Significance of bacteria in oviposition and larval development of the sand fly Lutzomyia longipalpis

Abstract

Background: Microbial ecology of phlebotomine sand flies is not well understood although bacteria likely play an important role in the sand fly biology and vector capacity for Leishmania parasites. In this study, we assessed the significance of the microbial community of rabbit feces in oviposition and larval development of Lutzomyia longipalpis as well as bacterial colonization of the gut of freshly emerged flies.

Methods: Sterile (by autoclaving) and non-sterile (control) rabbit feces were used in the two-choice assay to determine their oviposition attractiveness to sand fly females. Bacteria were identified by amplification and sequencing of the 16S rRNA gene with universal eubacterial primers. Sterile, control (non-sterile), and sterilized and inoculated rabbit feces were used to assess the significance of bacteria in L. longipalpis development. Newly emerged adult flies were surface-sterilized and screened for the bacterial population size and diversity by the culturing approach. The digestive tract of L4 sterile and control larvae was incubated with Phalloidin to visualize muscle tissues and DAPI to visualize nuclei.

Results: Two-choice behavioural assays revealed a great preference of L. longipalpis to lay eggs on rabbit feces with an active complex bacterial community (control) (85.8 % of eggs) in comparison to that of sterile (autoclaved) rabbit feces (14.2 %). Bioassays demonstrated that L. longipalpis larvae can develop in sterile rabbit feces although development time to adult stage was greatly extended (47 days) and survival of larvae was significantly lower (77.8 %) compared to that of larvae developing in the control rabbit feces (32 days and 91.7 %). Larval survival on sterilized rabbit feces inoculated with the individual bacterial isolates originating from this substrate varied greatly depending on a bacterial strain. Rhizobium radiobacter supported larval development to adult stage into the greatest extent (39 days, 88.0 %) in contrast to that of Bacillus spp. (76 days, 36.0 %). From the complex natural bacterial community of rabbit feces, R. radiobacter survived pupation and colonized the newly emerged females most successfully (82.6 % of all bacteria cultured); however, only 25 % of females were positive for bacteria in the digestive tract upon emergence. Immunohistochemistry did not reveal any obvious differences in anatomy of the digestive tract between control and axenic larvae.

Conclusions: The bacterial community in the sand fly larval habitat affects oviposition and larval development although bacteria are not essential for successful development of L. longipalpis. Different bacteria contribute to larval development to various degrees and some, e.g. Rhizobium radiobacter, survive pupation and colonize the digestive tract of newly emerged females. With the establishment of the axenic rearing system, this study opens new venues to study the effect of bacteria on the gut epithelial immunity and vector competence of sand flies for Leishmania parasites with a goal to develop paratransgenic approaches for Leishmania control.

Figure 1

Sand fly oviposition preference for the control and sterile rabbit feces (RF). Thirty-nine ovipots (X axis) containing sterile and non-sterile rabbit feces were used for the analyses.

Figure 2

Development time (mean + SEM) of sand flies reared on sterile rabbit feces inoculated with various bacterial isolates. RF rabbit feces; M. m. - Morganella morganii; P. sp. KK-2 - Pseudomonas sp. KK-2; P. sp. KK-1 - Pseudomonas sp. KK-1; R .r. - Rhizobium radiobacter; E. g. - Enterococcus gallinarum; B. sp. KK-1 - Bacillus sp. KK-1. Different letters above error bars indicate significant differences (P ≤ 0.05).

Figure 3

Schematic drawing of L4 larva digestive tract, salivary glands, fat body, and Malpighian tubules.

Figure 4

Immunohistochemistry of the larval (L4) digestive tract: Control larva: (A1) regular light, (A2) DAPI staining, (A3) phalloidin staining; Axenic larva: (B1) regular light, (B2) DAPI staining, (B3) phalloidin staining.

Figure 5

Diversity and prevalence of bacteria in the digestive tract of newly emerged sand flies that developed in control (A) and sterile rabbit feces inoculated with a mixture of six bacterial isolates (B).