Rahnella aquatilis Isolated from Aedes albopictus Impairs Mosquito Reproduction Capacity

Abstract

Aedes albopictus is one of the most important vectors of Dengue, which poses a serious threat to public health. The bacterial microbiota has an effect on the parameters of mosquitos, such as larval development and fecundity, and it has emerged as a promising field to be explored for novel environmentally friendly control strategies. Rahnella sp. are present in many insects, including Ae. Albopictus, and play a role in bacterial-insect interactions; however, the role of the bacteria in mosquito biology has not yet been characterized. In this study, we characterized the Rahnella isolate RAeA1 obtained from Ae. albopcitus, and its colonization stability in Ae. albopictus was investigated by generating GFP-tagged bacteria. The influences of the bacteria on larval development and mosquito reproductive capacity were evaluated by inoculating RAeA1 in axenic larvae and antibiotic-treated adult mosquitoes, respectively. The results indicated that RAeA1, which is widespread in the field population of Ae. albopictus, can be transmitted directly from the parental strain to the progeny and can rescue axenic larvae developing into adults with a prolonged development time to pupation. RAeA1 inoculation can impair egg production and ovary maturation, as well as reducing the synthesis of ecdysteroids and vitellogenin in Ae. albopictus females. Overall, our results provide a thorough study of bacterium function characterization that will facilitate the development of potential strategies in relation to the design of microbiomes for vector control.

Keywords: Aedes albopictus; Rahnella aquatilis; microbiota; mosquito reproduction.

Figure 1

The microscopic and phylogenetic analysis of RAeA1 isolated from Ae. albopictus. (A) A scanning electron microscopic photo indicating group bacteria structure. (B) The phylogenetic tree of RAeA1. The tree was constructed using the neighbor-joining method without distance correction based on the 16S rRNA sequence using MEGA and was visualized using iTOL [30]. The 16S rRNA sequences of other Rahnella strains were obtained from the National Center for Biotechnology Information. Bootstrap support percentages (>50%) are shown at nodes and the bar represents 0.001 substitutions per nucleotide position.

Figure 2

The location and distribution of the R. quantilis RAeA1 isolate at different developmental stages in field populations in Jiangsu Province (A–D), the laboratory (E), and different tissues (F). (A) The geographic location of Tai’zhou, Hai’an, and Lian’yungang in Jiangsu Province. Geographic information system (GIS)-based spatial analysis was conducted to demonstrate the study sites at a prefectural level in Jiangsu Province. All spatial analyses were carried out via QGIS (Quantum GIS, v2.10.1). (B) The infection of RAeA1 was observed using the relative qPCR method in different field-caught A. albopictus populations (F0 generations) from Tai’zhou (F _{(3, 7)} = 31.51, p = 0.0002), (C) Hai’an (F _{(2, 6)} = 25.35, p < 0.0001), (D) and Lian’yungang (F _{(3, 8)} = 29.38, p = 0.0001), Jiangsu, China. (E) The abundance of RAeA1 was determined using the relative qPCR method in different developmental stages from 3rd instar (L3) to 4th instar (L4) larvae, pupae, and male and female adults from the laboratory A. albopictus colony (F _{(3, 8)} = 3292, p < 0.0001). (F) The distribution of RAeA1 in different tissues (F _{(4, 18)} = 14.86, p < 0.0001) was also quantified using the same method. Values are means ± SEMs (error bar) of triplicate biological samples. Means that share the same letter are not significantly different and different letters indicate significant differences as determined by one-way ANOVA (with Holm-Šídák’s multiple comparisons test) analysis (p < 0.05). All the analysis was carried out using GraphPad Prism 10.

Figure 3

Distribution of Rah/hupB-GFP-Apr in various tissues of F0 generation and F1 generation Ae. albopictus (representative images). (A) One-day-old mosquitoes were fed Rah/hupB-GFP-Apr bacteria, and distribution of bacteria in midgut, ovary, and salivary glands was determined (scale bar: 100 μm or scale bar: 50 μm). (B) Distribution of Rah/hupB-GFP-Apr in eggs and larvae laid by female mosquitoes that were fed Rah/hupB-GFP-Apr bacteria for three days.

Figure 4

The development of axenic Ae. albopictus larvae supplied with RAeA1 and E. coli. (A) The percentage of axenic (AX) larvae that develop into pupae when supplied with RAeA1 (AX + RAeA1) and E. coli XL-10 (AX + E. coli) versus the percentage of conventional (CN) larvae that develop into adults over time (days). * p < 0.05; ** p < 0.01; ns: not significant. (B) Development times (days) from larvae to pupation in the CN, AX + RAeA1, and AX + E. coli groups. (C) The percentage of adults that emerged from the CN, AX + RAeA1, and AX + E. coli groups (Chi-square test and Fisher’s exact test). (D) Scatter plots showing the median wing length of adults (males and females) that emerge when supplied different bacteria. (E) The sex ratio of adult mosquitoes emerged from the groups when fed different bacteria (Chi-square test and Fisher’s exact test). Data are represented as means ± SEMs, and Student’s t-test was used in the analysis. Means that share the same letter are not significantly different, and different letters indicate significant differences (p < 0.05). All analysis was carried out using GraphPad Prism 10.

Figure 5

The egg production and ovary maturation of Ae. albopictus females supplied with RAeA1 and E. coli. (A) The total number of eggs laid per Ae. albopictus adult (F _{(5, 246)} = 19.91, p < 0.0001). Control: RAeA1 and E. coli (n = 34); antibiotic: antibiotic + RAeA1 and antibiotic +E. coli (n = 50). Control: conventionally reared; RAeA1: conventionally reared plus RAeA1; E. coli: conventionally reared plus E. coli; antibiotic: antibiotic treated; antibiotic + RAeA1: antibiotic treated before being fed with RAeA1; antibiotic + E. coli: antibiotic treated before being fed with E. coli. (B) Average ovary size (length of the long axis) isolated from randomly selected female mosquitoes in each group with different treatment (n = 20) after 24 h PBM (F _{(5, 114)} = 31.01, p < 0.0001). (C) The ratio of yolk to ovary by length 24 h PBM (F _{(5, 401)} = 154, p < 0.0001); control (n = 70), RAeA1 (n = 56), E. coli (n = 54), antibiotic (n = 76), antibiotic + RAeA1 (n = 75), and antibiotic + E. coli (n = 90). (D) Representative images of ovaries dissected from conventionally reared, RAeA1-inoculated, E. coli-infected, antibiotic-treated, antibiotic treatment before RAeA1 infection, or E. coli-infected female mosquitoes at 6 h, 24 h, 48 h, and 72 h PBM, respectively. All images were taken with LAS X Life Science under the DVM6 Digital Microscope (scale bar: 100 μm). (E) Ecdysteroids produced per ovary by females in each group at 6 h, 24 h, and 30 h PBM. A two-way ANOVA (with Holm-Šídák’s multiple comparisons test) was used in the analysis (comparing cell means regardless of rows and columns with a row factor (time) of F _{(2, 202)} = 87.47, p < 0.0001; column factor (groups): F _{(5, 202)} = 102.7, p < 0.0001). (F) Vitellogenin produced per fat body by females in each group (F _{(5, 36)} = 149.6, p < 0.0001). In (A–C,F), one-way ANOVA (with Holm-Šídák’s multiple comparisons test) was applied in the analysis. All data are presented as means ± SEMs. Means that share the same letter are not significantly different, and different letters indicate significant differences (p < 0.05). All analysis was carried out using GraphPad Prism 10.