Epigenetic manipulation can alter phenotype
There is little information on the epigenetic architecture of mosquitoes, especially in terms of links between epigenetics and phenotypic expression.
The effect of the epigenetic modulator DZNep on fertility has been examined in An. gambiae , while the reductive effects of genistein and vinclozolin on imidacloprid sensitivity have been described in Aedes albopictus . These epigenetic modulators exerted phenotypic effects on the SENN and SENN DDT strains as well. Despite the differences in administration of DZNep (shorter exposure time than in other protocols), treatment of SENN males resulted in a significant decrease in egg numbers, although hatching was unaffected. This shows that the insecticide susceptible SENN strain is more sensitive to DZNep, which appears to primarily affect males. The reduced hatching after SENN males and females were treated with genistein is noteworthy for the same reasons as DZNep.
Although DZNep, genistein and vinclozolin did not affect the fertility of SENN-DDT, they did affect the expression of insecticide resistance in this strain. Exposure to these modulators generally decreased insecticide-induced mortality, with all modulators decreasing deltamethrin-induced mortality in males. The exception was a DZNep-induced increase in malathion mortality in females. Vinclozolin induced the greatest effect on insecticide resistance in males and females. These data suggest that the epigenetic modulators may be susceptible to generalised enhanced metabolism of xenobiotics such as insecticides.
Epigenetic modulators produce a change in 5-mC and 5-hmC methylation patterns
DNA methylation can by subdivided into 5-mC and 5-hmC modifications, each of which have various implicated roles. 5-mC is implicated in DNA methylation while 5-hmC is implicated in demethylation and gene activation amongst other functions [50, 51]. Of course, only some instances of increased methylation decrease gene expression and vice versa , as the change in gene expression is highly dependent on the methylation taking place at particular genomic regions (promoters versus exons versus silencers etc.). This therefore makes accurately predicting changes in gene expression by methylation versus demethylation on a global scale very difficult without more sensitive techniques.
DNA methylation, as assessed by 5-mC methylation levels, was affected by dietary supplementation with the epigenetic modulators genestein, vinclozolin, and DZNep. The 5-mC levels in the SENN strain were unaffected by the modulators. In contrast to the SENN strain, all the modulators increased 5-mC levels in SENN-DDT females, but only genestein increased levels in SENN-DDT males. It is worth noting that this pattern is opposite to the5-hmC levels, where the modulators induced changes in SENN, but not SENN-DDT.
Dietary supplementation with the three epigenetic modulators did alter DNA demethylation levels. This may underlie some of the observations in the phenotypic studies. SENN was more sensitive to epigenetic modulation than SENN DDT, although all three modulators caused a significant change in 5-hmC methylation in at least one of the sexes, with genestein altering both male and female methylation. Where there were changes in the SENN strain, the modulators increased methylation in females, while decreasing it males. This combination (genestein treated male and female) evidently induced a significantly reduced larval hatch percentage. By contrast, genestein had no effect on 5-hmC methylation in SENN-DDT at all, unlike DZNep and vinclozolin exposures which were associated with, significant increases in female 5-hmC methylation. The increase in vinclozolin-induced 5-hmC methylation may be linked to the marked decrease in female DDT and pyrethroid resistance observed after treatment.
Larval heavy metal exposure alters the epigenetic profile of adult mosquitoes
The precise role of RNA methylation in epigenetic regulation is poorly understood. It is believed to be associated with physical changes in the messenger RNA (mRNA) structure, namely N6-methyl adenosine (m6A) addition. Such additions occur on both mRNA and long non-coding RNA transcripts and have implications in mRNA splicing, cellular transport, stability and immune tolerance . mRNA modification is therefore proposed to act as an epigenetic marker and may potentially allow for mediation by DNA and histone modification mechanisms . It is possible that because RNA methylation effects may be seen in downstream epigenetic modifications, this is the epigenetic architecture that is least affected by metals. What is noteworthy are the significant differences between males and females in SENN, where with the exception of treatment with cadmium, males always had significantly higher levels of m6A mRNA methylation. mRNA methylation is the only type of methylation where metal treatment resulted in more changes in SENN-DDT than in SENN, and while lead nitrate induced an increase in RNA methylation in SENN, copper nitrate treatment reduced methylation in SENN-DDT males and lead nitrate reduced methylation in males. Metal treatments therefore did not alter RNA methylation levels to a great extent.
Nucleic acid methylation varies greatly in invertebrates, and this is also true for mosquitoes. Although Aedes aegypti has been reported to be unmethylated , 5-mC methylation patterns were well detected in both strains of An. arabiensis. This supports previous detection of 5-mC detection in An. gambiae , which showed detectable levels of this methylation type using blotting techniques. In the SENN strain, 5-mC methylation levels always differed between males and females, with control and cadmium chloride treated females being higher, while copper and lead nitrate treated males showing higher levels of 5-mC. For both SENN and SENN-DDT, all treatments resulted in significant changes in 5-mC methylation in males. In SENN males, all treatments increased methylation, while only cadmium chloride treatment increased methylation with lead and copper nitrate exposure resulting in a significant reduction in 5-mC methylation. This further suggests that SENN is particularly susceptible to heavy metal-induced change on an epigenetic level and that exposure to such pollutants heavily alters the DNA epigenetic architecture of this strain.
The 5-hmC methylation patterns observed after modulator exposure were congruent with the patterns observed after larval metal exposure, where SENN-DDT was largely unaffected by metal treatment, except after copper nitrate treatment where female SENN-DDT 5-hmC methylation was higher than that of the males. All treatments reduced methylation in SENN males, but only lead nitrate induced a significant decrease in females.
As 5-mC and 5-hmC methylation are generally associated with methylation and demethylation, respectively, and, therefore, broad silencing and expression [50, 51], their levels could roughly be the inverse of each other within the same individual. In these data, there was a generalised pattern of lower 5-hmC patterns where the 5-mC was high, suggesting that this methylation-silencing/demethylation-activation pattern may also be a regulatory mechanism in An. arabiensis.
HAT activity reveals the most marked pattern changes due to metal exposure. There was a clear suppression of HAT activity with metal treatment in both males and females of the SENN strain. This pattern was inverted in SENN-DDT, with metals increasing HAT activity. Lead nitrate exposure resulted in the lowest HAT activity for SENN, but the highest for SENN-DDT.
Significance of changes in epigenetic architecture
A key discovery of this study on a basal level was that both SENN and SENN-DDT showed sex differences in methylation at the RNA, but not the DNA level. This may have implications for regulation of sex-specific behaviours such as blood feeding. There may therefore be numerous sex-specific differences in An. arabiensis that have an epigenetic basis.
The marked differences in methylation patterns in SENN and SENN-DDT support the work of Oppold et al  who demonstrated an epigenetic element to insecticide resistance in Aedes albopictus. The insecticide susceptible SENN strain is also far more sensitive to alteration in epigenetic signatures after pollutant stress. This may be the underlying reason why metal pollution results in more rapid transgenerational selection for insecticide tolerance than the SENN-DDT strain .
5-mC methylation is associated with gene silencing. In the SENN strain, this methylation pattern was generally increased after larval metal exposure. 5-hmC methylation, associated with gene activation, was generally decreased in this strain. In addition to reduced detoxification enzyme activity in SENN, this may explain the toxicity of first generation metal exposure in this strain as the general shift in methylation signatures point to reduced transcription. Conversely, 5-mC methylation is decreased in the insecticide-resistant SENN-DDT strain, but without a concomitant increase in 5-hmC methylation. This suggests that the epigenetic mechanism for metal tolerance may be reduced gene silencing rather than a de novo drive to gene expression. HAT activity partially supports this hypothesis, with larval metal exposure reducing HAT activity in SENN and increasing it in SENN-DDT. It is worth mentioning that the patterns of HAT activity reduction are inverse to the toxicity of the metals in SENN [29, 45], i.e. the less toxic the metal, the more exposure to it suppresses HAT activity, which is not congruent with the defence against toxicity hypothesis suggested above. Studies of An. gambiae demonstrated that the presence of the H3K27ac modification, a mark of acetylation on Histone H3, is associated with active transcription . This suggests that the HAT activity observed in this study may be associated with increased transcription.
The exact mechanism of histone modification is contentious. It is generally accepted that histone modification plays a more important role in recruiting non-histone proteins which bind to the areas surrounding these modifications and allow for dynamic changes in chromatin structure . This may explain the lack of changes in methylation patterns in SENN-DDT, suggesting that the metal stress induces a distinctly different epigenetic mechanism in the insecticide resistant strain than the insecticide resistant strain.