Laboratory-reared Sand Flies Research Articles

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

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

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts

BackgroundIn eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts.MethodsField-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve.ResultsThe qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10−3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents.ConclusionsThis study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Establishment of a Colony of Phlebotomus argentipes under Laboratory Conditions and Morphometric Variation between Wild-Caught and Laboratory-Reared Populations.

The field-based studies on sand flies are not adequate to uncover information required for the control of the leishmaniasis through reduction of vector populations. Therefore, establishment and maintenance of laboratory colonies of sand flies is an essential step in leishmaniasis research. In the current study, a colony of P. argentipes was established from wild-caught sand flies following standard procedures from the published literature. Morphological measurements of laboratory-reared and wild-caught individual sand flies were compared to assess the difference between two groups. The colony was successfully established under confined laboratory conditions. The comparison of morphometric parameters revealed that the laboratory-reared sand flies are significantly larger than those caught from wild, suggesting a possibility of increased fitness of sand flies under favorable environmental conditions which may cause higher prevalence in the disease. The current study reports the first successful attempt in colonizing sand flies under laboratory conditions. However, the colony data suggest that the conditions extracted from the published literature need to be optimized to suit local settings in order to achieve maximum population sizes within the available amount of resources.

Experimental infection of Phlebotomus perniciosus and Phlebotomus tobbi with different Leishmania tropica strains

Cutaneous leishmaniasis due to Leishmania tropica is increasingly documented in Europe and the Middle East. Besides its specific vector, Phlebotomus sergenti, permissive Phlebotomus sand flies are suspected as potential vectors of L. tropica. We investigated the susceptibility of two widely distributed species, Phlebotomus perniciosus and Phlebotomus tobbi. Laboratory-reared sand flies were infected experimentally with L. tropica strains differing in lipophosphoglycan epitopes, geographical distribution and epidemiology. High infection rates, heavy parasite loads and fully developed late-stage infections including colonization of the stomodeal valve were observed in all parasite-vector combinations. Our findings demonstrate that P. perniciosus and P. tobbi are susceptible to different L. tropica strains and may play a role in their circulation in endemic foci of Europe, the Middle East and North Africa.

Laboratory transmission of an Asian strain of Leishmania tropica by the bite of the southern European sand fly Phlebotomus perniciosus

Imported cases of anthroponotic cutaneous leishmaniasis due to Leishmania tropica are increasingly documented in Europe. We investigated the ability of Phlebotomus perniciosus, a competent vector of Leishmania infantum widespread in southwestern Europe, to support the growth and transmissibility of an Asian strain of L. tropica recently isolated from a refugee. Parasite growth behavior was investigated in laboratory-reared sand flies fed artificially with promastigotes as well as in sand flies infected after biting on footpad lesions induced in hamsters by promastigote inoculation. The evolution of infection was checked by gut microscopy and quantitative real-time PCR, and it was found to be similar between promastigote- and amastigote-initiated infections. In 80% of infected sand flies, despite survival and flourishing growth of promastigotes after blood digestion and defecation, either the parasites died, or failed to migrate to the foregut and/or to mature into infective forms. However, in the remaining 20% L. tropica developed into abundant metacyclic promastigotes. The quantitative real-time PCR assay detected variable loads of gut promastigotes irrespective of morphological evidence of viability or progressive/final death. Parasite transmissibility was investigated by exposing naive hamsters to P. perniciosus previously infected on chronic lesions induced in hamsters which survived to take a second blood meal. Two months post exposure, lesions developed in skin sites bitten by sand flies confirmed to harbor metacyclic promastigotes; in the following months, the presence of viable and transmissible L. tropica parasites in lesions was demonstrated by xenodiagnosis assays. Our findings support the hypothesis that, in particular epidemiological situations, P. perniciosus may play the role of an occasional L. tropica vector.

Leishmania major-Phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes.

To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut lysates have shared epitopes. A laboratory based study. Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x 10(6) amastigotes in 20 microl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100 microl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100 microl of 1:100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37 degrees C and growth at 26 degrees C, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms. 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (p<0.05). The control cultures or sand flies that fed on amastigotes alone and no MABS supported full parasite development up to the metacyclic stage. Results also showed that flies, which had fed on MABS, showed low parasitemia levels of 2+, compared to a high density of 4+ for their controls (p<0.5). These findings showed that anti-LPG MABS were effective in reducing sand fly infections. This study also showed that P. duboscqi gut lysates and proteins present in L. major-derived LPG share two common proteins of molecular weights 105 kDa and 106 kDa. Further analysis of these individual proteins from the gut should be studied with a view of determining their vaccine potential.

Development of Endotrypanum (Kinetoplastida:Trypanosomatidae) in experimentally infected phlebotomine sand flies (Diptera:Psychodidae).

The developmental biology (parasite establishment, migration, and differentiation) of Brazilian strains of Endotrypanum are reported for 3 sand fly species: Lutzomyia longipalpis Lutz & Neiva, L. shannoni Dyar, and Phlebotomus papatasi Scopoli. Laboratory-reared sand flies were infected by feeding on a promastigote suspension through a chick-skin membrane. Infections within the insect gut were examined at various times after feeding by staining fresh and fixed specimens. Development of Endotrypanum varied for each parasite-host species association. After feeding on culture forms of E. schaudinni Mesnil & Brimont (strain ISHA/BR/80/IM1111), significantly more L. shannoni (100%, 9/9) became infected than did L. longipalpis (62.3%, 33/53) or P. papatasi (27.3%, 15/55). The greatest number of infections were in the midgut and hindgut from 6 to 16 d after feeding, but flagellates also were present in the Malpighian tubules. Moreover, distinct development patterns in the sand fly gut were obtained when the Callejon L. longipalpis colony was fed on cultures of other Endotrypanum strains. Significantly fewer sand flies became infected with strain MCHO/BR/85/IM2259 (18.2%, 4/22) than with strain ISHA/BR/80/IM1111 (55.6%, 20/36). There were also individual variation in the distribution and survival of parasites within the guts of flies in each group. These data indicate that there is variation in the susceptibility to infection with Endotrypanum among and within sand fly species.

Natural infection of humans, animals, and phlebotomine sand flies with the Alagoas serotype of vesicular stomatitis virus in Colombia.

Five isolations of the Alagoas serotype of vesicular stomatitis virus (Rhabdoviridae: Vesiculovirus) were made from naturally infected phlebotomine sand flies (Lutzomyia spp.) collected in Colombia. These are the first isolations of Alagoas virus from an arthropod. Replication of the virus occurred in laboratory-reared sand flies (Lutzomyia longipalpis) after inoculation. Bite and transovarial transmission of the virus was also demonstrated in experimentally infected sand flies. Alagoas virus neutralizing antibodies were found in sera of humans and animals living near the insect collection site; antibody rates among human residents of two nearby towns were 63% and 83%, respectively. Results of comparative serologic studies demonstrated that Alagoas virus is closely related antigenically to Indiana, Cocal, and Maraba viruses and that these four agents form a complex within the vesicular stomatitis virus serogroup. The antigenic similarity among these four viruses makes their differentiation difficult; it also raises doubts about the accuracy of current laboratory methods used for identifying isolates in this serogroup. A discussion follows on the significance of human antibodies to these agents and on the role of sand flies in their ecology.

Leishmania in kenyan phlebotomine sandflies—IV: Artificial feeding and attempts to infect six species of laboratory-reared sandflies with Leishmania donovani

Five ( Sergentomyia antennatus, S. garnhami, S. schwetzi, S. ingrami and S. adleri ) of the six species of sandflies fed on mammalian blood containing cultured Leishmania donovani promastigotes on day 0, were able to feed through cockerel skin membranes at 5.5, 10, 34.3, 45.7 and 95% feeding rates, respectively. Sergentomyia bedfordi was able to feed on mammalian blood only through a lizard skin membrane at 100% feeding rate. Promastigotes were found in 9.3, 25 and 37.5% of S. schwetzi, S. adleri and S. ingrami respectively, dissected on days 3–9. The results open way for artificial feeding of Kenyan sandflies on mammalian blood through membranes, and indicate that it is necessary to select the type of membrane depending on sandfly species. The ability of L. donovani to develop in Kenyan sandflies is being investigated to experimentally establish and confirm the importance of various genera and species in the epidemiology of leishmaniasis.

Studies on the Biology of Phleboviruses in Sand Flies (Diptera: Psychodidae)

This paper describes a series of experiments which were done to determine the behavior of 14 different phleboviruses in laboratory-reared sand flies (Phlebotomus papatasi, P. perniciosus and Lutzomyia longipalpis) after oral and parenteral infection. Most of the viruses replicated in the sand flies after intrathoracic inoculation; however, the insects were quite refractory to oral infection. Six of 11 phleboviruses tested were transovarially transmitted in one or more sand fly species. The percentage of infected F1 offspring produced by parenterally infected female parents ranged from 1.5-60%, depending on the virus type used. These data support the hypothesis that some of the phleboviruses are maintained in sand flies by transovarial transmission.

Susceptibility of sand flies (Diptera: Psychodidae) to Trypanosomatidae from two-toed sloths (Edentata: Bradypodidae).

Xenodiagnostic trials consisted of 1665 laboratoryreared sand flies (Lutzomyia sanguinaria, Lu. gomezi, and Lu. trapidoi) in 114 lots fed on 72 Two-toed Sloths (Choloepus hoffmanni). Fifty-eight (80.6%) of the sloths had trypanosomatid infections as follows: Leishmania braziliensis, 20 (27.8%); Endotrypanum schaudinni, 39 (54.2%); Trypanosoma rangeli, 15 (20.8%); and T. Cruzi, 1 (1.4%), as determined by the biopsy-culture technique. Nineteen animals (26.4%) had multiple infections. Seven (7.7%) of 91 Lu. sanguinaria, which fed on 7 animals infected with Le. braziliensis, acquired the parasite. A total of 187 (31.1%) of 601 sand flies of 3 species developed flagellates after feeding on 21 animals infected with E. schaudinni. Only 1% of 292 flies developed parasites after feeding on 8 sloths with T. rangeli.

Neotropical Sand Flies (Diptera: Psychodidae), Invertebrate Hosts of Endotrypanum Schaudinni (Kinetoplastida: Trypanosomatidae)1

Trypanosomatid flagellates were recovered from 58 (81%) of 72 Panamanian sloths by using the biopsyculture technique; 21 (29%) of the animals were infected with Endotrypanum schaudinni to the exclusion of other trypanosomatids. Three species of laboratory-reared sand flies, Lutzomyia sanguinaria, L. gomezi and L. trapidoi , acquired E. schaudinni from the sloths during xenodiagnostic studies. Infections in the sand flies were verified in dissections from 1 to 23 days following acquisition feedings. The flagellates, all of which appeared to be promastigotes, were distributed from the cardia to the rectal ampula. Highest concentrations of flagellates were found consistently in the pylorus. Ovoid motile and nonmotile forms, attached to the gut wall, dominated the infections. Lesser numbers of elongated free-swimming forms were present in the gut lumen of most infected flies. Laboratory and field data indicate that several species of Lutzomyia are involved in the transmission cycle of this parasite.

Isolation of phlebotomus fever virus from Phlebotomus papatasi.

Summary A total of 24,179 female Phlebotomus flies collected in suburban Cairo, Egypt, were examined for phlebotomus fever virus. Virus was recovered from 4 of 59 sandfly pools tested; one pool of Phlebotomus papatasi and 3 pools of unindentified Phlebotomus, presumably consisting solely of P. papatasi, yielded virus. All four isolates were identified by complement-fixation tests as the Sicilian type of virus. Wild-caught engorged P. papatasi flies were shown to contain substances which neutralize the Sicilian type of phlebotomus fever virus. Failure to demonstrate similar activity in laboratory-reared sandflies suggests that the active principle is antibody contained in ingested human blood.