Anthropophagy and aggregation behaviour of the sandfly Phlebotomus argentipes in Sri Lanka

ABSTRACT. The visceral leishmaniasis (VL) vector Phlebotomus argentipes Annandale & Brunetti is widely distributed throughout the Indian sub-continent and S.E. Asia. The absence of VL in areas such as Sri Lanka has been attributed to the zoophilic nature of P.argentipes , since they were not recorded biting man.
Field studies on P. argentipes were undertaken in the central highlands of Sri Lanka, near Kandy, in May 1988. Male sandflies outnumbered females on cows by 19:1, and were regularly spaced at all densities. This behaviour is considered analagous to swarming in other Nematocera. However, all-night human-biting catches show the biting rate to be similar (mean=8.4, range 2–25 bites per night over ten consecutive nights) to that in N.E. India where VL is endemic. This anthropophagy was maintained during laboratory colonization.     

The efficacy of indoor CDC light traps for collecting the sandfly Phlebotomus argentipes, vector of Leishmania donovani

The efficacy of light traps for collecting sandflies (Diptera: Psychodidae) varies both inter-specifically and intra-specifically (by gender and physiological status) as a result of significant differences in phototropic and other behavioural characteristics. The efficacy of miniature CDC light traps for collecting Phlebotomus argentipes Annandale & Brunetti, a vector of Leishmania donovani Laveran & Mesnil (Kinetoplastida: Trypanosomatidae), was assessed in the Indian state of Bihar. Sandflies were collected during the night from 16 houses in each of three villages over 3 months (four times at fortnightly intervals) using CDC light traps indoors, and by aspirator collection (carried out by one person for 30 min/house) from the walls of the same houses the following morning. Incidence rate ratios (IRRs) between CDC light trap collections and aspirator collections were obtained through a negative binomial regression with household as random effect. CDC light traps were especially effective in catching males (IRR 3.08, 95% confidence interval [CI] 2.12-4.46) and unfed females (IRR 3.50, 95% CI 2.37-5.16) of P. argentipes, and to a lesser extent gravids (IRR 1.77, 95% CI 1.07-2.93). However, only a relatively small proportion of all blood-fed P. argentipes were collected by light trap (IRR 0.45, 95% CI 0.28-0.73). Despite its limitations in collecting blood-fed female sandflies, the CDC light trap appears to trap a sufficient proportion of the indoor population of sandflies for sampling purposes, and as this light trap is also more convenient and more easily standardized than the aspirator method, we conclude that it is the most efficient method for monitoring P. argentipes populations in the Indian subcontinent.     

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar,India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.     

Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle

Systemic control uses the vertebrate hosts of zoonotic pathogens as “Trojan horses,” killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.     

Morphometric and morphological variation between two different populations of Phlebotomus major s.l. from endemic and non‐endemic foci of visceral leishmaniasis in Iran

Populations of Phlebotomus major were examined in two endemic and nonendemic foci of visceral leishmaniasis in Iran. Based on the shape of the aedeagus and ventrally located hairs of coxite and pharyngeal armatures, two morphotypes were found sympatrically in the endemic area of Borazjan. Significant differences in morphometric survey were observed in at least 11 measured characters. The aedeagus of the non‐endemic Miyandoab morphotype, and also of a few specimens from Borazjan, is completely parallel throughout its length with a slightly expanded end. Ventrally located hairs of the middle coxite were longer and more compact. It is close morphologically to P. major neglectus (P. neglectus), which was recently recorded from Iran. It is also morphologically similar to P. notus, which has not yet been reported from Iran and needs further investigation. The aedeagus of the morphotype occurring only in Borazjan is narrower in the middle and the hairs are closer to the base of the coxite and are shorter and more outspread, which makes it similar to P. major krimensis or P. neglectus. The two morphotypes occurring sympatrically in Borazjan do not appear to be subspecies and it may be premature to propose them as separate species. Further investigation is needed to clarify the actual status of P. major s. l. in Iran.     

High nocturnal CO2 emanation guides the sand fly Phlebotomus papatasi to sugar‐rich plants

Abstract In arid areas, Phlebotomus papatasi obtains essential carbohydrates by feeding on green tissues of plants. There is a great variation in sugar content, metabolic rates and dark respiration between conspecific plants, and also between branches and leaves of the same plant. It is hypothesized that high nocturnal release of CO₂ may guide the sand flies to rich sugar sources. Comparisons of Ph. papatasi feeding on branches of different plant species demonstrate a significant positive correlation between the level of sugar in leaves, the rate of sand fly feeding on them and the size of ingested sugar meals. Mean nocturnal CO₂ emanation of low‐sugar Ricinus communis branches is 26.5 ppm and that of sugar rich branches is 86.3 ppm above room level. Low‐sugar Capparis spinosa branches release 45.0 ppm and the emission from sugar rich branches is 76.0 ppm above room level. Branches with similar emissions, placed behind net partitions, are used in no‐choice orientation experiments: R. communis branches releasing high CO₂ levels are approached by 53.7% of the flies compared with 3.0% of flies that orientate to low CO₂ branches. Capparis spinosa with high CO₂ emission are approached by 9.8% of the flies compared with 0.95% of flies that orientate to low CO₂ branches.     

Sand fly species of Sanliurfa province in Turkey

The species composition and seasonal abundance of sand flies (Diptera: Phlebotominae) were studied in the years 2000-2002 in the Sanliurfa region, which is the largest focus of cutaneous leishmaniasis in south-eastern Turkey. Sixteen species were identified among 29 771 specimens collected at 17 different sites by light traps, sticky papers and aspirators. The most common species were Phlebotomus papatasi (Scopoli) (45.4%), P. perfiliewi Parrot (21.9%), and P. sergenti Parrot (19.4%). The other species found were P. major Adler & Theodor (3%), P. neglectus Leger & Pesson (2.2%), P. brevis Theodor & Mesghali (2%), P. alexandri Sinton (1.9%), P. galilaeus Theodor (1.6%), P. halepensis Theodor (0.84%), Sergentomyia adleri Theodor (0.78%), S. dentata Sinton (0.49%), S. minuta Rondani (0.42%), S. theodori Parrot (0.16%), P. kazeruni Theodor & Mesghali (0.001%) and P. mascitti Grassi (0.001%) and one unidentified Phlebotomus species. Among these species P. galilaeus, S. minuta and S. dentata are the first records for this area. All species showed seasonal fluctuations, with the period of highest abundance between May and October.     

Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae)

Phlebotomus papatasi is a proven vector of Leishmania major which is one of the causative agents of cutaneous leishmaniasis in the Old World. Although it has a wide geographical range, its population structure is not yet well understood. In an effort to better understand the population dynamics of this vector, we developed a panel of di‐ and trinucleotide microsatellite markers, using a magnetic bead hybridization enrichment protocol. These microsatellite loci showed three to seven alleles with an expected heterozygosity range between 0.702 and 0.876. The level of polymorphisms found in this study suggests that these microsatellite loci can be used for population analysis of P. papatasi.     

Impact of control measures and dynamics of sand flies in southern Brazil

We report the results of control measures introduced to reduce the density of sand flies in domiciles and subsequent monitoring of the effects of these measures on the sand fly populations. The most common species of sand flies were Nyssomyia neivai and Nyssomyia whitmani, which are naturally infected by Leishmania. A total of 268,382 (93.4%) sand flies were collected in ecotypes constructed with the aim of attracting sand flies, and 19,091 (6.6%) sand flies were collected in the ecotypes consisting of residences and other buildings. Human actions determine the growth or reduction of the sand fly population in human‐occupied space. Understanding the dynamics of sand flies in this environment can substantially contribute to the prevention of cutaneous leishmaniasis.     

Associations of Phlebotomus orientalis and other sandflies with vegetation types in the eastern Sudan focus of kala-azar

Abundances of Phlebotomus orientalis, P. papatasi (Diptera: Psychodidae) and other sandflies were investigated in relation to different types of vegetation associated with endemic foci of visceral leishmaniasis (kala-azar) in eastern Sudan. Sandfly collection sites were in Umsalala Village (Galabat Province) and Dinder National Park (Dinder Province). Sandfly species found in the area were P. orientalis, P. papatasi, Sergentomyia africana, S. antennata, S. clydei, S. schwetzi and S. squamipleuris. Among the types of trees in the Dinder area, P. orientalis was more abundant in forests dominated by Acacia seyal than where Combretum kordofanum, Balanites aegyptiaca, Hyphaena thaibaica or Ziziphus spinachristi were the dominant trees. At Umsalala, P. papatasi was more abundant in the village than at any site with dense vegetation, but P. orientalis was uncommon. Sergentomyia spp. were distributed fairly evenly amongst most types of vegetation investigated at both localities. The ecological distribution of sandflies and epidemiology of kala-azar in Sudan are discussed with emphasis on the association of P. orientalis with A. seyal woodlands.     

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

The breeding habitat of sandflies is a little studied and poorly understood phenomenon. More importantly, oviposition behaviour is a largely neglected aspect of sandfly biology and this knowledge gap further undermines our understanding of the biology of sandflies. Pheromones released by the eggs play an important role in identifying good sites for oviposition by female insects. Several recent studies have examined the oviposition pheromone. The present study provides a preliminary report on the oviposition behaviour of Phlebotomus argentipes, the only vector of kala-azar (or visceral leishmaniasis) on the Indian sub-continent. Sandflies prefer to oviposit their eggs on surfaces that contain organic substances, especially substances with an odour of decaying animal products and the remains of conspecific eggs. The results presented here suggest that the odour released by the organic substances of old sandfly colony remains that contain dead flies, old unhatched eggs, larval food containing vertebrate faeces, frass and other organic matter serves as an attractant for the ovipositing females of P. argentipes and hence greatly increases the number of oviposited eggs compared to eggs deposited in controlled oviposition pots. This result will be helpful in maintaining an efficient colony of P. argentipes and may be a promising tool for monitoring and controlling the target insect as part of a synergistic approach.     

Honeydew of aphids as a source of sugar for Phlebotomus ariasi

Wild-caught Phlebotomus ariasi Tonnoir, starved until their sugar meals had been digested, were caged for 24 h with plants or plants infested with aphids and then tested for fructose, a constituent of plant sap. No evidence was found that the flies took sap directly from nine types of plants present in their habitat. About two-thirds of flies caged with oak (Quercus ilex L.) infested with an aphid [Lachnus roboris (L.)] and about a fifth kept with leaves of the french bean (Phaseolus vulgaris L.) infested with an aphid took honeydew. Experiments with three other aphid species gave negative results. Of sandflies caught in an oak tree infested with aphids, half of the females and three-quarters of the males contained fructose. Tests on females caught in a house and grouped according to state of engorgement showed that, having taken blood, the females stop taking sugar until the bloodmeal is completely digested. It is predicted that honeydews are important in the development of Leishmania infantum Nicolle in the gut of P. ariasi.     

Protective efficacy of lambdacyhalothrin-impregnated bednets against Phlebotomus orientalis, the vector of visceral leishmaniasis in Sudan

Field investigations on the sandfly Phlebotomus orientalis (Diptera: Psychodidae), the vector of Leishmania donovani causing visceral leishmaniasis (VL) in Sudan, were undertaken in two villages (Bellow and Elgamel) and Dinder National Park, to determine the protective value of bednets (polyester, 100 denier) impregnated with lambda-cyhalothrin 10 mg a.i./m2 pyrethroid insecticide. After exposure to treated netting for 30 s, P. orientalis females all died within 1 h. When field-tested in Acacia woodland, treated bednets provided complete protection from bites of the vector. Numbers of P. orientalis females landing on human collectors without bednets or using untreated bednets averaged 32.0 +/- 8.3 or 6.9 +/- 2.7 per man-night, respectively, whereas collectors using treated bednets experienced no sandfly bites during the same period (18.00-06.00 hours, 12 nights in June 1995). Socio-behavioural observations on the bed-time of people living in both study villages indicated that the use of impregnated bednets against P. orientalis would give more potential protection for women and children than for male adults. Overall the proportions of people and their durations of exposure to the risk of sandfly bites (i.e. after sunset until they went to bed) were 40% unprotected for< 1h, 50% for 1-2h and >10% for > or = 2h. Because visceral leishmaniasis in Sudan occurs mainly in children, the use of impregnated bednets (outdoors as well as indoors), and going to bed early could provide a high degree of personal protection against this zoonotic infection.     

Phlebotomine sandfly ecology on the Indian subcontinent: does village vegetation play a role in sandfly distribution in Bihar,India?

Visceral leishmaniasis (VL) is a disease that results in approximately 50 000 human deaths annually. It is transmitted through the bites of phlebotomine sandflies and around two‐thirds of cases occur on the Indian subcontinent. Indoor residual spraying (IRS), the efficacy of which depends upon sandfly adults resting indoors, is the only sandfly control method used in India. Recently, in Bihar, India, considerable sandfly numbers have been recorded outdoors in village vegetation, which suggests that IRS may control only a portion of the population. The purpose of this study was to revisit previously published results that suggested some sandflies to be arboreal and to rest on outlying plants by using Centers for Disease Control light traps to capture sandflies in vegetation, including banana plants and palmyra palm trees, in two previously sampled VL‐endemic Bihari villages. Over 3500 sandflies were trapped in vegetation over 12 weeks. The results showed the mean number of sandflies collected per trap night were significantly higher in banana trees than in other vegetation (P = 0.0141) and in female rather than male palmyra palm trees (P = 0.0002). The results raise questions regarding sandfly dispersal, oviposition and feeding behaviours, and suggest a need to refine current control practices in India and to take into account an evolving understanding of sandfly ecology.     

The roles of Phlebotomus martini and P. celiae (Diptera: Phlebotominae) as vectors of visceral leishmaniasis in the Aba Roba focus, southern Ethiopia

Abstract. During field studies (December 1988 to April 1990) to determine the vectors of visceral leishmaniasis (kala-azar) in the Aba Roba (Segen Valley) focus of southern Ethiopia, a total of 40,770 sandflies (Diptera: Psychodidae: Phlebotominae) were collected and identified as six species of Phlebotomus and seventeen Sergentomyia spp. Nine of these species were anthropophilic (four Phlebotomus and five Sergentomyia spp.), the dominant being Phlebotomus (Synphlebotomus) martini, P.(S.)celiae and Sergentomyia (Sergentomyia) schwetzi. P. celiae adult populations increased during the rainy season. Of 2326 P. martini and 1044 P. celiae dissected, the parous rate was 29.9% and 24.4%, respectively. Leishmania promastigotes were detected in sixteen P. martini (eleven identified as L. donovani by isoenzyme analysis and/or a DNA probe) and in three P. celiae (two identified by DNA probe), giving overall infection rates of 0.7% (1.9% parous infection rate) and 0.3% (1.2% parous infection rate), respectively. Four isolates from P. martini were typed by their isoenzyme profile as L. donovani zymodeme MON-37 = LON-44, identical to one of the zymodemes isolated from patients in this focus. This is the first evidence that P. celiae is a vector and the first time that P. martini has been implicated in Ethiopia. Based on observations of abundance, seasonality and prevalence of infection of the two Synphlebotomus spp., it is concluded that P. martini is the major vector and P. celiae a secondary vector in the Aba Roba focus. The risk of infection with L. donovani is greatest during the wet season.     

Protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for control of canine leishmaniasis

Abstract. Dog collars made of PVC plastic impregnated with the pyrethroid insecticide deltamethrin at 40 mg/g were investigated for their protective efficacy against phlebotomine sandflies. Collared dogs were kept separately (two untreated control dogs lived together) in outdoor enclosures, each with a kennel, in the Cevennes, southern France. To measure sandfly mortality and anti-feeding effects due to the deltamethrin-impregnated collars worn continuously by the dogs for up to 8 months, each dog was periodically sedated and exposed for 2h to 150–200 laboratory-reared Phlebotomus perniciosus females (plus c. 25 males) inside a net (1.2m square, 1.8 m high) indoors. After dogs were removed from the nets, allowed to recover and returned to their kennels, any dead sandflies were collected from inside the net and counted. Surviving flies were kept overnight, then scored according to whether they were still alive or dead, unfed or blood-fed. From tests 2, 3, 4, 13, 20, 26 and 34 weeks after the dogs began wearing collars, the overall numbers of blood-fed female sandflies recaptured were 75 from two dogs with collars, compared with 1911 from two collarless dogs. Thus, for every 100 flies which fed on collarless dogs, only 4 fed on collared dogs, i.e. the collars protected dogs from 96% of the bites and this activity was maintained for up to 34 weeks. During the same period, the percentage of recaptured female sandflies that had fed on collared dogs was 0–12% compared to 55–95% on collarless dogs. Immediately after dogs were taken out of the nets, 21–60% of flies confined with the collared dogs were found dead, compared to 0–12% with the controls. It is concluded that, at least in the Mediterranean subregion, this insecticidal collar would protect a dog from the majority of sandfly bites and retain a killing effect for a complete sandfly season. Moreover, it seems likely that the use of collars on all dogs in a focus of Leishmania infantum would reduce contact between sandfly vectors and canine reservoir hosts sufficiently to diminish the risk of infection for humans as well as dogs.     

Susceptibility status of the wild-caught Phlebotomus argentipes (Diptera: Psychodidae: Phlebotominae), the sand fly vector of visceral leishmaniasis,to different insecticides in Nepal

BackgroundVisceral leishmaniasis (VL) is targeted for elimination as a public health problem in Nepal by 2023. For nearly three decades, the core vector control intervention in Nepal has been indoor residual spraying (IRS) with pyrethroids. Considering the long-term use of pyrethroids and the possible development of resistance in the vector Phlebotomus argentipes sand flies, we monitored the susceptibility status of their field populations to the insecticides of different classes, in villages with and without IRS activities in recent years.Methodology/Principal findingsSand flies were collected from villages with and without IRS in five VL endemic districts from August 2019 to November 2020. The WHO susceptibility test procedure was adopted using filter papers impregnated at the discriminating concentrations of insecticides of the following classes: pyrethroids (alpha-cypermethrin 0.05%, deltamethrin 0.05%, and lambda-cyhalothrin 0.05%), carbamates (bendiocarb 0.1%) and organophosphates (malathion 5%). Pyrethroid resistance intensity bioassays with papers impregnated with 5× of the discriminating concentrations, piperonyl butoxide (PBO) synergist-pyrethroid bioassays, and DDT cross-resistance bioassays were also performed. In the IRS villages, the vector sand flies were resistant (mortality rate <90%) to alpha-cypermethrin and possibly resistant (mortality rate 90–97%) to deltamethrin and lambda-cyhalothrin, while susceptibility to these insecticides was variable in the non-IRS villages. The vector was fully susceptible to bendiocarb and malathion in all villages. A delayed knockdown time (KDT₅₀) with pyrethroids was observed in all villages. The pyrethroid resistance intensity was low, and the susceptibility improved at 5× of the discriminating concentrations. Enhanced pyrethroid susceptibility after pre-exposure to PBO and the DDT-pyrethroid cross-resistance were evident.Conclusions/SignificanceOur investigation showed that P. argentipes sand flies have emerged with pyrethroid resistance, suggesting the need to switch to alternative classes of insecticides such as organophosphates for IRS. We strongly recommend the regular and systematic monitoring of insecticide resistance in sand flies to optimize the efficiency of vector control interventions to sustain VL elimination efforts in Nepal.     

Sugar meals and longevity of the sandfly Phlebotomus papatasi in an arid focus of Leishmania major in the Jordan Valley

The sugar diet and life-span of Phlebotomus papatasi were studied in a typical zoonotic focus of Leishmania major in an arid area of the Jordan Valley during 1996-1997. Plant-tissue residues (cellulose particles) were identified in the stained guts of 23% of P. papatasi and significant amounts of sugar were found in the gut of 16%. Feeding on different plants was demonstrated by using their branches, suffused with cellulose stain, as baits in the field. Ingested, stained cellulose was detected in 10% of the sandflies (6% of males, 12.5% of females) caught near bait-branches of common local plants, mostly Chenopodiaceae. The similar rates of plant and sugar feeding, with the observed absence of aphids (ruling out the availability of honeydew), implied that the sugar meals of sandflies were obtained directly from plants. The relative paucity of sugar meals in P. papatasi coincided with a short life-span, evaluated by daily growth lines in the cuticle. The age of the oldest females was estimated to be 8 days, and 6 days for males. Under local conditions, the first gonotrophic cycle can be completed in 6 days and the usual transmission of L. major is apparently afterwards, when females ingest blood to initiate another cycle. Only about 9% of P. papatasi females survived > 6 days.     

Two populations of Phlebotomus ariasi in the Cévennes focus of leishmaniasis in the south of France revealed by analysis of cuticular hydrocarbons

Two distinct populations of Phlebotomus ariasi Tonnoir have been identified in the Cévennes focus of leishmaniasis in the south of France using gas-liquid chromatography (GLC) of cuticular hydrocarbons extracted from individual dried female flies. Results were obtained after analysis of flies collected from CDC light traps from a domestic and a sylvatic site separated by a distance of 900 m. Flies were provided for GLC analysis as six blind samples. Using cluster and discriminant analysis techniques, five of the samples were shown to form two distinct groups, while a sixth was identified as a mixture. These findings were subsequently confirmed to correspond exactly with the way the samples had been presented. Samples grouped together on the basis of the flies' cuticular hydrocarbon profiles had been taken from the same site, while the ungrouped sample had been deliberately mixed. Using a jack-knifed estimator, it is shown that specimens can be correctly allocated to the population to which they belong with a 92% success rate. These results confirm the value of the technique for the identification of populations of medical vectors. The implication of the findings for the epidemiology of leishmaniasis is discussed with special reference to the need to determine if both populations are vectors and to study differences in behaviour. In addition to adults, profiles of larval sandflies have been obtained for the first time.     

Polymerase chain reaction‐based assay for the detection and identification of sand fly gregarines in Lutzomyia longipalpis,a vector of visceral leishmaniasis

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly host‐specific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina).