Leishmania manipulation of sand fly feeding behavior results in enhanced transmission

Abstract

In nature the prevalence of Leishmania infection in whole sand fly populations can be very low (<0.1%), even in areas of endemicity and high transmission. It has long since been assumed that the protozoan parasite Leishmania can manipulate the feeding behavior of its sand fly vector, thus enhancing transmission efficiency, but neither the way in which it does so nor the mechanisms behind such manipulation have been described. A key feature of parasite development in the sand fly gut is the secretion of a gel-like plug composed of filamentous proteophosphoglycan. Using both experimental and natural parasite-sand fly combinations we show that secretion of this gel is accompanied by differentiation of mammal-infective transmission stages. Further, Leishmania infection specifically causes an increase in vector biting persistence on mice (re-feeding after interruption) and also promotes feeding on multiple hosts. Both of these aspects of vector behavior were found to be finely tuned to the differentiation of parasite transmission stages in the sand fly gut. By experimentally accelerating the development rate of the parasites, we showed that Leishmania can optimize its transmission by inducing increased biting persistence only when infective stages are present. This crucial adaptive manipulation resulted in enhanced infection of experimental hosts. Thus, we demonstrate that behavioral manipulation of the infected vector provides a selective advantage to the parasite by significantly increasing transmission.

Figure 1. Metacyclogenesis and fPPG Synthesis in L. mexicana and L. infantum

(A) Flies were infected by membrane feeding with amastigotes of L. mexicana (filled squares) or L. infantum (filled circles), ten flies/group harvested daily, and the average number of metacyclics/fly determined. Error bars represent 1 s.e.m. (B) Accumulation of fPPG within the midguts of infected sand flies. Midgut homogenates from flies infected with L. mexicana (upper panel) or L. infantum analyzed by SDS-PAGE and immunoblotting using AP3 mAb to detect the presence of fPPG glycans. Lane C is a day 7 uninfected blood-fed control sample. (C) Metacyclogenesis in sand flies infected with L. mexicana exponential phase– (closed squares) or stationary phase– (half squares) cultured amastigotes. Error bars represent 1 s.e.m. Asterisks indicate statistically significant differences (p < 0.05) from stationary infections using an unpaired t-test. (D) PSG accumulation in sand flies infected with L. mexicana exponential phase (upper panel) or stationary phase (lower panel) amastigotes analyzed by SDS-PAGE and immunoblotting using AP3.

Figure 2. Kaplan–Meier Plots Showing Survival of Lutzomyia longipalpis Under Various Experimental Conditions.

(A) Normal insectary conditions. Flies were fed by membrane feeding on rabbit blood alone (filled squares), blood containing L. mexicana lesion amastigotes (open triangle), or L. infantum splenic amastigotes (open circle). Groups of 300 fed female flies were monitored daily for the occurrence of new deaths. (B) Stress conditions. The experiment was performed as in (A), except that flies were stressed on a daily basis by incubating them at 16 °C without a sugar meal for 2 h and forcing them to fly during a 30-min period by continuously agitating the cage. (C) Effect of L. mexicana developmental rate on survival of sand fly vector. Flies were infected with L. mexicana exponential phase– (up-ended open triangle) or stationary phase– (down-ended open triangle) cultured amastigotes and maintained under normal insectary conditions. (D) The experiment was performed as in (C), except that flies were stressed on a daily basis as described for (A).

Figure 3. Feeding Persistence of Sand Flies

(A) Relationship between numbers of metacyclic promastigotes in Lu. longipalpis and feeding persistence. Flies were infected with L. mexicana (closed circle) or L. infantum (open triangle) amastigotes. From 4 d post-infection flies were assayed for their feeding persistence in a 10-min behavioral assay and then dissected to determine the total number of metacyclic promastigotes present. The feeding persistence of each individual fly is plotted against the number of metacyclic promastigotes they harbored. Linear correlation coefficient, r ² = 0.504 for L. mexicana (L. m), and 0.705 for L. infantum (L. i). (B) Feeding persistence of L. infantum–infected flies fed rabbit blood alone (open square) or infected with L. infantum amastigotes (closed square). The persistence of flies exposed individually to an anaesthetized mouse was observed daily. n = 16 sand flies per point, representing the combination of two independent experiments. Error bars 1 s.e.m. (C) Feeding persistence of L. mexicana–infected flies, experimental design as in (B). Flies were fed rabbit blood alone (open square) or infected with L. mexicana (closed circle). (D) Effect of Leishmania development on sand fly feeding persistence, experimental design as in (B). Flies were infected with exponential (closed square) or stationary (half square) phase L. mexicana-cultured amastigotes. Asterisks indicate values from exponential and stationary infections that are statistically significant (*p < 0.05, **p < 0.005) from blood-fed control flies using an unpaired t-test.

Figure 4. Feeding Persistence and Transmission of Leishmania to Multiple Hosts

Flies were fed blood alone (open square), L. infantum (dotted square), L. mexicana stationary phase (shaded square), or exponential phase amastigotes (closed square). The number of flies feeding on a different host when interrupted was determined on days 5, 7, and 10 post-infection/feeding in a behavioral assay. n = 12 per point, representing the combination of two independent experiments. Error bars 1 s.e.m. Asterisks indicate values that are statistically significant (*p < 0.05, **p < 0.005) from blood fed control flies using an unpaired t-test.

Figure 5. Feeding Persistence and Infectivity

(A) Infectivity of fly-derived L. mexicana metacyclic promastigotes. Flies were infected with exponential phase or stationary phase L. mexicana-cultured amastigotes, infections allowed to mature (day 7), then metacyclic promastigotes were obtained, washed, and 10³ parasites injected into the right foot of each BALB/c mouse. Data from two groups of six mice are shown, infected with metacyclic promastigotes from exponential phase– (closed square) or stationary phase– (half-square) derived infections. (B and C) Influence of feeding persistence on Leishmania infection in mice. Flies were infected with exponential (closed square or open square) or stationary phase (closed circle or open circle) L. mexicana-cultured amastigotes. Day 7–infected flies were exposed individually to the right paw of anaesthetized BALB/c mice and allowed to feed for 15 min with ([B], closed circle or closed square) or without ([C], open square or open circle) interruption.