Female Phlebotomus Research Articles

Evidence for a lectin specific for sulfated glycans in the salivary gland of the malaria vector, Anopheles gambiae.

Salivary gland homogenate (SGH) from the female mosquitoes Anopheles gambiae, An. stephensi, An. freeborni, An. dirus and An. albimanus were found to exhibit hemagglutinating (lectin) activity. Lectin activity was not found for male An. gambiae, or female Ae aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, and Lutzomyia longipalpis. With respect to species-specificity, An. gambiae SGH agglutinates red blood cells (RBC) from humans, horse, sheep, goat, pig, and cow; it is less active for rats RBC, and not detectable for guinea-pigs or chicken RBC. Notably, lectin activity was inhibited by low concentrations of dextran sulfate 50–500 K, fucoidan, heparin, laminin, heparin sulfate proteoglycan, sialyl-containing glycans (e.g. 3′-sialyl Lewis X, and 6′-sialyl lactose), and gangliosides (e.g. GM3, GD1, GD1b, GTB1, GM1, GQ1B), but not by simple sugars. These results imply that molecule(s) in the salivary gland target sulfated glycans. SGH from An. gambiae was also found to promote agglutination of HL-60 cells which are rich in sialyl Lewis X, a glycan that decorates PSGL-1, the neutrophils receptor that interacts with endothelial cell P-selectin. Accordingly, SGH interferes with HL-60 cells adhesion to immobilized P-selectin. Because An. gambiae SGH expresses galectins, one member of this family (herein named Agalectin) was expressed in E. coli. Recombinant Agalectin behaves as a non-covalent homodimer. It does not display lectin activity, and does not interact with 500 candidates tested in a Glycan microarray. Gel-filtration chromatography of the SGH of An. gambiae identified a fraction with hemagglutinating activity, which was analyzed by 1D PAGE followed by in-gel tryptic digestion, and nano-LC MS/MS. This approach identified several genes which emerge as candidates for a lectin targeting sulfated glycans, the first with this selectivity to be reported in the SGH of a blood-sucking arthropod. The role of salivary molecules (sialogenins) with lectin activity is discussed in the context of inflammation, and parasite-vector-host interactions.

Diversity and incrimination of sandflies (Psychodidae: Phlebotominae) captured in city and suburbs of Hamadan, Hamadan province, west of Iran

ObjectiveTo investigate Phlebotomus sand fly fauna and their vectorial capacity in Hamadan, Iran. MethodsDuring the three consecutive years (2010-2012), from May to October, sandflies were sampled monthly from residential areas of Hamadan and selected villages using sticky traps. ResultsA total of 3 089 sandflies comprising 12 species from 2 genera were collected, of which 6 species are known vectors of leishmaniasis in Iran and/or other parts of the world. About 75% of the collected sandflies were of two dominant species, Phlebotomus kandelaki (38%) and Phlebotomus papatasi (36.6%). These species in combination with Phlebotomus major (6.8%), Phlebotomus sergenti (6.3%), Phlebotomus caucasicus (5.3%), Phlebotomus wenyoni (1.3%), Sergentomyia sintoni (2.9%) and Sergentomyia dentate (2%) collectively comprised the majority of the sandflies. Other species including Phlebotomus halpensis, Phlebotomus longiductus, Phlebotomus comatus and Phlebotomus alexandri each comprised only less than 1% of collected samples. ConclusionsAlthough none of the 158 dissected female Phlebotomus kandelaki and Phlebotomus papatasi were infected with leishmania promastigote, it is probable that in the future, factors such as environmental changes, migration and urbanization can ease the transmission of leishmaniases in this area.

Sand fly surveillance and control on Camp Ramadi, Iraq, as part of a leishmaniasis control program

Abstract : Vector-borne diseases are a serious health threat to U.S. troops stationed around the world. U.S. forces deployed to Iraq following the 2003 invasion experienced serious risk of infection by several vector-borne pathogens, specifically cutaneous (CL) and visceral leishmaniasis (VL) (Aliaga and Aronson 2007). Camp Ramadi, a U.S. military Forward Operating Base, was established in 2003 at the Al Anbar provincial capital of Ramadi, approximately 110 km west of Baghdad. In Iraq the total number of cases of CL reported per year from 2004 to 2008 was 1,655 and for VL was 1,711 (Alvar et al. 2012). In Al Anbar province in 2008, the estimated incidence of CL and VL was 1 per 10,000 (Alvar et al. 2012). Because of the war, the number of cases of both CL and VL was most likely underreported in Al Anbar and all provinces of Iraq (Alvar et al. 2012). Because of the risk of vector-borne diseases, each large U.S. military facility, such as Camp Ramadi, had a vector control program operating throughout the year. As part of a base-wide vector control program, unbaited Center for Disease Control and Prevention (CDC) light traps were placed at three locations on Camp Ramadi between April and August, 2009 to gather baseline population estimates of adult sand flies and monitor the success of subsequent control measures. This paper reports sand fly collections over the five-month period, whether they were positive for Leishmania, and describes efforts to control sand fly populations using ultra-low volume (ULV) insecticide applications. Three sites on Camp Ramadi were established for trap placement in areas where U.S. troops worked and lived. Site one was in a grove of palm trees near troop living quarters and the base medical clinic. Site two was in a plot of scrub vegetation near the helicopter landing pad, and site three in tall vegetation near buildings occupied by the base Commander. All three sites had active rodent burrows.

Seasonal Variation in the Prevalence of Sand Flies Infected with Leishmania donovani

BackgroundVisceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures.MethodologyWe collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR.ConclusionThough sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.

Identifying salivary antigens of Phlebotomus argentipes by a 2DE approach

In the Indian subcontinent visceral leishmaniasis, also known as kala-azar, is caused by the protozoa Leishmania donovani and is transmitted to humans by the bite of infected female sand flies Phlebotomus argentipes in an anthroponotic cycle. Sand fly saliva is known to play an important role in host infection outcome after an infective bite. Immunogenicity of P. argentipes saliva has already been described. However, specific antigens that can contribute to these immunogenic properties are unknown. This work focuses on the identification of antigens present in P. argentipes saliva through the combination of two-dimensional electrophoresis (2DE) and Western blot (WB). Analysis of the salivary protein profile showed a gradual increase of the protein content in relation to the age of sand flies, reaching the complete salivary protein pattern at day five, which marked the minimum age for dissections. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). The resulting salivary proteomic map consisted of at least 30 spots located between 10 and 60kDa. According to their isoelectric points, spots were mostly distributed around pH ranges: 5–6 and 9–10. In the proteomic maps, the presence of isoforms or posttranslational modifications was also highlighted since several spots were identified as the same protein. Analysis by in silico prediction programs located several potential glycosylation and phosphorylation sites in the aminoacidic sequences. On the other hand, pooled sera of immunized hamsters through the bite of uninfected sand flies showed elevated anti-saliva IgG levels. These sera permitted the detection of 4 protein bands and at least 20 protein spots in 1DE and 2DE respectively, followed by WB. The antigens were identified by MALDI-TOF, MALDI-TOF/TOF and de novo sequencing as D7-related proteins, PpSP15-like proteins, antigen 5-related proteins, apyrases, and several proteins without assigned protein family. Absence of cross-reactivity between P. argentipes and Phlebotomus perniciosus saliva antibodies determined by ELISA and WB was highlighted in this study, confirming that specific salivary antigens from different sand fly vectors need to be sought when designing vector-borne vaccines and markers for vector exposure assays.

Phlebotomine sand flies from Madagascar (Diptera: Psychodidae). VII. An identification key forPhlebotomuswith the description ofPhlebotomus(Anaphlebotomus)vaomalalaen. sp.

An identification key of the Phlebotomus in Madagascar is proposed as well as the description of the male and female Phlebotomus (Anaphlebotomus) vaomalalae n. sp. from Mikea Forest in the south-west of Madagascar. The assignation of this new species to the genus Phlebotomus is based on the presence of mesanepisternal setae. Its inclusion in the subgenus Anaphlebotomus is based on the males on the presence of four spines on the style, the lack of a coxite basal process and the existence of a bifurcated paramere. The female has cibarial and pharyngeal armature and spermathecal architecture similar to Phlebotomus fertei and Phlebotomus berentiensis, two other Malagasy species which belong to Anaphlebotomus. Male and female are held to belong to the same species because of their morphological characters, the homology (100%) of their partial cytochrome b mtDNA sequences and their capture in the same trap. P. vaomalalae n. sp. is a small species compared to the other Phlebotomus species of Madagascar. The cibarium of the male and the female of P. vaomalalae n. sp. is armed with teeth, like those of other Malagasy Phlebotomus. However, it differs in the arrangement and shape of the respective teeth and denticles. The male of P. vaomalalae n. sp. looks like that of P. fontenillei due to its tuft of coxal setae (lacking in P. berentiensis and P. fertei) but differs from this species by the location of this tuft. As P. fertei and P. berentiensis, there is no spermathecal common duct in P. vaomalalae n. sp.

PCR-RFLP Based Method for Molecular Differentiation of Sand Fly Species <I>Phlebotomus argentipes</I>, <I>Phlebotomus papatasi</I>, and <I>Sergentomyia babu</I> Found in India

PCR-Restriction fragment length polymorphism is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis in India, various species of morphological similar sand fly exist but only female Phlebotomus argentipes is the vector for visceral leishmaniasis. In the current study primers were designed targeting the 18S rRNA encoding gene that showed amplification in all the major sand fly species found in India. The amplified fragments were further digested using the HinfI or HpaII restriction enzymes. Each of the restriction enzyme produced a species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be used in the identification of sand fly species.

RISK ZONE MODELLING AND EARLY WARNING SYSTEM FOR VISCERAL LEISHMANIASIS (KALA-AZAR) DISEASE IN BIHAR, INDIA USING REMOTE SENSING AND GIS

Abstract. Visceral Leishmaniasis (VL) commonly known as Kala-azar is one of the most neglected tropical disease affecting approximately 200 million poorest populations 'at risk in 109 districts of three endemic countries namely Bangladesh, India and Nepal at different levels. This tropical disease is caused by the protozoan parasite Leishmania donovani and transmitted by female Phlebotomus argentipes sand flies. The analysis of disease dynamics indicate the periodicity at seasonal and inter-annual temporal scale which forms the basis for development of advanced early warning system. Study area of highly endemic Vaishali district, Bihar, India has been taken for model development. A Systematic study of geo-environmental parameters derived from satellite data in conjunction with ground intelligence enabled modelling of infectious disease and risk villages. High resolution Indian satellites data of IRS LISS IV (multi-spectral) and Cartosat-1 (Pan) have been used for studying environmentally risk parameters viz. peri-domestic vegetation, dwelling condition, wetland ecosystem, cropping pattern, Normalised Difference Vegetation Index (NDVI), detailed land use etc towards risk assessment. Univariate analysis of the relationship between vector density and various land cover categories and climatic variables suggested that all the variables are significantly correlated. Using the significantly correlated variables with vector density, a seasonal multivariate regression model has been carried out incorporating geo-environmental parameters, climate variables and seasonal time series disease parameters. Linear and non-linear models have been applied for periodicity and interannual temporal scale to predict Man-hour-density (MHD) and 'out-of-fit' data set used for validating the model with reasonable accuracy. To improve the MHD predictive approach, fuzzy model has also been incorporated in GIS environment combining spatial geo-environmental and climatic variables using fuzzy membership logic. Based on the perceived importance of the geoenvironmental parameters assigned by epidemiology expert, combined fuzzy membership has been calculated. The combined fuzzy membership indicate the predictive measure of vector density in each village. A γ factor has been introduced to have increasing effect in the higher side and decreasing effect in the lower side which facilitated for prioritisation of the villages. This approach is not only to predict vector density but also to prioritise the villages for effective control measures. A software package for modelling the risk villages integrating multivariate regression and fuzzy membership analysis models have been developed to estimate MHD (vector density) as part of the early warning system.

Prevalence of Sand Flies andLeishmania donovaniInfection in a Natural Population of FemalePhlebotomus argentipesin Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.

Colonization of Phlebotomus papatasi changes the effect of pre-immunization with saliva from lack of protection towards protection against experimental challenge with Leishmania major and saliva

BackgroundSand fly saliva has been postulated as a potential vaccine or as a vaccine component within multi component vaccine against leishmaniasis. It is important to note that these studies were performed using long-term colonized Phlebotomus papatasi. The effect of sand flies colonization on the outcome of Leishmania infection is reported.ResultsWhile pre-immunization of mice with salivary gland homogenate (SGH) of long-term colonized (F5 and beyond) female Phlebotomus papatasi induced protection against Leishmania major co-inoculated with the same type of SGH, pre-immunization of mice with SGH of recently colonized (F2 and F3) female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Our data showed for the first time that a shift from lack of protection to protection occurs at the fourth generation (F4) during the colonization process of P. papatasi.ConclusionFor the development of a sand fly saliva-based vaccine, inferences based on long-term colonized populations of sand flies should be treated with caution as colonization of P. papatasi appears to modulate the outcome of L. major infection from lack of protection to protection.

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies

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.

Comparison of various configurations of CDC-type traps for the collection of Phlebotomus papatasi Scopoli in southern Israel

We conducted two experiments to determine the best CDC-trap configuration for catching male and female Phlebotomus papatasi. First, visual features were evaluated. Standard CDC traps were modified to have black or white catch bags, black or white lids, or no lids and these were tried in different combinations. Significantly more male sand flies were caught by darker traps; significantly more females were captured by traps with either all black or a combination of black and white features. Attraction may be due to dark color or contrast in colors. CDC traps with suction and the following features were also evaluated: no light; incandescent light; ultraviolet (UV) light; combination of black color, heat and moisture; CO(2) alone, or a combination of black color, heat, moisture, and CO(2) simultaneously, all in upright and inverted positions, with the opening for insect entry always 50 cm above the ground. Significantly more females than males were caught by all traps (standard and inverted) except the control traps with suction only. Traps with CO(2) caught more sand flies than traps without CO(2.) Traps with black color, heat and moisture captured significantly more sand flies than the control traps, but with the addition of CO(2) , these traps catch significantly more sand flies than the other traps evaluated. Inverting traps increased the catch for like traps by about two times.

Evaluation of propane combustion traps for the collection of Phlebotomus papatasi (Scopoli) in southern Israel

In this study, we evaluated the efficacy of eleven commercial models of propane combustion traps for catching male and female Phlebotomus papatasi. The traps differed in physical appearance, amount of carbon dioxide produced and released, type and location of capturing device, and the method by which the trap suction fans were powered. The traps tested were the Mosquito Magnet™(MM)-Pro, MM-Liberty, MM-Liberty Plus, MM-Defender, SkeeterVac®(SV)-35, SV-27, Mosquito Deleto™(MD)-2200, MD-2500, MT150-Power Trap, and two models of The Guardian Mosquito Traps (MK-01 and MK-12). All trap models except the SV-35, the SV-27, the MD-2500, and the MK-12 attracted significantly more females than males. The SV-35 was the most efficient trap, catching significantly more females than all the other models. The MD-2200 and MK-12 models were the least effective in catching either female or male sand flies. These data indicate that several models of propane combustion traps might be suitable substitutes for either CO(2) -baited or unbaited light traps for adult sand fly surveillance tools. One advantageous feature is the traps' ability to remain operational 24/7 for ca. 20 days on a single tank of propane. Additionally, the models that produce their own electricity to power the trap's fans have an important logistical advantage in field operations over light traps, which require daily battery exchange and charging.

Imidacloprid as a potential agent for the systemic control of sand flies

Our goal was to study the effectiveness of the insecticide imidacloprid as a systemic control agent. First, to evaluate the blood-feeding effect, we fed adult female Phlebotomus papatasi with imidacloprid-treated rabbit blood and monitored blood-feeding success and survival. Second, to evaluate the feed-through effectiveness of this insecticide, we fed laboratory rats and sand rats with insecticide-treated food and evaluated the survival of sand fly larvae feeding on rodents' feces. In the blood-feeding experiment, 89.8% mortality was observed with the higher dose (5 mg/ml) and 81.3% with the lower dose (1 mg/ml). In the larvicide experiments, both sand fly species demonstrated a typical dose-response curve with the strongest lethal effect for the 250 ppm samples. Lutzomyia longipalpis larvae, however, were less sensitive. In all experiments, 1(st) instar larvae were more sensitive than the older stages. First instar P. papatasi larvae feeding on sand rat feces passed the larvicidal threshold of 90% mortality at doses higher than 50 ppm. In comparison, in older stages 90% mortality was obtained with a dose of only 250 ppm. Overall, results support the feasibility of imidacloprid as a systemic control agent that takes advantage of the tight ecological association between the reservoir host and the sand fly vector.

Les leishmanioses viscérales méditerranéennes

Mediterranean visceral leishmaniasis is a parasitic zoonosis due to Leishmania infantum. The dog is the reservoir species and also the main victim. The vector is the female Phlebotomus sand fly. In the southern Mediterranean region the disease is most frequent in children, whereas in Europe, and particularly in France, it is mostly an opportunistic infection associated with immunosuppression. Frequent asymptomatic carriage has been detected in southern Europe. The classic symptom triad consists of fever, pallor and splenomegaly. Biological signs include low cell blood counts (anemia, leukoneutropenia, and thrombocytopenia) and an inflammatory syndrome. Commercial serologic tests such as those based on immunoblotting are very useful. The gold standard for diagnosis is parasite detection in bone marrow or blood. PCR is useful for therapeutic follow-up. Treatment is currently based on liposomal amphotericin B (AmBisome).

Differences in the Salivary Effects of Wild-Caught Versus Colonized <I>Phlebotomus papatasi</I> (Diptera: Psychodidae) on the Development of Zoonotic Cutaneous Leishmaniasis in BALB/c Mice

Preimmunization of mice with salivary gland homogenate (SGH) of long-term colonized (F29) female Phlebotomus papatasi Scopoli (Diptera: Psychodidae) induced protection against Leishmania major Yakimoff & Schokhor (Kinetoplastida: Trypanosomatidae) co-inoculated with the same type of SGH. In contrast, preimmunization of mice with SGH of wild-caught female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Similarly, SGH from recently colonized (F1) female P. papatasi did not protect mice against L. major. These results suggest that when developing a sand fly saliva-based vaccine, the natural vector populations should be considered.

Efficacy of Olyset long-lasting bednets to control transmission of cutaneous leishmaniasis in Iran

A large-scale intervention field trial of the effect of Olyset long-lasting insecticide-treated bednets on transmission of cutaneous leishmaniasis was carried out in 2 cities in the Islamic Republic of Iran from October 2003 to July 2005. We enrolled 8620 individuals in 3000 households in 6 pairs of sectors in each city. Epidemiological and entomological surveys were carried out pre- and post-intervention. In both cities a statistically significant reduction was found in the incidence of new cases in intervention sectors who received bednets compared with control areas. Entomological surveys showed a reduction in numbers of female Phlebotomus sergenti captured indoors in intervention sectors.

Immunolocalization of a lipase-like protein in the reproductive apparatus of femalePhlebotomus papatasi, at various stages of the gonotrophic cycle

In female phlebotomine sandflies, little is known about the reproductive accessory glands that presumably contribute to egg production and/or oviposition. The main protein secreted in the accessory glands of female Phlebotomus papatasi was recently characterised as a lipase-like protein, the first to be found in the female accessory glands of any insect. This protein, named PhpaLIP (for Phlebotomus papatasi lipase), has now been detected and localized in the reproductive tissues of female P. papatasi, at different stages of the gonotrophic cycle, using a polyclonal anti-PhpaLIP serum and both confocal scanning laser and immuno-electron microscopy. PhpaLIP appears to be always present in the accessory glands (with a secretory peak shortly before oviposition) but was also detected in the follicle cells of the ovarioles, within the developing vitelline envelope, and in the oviducts. The results are discussed in relation to the functions that PhpaLIP could have during the gonotrophic cycle, in the various reproductive structures of female P. papatasi.

Wilson Disease with Visceral Leishmaniasis: An Extremely Uncommon Presentation

Visceral leishmaniasis (VL), which is caused by the protozoa Leishmania donovani and transmitted by the bite of the female sand fly Phlebotomus argentipes, is common in Bihar, India. Wilson disease is an autosomal recessive disorder of copper metabolism in which copper is deposited in the brain and liver. We report a case of an extremely uncommon combination of these diseases in a patient. Treatment options for such a combination of diseases are limited and difficult.

Exploring the midgut transcriptome of Phlebotomus papatasi: comparative analysis of expression profiles of sugar-fed, blood-fed and Leishmania major-infected sandflies

BackgroundIn sandflies, the blood meal is responsible for the induction of several physiologic processes that culminate in egg development and maturation. During blood feeding, infected sandflies are also able to transmit the parasite Leishmania to a suitable host. Many blood-induced molecules play significant roles during Leishmania development in the sandfly midgut, including parasite killing within the endoperitrophic space. In this work, we randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR.ResultsTranscriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4), chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major.ConclusionThis tissue-specific transcriptome analysis provides a comprehensive look at the repertoire of transcripts present in the midgut of the sandfly P. papatasi. Furthermore, the customized bioinformatic analysis allowed us to compare and identify the overall transcript abundance from sugar-fed, blood-fed and Leishmania-infected sandflies. The suggested upregulation of specific transcripts in a blood-fed cDNA library were validated by real-time PCR, suggesting that this customized bioinformatic analysis is a powerful and accurate tool useful in analysing expression profiles from different cDNA libraries. Additionally, the findings presented in this work suggest that the Leishmania parasite is modulating key enzymes or proteins in the gut of the sandfly that may be beneficial for its establishment and survival.