The contribution of gut bacteria to insecticide resistance and the life histories of the major malaria vector Anopheles arabiensis (Diptera: Culicidae)

Abstract

The gut microbiota of mosquitoes is a crucial determinant of their fitness. As such, the biology of the gut microbiota of Anopheles arabiensis, a major malaria vector of Southern Africa, was investigated. Two laboratory strains of An. arabiensis were used; SENN, an insecticide susceptible strain, and SENN-DDT, a resistant strain. The strains were supplemented with either non-commensal bacteria or antibiotics via a sucrose source to sterilize the gut. The strains were fed the broad-spectrum bactericidal antibiotic gentamicin, or a preferentially gram-positive bactericidal (vancomycin), gram-negative bactericidal (streptomycin) or broad-spectrum bacteriostatic (erythromycin), either by sugar supplementation or by artificially-spiked blood-meal. The effects on adult mosquito longevity and insecticide resistance phenotype were assessed. Bacteria from the midgut of both strains were characterised by MALDI-TOF mass spectroscopy. Bactericidal antibiotics increased longevity in SENN-DDT. Bacterial supplementation increased insecticide tolerance. Antibiotic supplementation via sugar decreased tolerance to the insecticides deltamethrin and malathion. Blood-supplemented vancomycin decreased insecticide resistance, while gentamicin and streptomycin increased resistance. SENN showed a greater gut bacterial diversity than SENN-DDT, with both strains dominated by Gram-negative bacteria. This study suggests a crucial role for bacteria in An. arabiensis life history, and that gut microflora play variable roles in insecticide resistant and susceptible mosquitoes.

Figure 1

The effect of antibiotic supplementation on Anopheles arabiensis longevity. (A) For the insecticide-susceptible Anopheles arabiensis SENN strain control treatments (SFCon-dark blue and SMCon-purple) lived significantly longer than erythromycin-treated individuals (SFEry-red and SMEry-green). (B) Untreated control females of the Anopheles arabiensis insecticide-resistant SENN-DDT strain (DFCon-dark blue) lived for a significantly shorter time than gentamicin- (DFGent-red), vancomycin- (DFVan-green), or streptomycin-treated individuals (DFStrep-purple). Vancomycin treated females lived the longest. (C) Untreated control males of the Anopheles arabiensis insecticide-resistant SENN-DDT strain (DMCon-dark blue) lived for a significantly shorter time than gentamicin- (DMGent-red), vancomycin- (DMVan-green), or streptomycin-treated individuals (DMStrep-purple). Gentamicin-treated males lived the longest. Significance was determined by the Log-Rank test. Insecticide susceptible strains are denoted by a green block and insecticide resistant strains are denoted by a red block.

Figure 2

The effect of the supplementation of non-commensal bacteria on the insecticide resistance phenotype of Anopheles arabiensis SENN-DDT. When the insecticide resistant SENN-DDT strain was supplemented with either gram-positive (Streptococcus pyrogenes) or gram-negative bacteria (Escherichia coli), deltamethrin-induced mortality decreased in both males and females. Bacterial supplementation had no effect on male malathion-induced mortality, while only S. pyrogenes supplementation significantly reduced malathion-induced mortality in females. Asterisks indicate a significant difference from the untreated control of the same sex and insecticide exposure. The treatment label is in red to indicate insecticide resistance.

Figure 3

The effect of antibiotic supplementation on the insecticide resistance phenotype of Anopheles arabiensis SENN-DDT. Vancomycin treatment significantly increased malathion an deltamethrin-induced mortality in males and females. Gentamicin increased deltamethrin-induced mortality in females. Streptomycin increased malathion and deltamethrin-induced mortality in males and deltamethrin-induced mortality in females. Asterisks denote a significant change from untreated controls of the same sex of the same sex and insecticide exposure. The treatment label is in red to indicate insecticide resistance.

Figure 4

The effect of bacterial and antibiotic supplementation on insecticide-induced LT50 (time to 50% mortality) values in Anopheles arabiensis SENN. (A) Supplementation with gram-positive bacteria (Streptococcus pyrogenes) increased deltamethrin and malathion LT50s in females, but only malathion LT50 in males. Gram-negative (Escherichia coli) treatment increased male and female deltamethrin LT50, but had no effect on malathion lethal time. Asterisks indicate a significant difference from the exposed, untreated control of the same sex and insecticide exposure. (B) The effect of antibiotic-supplemented sugar on female deltamethrin LT50. Asterisks indicate a significant difference from the exposed, untreated control. (C) The effect of antibiotic-supplemented sugar on female malathion LT50. (D) The effect of antibiotic-supplemented sugar on male deltamethrin LT50. (E) The effect of antibiotic-supplemented sugar male malathion LT50. The treatment label is in green to indicate insecticide susceptibility.

Figure 5

The effect of antibiotic-treated blood on the insecticide resistance phenotype of Anopheles arabiensis SENN-DDT. (A) Malathion-induced mortality 4 hours post antibiotic supplemented blood-meal. Asterisks indicate a significant difference from the exposed, untreated control. (B) Deltamethrin-induced mortality 4 hours post antibiotic supplemented blood-meal. Symbols indicate a significant difference from the exposed, untreated control, with the different symbols indicative a significant difference between treatments. (C) Malathion-induced mortality 24 hours post antibiotic supplemented blood-meal. Asterisks indicate a significant difference from the exposed, untreated control. (D) Deltamethrin-induced mortality 4 hours post antibiotic supplemented blood-meal. Symbols indicate a significant difference from the exposed, untreated control, with the different symbols indicative a significant difference between treatments. The treatment label is in red to indicate insecticide resistance.

Figure 6

The effect of antibiotic and bacterial-sugar supplementation on the detoxification enzyme activity of Anopheles arabiensis SENN and SENN-DDT. (A) GST activity in SENN and SENN-DDT. Asterisks indicate a significant difference from the untreated control of the same sex and strain. Vancomycin and Streptomycin reduced GST activity in SENN females, but this was the only significant difference. (B) Cytochrome P450 activity in SENN and SENN-DDT. Asterisks indicate a significant difference from the untreated control of the same sex and strain. No consistent pattern was observable in the activity of this class of enzyme. (C) Alpha-esterase activity in SENN and SENN-DDT. Asterisks indicate a significant difference from the untreated control of the same sex and strain. Where significant differences were observed, the activity was significantly lower. (D) Beta-esterase activity in SENN and SENN-DDT. Asterisks indicate a significant difference from the untreated control of the same sex and strain. For the antibiotic treatment, all the significant changes were significantly lower. For live gram-negative bacterial treatment, the significant changes were significantly higher, except for SENN-DDT females, were the activity was significantly lower. Dead gram-positive bacteria resulted in significantly lower activity where activity was significantly different. Treatment with dead gram-negative bacteria resulted in significantly higher beta esterase activity in SENN females, but significantly lower activity in SENN-DDT males. No consistent patterns in enzyme activity were observed, and the changes in enzyme activity were not congruent with the observed bioassay results.