Parental effects of Bt toxin and vitamin A on Helicoverpa armigera.

The increase in the area cultivated with vitamin-enriched transgenic crops producing Bt toxin raises the question of whether the addition of vitamins will in any way mitigates the effect of the toxin on the phytophagous insects that feed on those crops. On the other hand, the parental effect that feeding on these enriched transgenic crops may have on the offspring of the phytophagous that survive on them is not well known. In this work, the effect of vitamin A (β-carotene) addition to diets with or without Bt toxin on Helicoverpa armigera larvae and their offspring was determined. The addition of vitamin A did not have any beneficial effect either for the larvae fed on enriched diets nor for their offspring. However, parental effects due to dietary feeding with the toxin were detected since adults from larvae fed on the Bt diet had higher mating success than those fed on the toxin-free diet, although there were no differences on the fertility of mated females regardless of whether their previous larvae fed on the Bt or non-Bt diet. A certain adaptive effect to the toxin was also noted since the mortality of larvae whose previous generation fed on diet with Bt was lower than that of the larvae that came from larvae fed on a non-Bt diet. It would be interesting to determine if H. armigera adults prefer to mate and lay eggs in the same type of crops in which they have developed or if feeding on different crops, such as corn or alfalfa, causes different paternal effects on the offspring. These aspects can be of great importance in the development of resistance of this species to the Bt toxin.

Introduction

The portion of cultivated land devoted to transgenic crop production has been steadily increasing since being introduced in 1996. Since the introduction of transgenic crops, their characteristics have diversified to include some that incorporate the toxin produced by Bacillus thuringiensis (Bt), an entomopathogenic bacterium. In 2018, 26 countries, 21 developed and 5 industrialized, planted 191.7 million hectares of transgenic crops, most of which had two or more Bt toxins or were tolerant to herbicides [1]. Furthermore, many new biofortified conventional and transgenic crops have been developed, with improved nutritional characteristics such as high levels of vitamin A (β-carotene) or vitamin C (Ascorbic acid: AsA), and are now becoming commercialized [2, 3]. Some examples of these biofortified transgenic crops under development are biofortified transgenic sorghum for nutritional enhancement [4] and the "golden" rice, which has been recently authorized for consumption in the Philippines [5].

The increasing growth of vitamin-biofortified crops raises questions about the potential effects on the development and mortality of phytophagous insects that feed on these enriched crops. One of these questions is whether biofortified crops favor populations of phytophagous pests. To date, these concerns have received little attention. Unsurprisingly, little is known about the effects of biofortified Bt crops on phytophagous insects, or how an increase in vitamins could affect their survival, development, and behavior. Zanga et al. [6] found that the addition of β-carotene to a Bt diet moderated the efficacy of the Bt toxin, reducing larval mortality in the Bt target pest, Ostrinia nubilalis Hübner (Lepidoptera: Crambidae). It was hypothesized that the Bt toxin was less effective in the β-carotene groups as a result of the increased activity of enzymes involved in detoxification mechanisms, such as catalase (CAT), superoxide dismutase (SOD), and glutathione S-transferase (GST). Conversely, Girón-Calva et al. [7] demonstrated that newborn O. nubilalis larvae fed Bt diets exhibited higher mortality when supplemented with β-carotene. Similar studies focusing on caterpillars that are less susceptible to Bt toxins are particularly interesting, as these organisms may still be favored by the higher vitamin content of Bt maize plants. For instance, Lopez et al. [8] determined that the response to a vitamin A- and C-enriched Bt diet was somewhat different between two secondary maize pests, Helicoverpa armigera (Hübner) and Mythimna unipuncta (Haworth) (Lepidoptera: Noctuidae). Ingestion of the Bt diet caused oxidative stress in both species and resulted in longer larval development and lower pupal weight. These effects were not mitigated by the presence of vitamins in the diet; however, vitamin enrichment reduced the mortality of Bt-fed H. armigera. In addition, β-carotene decreased the activity of GST in both species, suggesting that this enzyme has an antioxidant role. The study demonstrated that biofortified Bt crops should not enhance the development of H. armigera or affect the development of M. unipuncta. However, the effects of increased vitamins could be highly variable amongst different species and should, therefore, be studied in each specific phytophagous.

Eizaguirre et al. [9] elucidated that the larvae of H. armigera, a polyphagous [10, 11] cosmopolitan pest whose larvae have a high capacity to develop resistance to insecticides [12, 13] and Bt toxins [14, 15], could survive and complete their development even when feeding on Bt maize. The introduction of biofortified Bt cultures could thereby improve the fitness of this low-susceptibility species when it feeds on these crops.

Due to the increase in temperature and irrigated areas in the region where this study was conducted, maize has changed from being cultivated in an annual cycle to more intense alternatives [16]. These include long-cycle grain maize sown in early spring and shorter-cycle forage maize sown after the harvest of winter cereal. This change in cultivation favors the presence of ears with tender silks from early spring to late fall and allows H. armigera to complete several successive generations. Despite scientists having recognized the immense influence of parental nutrition on progeny phenotype [17], there are few studies assessing the mating behavior of adults and the development of the next generation of larvae when fed Bt or non-Bt crops.

Similarly, studies assessing the potential consequences of a diet containing biofortified Bt-maize on the development, mortality, and final weight of the next generation of larvae are uncommon. Studies of this nature are significant because a portion of the H. armigera larvae that feed on Bt transgenic maize survives and pupates. These pupae are usually smaller in size and weight than those fed a non-Bt diet [8]. Because the weight of lepidopteran pupae has been associated with the fertility of the resulting adults [18], it is conceivable that adults from heavier pupae (larvae fed non-Bt maize) will present with higher fertility than adults from lighter pupae (larvae fed Bt maize). However, under field conditions, it is expected that adults emerging in a determinate field (Bt or non-Bt) mate in the same field and then disperse to lay eggs in neighboring fields [19] (Bt or non-Bt). Studies about whether the fact that the larvae fed on Bt or non-Bt maize enriched or not with vitamins will have consequences on development, mortality or final weight of larvae of the next generation are uncommon. Accordingly, this study aimed to determine the possible effects of parental diets, (i) with or without Bt toxin (ii) supplemented or not supplemented with vitamin A, on the fertility of the next generation of adults and the performance of the subsequent larvae fed again on a diet with Bt or non-Bt maize supplemented or not with vitamin A.

Materials and methods

Insect general rearing

To establish the population required for the experiments, H. armigera larvae were collected from alfalfa crops, with permission of the owner (Josep Piqué), in Lleida (GPS coordinates 41°37’39.1”N, 0°35’39.5”E). Research on insects does not need approval from an Institutional Animal Care and Use Committee (IACUC) [20]. All field-collected larvae (F0) were individually collected to avoid cannibalism. The general rearing conditions were 25°C temperature and 16:8 light: dark (LD) (h) photoperiod and relative humidity (> 60%). Larvae were fed until pupae emergence with the semi artificial diet (Fig 1) [21]. The pupae were maintained at 15°C and 16:8 LD photoperiod to synchronize adult emergence. When the number of females and males was considered sufficient to full a mating cage (6 females and 5 males per cage, [8]) the pupae were placed in the general rearing conditions in pots with daily moistened soil. Adults (F0) from these pupae were placed in the mating cages (cylindrical methacrylate tubes of 25 cm diameter and 40 cm height, closed at the base with an expanded polystyrene lid, and sealed at the top with white gauze) and given a drinker of honey solution and a cotton ball for egg laying. Eggs were maintained until hatching in the same rearing conditions.

Fig 1

Scheme of the performed experiments and the diets used for each generation.

Bt indicates diets with lyophilized leaves of Bt maize; non-Bt indicates diets with lyophilized leaves of non-Bt maize; +β indicates the addition of vitamin A (β-carotene: 0.6% in weight) to the corresponding diets.

Lyophilized maize

Leaves from Bt (DKC6667Y, with Cry1Ab, MON810) and non-Bt (DKC6666, isogenic) maize were collected from unsprayed maize crops near Lleida during the summer of 2017. The leaves were cut into small strips, and the main veins were removed. The plant material was freeze-dried in a vacuum drier (Gamma 2–16 LSC plus, CHRIST, Osterode am Harz, Germany) and ground in a Thermomix® until a fine powder was obtained. The lyophilized material was stored at -80°C for later use.

Diets

The diets used in these experiments were prepared by modifying the diet of noctuids from Eizaguirre and Albajes [21]. To obtain diets with sublethal doses of the Bt toxin, 25% of maize flour was replaced with lyophilized Bt or non-Bt maize leaves, with or without 0.6% of β-carotene. The amount of lyophilized Bt was chosen according to our previous study [22] in order to obtain a low toxin concentration in the diet (approximately 160 ng Cry 1Ab g-1 fresh weight) in the range of commercial transgenic maize [23]. The β-carotene concentration was decided according to our previous works [6, 8]. The resulting diets (Fig 1) were: diet made with non-Bt leaves (non-Bt), non-Bt leaves with 0.6% β-carotene (non-Bt+β), Bt leaves (Bt), and Bt leaves with 0.6% β-carotene (Bt+β).

Experiments

Experiments with the F1 larvae and adults

The newly hatched F1 larvae were divided into 4 groups (≈ 30 larvae per group) and fed 1 of the 4 experimental diets. Larvae were individually placed in small rearing boxes (5.5 cm diameter × 3 cm high) with a small portion of the corresponding diet. Larvae were checked every 2 or 3 days, and the diet was replaced when necessary. Larval mortality, larval development duration, and pupal weight were daily recorded. The mortality due to rearing management was not included in the calculations (e.g., larvae dead before eating the diet).

The resulting F1 pupae were characterized based on sex and parental diet, and kept at 15°C and 16:8 (LD) photoperiod to synchronize adult emergence. Once there was sufficient pupae for a mating cage, the procedure was the previously described for the F0. The emerging F1 adults were transferred to mating cages with a honey solution for feeding and a piece of hydrophilic cotton for egg laying. The mating experiment was performed using 36 females (6 cages with 6 females per /cage) and 30 males (6 cages with 5 males per /cage) for each parental diet.

Cotton pieces and drinkers were changed every two days. Cotton pieces containing eggs were identified and placed in separate boxes at 25°C to evaluate egg evolution. The neonate larvae (F2) and fertilized eggs were counted once the first larvae emerged (4 to 5 days) and the fertilized eggs were distinguishable by the obvious presence of the head. As each cotton piece had eggs of only two consecutive days, larval emergence was synchronized to avoid sampling error (resulting from counting the many infertile eggs of unmated females). Hatched larvae were placed in rearing boxes with the appropriate diet if it was necessary to continue the experiment, if not, they were frozen.

Once the F1 adults died, they were frozen for later verification of mating status. The number of mated females (F1) and the total number of larvae (F2) obtained from the mated females were analyzed.

Experiments with F2 larvae

At least 53 F2 larvae were randomly selected from the different mating cages of each treatment and used for the F2 experiments. To determine the effect of the different progeny diets (in addition to the effect of different parental diets) on the F2 larval development, F2 larvae were divided into 4 groups based on parental diet and each of these 4 groups was further divided based on the 4 diets used in the previous experiment (Fig 1). As a result, we obtained 16 different groups, with combinations considering the feeding of parental and progeny. Additionally, larval mortality, larval development duration, and pupal weight were recorded.

Statistical analysis

To assess the effect of diet and sex on development duration and F1 pupal weight, a two-way analysis of variance (ANOVA) was used (diet * sex). To analyze the effect of each component of the diet on the same biological traits, a three-way ANOVA was used (Bt * β-carotene * sex), and a student’s t-test was used for least squares means comparison.

Mortality was calculated as a percentage in each trial and was analyzed using a generalized linear model (GLM) with a binomial distribution. Mortality was assessed in relation to the influence of diet and dietary components (Bt and β-carotene).

Mating rate was calculated as the percentage of mated females in each mating cage and analyzed using a GLM with a binomial distribution. Fertility was calculated as the number of fertile eggs from mated females.

For the second-generation study, we added the parent factor (i.e., the diet ingested by the parents of the studied larvae). In this way, the analysis performed was a two-way (parents * diet) ANOVA on development duration and pupal weight, and a two-way ANOVA (Bt * β-carotene) for the effect of the dietary components. The effect of parental diet was also included in the mortality analyses.

All statistical tests were performed using JMP® Pro 15.2.0 [24]. In all statistical analyses, statistical significance was set at P < 0.05.

Results

F1 development and mortality

The larval development duration (in days) of H. armigera F1 larvae fed diets with or without Bt and with or without β-carotene is shown in Fig 2.

Fig 2

Development of Helicoverpa armigera F1 larvae.

Effect of the addition of vitamin A (β-carotene: 0 or 0.6% in weight) to non-Bt or Bt diets on F1 larval duration (in days) of Helicoverpa armigera. Bt (grey boxplot) indicates diets prepared with lyophilized leaves of Bt maize while non-Bt (white boxplot) indicates diets prepared with lyophilized leaves of non-Bt maize. Lined boxplots represent the addition of β-carotene to the diets while plots without lines represent diets without β-carotene. Statistical test: Two-way ANOVA (P < 0.005), means were compared by the LS Means Student’s test.

The duration of larval development of H. armigera depended on the type of diet ingested (F101, 3 = 66.83, P = 0.0001); however, there were no differences in the duration according to the sex of the resulting pupae (F101, 1 = 0.0158, P = 0.9003). Diet component analysis indicated that the presence of Bt toxin in the diet prolonged larval stage development (F101, 1 = 197.25; P = 0.0001). The addition of β-carotene alone did not influence this duration (F101, 1 = 0.6736; P = 0.4138); however, there was an interaction between Bt and β-carotene (F101, 1 = 6.0852; P = 0.0154) (Fig 2), in that β-carotene did not affect larval duration when they were fed Bt diets but prolonged larval development when they were fed non-Bt diets (Fig 2)

Statistical analysis of the weight (Fig 3) of the resulting F1 pupae indicated that larval diet had a significant effect on weight (F101, 3 = 5.86; P = 0.0010) but not on sex (F101, 1 = 0.26; P = 0.6099). Analysis of larval dietary components demonstrated that the presence of β-carotene affected the final pupal weight (F101, 1 = 7.3867; P = 0.0078). Similarly, the presence of the Bt toxin affected pupal weight (F101, 1 = 10.97; P = 0.0013) (Fig 3). These analyses indicate that pupae from larvae fed Bt diets weighed less than those fed non-Bt diets. Moreover, pupae from larvae fed diets with β-carotene weighed less than pupae from larvae fed diets without β-carotene.

Fig 3

Weight of Helicoverpa armigera F1 pupae.

Effect of the addition of vitamin A (β-carotene: 0 or 0.6% in weight) to non-Bt or Bt diets on the weight of the resulting F1 pupae of Helicoverpa armigera. Bt (grey boxplot) indicates diets prepared with lyophilized leaves of Bt maize while non-Bt (white boxplot) indicates diets prepared with lyophilized leaves of non-Bt maize. Lined boxplots represent the addition of β-carotene to the diets while plots without lines represent diets without β-carotene. Statistical test: Two-way ANOVA (P < 0.005), means were compared by the LS Means Student’s test.

Mortality, calculated as the percentage of dead larvae in each group, was analyzed using a GLM with a binomial distribution (Table 1). The results indicated that there were no differences in larval mortality based on diet type (Bt or non-Bt, with or without β-carotene) [Diet (χ2 = 3.7410; P = 0.2908); Bt toxin (χ2 = 0.00296; P = 0.9566); β-carotene (χ2 = 0.8531; P = 0.3557)].

Table 1

Mortality of the Helicoverpa armigera F1 larvae.

Diet	Mortality (%)
Bt	7
Bt+β	23
non-Bt	16
non-Bt+β	11

Percentage mortality of Helicoverpa armigera F1 larvae fed on Bt or non-Bt diets with or without β-carotene supplementation during all stages.

F1 mating and fertility

The percentage of F1 mated females (Fig 4) in each trial was analyzed using a GLM with a binomial distribution. The results indicated a statistically significant effect of diet (χ2 = 9.14, fd = 3, P = 0.0275). The diet component analysis indicated that the presence of the Bt toxin had a positive effect (χ2 = 8.195, fd = 1, P = 0.0042). The presence of β-carotene (χ2 = 0.2862, fd = 1, P = 0.5962) and the Bt–β-carotene interaction (χ2 = 0.758499, fd = 1, P = 0.3838) had no effects on mating percentage.

Fig 4

Mating of the Helicoverpa armigera F1 adults.

Mating percentage of the Helicoverpa armigera F1 adults of larvae fed Bt or non-Bt diets with or without β-carotene supplementation during all stages. Bt (grey boxplot) indicates diets prepared with lyophilized leaves of Bt maize; non-Bt (white boxplot) indicates diets prepared with lyophilized leaves of non-Bt maize. As the presence of β-carotene had no effects on mating percentage, column Bt includes Bt and Bt+β-carotene results and column non-Bt includes non-Bt and non-Bt+β-carotene results.

Fertility was calculated as the number of fertile eggs from mated females. Assessment of fertility using ANOVA revealed no differences in the number of eggs laid based on larval diet type (F23, 3 = 0.9171; P = 0.4505) (Table 2).

Table 2

Mating and fertility of Helicoverpa armigera F1 females.

DIET	% Mated females (X¯ + se)	Fertile eggs/mated female (X¯ + se)
Bt	61.1 ± 10.24	459.9 ± 178
Bt+β	58.3 ± 8.33	296.8 ± 76.8
non-Bt	30.6 ± 13.89	508.11 ± 91.66
non-Bt+β	41.7 ± 19.12	373.63.8 ± 60.38

Percentage of mated Helicoverpa armigera F1 females of larvae fed Bt or non-Bt diets with or without β-carotene supplementation and number of fertile eggs of the mated F1 females.

F2 Development and mortality

No significant differences were found in the duration of larval development (F617, 1 = 0.6205; P = 0.4312) or weight of the resulting pupae (F617, 1 = 0.2510; P = 0.6166) due to the sex factor.

The diet ingested by both the F2 and F1 larvae affected the duration of development. Analysis of the F2 dietary components demonstrated that the Bt toxin was the only component that influenced F2 larval development (Fig 5 and Table 3).

Fig 5

Development of Helicoverpa armigera F2 larvae.

Larval duration (in days) of Helicoverpa armigera F2 larvae fed Bt or non-Bt diets with or without β-carotene supplementation coming from F1 larvae fed 1 of the 4 different diets. F2 diet indicates the diet of the experimental larvae; F1 diet indicates the diet of the parental larvae. Data were analyzed by a two-way ANOVA (P < 0.005); means were compared by LSD means.

Table 3

Statistics of the two-way ANOVA of larval development and pupal weight of Helicoverpa armigera F2.

	Larval development			Pupal weight
	F	fd	P	F	fd	P
Diet F1	22.55	632; 3	< 0.0001	1.0380	629;3	0.3752
Diet F2	12.97	632;3	< 0.0001	180.82	629;3	< 0.0001
Diet F1 * Diet F2	10.2329	632;9	< 0.0001	0.9307	629;3	0.4976
Components F2 diet
Bt	23.2974	632;1	< 0.0001	600.924	629;1	0.0001
β-carotene	0.0421	632;1	0.8374	7.55	629;1	0.0062

Statistics (F, degrees of freedom, and P) were obtained using two-way ANOVA to analyze the influence of parental F1 larval diet and experimental F2 larval diet on F2 larval development and pupal weight. The last three rows show statistics from the two-way ANOVA of the F2 dietary components.

F2 pupal weight was affected by the diet consumed by the experimental F2 larvae but not the diet consumed by the parental F1 larvae. (Table 3 and Fig 6).

Fig 6

Weight of the Helicoverpa armigera F2 pupae.

Weight of the Helicoverpa armigera pupae of F2 larvae fed Bt or non-Bt diets with or without β-carotene, from parental F1 larvae also fed 1 of the 4 experimental diets. F2 diet indicates the diet received by the experimental larvae while the F1 diet indicates the diet received by the parental larvae. Data were analyzed by two-way ANOVA (P < 0.005), and means were compared by an LSD test.

ANOVA of the dietary components indicated that pupal weight decreased in the presence of the Bt toxin or β-carotene.

The diet of the parental F1 larvae affected the mortality of the F2 larval progeny (χ2 = 38.841; P < 0.0001), as did the diet of these experimental larvae (χ2 = 158.37; P < 0.0001) (Fig 7). Mortality was also affected by the interaction between the F1 larval diet and the experimental F2 diet (χ2 = 29.63; P < 0.0001). Furthermore, mortality was increased in the F2 larvae that ingested the Bt toxin in comparison to those that did not. The mortality of the F2 larvae descending from F1 larvae fed on Bt diets decreased in both F2 larvae that were fed on Bt diets and non-Bt diets (Fig 7). Analysis of dietary components indicated that only the presence of the Bt toxin in the diet affected the resulting mortality (χ2 = 140.26; P < 0.0001) (Fig 7). β-carotene (χ2 = 0.20488; P < 0.6508), and the interaction (χ2 = 1.08321; P < 0.298) did not affect the mortality.

Fig 7

Mortality of Helicoverpa armigera F2 larvae.

Percentage of the mortality of F2 larvae fed on Bt or non-Bt diets. The grey boxplots indicate F2 diets with Bt toxin; white boxplots indicate non-Bt diets. The lined grey boxplot indicates F2 larvae fed on Bt diet descending from F1 larvae fed on non-Bt diet; the lined white boxplot indicates F2 larvae fed on non-Bt diet descending from F1 larvae fed on Bt diet. As the presence of β-carotene had no effects on mortality percentage, columns Bt include Bt and Bt+β-carotene results and columns non-Bt include non-Bt and non-Bt+β-carotene results.

Discussion

Numerous studies on phytophagous species have analyzed life-history traits that are affected by the nutritional quality of the parents and the transgenerational effects thereof [25]. Parental and maternal effects have been detected in response to various biotic environmental factors, such as the quality and quantity of the host plant and the presence of pathogens. Previous research has assessed life traits such as mortality, developmental duration, pupal mass, mating success, fertility, and offspring development and mortality [18, 25–27]. However, the effect of feeding on Bt crop on phytophagous life-history traits and the transgenerational effects thereof have received far less attention, perhaps due to the difficulty of collecting Bt-resistant larvae from Bt crops. To study these transgenerational effects, Guo et al. [28] collected H. zea larvae from seed blend refuges cops (RIB: seed blend refuges), sown with a mixture of Bt and non-Bt seeds, where the supposed that surviving larvae feed on sublethal doses of the toxin. Alternatively, Paula et al. [26] studied the intergenerational transfer of Bt proteins by feeding adults of non-target species on nectar with sublethal amounts of Bt toxin and by feeding larvae on leaves immersed in the Bt toxin. In the present study, neonate H. armigera larvae were fed, for 2 successive generations, on diet with a known concentration of Bt toxin [22] (a low-quality diet resulting in a sublethal dose of the Bt toxin) [29], or non-Bt leaves (a high-quality diet). The diets were or no supplemented with vitamin A. Using this model, the parental and transgenerational effects of diet quality on larval development, pupal weight, mating success, and female fertility were determined. The results confirmed that feeding on a Bt toxin diet throughout the larval instar prolongs development, producing lighter pupae from the larvae that survive. These findings have also been observed in a previous work on the same species, with a similar procedure (larvae fed on lyophilized Bt diet) [8] and on other related species, such as H. zea [28] but with larvae from field conditions, fed on sublethal amounts of the toxin. The parental effects of environmental nutritional factors on mating success have been studied scarcely; in this sense, Moreau et al. [25] related mating success with the quality of the parental diet. However, the present study demonstrated that H. armigera larvae that are fed on low-quality Bt diet produce lighter pupae and adults with greater mating success, while larvae that are fed a high-quality non-Bt diet produce heavier pupae and adults with lower mating success. Moreover, although Rossiter et al. [18] indicated that pupal weight is strongly correlated with fecundity in many Lepidoptera, this study shows that the fertility of H. armigera mated females is not affected by pupal weight. This was also observed in the fertility studies performed by Guo et al. [28, 30] on H. zea, and by Paula et al. [26] on Chlosyne lacinia (Geyer). Mating success and fertility could be two crucial factors that affect the performance and growth of H. armigera populations in Bt crops. According to Cahenzli and Erhardt [17], parental effects are one of the most important influences on the progeny phenotype. Regarding transgenerational effects, the results of this study indicate that the duration of larval development is longer when the parental larvae are fed a Bt diet instead of a non-Bt diet. Similar findings were obtained by Rossiter et al. [18], who fed Lymantria dispar diets of low nutritional quality and high phenolic content. The parental effect was also clearly seen in the lower mortality of F2 individuals whose parents were fed a Bt diet instead of a non-Bt diet. This decrease in larval mortality could be considered an adaptive effect to the toxin or the low-quality diet, as noted by Quezada-Garcia et al [27] in Chroristoneura fumiferana. This adaptive effect may favor the development of resistance to Bt, which has been observed in H. armigera [14, 15], as well as H. zea [28].

Of importance, the addition of β-carotene to the diet was not beneficial for the pest in terms of larval development. On the contrary, it produced a reduction in pupal weight and prolonged developmental duration, confirming the conclusions of Lopez et al. [8], that the addition of vitamins to Bt crops should not be an advantage for the pests of these crops.

Maternal and paternal effects due to environmental factors on offspring are complex phenomena that have been studied from various viewpoints, such as the induction of diapause, nutritional or reproductive responses, causes of certain pest outbreaks, immune responses to pathogens, and acclimatization to host plants. Transgenic crops should, therefore, be considered from the perspective of the consequences involving the pests that feed on them. H. armigera is highly polyphagous that feeds on various crops from different coexisting botanical families in the region of the present study. Therefore, it would be interesting to study the effects of parental host crop type on the mating success and mortality of the subsequent generation. Similarly, it would be interesting to study the mating and ovipositor preferences of females developed in transgenic maize, particularly if they prefer to mate with males of the same crop and if, once mated, they preferred to oviposit in the transgenic maize, as described by Hopkins’ host selection principle (HHSP), or by the chemical legacy hypothesis [31]. All of these factors play a part in determining the adaptability of H. armigera to Bt maize, thereby facilitating the development of resistance, which has already been detected in this species.

The file contains data set needed to draw Figs 2–7 and Tables 1–3 Parental effects of Bt toxin and vitamin A on Helicoverpa armigera.

(PDF)

Click here for additional data file.

6 Apr 2022

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4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments:

This paper by López et al. “Parental effects of Bt toxin and vitamin A on Helicoverpa armigera” has researched the effect of vitamin A (β-carotene) and Bt protein on H. armigera larvae and their offspring. The work could be helpful to better understand the transgenerational effect of vitamin and Bt protein, and the potential resistance of H. armigera to Bt crops. In general, the experimental design of the paper is good and the data provided consequent, the information amassed is interesting. However, the manuscript is not acceptable before some experiment should be added. Meanwhile, the language quality of the MS should be improved. I hope my comments could help this interesting work

Introduction

The paragraphs of the Introduction is much too long. And there are too many long sentences, many of them are hard to understand.

Materials and methods

Insect rearing

“H. armigera larvae were collected in maize or alfalfa crops in Lleida”. Host adaptability of insect has been found to be dependent on many factors, the authors need to be clear that the larvae were collected from maize? or alfalfa crops?

Diets

“……, a 25% of maize flour was replaced by lyophilized Bt or non-Bt maize leaves, adding or not 0.6 % of β-Carotene”. The author needs to explain that why diets and 25% maize folur, 0.6% β-Carotene. Is there any literature on this? What was the dosage of Cry1Ab protein in 25% of Bt maize flour?

Experiments

Experiments with the F1 larvae and adults

“Larval mortality, duration of larval development and pupal weight were recorded”. Were the insect observed and recorded the mortality daily? What is the standard of death? Why were there 6 females and 5 males in the mating cage?

Please clearly state the number of insect for each experiment.

Statistical analysis

“using a generalized linear model with a binomial distribution” corrected to “using a generalized linear model (GLM) with a binomial distribution”. I recommend that you use this symbol instead of the common abbreviations throughout the manuscript.

Results

F1 mating and fertility

“……but neither β-carotene nor the interaction Bt-β-carotene had any effect”, the result of this sentence was not show in Fig. 4.

F2 Development and Mortality

Can the authors clarify why they did not research on the β-carotene to F2 development and mortality? Figure note should be added in Fig. 7.

Discussion

The paragraphs of the Discussion is much too long

“However, the life-history-traits affected by phytophagous feeding……”, please cite literature references.

“……in previous work with the same species [8] and with other nearby species such as H. zea [22]”, the authors should be discussed the differences between references and this research.

“Maternal o paternal effects due to……” corrected to “Maternal or paternal effects due to……”

Reviewer #2: Reviewer Comments for PONE-D-22-04499

The manuscript entitled “Parental effects of Bt toxin and vitamin A on Helicoverpa armigera” by

Carmen López, Daniela Zanga, Alejandro Juárez-Escario, Pilar Muñoz, Matilde Eizaguirre provides advances in the existing knowledge on Bt toxins for crop protection.

Introduction

developed instead of development

Add more reference against each already mentioned “H. armigera is a serious cosmopolitan insect pest whose larvae are polyphagous [9] and have a high capacity to develop resistance to insecticides [10] and to the Bt toxin [11].

Rephrase “that feed on maize mainly on the silks (styles) among many other crops such as cotton or alfalfa.”

(to the Bt toxin) and although part of the larvae feed on transgenic maize diet, another considerable part survives and pupates.

were collected from maize or alfalfa crops in Lleida

16:8 light: dark (h) photoperiod

relative humidity (>60%)

the pupae were placed in the adult-emerging conditions at 25ºC

Rephrase this “(cylindrical methacrylate tubes of 25 cm Ø and 40 cm high, closed at the base with an expanded polystyrene lid and at the top with a white gauze) with a drinker with honey solution and a cotton ball for egg laying.”

80⁰C until use.

diet made with non-Bt leaves (non-Bt),

rewrite in scientific way (5.5 cm ø x 3cm high) and use X from symbols not from alphabets

correct the space between degree and Celsius throughout the text of manuscript (e.g., 25 º C)

Better to use symbolic multiplication instead of others……for (6 cages* 6 females/cage) and 30 males (6 cages* 5 males/cage) for each condition. & ANOVA was used (the factors were, diet * sex)

Maintain space with * for two-way (parents*diet) ANOVA on development duration

Maintain space (Bt * β-carotene).

Space between fig and number (e.g., Fig1 = Fig 1)

Results

Percent mortality of the Helicoverpa

Discussion

Please update with latest references

Overall, please carefully crosscheck the reference mentioned in the text and list.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Abid Ali

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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19 May 2022

PONE-D-22-04499

Parental effects of Bt toxin and vitamin A on Helicoverpa armigera

PLOS ONE

Patrizia Falabella Academic Editor and Reviewer's Questions

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Now Corrected

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

[We are grateful to Ministerio de Ciencia e Innovación (Spanish government) for financial support. The authors thank Teresa Estela her administrative support and Joan Safont for his invaluable help in the general rearing of insects. Thanks to Professor Ramon Albajes for his unconditional support.]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

[The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Done L 394

4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Done, Line 108

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No The document has now been revised by an official English Editing services. We have the certificate showing it.

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments:

This paper by López et al. “Parental effects of Bt toxin and vitamin A on Helicoverpa armigera” has researched the effect of vitamin A (β-carotene) and Bt protein on H. armigera larvae and their offspring. The work could be helpful to better understand the transgenerational effect of vitamin and Bt protein, and the potential resistance of H. armigera to Bt crops. In general, the experimental design of the paper is good and the data provided consequent, the information amassed is interesting. However, the manuscript is not acceptable before some experiment should be added. Meanwhile, the language quality of the MS should be improved. I hope my comments could help this interesting work

Introduction

The paragraphs of the Introduction is much too long. And there are too many long sentences, many of them are hard to understand. Done. The document has now been revised by an official English Editing services

Materials and methods

Insect rearing

“H. armigera larvae were collected in maize or alfalfa crops in Lleida”. Host adaptability of insect has been found to be dependent on many factors, the authors need to be clear that the larvae were collected from maize? or alfalfa crops?

The larvae of all experiments were collected only in alfalfa fields. Now this point has been corrected in M&M Line 108

Diets

“……, a 25% of maize flour was replaced by lyophilized Bt or non-Bt maize leaves, adding or not 0.6 % of β-Carotene”. The author needs to explain that why diets and 25% maize folur, 0.6% β-Carotene. Is there any literature on this? What was the dosage of Cry1Ab protein in 25% of Bt maize flour? Done. We have rewritten lines 129-136 adding the data and necessary references

Experiments

Experiments with the F1 larvae and adults

“Larval mortality, duration of larval development and pupal weight were recorded”. Were the insect observed and recorded the mortality daily? Yes, daily, line 149.

What is the standard of death? We consider the standard mortality, the mortality on the non-Bt diet

Why were there 6 females and 5 males in the mating cage? In the M&M, Insect General Rearing we have added the explanation with the reference of why we used 6 females and 5 males per cage, lines 114-115

Please clearly state the number of insect for each experiment. Each experiment indicates the number on insects tested. Lines 145, 171

Statistical analysis

“using a generalized linear model with a binomial distribution” corrected to “using a generalized linear model (GLM) with a binomial distribution”. I recommend that you use this symbol instead of the common abbreviations throughout the manuscript. Done

Results

F1 mating and fertility

“……but neither β-carotene nor the interaction Bt-β-carotene had any effect”, the result of this sentence was not show in Fig. 4. We apologized for the unclear presentation of these results. Now, we have added the statistics of the effect of β-carotene in lines 244-246, we have also modified the X-axis the Figure 4 and the Figure 4 caption (lines 251-253) to explain in a better way.

F2 Development and Mortality

Can the authors clarify why they did not research on the β-carotene to F2 development and mortality? Figure note should be added in Fig. 7. Again, we apologized for the unclear presentation of these results. Now, we have added the statistics of the effect of β-carotene in lines 298-299, we have also modified the X-axis in the Figure 7, and the Figure 7 caption (lines 304-306) to explain in a better way.

Discussion

The paragraphs of the Discussion is much too long

Done. The document has been revised.

“However, the life-history-traits affected by phytophagous feeding……”, please cite literature references.

The literature references appear in line 314

“……in previous work with the same species [8] and with other nearby species such as H. zea [22]”, the authors should be discussed the differences between references and this research. Done, lines 329-332

“Maternal o paternal effects due to……” corrected to “Maternal or paternal effects due to……” Changed in the new version, line 360

Reviewer #2: Reviewer Comments for PONE-D-22-04499

The manuscript entitled “Parental effects of Bt toxin and vitamin A on Helicoverpa armigera” by

Carmen López, Daniela Zanga, Alejandro Juárez-Escario, Pilar Muñoz, Matilde Eizaguirre provides advances in the existing knowledge on Bt toxins for crop protection.

Introduction

developed instead of development Changed in the new version

Add more reference against each already mentioned “H. armigera is a serious cosmopolitan insect pest whose larvae are polyphagous [9] and have a high capacity to develop resistance to insecticides [10] and to the Bt toxin [11]. Done, added more references. L74-77

Rephrase “that feed on maize mainly on the silks (styles) among many other crops such as cotton or alfalfa.” Eliminated in the new version

(to the Bt toxin) and although part of the larvae feed on transgenic maize diet, another considerable part survives and pupates. Rewritten in the new version

were collected from maize or alfalfa crops in Lleida Changed, line 108

16:8 light: dark (h) photoperiod Corrected

relative humidity (>60%) Corrected

the pupae were placed in the adult-emerging conditions at 25ºC

Rephrase this “(cylindrical methacrylate tubes of 25 cm Ø and 40 cm high, closed at the base with an expanded polystyrene lid and at the top with a white gauze) with a drinker with honey solution and a cotton ball for egg laying.” All M&M have been rewritten

80⁰C until use. -80 º C Corrected

diet made with non-Bt leaves (non-Bt), M&M have been rewritten

rewrite in scientific way (5.5 cm ø x 3cm high) and use X from symbols not from alphabets Done

correct the space between degree and Celsius throughout the text of manuscript (e.g., 25 º C) Done

Better to use symbolic multiplication instead of others……for (6 cages* 6 females/cage) and 30 males (6 cages* 5 males/cage) for each condition. & ANOVA was used (the factors were, diet * sex) Maintain space with * for two-way (parents*diet) ANOVA on development duration Done

Maintain space (Bt * β-carotene). Done

Space between fig and number (e.g., Fig1 = Fig 1) Done

Results

Percent mortality of the Helicoverpa Corrected

Discussion

Please update with latest references We have used the latest references we have found

Overall, please carefully crosscheck the reference mentioned in the text and list.

________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Abid Ali

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 May 2022

Parental effects of Bt toxin and vitamin A on Helicoverpa armigera

PONE-D-22-04499R1

Dear Dr. Eizaguirre,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Patrizia Falabella

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

23 Jun 2022

PONE-D-22-04499R1

Parental effects of Bt toxin and vitamin A on Helicoverpa armigera

Dear Dr. Eizaguirre:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Prof. Patrizia Falabella

Academic Editor

PLOS ONE