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

Abstract

BackgroundMicrobial 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.MethodsSterile (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.ResultsTwo-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.ConclusionsThe 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 f...

Diversity of bacteriome associated with Phlebotomus chinensis (Diptera: Psychodidae) sand flies in two wild populations from China

“…Interestingly, in L. longipalpis , an isolate of Pseudomonas , Pseudomonas sp. KK-1, has been shown to support larval development and to be able to pass through pupation and remain in the digestive tract of newly emerged females17. Taxa in Enterobacteriaceae are commonly associated with sand flies, such as Serratia, Enterobacter, Pantoea, Klebsiella , and Citrobacter 781112141731.…”

“…perfiliewi and Lutzomyia longipalpis 78910111213141516. Regarding the microbial contributions to the physiology of sand flies, gut bacteria have been shown to play roles in development17 and immunity1819. In addition, evidence has become available that resident microorganisms in the gut are able to inhibit Leishmania infections2021.…”

Phylogeographic Structure in Anastrepha ludens (Diptera: Tephritidae) Populations Inferred With mtDNA Sequencing

“…In insects, M. morganii was found in the intestines of house fly larvae (Musca domestica [Diptera: Muscidae]) (Zurek et al 2000), gastrointestinal tracts of Drosophila melanogaster (Diptera: Drosophilidae) (Cox & Gilmore 2007), adults and eggs of horn fly (Haematobia irritans [Diptera: Muscidae]) (Palavesam et al 2012), midgut of phlebotomine sand fly (Lutzomyia longipalpis [Diptera: Psychodidae]) (Gouveia et al 2008;Peterkova-Koci et al 2012), gastrointestinal tracts of the Mediterranean fruit fly (Ceratitis capitata [Diptera: Tephritidae]) (Ami et al 2009;Yuval et al 2013), in isolations made from the whole body of the guava fruit fly (Anastrepha striata [Diptera: Tephritidae]) (Martinez et al 2012), and in the gastrointestinal tracts of the oriental fruit fly (Bactrocera dorsalis [Diptera: Tephritidae]) (Pramanik et al 2014;Liu et al 2016). To our knowledge, no pathogenicity tests were conducted in any of these studies on fruit fly species, especially in regards to fruit fly larvae.…”

“…This implies that a tiny, point contamination of diet by Morganella could cause the dark rings of death. Only 1 other study has found a Morganella isolate pathogenic to insects: the larvae of phlebotomine sand flies L. longipalpis (Lutz and Neiva) (Peterkova-Koci et al 2012). Unfortunately, previous researchers did not report if Morganella was pathogenic or non-pathogenic when found.…”

Oviposition behaviour of Phlebotomus argentipes - A laboratory-based study

“…longipalpis preferred sites for oviposition that contain frass or rabbit faeces, which act as its larval rearing medium. More recently, Wasserberg and Rowton (2011) demonstrated that in choice and no-choice oviposition chambers, the effect of old sandfly colony remains (frass), conspecific eggs and their combination affected the oviposition rates of sandflies (Peterkova-Koci et al 2012). The present study will contribute to the development of a methodology for the maintenance of sandfly colonies, which is a major challenge for scientists working on different biological aspects of sandflies.…”