The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major

Abstract

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

Keywords: Leishmania; microbiota; osmotic stress; sand flies; vector competence.

Figure 1

Effect of penicillin/streptomycin (P/S) on L. major development in P. duboscqi. Flies were artificially fed through a membrane on mouse blood seeded with 4×10⁶/ ml LmRy promastigotes and dissected at the indicated times post-infection and scored for the number and developmental stage of promastigotes under a hemocytometer. Flies were treated or not with (A) P/S in the blood and sugar meals, (B) P/S in the blood and/or sugar meals, (C) Quantification of ear dermal parasite loads in BALB/c mice 4 weeks after exposure to the bites of sand flies infected 2 weeks previously by artificial feeding on mouse blood seeded with 4×10⁶/ ml LmRy promastigotes and treated or not with P/S in the blood and sugar meals. Values shown are parasite numbers per individual ears with geometric means ± 95% confidence interval (CI). Data shown in (A) are representative of more than 5 independent experiments, (B) 3 independent experiments, (C) one experiment involving the same population of infected flies.

Figure 2

Changes in the size and diversity of bacterial communities in P. duboscqi following infection and treatment with P/S. Flies were artificially fed through a membrane on mouse blood seeded with 4×10⁶/ ml LmRy promastigotes and treated or not with P/S in the blood and sugar meals. (A) Dissected midguts at 14 days p.i. were cultured on TSA and MacConkey agar. Bar graphs show mean CFUs + SD, 10 midguts/group. (B) Culture dependent, and (C) culture independent analysis of the gut bacteria at 14 days p.i. based on PCR amplification and cloning of the 16S DNA prepared from pools of 10 flies in each group.

Figure 3

Effect of antibiotic treatment and/or bacterial feeds on the development of metacyclic promastigotes in P. duboscqi. Flies were artificially fed through a membrane on mouse blood seeded with 4×10⁶ / ml LmRy promastigotes. Flies were treated or not with P/S in the blood and sugar meals, and fed or not on blood meals containing 1000 CFU / ml of (A) Leifsonia, (B) Rahnella aquatilis, (C) Enterobacter cloacae, and (D) Seratia rubidaea. Midguts were dissected at 13-15 days p.i. except for flies infected with Rahnella that were dissected 19-20 days p.i., and scored for promastigote numbers and developmental stages under a hemocytometer. D also shows groups of flies that were treated with P/S and fed on Seratia rubidaea (1000 CFU / ml) in the sugar meal at 6 days and 3 days, respectively, prior to blood feeding and infection. Values shown are numbers of metacyclic promastigote per individual flies with geometric means ± 95% CI. Data shown in (A) is one of two representative independent experiments, (B) pooled data from two independent experiments, (C) one of three representative experiments, (D) one of two representative experiments.

Figure 4

Population size and diversity of bacterial communities in infected P. duboscqi that were fed live bacteria. Midgut homogenates from infected control or antibiotic treated flies fed on (A) Rahnella aquatilis and dissected at 19 days p.i., or fed on (B) Enterobacter cloacae and dissected at 14 days p.i, were cultured on LB agar. Values shown are CFUs per individual midgut with geometric means ± 95% CI. Culture independent analysis of the gut bacteria in flies fed on (C) Rahnella aquatili, and recovered 19 days p.i, or (D) Enterobacter cloacae, and recovered 14 days p.i, based on 16S amplicon sequencing of cDNA prepared from pools of 10 flies in each group.

Figure 5

Effect of bacterial culture supernatants and replete medium on the development of metacyclic promastigotes in P. duboscqi. Flies were artificially fed through a membrane on mouse blood seeded with 4×10⁶ / ml LmRy promastigotes. Beginning on day 5 p.i., flies were treated or not with P/S and/or culture supernatant from Ochrobactrum anthropi and/or Rahnella aquatilis, in the sugar meal (A & B), or fed on M199 or Grace’s Insect Medium supplemented with BME, folate, hemin, and adenosine with or without P/S (C&D). Values shown are numbers of metacyclic promastigote per individual flies with geometric means ± 95% CI on days 13-14 p.i. Data shown in A,B, &D are each representative of two independent experiments; C is representative of three independent experiments.

Figure 6

Effect of reduced sucrose concentration on the development of metacyclic promastigotes in P. duboscqi. Flies were artificially fed through a membrane on mouse blood seeded with 4×10⁶ / ml LmRy promastigotes. Beginning on day 5 p.i., flies were treated or not with P/S in sugar meals composed of (A) 30% or 15% sucrose, or (B) 30% or 10% sucrose. Values shown are numbers of metacyclic promastigote per individual flies with geometric means ±95% CI on days 13-14 p.i. Data in (A) and (B) are representative of three and two independent experiments, respectively.

Figure 7

Survival and differentiation of L. major promastigotes cultured in sucrose solutions in vitro. LmRy promastigotes were cultured at 26°C in complete medium 199 to mid-logarithmic phase, washed × 3 in PBS and resuspended to 2 × 10⁸ / ml in an isosmotic sucrose solution (10.4%). Ten μl of the parasite suspension was added to 500 μl of different concentrations of sucrose in microwell plates, and the number and developmental stage of viable promastigotes were scored under a hemocytometer after different times in culture at 26°C. Data shown are the total number of viable promastigotes (A) or percent metacyclics (B). The data are representative of three independent experiments.