distinguish the two species of sand flies. This method should be very useful for increasing our knowledge of sand fly larval ecology in the field, and thus help develop more efficient, targeted control strategies against Phlebotomus sand flies.

Remotely identifying potential vector habitat in areas of refugee and displaced person populations due to the Syrian civil war.

Historically leishmaniasis is most prevalent in established urban centres but this research shows that refugees and, most significantly, internally displaced persons are now commonly in areas characterized by the presence of fly habitats potentially leading to higher prominence of Leishmania infection. Areas engulfed by the Syrian civil war has thus caused the dispersal of humans into previously unpopulated areas amid habitats of the sand fly Phlebotomus papatasi that hosts the parasite Leishmania. The addition of new places of exposure to this disease add to difficulties with respect to diagnosis as well as provision of care and treatment. We used geospatial methodology adapting it to remotely identifying and analyzing sand fly habitats with the aim of measuring how common it is. Our methodology helps avoid the issue of resolution in satellite imagery by measuring likelihood rather than strictly known locations. We followed up this information with spatial analysis identifying which civilian populations are most prone to sand fly exposure, and therefore leishmaniasis, due to their geographical situation. Our results suggest that those most likely to be exposed to Leishmania are internally displaced persons, those camps less likely to receive medical relief and typically having temporary residents migrating elsewhere.

Laboratory assessment of the anti-feeding effect for up to 12 months of a slow release deltamethrin collar (Scalibor\xae) against the sand fly Phlebotomus perniciosus in dogs

BackgroundLeishmaniosis/leishmaniasis consists of a wide group of diseases, caused by different Leishmania species and having different hosts. Leishmaniosis caused by Leishmania infantum, a disease primarily of dogs and humans, occurs after susceptible hosts are exposed to the feeding behavior of infected sand flies. A one-year laboratory study in dogs was designed to determine the 364-day anti-feeding efficacy of a slow release deltamethrin collar against the sand fly P. perniciosus, a common host of L. infantum in the Mediterranean basin.MethodsIn this assessor-blinded study, 16 Beagle dogs were randomized into two groups using P. perniciosus engorgement rates from a Day -7 challenge. On Day 0, dogs in Group 1 received a placebo collar, while dogs in Group 2 received a deltamethrin collar (Scalibor® Protector Band). All dogs were caged, sedated and then exposed for 1 h to 85 (± 10) female and 15 (± 5) male P. perniciosus on Day 7 and every 28 days through Day 364. All flies, alive and dead, were aspirated from cages and from dogs, immediately counted and then frozen for assessment of blood engorgement. Anti-feeding efficacy was determined by comparing the arithmetic means of engorged female flies (alive, dead and moribund) in the deltamethrin group to the control group means. Insecticidal efficacy at the time flies were retrieved was assessed by comparisons between groups of mean live female fly counts.ResultsIn the deltamethrin group, relative to the control group, there was a significant reduction in arithmetic mean numbers of engorged P. perniciosus of 94–98% from Day 7 through Day 364. On Day 28, in the treated group relative to the control group, there was a 74% reduction in mean live fly counts, with between-group differences significant from Days 7 through 196, although insecticidal activity remained less than 50% from Day 56.ConclusionDeltamethrin collar application to dogs reduced sand fly feeding by ≥ 94%, relative to unprotected control dogs, for 364 days. Thus, one collar applied to a dog can prevent or reduce the risk of sand fly transmission of Leishmania for one full year.

Machine learning approaches in GIS-based ecological modeling of the sand fly Phlebotomus papatasi, a vector of zoonotic cutaneous leishmaniasis in Golestan province, Iran

The distribution and abundance of Phlebotomus papatasi, the primary vector of zoonotic cutaneous leishmaniasis in most semi-/arid countries, is a major public health challenge. This study compares several approaches to model the spatial distribution of the species in an endemic region of the disease in Golestan province, northeast of Iran. The intent is to assist decision makers for targeted interventions. We developed a geo-database of the collected Phlebotominae sand flies from different parts of the study region. Sticky paper traps coated with castor oil were used to collect sand flies. In 44 out of 142 sampling sites, Ph. papatasi was present. We also gathered and prepared data on related environmental factors including topography, weather variables, distance to main rivers and remotely sensed data such as normalized difference vegetation cover and land surface temperature (LST) in a GIS framework. Applicability of three classifiers: (vanilla) logistic regression, random forest and support vector machine (SVM) were compared for predicting presence/absence of the vector. Predictive performances were compared using an independent dataset to generate area under the ROC curve (AUC) and Kappa statistics. All three models successfully predicted the presence/absence of the vector, however, the SVM classifier (Accuracy = 0.906, AUC = 0.974, Kappa = 0.876) outperformed the other classifiers on predicting accuracy. Moreover, this classifier was the most sensitive (85%), and the most specific (93%) model. Sensitivity analysis of the most accurate model (i.e. SVM) revealed that slope, nighttime LST in October and mean temperature of the wettest quarter were among the most important predictors. The findings suggest that machine learning techniques, especially the SVM classifier, when coupled with GIS and remote sensing data can be a useful and cost-effective way for identifying habitat suitability of the species.

Leishmaniosis in dogs and cats

Leishmania are vector-borne protozoan parasites. Infection with these parasites can result in a range of clinical diseases, dependent upon the infecting species. Leishmania infantum is the main species causing leishmaniosis in dogs and cats in Europe, as well as visceral and cutaneous forms of leishmaniosis in humans. Dogs are the main reservoir, but cats and other potential vertebrate reservoirs have been also reported. Sand flies (Phlebotomus spp.) are the main vectors, but non-vectorial transmission (e.g. venereal, transplacental) is possible. Lacking a gold-standard diagnostic test, diagnosis of leishmaniosis is achieved mainly based on clinical signs, skin histopathology, serological detection of specific immune responses against Leishmania spp., and molecular detection of the parasite DNA in tissues using polymerase chain reaction. Correct and early diagnosis is essential for timely institution of treatment and for minimising the transmission of Leishmania from infected animals to vectors. Meglumine antimoniate and allopurinol are the most widely used anti-leishmanial drugs, although miltefosine is emerging as a useful alternative to meglumine, especially in the renally-compromised patient. Vaccination to prevent disease is also available, but only for dogs. The advent of effective insecticide-impregnated collars and topical (‘spot-on’) formulations, and insights into the appropriate management of leishmaniosis, lends a hopeful outlook for the future. This article discusses biology, epidemiology, diagnosis, and management of leishmaniosis in dogs and cats, and explains the importance of connecting clinical and research communities in a ‘One Health’ approach for effective surveillance and control of this disease.

Do the rodents have a role in transmission of cutaneous leishmaniasis in Turkey?

Leishmaniasis is a zoonotic/anthroponotic vector borne parasitic infection which is caused by Leishmania species and transmitted by sand flies (Phlebotomus spp.) The reservoirs of Leishmania species in nature are various wild and domestic carnivores, rodents and human. The aim of this study was to investigate whether the rodents in genera Meriones, Mesocricetus, Rattus and Mus which inhabit in the natural habitat of our country could be natural reservoirs of Leishmania tropica, Leishmania infantum, Leishmania major and Leishmania donovani for cutaneous Leishmaniasis (CL)., The rodents Mus musculus (Balb/C mouse), Mesocricetus auratus (hamster), Meriones unguiculatus (gerbil) and Rattus norvegicus (rat) which are part of the natural habitat in Turkey were used in the study. L.tropica, L.infantum, L.major and L.donovani promastigote isolates obtained from CL patients and cultured in enriched media were injected in the footpads of the animals intradermally using the density of 108 promastigote/ml. The scale of the lesions on the footpads of the animals were measured for 12 weeks. At the end of the experiment, the animals were sacrificed and "touch preparations" were prepared using footpad, liver, spleen and testicles of the sacrified animals and were examined using Giemsa stained slides following culturing in enriched NNN medium. Leishmania amastigotes were seen in the slides prepared from the footpads of the all experimental animals and all cultures were positive for promastigotes prepared from the same clinical material. But not all the experiment groups were positive for the liver, spleen and testicle preparations. According to these results it was concluded that while all rodents in the experiment groups were positive for CL, only a part of the experiment groups were positive for internal organ involvement. Accordingly, (a) All Leishmania strains caused both CL and internal organ involvement in M.unguiculatus and M.musculus, (b) only L.tropica caused CL and internal organ involvement in R.norvegicus, while other Leishmania strains only caused CL in this group, (c) in M.auratus only L.donovani caused CL while other strains caused both CL and internal organ involvement. In our study, it was determined that the rodents Meriones, Mesocricetus, Rattus and Mus genera which are part of our country's natural habitat could serve as natural reservoirs of L.tropica, L.infantum, L.major and L.donovani, thus having the potential for the spreading of Leishmaniasis in our country and important information were gathered concerning the clinical aspects of the infection caused by Leishmania species in their potential reservoir hosts.

Exploring new immunological insight on SP15 (∼14 kDa) family protein in saliva of Indian sand-fly (Phlebotomus argentipes) in experimental visceral leishmaniasis

Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6 kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6 kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.

Isolation of a Trypanosome Related to Trypanosoma theileri (Kinetoplastea: Trypanosomatidae) from Phlebotomus perfiliewi (Diptera: Psychodidae).

The Trypanosoma theileri group includes several trypanosome species hardly distinguishable due to the lack of discriminating morphological characters. Trypanosomes belonging to this group have been isolated from different bovine, ovine, and cervids in Europe, Africa, Asia, and Americas. The principal vectors of the T. theileri group are considered tabanid flies; however, T. melophagium is transmitted exclusively by sheep keds. In 2016, 128 sand flies out of 2,728 trapped in Valsamoggia municipality, Italy, were individually dissected and an unknown trypanosome strain, named TrPhp1, was isolated from a female of the sand fly Phlebotomus perfiliewi. Sequence analysis placed this trypanosome in the T. theileri group with very high homology to other trypanosomes detected in European cervids. This is the first report of the T. theileri group isolation from a sand fly, and the possible role of this insect group in the trypanosome transmission cycle is discussed. Within the T. theileri group, the phylogenetic analysis distinguished several lineages, which, unfortunately, do not correspond with their host specificity and their taxonomic status remains ambiguous.

Multilocus microsatellite typing (MLMT) reveals host-related population structure in Leishmania infantum from northeastern Italy.

BackgroundVisceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic.Methodology/Principal findingsMultilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs.Conclusions/SignificanceA peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities.

Repurposing isoxazoline veterinary drugs for control of vector-borne human diseases

Isoxazolines are oral insecticidal drugs currently licensed for ectoparasite control in companion animals. Here we propose their use in humans for the reduction of vector-borne disease incidence. Fluralaner and afoxolaner rapidly killed Anopheles, Aedes, and Culex mosquitoes and Phlebotomus sand flies after feeding on a drug-supplemented blood meal, with IC50 values ranging from 33 to 575 nM, and were fully active against strains with preexisting resistance to common insecticides. Based on allometric scaling of preclinical pharmacokinetics data, we predict that a single human median dose of 260 mg (IQR, 177-407 mg) for afoxolaner, or 410 mg (IQR, 278-648 mg) for fluralaner, could provide an insecticidal effect lasting 50-90 days against mosquitoes and Phlebotomus sand flies. Computational modeling showed that seasonal mass drug administration of such a single dose to a fraction of a regional population would dramatically reduce clinical cases of Zika and malaria in endemic settings. Isoxazolines therefore represent a promising new component of drug-based vector control.

Susceptibility of field-collected Phlebotomus argentipes (Diptera: Psychodidae) sand flies from Bangladesh and Nepal to different insecticides

BackgroundThe sand fly Phlebotomus argentipes is the vector for visceral leishmaniasis (VL) in the Indian sub-continent. In Bangladesh since 2012, indoor residual spraying (IRS) was applied in VL endemic areas using deltamethrin. In Nepal, IRS was initiated in 1992 for VL vector control using lambda-cyhalothrin. Irrational use of insecticides may lead to vector resistance but very little information on this subject is available in both countries. The objective of this study was to generate information on the susceptibility of the vector sand fly, P. argentipes to insecticide, in support of the VL elimination initiative on the Indian sub-continent.MethodsSusceptibility tests were performed using WHO test kits following the standard procedures regarding alpha cypermethrin (0.05%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%), permethrin (0.75%), malathion (5%) and bendiocarb (0.1%) in six upazilas (sub-districts) in Bangladesh. In Nepal, the tests were performed for two insecticides: alpha cypermethrin (0.05%) and deltamethrin (0.05%). Adult P. argentipes sand flies were collected in Bangladesh from six VL endemic upazilas (sub-districts) and in Nepal from three endemic districts using manual aspirators.ResultsThe results show that VL vectors were highly susceptible to all insecticides at 60 minutes of exposure in both countries. In Bangladesh, corrected mortality was 100% at 15 minutes as well as 30 minutes of exposure. The study sites in Nepal, however, showed some diverse results, with a mortality rate less than 90% for 15 minutes of exposure with alpha cypermethrin and deltamethrin in two districts but was above 95% after 30 minutes of exposure.ConclusionsThese results suggest that the insecticides tested can still be used in the national programmes of Bangladesh and Nepal. However, insecticide rotation should be performed to mitigate the possible development of insecticide resistance. Periodic susceptibility tests should be performed by the countries to get timely alerts regarding insecticide resistance.

Study of the efficacy of Lambdacyhalothrin 10% wp and the Fruits extracts of Datura innoxia plant in the fourth larval instar and adults of sand fly Phlebotomus papatasi (Diptera: Psychodidae)

The results of the present study, which aimed to demonstrate the efficacy of Lambdacyhalothrin 10% and alcoholic extracts of Datura innoxia fruits in the control of fourth larval instar and adults of the phlebotomus papatasi , indicated that the highest mortality percentage of the larval instar was 100% in concentration 3 μL / ml, and the lowest mortality percentage of 33.3% at the lowest concentration of 0.05 μL / ml. The results of adult treatment were the highest mortality percentage of 93.33% at the concentration of 3 μL / ml and the lowest mortality percentage were 20% at concentration of 0.05 μL / ml after 24 Hour of treatment.
 The results of the treatment of the fourth larval instar with the Datura innoxia fruits extracts showed that the highest mortality percentage was 100% for the concentration of 2000 ppm and the lowest of 13.3% at 75 ppm. The highest mortality percentage of adults insects was 100% when treated with the most effective concentrations (1600, 2000ppm). Thus, the results are shown to be affected by the control method used and the interaction of the insect larval instar to this method. In the end, there was a convergence of the effect of the insecticide and extract in the larval instar and adults insect.

The mycobiota of the sand fly Phlebotomus perniciosus: Involvement of yeast symbionts in uric acid metabolism.

The knowledge of the fungal mycobiota of arthropods, including the vectors of human and animal diseases, is still limited. Here, the mycobiota associated with the sand fly Phlebotomus perniciosus, the main vector of leishmaniasis in the western Mediterranean area, by a culture-dependent approach (microbiological analyses and sequencing of the 26S rRNA gene), internal transcribed spacer (ITS) rRNA amplicon-based next-generation sequencing, fluorescence in situ hybridisation (FISH), and genome sequencing of the dominant yeast species was investigated. The dominant species was Meyerozyma guilliermondii, known for its biotechnological applications. The focus was on this yeast and its prevalence in adults, pupae and larvae of reared sand flies (overall prevalence: 57.5%) and of field-collected individuals (overall prevalence: 9%) was investigated. Using whole-mount FISH and microscopic examination, it was further showed that M. guilliermondii colonizes the midgut of females, males and larvae and the distal part of Malpighian tubules of female sand flies, suggesting a possible role in urate degradation. Finally, the sequencing and analysis of the genome of M. guilliermondii allowed predicting the complete uric acid degradation pathway, suggesting that the yeast could contribute to the removal of the excess of nitrogenous wastes after the blood meal of the insect host.

Identification of wild-caught phlebotomine sand flies from Crete and Cyprus using DNA barcoding

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are vectors of Leishmania spp., protozoan parasites responsible for a group of neglected diseases called leishmaniases. Two sand fly genera, Phlebotomus and Sergentomyia, contain species that are present in the Mediterranean islands of Crete and Cyprus where the visceral (VL), cutaneous (CL) and canine (CanLei) leishmaniases are a public health concern. The risk of transmission of different Leishmania species can be studied in an area by monitoring their vectors. Sand fly species are traditionally identified using morphological characteristics but minute differences between individuals or populations could be overlooked leading to wrong epidemiological predictions. Molecular identification of these important vectors has become, therefore, an essential tool for research tasks concerning their geographical distribution which directly relates to leishmaniasis control efforts. DNA barcoding is a widely used molecular identification method for cataloguing animal species by sequencing a fragment of the mitochondrial gene encoding cytochrome oxidase I.ResultsDNA barcoding was used to identify individuals of five sand fly species (Phlebotomus papatasi, P. similis, P. killicki, Sergentomyia minuta, S. dentata) circulating in the islands of Crete and Cyprus during the years 2011–2014. Phlebotomus papatasi is a known vector of zoonotic CL in the Middle East and it is found in both islands. Phlebotomus similis is the suspected vector of Leishmania tropica in Greece causing anthroponotic CL. Phlebotomus killicki was collected in Cyprus for the first time. Sergentomyia minuta, found to present intraspecific diversity, is discussed for its potential as a Leishmania vector. Molecular identification was consistent with the morphological identification. It successfully identified males and females, which is difficult when using only morphological characters. A phylogenetic tree was constructed based on the barcodes acquired, representing their genetic relationships along with other species from the area studied. All individuals identified were clustered according to their species and subgenus.ConclusionsMolecular identification of sand flies via DNA barcoding can accurately identify these medically important insects assisting traditional morphological tools, thus helping to assess their implication in Leishmania transmission.