Dataset Information

Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of Phlebotomus (Larroussius) perfiliewi.

ABSTRACT: Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.

SUBMITTER: Chelbi I

PROVIDER: S-EPMC9696294 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of Phlebotomus (Larroussius) perfiliewi.

Chelbi Ifhem I Abdi Amani A Depaquit Jérôme J Fares Wasfi W Abbas Mohammed Abdo Saghir MAS Dachraoui Khalil K Zhioua Elyes E

Insects 20221116 11

Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage ...[more]

PMID: 36421960

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Project description:BackgroundPhlebotomus chinensis is a primary vector of visceral leishmaniasis; it occurs in various biotopes with a large geographical distribution, ranging from Yangtze River to northeast China. Phlebotomus sichuanensis, a species closely related to P. chinensis in high altitude regions, has a long term disputation on its taxonomic status. Both species occur in the current epidemic regions and are responsible for the transmission of leishmaniasis. Population genetic analysis will help to understand the population structure and infer the relationship for morphologically indistinguishable cryptic species. In this study, microsatellite markers were used for studying the genetic differentiation between P. chinensis and P. sichuanensis.MethodsSandflies were collected in 6 representative localities in China in 2005-2009. Ten microsatellite loci were used to estimate population genetic diversity. The intra-population genetic diversity, genetic differentiation and effective population size were estimated.ResultsAll 10 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy-Weinberg disequilibrium was found in 23 out of 60 (38.33%) comparisons associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed three clusters. The cluster I included almost all specimens in the sample SCD collected at high altitude habitats in Sichuan. The other two clusters were shared by the remaining 5 populations, SCJ in Sichuan, GSZ in Gansu, SXL and SXX in Shaanxi and HNS in Henan. The diversity among these 5 populations was low (FST = -0.003-0.090) and no isolation by distance was detected. AMOVA analysis suggested that the variations were largely derived from individuals within populations and among individuals. Consistently, the analysis of ribosomal DNA second internal transcribed spacer (ITS2) sequence uncovered three types of variants, which corresponded with the three gene pools revealed by microsatellites.ConclusionsThe data suggested that the SCD population carried a distinct gene pool, which was differentiated from the other populations. The high altitude ecological habitats, distinctive ITS2 and herein divergence inferred by microsatellite loci support the species status of P. sichuanensis. The P. chinensis populations did not have a significant divergence from each another.

Project description:BackgroundPhlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs.Methodology and findingsA total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively.ConclusionOur results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.

Project description:Phlebotomus perniciosus is one of the major vectors of Leishmania infantum in the Mediterranean basin. The aim of this work was (i) to provide information about abundance and temporal dynamics of this Larroussius species in a hot spot area of visceral leishmaniasis in Tunisia, (ii) to detect L. infantum DNA in wild caught female sandflies and (iii) to measure Phlebotomus perniciosus infection rate throughout the active season. Sandflies were collected monthly during one year using CDC miniature light-traps in house and in animal shelters. Male specimens were identified at species level according to morphological characters. Female specimens were conserved individually for molecular study. Leishmania infection was tested by kinetoplast DNA real-time PCR and ITS-1 PCR-sequencing. Subsequent sandfly species identification of infected specimens was done by mitochondrial cytochrome b sequencing. In one year period, overall 4,441 specimens (2230 males and 2211 females) were collected. Sandfly activity started in end-April and ended in early-November. Mean sandfly density in house was significantly lower than in animal shelters (51 ± 50 versus 504 ± 460 sandflies /CDC night, p<0.05). However, a higher proportion of females was found in house (58.4% versus 49.2%, p<0.001). Based on species identification of male specimens, Phlebotomus perniciosus was the dominant species (56% of the whole male sandfly fauna, p<0.0001). It showed two peaks of density in the active season, a sharp one in early May and a higher long lasting one from end-July to end-September. DNA was extracted from 190 female specimens randomly sampled and corresponding to 96 specimens from house and 94 from animal shelters. Twenty four female sandfly were infected by Leishmania infantum. All infected specimens were recognized as Phlebotomus perniciosus. Leishmania infantum infection rate in female sandflies was 2.3 fold higher in house than in animal shelters (17.7% versus 7.4%, p<0.05). In house, estimated number of infected specimens was the highest at the end of the active season. Abundance, dynamics of density and Leishmania infantum infection prevalence of Phlebotomus perniciosus in Tunisian hot spot of visceral leishmaniasis highlight the major role of this Phlebotominae species in L. infantum transmission.

Project description:BackgroundParasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles.ResultsA total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal.ConclusionThis midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Dataset Information

Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of Phlebotomus (Larroussius) perfiliewi.

Publications

Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of <i>Phlebotomus</i> (<i>Larroussius</i>) <i>perfiliewi</i>.

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