Evaluation of sugar meal administered anti-Leishmania compounds on the vectorial capacity of the vector, Lutzomyia longipalpis

Abstract

Multiple strategies involving the parasite-host-vector triad are necessary to control leishmaniasis. One option is to prevent or reduce transmission of the pathogen by the phlebotomine sand fly vectors. In this sense, it is essential to explore compounds that may influence the vectorial capacity of the insect and reduce its longevity. We investigated the effect of anti-Leishmania drugs administered via the sugar meal on longevity, blood feeding, oviposition, and parasite load on the third day of infection of the sand flies, to identify the most promising candidates for vector infection tests. We identified compounds that affected the longevity of sand flies (three pterocarpanquinones - LQB-475, LQB-181, and LQ-03; one hydroxyethylpiperazine, PMIC-4, and Pentamidine), reduced oviposition of females after blood feeding (LQB-181 and PMIC-4), but did not decrease infection rates or parasite loads. The results provide the effect of antiparasitic drugs from the perspective of the insect vector.

Fig 1. Effect of anti-Leishmania drugs added to a sugar diet on the longevity of Lu. longipalpis males.

Insects were kept under controlled conditions (26 ºC and 70% humidity), and mortality was checked daily. In the control group, insects were fed with a solution containing only sucrose 70% (w/v). All results are the mean ± SEM. Comparison test between log-rank survival curves (Mantel-Cox). Experiments were performed at least three times independently, with n = 20 each. See Material and Methods and Table 2 for details.

Fig 2. Effect of anti-Leishmania drugs added to a sugar diet on the longevity of Lu. longipalpis females.

Insects were kept under controlled conditions (26 ºC and 70% humidity), and mortality was checked daily. In the control group insects were fed with sucrose 70% (w/v). All results are the mean ± SEM. Comparison test between log-rank survival curves (Mantel-Cox). Experiments were performed at least three times independently, with n = 20 each. See Material and Methods and Table 2 for details.

Fig 3. Effect of anti-Leishmania compounds added to a sugar diet on females of Lu. longipalpis pre and post-blood feeding.

(a) Mortality of females before blood feeding. These females were offered the anti-Leishmania compound in a sugar diet for seven days before blood feeding. 7d PE: 7 days post-emergence. (b) Percentage of females that are not engorged after blood feeding. Females were exposed to the source of blood for 30 min. (c) Mortality of females three days after blood feeding. Females continued to be offered anti-Leishmania compounds in the sugar diet after blood feeding. 3d PBF: 3 days post-blood feeding. All percentage results are the mean ± SEM. For LQB-181, 5 replicates were performed, 1 with 30 insects and 4 with 50 insects each. For Controls and PMIC-4, 5 replicates with 30 insects each, and for the remaining compounds, 3 replicates with 30 insects each were performed. Dunn’s multiple comparison test, ** p = 0.007.

Fig 4. Effect of anti-Leishmania compounds on fecundity of females and egg fertility.

The compounds were added to a sugar diet and offered to females seven days before blood feeding and three days after when females were separated for oviposition. (a) Fecundity (eggs laid per female). The number of eggs was accessed seven days after oviposition. (b) Fertility (whether the eggs hatched or not). The hatching was evaluated 14 days after oviposition. The percentage results consider the total number of eggs hatching or not in each replicate (three to four repetitions). Different letters beside the bars indicate groups that are statistically different from each other, according to Fisher’s exact test (p < 0.05). For DMSO and PMIC-4, 4 replicates with 30 insects were performed. For LQB-181, 4 replicates with 50 insects were performed. For the other compounds, 3 replicates with 30 insects each were performed.

Fig 5. Quantification of ingested proteins by females after 30 minutes of blood feeding.

Females were previously fed with a sugar solution containing anti-Leishmania compounds for seven days. Proteins were quantified from the blood present in the intestinal contents of females immediately after blood feeding, using the BCA method. The percentage results are the mean ± SEM of three to five (LQB-181 and PMIC-4) biological replicates. Dunn’s multiple comparisons test, **p < 0.01. Each symbol (square, triangle, diamond or circle) shows the measurement from one individual insect.

Fig 6. Influence of anti-Leishmania compounds added to the sugar diet on the infection of Lu. longipalpis with Le. amazonensis.

Parasite counts were measured on the 3rd, 5th, and 7th day after infection. Significant differences were investigated by Dunn’s multiple comparisons test and Mann-Whitney test.