Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

Abstract

b Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut. E lizabethkingia species have attracted much interest because of their close biologic associations with Anopheles malaria vector and Aedes dengue fever vector mosquitoes (1-5). Elizabethkingia was detected in diverse sources of mosquitoes (field caught, seminatural reared, and insectary reared) sampled in different regions (Africa, Europe, and North America) (1, 3, 4). For instance, Elizabethkingia or Elizabethkingia-like bacteria were detected in 68% of field-caught mosquito populations collected in Cameroon (3). E. anophelis was isolated by Kämpfer et al. from the midgut of Anopheles gambiae G4 reared in an insectary as a predominant bacterial species (6). Wang et al. conducted a dynamic microbial community analysis of mosquitoes reared in seminatural microcosms (in Kenya) by using pyrosequencing methods and showed that Elizabethkingia spp. were more abundant in mosquitoes...

Larval diet affects mosquito development and permissiveness to Plasmodium infection

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Diet-Induced Nutritional Stress and Pathogen Interference in Wolbachia-Infected Aedes aegypti

“…The composition of the host microbial community can be affected by host diet [100, 101], can alter host metabolic profile [102–104], and can affect response to pathogen infection [105–108]. Interestingly, the microbiota induce production of ROS, which can influence susceptibility to Plasmodium infection, and offers a potential explanation for the diet-induced changes we observed in oxidative stress response[109].…”

Altered gut microbiota and immunity defines Plasmodium vivax survival in Anopheles stephensi

“…Paratransgenesis approaches for manipulating gut endosymbionts such as Elizabethkingia, Serratia to block parasite development are under progress (Chen, Bagdasarian et al 2015;Wang, Dos-Santos et al 2017;Koosha, Vatandoost et al 2018). A dominant association of tested Elizabethkingia, Pseudomonas, Serratia with mosquito ovaries/eggs, and subsequent validation of trans ovarian transfer from F1, F2 and F3 generation (See Supplementary data S7), supports an idea to select and target them for future manipulation Alternatively, manipulating intracellular endosymbiont such as Wolbachia induced male sterility and pathogen development inhibition, are rapidly gaining much attention for vector borne disease control program (Bourtzis, Dobson et al 2014;Jayakrishnan, Sudhikumar et al 2018).…”