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Survival and development of Spodoptera eridania, Spodoptera cosmioides and Spodoptera albula (Lepidoptera: Noctuidae) on genetically‐modified soybean expressing Cry1Ac and Cry1F proteins

Abstract BACKGROUND Spodoptera eridania (Stoll), S. cosmioides (Walker) and S. albula (Walker) (Lepidoptera: Noctuidae) are considered secondary pests of soybean in South America. The genetically‐modified soybean DAS‐444Ø6‐6 × DAS‐81419‐2 with tolerance to 2,4‐dichlorophenoxyacetic acid (2,4‐D), glyphosate and ammonium glufosinate herbicides (event DAS‐444Ø6‐6) and insect‐resistance due to expression of Cry1Ac and Cry1F Bt proteins (event DAS‐81419‐2) may provide a potential tool for integrated pest management (IPM) of these species in soybean fields. Based on this, we conducted bioassays to evaluate the survival and development of S. eridania, S. cosmioides and S. albula fed on Cry1Ac/Cry1F‐soybean leaf tissue. RESULTS Spodoptera eridania and S. cosmioides fed on Cry1Ac/Cry1F‐soybean showed longer developmental time, lower larval and egg to adult survival compared to those fed on non‐Bt soybean, reducing the population growth of these species. Spodoptera albula also had lower larval survival and number of insects that reached adulthood on Cry1Ac/Cry1F‐soybean. However, no significant effects of Cry1Ac/Cry1F‐soybean on population growth parameters were detected in this species. CONCLUSIONS Soybean with stacked events DAS‐444Ø6‐6 × DAS‐81419‐2 expressing Cry1Ac/Cry1F Bt proteins provide population suppression of S. eridania and S. cosmioides. However, this Bt soybean had minimal effects on S. albula, and is unlikely to have negative population‐level effects on this species. It is expected that under field conditions, other control tactics must be integrated with Cry1Ac/Cry1F‐soybean for the management of these Spodoptera species. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


INTRODUCTION
Soybean, Glycine max (L.) Merrill (Fabaceae: Phaseoleae), is the most important agricultural crop in Brazil, Argentina, Paraguay and Uruguay, being cultivated on approximately 59 million ha per season (35 million ha in Brazil). 1,2 The main soybean Lepidopteran pests are velvetbean caterpillar, Anticarsia gemmatalis (Hübner), soybean looper, Chrysodeixis includens (Walker), Old World bollworm, Helicoverpa armigera (Hübner), and tobacco budworm, Chloridea virescens (F.). [3][4][5][6] However, outbreaks of Spodoptera species have been reported in soybean fields, including S. frugiperda (Smith), S. eridania (Stoll), S. cosmioides (Walker) and S. albula (Walker). [7][8][9] Of these species, S. frugiperda is an emerging soybean pest due to increased infestations and damage observed on soybean in recent seasons in Brazil. Spodoptera eridania is the most important Spodoptera pest species on soybean in the Cerrado region, and southern Brazil. 9,10 In contrast, S. cosmioides and S. albula occur at relatively lower frequencies. 9 However, S. cosmioides can consume almost twice the soybean leaf area as other species from this genus. 11 Spodoptera species are considered secondary soybean pests in South American countries. 7,8 However, sporadic infestations of these species can cause significant yield loss to soybean due to their defoliation capacity 11 and damage to reproductive structures (flowers and pods). 9,12 Outbreaks of Spodoptera species in Brazilian soybean fields may be influenced by the widespread deployment of transgenic soybean plants expressing Bacillus thuringiensis Berliner (Bt) proteins. 13 Transgenic Cry1Ac-soybeans provide effective control of key defoliating pests (A. gemmatalis, C. includens, C. virescens and H. armigera), but only minimal activity against Spodoptera species. 3,13-18 The Bt soybeans with sustained efficacy against primary pests can create opportunities for secondary pests to become more prevalent. Outbreaks of these species may also be associated with the crop production landscape, such as cropping systems where there is an overlap and/or succession of cultivated host plants across seasons that provides available food resources for their survival and development. 12,13 In 2018, Bt soybean was the main control tactic for management of lepidopteran pests in South America, representing 41% of the total soybean plantings in Brazil, Argentina, Paraguay and Uruguay. 2,19 Recently, a new Bt soybean (event DAS-444Ø6-6 × DAS-81419-2) was approved for commercialization in Brazil. 20 The DAS-444Ø6-6 event (Enlist E3®, Corteva Agriscience, Wilmington, DE, USA) expresses the enzymes 5-enolpyruvyl shikimate-3phosphate synthase (2mEPSPS), phosphinothricin acetyltransferase (PAT), and aryloxyalkanoate dioxygenase 12 (AAD-12) that confer tolerance to the herbicides glyphosate, glufosinate ammonium, and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. The DAS-81419-2 event (Conkesta®, Corteva Agriscience) consists of insect-resistant technology that expresses Cry1Ac and Cry1F Bt proteins 20,21 and PAT that confers tolerance to the herbicide glufosinate ammonium as a selectable marker. This Bt technology demonstrates efficacy against A. gemmatalis, C. includens, C. virescens and H. armigera, 22,23 which are the main pest species for the development of Bt soybean events.
Transgenic plants expressing Cry1Ac and Cry1F Bt proteins have shown good activity against some Spodoptera species. [24][25][26] Thus, understanding the survival and development of Spodoptera species on Cry1Ac/Cry1F-soybean technology is particularly important to support integrated pest management (IPM), insect resistance management (IRM) programs and best agricultural practices to manage these species in soybean fields. In this study, we present data from the first studies that evaluate the survival and development of S. eridania, S. cosmioides and S. albula on Bt soybeans expressing both Cry1Ac and Cry1F proteins (event DAS-444Ø6-6 × DAS-81419-2).

Populations of Spodoptera species
Two populations of S. eridania and S. cosmioides and a single population of S. albula were collected in commercial plantings of non-Bt soybean (more than 200 larvae per species per location) in distinct soybean growing regions of Brazil (Table 1). After collection, larvae were taken to the laboratory and placed on an artificial diet consisting of white bean, wheat germ and yeast (adapted from Greene et al.), 27 and maintained at 25 ± 2°C with a 14 h:10 h light/ dark photoperiod. Neonates from the F 1 generation of each species and population were used to perform these studies.

Soybean plants
Plants of Cry1Ac/Cry1F-soybean (event DAS-444Ø6-6 × DAS-81419-2) and a non-Bt soybean isoline (experimental varietymaturity group 5.0) were cultivated in a glasshouse in 12-L plastic pots (four seeds/pot) containing two parts of soil and one part of composted plant material from January to March of 2019. Before the bioassays, Bt and non-Bt plants were checked for Bt protein expression using detection kits for Cry1Ac and Cry1F (Envirologix, São Paulo, SP, Brazil).

Biological parameters of Spodoptera species on
Cry1Ac/Cry1F-soybean Bioassays were performed with soybean leaves of Cry1Ac/Cry1Fsoybean (event DAS-444Ø6-6 × DAS-81419-2) and non-Bt soybean isolines from the R 1 to R 3 growth stages. Leaves were removed from the middle third and top of the plants and placed on a gelled mixture of agar-water at 2.5% in 50-mL plastic cups. Each cup was infested with a single neonate and then sealed and maintained in a room at 25 ± 2°C with a 14 h:10 h light/dark photoperiod. The experimental design was completely randomized with ten replicates of ten larvae, totaling 100 larvae/species/treatment. Leaves were replaced every 48 h. The following biological parameters were evaluated: duration and survival of egg, larva, pupa and total development (egg to adult); larval weight at 14 days; pupal weight 24 h after pupal formation and number of eggs per female. The number of eggs were assessed daily from 18 couples kept in polyvinyl chloride (PVC) cages (23cm height × 10-cm diameter) internally coated with a paper towel and closed at the top with a voile-type fabric. To determine the embryonic period and survival, 50 to 100 eggs of the second oviposition were obtained from each couple. The biological parameters of each Spodoptera species on Cry1Ac/Cry1F-soybean and non-Bt soybean isolines were compared by t-test (P < 0.05) using the PROC TTEST procedure in SAS® 9.1. 28 2.4 Population growth parameters of Spodoptera species on Cry1Ac/Cry1F-soybean To estimate parameters related to population growth potential, fertility life tables were estimated for Spodoptera species on Cry1Ac/Cry1F-soybean (event DAS-444Ø6-6 × DAS-81419-2) and non-Bt soybean isolines. The mean length of a generation (T), the net reproductive rate (R oaverage number of female offspring produced from a cohort of females), and the intrinsic rate of increase (r mrate of natural increase of a population) were estimated by the jackknife technique using 'lifetable.sas' protocol developed by Maia et al. 29 in SAS® 9.1. 28 This protocol calculates confidence intervals for all estimated parameters, and applies one-sided and two-sided t-tests to perform pairwise comparisons between groups (P ≤ 0.05).
wileyonlinelibrary.com/journal/ps development was significantly longer for S. eridania and S. cosmioides on Cry1Ac/Cry1F-soybean (31 and 32 days, respectively) than on non-Bt (20 and 18 days, respectively). These species took 10 days longer for development into adults when feeding on Cry1Ac/Cry1F-soybean. Larval survival of S. eridania and S. cosmioides feeding on Cry1Ac/Cry1F-soybean (< 45% survival) was lower than those feeding on non-Bt soybean (> 70% survival). This significantly reduced the numbers of S. eridania and S. cosmioides that completed their life cycle on Cry1Ac/Cry1F-soybean (10 and 39% survival, respectively), compared with those on non-Bt soybean (31 and 65% survival, respectively) ( Fig. 1). In contrast, durations of egg, larval, pupal and egg to adult periods of S. albula were similar on Bt and non-Bt soybean. However, survival of larvae, pupae and insects that completed their life cycle were reduced when fed Cry1Ac/Cry1F-soybean compared to non-Bt soybean.
When fed on Cry1Ac/Cry1F-soybean, significant reductions in larval weight of S. eridania, S. cosmioides and S. albula (from 52 to 71%, respectively) were observed ( Table 2). Similar effects were observed for pupal weight of S. eridania and S. cosmioides, which weighed 16 and 25% less than pupae from larvae fed on non-Bt soybean. Spodoptera albula had similar pupal weights when larvae were fed Bt and non-Bt soybean (165.7 versus 167.5 mg, respectively). Females from S. albula and S. cosmioides produced similar numbers of eggs when their larval development occurred on Bt and non-Bt soybean. By contrast, S. eridania females oviposited 55% fewer eggs when larvae developed on Cry1Ac/Cry1Fsoybean (Table 2).
3.2 Population growth parameters of Spodoptera species on Cry1Ac/Cry1F-soybean A summary of the life table statistics for each Spodoptera species evaluated are shown in Table 3. The estimated population growth parameters indicated that S. eridania and S. cosmioides populations had higher development time (due to longer development time from neonate to adulthood on Bt soybean) and lower population increases (due to low larval survival and less eggs per female on Bt soybean) when fed on Cry1Ac/Cry1F-soybean (event DAS-444Ø6-6 × DAS-81419-2) compared to non-Bt soybean. In contrast, S. albula presented similar development time and population increases on Bt and non-Bt soybean. Based on these results, females of S. eridania and S. cosmioides originating from larvae that fed on Cry1Ac/Cry1F-soybean produced less than 34 and 415 females per generation (R o ), respectively, in an average generation time (T) up to 58 days, while females of these species produced more than 178 and 607 females, respectively, in less than 49 days when fed on non-Bt soybean. Spodoptera albula produced a similar number of females on Cry1Ac/ Cry1F-soybean as on non-Bt soybean over the same amount of time (156 versus 217 females per female, respectively). These results indicate that females from S. eridania and S. cosmioides produced 93 and 51% less females per generation, respectively, on Cry1Ac/Cry1F-soybean. When exposed to Cry1Ac/Cry1Fsoybean, S. eridania and S. cosmioides populations also presented a rate of natural population increases lower than 0.07 and 0.10 (Table 3). These parameter values are indicative of population suppression of these species by Cry1Ac/Cry1F-soybean. In contrast, S. albula had similar intrinsic rates of population increase on Bt and non-Bt soybean.

DISCUSSION
The Cry1Ac/Cry1F-soybean variety tested in this study (containing events DAS-444Ø6-6 × DAS-81419-2) reduced the survivorship, development and population growth of S. eridania and S. cosmioides. However, this Bt soybean had lower biological activity against S. albula. These results also indicated that all three Spodoptera species evaluated had lower susceptibility to Cry1Ac/ Cry1F-soybean than other lepidopteran species, such as A. gemmatalis, C. includens, C. virescens, and H. armigera 22,23 which are the main target pests of this transgenic event. The relatively low biological activity of Cry1Ac/Cry1F Bt soybean on these Spodoptera species reflects their inherently low susceptibility to the Cry1Ac and Cry1F Bt proteins.
Similar results were observed for S. eridania fed on Cry1Ac/ Cry1F-cotton. 30 Previous studies also reported that S. eridania and S. cosmioides had naturally low susceptibility to Cry1Ac and Cry1F proteins in diet bioassays 13,[31][32][33][34] and to Cry1Ac-soybean 13,35 and Cry1Ac-cotton. 36,37 Consistent with these findings, other species from the Spodoptera genus including S. frugiperda, S. exigua (Hübner) and S. litura (F.) exhibited low susceptibility to Cry1Ac-soybean, 13,37 Cry1Ac-cotton 24,25,38,39 and Cry1Ac/Cry1Fcotton. 24,26 The relative low susceptibility of S. eridania, S. cosmioides and S. albula to Cry1Ac and Cry1F may be influenced by the low affinity of these Bt proteins to midgut receptors, and faster protein degradation in the larval midgut. 40,41 However, this may also be associated with the insufficient in planta expression of Bt proteins to control these species.
Our findings also indicate that distinct populations of S. eridania and S. cosmioides show similar but significant impacts on biological parameters when exposed to Cry1Ac/Cry1F-soybean. Similar effects on survivorship and development were also observed in populations of S. frugiperda fed on Cry1Ac-soybean, 13 Cry1Ac-cotton 42 and Cry1Ab-maize. 43,44 In contrast, S. eridania and S. cosmioides had similar survivorship and development on Cry1Acsoybean, 13,31 Cry1Ac-cotton 33 and non-Bt plants. The effects of Cry1Ac/Cry1F-soybean on immature stages of S. eridania and S. cosmioides, such as longer development time, lower survival and reduced eggs per female, affected population growth parameters, causing population suppression of these species. In contrast, Cry1Ac/Cry1F-soybean did not significantly affect S. albula biological parameters compared to non-Bt plants.
Within the IPM context, alternative strategies will be necessary to control Spodoptera species when significant infestation occurs on Cry1Ac/Cry1F-soybean under field conditions. The fact that S. eridania and S. cosmioides fed Cry1Ac/Cry1F-soybean had reduced larval weight and increased developmental time to adulthood is favorable for management. Less healthy larvae with longer development times may make them more susceptible to natural enemies 17 and entomopathogenic agents, 45 which consequently would further reduce population growth, reducing the population density and outbreaks of S. eridania and S. cosmioides in Brazilian agricultural landscapes. Alternatively, the high adoption of Bt soybean with relatively low efficacy against secondary pests such as Spodoptera species may provide opportunities for these species  Note: T is the mean length of a generation (in days); R o is the net reproductive rate (females per female per generation); r m is the intrinsic rate of population increase (per day). Means ± standard error within a column followed by the same letter in each Spodoptera species are not significantly different (t-tests for pairwise group comparisons, P > 0.05).
to become more important in soybean. This can be favored by the competitive advantage of some Spodoptera species when competing with other noctuids, 46,47 including S. frugiperdawhich appears to show increased incidence in Bt soybean fields in recent seasons in Brazil. Under field conditions, the Cry1Ac/Cry1F-soybean may provide population suppression of S. eridania and S. cosmioides, but little protection against outbreaks of S. albula. Thus, scouting for larvae and damage from these species on Cry1Ac/Cry1F-soybean will help decision making regarding the use of other IPM tactics. Chemical insecticides will likely be the main control tactic against Spodoptera species on this Bt soybean. Accordingly, if outbreaks of these species are verified it is recommended that insecticides be applied when local action thresholds are reached. In summary, to extend the benefits of Cry1Ac/Cry1F-soybean against lepidopteran pests that attack soybean, this biotech event must be combined with other IPM tools and robust IRM plans 48 to prolong the durability of this soybean technology.