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. 2013 Apr 25;7(4):e2187.
doi: 10.1371/journal.pntd.0002187. Print 2013.

Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani

Affiliations

Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani

Veronika Seblova et al. PLoS Negl Trop Dis. .

Abstract

Background: Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ).

Methodology/principal findings: Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (×ばつ10(3) per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding.

Conclusions: Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 μl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of nutrition on the life cycle of two P. orientalis colonies.
Data originate from the offspring of about 4,600 ovipositing females (2,200 MW and 2,400 AZ) during a 3 month period. 1A: On the non-autoclaved food the number of adults emerging from pupae peaked on week 8 PBM in MW, and week 9 PBM in AZ. All individuals completed the life cycle within 13 and 20 weeks for MW and AZ, respectively. 1B: On the autoclaved food the life cycle was prolonged and the larval growth appeared less synchronized in both colonies. The impact was more significant in the AZ colony: emergence of AZ adults peaked on week 13 (four weeks later than on non-autoclaved food).
Figure 2
Figure 2. RAPD analysis of two P. orientalis colonies.
RAPD analysis was based upon PCR results using five random primers (OPI12, 13, OPO20, OPE16, OPL5; in total 58 characters), electrophoretogram for OPL5 is shown as an example. Dendrogram was constructed by the Neighbor-joining method.
Figure 3
Figure 3. Development of L. donovani (GR 374) in females of two P. orientalis colonies.
Sand flies were infected by feeding on a suspension of 105 promastigotes/ml of blood and kept at 26°C. 3A: Infected females of P. orientalis were examined microscopically 2, 5–6 and 8–11 days post-bloodmeal (PBM). The infection intensities were classified into three categories according to their intensity: heavy (more than 1,000 parasites per gut [black]), moderate (100–1,000 parasites [grey]) and light (1–100 parasites [white]). Numbers above the bars indicate the number of dissected females. 3B: Parasite numbers from 40–50 individual females were quantified by Q-PCR targeted on amplification of Leishmania kDNA 10 days PBM.
Figure 4
Figure 4. Effect of initial infective dose on development of L. donovani (GR 374) in P. orientalis.
4A: Infected females of P. orientalis (MW colony) were examined microscopically 2–3, 6 and 10 days post-bloodmeal (PBM). The infection intensity was classified as described in Fig. 3. 4B: Parasite numbers were determined using Q-PCR at 10 days PBM. Twenty females were used per group.

References

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