Effects of host species on microbiota composition in Phlebotomus and Lutzomyia sand flies.

BACKGROUND: Blood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions. METHODS: Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis. RESULTS: Our results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch. CONCLUSIONS: The present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection.

A rare sugar, allose, inhibits the development of Plasmodium parasites in the Anopheles mosquito independently of midgut microbiota.

A rare sugar, allose, was reported to inhibit the development of Plasmodium parasites in Anopheles mosquitoes; however, the mechanism remains unknown. The present study addressed the inhibitory mechanism of allose on the development of the Plasmodium parasite by connecting it with bacteria involvement in the midgut. In addition, further inhibitory sugars against Plasmodium infection in mosquitoes were explored. Antibiotic-treated and antibiotic-untreated Anopheles stephensi were fed fructose with or without allose. The mosquitoes were infected with luciferase-expressing Plasmodium berghei, and parasite development was evaluated by luciferase activity. Bacterial composition analysis in gut of their mosquitoes was performed with comprehensive 16S ribosomal RNA sequencing. As the result, allose inhibited the development of oocysts in mosquitoes regardless of prior antibiotic treatment. Microbiome analysis showed that the midgut bacterial composition in mosquitoes before and after blood feeding was not affected by allose. Although allose inhibited transient growth of the midgut microbiota of mosquitoes after blood feeding, neither toxic nor inhibitory effects of allose on the dominant midgut bacteria were observed. Ookinete development in the mosquito midgut was also not affected by allose feeding. Additional 15 sugars including six monosaccharides, four polyols, and five polysaccharides were tested; however, no inhibitory effect against Plasmodium development in mosquitoes was observed. These results indicated that allose inhibits parasite development in midgut stage of the mosquito independently of midgut microbiota. Although further studies are needed, our results suggest that allose may be a useful material for the vector control of malaria as a "transmission-blocking sugar."

Ayaconin, a novel inhibitor of the plasma contact system from the sand fly Lutzomyia ayacuchensis, a vector of Andean-type cutaneous leishmaniasis.

Transcriptome analysis of the salivary gland cDNA library from a phlebotomine sand fly, Lutzomyia ayacuchensis, identified a transcript coding for the PpSP15/SL1 family protein as the second most abundant salivary component. In the present study, a recombinant protein of the PpSP15/SL1 family protein, designated ayaconin, was expressed in Escherichia coli, and its biological activity was characterized. The recombinant ayaconin purified from the soluble fraction of E. coli lysate efficiently inhibited the intrinsic but not extrinsic blood coagulation pathway. When the target of ayaconin was evaluated using fluorescent substrates of coagulation factors, ayaconin inhibited factor XIIa (FXIIa) activity more efficiently in a dose-dependent manner, suggesting that FXII is the primary target of ayaconin. In addition, incubation of ayaconin with FXII prior to activation effectively inhibited FXIIa activity, whereas such inhibition was not observed when ayaconin was mixed after the production of FXIIa, indicating that ayaconin inhibits the activation process of FXII to produce FXIIa, but not the enzymatic activity of FXIIa. Moreover, ayaconin was shown to bind to FXII, suggesting that the binding of ayaconin to FXII is involved in the inhibitory mechanism against FXII activation. These results suggest that ayaconin plays an important role in the blood-sucking of Lu. ayacuchensis.

Prevalence of Genetically Complex Leishmania Strains With Hybrid and Mito-Nuclear Discordance.

Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis. Although Leishmania species have been conventionally identified by multilocus enzyme electrophoresis, genetic analysis targeting kinetoplast and nuclear DNA (kDNA and nDNA, respectively) is now widely used for this purpose. Recently, we conducted countrywide epidemiological studies of leishmaniasis in Ecuador and Peru to reveal prevalent species using PCR-RFLP targeting nDNA, and identified unknown hybrid parasites in these countries together with species reported previously. Furthermore, comparative analyses of kDNA and nDNA revealed the distribution of parasites with mismatches between these genes, representing the first report of mito-nuclear discordance in protozoa. The prevalence of an unexpectedly high rate (~10%) of genetically complex strains including hybrid strains, in conjunction with the observation of mito-nuclear discordance, suggests that genetic exchange may occur more frequently than previously thought in natural Leishmania populations. Hybrid Leishmania strains resulting from genetic exchanges are suggested to cause more severe clinical symptoms when compared with parental strains, and to have increased transmissibility by vectors of the parental parasite species. Therefore, it is important to clarify how such genetic exchange influences disease progression and transmissibility by sand flies in nature. In addition, our aim was to identify where and how the genetic exchange resulting in the formation of hybrid and mito-nuclear discordance occurs.

Salivary gland transcriptome of the Asiatic Triatoma rubrofasciata.

Salivary gland transcriptome analysis of the Asiatic Triatoma rubrofasciata was performed by high-throughput RNA sequencing. This analysis showed that the majority of reads accounting for 85.38% FPKM (fragments per kilobase of exon per million mapped fragments) were mapped with a secreted class. Of these, the most abundant subclass accounting for 89.27% FPKM was the lipocalin family. In the lipocalin family, the most dominant molecules making up 70.49% FPKM were homologues of procalin, a major allergen identified from T. protracta saliva, suggesting an important role in blood-sucking of T. rubrofasciata. Other lipocalins showed similarities to pallidipin and triplatin, inhibitors of collagen-induced platelet aggregation identified from T. pallidipennis and T. infestans, respectively, Td38 from T. dimidiata with unknown function, triatin-like lipocalin with unknown function, and triafestin, an inhibitor of the activation of the kallikrein-kinin system, identified from T. infestans saliva. Other than lipocalin family proteins, homologues of antigen-5 (3.38% FPKM), Kazal-type serine protease inhibitor (1.36% FPKM), inositol polyphosphate 5-phosphatase (1.32% FPKM), and apyrase/5'-nucleotidase (0.64% FPKM) were identified as abundant molecules in T. rubrofasciata saliva. Through this study, de novo assembly of 42,580,822 trimmed reads generated 35,781 trinity transcripts, and a total of 1,272 coding sequences for the secreted class were deposited in GenBank. The results provide further insights into the evolution of salivary components in blood-sucking arthropods.

Transcriptome data on salivary lipocalin family of the Asiatic Triatoma rubrofasciata.

The dataset in this report is related to the research article entitled: "Salivary gland transcriptome of the Asiatic Triatoma rubrofasciata" [1]. Lipocalin family proteins were identified as the dominant component in T. rubrofasciata saliva, and phylogenetic analysis of the salivary lipocalins resulted in the formation of five major clades (clade I-V). For further characterization, each clade of T. rubrofasciata lipocalin was subjected to alignment and phylogenetic analyses together with homologous triatomine lipocalins: procalin, a major allergen in T. protracta saliva and its homologue Td04 from T. dimidiata (clade I), pallidipin and triplatin, inhibitors of collagen-induced platelet aggregation identified from T. pallidipennis and T. infestans, respectively, and their homologue Pc20 identified from Panstrongylus chinai (clade II), Td30 and Td38 from T. dimidiata with unknown functions (clade III), triatin-like salivary lipocalins, Pc58 and Pc226 identified from P. chinai and Td18 from T. dimidiata (clade IV), and triafestin, an inhibitor of the activation of the kallikrein-kinin system, identified from T. infestans saliva and its homologues, Td25 and Td40 from T. dimidiata and Pc64 from P. chinai (clade V).

Comparative Analysis of Bacterial Communities in Lutzomyia ayacuchensis Populations with Different Vector Competence to Leishmania Parasites in Ecuador and Peru.

Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.

Nuclear and kinetoplast DNA analyses reveal genetically complex Leishmania strains with hybrid and mito-nuclear discordance in Peru.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered.

Review of Leishmaniasis in the Middle East and North Africa.

BACKGROUND: Cutaneous and visceral forms of leishmaniasis are the most important protozoan infection in the Middle East and North Africa (MENA). OBJECTIVES: Review the current knowledge on leishmaniasis in the MENA. METHODS: The data presented in this review are gathered primarily from WHO reports and from an extensive literature search on PubMed. RESULTS: There are four cycles of transmission of leishmaniasis: zoonotic cutaneous leishmaniasis (ZCL), induce by Leishmania (L.) major, transmitted by Phlebotomus (P.) papatasi, with rodent species of Psammomys obesus, Meriones libycus, Nesokia indica, and Rhombomys opimus are considered as host reservoirs. Zoonotic visceral leishmaniasis (ZVL) is inducing by L. infantum, transmitted by several Phlebotomus spp. of the sub-genus Larroussius and mainly P. perniciosus in more than one-half of the MENA countries and the dog species of Canis familiaris are considered as the main reservoirs. Anthroponotic cutaneous leishmaniasis (ACL), induce by L. tropica and transmitted by P. sergenti, without any non-human reservoir in most cases. Anthroponotic visceral leishmaniasis (AVL) induces by L. donovani spreads through P. alexandri, circulates exclusively in humans. CONCLUSION: There are many challenges facing the successful control of leishmaniasis. However, there is continuing research into the treatment of leishmaniasis and potentially vaccinations for the disease.

Evaluation of resistance to temephos insecticide in Culex pipiens pipiens larvae collected from three districts of Tunisia.

BACKGROUND: Mosquitoes are considered as the main groups of arthropods that cause nuisance and public health problems. OBJECTIVES: Evaluation of resistance to temephos insecticide in Culex pipiens pipiens larvae collected from three districts of Tunisia. METHODS: Late third and early fourth instars larvae of Culex pipiens pipiens were collected in three localities of Northern and Southern Tunisia. Field collected populations were tested against temephos insecticide and compared to bioassays of a susceptible reference strain. The cross-resistance between temephos and propoxur, and the polymorphism of over-produced esterases and AChE 1 were investigated. RESULTS: Studied populations exhibited tolerance to temephos with low and high levels of resistance. The resistance ratio (RR50) values of temephos ranged from 1.34 to 114. Synergists and starch electrophoresis showed that the metabolic resistances were involved in the recorded resistance. Likewise, the resistant target site (acetyl cholinesterase: AChE 1) was responsible for the recorded resistance to temephos compound in Culex pipiens pipiens. CONCLUSION: The low and high resistance recorded to temephos insecticides is particularly interesting, because it leaves a range of tools useable by vector control services. However, further studies are needed to determine its spread and anticipate vector control failure where these insecticides are used.

The potential role of urbanization in the resistance to organophosphate insecticide in Culex pipiens pipiens from Tunisia.

OBJECTIVE: To examine the effects of urbanization on the resistance status of field populations of Culex pipiens pipiens to organophosphate insecticide. METHODS: Bioassays and biochemical assays were conducted on Tunisian field populations of Culex pipiens pipiens collected in four various areas differing in the degree of urbanization. Late third and early fourth larvae were used for bioassays with chlorpyrifos and adults mosquitoes for biochemical assays including esterase and acetyl cholinesterase (AChE) activities. RESULTS: The distribution of resistance ratios in this study appears to be influenced by the degree of urbanization. The highest resistance was recorded in the population from most urbanized areas in Tunisia whereas the lowest resistance was found in relatively natural areas. Both metabolic and target site mechanisms were involved in the recorded resistance. CONCLUSION: This is the first study in Tunisia showing evidence of the impact of urbanization on the resistance level in Culex pipiens pipiens. Proper management of the polluted breeding sites in the country and effective regulation of water bodies from commercial and domestic activities appear to be critical for managing insecticide resistance.

PCR-RFLP analyses of Leishmania species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador.

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.

Further insight into the geographic distribution of Leishmania species in Peru by cytochrome b and mannose phosphate isomerase gene analyses.

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.

First studies showing high temephos resistance in Anopheles labranchiae (Diptera: Culicidae) from Tunisia.

BACKGROUND: Despite the public health importance of Anopheles (An.) labranchiae, their resistance status to temephos insecticide has not, to our knowledge, been explored. OBJECTIVES: The present study was carried out to determine the temephos resistance status of field populations of An. labranchiae from Tunisia. METHODS: Six field populations of An. labranchiae were collected as larvae from breeding sites of Northern and Central Tunisia. All the tests were carried out according to the WHO method. RESULTS: Results reported that the majority of field populations showed low and medium resistance ratios (6.2

First study of susceptibility and resistance status to pyrethroids insecticides in Anopheles (Cellia) sergentii (Theobald, 1907) from Southern Tunisia.

BACKGROUND: Insecticide resistance is an important threat to malaria control. Anopheles (An.) sergentii proved to be the number one vector in the oases and may be of a particular interest in projection of the future trends of the disease in Tunisia. OBJECTIVES: Resistance status to pyrethroids insecticides in An. sergentii was evaluated for the first time in Tunisia. METHODS: Diagnostic resistance tests to pyrethroids insecticides were conducted on late third and early fourth larvae of An. sergentii collected in Southern Tunisia. RESULTS: The level of resistance to permethrin and deltamethrin varied from 1.9 to 5.77 and from 2.75 to 4.63, respectively. The highest resistance was recorded in sample # 3 to the two used insecticides. Synergists showed that esterases and glutathione-S-transferase were not involved in the resistance to any of the evaluated insecticides. In contrast, cytochrome-P450 monooxygenases played a role in the detoxification of two among three studied samples. Positive correlations between larval tolerance to both Permethrin/DDT and Deltamethrin/DDT were recorded indicated target site insensitivity. CONCLUSION: Continued monitoring of insecticide susceptibility and generating complementary data on mechanisms of resistance using molecular and biochemical methods is essential to ensure early detection of insecticide resistance in potential malaria vectors in Tunisia.

Resistance status to deltamethrin pyrethroid of Culex pipiens pipiens (Diptera: Culicidae) collected from three districts of Tunisia.

OBJECTIVES: The aim of the present study was to determine the susceptibility status of Culex pipiens pipiens populations against deltamehtrin insecticide. METHODS: Larvae of Culex pipiens pipiens were collected from three breeding places in Northern and Southern Tunisia between 2003 and 2005. Early third and late fourth instars were tested against deltamethrin pyrethroid insecticide. Cross-resistance with DDT resistance was evaluated in studied samples to estimate the role of target site insensitivity and two synergists including piperonyl butoxide (Pb) and S,S,S-tributyl phosphorotrithioate (DEF) were used to estimate the role of detoxification enzymes. RESULTS: Our results revealed that the level of deltamehtrin resistance ranged from 0.67 to 31.4. We also showed the non-involvement of kdr resistance in pyrethroid resistance and no cross-resistance with DDT resistance was detected in all studied populations including the most resistant one. Synergists study on the resistant population (sample # 1) showed the involvement of CYP450 in the recorded resistance to the deltamethrin insecticide. CONCLUSION: The results obtained from this study should be considered in the current control programs to combat mosquitoes in Tunisia.

Resistance development and insecticide susceptibility in Culex pipiens pipiens, an important vector of human diseases, against selection pressure of temephos and its relationship to cross-resistance towards organophosphates and pyrethroids insecticides.

BACKGROUND: Culex pipiens pipiens is an important vector of human diseases. OBJECTIVE: To determine the insecticide resistance development in Culex pipiens pipiens against selection pressure of temephos.. METHODS: A field population of Culex pipiens pipiens was collected from Northwestern Tunisia with a medium level of temephos resistance (LC50 = 0.0069). It was subjected to six generations of temephos pressure selection to evaluate its relationship to cross-resistance towards organophosphates (OPs) and pyrethroids (PYR) insecticides. RESULTS: The selection was initiated at the dose 0.0266, 0.0748 and 0.0069 which were increased during successive generations up to 0.1488, 3.8747 and 0.0086 after sixth generation for temephos, chlorpyrifos and permethrin insecticides, respectively. It is important to noted that high cross-resistance to chlorpyrifos insecticide (OP) was detected (51.88×). However, little or no cross-resistance to the pyrethroid permethrin (PYR) was recorded (1.24×). Contrary to metabolic resistance, it seemed that acetylcholinesterases AChE 1 was fixed under pressure selection. CONCLUSION: The high cross-resistance to temephos and chlorpyrifos is reasonable because they belong to the same class of insecticide (OP). However, the little cross-resistance to the pyrethroid permethrin could support its use alternately for Culex pipiens pipiens control.

First detection of Leishmania major DNA in Sergentomyia (Sintonius) clydei (Sinton, 1928, Psychodidae: Phlebotominae), from an outbreak area of cutaneous leishmaniasis in Tunisia.

In recent years there has been growing interest in Sergentomyia species. Their role in the spread of mammalian leishmaniasis appears repeatedly in the literature and the possibility of its implication in Leishmania transmission to humans remains controversial. Sergentomyia (Sintonius) clydei is one of several cryptic species sharing therefore common morphologic criteria with others species of the subgenera Sintonius. Little is known about this specie in Tunisia. We sampled and identified different specimens including four specimens of S. clydei collected from Sidi Bouzid and Kairouan areas (center of Tunisia) using morphological tools. Male Sergentomyia clydei and Sergentomyia christophersi are known to share several morphological characters and can be mistaken for. Consequently we took advantage of 5 male S. christophersi available in our collection (Tataouin, South of Tunisia). In our study morphological tools were completed by molecular study of cytochrome b gene to identify S. clydei. For the detection of Leishmania spp. that might infect our specimens, Leishmania DNA was analyzed by amplification of kinetoplast minicircle DNA using real-time PCR and nested-PCR. Obtained result was confirmed by restriction analysis of the amplified ribosomal internal transcribed spacer 1 (ITS1). We provide in our study, the first molecular identification of S. clydei, in Tunisia. Our Neighbor Joining tree based on mitochondrial cytochrome b gene shows two different clusters. The first includes the Tunisians S. clydei and other specimens from Africa, Middle East and the Arabic peninsula, and the second cluster containing the specimens from Seychelle. Unexpectedly, we also demonstrate the infection of one anthropophilic female S. clydei by Leishmania major DNA. This finding shows that more attention should be paid when identifying parasites by molecular tools within sandfly vector.

Larval habitats characterization and species composition of Anopheles mosquitoes in Tunisia, with particular attention to Anopheles maculipennis complex.

In Tunisia, malaria transmission has been interrupted since 1980. However, the growing number of imported cases and the persistence of putative vectors stress the need for additional studies to assess the risk of malaria resurgence in the country. In this context, our aim was to update entomological data concerning Anopheles mosquitoes in Tunisia. From May to October of 2012, mosquito larval specimens were captured in 60 breeding sites throughout the country and identified at the species level using morphological keys. Environmental parameters of the larval habitats were recorded. Specimens belonging to the An. maculipennis complex were further identified to sibling species by the ribosomal deoxyribonucleic acid (rDNA)-internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) technique. In total, 647 Anopheles larvae were collected from 25 habitats. Four species, including An. labranchiae, An. multicolor, An. sergentii, and An. algeriensis, were morphologically identified. rDNA-ITS2 PCR confirmed that An. labranchiae is the sole member of the An. maculipennis complex in Tunisia. An. labranchiae was collected throughout northern and central Tunisia, and it was highly associated with rural habitat, clear water, and sunlight areas. Larvae of An. multicolor and An. sergentii existed separately or together and were collected in southern Tunisia in similar types of breeding places.