The effect of multiple blood-feeding on the longevity and insecticide resistant phenotype in the major malaria vector Anopheles arabiensis (Diptera: Culicidae)

Abstract

Background: Anopheles arabiensis is a major malaria vector in Africa. Adult females are likely to imbibe multiple blood meals during their lifetime. This results in regular exposure to potential toxins and blood-meal induced oxidative stress. Defence responses to these stressors may affect other factors of epidemiological significance, such as insecticide resistance and longevity. The aims of this study were to examine the effect of multiple blood-feeding on insecticide tolerance/resistance with increasing age, to assess the underlying biochemical mechanisms for the responses recorded, and to assess the effect of multiple blood-feeding on the life histories of adult females drawn from insecticide resistant and susceptible laboratory reared An. arabiensis.

Methods: Laboratory reared An. arabiensis females from an insecticide resistant and an insecticide susceptible colony were offered either a single blood meal or multiple blood meals at 3-day intervals. Their tolerance or resistance to insecticide was then monitored by WHO bioassay four hours post blood-feeding. The biochemical basis of the phenotypic response was assessed by examining the effect of blood on detoxification enzyme activity and the effect of blood-meals on detoxification enzyme activity in ageing mosquitoes.

Results: Control cohorts that were not offered any blood meals showed steadily decreasing levels of insecticide tolerance/resistance with age, whereas a single blood meal significantly increased tolerance/resistance primarily at the age of three days. The expression of resistance/tolerance in those cohorts fed multiple blood meals generally showed the least variation with age. These results were consistent following exposure to DDT and pyrethroids but not to malathion. Multiple blood-meals also maintained the DDT and permethrin resistant phenotype, even after treatment females had stopped taking blood-meals. Biochemical analysis suggests that this phenotypic effect in resistant females may be mediated by the maintenance of increased glutathione s-transferase activity as a consequence of multiple blood-feeding. Multiple blood-feeding increased the longevity of insecticide resistant females regardless of their mating status, but only increased the longevity of unmated susceptible females.

Conclusion: These data suggest that multiple blood-feeding confers a competitive advantage to insecticide resistant females by increased longevity and maintenance of the expression of resistance with age.

Figure 1

The effect of blood feeding on insecticide induced mortality in Anopheles arabiensis SENN-DDT females. Mean percentage mortalities 24 hours post exposure are given for DDT (A), permethrin (B), deltamethrin (C) and malathion (D). Insecticide bioassays were conducted 4 hours post blood meal and mortalities are plotted by age and blood feeding regimen i.e. sugar fed only (0 blood), single blood meal (1 blood), multiple blood meals (multiblood). P-values obtained by 2-sample test indicate degree of significance in differences in mortalities between blood-fed cohorts and their respective sugar fed control cohorts.

Figure 2

Linear regression analysis of insecticide-induced mortality by age by blood feeding regimen (sugar fed only - 0 blood, single blood meal - 1 blood, multiple blood meals – multiblood) in Anopheles arabiensis SENN-DDT females. Insecticide bioassays were conducted 4 hours post blood meal at each age interval indicated and mean percentage mortalities 24 hours post exposure are shown for DDT (A), permethrin (B), deltamethrin (C) and malathion (D). Goodness-of-fit for each regression is indicated by R² values. No R² value is given for multiple-feeding for the malathion treatment as only two points were available for analysis.

Figure 3

The lasting effects of multiple blood-feeding on the expression of insecticide resistance in Anopheles arabiensis SENN-DDT females. Mean percentage mortalities of 18 day old SENN-DDT females that either had no blood, a single blood meal (1 blood) or their fifth blood meal (multiblood) 4 hours prior to exposure were compared to that of 18 day old females that acquired their last blood meal at the age of 15 days (15 day blood; 18 day exposed). Insecticide bioassays were conducted 4 hours post blood meal except for the last cohort which was assayed against listed insecticides 3 days post blood-feeding (15 day blood; 18 day exposed). Only the mean mortality induced by the delayed deltamethrin treatment differs significantly from the mean mortality of the multiple blood meal cohort (multiblood) of the same age (p < 0.05). The effect of multiple blood-feeding on the expression of resistance was maintained for the DDT and permethrin resistant phenotypes 3 days post blood-feeding. Perm = permethrin; Delta = deltamethrin. The asterisks denote significant differences between 18 day multiple blood meal treatments and those exposed at 18 days but last fed 3 days prior to exposure. Lower case letters indicate no significant differences.

Figure 4

Comparison of detoxification enzyme activity of 18 day old SENN and SENN-DDT Anopheles arabiensis females. 15 day old females either deprived of blood (15 day 0 blood), offered only one blood meal at 15 days (15 day 1 blood) or fed multiple blood meals at three day intervals up to and including 15 days (15 day multiblood) were allowed 72 hours to digest their final blood meal (if offered) before being assayed for detoxification enzyme activity. Block A represents haeme peroxidase activity and shows a comparison of SENN-DDT females that had no blood meals compared to those that had single or multiple meals. The asterisk denotes a significantly lower enzyme activity in multiple blood-fed SENN-DDT compared to all other cohorts. SENN is not represented as there were no significant changes in haeme peroxidise activity in this strain regardless of dietary treatment. Block B represents Glutathione S-transferase (GST) activity of SENN and SENN-DDT females. The asterisks denote a significant difference between GST activities of the different strains at the same age. A circle denotes a significant increase in activity of a 15 day old SENN-DDT cohort compared to their 3 day old counterparts.

Figure 5

Detoxification enzyme activities of Anopheles arabiensis SENN-DDT by age and as a function of blood feeding regimen in females. Block A represents Cytochrome P450 activity as measured by haeme peroxidase activity, Block B represents GST activity, Block C represents α esterase activity and Block D β esterase activity. Enzyme activities in males were measured as baseline levels against which the enzyme activities of unfed, single blood meal and multiple blood meal cohorts of females were compared.

Figure 6

The effect of multiple blood-feeding on the longevity (survivorship) of Anopheles arabiensis SENN and SENN-DDT females (Blocks A and B respectively). Light blue and pink lines represent unmated and mated respectively. The effect of Bovine Serum Albumin (BSA) on the longevity of SENN and SENN-DDT females are also displayed, with the BSA treated females represented by the dark blue line. The longevity of all the treatments were compared to that of the sugar fed controls (red line).