Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity

Abstract

A critical stage in malaria transmission occurs in the Anopheles mosquito midgut, when the malaria parasite, Plasmodium, ingested with blood, first makes contact with the gut epithelial surface. To understand the response mechanisms within the midgut environment, including those influenced by resident microbiota against Plasmodium, we focus on a midgut bacteria species' intra-specific variation that confers diversity to the mosquito's competency for malaria transmission. Serratia marcescens isolated from either laboratory-reared mosquitoes or wild populations in Burkina Faso shows great phenotypic variation in its cellular and structural features. Importantly, this variation is directly correlated with its ability to inhibit Plasmodium development within the mosquito midgut. Furthermore, this anti-Plasmodium function conferred by Serratia marcescens requires increased expression of the flagellum biosynthetic pathway that is modulated by the motility master regulatory operon, flhDC. These findings point to new strategies for controlling malaria through genetic manipulation of midgut bacteria within the mosquito.

Figure 1. Serratia marcescens reduces the Plasmodium parasite load in Anopheles mosquitos.

(A) P. berghei oocyst counts in the midgut 8 days after An. stephensi mosquitoes were fed on a P. berghei-infected mouse. Circles (red) represent the number of parasites from an individual mosquito, and horizontal lines indicate the median. ***p < 0.001, Mann-Whitney test. (n = 100/group). (B and C) P. berghei ookinete counts in the midgut lumen 16 and 20 hours (B) and midgut epithelium 20 and 24 hours (C) after mosquitoes were fed on a P. berghei-infected mouse. Error bars show standard deviation. ***p < 0.001; n.s., not significant, unpaired t-test. (n = 50/group). (D) Effect of heat-inactivated S. marcescens HB3 on P. berghei load in An. stephensi mosquitoes. Oocyst numbers were determined as in (A). ***p < 0.001; n.s., not significant, Mann-Whitney test. (n = 50/group). anti-, antibiotics treatment; HB3, S. marcescens HB3 treatment; HIA-HB3, heat-inactivated S. marcescens HB3. The experiments in (A) to (D) were performed more than once with similar results.

Figure 2. Serratia marcescens without phenotypic diversity loses its Plasmodium inhibitory ability.

(A) P. berghei oocyst counts on the An. stephensi midgut 8 days after mosquitoes fed on P. berghei-infected mice. Circles (red) represent the number of parasites from an individual mosquito, and horizontal lines indicate the median. ***p < 0.001; n.s., not significant, Mann-Whitney test. (n = 100/group). The experiment was repeated at least three times. (B) (left) Frequency distribution of the bacterial major axis lengths 10 hours after S. marcescens inoculation. (n = 500/group). (right) Gram-staining of S. marcescens HB3 and HB18 reveals their size difference. Bars = 3 μm. (C) (left) Bacterial flagella on S. marcescens HB3 and HB18 ((a)–(d)). S. marcescens HB3 contained many mature flagella. (a) and (b) Bars = 1 μm, (c) and (d) Bars = 250 nm. (right) Relative intensity of flagella per bacterium measured using Scion Imaging software ((e)). Error bars show standard deviation. (n = 208/group). ***p < 0.001, student's t-test. (D) Expression of flhDC genes in S. marcescens HB3 and HB18. flhDC mRNA expression levels were determined by northern blot analysis using a probe corresponding to flhDC 2, 8, and 24 hours after cultivation. The amounts of mRNA were confirmed using 16S ribosomal RNA. anti-, antibiotics treatment; HB3, S. marcescens HB3 treatment; HB18, S. marcescens HB18 treatment.

Figure 3. flhDC-regulated bacterial phenotypes correlate with their anti-Plasmodium function.

(A) Essential flhDC promoter region in S. marcescens HB3 and HB18. The flhDC genes from HB3 and HB18 were obtained by PCR with primers (flhDF2 and flhCR2) (Table S4) and sequenced from the PCR product. The flhDC gene (GenBank Accession Number AF077334) contains a −10 box (TAAATT; nucleotide position 413–418) and −35 box (TTGCGC; nucleotide position 387–392). The −35 box of HB18 had one nucleotide substitution (blue) and two insertions (red) compared to HB3. (B) (left) Rescued flhDC gene expression in HB18 (flhDC+). The flhDC mRNA levels were determined by northern blot 10 hours after cultivation, as in Fig. 2D, and confirmed using 16S ribosomal RNA. (right) Relative mRNA signal intensities measured using ImageJ (NIH). Error bars: standard deviation (n = 3/group). ***p < 0.001 *p < 0.05; n.d., not detected, student's t-test. (C–E) Cellular phenotypes of HB18 are caused by loss of flhDC expression. (C) Length of individual bacteria 10 hours after S. marcescens inoculation. Dashed lines indicate the mean (μm). (n = 100/group). (D) Distribution of bacterial length. ***p < 0.001, Mann-Whitney test. Box plots in which whiskers indicate the most distant data point no more than 2.5 times the interquartile distance from the median. (n = 100/group). (E) Cell-size diversity compared using the Shannon-Weaver diversity index (H′). (n = 50/group). (F) P. berghei oocysts in the An. stephensi midgut 8 days after feeding on P. berghei-infected mice. Circles (red) represent the number of parasites from an individual mosquito, and horizontal lines indicate the median. ***p < 0.001, *p < 0.05; n.s., not significant, Mann-Whitney test. (n = 50/group). anti-, antibiotics; HB3, HB18, HB18 (flhDC+): S. marcescens HB3, HB18, and HB18 (flhDC+) treatment, respectively. The experiment was performed at least three times with similar results.

Figure 4. Intra-species variation of S. marcescens is associated with the vectorial competency of Plasmodium-transmitting mosquitoes in a malaria endemic area.

(A–C) S. marcescens from wild mosquitoes (An. gambiae) collected in Burkina Faso exhibit large fluctuations in cell size. (A) Length of individual bacteria 10 hours after S. marcescens inoculation. Dashed lines indicate the mean (μm) (n = 100/strain). (B) The distribution of bacterial lengths compared using the Mann-Whitney test (different letters signify distinct statistical groups; p < 0.01). Box plots were as in Fig. 3D (n = 100/strain). (C) Cell-size diversity (Purple bars) was compared using the Shannon-Weaver diversity index (H′) (n = 500/strain). Orange line chart shows average cell length (μm). (D) P. berghei oocysts in the An. stephensi midgut 8 days after feeding on P. berghei-infected mice. Circles (red): number of parasites from an individual mosquito. Horizontal lines: median. Distributions compared using the Mann-Whitney test (letters signify different statistical groups; p < 0.01) (n = 50/strain). The experiment was performed at least three times with similar results. (E) Pearson correlation analysis comparing cell-size fluctuation and anti-Plasmodium function. Purple: cell-size diversity index (H′) versus average number of oocysts (r = 0.7449, p < 0.01). Orange: average cell length (μm) versus average number of oocysts (r = 0.7142, p < 0.01).