Transgenerational effects of insecticides - implications for rapid pest evolution in 1 agroecosystems

19 Although pesticides are a major selective force in driving the evolution of insect pests, 20 the evolutionary processes that give rise to insecticide resistance remain poorly understood. 21 Insecticide resistance has been widely observed to increase with frequent and intense insecticide 22 exposure, but can be lost following the relaxation of insecticide use. One possible but rarely 23 explored explanation is that insecticide resistance may be associated with epigenetic 24 modifications, which influence the patterning of gene expression without changing underlying 25 DNA sequence. Epigenetic modifications such as DNA methylation, histone modifications, and 26 small RNAs have been observed to be heritable in arthropods, but their role in the context of 27 rapid evolution of insecticide resistance remain poorly understood. Here, we discuss evidence 28 supporting how: 1) insecticide-induced effects can be transgenerationally inherited, 2) epigenetic 29 modifications are heritable, and 3) epigenetic modifications are responsive to pesticide and 30 xenobiotic stress. Therefore, pesticides may drive the evolution of resistance via epigenetic 31 processes. Moreover, insect pests primed by pesticides may be more tolerant of other stress, 32 further enhancing their success in adapting to agroecosystems. Resolving the role of epigenetic 33 modifications in the rapid evolution of insect pests has the potential to lead to new approaches


Highlights
• Insecticide-induced effects can be transgenerationally inherited.
• Epigenetic modifications are responsive to insecticide-induced stress.
• Pesticide use may directly and indirectly drive the evolution of insect pests in agroecosystems via epigenetic processes.

Abstract
Although pesticides are a major selective force in driving the evolution of insect pests, the evolutionary processes that give rise to insecticide resistance remain poorly understood.
Insecticide resistance has been widely observed to increase with frequent and intense insecticide exposure, but can be lost following the relaxation of insecticide use.One possible but rarely explored explanation is that insecticide resistance may be associated with epigenetic modifications, which influence the patterning of gene expression without changing underlying DNA sequence.Epigenetic modifications such as DNA methylation, histone modifications, and small RNAs have been observed to be heritable in arthropods, but their role in the context of rapid evolution of insecticide resistance remain poorly understood.Here, we discuss evidence supporting how: 1) insecticide-induced effects can be transgenerationally inherited, 2) epigenetic modifications are heritable, and 3) epigenetic modifications are responsive to pesticide and xenobiotic stress.Therefore, pesticides may drive the evolution of resistance via epigenetic processes.Moreover, insect pests primed by pesticides may be more tolerant of other stress, further enhancing their success in adapting to agroecosystems.Resolving the role of epigenetic modifications in the rapid evolution of insect pests has the potential to lead to new approaches for integrated pest management as well as improve our understanding of how anthropogenic

Introduction
The pesticide treadmill describes how agricultural insect pests evolve resistance in response to frequently used pesticides, rendering them ineffective.Pesticides are pervasive in agriculture, and are a major selective force driving the evolution of insect pests in agroecosystems [1].Although insecticide resistance has been documented in a wide range of insect pests [2] and the genetic basis of major gene resistance has been mapped in key pests for select insecticides [3], the broader evolutionary processes that give rise to insecticide resistance remain poorly understood [4,5].Farmers and entomologists have observed that insecticide resistance increases with the frequency of exposure to particular insecticides [6][7][8], but can be lost following the relaxation of insecticide use [9][10][11].The rapid gain and loss of resistance appears to occur far more rapidly than expected based upon mutation rates [12,13], suggesting that insecticides themselves may increase the rate of mutation or cause physiological changes in pest organisms [5].One possible explanation that has been relatively unexplored is that the evolution of insecticide resistance results from epigenetic modifications, which are heritable and influence gene expression without changing the underlying DNA sequence.
The evolution of insecticide resistance has been considered an evolutionary paradox [5], in that pest species which have experienced repeated genetic bottlenecks due to invasion and selection remain able to adapt very rapidly, despite limited genetic diversity.The same insect pests have evolved resistance to insecticides in all of the major classes [14], and are expected to evolve resistance to future chemistries [15].Extreme genetic bottlenecks also do not appear to limit the likelihood that insecticide resistance evolves.For example, all Colorado potato beetle (Leptinotarsa decemlineata Say) populations in Europe are descended from the introduction of a A c c e p t e d M a n u s c r i p t single female, or single mtDNA haplotype [16].Despite this strong historic bottleneck, L. decemlineata populations in Eurasia have evolved resistance to a wide range of insecticides in Europe, the Middle East, and East Asia [6,17,18].There is a seeming inevitability of insecticide resistance developing in pests, where new phenotypes arise following environmental stress at rates that may not be explained by natural selection.Indeed, Skinner et al. [19] argued how epigenetic processes fit within a neo-Lamarckian framework, because environmental epigenetic patterning can influence transgenerational transmission of phenotypic variation.By influencing epigenetic modifications, xenobiotic and environmental stressors can directly influence the phenotypic responses of organisms to their environment.
Epigenetics is the field of study that examines how environmental factors influence heritable changes in gene expression.There are several epigenetic mechanisms that are heritable and could underlie transgenerational effects of insecticides: DNA methylation [20], histone modifications [21], and heritable noncoding RNA [22].Here, we discuss evidence supporting how 1) insecticide-induced effects can be transgenerationally inherited, 2) epigenetic modifications are heritable, and 3) epigenetic modifications are responsive to insecticide-induced stress.We draw on other model systems from a diverse body of literature, including genetics, epigenetics, and toxicology to identify gaps in our understanding around the evolution of insecticide resistance in insect pests.We close with a discussion of the implications of epigenetic processes for insect fitness in intensively-managed agroecosystems.

Insecticide-induced hormetic effects can be heritable
Insecticides not only select for insecticide resistance and point mutations at target sites, but they can also affect physiological and life-history traits [23].In particular, exposure to sublethal dosages of insecticides can incur stress and lead to increased phenotypic variation [24].
Stress responses can lead to hormesis, a well-known phenomenon from toxicological literature, where small dosages can stimulate biological functions whereas large dosages are detrimental or lethal [24].Hormetic responses include activation of stress response pathways in a variety of taxa from microbes, plants, and animals.They are not related to any special class of compounds, as hormetic effects have been reported for over 240 different chemical classes [23].Sublethal exposure to insecticides can induce hormetic effects and lead to variety of positive life history effects, such as mating success [25], fecundity [26], and body size [27].By positively influencing traits associated with fitness, hormetic effects may play an important role in pest evolution.
There is evidence that individuals exposed to stressful conditions, either abiotic or biotic, can prime gene expression in their offspring to be able to better tolerate stress [28,29].
Insecticides have been shown to induce transgenerational insecticide induced hormetic effects, but thus far the results have been difficult to interpret.For example, Myzus persicae aphids treated with sublethal levels of imidacloprid produce offspring that survive longer when exposed to food/water stress, but tolerance to insecticide stress is unchanged [30].Similarly, although sublethal levels of precocene (an antagonist to Juvenile hormone) stimulate reproduction in M. persicae, the results are not passed on to subsequent generations [31*].Although chemicalinduced hormesis has been reported from many groups and these changes have also been reported to be inherited [23] the genetic, epigenetic, and toxicological basis of hormesis is still poorly understood [5,32].

A c c e p t e d M a n u s c r i p t
Transgenerational effects on pest evolution Epigenetic modifications have been shown to be heritable [20].DNA methylation, the addition of a methyl group to the 5 carbon position of cytosine a nucleotide (usually the cytosine in CpG dinucleotides), is a well-documented mechanism of epigenetic inheritance that can influence phenotypic variation (Table 1), and is found in most, if not all, orders of insects [32].
Methylation in insects is largely found within coding regions, and is closely linked with gene expression and alternative splicing -where a single gene can generate a diversity of gene transcripts of differing length, based on which exons are translated [33].Methylation can occur at any location in the genome, but the effects of DNA methylation vary based on its location in the genome (Figure 1): A) changes in DNA methylation at the promoter region can influence gene expression in downstream genomic regions [34], B) methylation suppresses gene expression of transposable elements (TEs, which are mobile genetic elements responsible for the majority of mutations in many genomes) and prevent TE mobilization [35], and C) Gene body methylation can increase gene expression [32], as well as an increase in the number of alternative splice variants [36].Changes in methylation patterns in arthropods can be associated with changes in levels of resistance to insecticides.Myzus persicae, can gain insecticide resistance through the duplication of esterase genes and subsequent overexpression of esterases [37].After suspending insecticide exposure, extra copies of esterase genes can be methylated, leading to a loss of resistance.It is possible that these aphid populations could quickly become resistant again following demethylation of these amplified genes.
Histone modifications include additions of acetyl or methyl groups on the histone proteins around which nuclear DNA is wrapped, which can influence gene regulation and expression [38].The full effects of these modifications are not well known, especially in arthropods.However, it does appear that some histone modifications are able to be transmitted

A c c e p t e d M a n u s c r i p t
Transgenerational effects on pest evolution transgenerationally [39].Different noncoding RNA (ncRNA) [22] can be inherited through either the male or female gametes, though most current research does not incorporate analysis of heritable RNA.Certain types of small RNA can direct and maintain DNA methylation and histone modification, and therefore affect chromatin structure [40].DNA methylation, histone modifications, and ncRNAs form a constellation of interacting effects that result in a phenotypic response [41].To fully understand how epigenetic modifications influence transgenerational phenotypic inheritance, it would be optimal to assess all three mechanisms simultaneously through concurrent small RNA-seq, bisulfate-treated DNA-seq, and histone modification assays, in as many tissues and individuals as possible.Ideally, multiple generations would be sequenced, to determine if changes in epigenetics and gene expression differ consistently between treatments.Because the cost of sequencing is the major limiting factor for these studies, projected lower sequencing costs in the future should enable these types of studies.

Epigenetic modifications are responsive to xenobiotic stress
Exposure to insecticides and other xenobiotic compounds can alter DNA methylation status in arthropods, and these epigenetic changes can persist for at least several generations [20,42,43].Table 1 lists a number of examples of stress leading to epigenetic changes in arthropods.Studies focusing on insects are few in number, so our scope is broadened to include examples from aquatic ecotoxicology literature, which includes a number of non-insect arthropods.Oppold (2015) found that exposure of mosquitoes to a fungicide leads to heritable changes in methylation and decreases in sensitivity to imidacloprid, an insecticide.Methylated cytosines also spontaneously deaminate, becoming thymines, at a higher rate than nonmethylated cytosines, which can lead to higher mutation rates in methylated regions [44].If A c c e p t e d M a n u s c r i p t genes that are associated with resistance are methylated, which leads to increased expression and increased mutation rate, then genes that are most upregulated in response to insecticide resistance may also be the most likely to experience spontaneous deamination.
Both the role of histone modifications and small RNA in modifying epigenetic responses to toxins are less understood than DNA methylation in arthropods, though it has been shown that methylation and histone modifications tend to be co-located in the genome [38].Kishimoto et al.
[45*] showed that parental hormetic responses to oxidative stress can be epigenetically transmitted to descendants via histone modifications.A wide range of environmental chemicals, such as heavy metals, air pollutants, dioxins, and endocrine disrupters, can alter histone modifications [46], but it is unknown whether these changes are heritable.We have not found any studies on arthropods examining if insecticides can induce transgenerational small RNAs responses.Small RNAs have been found to interact with histone modifications [47], so changes in small RNAs may be implicated in the transgenerational inheritance of stress phenotypes as well.

Implications for transgenerational effects on insect fitness in agroecosystems
We hypothesize that pesticide use can directly and indirectly drive the evolution of insect pests in agroecosystems via epigenetic processes (Figure 2).Pesticides may directly stimulate the expression of advantageous phenotypes, which may be underwritten by epigenetic modifications.
Continued insecticide use on populations developing resistance would thus operate as 'natural selection' and selectively increase the frequency of insect phenotypes that are adaptive to pesticides.Indirectly, pesticide use may maintain stressful environments that hormetically prime insect pests to become more tolerant of stressful conditions.For instance, sublethal exposure to

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Transgenerational effects on pest evolution 10 insecticides can influence adult body size of the L. decemlineata [23], which may allow insect pests to be better able to tolerate overwintering conditions [48].Insecticides can also increase female fecundity [49] or propensity to mate [25], which can increase population size.
The phenotypic traits of insect pests that allow them to thrive under insecticide exposure may also facilitate global invasions.For example, L. decemlineata is a globally-invasive pest that is expanding its range northwards into the Arctic Circle [48].Insecticide exposure appears to stimulate the beetle to invest more in fat bodies and have a higher metabolic rate than control beetles [50].While the higher metabolic rate and larger fat bodies may enable beetles to better detoxify chemicals, higher fat body reserves enable small individuals to overwinter successfully [51,52].For example, sublethal applications of the pyrethroid deltamethrin on resistant L. decemlineata populations can have stimulatory effects rendering exposed individuals larger which is also inherited to the next generation (Lindström, unpublished data).
To date, most of the research examining the role of pesticides or xenobiotics in epigenetic change come from the field of aquatic toxicology [53**], where environmental exposure to toxins can be highly variable and difficult to predict.In contrast, pesticide use in agroecosystems is intentionally part of an active pest management system, where insect responses to stresses can cause positive feedbacks on subsequent management decisions.Agroecosystems are also highly controlled systems, which allows for greater experimental control for field and landscape level studies.Along these lines, it would be important to know how epigenetic responses to the same insecticides may vary among individuals, populations, and species.Such information would help provide insight on whether epigenetic responses can be broadly predictable across individuals and species, and possibly, how pesticide resistance may be better managed.A combination of new genomic tools, epigenetic assays, and computationally-intensive approaches may allow us to

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c c e p t e d M a n u s c r i p t Transgenerational effects on pest evolution 3 stress may drive the evolution of insect pests.

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c c e p t e d M a n u s c r i p t Transgenerational effects on pest evolution 6

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photoperiod in the invasive Colorado potato beetle (Leptinotarsa decemlineata).

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c c e p t e d M a n u s c r i p t Transgenerational effects on pest evolution 18 **56.Asselman J, De Coninck DI, Beert E, Janssen CR, Orsini L, Pfrender ME, Decaestecker E, De Schamphelaere KA: Bisulfite sequencing with Daphnia highlights a role for epigenetics in regulating stress response to Microcystis through preferential differential methylation of serine and threonine amino acids.Environ.Sci.Technol.2017, 51:924-931.This study describes how stress-induced methylation patterns can be associated with certain codon and genes with certain functions relevant to a stress response.57.Kumar S, Kim Y: An endoparasitoid wasp influences host DNA methylation.Sci.Rep. 2017, 7:43287.58.Seong K-H, Li D, Shimizu H, Nakamura R, Ishii S: Inheritance of stress-induced, ATF-Dependent Epigenetic Change.Cell 2011, 145:1049-1061.

Figure 1 .
Figure 1.Examples of how changes in methylation status of in different gene regions can effect

Figure 2 .
Figure 2. How exposure to a stressor may lead to heritable epigenetic changes that could lead to

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Transgenerational effects on pest evolution

Table 1 .
Examples of epigenetic alterations following exposure to anthropogenic and "natural", (non-anthropogenic) stress.