Natural breeding sites of phlebotomine sand flies (Diptera: Psychodidae) on Marambaia Island,Rio de Janeiro State,Brazil

Immature phlebotomine sand flies develop in soils with essential and ideal characteristics for their life cycle, such as organic matter, humidity, temperature and low levels of light. Information regarding the potential breeding places of these dipterans is fundamental to understand the epidemiology and ecology of leishmaniasis, in addition to its importance to control them. In the present study, we aimed to find natural breeding sites of sand flies on Marambaia Island with the aid of emergence traps and direct search of immature forms using the flotation technique with saturated sugar solution in organic substrates of the region. Both methods were effective, with a total of 42 specimens of six different species – including some species that participate in the transmission cycle of American Tegumentary Leishmaniasis – collected by the emergence traps, and five immature forms obtained by floatation technique. However, further studies are still necessary, mainly with respect to the ecology and biology of immature sandfly stages, so that control measures focused on breeding sites can produce positive sustainable results in natural environments.     

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) in urban rainforest fragments,Manaus – Amazonas State,Brazil

The non-flooded upland rainforest fragment in the Federal University of Amazonas Campus is considered one of the world's largest urban tropical woodland areas and Brazil's second largest one in an urban setting. It is located in the city of Manaus, State of Amazonas at 03° 04′ 34″ S, 59° 57′ 30″ W, in an area covering nearly 800 hectares. Forty-one (41) sand fly species belonging to genus Lutzomyia were found attaining a total of 4662 specimens collected. Lutzomyia umbratilis was the dominant species at all heights, followed by Lutzomyia anduzei and Lutzomyia claustrei. The fauna alpha diversity index showed to be 6.4, which is not much lower than that reported for areas of continuous forest in this Amazonian region. This data provides additional evidence on Phlebotomine sand flies found to transmit Leishmania and other trypanosomatids to humans and other animals circulating in this area. This is the first study being reported on sand flies collected in an urban rainforest fragment in Amazonia.     

Natural Leishmania infantum infection in Migonemyia migonei (França, 1920) (Diptera:Psychodidae:Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil

A study of the natural infection of phlebotomine sand flies by Leishmania (Leishmania) infantum was conducted in an area of visceral leishmaniasis in São Vicente Férrer, located in the northern part of the Atlantic rain forest region in the State of Pernambuco, Brazil. In a previous study, Migonemyia migonei have been found predominantly in peridomiciles and houses in this endemic area. The analysis of M. migonei, collected by CDC light trap, by multiplex PCR assay coupled to non-isotopic hybridization showed that 2 females out of 50 were infected by L. infantum. This is the first finding of natural infection of M. migonei by L. infantum suggesting that M. migonei may be the vector of L. infantum in areas of visceral leishmaniasis where Lutzomyia longipalpis, the usual vector, is absent.     

Anthropic effects on sand fly (Diptera: Psychodidae) abundance and diversity in an Amazonian rural settlement,Brazil

Sand flies (Diptera: Psychodidae) are responsible for the transmission of protozoan parasites that cause leishmaniases. They are found predominantly in forests, but some species exploit environments that have been subject to deforestation and subsequent human colonization. Studies conducted in Brazil over the past 30 years show that some species are adapting to peri-urban and urban settings. We evaluated sand fly diversity and abundance in the rural settlement of Rio Pardo, Presidente Figueiredo Municipality, Amazonas State, Brazil. Settlement households were divided into four categories. These categories were determined by the human population density and the degree of deforestation in the immediate area. We used CDC light traps to sample the area surrounding 24 households (6 households in each category). Samples were taken on six occasions during September–November 2009 and June–August 2010. A total of 3074 sand fly specimens were collected, including 1163 females and 1911 males. These were classified into 13 genera and 52 species. The greatest abundance of sand flies and the greatest richness of species were observed in areas where human population density was highest. Our results show that changes in the human occupancy and vegetation management in rural settlements may affect the population dynamics and distribution of sand fly species, thereby affecting the local transmission of cutaneous leishmaniases.     

Seasonal fluctuations of phlebotomine sand fly populations (Diptera: Psychodidae) in the urban area of Marrakech, Morocco

Phlebotomine sand flies (Diptera: Psychodidae) were collected continuously, using sticky traps, during 1 year from October 2002 to September 2003, in an urban area of Marrakech city (Morocco). A total of 3277 specimens were collected belonging to five species. Phlebotomus (Phlebotomus) papatasi (54.6%) is the predominant species followed by Sergentomyia (Sergentomyia) minuta (20%), S. (S.) fallax (11.3%), P. (Paraphlebotomus) sergenti (10.3%) and P. (Larroussius) longicuspis (3.8%). Data analyses showed a mono-modal annual pattern for P. sergenti and a bi-modal one for the other species. P. papatasi, the proven vector of Leishmania major in Morocco, was active throughout the year. This species did not diapause in this region. P. papatasi population peaked in June and November, which relating to the periods of risk in this area. Its preferred temperature ranged between 32 and 36 degrees C but no significant correlation was found between its density and the temperature. Considering the high density and long activity period of P. papatasi, the area of Marrakech should be regarded as a potential focus for L. major. This suggests the need for a continuously surveillance to prevent risk of zoonotic cutaneous leishmaniasis.     

Spatial and temporal distributions of phlebotomine sand flies (Diptera: Psychodidae), vectors of leishmaniasis,in Iran

Leishmaniasis is a major vector-borne disease and health problem in Iran. Studies on sand flies, as the vectors of the disease, began in the Northern and Western parts of the country in 1930 and have been continued up to now. Concerning many published information in the field of sand flies, providing a digital database for the country will help the public health authorities to make more correct and prompt decisions for planning leishmaniasis control programs as well as modeling and forecasting of transmission potential across the country.     

Lutzomyia (Pintomyia) fischeri (Diptera: Psychodidae: Phlebotominae), a probable vector of American cutaneous leishmaniasis: detection of natural infection by Leishmania (Viannia) DNA in specimens from the municipality of Porto Alegre (RS), Brazil, using multiplex PCR assay

In order to determine natural Leishmania (Viannia) infection in Lutzomyia (Pintomyia) fischeri, a multiplex PCR methodology coupled to non-isotopic hybridization was adopted for the analysis of sand fly samples collected by CDC light traps in an endemic area of American Cutaneous Leishmaniasis (ACL) in the periurban region of the municipality of Porto Alegre, Rio Grande do Sul State, Brazil. We analyzed by PCR methodology 560 specimens of Lutzomyia (Pintomyia) fischeri (520 females and 40 males). The wild sand flies were grouped into 56 pools (52 females and 4 males) of 10 each, and positive results were detected in 2 of the 52 female pools, representing a minimum infection rate of 0.38% based on the presence of at least 1 infected insect in the pool. This result associated with some local evidence such as anthopophily, spatial distribution in accordance with the transmission area and human case incidence, suggests that L. (P.)fischeri may be considered as a secondary vector of ACL in the studied locality.     

Canine visceral leishmaniasis in Teresina, Brazil: Relationship between clinical features and infectivity for sand flies

Leishmania chagasi is an intracellular parasite transmitted by the bite of the phlebotomine sand fly Lutzomyia longipalpis, which is the most important of American visceral leishmaniasis. In the gut of the vector, amastigoste forms of the parasite transform into metacyclic promastigotes, from there to the foregut, where they could be transmitted in the next blood meal. Xenodiagnosis is an important tool for the detection of Leishmania, especially when associated to molecular techniques, both being useful for the monitoring and evaluation of dog infectivity in endemic areas. In this study, direct search of Leishmania from material obtained through xenodiagnosis performed in dogs captured in Teresina (Piauí State, Brazil) identified that the predominant forms of the parasite were the procyclic and metacyclic forms located in the hindgut, detected between the 5th and 6th day after the blood meal. Using polymerase chain reaction (PCR), we revealed that dogs with different clinical status were able to infect phlebotomines, the rates of sand fly infection being higher for symptomatic dogs (13%) as compared to asymptomatic ones (3.5%). The direct search was able to demonstrate infection only in phlebotomines in which the blood meal was performed on symptomatic dogs, with a rate of infection of 1.6%. The results underline the importance of using PCR and xenodiagnosis for the detection of Leishmania sp. And for the evaluation of infectivity of dogs in endemic areas, especially those that are asymptomatic.     

Seasonal variation of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in endemic area of visceral leishmaniasis, Campo Grande, state of Mato Grosso do Sul, Brazil

The seasonal distribution of Lutzomyia longipalpis was studied in two forested and five domiciliary areas of the urban area of Campo Grande; MS, from December 2003 to November 2005. Weekly captures were carried out with CDC light traps positioned on ground and in the canopy inside a residual forest and on the edge (ground) of a woodland and in at least one of the following ecotopes in peridomiciles-a cultivated area, a chicken coop, a pigsty, a kennel, a goat and sheep shelter and an intradomicile. A total of 9519 sand flies were collected, 2666 during the first year and 6853 during the second. L. longipalpis was found throughout the 2-year period, presenting smaller peaks at intervals of 2-3 months and two greater peaks, the first in February and the second in April 2005, soon after periods of heavy rain. Only In one of the woodlands was a significant negative correlation (p<0.05) between the number of insects and temperature during the first year and the climatic factors (temperature, RHA and rain) was observed. In the domiciliary areas in four domiciles some positive correlations (p< or =0.05) occurred in relation to one or more climatic factors; however, the species shows a clear tendency to greater frequency (72%) in the rainy season than in the dry (28%). Thus, we recommend an intensification of the VL control measures applied in Campo Grande, MS, during the rainy season with a view to reducing the risk of the transmission of the disease.     

Observations on the feeding habits of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Campo Grande, an endemic area of visceral leishmaniasis in Mato Grosso do Sul, Brazil

Sand flies were captured weekly with CDC light traps from December 2003 to November 2005 in three areas of Campo Grande, in the Brazilian state of Mato Grosso do Sul. These areas incorporated two patches of remnant forest and five houses. The blood meals of engorged female sand flies were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Most (327/355) of the females analysed were Lutzomyia longipalpis, of which 66.4% reacted with human blood, 64.8% with that of birds and 8.9% with that of dogs. Females that had taken human blood predominated in the residential areas and two forest patches. The following combinations of blood were also detected for L. longipalpis in some of the samples analysed: bird + human (43.4%), bird + human + dog (6.1%). The combination bird + human + dog + pig was also found for Nyssomyia whitmani. Dogs and pigs appear to have little attractiveness for L. longipalpis. The results obtained demonstrate the eclecticism and high anthropophily of L. longipalpis and raise new questions with regard to the importance of dogs in VL epidemiology and the possible role of man as a source of infection for sand flies.     

Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.     

Molecular detection of the blood meal source of sand flies (Diptera: Psychodidae) in a transmission area of American cutaneous leishmaniasis,Paraná State,Brazil

The feeding behavior of sand flies provides valuable information about the vector/host interactions and elucidates the epidemiological patterns of American cutaneous leishmaniasis (ACL) transmission. The aim of this study was to identify the blood meal sources of sand flies in endemic areas of leishmaniasis in Paraná State through polymerase chain reaction (PCR) amplification of a prepronociceptin (PNOC) gene fragment and its subsequent DNA sequencing. Moreover, molecular assays were conducted to evaluate the sensitivity and reproducibility of the PNOC gene amplification. Besides that, a time-course digestion test of the blood using sand flies that fed artificially on BALB/c mice was performed. Of 1263 female sand flies collected in the field, 93 (3.6%) specimens were engorged and 27 allowed efficient amplification of the PNOC gene. These flies had fed on equine (Equus caballus), porcine (Sus scrofa) and canine (Canis lupus familiaris) species. The results also showed that the identification of the blood meal sources of the sand flies using the molecular method was directly linked to the level of digestion of the blood (time-course) and not to the amount of blood that had been ingested or to the presence of inhibitors in the blood.     

Morphological and isoenzymatic differentiation of Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) in Southern Morocco

Morphological and enzymatic characterization of Phlebotomus perniciosus and Phlebotomus longicuspis in Morocco is reported. Twenty-nine localities in central and southern of Morocco were sampled and compared with three localities from the Rif (northern Morocco). For morphological study, sand flies were collected by sticky-paper traps. For males, the morphology of the copulatory valves (aedeagi) was examined and the number of coxite hairs was recorded. For isoenzyme analyses, specimens were collected in CDC light traps and immediately conserved at -80 degrees C. P. perniciosus samples from the south of Morocco, up to 150 km from Marrakech, showed single-pointed aedeagi curved at their apices, indistinguishable from the atypical morph of P. perniciosus, previously described in northern Morocco. Twelve enzyme systems were tested and the qualitative analysis of zymogram profiles revealed eight polymorphic loci (glucosephosphate isomerase (GPI), phosphoglucomutase (PGM), hexokinase (HK), fumarate hydratase (FUM), malate dehydrogenase 1 (MDH1), malate dehydrogenase 2 (MDH2), 6 phosphogluconate dehydrogenase (6PGD) and aconitase (ACO)). Enzyme loci showed fixed alleles diagnostic for P. perniciosus (aconitase) and P. longicuspis (aconitase and hexokinase).     

Sergentomyia (Neophlebotomus) monticola, a new species of sand fly (Diptera: Psychodidae) from the Western Ghats,Thiruvananthapuram District,Kerala, India

Sergentomyia (Neophlebotomus) monticola, a new species of sand fly (Diptera: Psychodidae), from the Kani tribal settlements, Thiruvananthapuram District, Kerala, southern India was described. These settlements were located in the Western Ghats, which is one of the 25 biodiversity hotspots in the world. Morphological characters of male and female specimens of Sergentomyia (Neophlebotomus) monticola were described with illustrations and its taxonomic position is defined within the genus. The DNA barcode analysis showed that both male and female specimens of the species were belonging to a single taxonomic category. The genetic distance with the most similar taxonomic neighbour was 14.61%, which confirms its distinctness from its congeners. Voucher specimens of the new species were deposited at the museum, Vector Control Research Centre (Indian Council of Medical Research), Puducherry, India, Zoological Survey of India, India and Smithsonian National Museum of Natural History (NMNH), Washington, D.C., USA.     

Molecular characterization of the invasive Asian tiger mosquito, Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Corsica

The Asian tiger mosquito Aedes albopictus, vector of various human viruses and parasites, has recently spread and established in many temperate regions including European countries. In the present study, we developed a simple PCR-based assay (the amplification of the internal transcribed spacer ITS2 within nuclear ribosomal rDNA) for molecular identification of A. albopictus and confirmed its presence in Corsica island. This assay may (i) facilitate future large scale studies and avoid misidentifications, especially because of the presence of co-occurring close species in this island and (ii) contribute to the monitoring of A. albopictus populations required for targeted control programs.     

Effect of temperature on life history traits during immature development of Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) from Córdoba city,Argentina

We investigated how ambient temperature under fluctuating conditions affects the larval–pupal immature traits of Aedes aegypti and Culex quinquefasciatus mosquitoes from Córdoba city, Argentina, and established each species development threshold and physiological time. Based on life tables, three cohorts of each mosquito species were reared in the laboratory under small fluctuating temperatures conditions of 15.2 ± 1.7 °C, 17.9 ± 1.6 °C, 21.6 ± 0.7 °C and 25.3 ± 0.4 °C for Ae. aegypti, and 16.6 ± 1.7 °C, 18.7 ± 1.7 °C and 25.2 ± 0.3 °C for Cx. quinquefasciatus. Immature development time and survival values, and also thermal development threshold and physiological time were estimated. Development times of all larval and pupal stages of Ae. aegypti and Cx. quinquefasciatus were significantly affected by the rearing temperatures, decreasing when temperature increased. Mean Ae. aegypti total (larva + pupa) development time ranged from 21.9 to 8.6 days, at 15.2 and 25.3 °C, whereas, for Cx. quinquefasciatus varied between 23.5 to 9.2 days at 16.6 and 25.2 °C, respectively. Larval and pupal survival of both species was affected by different rearing temperatures, increasing in general as temperature increased. For Ae. aegypti the total immature survival ranged from 26% at 15.2 °C to 92% at 21.6 °C; however, temperature did not have significant effect on this variable. The total immature survival of Cx. quinquefasciatus was significantly and positively affected by temperatures, ranging from 32 to 88%, at 16.6 and 25.2 °C. The temperature development threshold and the physiological time estimated for Ae. aegypti and Cx. quinquefasciatus were 11.11 °C and 93.74 degree-days, and 10.96 °C and 136.87 degree-days, respectively. The results of the present study showed that temperature significantly affects the larval–pupal immature traits of these mosquito species of sanitary importance, from the central region of Argentina. All the parameters recorded are useful for the development of mosquito management models.     

The effect of immune sera from hamsters immunized with sandfly gut and whole body extract antigens on the fecundity and mortality of Phlebotomus duboscqi (Diptera: Psychodidae)

Phlebotomus duboscqi were fed on hamsters previously immunized with different concentrations of homogenized crude sandfly gut antigen and supernatant obtained from whole body extract. The humoral response in the rodents was quantified at different times post-immunization by the enzyme-linked immunosorbent assay. Sandflies were fed on either immunized or saline control hamsters and the effect of the blood meals on sandfly feeding, survival and fecundity was investigated. The humoral response in immunized hamsters as measured by the presence of P. duboscqi-specific IgG antibodies was significantly greater (P < 0.05) as compared to the controls. This difference was noted in sera collected on 15 and 25 days post-immunization. Sandflies fed on immunized hamsters had a significantly higher mortality (P < 0.05) and decreased egg production (P < 0.05) than those fed on unimmunized control hamsters.     

Effect of niloticin,a protolimonoid isolated from Limonia acidissima L. (Rutaceae) on the immature stages of dengue vector Aedes aegypti L. (Diptera: Culicidae)

The aim of the present study was to evaluate the mosquitocidal activity of fractions and a compound niloticin from the hexane extract of Limonia acidissima L. leaves on eggs, larvae and pupae of Aedes aegypti L. (Diptera: Culicidae). In these bioassays, the eggs, larvae and pupae were exposed to concentrations of 2.5, 5.0, 7.5 and 10.0 ppm for fractions and 0.5, 1.0, 1.5 and 2.0 ppm for compound. After 24 h, the mortality was assessed and the LC₅₀ and LC₉₀ values were calculated for larvae and pupae. Per cent ovicidal activity was calculated for eggs after 120 h post treatment. Among the sixteen fractions screened, fraction 8 from the hexane extract of L. acidissima generated good mosquitocidal activity against Ae. aegypti. The LC₅₀ and LC₉₀ values of fraction 8 were 4.11, 8.04 ppm against Ae. aegypti larvae and 4.19, 8.10 ppm against Ae. aegypti pupae, respectively. Further, the isolated compound, niloticin recorded strong larvicidal and pupicidal activities. The 2 ppm concentration of niloticin showed 100% larvicidal and pupicidal activities in 24 h. The LC₅₀ and LC₉₀ values of niloticin on Ae. aegypti larvae were 0.44, 1.17 ppm and on pupae were 0.62, 1.45 ppm, respectively. Niloticin presented 83.2% ovicidal activity at 2 ppm concentration after 120 h post treatment and niloticin exhibited significant growth disruption and morphological deformities at sub lethal concentrations against Ae. aegypti. The structure of the isolated compound was identified on the basis of single XRD and spectral data (¹H NMR and ¹³C NMR) and compared with literature spectral data. The results indicate that niloticin could be used as a potential natural mosquitocide.     

Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus (Diptera, Psychodidae) in a high-endemic focus of canine leishmaniasis in Rome province, Italy

A 2-year survey aimed to study seasonal phenology, host-blood feeding preferences and Leishmania infections of Phlebotomus perniciosus, was carried out in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Sandfly densities were monitored by sticky traps, while CDC light traps and hand collections were used for individual sandfly analyses. Four species were identified, three belonging to Phlebotomus (P. perniciosus, Phlebotomus mascittii and Phlebotomus papatasi) and one to Sergentomyia (Sergentomyia minuta) genera. In sticky traps, P. perniciosus (53.5%) and S. minuta (46.0%) were the prevalent species, whereas specimens collected by CDC light traps and hand collections consisted mostly of P. perniciosus. The study showed a markedly different sandfly density between 2003 and 2002 collections (5024 specimens versus 644 specimens, respectively), which was attributed to different climatic conditions. In 2003 adults were active during 7 months (May-November) with a typical bimodal distribution, while in 2002 their activity was shorter (5 months, June-October) without clear density peaks. In this low-density year, the prevalence of P. perniciosus was significantly higher in domestic than in wild resting sites. Blood meal tests showed that P. perniciosus had no preferences for any of the two Leishmania susceptible hosts, being the forage ratio rate 0.7 and 1.0 for man and dog, respectively. A significant number of P. perniciosus was found with avian (60.3%) or ovine (24.2%) blood, that was in relation with the habitats where fed specimens were collected. The rate of specimens of P. perniciosus without blood and found positive for Leishmania DNA, detected by a sensitive nested-PCR technique, was much higher (27.6%) than the promastigote infection rate determined by microscopy in the dissected females (1.4%). The intense Leishmania transmission in the study area was confirmed by the high prevalence of anti-leishmanial antibodies in dogs (33.3%).     

Laboratory and field evaluation of neem (Azadirachta indica A. Juss) and Chinaberry (Melia azedarach L.) oils as repellents against Phlebotomus orientalis and P. bergeroti (Diptera: Psychodidae) in Ethiopia

The study evaluated the efficacy of neem (Azadirachta indica A. Juss.) and Chinaberry (Melia azedarach L.) seed oils as repellents against laboratory and field populations of some sandflies in Ethiopia. In the laboratory, concentrations of 2% and 5% neem oil in coconut oil tested against Phlebotomus orientalis (vector of visceral leishmaniasis) provided 96.28% (95% CI = 95.60-96.97) protection up to a mean time of 7 h and 20 min and 98.26% (95% CI = 93.46-104. 07) protection up to 9 h, respectively. Similarly, M. azedarach oil at 2% concentration produced 95.13% (95% CI = 90.74-99.52) protection for the same duration (7 h and 20 min), while the 5% oil gave 96.20 (95% CI = 86.98-105.41) protection for 8 h and 20 min against the same species with no significant difference in percentage protection between the two oils at 2% and 5% concentrations. In the field tests with only neem oil (A. indica) against field populations of P. orientalis and P. bergeroti, similar high level of repellencies were recorded with about the same duration of protection. Application of both neem and Chinaberry oils can be safe and low-cost means of personal protection against sandfly bites in endemic areas of Ethiopia, if the community is advised and encouraged to grow the plants abundantly.