Hormetic and transgenerational effects in spotted-wing Drosophila (Diptera: Drosophilidae) in response to three commonly-used insecticides.

Although insecticide formulations and spray rates are optimized to achieve lethal exposure, there are many factors in agricultural settings that can reduce the effective exposure of insect pests. These include weather patterns, timing of application, chemical degradation/volatilization, plant structural complexity, and resistant populations. While sub-lethal exposure to insecticides can still have negative impacts on pest populations, they can also lead to stimulatory, or hormetic, responses that can increase the fitness of surviving insects. Sub-lethal concentrations may also produce increased tolerance in the offspring of surviving adults through transgenerational effects. Sub-lethal effects are pertinent for the invasive fruit pest, spotted-wing Drosophila, Drosophila suzukii (Matsumura), because its small size, diurnal movement patterns, and utilization of hosts with complex plant structures, such as caneberries and blueberries, make effective insecticide applications tenuous. In this study, we measured spotted-wing Drosophila survivorship, reproductive performance, and offspring tolerance in flies exposed to sub-lethal concentrations of three commonly-used insecticides (zeta-cypermethrin, spinetoram, and pyrethrin). We found some evidence for hormesis, with survival effects being sex- and concentration-dependent for all insecticides. Males were far more susceptible to insecticides than females, which in some cases exhibited higher eclosion success and reproductive rates when exposed to sub-lethal doses. We did not observe significant transgenerational effects at sub-lethal concentrations, despite trends of increased offspring viability for zeta-cypermethrin and spinetoram. More research, however, is needed to fully understand the role that sub-lethal effects may play in pest population dynamics, insecticide efficacy, and the development of genetic resistance.

Introduction

Spotted-wing Drosophila, Drosophila suzukii (Matsumura), an invasive species native to Asia, has become a serious pest in fruit-producing areas throughout the United States and Europe, following its establishment in the early 2000s [1-4]. The ability of spotted-wing Drosophila to infest ripening intact fruit has allowed the pest to occupy unique niches in invaded regions, while its ability to utilize non-crop hosts and overwinter has also led to the establishment of stable populations in regions of large-scale fruit production. High population densities, coupled with strict zero-tolerance infestation limits in fresh berry markets, have led to the increased use of broad-spectrum insecticides for control. Many commercial conventional and organic insecticides are available for use on spotted-wing Drosophila, and overall, these products are effective [5-9] but it is not uncommon for growers to perform 4–9 seasonal applications depending on the crop [10-12]. Reductions in efficacy may be due to resistance, which has been detected for some chemistries in California [13], Michigan [14], and Georgia [15]. However, other factors, such as weather [12, 16], timing of application [12, 17–19], pesticide volatilization/degradation [20-22], and penetration into cultivars with high structural complexity [23], plant nutrient content [24-26], and insecticide resistance [14, 27, 28], can also reduce insecticide efficacy. This can occur by impeding contact between the insecticide and spotted-wing Drosophila adults, considering that spotted-wing Drosophila are small, highly mobile, crepuscular insects that are documented to exhibit daily movement patterns [29-33]. It can also occur due to environmental factors that degrade insecticidal compounds or cause run-off, which can reduce the residual activity or effective exposure of insect pests. These factors can cause spotted-wing Drosophila populations to be occasionally or routinely subjected to sub-lethal doses of insecticides, even when growers adhere to the recommended concentrations and spray rates. While sub-lethal exposure to insecticides may still produce negative effects on insect performance [34-37], evidence of stimulatory effects, via hormetic and/or transgenerational effects, have also been documented. Hormetic and transgenerational effects can not only directly increase plant damage through stimulatory effects, they may also promote the evolution of insecticide resistance through their impacts on phenotypic diversity [38-45].

Hormesis occurs when a stressor, such as an insecticide, produces a stimulatory effect at low doses and an inhibitory effect at higher doses. It is typified by a J- or U-shaped dose-response curve [46, 47]. Hormetic responses in insects have been documented since the mid-1940’s [48-50], corresponding with the rise in synthetic insecticide use. However, hormetic research didn’t increase substantially until the 1980’s [51-54]. Hormesis, as measured by increases in longevity [50, 54–56], reproductive rate [51, 52, 57–68], body size [53, 54, 56, 69–72], oviposition [63, 73–77], and growth rate [78-80] have been reported for a wide range of different insect genera and chemical compounds [80]. Despite the wealth of studies documenting hormetic effects at low dose ranges for different insecticides, the implication of these effects for pest control efficacy have been largely under-explored, but see [80]. Though addressed by some [80-89], it is surprising that the effects of hormesis have not been more thoroughly discussed in agricultural pest management, considering that there are a multitude of factors in agricultural settings that may reduce effective exposure and there are significant costs associated with insecticide failures due to increases in insect tolerance or genetic resistance. Another, often-overlooked, impact of sub-lethal exposure is transgenerational tolerance [90, 91]. Transgenerational effects, sometimes referred to as maternal or paternal effects, occur when sub-lethal parental exposure to a toxin leads to increased tolerance in their offspring [40]. In agricultural systems transgenerational effects have been documented for aphids exposed to imidacloprid [81] and thiamethoxam [89] nitenpyram [92], and carbamate [93]. Transgenerational response to insecticides have also been reported for white flies [94] caterpillars [95], and other insect predators [96-98] in agricultural systems. These effects are purported to be mediated by epigenetic mechanisms, such as heritable gene methylation and/or histone modification, or the transfer of cytoplasmic molecules like RNAs [40, 99, 100]; however, the underlying mechanisms of these response are rarely studied.

While the development of genetic resistance arguably poses a greater threat to the viability of current chemical controls, gene-by-environment interactions associated with sub-lethal exposure, including hormesis and transgenerational effects, may also pose significant threats to insecticide efficacy. This is particularly true given that a multitude of different factors can reduce effective insecticide exposure in agricultural systems, that, when taken together, can lead to cumulative effects that are potentially more widespread. Sub-lethal effects can also generate diverse phenotypes that may serve as adaptive intermediate stages in the evolution of insecticide resistance [40, 101–103]. When taken together, the impact of these issues may rival that of genetic resistance.

In this study, we measured the sub-lethal effects of three commonly used insecticides for spotted-wing Drosophila, including a synthetic pyrethroid, zeta-cypermethrin (Mustang Maxx), a spinetoram (Delegate), and an organic broad-spectrum pyrethrin (Pyganic). More specifically, we aimed to determine whether hormetic and/or transgenerational effects were detectable when spotted-wing Drosophila adults were exposed to sub-lethal concentrations. Spotted-wing Drosophila is a relatively new invasive pest that utilizes a broad range of fruit cultivars that vary in structural complexity [3]. It is also a small, highly mobile pest that displays distinct diurnal movement patterns [30-32]. These characteristics, in addition to other environmental factors, may act as obstacles to achieving lethal practical exposures in the field. Therefore, determining the impact that sub-lethal exposure may have on spotted-wing Drosophila survivorship and/or performance is important for maintaining and/or optimizing insecticide protocols. Furthermore, documenting the prevalence of hormetic and transgenerational effects is needed for further understanding the extent to which gene-by-environment interactions may contribute to the development of insecticide tolerance and/or resistance.

Materials and methods

Fly culture

All flies used in the experiments were from a lab colony established in 2018 from infested raspberry samples collected at the UMORE Station in Rosemount, MN. Flies were kept in clear narrow polystyrene vials with foam plugs (Genesee Scientific, San Diego, CA), and each vial contained approximately 5ml of a standard cornmeal-based oligidic diet (cornmeal, sugar, agar, nutritional yeast, propionic acid, methyl paraben, ethanol), as well as a strip of filter paper to reduce condensation. Each vial contained approximately 50 adults, which were transferred to new diet every 2–3 days and kept in a walk-in chamber at ambient lab temperature (20–22°C) under a 14:10 light-day cycle [104].

Insecticide exposure

For all insecticide exposures, newly-eclosed flies (24–48 hrs old) were anaesthetized with CO2 and placed in glass scintillation vials that were coated with a specific concentration of insecticide residue. For all insecticide exposures, including the initial dose-response assays, we used Mustang Maxx (FMC Corporation, Philadelphia, PA) as a source of zeta-cypermethrin, Delegate (Corteva Agriscience, Indianapolis, IN) as a source of spinetoram, and Pyganic (MGK, Minneapolis, MN) as a source of pyrethrin. Zeta-cypermethrin formulations used acetone as a solvent, while spinetoram and pyrethrin formulations were water-based. Control flies were placed in vials with no insecticide coating to standardize for CO2 anaesthetization and vial conditions. The exposure period for all experiments was 4 hours, after which flies were transferred to rearing vials containing standard fly diet.

Dose-response assays

Before each experiment, dose-response assays were conducted for each insecticide. Mortality was recorded after 4 hours of exposure to zeta-cypermethrin (6 concentrations: 0, 0.05, 0.1, 0.5, 1, 20), spinetoram (7 concentrations: 0, 0.01, 0.1, 1, 10, 100, 1000), and pyrethrin (8 concentrations: 0, 0.001, 0.01, 0.1, 1, 10, 50, 140), respectively. Six replicates were used for each concentration and each vial contained 3 male and 3 female flies. The results of these initial assays were used to select the doses used for the sub-lethal and transgenerational treatments and were based on a probit analysis that estimated the concentrations associated with varying degrees of lethality.

Hormesis protocol

We measured survival and performance across five sub-lethal concentrations of three of the most commonly used insecticides for spotted-wing Drosophila control in the Midwest United States [105]: zeta-cypermethrin, spinetoram, and pyrethrin. We tested concentrations that corresponded to the LC0, LC10, LC20, LC30, LC40 values determined by our initial dose-response assays. We tested single-exposure regimes for all three insecticides but also a double-exposure regime for spinetoram and pyrethrin. Table 2 shows the exact concentrations of each insecticide used for each treatment. Flies in the single-exposure treatments were exposed at the start of the experiment, while flies in the double-exposure treatments were exposed at the start of the experiment and again 5 days later. During the treatment exposures, flies were placed in insecticide-treated vials for 4 hours then transferred into new insecticide-free vials with fresh diet. Flies were subsequently transferred to new vials with new diet three times for the rest of their lifespan. Each treatment consisted of 10 replicate vials containing 5 male and 5 female flies. Mortality was recorded daily for 10 days. The total number of pupae and emerging adults, as well as the average adult mass of F1 flies per day, were recorded for each vial throughout the entire lifecycle.

Table 2

Concentrations tested for each insecticide treatment and their corresponding lethal concentrations.

Confidence intervals (95%) are shown in parentheses.

Treatment	zeta-cypermethrin (ppm)	spinetoram (ppm)	pyrethrin (ppm)
LC0	0	0	0
LC10	0.005 (0.001–0.039)	0.05 (0.10–0.227)	0.001 (0.0001–0.004)
LC20	0.050 (0.009–0.391)	0.35 (0.073–1.637)	0.005 (0.001–0.017)
LC30	0.300 (0.045–2.056)	1.43 (0.302–6.804)	0.014 (0.004–0.048)
LC40	1.20 (0.186–8.497)	4.84 (1.019–22.989)	0.033 (0.009–0.115)

Transgenerational protocol

To determine if parental exposure had any impact on offspring susceptibility, we exposed adult parental flies (P) to one of 6 sub-lethal concentrations (LC0, LC10, LC15, LC20, LC25, LC30 doses) of cypermethrin, spinetoram, or pyrethrin and then measured their offspring’s (F1) susceptibility to a single diagnostic dose. Newly eclosed male and female P flies were exposed via a 4-hr vial assay then moved into new untreated diet vials containing 4 males and 4 females from the same exposure treatments. They were allowed to mate and lay eggs for 7 days. Each vial was transferred to new diet every 2–3 days for one week. Once enough F1 adults were available (~15 days after P exposure) groups of newly-eclosed 5 males/5 females were exposed to a ~LC25 dose of their respective insecticide, except for pyrethrin, which was exposed to a higher dose (~LC50) after observing unexpectedly high survivorship across the sub-lethal parental doses (0.1 ppm for zeta-cypermethrin, 1.0 ppm for spinetoram, and 0.1 ppm for pyrethrin. Mortality was recorded after a 4-hour vial exposure period. The mortality of F1 flies from the P control treatment, which was not exposed to insecticide, was compared to the F1 mortality across the other P treatments do determine if their susceptibility was statistically different.

Data analysis

A probit analysis was used to analyze the dose-response results and determine the lethal concentrations or each mortality rate. For the hormesis survival data, survivorship at the end of 11 days was analyzed in a three-way ANOVA for the spinetoram and pyrethrin datasets, with sex, exposure (single or double), and treatment as main factors. For the zeta-cypermethrin data, only sex, treatment, and sex*treatment were included as factors. The same ANOVA models were used for the performance data, which included the number of pupae (adjusted for the number of living females per vial per day, or pupae/female/day), eclosion success (the proportion of pupae that eclosed as adults), and average adult mass (the mass of all eclosing adults each day divided by the total number of adults). Each insecticide was analyzed separately, as experiments were done at different times. Datasets were rank-transformed when necessary to meet normality assumptions and a Tukey’s HSD test (with a Bonferroni correction) was used for post-hoc comparisons. For the transgenerational data, the effects of sex and parental treatment on the mortality of F1 adults were analyzed using an ANOVA, with a Tukey’s HSD test for post-hoc analyses (data were rank-transformed to meet normality assumptions). All statistical analyses were performed in SPSS v.27 (IBM Corp, 1989, 2020).

Results

Table 1 shows the summary statistics for each initial dose-response probit analyses. We achieved good fits for each curve, with all Chi-square analyses being non-significant, and all R2 values being greater than 0.80. Although the susceptibility of the lab colony to the different insecticides did vary, the dose-response assays were performed at different times, so no statistical comparisons were made. Adult flies were initially more susceptible to pyrethrin and less susceptible to spinetoram, with susceptibility to zeta-cypermethrin being intermediate. Fig 1A–1C shows the dose-response curves for each insecticide, which vary in the magnitude of the hormetic responses and dose ranges over which it occurs. Both the hormetic response and dose range for zeta-cypermethrin (Fig 1A) is considerable smaller than that for spinetoram (Fig 1B) and pyrethrin (Fig 1C). Our LC50 values for zeta-cypermethrin and spinetoram were comparable to those found for Michigan populations and reported in [9], with the zeta-cypermethrin LC50 being slightly higher and the spinetoram LC50 being comparable to Michigan populations before increases in insecticide use. Insecticide concentrations corresponding to specific mortality levels based on the initial probit analyses are shown for each insecticide in Table 2.

Table 1

Statistics for the initial dose-response probit analysis for each insecticide.

Insecticide	X2	df	P-value	slope	intercept	R2
zeta-cypermethrin	0.141	2	0.932	0.444	4.702	0.842842
spinetoram	0.231	3	0.972	0.549	4.370	0.812
pyrethrin	0.700	2	0.705	0.725	5.817	0.972

Fig 1

Initial dose-response curves.

Initial dose-response curves and equations for zeta-cypermethrin (a), spinetoram (b), and pyrethrin (c) with the dotted lines representing the zero equivalent point. Log transformations for zeta-cypermethrin and spinetoram were log10(ppm + 0.001) and log10(ppm + 0.0001) for pyrethrin.

Hormetic effects

The hormesis data for zeta-cypermethrin showed significant effects of sex and treatment but no impact of exposure type or any interactions (Table 3). Overall, female mortality was 1.4 times higher than that of male flies. Fig 2A shows that survivorship was highest in the LC10 treatment for males and in the LC20 treatment for females. Despite this, survivorship across the LC0, LC10, and LC20 concentrations was statistically similar but decreased significantly at the LC30 and LC40 concentrations (S1 Table). S1A and S1D Fig shows that survivorship for both sexes was high across all treatments until day 8, when mortality sharply increased for the controls and the higher concentration treatments but less so for the LC10 and LC20 treatments. Table 4 shows that sub-lethal exposure to zeta-cypermethrin had no effect on pupal rate (Fig 3A), on the eclosion success of F1 flies (Fig 4A), or on the adult mass of F1 flies (Fig 5A).

Table 3

Three-way ANOVA results for the effects of fly sex (male or female), treatment concentration (LC0, LC10, LC20, LC30, LC40) and type of exposure (single or double) on survival for each insecticide experiment.

Bolded values indicate statistically significant p-values (P ≤ 0.05).

Factor	zeta-cypermethrin		spinetoram		pyrethrin
sex	F1,180 = 15.90	P<0.0001	F1,180 = 10.67	P<0.001	F1,180 = 15.33	P<0.0001
treatment	F4,180 = 5.38	P<0.0001	F4,180 = 9.55	P<0.0001	F4,180 = 5.03	P<0.001
exposure	-	-	F1,180 = 1.01	P = 0.314	F1,180 = 1.19	P = 0.277
sex* treatment	F4,180 = 0.70	P = 0.596	F4,180 = 2.23	P = 0.068	F4,180 = 5.22	P<0.001
sex*exposure	-	-	F1,180 = 0.053	P = 0.818	F1,180 = 3.58	P = 0.060
treatment*exposure	-	-	F4,180 = 1.88	P = 0.116	F4,180 = 1.43	P = 0.225
sex*treatment*exposure	-	-	F4,180 = 0.23	P = 0.924	F4,180 = 0.49	P = 0.744

Single- and double-exposure were only tested for the spinetoram and pyrethrin experiments, hence the exposure factors are not included in the model for zeta-cypermethrin.

Fig 2

Survivorship.

Average survivorship for male and female flies across sub-lethal concentrations of (a) zeta-cypermethrin, (b) single-exposure and (c) double-exposure to spinetoram, (d) single-exposure and (d) double-exposure to pyrethrin (N = 10). Different letters indicate significant post-hoc differences between treatments or sex where significant main effects or interactions were found (S1 and S2 Tables; Tukey’s Test, P ≤ 0.05).

Table 4

Three-way ANOVA results for the effects of treatment concentration (LC0, LC10, LC20, LC30, LC40) and type of exposure (single or double) on female reproduction, including pupal rate (pupae rate/female), the eclosion success of F1 offspring, and the average mass of F1 offspring for each insecticide experiment.

Bolded values indicate statistically significant p-values (P ≤ 0.05).

Variable	Factor	zeta-cypermethrin		spinetoram		pyrethrin
Pupal Rate	treatment	F4,45 = 0.46	P = 0.761	F4,89 = 0.96	P = 0.435	F4,90 = 2.87	P = 0.027
	exposure	-	-	F1,89 = 2.38	P = 0.128	F1,90 = 11.61	P = 0.001
	treatment*exposure	-	-	F4,89 = 1.33	P = 0.266	F4,90 = 0.97	P = 0.427
F1 Eclosion Success	treatment	F4,45 = 2.16	P = 0.089	F4,85 = 3.33	P = 0.014	F4,83 = 0.42	P = 0.796
	exposure	-	-	F1,85 = 2.77	P = 0.100	F1,83 = 3.13	P = 0.081
	treatment*exposure	-	-	F4,85 = 0.61	P = 0.659	F4,83 = 2.01	P = 0.100
F1 Adult Mass	treatment	F4,45 = 0.62	P = 0.650	F4,79 = 1.54	P = 0.198	F4,83 = 0.28	P = 0.893
	exposure	-	-	F1,79 = 0.67	P = 0.416	F1,83 = 1.76	P = 0.188
	treatment*exposure	-	-	F4,79 = 5.33	P = 0.001	F4,83 = 0.65	P = 0.626

Single- and double-exposure were only tested for the spinetoram and pyrethrin experiments, hence the exposure factors is not included in the model for zeta-cypermethrin.

Fig 3

Pupation success.

Pupal rate across single- and double-exposures for (a) zeta-cypermethrin (N = 19–20), (b) spinetoram (N = 9–10), and (c) pyrethrin (N = 10). The last collection of pupae from the pyrethrin treatment were too numerous to count accurately, so data only include the first two collections. Different letters indicate significant post-hoc differences between treatments (Tukey’s Test, P ≤ 0.05).

Fig 4

Eclosion success.

Eclosion success of offspring across single- and double-exposures for (a) zeta-cypermethrin (N = 17–20), (b) spinetoram (N = 8–10), and (c) pyrethrin (N = 5–10). Different letters indicate significant post-hoc differences between treatments (Tukey’s Test, P ≤ 0.05).

Fig 5

Adult mass.

Adult mass of offspring across single- and double-exposures for (a) zeta-cypermethrin (N = 11–20), (b) spinetoram (N = 8–10), and (c) pyrethrin (N = 5–10). Different letters indicate significant post-hoc differences between each exposure group across treatments (Tukey’s Test, P ≤ 0.05).

Despite having the highest initial LC50 of all the insecticides, the spinetoram treatment had the lowest survivorship across the sub-lethal concentrations. Survivorship in the controls only averaged ~40% for females and 13% for males. A significant effect of sex and treatment was found, as well as a marginally significant sex*treatment interaction, but no effect of exposure type (Table 3). Female flies had significantly higher survivorship than males, but the overall trends were similar. Fig 2B and 2C shows that survivorship was highest in the LC0 and LC10 concentrations for males and in the LC10 and LC20 treatments for females. Again, however, the LC0, LC10, and LC20 concentrations were statistically similar, with a significant decline in survivorship occurring at the LC30 and LC40 concentrations for both sexes (S1 Table). S1B and S1E Fig shows that survivorship sharply declined around day 6 for the LC40 treatment but not until day 9 for the other treatments. Sub-lethal concentrations of spinetoram did not impact pupal rate (Fig 3B) but did significantly impact eclosion success. Fig 4B shows that eclosion success was highest for the LC10 treatment and significantly lower for the LC40 treatment (S2 Table). There was a significant interaction between treatment concentration and exposure type on F1 fly mass (Table 4). Average adult mass did not differ across concentrations in the single-exposure group; however, adult mass was significantly higher in the LC30 and LC40 treatments for the chronic exposure group (S4 Table, Fig 5B).

For pyrethrin, survivorship was high across all treatments and there was a significant interaction between sex and treatment (Table 3). Fig 2D and 2E shows that female survivorship was comparable across all concentrations in both the single- and double-exposure treatments. Male survivorship was high and comparable across the 0 to LC30 treatments but significantly lower in the LC40 treatment for both single- and double-exposures (Fig 2D and 2E). Survivorship was statistically similar for males and females in the LC0-LC30 treatments but males exhibited significantly lower survivorship than females in the LC40 treatments (S4 Table), which S1C and S1F Fig shows was due to a decline in male survivorship around day 5. There was a significant effect of treatment and exposure type on pupal rate (Table 4, Fig 3C) but no other significant effects on F1 eclosion success (Fig 4C) or adult mass (Fig 5C). Interestingly, pupal rate was highest in the LC30 treatment for both single- and double-exposure treatments and higher overall in the double-exposure treatments (S5 Table).

Across all insecticides, which can only be compared qualitatively, pupal rate was similar in the zeta-cypermethrin and spinetoram experiments but considerably higher in the pyrethrin experiment (Fig 3A–3C). Eclosion success of F1 flies was also similar for the zeta-cypermethrin and spinetoram insecticides but considerably lower in the pyrethrin treatments (Fig 4A–4C), ranging from 7.5–41.5% compared to 24.0–44.7% for zeta-cypermethrin and 21.8–45.8% for spinetoram. Adult mass of F1 flies was similar across insecticides (Fig 5A–5C).

Transgenerational effects

For all insecticides, and in corroboration with the sub-lethal data, males had higher overall mortality than female flies (Fig 6A–6C). Table 5 shows that a significant parental treatment*sex interaction was found for pyrethrin, with Fig 4C indicating that F1 male morality was only higher in the LC15 and LC20 parental treatments but similar across the other treatments. Overall, mortality was considerably lower in the pyrethrin experiment (Fig 6C), despite using a higher LC50 diagnostic dose.

Fig 6

F1 Mortality.

Average mortality of male (darker bars) and female (lighter bars) F1 flies from (a) zeta-cypermethrin (N = 6–10), (b) spinetoram (N = 1–10), and (c) pyrethrin (N = 6–10) parental exposure treatments after exposure to a diagnostic dose. Mortality of parental flies exposed to no insecticides are shown in blue, all other exposure concentrations are shown in orange. Different letters indicate significant post-hoc differences between the sexes for each treatment (Tukey’s Test, P ≤ 0.05).

Table 5

Two-way ANOVA results for the impacts of sex and parental exposure to zeta-cypermethrin, spinetoram, or pyrethrin on adult F1 mortality after exposure to a diagnostic dose.

Bolded values indicate statistically significant p-values (P-value ≤ 0.05).

Factor	zeta-cypermethrin	spinetoram	pyrethrin
parental treatment	F5,96 = 0.265, P = 0.931	F5,105 = 0.740, P = 0.595	F5,94 = 0.931, P = 0.918
sex	F1,96 = 11.36, P = 0.001	F1,105 = 18.60, P<0.001	F1,94 = 0.1.222, P = 0.272
parental treatment*sex	F5,96 = 0.545, P = 0.742	F5,105 = 0.664, P = 0.651	F5,94 = 2.676, P = 0.026

Overall, results showed comparable mortality with a weak trend of slightly higher mortality for males and slightly lower mortality for females in some sub-lethal parental treatments. For instance, Fig 6A shows that female mortality was lower than the controls in the zeta-cypermethrin LC10 and LC25 treatments. Fig 6B also shows lower female mortality than the controls in the spinetoram LC15 treatment. For pyrethrin, Fig 6C, mortality is lower than controls in the LC10 and LC30 treatments for males and in the LC15 and LC20 treatments for females (S6 Table). However, despite showing a significant effect of sex on F1 survivorship for the zeta-cypermethrin and spinetoram treatments, there was no significant effect of parental exposure for either sex (Table 4).

Discussion

Overall, we found strong sex-specific effects of sub-lethal concentrations on spotted-wing Drosophila survival (Table 3). In general, male mortality was higher than female mortality, even across control treatments, and this pattern was evident in both the hormesis and transgenerational experiments. Sub-lethal exposure had largely similar effects on survival across insecticides and showed a trend of hormesis, as survivorship was the same or higher than the control treatments in at least one sub-lethal dose for each insecticide (Fig 2A–2E). For instance, survivorship was higher than the control in the LC10 and LC20 treatments for males and the LC20 treatment for females in the zeta-cypermethrin experiment (Fig 2A). A similar pattern was evident for spinetoram (Fig 2B and 2C) and pyrethrin (Fig 2D and 2E). Despite this trend, the differences between the controls and other concentrations were not statistically significant. These results, however, advocate further testing, as it is possible that the number of doses we tested limited our ability to detect a maximized hormetic response. It is also possible that our sample size limited our statistical power to detect differences [106]. After assessing over 11,000 dose-responses in animal, microbe, and plant systems, [107] showed that the maximum hormetic stimulation increased with the number of doses assessed below the zero equivalent point (ZEP), i.e., the dose where the response is the same as the control group. This indicates that the number of doses tested and the dose range surveyed have a significant impact on the ability to detect hormesis and quantify its magnitude of stimulation. As shown in Fig 1A–1C, the ZEP for the initial dose-response curves for zeta-cypermethrin, spinetoram, and pyrethrin were 0.05, 1.67, 0.0085 respectively, resulting in only one dose for zeta-cypermethrin, three doses for spinetoram, and two doses for pyrethrin being tested below the ZEP. Given that [107] suggest testing six doses below the ZEP, it is likely that the doses tested were too restrictive to elicit the maximum hormetic stimulation for all insecticides. Despite this, we tested a comparable or greater number of doses than several other studies that found evidence for insecticide hormesis [81, 85, 88]. Ultimately, higher-resolution studies will be needed to better determine if these insecticides are likely to produce hormetic effects in spotted-wing Drosophila and what those dose ranges would be.

For female reproductive performance the data also exhibited some evidence of hormesis. The eclosion success data exhibited an inverted-U shape curve for all insecticides, but differences between controls and other concentrations were only evident for spinetoram (Fig 4B). There were three specific instances where hormetic effects were statistically supported by the performance data. Pyrethrin pupal rates were higher overall for the double- versus the single-exposure treatments. Also, the LC30 treatments for both exposure groups showed significantly higher pupal rates than the controls (Fig 3C). For spinetoram, average adult mass was also significantly higher in the LC30 treatment than the controls but only for the chronic treatment (Fig 5B). These results show that some spotted-wing Drosophila performance variables do respond hormetically to sub-lethal exposure and suggest that female reproductive performance may be stimulated at low dose ranges. Of course, in order to understand the effect that these responses may have on fly population dynamics, more work will be required to determine their ultimate impact on overall fecundity and offspring viability.

An interesting result from hormesis experiments was that the mortality observed across the sub-lethal treatments was somewhat discordant from what was expected based on the initial dose-response bioassays for each insecticide. For example, zeta-cypermethrin survivorship was slightly lower than expected across all sub-lethal doses, while pyrethrin survivorship was much higher than expected, exhibiting ~90% survival across all sub-lethal treatments (Fig 2D and 2E). Spinetoram survivorship was markedly lower than expected, with 100% mortality observed in the LC40 treatment (Fig 2C and 2D). These results are particularly surprising given that the initial dose-response assays were performed only a month before the sub-lethal experiments, limiting the likelihood that the populations used for the initial dose-responses and the sub-lethal experiments were genetically different due to lab selection or drift. Overall, these results suggest that either an external unknown factor may have impacted the lab colony’s susceptibility to spinetoram or that the colony contained a large amount of genetic variability across genes related to insecticide resistance and detoxification. The genetic variability hypothesis is bolstered by the fact that the discrepancy between the initial dose-response curves and experimental results were similar for the insecticides with the same mode of action. Zeta-cypermethrin and pyrethrin are both sodium channel modulators [108, 109], while spinetoram is a spinosad that acts on GABA-gated chloride channels [110, 111]. The sex-specific differences in survivorship, on the other hand, seem to be impacted by the intensity of the insecticide, as differences between male and female flies were greater in the zeta-cypermethrin and spinetoram treatments, both synthetic insecticides, than the pyrethrin treatment, an organic insecticide. However, this likely a reflection of the fact that pyrethrin had more limited effects overall on mortality than the other two insecticides, at least at the sub-lethal doses tested (Fig 2). The higher survivorship exhibited by female versus male flies has important implications for population dynamics and pest management, particularly for spotted-wing Drosophila in fruit production systems where oviposition in high-value crops is the primary driver of reductions is marketable yields.

While the transgenerational results did show a trend of higher mortality in male F1 flies from parents exposed to zeta-cypermethrin and spinetoram, differences between the controls and treatments were not statistically different (Fig 6). These data do not indicate that parental exposure had any positive impact on offspring susceptibility; however, we only subjected F1 flies to one diagnostic dose. Without a full dose-response assay, which was not possible in this study due to the limited number of F1 flies available, we cannot determine whether susceptibility at other doses was affected. It is also unknown how parental exposure may have impacted other fitness characteristics in F1 flies, such as performance variables. Several other studies that have looked at and detected transgenerational effects exposed parental populations for multiple generations [81, 112, 113], which may be necessary for hormetic traits to exhibit in offspring. To better assess the potential for transgenerational effects to play a role in spotted-wing Drosophila population dynamics, future research should focus on multiple fitness characteristics, including survival and performance, measured over a broader range of insecticide concentrations administered in acute and chronic exposures.

In conclusion, the results of this study show variable effects of sub-lethal insecticide exposure on spotted-wing Drosophila survival, with potential hormetic effects on survival and demonstrable impacts on female reproductive traits. Additionally, the methodological uncertainties discussed above highlight the difficulties that exist in studying hormetic effects. Unlike the stabilizing responses to lethal insecticide exposure, i.e., the inevitability of 100% mortality, hormetic and transgenerational effects represent transient maxima occurring at some point along an insect’s dose-response curve; a point that is often unknown a priori and can be easily missed if the dose range and/or number of concentrations tested do not provide adequate resolution. This requires testing a large number of treatments with high replication and makes identifying these responses logistically challenging. Despite this, it is important to account for the impact that these lesser-studied responses have on pest population dynamics, particularly in agricultural systems. While the impact that sub-lethal effects have on insecticide efficacy may be indirect, ambiguous, and seemingly minor, their cumulative effects are potentially significant and widespread [37, 40, 114, 115].

There are many factors that can reduce effective exposure in the field, including effective spraying procedure, the structural complexity of the host, insecticide degradation due to environmental factors, and the movement of insects in-and-out, as well as, within the system. Depending on the level of sub-lethal exposure, results can have negative, neutral, or positive impacts on insect fitness, which can directly affect pest population dynamics but can also have indirect effects. For example, sub-lethal exposure, by definition, doesn’t lead to instant mortality, and therefore, doesn’t directly contribute to the evolution of genetic resistance through natural selection. However, it can produce resistant phenotypes intra-generationally through hormesis and inter-generationally via epigenetic effects. Poor nutrition [116-118], exposure to various environmental stressors [119-123] and insecticides have been shown to produce transgenerational tolerance in many different taxa, including insects. Given that epigenetic changes can be instantaneous and widespread, transgenerational effects have the capacity to have significantly impacts on insecticide efficacy in agricultural systems. However, because epigenetic marks are reversible, transgenerational effects can be short lived in the absence insecticide use [124], manifesting in short outbreaks, or they can have longer-term effects on the development of resistance traits [40, 124]. The creation and perpetuation of phenotypic variability can impact natural selection by serving as adaptive intermediate stages [40, 101–103]. Yet, the relative contribution of these responses to the development of insecticide resistance is not well understood. In fact, even the evolution of genetic resistance in the field is still quite mysterious, as resistance can readily develop to multiple unique chemistries within the same species but is often lost at rates faster than predicted by natural selection [40, 125–127]. Genetic bottlenecks also do not appear to limit the evolution of resistance, as would be expected [40, 128, 129]. In light of this, researchers have hypothesized that plastic responses to insecticides may play an important role, either through direct impacts on mutation rates or via epigenetic effects [40, 128, 129]. In any case, much more research is needed to better understand this role, as well as the more generalized impacts of sub-lethal exposure on insecticide viability, particularly for pests that are difficult to control, such as spotted-wing Drosophila.

Time-dependent mortality.

Survival curves for male (a-c) and female (d-f) flies from the (a, d) zeta-cypermethrin, (b, e) spinetoram, and (c, f) pyrethrin experiments (N = 10).

(TIF)

Click here for additional data file.

Survivorship post-hoc results for treatment effects.

Tukey’s post-hoc test P-values for the significant treatment effects on survival from the three-way ANOVA. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Eclosion success post-hoc results for spinetoram.

Tukey’s post-hoc test P-values for the significant treatment effects on eclosion success for the spinetoram treatment. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Treatment*exposure post-hoc results for spinetoram.

Tukey’s post-hoc test P-values for the significant treatment*exposure interaction on adult mass for the spinetoram treatment. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Survivorship post-hoc results for pyrethrin.

Tukey’s post-hoc test results for the significant sex*treatment interaction on lifespan for the pyrethrin treatment. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Pupal rate post-hoc results for pyrethrin.

Tukey’s post-hoc test P-values for the significant treatment effects on pupal rate for the pyrethrin treatment. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Treatment*sex interaction post-hoc results for F1 mortality.

Tukey’s post-hoc test P-values for the significant parental treatment*sex interaction on F1 mortality for the pyrethrin treatment. Bolded values indicate statistically significant P-values (P-value ≤ 0.05).

(PDF)

Click here for additional data file.

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

31 May 2022

17 Jun 2022

Editor:

Our response to specific Reviewer comments, are noted below (Italics):

Reviewer #1: General comments

This is a good paper. Good hypotheses, good methods, good interpretation, although things get unwieldy in places (too much into the weeds on minor results that distract from key findings). I have offered some minor edits and a few papers that may or may not be of interest related to the topics discussed.

Specific comments

1. L80. It is usually advisable to avoid the word “beneficial” and instead use the word “stimulatory” when referring to hormetic effects. Beneficial effects of one endpoint measure can only be assessed in the context of other effects over the long-term, which are rarely measured in published paper. The word “stimulatory” will be more precise and accurate in most instances.

I don’t see the term “beneficial” used here, but perhaps you are referring to the use of “negative” and “positive” effects. We have changed these terms to “inhibitory” and “stimulatory”.

2. L95-99. Yes, under-explored but potential impacts in agriculture are becoming more appreciated, e.g. Sci Total Environ, 2022, 825, 153899

The Cutler et al., 2022 (ref 51) was added here.

3. L100-112. Links of insecticide induced hormesis with transgenerational effects, epigenetics, resistance, and enzyme induction have been studied, e.g. papers on hormesis by Rix, Ayyanath, Cutler

Three more citations were added on line 103 (Ayyanath et al., 2013; Rix et al., 2015; Ullah et al., 2020).

4. L162-164. Modify. You are not calculating exact concentrations. You are using the data to generate a linear model that estimates LC values, some of which you hypothesize will induce stress responses reflective of hormesis

The wording was changed to emphasize that the concentrations were generated from a model and used to estimate dose-mortality relationships.

5. L170. You are exposing insects to prescribed concentrations, not doses

The word “dose” was removed.

6. L171. Suggest using different terminology. Acute vs chronic is not differentiated one exposure vs two exposures, but rather short vs long-term exposure. Just call this what it is, being more precise and accurate: one exposure vs two exposures.

We changed the treatment names to “single-exposure” and “double-exposure” regimes rather than acute and chronic (line 174-177).

7. L171. Why was the two-time exposure not done with zeta-cypermethrin?

We made the decision to add another treatment after we had already completed the zeta-cypermethrin trials.

8. L176. Clarify. In the one-time exposure, flies were exposed for 4 h, and thereafter unexposed to insecticide for the remainder of their lives. In two-time exposure, flies were immediately exposed for 4 h, then unexposed for 116 h following, and then exposed for another 4 hours – correct? Were the “new vials” untreated with insecticide.

Yes, after the 4-hr exposure period the flies were moved into new vials containing no insecticide and remained unexposed for the rest of their lifespan. The double-exposure flies were treated for 4-hrs, then moved into clean vials and treated again in the same manner 5 days later. We changed the wording in this section to make these details clearer (line 179-182).

9. L183. Parental (P) generation adult flies?

We specified that parental treatments will be indicated by P and made changes throughout the document.

10. L186. New untreated vials?

We added the term “untreated” (line191).

11. L191-192. Why do you say approximate LC25 or LC50 DD concentration. Had you not previously determined ‘exact’ estimates of LC25 and LC50 values?

12. L193. … pyrethrin).

End parenthesis was added.

13. L219. More complete results of the probit analyses should be presented: Chi-square, confidence intervals, slopes, df, etc. These data may be informative in interpreting hormesis results, e.g. lower slope is indicative of a more heterogeneous response to the insecticide, which in turn might correspond to a more pronounced hormetic response in the population, particularly across generations where interactive tolerance/resistance factors might be more likely to come into play. Even as is table 1 lack proper headings and is presented bass-ackwards. (‘Insecticide’ should be the left-most column, thereafter with sequential presentation of lethal concentrations)

Table 1 was intended to show the concentrations that we used for each sub-lethal treatment and their justification based on their associated mortalities but we did add another table that shows more information about the initial dose-response bioassays we performed (line 223-225). Table 1 show the information regarding the dose-response curves and Table 2 now shows the treatment concentrations.

14. L243. The very low control survival in spinetoram treatments relative to the other insecticides is striking. So low that I wonder if these data should even be included. I assume this is discussed later in the Discussion.

We do discuss this result in the Discussion (line 354-361), although we cannot explain really it. While it is a surprising result, it is a consistent result across the sexes and the single- and double-exposure treatments so we will leave it in the manuscript but discuss its anomalous nature.

15. L311. Section 3.1 of this paper talks about some the experimental considerations for detecting hormesis. Perhaps more important in the number of doses/concentrations is replication within treatments to reduce type 2 error: Calabrese EJ. 2005. Paradigm lost, paradigm found…. Environ Poll 138, 378–411

We do briefly discuss this point on line 320 but have added this reference there.

16. L351-353. Seems a more likely and parsimonious explanation is that something unknown and external to the fly population, in and of itself, affected the fly population. It is doubtful genetic variability in a lab colony is so great to account for this. I bet if you did the experiment again today you’d get very different (better) survival for spinetoram treatments.

True. We included a sentence about external impacts (line360).

17. L391. Yes. I suspect hormetic responses, though subtle in many (not all) cases, are ubiquitous in the field and are simply not realized due to lack of study.

18. L412. These papers may be of interest as it relates to hormesis, tolerance to insecticides, resistance, etc. Pest management science 74 (2), 314-322; Journal of pest science 89 (2), 581-589; Science of The Total Environment 827, 154085

Yes, thank you. These references were added on line 423.

Reviewer #2: Hormetic and Transgenerational Effects in Spotted-wing Drosophila (Diptera: Drosophilidae) in Response to Three Commonly-Used Insecticides

Dear Authors:

I am glad to see more researchers examining hormesis in the context of agriculture. I have provided some specific comments to assist in editing the manuscript. By way of summary, there are areas of the introduction, methods, results, and discussion that need to be amended. The authors spend time in the introduction discussing epigenetic mechanisms of hormesis, however this is not an aspect of their paper, therefore this should be edited to be proportionate with their work and proposed potential future work. There are a number of areas in methods where not enough detail is included, and I have provided specific comments on these areas. There are a number of errors in describing the results. The results from the probit analysis are missing. This should be included. There is some confusion with mortality and survival, which confounds the findings. In addition, the figures need to be edited for consistency, and colour scheme changed. The discussion is heavily lacking in comparison and discussion of other works examining insecticide induced hormesis in insects. This is surprising as this field has grown considerably in the last 5-10 years. I have provided the authors with a number of examples of papers in this field that may be pertinent to their work, which also have works cited in this field, and I highly suggest the authors search the literature for more work on insecticide induced hormesis. Despite this, I think the authors are on solid footing with their work, and following edits, this paper should be ready for publication.

Review Comments

The abstract is well constructed and clear. I like that the authors began by discussing factors that can alter the effective exposure of pests to insecticides. This is key to understanding the legitimate implications of hormesis in agricultural systems.

Keywords

I would suggest the authors replace epigenetics with transgenerational effects, where epigenetics is often more associated with the examination of heritable effects on the genome not involving changes in nucleotide sequences, such as gene expression, gene methylation, histone modification etc, rather than strictly phenotypic changes. This could be misleading as to what was specifically studied, if interpreted as studying effects on the molecular level.

Respectfully, we feel that transgenerational effects appropriately fall under the broader field of epigenetics, in that they are mediated by the epigenetic mechanisms described above. Although this study does not directly address the molecular aspects of epigenetics, it is important to us that transgenerational effects are acknowledged as epigenetic phenomenon because it highlights the ability of responses that are not strictly genetically-determined to play a role in resistance, which is an underappreciated point.

Introduction

Line 57: I think here it would be better to start this as follows:

Spotted-wing Drosophila (Drosophila suzukii Matsumura), an invasive species native to Asia, has become a serious pest in fruit-producing areas throughout the United States and Europe, following its establishment in the early 2000s.

Ok, revised; however, as the authority name was placed in parentheses, as recommended by the ESA (Entomol. Soc. of America).

Line 61: What is the purpose of saying “certain climates”? It is a bit too vague to provide pertinent information. Be a bit more specific, or do not include at all.

We were referring to climates where winter temperatures do not allow for overwintering but have removed the phrase to improve clarity.

Line 63: You might want to include some specifics on regions and specific crops where D. suzukii outbreaks have been major issues.

It's difficult to pinpoint different spots because spotted-wing has become established in every U.S. state except Arizona, and despite this, data relating to specific economic costs is still rather limited (e.g., CA, and MN). As noted in Asplen et al. (2015), (and Refs # 3-5), SWD has become a major pest in nearly every U.S. state and EU country, where preferred host crops are available (raspberry, strawberry, cherry, etc.). With such low tolerances for fruit damage, rather low to moderate populations can have economic impacts (Refs #10-12). Because of this we feel it is more productive to discuss the areas where resistance is becoming a problem rather than its general pest status, which we discuss on line 70.

Line 71:

“This can occur by impeding contact between the insecticide and spotted-wing Drosophila adults, especially considering that spotted-wing Drosophila are small, highly mobile, and crepuscular insects that have been documented to exhibit daily movement patterns in 74 several crops.”

Be careful of long or run-on sentences. The writing of this manuscript is very good, but when sentences are too long it confounds their meaning. I would suggest reading back through the manuscript sentence by sentence, and look for areas where you can break up your sentences, or adjust your sentences.

We simplified the sentence and scanned for other run-on sentences.

Line 84: “Hormetic and transgenerational effects can not only increase plant damage through direct reductions in insecticide efficacy, they may also promote the evolution of insecticide resistance through their impacts on phenotypic diversity”

Be careful of wording here. Any increases in plant damage that would be associated directly with hormesis would be due to stimulatory effects induced on the insect, such as stimulation in fecundity/fertility (thus increased feeding on the plant), stimulation of longevity (thus longer lifetime of feeding), stimulation of feeding (thus more feeding), etc. The reduced efficacy of the insecticide is presumably already in existence if the insect is undergoing hormesis as hormesis occurs in concentration ranges generally below the NOAEL. Hormesis and transgenerational effects in this context are a function of reduced insecticide efficacy, hormesis does not act through reducing insecticide efficacy, which is how your statement is worded.

Good point. We reworded the sentence.

With regard to information on hormesis and pesticide resistance and impacts on phenotypic diversity or plasticity, see works by Costantini. Some examples:

Costantini D. Hormesis Promotes Evolutionary Change. Dose Response 2019; 17: 1559325819843376.

Costantini D. Does hormesis foster organism resistance to extreme events? Frontiers in Ecology and the Environment 2014; 12: 209-210.

Thanks. References were added.

Lines 96: “It is particularly surprising that the effects of hormesis have not been more thoroughly addressed in agricultural pest management, considering that there are a multitude of factors in agricultural settings that may reduce effective exposure and there are significant costs associated with insecticide failures due to increases in insect tolerance or genetic resistance.”

This has been examined and discussed by authors such as Cutler, Guedes, Rix, Ullah, amongst some others. Transgenerational effects are also addressed in these. For some examples see:

Cutler, G.C., Amichot, M., Benelli, G., Guedes, R.N.C., Qu, Y., Rix, R.R., Ullah, F., Desneux, N. (2022). Hormesis and insects: Effects and interactions in agroecosystems. Science of the Total Environment. doi: 10.1016/j.scitotenv.2022.153899

Ullah F, Gul HN, Desneux N, Gao XW, Song DL. Imidacloprid-induced hormesis effects on demographic traits of the melon aphid, Aphis gossypii. Entomologia Generalis 2019; 39: 325-337.

Ullah F, Gul H, Tariq K, Desneux N, Gao XW, Song DL. Thiamethoxam induces transgenerational hormesis effects and alteration of genes expression in Aphis gossypii. Pesticide Biochemistry and Physiology 2020; 165: 11.

Rix, R.R., Cutler, G.C. (2018). Does multigenerational exposure to hormetic concentrations of imidacloprid precondition aphids for increased insecticide tolerance? Pest Management Science. 74: 314-322.

Cutler GC, Guedes RNC. Occurrence and Significance of Insecticide-Induced Hormesis in Insects. Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms. 1249. American Chemical Society, 2017, pp. 101-119.

Rix, R.R., Ayyanath, M.M., Cutler, G.C. (2016). Sublethal concentrations of imidacloprid increase reproduction, alter expression of detoxification genes, and prime Myzus persicae for subsequent stress. Journal of Pest Science. 89: 581-589.

Guedes RN, Cutler GC. Insecticide-induced hormesis and arthropod pest management. Pest Manag Sci 2014; 70: 690-7.

Ayyanath MM, Cutler GC, Scott-Dupree CD, Sibley PK. Transgenerational shifts in reproduction hormesis in green peach aphid exposed to low concentrations of imidacloprid. PLoS One 2013; 8: e74532

Cutler GC. Insects, insecticides and hormesis: evidence and considerations for study. Dose Response 2013; 11: 154-77.

Thanks. We have added all of these references and cited them on line 97.

Line 101: Transgenerational effects can also refer to hormetic stimulatory effects (such as increased fecundity) occurring across generations, not just increased tolerance in offspring. The papers cited above also have examples of this.

While other researchers may categorize increased fecundity and other hormetic effects on reproduction as transgenerational effects, we feel that this can be misleading, given that these responses occur within the parent, not the offspring. It is important to make the distinction between processes occurring in the parent due to the exposure of the parent and those occurring in the offspring due to the exposure of the parent but not the offspring themselves (or the exposure of the offspring at early developmental stages, i.e., egg exposure). This is an important point because it specifies the mechanisms involved, which often get convoluted based on semantical differences in definitions. In our view, transgenerational effects are reserved for situations where expressional changes in the offspring are caused by parental exposure to a stimulus and the absence of direct exposure of the offspring. These responses implicate epigenetic processes, as they persist across meiosis/mitosis.

Lines 100-122: These paragraphs include some excellent information on potential epigenetic mechanisms of hormesis. As mentioned above regarding the abstract, the authors are not examining epigentic mehcnaisms, thus the information might be more suited to a small area of the discussion or conclusion as something that might need to be focused on in the future. Mentioning it at the outset of the paper and in a large section is misleading the reader into thinking it is part of your experimental focus. Your focus should be on the phenotypic effects observed from other studies. Given the fact the authors have missed a number of key studies/authors in this field, I would suggest perhaps searching through the agricultural hormesis literature further and focus there, rather than brining in the extraneous epigenetic work.

Ok. We moved a large portion of this paragraph to the Discussion and included more specific content about transgenerational effects reported in agricultural systems in the Introduction (line 107).

Methods

Fly Culture

Can the authors provide more details on the fly rearing? Were they kept in containers? Approximately how many flies per container? What are the specific details of the diet? Etc. My suggestion would be to describe it in as much detail as you would if you were giving someone an SOP (Standard Operating Procedure).

We added a more detailed description on line 146.

Insecticide Exposure

Make sure you include any necessary trademarks and company/manufacturer names and locations when using the brand names of the insecticides. Examples for how this may look can be found in the publications suggested in previous comments.

This information was added on line 157.

Dose-response assays

Can the authors list the concentrations used for each insecticide?

This information was added on line 167.

Hormesis protocol

The authors refer to their two-time exposure as a chronic exposure. I would suggest just calling it two-time exposure. Chronic exposure can more often be interpreted as being a constant exposure or constantly reoccurring exposure. I would not consider a two-time exposure to be chronic.

Noted. We changed the terminology to single- and double-exposure for these treatments.

Transgenerational protocol

Were the F1 adults of the same age or close in age when exposed to the diagnostic/discriminating dose?

Yes, we added the term “newly-eclosed” to indicate this on line 201.

Also, some punctuation is missing throughout the paragraph. The copy editor may pick up on this as well.

We fixed the punctuation issues.

Results

Dose-response assays

The authors should include the statistics associated with a probit analysis, ex: chi-square, fiducial limits for endpoints of interest (LC50, hormetic LC, etc).

Yes, we added a new table (Table 1) that contains this information and a brief discussion about the probit analysis on line 229.

Hormetic effects

I think the authors are mixing up mortality and survival here. Fig 2a shows that the LC10 and LC20 treatments had the highest survival for males and females, not the highest mortality as stated in the text. And survivorship decreases significantly at the LC30 and LC40 concentrations in Fig 2a. It does not increase as was stated in the text.

We corrected the results to indicate that survivorship was highest, not mortality.

I would also suggest the authors include the supplementary material as main material.

Respectfully, we feel that with 5 tables and 6 figures already in the main document, the best place for the remaining tables and figures are in the supplemental information section, particularly because most of the information contained in them is denoted on the figures (posthoc letters) or discussed in text (time course of mortality).

Line 243: “Despite having the highest initial LC50 of all the insecticides, the spinetoram treatment had the lowest survivorship across the sub-lethal concentrations. Survivorship in the controls only averaged ~40% for females and 13% for males.”

Given the large control mortality (40%) for females, it may not actually be that the spinetoram has the lowest survivorship, it may simply be a weak group of insects. There seems to be a vast difference between survivorship in controls with experiments corresponding to Figs 2b,c. From close to 80-100% survival to less than 10% in some cases, although it does seem proportional to the treatments. The authors should discuss and try to account for this.

All the insecticide trials were done at the same time with the same group of flies, so it is not likely that the insects were weaker. We do discuss this anomalous result in the Discussion with potential explanations (line 360).

Transgenerational effects

Line 287: LC15 should be LC10

The posthoc letters in Fig 4c indicate that male mortality was higher than females in the LC15 and LC20 treatments.

Line 290: “Results showed a trend toward comparable or lower mortality for some sub-lethal parental treatments”. While there are some very small trends, if anything, there appears to be slightly increased mortality in most concentrations compared with controls. However, there are no statistically significant differences.

Correct. Upon reassessment, the text was changed to better reflect the general trends.

Line 291: “For instance, Fig 6a shows that male mortality was lower than controls in the zeta-cypermethrin LC10 and LC25 treatments for female flies.” This is not what I see in this figure, although the trends mentioned in the other figures are visible. The legend is also in grey, while the bars in the figure are in orange, which may be confounding the authors findings.

The grey legend is to show that the male are in a darker shade than the females and the figure does show that female mortality was slighter lower than the female control values for the LC10 and LC25 treatments.

Discussion

Line 310: Despite this trend, the differences between the controls and other concentrations were not statistically significant. These results, however, advocate further testing, as it is possible that the number of doses we tested limited our ability to detect a maximized hormetic response.

Yes, indeed your results warrant further testing, you did see some small trends. I would agree that your concentration ranges may have limited your ability to detect the maximized hormetic dose. The range in which the maximal hormetic responses occur can be wide ranging, thus it can be difficult to detect in a first experiment. You may want to consider next time testing a number of concentrations below the LC10 and even LC1. Calabrese indicates that frequently the hormetic concentration falls below the NOAEL, however, with insects, studies have shown that hormetic effects can occur outside the NOAEL. Looking at transgenerational effects as well could certainly impact where the hormetic concentration could fall. A concentration that is not hormetic in one generation, could in fact be hormetic in another generation (as you saw with your eclosion data).

I like the fact the authors include a lot of analysis of what they found, unwinding their findings for the reader. I would suggest the authors find more works on hormesis in insects in the literature (there are lots) and weave this into the discussion, to perhaps assist their insights. For example, the authors could discuss other works on transgenerational effects, the ranges of concentrations other studies used compared with theirs. This would be a nice addition to the authors’ existing discussion on concentration ranges. The discussion is highly lacking in examples of hormesis in insects in response to insecticides, and the connection to agriculture. There is solid work on this. I have provided a number of examples, however there are many others, and the authors should provide some analysis of their work in relation to other studies.

Given the current length of our Discussion, and several new references already added, we do not feel that we have space to add many more examples from the literature. However, we have added a short comparison of our concentration range with other studies (line 337) and the methodology of other transgenerational studies (line 394). We believe additional examples, beyond what we now provide, is beyond the scope of this paper. Perhaps a future paper, such as a Review article, may be needed to fully update this topic for other researchers?

Tables and Figures:

Table 4: Represent the degrees of freedom with the F-value as per the previous tables.

Table 5 was edited to match Table 4.

I would suggest the authors use greyscale for their figures, rather than colour. If they want to use colour, I would suggest using darker colours. These colours are not aesthetically pleasing.

We decided to keep the colors but changed them to the darker colors used on Fig6 to maintain consistency.

The authors should be consistent with their formatting. For example, Fig 1 has the y-axis survivorship label on all the individual graphs, whereas the other figures do not. The graphs in Fig 6 are also boxed in. The top and right side lines should be removed from each graph to be consistent with the other graphs. Only the bottom x-axis, and left y-axis are necessary for these graphs.

We fixed the axes and removed the lines around Fig 6 to maintain consistency.

Thanks again for the excellent questions and suggestions for improvement of the manuscript.

Submitted filename: PLOS One reviewers response.docx

Click here for additional data file.

30 Jun 2022

Hormetic and transgenerational effects in spotted-wing Drosophila (Diptera: Drosophilidae) in response to three commonly-used insecticides

PONE-D-22-09651R1

Dear Dr. Deans,

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Additional Editor Comments (optional):

Dear authors:

I really appreciate the detailed effort put forth to address the comments brought by the reviewers. In my opinion, the MS was solid during the initial submission, but many of the changes made make aspects of this neat story clearer to the wider audience of this journal. Personally, and as a hormesis/transgenerational person, these results are really cool, and I really appreciate a thorough rebuttal letter.

Thank you,

GC

Reviewers' comments:

12 Jul 2022

PONE-D-22-09651R1

Hormetic and transgenerational effects in spotted-wing Drosophila (Diptera: Drosophilidae) in response to three commonly-used insecticides

Dear Dr. Deans:

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