High climatic ancestral affinity between the lineages of the Leishmania vector Psathyromyia shannoni sensu stricto (Diptera: Phlebotominae).

Psathyromyia (Psathyromyia) shannoni sensu stricto (Dyar) is a vector of Leishmania parasite and the second sandfly of medical importance with a wide geographical but discontinuous distribution in America. Preliminary genetic structure analysis using a mitochondrial marker shows that the species integrated by at least four lineages could be the result of ecological adaptations to different environmental scenarios, but this hypothesis had never been proven. The aim of the present study was to analyse whether the genetic structure that detected Pa. shannoni ss. is associated with divergence or conservatism niche. Using Ecological Niche Models (ENMs) theory, we estimated the potential distribution for each genetic lineage, and then, we evaluated the equivalency niche for assessing whether climatic niche was more different than expected. The ENMs identify different suitable distribution areas but the same climatic or ecological conditions for the genetic lineages of Pa. shannoni (conservatism niche). Our findings allow us to speculate that other potential processes or events could be related to the genetic differentiation of Pa. shannoni. These studies are important because they allow us to identify the factors that could restrict the potential distribution of the different lineages whose vectorial competence is still unknown.

Molecular investigation of Leishmania in sandflies (Diptera: Psychodidae) and rodents (Mammalia: Rodentia) in Nahavand, west of Iran.

Cutaneous leishmaniasis caused by different species of Leishmania is transmitted by Phlebotominae sandflies. This disease remains a public health concern in Iran. Therefore, the present study aimed to examine Leishmania infection in sandflies and reservoir rodents in six rural regions of Nahavand, located in western Iran. From May to October 2022, sandflies and rodents were collected and identified at the species level. Additionally, rodents' skin lesions and earlobe specimens were collected separately for microscopic and molecular examination. All specimens were tested for Leishmania DNA by PCRs targeting the parasite's ITS-2 and 18S rRNA gene and positive were Sanger sequenced. A total of 3396 sandflies belonging to seven subgenera and 11 species, i.e., Phlebotomus papatasi (42.7%), P. major (20.6%), P. mascitti (0.3%), P. neglectus (0.2%), P. alexandri (0.2%), P. turanicus (0.3%), Sergentomyia murgabiensis (18.1%), S. dentata (10.5%), S. theodori (5.8%), S. antennata (1.1%), and S. pawlowski (0.1%) were identified. Based on the species population, 29 pools of sandflies were examined for the presence of Leishmania DNA using conventional PCR (cPCR), and individual DNAs were tested when positive. Leishmania major DNA was detected in two P. papatasi and Leishmania sp. in one P. major individual sandfly. This is the first report of Leishmania infection in sandflies from Hamadan province. The captured rodents (n = 61) belonged to four families and seven species, i.e., Arvicola amphibius (37.7%), Mus musculus (29.5%), Microtus socialis (13.1%), Apodemus sylvaticus (11.5%), Talpa davidiana (4.9%), Apodemus witherbyi (1.6%), and Rattus norvegicus (1.6%). Microscopic and molecular examinations of the rodent lesions and earlobes scored negative results. The presence of Leishmania in the Phlebotominae sandflies in Nahavand indicates a potential threat to humans and animals in the region. Regular monitoring and examination of the sandflies' population and timely diagnosis and treatment of new patients are strongly recommended.

Identification of cuticle and midgut fungal microflora of phlebotomine sandflies collected in Tunisia.

Phlebotomine sand flies (Diptera: Psychodidae) are the proven vectors of Leishmaniases which are widespread parasitosis in many tropical and subtropical countries. The development of infective metacyclic Leishmania (Kinetoplastida: Trypanosomatidae) promastigotes stage is restricted to the vector midgut. Recently, several studies have assessed the influence of the sand fly midgut fungal microflora on the development of invective Leishmania stage. The aim of this study was to identify the fungal microflora from the cuticle and midgut of wild caught sandflies. A total of 50 sandflies were caught in two different leishmaniasis foci of center Tunisia and analyzed using an in vitro isolation of fungi followed by a morphological and molecular identification of fungal isolates. The morphological identification of sandflies specimens revealed five Species: Phlebotomus (P.) papatasi (n = 25), P. perniciosus (n = 15) P. riouxi (n = 6), P. longicuspis (n = 3) and P. sergenti (n = 1). Forty positive fungal cultures were isolated from 34 sand flies (19 males and 15 females) distributed as following: P. papatasi (n = 16), P. perniciosus (n = 11), P. riouxi (n = 4), P. longicuspis (n = 2) and P. sergenti (n = 1). Thirty-five cultures were isolated from the cuticles and five from the guts. A total of 15 fungi genera belonging to 8 families were identified with the predominance of Aspergillus genus followed by Penicillium genus. Among the 15 fungi genera, five were common between males and females specimens. Lecytophora canina and Leishmania major co-infection was detected in the gut of a female P. papatasi. Our preliminary findings highlight the high diversity of fungal microflora from the sand flies midguts.

Robust comparative performance of genomic DNA extraction methods from non-engorged phlebotomine sandflies.

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen® kit (Qiagen®, Hilden, Alemanha), salting out, HotShot and phenol-chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol-chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen® (73.3% [44/60]). The phenol-chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector-pathogen interactions.

[Tegumentary leishmaniasis and sandflies in Colonia Santa Rosa locality in northern Argentina]. / Leishmaniasis tegumentaria y flebótomos en la localidad de Colonia Santa Rosa del norte de Argentina.

Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania and transmitted by the sandfly species, insects belonging to the order Diptera, family Psychodidae. Historically, the most endemic area of TL in Argentina has been the northern region. The aim of this work was to analyze the presence and temporal variation of TL cases reported between 1985 and 2019 in Colonia Santa Rosa locality, northern Argentina. Furthermore, its clinical forms were characterized and sandflies were captured. Patients were diagnosed by smear and the Montenegro skin test. For sampling, CDC light traps were placed at 14 sites from 7pm to 7am. The correlation between vegetation cover and sandfly abundance was also studied. One hundred and twenty TL cases were diagnosed and the overall prevalence was 0.75% (≈16 000 inhabitants). Patients presented simple and multiple cutaneous leishmaniasis (88.79%) and the mucocutaneous form (10.83%). Skin lesions were more frequent on the lower extremities (46.73%). Of the total number of sandflies, Nyssomyia neivai (95%) was the predominant species followed by Migonemyia migonei (1.9%), cortelezzii complex (1.3%) and Evandromyia sallesi (0.09%). The persistent occurrence of cases and the presence of sandflies in the locality suggest the existence of endemic transmission in the area. This highlights the need to design prevention and control measures for TL in northern Argentina.

Insecticide susceptibility of Phlebotomus argentipes sandflies, vectors of visceral leishmaniasis in India.

OBJECTIVES: Indoor residual spraying (IRS) with insecticides is the main vector control intervention for the elimination of visceral leishmaniasis in India. After a change in IRS policy in 2015 due to widespread resistance of Phlebotomus argentipes to DDT, IRS with DDT was replaced with alpha-cypermethrin IRS in 2016. The objective of the present study was to evaluate the susceptibility of P. argentipes to DDT and its alternatives, namely malathion and pirimiphos-methyl (organophosphates); alpha-cypermethrin, deltamethrin, lambda-cyhalothrin and permethrin (pyrethroids), and bendiocarb and propoxur (carbamates), in support of visceral leishmaniasis elimination in India. METHODS: Phlebotomus argentipes sandflies were collected from the visceral-leishmaniasis endemic states of Bihar, Jharkhand and West Bengal. In the WHO tube tests, the phenotypic susceptibility of F1, 2-day old, non-blood fed females were determined against filter papers impregnated with DDT 4%, malathion 5%, pirimiphos-methyl 0.25%, alpha-cypermethrin 0.05%, deltamethrin 0.05%, lambda-cyhalothrin 0.05%, permethrin 0.75%, bendiocarb 0.1% and propoxur 0.1%, which were sourced from Universiti Sains Malaysia. The knockdown of sandflies after 1-h exposure and mortality at 24 h after the 1-h exposure period were scored. RESULTS: Mean mortality of P. argentipes 24 h after exposure in tube tests was 22.6% for DDT and ≥ 98% for other insecticide-impregnated papers tested. CONCLUSION: Phlebotomus argentipes continues to be highly resistant to DDT with no reversal of resistance after DDT's withdrawal from IRS. P. argentipes was fully susceptible to pyrethroid, organophosphate and carbamate insecticides tested. Regular monitoring is warranted for insecticide resistance management in sandfly vectors.

The potential future change of the suitability patterns of six leishmaniasis vectors in Iran.

BACKGROUND & OBJECTIVES: Visceral and cutaneous leishmaniasis are endemic in Iran. The aim of this study was to model the changing suitability patterns of five confirmed and one suspected leishmaniasis vector Phlebotomus species resident in the country. METHODS: The potential present and future suitability patterns of the sandfly species in Iran were modelled using climate envelope forecasting method for the reference period 1970-2000 and the future period 2041-2060. RESULTS: The reference period climate of Iran seemed to be the most suitable for Phlebotomus perfiliewi and Phlebotomus tobbi and less suitable for Phlebotomus simili, while Phlebotomus neglectus, Phlebotomus papatasi and Phlebotomus sergenti showed intermediate values among the studied sandfly species. The modelled changes in the suitability values show a similar pattern in the case of the six species, even the exact magnitude of the modelled values varied. The model results indicate that climate change could decrease the sandfly habitability in the present-day arid regions in Central Iran. The Iranian sandfly populations will move to higher elevation regions, and the suitability values of the sandfly species are predicted to increase in the foothills of the mountainous regions in the northern and the western part of the country. INTERPRETATION & CONCLUSION: The increase of the maximally suitable areas in Iran was found which was predicted to be accompanied by the parallel shrinkage of the sandfly-inhabited areas in the arid regions of the country. Topographical conditions could strongly influence the suitability patterns of the vectors in Iran.

Two new phlebotomine sandflies (Diptera: Psychodidae) from the forest edge in Madagascar: the anthropophilic Phlebotomus artemievi sp. nov. and Sergentomyia maroantsetra ensis sp. nov.

A few data are related to the anthropophily of Malagasy Phlebotomine sandflies. Prior studies focussed mainly to inventories and description of new species. Our goal was to emphasize the anthropophily of Malagasy Phlebotomine sandflies. We worked in the Makira region, using two simultaneous methods: human landing catches (HLC) and CDC light traps. We collected sandflies in three rural communities adjacent to the Makira Natural Park. In each community, three different biotopes were sampled: within community settlements; at the edge of forest, typically in agricultural land; and within the forest. We collected 61 sandflies belonging to two new species presently described: Phlebotomus artemievi sp. nov. and Sergentomyia maroantsetraensis sp. nov. These sandflies were caught exclusively in the forest edge biotope. None were captured within communities or within forests. HLC provided 97% of the collected sandflies, corresponding to a human-biting rate of 15 females per human per night. CDC provided only two females. Ph. artemievi sp. nov. was predominantly captured by HLC and appears to be highly anthropophilic. Here, we update the behavioural ecology of sandflies and describe two new species. Further research is required to understand their vector competence and their ability to transmit arboviruses and other pathogens such as Leishmania.

Phlebotominae in peri-domestic and forest environments inhabited by Alouatta caraya in northeastern Argentina.

Multiple species of Phlebotominae are vectors of Leishmania (Protozoa: Trypanosomatidae), which causes visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). To describe the Phlebotominae (Diptera: Psychodidae) related to the environments of black and gold howler monkeys Alouatta caraya (Humbodlt, 1812) (Primates: Atelidae), potential vectors were sampled in different landscapes and vertical strata of sleeping trees. Phlebotomine captured between December 2011 and March 2012 (2365 individuals) belonged to eight species, of which Nyssomyia neivai (Pinto, 1926) (61.4%) and Migonemyia migonei (França, 1920) (18.73%) were the most abundant, and Ny. withmani was recorded for the first time in the Chaco province. In the 'peri-domestic' landscape, the phlebotomine were mainly captured in henhouses (78.7%), whereas the tree canopy in 'rural' and 'wild' landscapes yielded 31.2% and 29.1% of the phlebotomine, respectively. A significant association between the type of landscape and the species of phlebotomine was observed by multivariate analysis. Lutzomyia longipalpis (Lutz & Neiva, 1912) and Mg. migonei were associated with 'peri-domestic' landscape, and Ny. neivai was associated with the 'wild' landscape. The results of this prospective study suggest that the interaction between phlebotomine and A. caraya could be a key factor with respect to understanding the epidemiology of leishmaniasis.

Inferring distributional shifts of epidemiologically important North and Central American sandflies from Pleistocene to future scenarios.

Nine sandfly species (Diptera: Psychodidae) are suspected or proven vectors of Leishmania spp. in the North and Central America region. The ecological niches for these nine species were modelled in three time periods and the overlaps for all time periods of the geographic predictions (G space), and of ecological dimensions using pairwise comparisons of equivalent niches (E space), were calculated. Two Nearctic, six Neotropical and one species in both bioregions occupied a reduced number of distribution areas. The ecological niche projections for most sandfly species other than Lutzomyia shannoni and Lutzomyia ovallesi have not expanded significantly since the Pleistocene. Only three species increase significantly to 2050, whereas all others remain stable. Lutzomyia longipalpis shared a similar ecological niche with more species than any other, although both L. longipalpis and Lutzomyia olmeca olmeca had conserved distributions over time. Climate change, at both regional and local levels, will play a significant role in the temporal and spatial distributions of sandfly species.

In Vitro and In Vivo Effectiveness of Carvacrol, Thymol and Linalool against Leishmania infantum.

BACKGROUND: One of the most important causative agents of visceral leishmaniasis (VL) is Leishmania infantum, which is mainly spread by Phlebotomus and Lutzomyia sandflies in the Old and New World, respectively. Novel and effective drugs to manage this neglected vector-borne disease are urgently required. In this study, we evaluated the toxicity of carvacrol, thymol and linalool, three common essential oil constituents, on amastigotes and promastigotes of L. infantum. Methods: in vitro experiments were performed by 24 h MTT assay. Carvacrol, thymol and linalool at concentrations ranging from 1.3 to 10 µg/mL were tested on promastigotes of L. infantum. For in vivo test, two groups of hamsters (Mesocricetus auratus) received 100 mg/kg of body weight/day of carvacrol and thymol as intraperitoneal injection on day 7 post-infection, followed by a 48 h later injection. The third group was treated with the glucantime as standard drug (500 mg/kg) and the last group (control) just received normal saline. On the 16th day, the number of parasites and histopathological changes in liver and spleen were investigated. RESULTS: 24 h MTT assay showed promising antileishmanial activity of thymol and carvacrol, with IC50 values of 7.2 (48 µM) and 9.8 µg/mL (65 µM), respectively. Linalool at all concentrations did not affect L. infantum promastigote viability. In vivo toxicity data of carvacrol and thymol showed that the former at 100 mg/kg was the safest and most effective treatment with little side effects on the liver. CONCLUSIONS: Overall, thymol and carvacrol are highly promising candidates for the development of effective and safe drugs in the fight against VL.

Visceral Leishmaniasis in Traveler to Guyana Caused by Leishmania siamensis, London, UK.

The parasite Leishmania siamensis is a zoonotic agent of leishmaniasis; infection in animals has been documented in Europe and the United States. Reported authochthonous human infections have been limited to Thailand. We report a case of human visceral Leishmania siamensis infection acquired in Guyana, suggesting colonization in South America.

Comments on "A Mathematical Study to Control Visceral Leishmaniasis: An Application to South Sudan".

Deterministic (ordinary differential equation) models for the transmission dynamics of vector-borne diseases that incorporate disease-induced death in the host(s) population(s) are generally known to exhibit the phenomenon of backward bifurcation (where a stable disease-free equilibrium of the model coexists with a stable endemic equilibrium when the associated reproduction number of the model is less than unity). Further, it is well known that, in these models, the phenomenon of backward bifurcation does not occur when the disease-induced death rate is negligible (e.g., if the disease-induced death rate is set to zero). In a recent paper on the transmission dynamics of visceral leishmaniasis (a disease vectored by sandflies), titled "A Mathematical Study to Control Visceral Leishmaniasis: An Application to South Sudan," published in Bulletin of Mathematical Biology, Vol. 79, Pages 1110-1134, 2017, Ghosh et al. (2017) stated that their deterministic model undergoes a backward bifurcation even when the disease-induced mortality in the host population is set to zero. This result is contrary to the well-established theory on the dynamics of vector-borne diseases. In this short note, we illustrate some of the key errors in the Ghosh et al. (2017) study.

Environmental risk modelling and potential sand fly vectors of cutaneous leishmaniasis in Chitral district: a leishmanial focal point of mount Tirich Mir, Pakistan.

OBJECTIVE: To provide baseline information about suspected vectors and the incidence, distribution and an active zone of transmission for cutaneous leishmaniasis (CL) in Chitral, Pakistan, using GIS tools; and to investigate the role of environmental factors in the disease dynamics. METHOD: Two surveys in 2014 and 2016 as a basis for choropleth and environmental risk mapping. RESULTS: A total of 769 captured specimens yielded 14 Phlebotomus and six Sergentomyia species including two potential vectors of CL, i.e. Phlebotomus papatasi and Phlebotomus sergenti. P. papatasi (71%) was dominant, followed by P. sergenti (18%). A choropleth map generated in Arcmap 10.1 based on 1560 CL case reports displayed maximum prevalence (0.92-2.5%) in Ayun, Broz, Charun, Chitral 1 and 2 and Darosh 1 and 2 union councils. An environmental risk map constructed by MaxEnt 3.3.3 defined an active zone of transmission based on leishmaniasis occurrence records (n = 315). The analysis of variable contribution in MaxEnt indicates significance of elevation (54.4%), population density (23.3%) and land use/land cover (6.6%) in CL disease dynamics. CONCLUSION: The probability of CL increases (0.6-1 on logistic scale) in severely deforested areas, in lowland valleys and in regions with high-population density.

The recombinant protein rSP03B is a valid antigen for screening dog exposure to Phlebotomus perniciosus across foci of canine leishmaniasis.

The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus, displaying the killer phenotype.

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory-reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito-isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp.

Assessing the importance of four sandfly species (Diptera: Psychodidae) as vectors of Leishmania mexicana in Campeche, Mexico.

Localized cutaneous leishmaniasis represents a public health problem in many areas of Mexico, especially in the Yucatan Peninsula. An understanding of vector ecology and bionomics is of great importance in evaluations of the transmission dynamics of Leishmania parasites. A field study was conducted in the county of Calakmul, state of Campeche, during the period from November 2006 to March 2007. Phlebotomine sandfly vectors were sampled using Centers for Disease Control light traps, baited Disney traps and Shannon traps. A total of 3374 specimens were captured in the two villages of Once de Mayo (93.8%) and Arroyo Negro (6.1%). In Once de Mayo, the most abundant species were Psathyromyia shannoni, Lutzomyia cruciata, Bichromomyia olmeca olmeca and Psychodopygus panamensis (all: Diptera: Psychodidae). The Shannon trap was by far the most efficient method of collection. The infection rate, as determined by Leishmania mexicana-specific polymerase chain reaction, was 0.3% in Once de Mayo and infected sandflies included Psy. panamensis, B. o. olmeca and Psa. shannoni. There were significant differences in human biting rates across sandfly species and month of sampling. Ecological niche modelling analyses showed an overall overlap of 39.1% for the four species in the whole state of Campeche. In addition, the finding of nine vector-reservoir pairs indicates a potential interaction. The roles of the various sandfly vectors in Calakmul are discussed.

DNA sequence analysis suggests that cytb-nd1 PCR-RFLP may not be applicable to sandfly species identification throughout the Mediterranean region.

Molecular methods are increasingly used for both species identification of sandflies and assessment of their population structure. In general, they are based on DNA sequence analysis of targets previously amplified by PCR. However, this approach requires access to DNA sequence facilities, and in some circumstances, it is time-consuming. Though DNA sequencing provides the most reliable information, other downstream PCR applications are explored to assist in species identification. Thus, it has been recently proposed that the amplification of a DNA region encompassing partially both the cytochrome-B (cytb) and the NADH dehydrogenase 1 (nd1) genes followed by RFLP analysis with the restriction enzyme Ase I allows the rapid identification of the most prevalent species of phlebotomine sandflies in the Mediterranean region. In order to confirm the suitability of this method, we collected, processed, and molecularly analyzed a total of 155 sandflies belonging to four species including Phlebotomus ariasi, P. papatasi, P. perniciosus, and Sergentomyia minuta from different regions in Spain. This data set was completed with DNA sequences available at the GenBank for species prevalent in the Mediterranean basin and the Middle East. Additionally, DNA sequences from 13 different phlebotomine species (P. ariasi, P. balcanicus, P. caucasicus, P. chabaudi, P. chadlii, P. longicuspis, P. neglectus, P. papatasi, P. perfiliewi, P. perniciosus, P. riouxi, P. sergenti, and S. minuta), from 19 countries, were added to the data set. Overall, our molecular data revealed that this PCR-RFLP method does not provide a unique and specific profile for each phlebotomine species tested. Intraspecific variability and similar RFLP patterns were frequently observed among the species tested. Our data suggest that this method may not be applicable throughout the Mediterranean region as previously proposed. Other molecular approaches like DNA barcoding or phylogenetic analyses would allow a more precise molecular species identification.

Paraphyly of the subgenus Anaphlebotomus and creation of Madaphlebotomus subg. nov. (Phlebotominae: Phlebotomus).

The systematic position of the Malagasy Phlebotomus (Diptera: Psychodidae) species was assessed in molecular phylogenetic studies. Three molecular markers were sequenced: cytochrome b of the mitochondrial DNA; ITS2, and the D8 domain of the ribosomal DNA. The following species were studied: Phlebotomus (Anaphlebotomus) berentiensis, Phlebotomus (Anaphlebotomus) fertei, Phlebotomus (Anaphlebotomus) fontenillei, Phlebotomus (Anaphlebotomus) vaomalalae and Phlebotomus (Anaphlebotomus) vincenti from Madagascar; Phlebotomus (Anaphlebotomus) stantoni from Asia, and Phlebotomus (Anaphlebotomus) rodhaini from Africa. The following outgroups were selected: Phlebotomus (Euphlebotomus) argentipes, Phlebotomus (Euphlebotomus) barguesae, Phlebotomus (Larroussius) perfiliewi s.l. and Phlebotomus (Adlerius) simici. Each marker analysed by maximum parsimony and maximum likelihood supports the monophyly of the Malagasy Phlebotomus spp. Consequently, we create a new subgenus for these species: Madaphlebotomus subg. nov. This molecular individualization is reinforced by the originality of their spermathecae and by the fact that their geographical distribution is limited to Madagascar, and considers the high level of endemism on this island.

Molecular Identification of Host Blood Meal from Phlebotomine Sandflies (Diptera: Psychodidae) from Kerala, India.

Female phlebotomine sandflies serve as vectors for the transmission of Leishmania parasites, perpetuating an enzootic cycle by disseminating between sylvatic and domestic animals. Humans form a part of this cycle because the sandflies search for a blood source required for egg development. The present study aimed to identify the feeding preferences of different sandfly fauna from six districts of Kerala, India, using molecular tools. An entomological survey was conducted during 2021-2023 in Kollam, Kottayam, Thiruvananthapuram, Thrissur, Malappuram, and Palakkad. Both indoor and outdoor habitats were targeted from sandfly collection using different standard tools and methods. Sandflies were identified using standard taxonomic keys, and DNA was extracted from blood meal collected from sandflies. A total of 7366 sandfly specimens were collected during the study period, which belonged to three different genera and 19 species. Blood source was successfully identified from 119 sandflies revealing that the Sergentomyia genus preferably fed on small reptiles and amphibians, whereas Phlebotomus genus was found to mainly feed on mammalian and avian blood. Sergentomyia zeylanica was an exception, as it primarily fed on mammalian blood sources. Interestingly, humans were the second feeding source for Phlebotomus species, which are the proven vectors. Comprehending the feeding patterns of sandflies is crucial, not just for public health but also for obtaining insights into the ecological dynamics between vectors and hosts, ultimately enabling more efficient strategies for disease control and prevention.