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Anopheles arabiensis (a member of the An. gambiae species complex) is a major vector of malaria in sub-Saharan Africa. Despite its disease vector status, there is currently a paucity of epigenetic information for this species. The aim this study was therefore to analyse global epigenetic markers and their response to metal exposure in insecticide susceptible and resistant laboratory strains of An. arabiensis. This was done using commercially available epigenetic marker quantification kits. In order to validate the efficacy of the kits, several kits were assessed to determine whether changes induced by known epigenetic modulators were detectable using these platforms. The efficacy of the dosages used were determined by examining the effect of the dosages used on insecticide resistant phenotypes. Upon confirmation that the dosages used were sufficient to induce a phenotypic change, the effect on epigenetic markers was assessed. Commercial kits were used to quantify 5-methylcysteine (5-mC) and 5-hydroxymethylcysteine (5-hmC) methylation in DNA, m6A methylation in mRNA as well as Histone Acetyl Transferase (HAT) activity. There was a marked difference in the phenotypic response in adult mosquitoes of the insecticide susceptible strain compared to that of its' resistant counterpart. For males and females of the resistant strain, exposure to nucleic acid modifying drugs typically increased their tolerance to insecticides. The patterns of changes in 5-mC methylation by epigenetic modulators was congruent with previous studies which quantified by mass spectrometry. The two strains differed in methylation patterns under control conditions and responded differentially to larval metal exposure. In the resistant strain, which previously was demonstrated to show increased detoxification enzyme activity and insecticide tolerance after the same treatment, the potential increase in transcriptional activity appeared to be modulated by reduced methylation and increased HAT activity. This study suggests that the commercial epigenetic quantification kits can be used to characterise phenotypic changes in An. arabiensis, and also shows that epigenetic regulation of the response to metal exposure is regulated at the DNA as opposed to the RNA level., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Members of the Anopheles gambiae complex breed in clean, sunlit temporary bodies of water. Anthropogenic pollution is, however, altering the breeding sites of the vectors with numerous biological effects. Although the effects of larval metal pollution have previously been examined, this study aims to assess the transgenerational effects of larval metal pollution on the major malaria vector An. arabiensis., Methods: Two laboratory strains of An. arabiensis, SENN (insecticide-susceptible) and SENN-DDT (insecticide-resistant), were used in this study. After being bred in water polluted with either cadmium chloride, copper nitrate or lead nitrate, several life history characteristics that can have epidemiological implications (fertility, apoptotic damage to reproductive structures, adult longevity and insecticide tolerance) were examined in the adults and compared to those of adults bred in clean water., Results: All metal treatments reduced fecundity in SENN, but only lead treatment reduced fertility in SENN-DDT. Cadmium chloride exposure resulted in apoptosis and deformation of the testes in both strains. After breeding generation F0 in polluted water, F1 larvae bred in clean water showed an increase in longevity in SENN-DDT adult females. In contrast, after breeding the F0 generation in polluted water, longevity was reduced after cadmium and copper exposure in the F1 generation. Larval metal exposure resulted in an increase in insecticide tolerance in adults of the SENN strain, with SENN-DDT adults gaining the greatest fold increase in insecticide tolerance., Conclusions: This study demonstrates that a single exposure to metal pollution can have transgenerational effects that are not negated by subsequent breeding in clean water.

Zoophilic members of the Anopheles gambiae complex are often associated with cattle. As such, it is likely that the immature aquatic stages will be exposed to cattle faeces as a pollutant. This study aimed to examine the effect of cattle manure on members of the An. gambiae complex found in South Africa. In this study, a commercial organic fertiliser originating from cattle manure was used as a proxy for cattle faeces. Laboratory strains of An. merus, An. quadriannulatus as well as four An. arabiensis strains (SENN and MBN: insecticide susceptible, MBN-DDT: insecticide resistant, unselected, SENN-DDT: insecticide resistant: selected for resistance) were used in this study. The effect of larval fertiliser exposure on larval development rate and adult longevity was assessed in all three species. The effect of larval fertiliser exposure on subsequent adult size, insecticide tolerance and detoxification enzyme activity of the four strains of the malaria vector An. arabiensis was also assessed. Following fertiliser treatment, all strains and species showed a significantly increased rate of larval development, with insecticide susceptible strains gaining the greatest advantage. The adult longevities of An. merus, An. quadriannulatus, insecticide susceptible and resistant An. arabiensis were significantly increased following fertiliser treatment. Insecticide susceptible and resistant An. arabiensis adults were significantly larger after larval organic fertiliser exposure. Larval fertiliser exposure also increased insecticide tolerance in adult An. arabiensis, particularly in the insecticide resistant, selected strain. This 4.7 fold increase in deltamethrin tolerance translated to an increase in pyrethroid resistance intensity, which could exert operational effects. In general, larval exposure to cattle faeces significantly affects the life histories of members of the An. gambiae complex., Competing Interests: The authors have declared that no competing interests exist.