Performance of Sesamia Nonagrioides on Cultivated and Wild Host Plants: Implications for Bt Maize Resistance Management Ana M

Research Article

Received: 28 March 2020 Revised: 2 May 2020 Accepted article published: 17 May 2020 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/ps.5913 Performance of Sesamia nonagrioides on cultivated and wild host plants: Implications for Bt maize resistance management Ana M. Camargo,a#,† María Arias-Martín,a#,‡ Pedro Castañeraa and Gema P. Farinósa*

Abstract

BACKGROUND: Sesamia nonagrioides is an important maize pest in the Mediterranean basin that is effectively controlled by Cry1Ab-expressing maize (Bt maize). The continued cultivation of Bt maize in Spain exerts high selection pressure on the target pests, which could lead to the development of resistance. Provision of refuges of non-Bt plants is an essential component in the high-dose/refuge (HDR) strategy to delay resistance evolution. Here we analyze the suitability of cultivated (rice and sorghum) and wild (Johnsongrass, cattail, common reed and giant reed) plants, reported as hosts of S. nonagrioides, for larval development and oviposition of this pest compared to maize, and we evaluate their potential role in delaying resistance development to Bt maize. RESULTS: Bioassays conducted with plant pieces or whole plants showed that the larval cycle could only be completed in the three cultivated plants and in Johnsongrass. Females showed a strong preference for ovipositing on maize in comparison with sorghum or rice. Although young larvae consumed more sorghum than maize in two-choice bioassays, both larvae and adults had a better performance (shorter larval period and higher pupal weight, fecundity and fertility) when larvae fed on maize throughout their larval stage than when they fed on sorghum or rice. CONCLUSION: None of the alternative hosts of S. nonagrioides tested here should be considered as natural unstructured refuges within the HDR strategy for Bt maize and this pest in Spain, as some of the necessary requirements to fulﬁll this strategy would not be met. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Keywords: Mediterranean corn borer; alternative hosts; resistance management; MON 810; HDR strategy; unstructured refuges

1 INTRODUCTION resistance evolution to Bt crops, involves (i) using crop varieties The Mediterranean corn borer, Sesamia nonagrioides Lefèbvre that produce high concentrations of the Bt toxin, capable of killing 10 (Lepidoptera: Noctuidae), is the most damaging pest of maize in all or nearly all individuals heterozygous for resistance, and (ii) fi the Mediterranean basin.1 First instar larvae of this species bore setting refuges close to the Bt elds. In principle, refuges can be into maize plants shortly after the eggs hatch and feed inside non-Bt varieties of the Bt crop or any other plant species, as long the stalks for the rest of the larval stage,2 which greatly limits as they produce large enough pest populations consisting of the efficacy of chemical sprays to control them.3 The introduction viable, susceptible insects that could mate with the potentially in Spain of genetically modified (GM) maize varieties that express resistant homozygous individuals that might survive in the Bt the Bacillus thuringiensis toxin Cry1Ab (Bt maize) in the late 1990s marked a breakthrough in the management of this pest, given * Correspondence to: GP Farinós, centre es Centro de Investigaciones Biológicas their high effectiveness in its control. This led to the rapid imple- Margarita Salas, Madrid 28040, Spain. E-mail: [email protected] mentation of this technology in Spain, especially in areas of high infestation like the Ebro Valley (northeast of Spain), where yield # These authors contributed equally to this work 4,5 losses were greatest, so that about 60% of all the maize grown † 6,7 Present address: Plant Ecology and Phytochemistry, Institute of Biology Leiden, in this area in the last 5 years is Bt. Leiden University, Leiden, The Netherlands The intensive and continuous cultivation of Bt maize exerts a ‡ high selective pressure over the pests they target and could lead Present address: Dept. of Plant Protection, Instituto Nacional de Investigación y to resistance evolution, considered as one of the main threats for Tecnología Agraria y Alimentaria, Madrid, Spain 8,9 its long-term sustainability. The strategy known as high-dose/ a Dept. of Microbial & Plant Biotechnology, centre es Centro de Investigaciones refuge (HDR), adopted in the European Union (EU) to delay Biológicas Margarita Salas, Madrid, Spain 1 © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. www.soci.org AM Camargo et al.

crop.11–13 Structured refuges, i.e. allotting an area near to the Bt reported to be potential hosts of S. nonagrioides, are suitable for field to growing varieties of the same crop not expressing the Bt the larval development and oviposition of this pest, as compared trait, are the most commonly used type of refuge. For instance, their to maize, its main host. The results obtained will shed light on the adoption is mandatory in the EU, where they have proved to suc- possibility of considering these plants as unstructured refuges for cessfully delay resistance development to Bt maize in the target Bt maize within the HDR strategy, which in turn will help to pests S. nonagrioides and Ostrinia nubilalis Hübner,14,15 and also in improve the management of resistance of S. nonagrioides to this the USA for the cultivation of Bt maize expressing one insecticidal GM crop and contribute to fine-tune the S. nonagrioides resistance protein and targeting Lepidopteran pests.16 Unstructured refuges, evolution model. on the other hand, consider alternative host plants of the target pest, such as other crops and weeds that grow close to the Bt crop, as the source of susceptible individuals.17 These unstructured refuges have 2 MATERIALS AND METHODS been shown to be effective in delaying resistance evolution to Bt cot- 2.1 Plant material 18,19 ton of different polyphagous pests in China and the USA, and We tested the suitability for oviposition and larval performance of their use is approved as part of the resistance management program S. nonagrioides of three cultivated plants [Zea mays (maize), Oryza 16 of pyramided Bt cotton in some areas of the USA. Moreover, some sativa (rice) and Sorghum bicolor (sorghum)] and four wild host authors have argued that in some cases using natural refuges could plants that are frequently found within maize fields [the weed Sor- reduce and sometimes even suppress the need to plant structured ghum halepense (Johnsongrass)] or close to them [Typha domin- 20 refuges composed of non-Bt varieties. The potential of alternative gensis (cattail), Phragmites australis (common reed) and Arundo plant species used by S. nonagrioides to serve as unstructured ref- donax (giant reed)]. All species belong to the family Poaceae, uges in Bt maize resistance management has not been studied to except cattail, which belongs to the family Typhaceae. the date. To evaluate whether alternative hosts could be useful in Maize (var. DKC4795) and sorghum (var. Express Rojo) plants insect resistance management (IRM) plans it is key to evaluate the were grown in potting soil (Compo Sana Universal, CompoAgri- performance of this noctuid pest on these plant species, as well as cultura S.L., Barcelona, Spain), whereas rice plants (var. Gleva) its preference between them and its main host, maize, to learn were grown on a mixture of 63.5% peat, 36.5% vermiculite and whether large and healthy populations of the pest could build up 0.63 g of CaCO3 per liter of soil. Johnsongrass (collected in San in the alternative hosts. In this context, determining the free amino Fernando de Henares, Madrid, Spain) and giant reed (Instituto acid and soluble sugars content in the tested plant species could Nacional de Tecnología Agraria y Alimentaria, Madrid, Spain) were provide important information, since these parameters have been also grown in potting soil, whereas a mixture of 50% potting soil fl reported to in uence oviposition preference and to stimulate larval and 50% river sand was used to grow cattail (collected in the 21,22 feeding in a range of insect species, including noctuids. stream Pantueña, Madrid, Spain) and common reed (Ecodena S.L., The inadequate or poor implementation of IRM strategies to delay Sevilla, Spain). All plants were grown in 25 cm diameter × 24 cm resistance evolution has led several insect pests to develop resistance high pots, and maintained in a greenhouse at 25 ± 3 °C, relative 23 to a range of Bt crops expressing different Bt toxins. Sesamia nona- humidity of 75 ± 10% and 16:8 (L:D) photoperiod. grioides, however, has remained susceptible to Cry1Ab expressing maize varieties, which have been cultivated in Spain for over 2.2 Insect rearing 15 years,15 even though a resistance allele to Bt maize has been reported in a population from the Ebro Valley.24 A resistance evolution Insects used in the assays came from a laboratory colony of S. non- model for S. nonagrioides was recently developed considering more agrioides of the Centro de Investigaciones Biológicas (Madrid, Spain) reared on a meridic diet, as described in González-Núñez than 20 variables known to affect the rate of resistance development, 3 including aspects of the pest biology and genetics as well as agro- et al. The oviposition cages consisted of a 12.5 cm diame- × – nomic practices in that area.4 This model considered S. nonagrioides ter 12 cm high pot with 8 10 V3 maize seedlings, enclosed by × as a functional monophagous species on maize, its primary host, in a 12 cm diameter 30 cm high see-through and colorless Plexi- the Ebro Valley. Nevertheless, despite its high level of specialization in glas cylinder covered on top by a mesh that allowed ventilation, – maize, S. nonagrioides has shown a certain degree of polyphagy, since and 8 10 pairs of adults were placed inside. After 7 days, egg clus- fi it has been recorded in a wide range of cultivated and wild host species ters were collected from the plants and placed on moistened lter of the Poaceae, Cyperaceae and Thyphaceae families across its distribu- paper for egg hatching. The whole rearing process took place in ° tion range.25,26 Regarding cultivated plants, S. nonagrioides is known to growth chambers (Sanyo MLR-350 H, Sanyo, Japan) at 25 ± 0.3 be a major pest of sorghum27,28 and rice.29,30 The degree of polyphagy C and 16:8 (L:D) photoperiod. has usually been considered an important factor for resistance development, although some studies have notfoundaclearcausalrelation- 2.3 Performance of S. nonagrioides on different host ship between both evolutionary phenomena.31 InthecaseofBtcrops, plants resistance evolution is expected to occur faster in monophagous pests 2.3.1 Preliminary oviposition bioassay that feed on the modified plant species, which are subjected to a A preliminary no-choice oviposition test using 10–20 replicates higher selective pressure in comparison with polyphagous pests.32,33 per plant species, each of them consisting of three confined pairs Gaining knowledge about the range of hosts that can be used of S. nonagrioides per arena, was performed to confirm that the by S. nonagrioides and its preference for them is essential for the seven hosts selected according to the available bibliography were management of this corn borer in Bt maize. Furthermore, the suitable for oviposition in the conditions used for this study. The use of different types of refuges (crop vs non-crop) is also impor- results showed that females laid a significant number of fertile tant in the context of IRM strategies, as changes in agronomic eggs in all the plants (on average, more than 400 eggs were recov- practices affecting the availability of these hosts may influence ered per replicate, data not shown). Therefore, larval performance the effectiveness of IRM programs.34 Thus, the aim of this study was assessed on all seven hosts in two ways: by using excised 2 was to investigate whether a range of cultivated and wild plants, parts of leaves and stems and using whole plants.

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2.3.2 Performance on excised parts of plants tested species, feeding preference was considered as a signifi- Individualized neonates (<24 h) were confined in plastic boxes cantly higher consumption of one of the two species used in the 4 cm diameter × 2 cm high and fed ad libitum with fresh pieces assay. The choice arena consisted of a Petri dish (60 mm diame- of leaves and stems of each plant species. All boxes were exam- ter × 5 mm high) coated on its bottom with a 2.5% agar solution. ined daily, and the dates of molting, pupation and adult emer- Leaf disks (8 mm diameter) containing the mid-rib were excised gence were recorded. Between 60 and 144 larvae were used for from rice, maize and sorghum plants with a cork borer and fitted each plant species. A control to ensure that the population was into the holes punched in the agar layer, alternating leaf disks of in optimal condition was set up with 102 larvae fed with the same the two species (maize-rice or maize-sorghum) in the agar arena. meridic diet used to rear the laboratory population. The length of A recently molted (<24 h) second instar larva weighing 0.50– the larval cycle was determined by counting the number of days it 1.25 mg (mean ± 1SD) was placed in the center of the dish after took each larva to reach the pupal stage from the start of the a 6-h starvation period. All dishes were sealed and placed in a experiment, and the longevity of the adults was calculated as growth chamber at 25 ± 0.3 °C and complete darkness for the the number of days between emergence and death of the adult. duration of the assay. Twenty replicates of each combination were Pupae were weighed 24 h after pupation. To evaluate dietary evaluated. The experiment concluded when larvae in an external effects on adult performance, individual pairs of adults were control that only contained maize disks had consumed approxi- placed in arenas consisting of 6 cm diameter × 6 cm high pots mately 50% of the plant material. Both the initial and final fresh with three V3 maize seedlings confined by a ventilated, see- weights of larvae and leaf disks, measured separately for each through plastic cylinder (5.4 cm diameter × 15.5 cm high) for mat- plant species, were recorded. Larvae were then frozen at −20 °C ing and oviposition. Egg clusters were collected 7 days later and and afterwards dried in an oven at 60 °C for 48 h to estimate their the number of eggs determined using a stereomicroscope (Leica dry weight. Uneaten leaf disks were cleaned of frass and oven- M125, Leica Mycrosystems, Germany). Eggs were placed on top of dried following the same procedure. moistened filter paper in plastic boxes for hatching and their via- The preference index proposed by Kogan and Goeden (1970)36 bility recorded. These assays were carried out in growth chambers was calculated as a measure of larval preference on a dry weight at 25 ± 0.3 °C and 16:8 (L:D) photoperiod. Additionally, the stan- (DW) basis: dardized growth index (SGI) was estimated for each larva of each preference index (C) = 2A/(M + A) where A is the consumption host species tested35: of alternative host (%DW) and M is the consumption of primary SGI = pupal weight (mg)/length of larval period (days). host (%DW). This index can range between 0 and 2, so that C = 1 indicates that larvae do not feed preferentially on either 2.3.3 Performance on whole plants plant, whereas values lower than 1 denote a preference for the Six neonates (<24 h) of S. nonagrioides were placed on the leaf primary host and values higher than 1 indicate larvae feed prefer- sheaths of leaves 3, 4 and 5 (two larvae per leaf), with the excep- entially on the alternative host. tion of P. australis, in which three neonates were used (one per Additionally, the nutritional indices described by Farrar et al.37 leaf) due to the narrow diameter of the stem in this species. The were calculated. The relative consumption rate (RCR) was esti- main stem of rice plants was considered for infestation. Plants mated separately for the two plant species in each two-choice were then confined within a ventilated methacrylate cylinder assay: and watered regularly during the running time of the experiment. RCR = DWi – DWf)/LWi × D where DWi is the initial dry weight of Between 25 and 27 days after infestation the plants were dis- leaf disks (mg), DWf is the final dry weight of leaf disks (mg), LWi is sected and the larval recovery rate, measured as the percentage the initial larval dry weight (mg) and D is the duration of the assay of initial larvae that were recovered at the end of the bioassay, (days). Initial dry weight of leaf disks was calculated from their was recorded in each plant, as well as larval weight and larval fresh weight using an equation that relates both parameters, stage (L1–L6 for first to sixth instars, respectively) of the recovered obtained for each plant species by weighing 10 batches of six larvae. The assays were performed with V6–V8 plants of all plant freshly excised leaf disks and weighing them again after 48 h at species except rice, which was used when the plants reached 60 °C. Similarly, LWi was calculated using an equation obtained the panicle formation phase, and they took place in a greenhouse, by measuring the fresh and dry weights of 373 L2 larvae in the using 7–22 plants per species, at 25 ± 3 °C and 16:8 (L:D) same weight range as those used in the assays. All weights were photoperiod. determined using an analytical balance (Mettler-Toledo AX205, Mettler-Toledo International Inc., Columbus, OH, USA). 2.4 Larval feeding preference Feeding preference was evaluated by two-choice and no-choice bioassays. Since maize is the primary host of S. nonagrioides in 2.4.2 No-choice assays Spain, this species was used as the reference host for comparison These tests were performed similarly to two-choice assays, but all with the other two cultivated hosts, rice and sorghum. The three disks in the agar arena corresponded to the same plant species. species were planted at the same time and offered to S. nona- Seventeen replicates were tested for maize, 16 for sorghum and grioides females when maize plants reached the V8 phenological eight for rice. In this case, the experiment concluded when larvae stage. These experiments were performed using leaf disks as an in the maize assay had consumed approximately 75% of leaf disks. appropriate proxy to assess feeding preferences of S. nonagrioides Three nutritional indexes were estimated: the RCR (described larvae. above), the relative growth rate (RGR) and the efficiency of conversion index (ECI),37 so that: 2.4.1 Two-choice assays RGR = (LWf – LWi)/LWi × D where LWf is the final larval dry Two-choice assays were conducted to examine feeding prefer- weight (mg), LWi is the initial larval dry weight (mg) and D is the ences of S. nonagrioides larvae between maize and rice or sor- duration of the assay (days), and ghum. Given that this corn borer is known to feed on the three ECI (%) = (RGR/RCR) × 100. 3

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2.5 Oviposition preference on cultivated hosts more times. The supernatants of each sample were pooled and Two-choice assays were carried out to determine the oviposition divided into two 750 μL replicates, which were dried in a Speed- preference of females between the primary cultivated host Vac Concentrator Savant SVC-100H for approximately 12 h. Each (maize) and rice or sorghum. For each replicate, two males and a sample was then resuspended in 500 μL of double-distilled water female were confined in a choice arena consisting of a 25 cm dia- and 1 mL of 0.2% anthrone in 95% sulfuric acid (v/v) was added to meter × 24 cm high pot with one maize plant and either a rice or each of them. After 15 min of incubation at 90 °C the absorbance a sorghum plant, covered with a mosquito net with a diameter of of each sample at 630 nm was measured in a VERSAmax micro- 56 cm and a height of 230 cm, attached by a cable to the roof of plate reader (Molecular Devices Corp., Sunnyvale,USA). the greenhouse, that gave the moths enough space to move freely. All host species were sown at the same time and exposed 2.7 Statistical analysis to S. nonagrioides adults when maize plants reached the V8 phe- Prior to the statistical analysis of the results, normality (Kolmogo- nological stage. After 7 days, all the adults were recovered and rov–Smirnov test) and homocedasticity (Levene test) were plants were examined. Fecundity was estimated as the total num- checked in all variables, and those that did not comply with these ber of eggs laid per female and plant and egg viability was esti- requirements were transformed to arcsin√x or log(x + 1) for per- mated a week later as described in section 2.3. Female moths centages or continuous variables, respectively. A significance level were dissected to check their mating status, and only replicates of ⊍ = 0.05 was considered, and all analyses were performed using in which a mated female was recovered were considered as valid. the statistical software SPSS (SPSS Statistics 24.0, IBM, USA). Twenty replicates of each option (maize-rice or maize-sorghum) In larval development assays using parts of plants, one-way were set up. These assays took place in the greenhouse at 25 ± 3 ° ANOVA followed by either a Dunnett's t-test (when variances C and 16:8 (L:D) photoperiod. were homogenous) or a Dunnett's T3 test (when variances were not homogenous)42 were carried out to study whether length of 2.6 Free amino acid and free sugar content of the the larval cycle, pupal weight, adult longevity and SGI in the differ- cultivated hosts ent plant species differed significantly from the values recorded in The free amino acid and free sugar content was assessed in maize, maize. A Student's t-test was carried out to check for differences sorghum and rice leaves used in the feeding assays to determine between host species in fecundity and fertility for adult pairs if they could have an effect on the choice of the plants, since these resulting from larvae fed on maize and sorghum, the only two compounds have been proved to influence insect preference and species in which adults could be set up for mating and oviposi- 38,39 performance between hosts in some lepidopteran species. tion. In assays that considered whole plants, one-way ANOVA fol- The extraction method followed to estimate the quantity of free lowed by Dunnett's tests were used as described above to study 40 amino acids was that used in Ximenez-Embún et al., based in the whether larval recovery rate and mean weight per instar were dif- 41 technique described in Hacham et al. Three samples (20–30 leaf ferent in the alternative hosts in comparison with maize. disks/sample) of each plant species were frozen at −80 °C and Differences in fecundity and fertility between maize and sor- grinded using a mortar to obtain approximately 100 mg of leaf ghum or rice were analyzed by paired Student's t-tests, compar- material per sample, whereupon 600 μL of water:chloroform: ing the values resulting from subtracting the value of the methanol (3:5:12 v/v/v) extraction buffer was added to each sam- variable measured in the alternative host from the value mea- ple. This was followed by a 4-min centrifugation at 4 °C and sured in maize. Likewise, differences in RCR between species in 14 000 rpm, after which the supernatant was transferred to a larval feeding two-choice assays were analyzed with paired Stu- new tube and the pellet was resuspended in 600 μL of extraction dent's t-tests following the same procedure. buffer and centrifuged again. The supernatant was pooled with In no-choice larval feeding assays, one-way ANOVA followed by that obtained in the previous centrifugation and 300 μL of chloro- a Dunnett's t- or T3 tests was performed to determine if host spe- form and 450 μL of double-distilled water were added to each cies had a significant effect on RCR, RGR and ECI. Differences sample. After a final 2-min centrifugation of the samples at the between alternative hosts and maize in their free sugar and total same temperature and speed, the top layer of the solution con- free amino acid content were analyzed with one-way ANOVA fol- taining the amino acids was transferred to a new tube and placed lowed by Dunnet's t-tests. in a SpeedVac Concentrator Savant SVC-100H (ThermoFisher Sci- entific, Wilmington, DE, USA) overnight. When all the solvent was evaporated, the samples were taken to the Protein Chemistry 3 RESULTS Service at the CIB (CSIC, Madrid), where their amino acid content 3.1 Performance of S. nonagrioides on different host was determined using a Biochrom 30 Amino Acid Analyser (Bio- plants chrom, Cambridge, UK). For this purpose, the samples were first 3.1.1 Performance on excised parts of plants resuspended in 100 μL of sodium citrate loading buffer at Sesamia nonagrioides only reached the adult stage when larvae pH 2.2 and 10 μL of each sample was injected in the analyzer. were fed with pieces of three out of the seven tested plant spe- The free amino acid content in each sample was estimated on a cies: maize, sorghum and Johnsongrass, with survival rates to dry weight basis. the adult stage of 63.7%, 23.6% and 9.9%, respectively. Larvae Determination of the plants' free sugar content was performed fed on common reed and cattail died mostly during the early lar- on dry plant material. Leaf disks excised from the leaves used in val stages, while larvae fed on rice and giant reed died at more the assays were dried at 75 °C for 48 h and then ground to obtain advanced larval stages, so that no pupae were obtained in any a fine powder. Three samples (≈3 mg of leaf powder per sample) of these four species (Fig. 1). Larvae feeding on maize usually were considered per plant species. Each sample was homoge- underwent five molts before they completed their larval stage, nized in 650 μL of 95% ethanol and heated at 80 °C for 20 min, whereas supernumerary molts were common in individuals fed followed by centrifugation at 10000 rpm for 10 min and collec- on the alternative hosts. A high survival rate to adulthood was 4 tion of the supernatant in a tube. This process was repeated two recorded in larvae fed on a meridic diet (91.2%), indicating the

100

80 Maize (102)

Sorghum (144) 60 Rice (60)

Johnsongrass (111) 40 Survival (%) Giant reed (100)

Common reed (110) 20 Cattail (116)

0 L1 L2 L3 L4 L5 L6-Ln Pupa Adult Figure 1 Survival rates of the different stages of S. nonagrioides fed on pieces of leaf and stem of seven host plants. Number of larvae tested per plant species is shown in brackets. good health of the S. nonagrioides population used in the recovered were L5 or bigger (22.5% L5, 57.5% L6 and 20.0% experiments. pupae). Nearly 69% of the larvae recovered from sorghum were Focusing on the three plant species where S. nonagrioides com- L2–L4, and around 31% were L5–L6 larvae. Likewise, 83.1% of pleted its development (maize, sorghum and Johnsongrass), it the larvae recovered from Johnsongrass were L2–L4 larvae, and had a better performance when fed with maize than with any 16.9% were L5–L6 larvae. Finally, 42.5% of the individuals recovered one of the others, as indicated by the significantly shorter dura- from rice plants were L3–L4 larvae, and 57.5% were L5–pupae tion of the larval development and the higher adult longevity, (Fig. 2). Average larval weight was significantly higher in L5 larvae SGI and pupal weight observed in larvae fed on maize (Table 1). recovered from maize than in those recovered from sorghum, rice Average fecundity and fertility were also higher in adult pairs and Johnsongrass. Sixth instar larvae and pupae were also heavier derived from larvae fed with maize. Since females and males when recovered from maize plants with regard to those from rice emerged at different times, no couples could be set up for mating plants, whereas the difference was not significant in the case of and oviposition in adults resulting from larvae fed with Johnson- L6 larvae recovered from sorghum (P = 0.054) (Fig. 3). grass (Table 1).

3.2 Larval feeding preference 3.1.2 Performance on whole plants 3.2.1 Two-choice assays Individuals of S. nonagrioides were recovered 25–27 days post When larvae of S. nonagrioides could choose between maize and infestation in six out of the seven plant species tested, whereas rice or sorghum, they exhibited different feeding preferences. Lar- no larvae were recovered from giant reed. The recovery rate of larvae chose to feed on maize rather than on rice when they were vae at the end of the bioassay was very low in common reed and provided with both species, as shown by the signiﬁcantly higher cattail (5.6% and 5.0%, respectively), so these plants were value of the relative consumption rate in maize (t = 3.78, excluded from the statistical analyses. When the experiment was P = 0.001). However, this index had a signiﬁcantly higher value stopped, nearly 4 weeks post infestation, all larvae were expected in sorghum when larvae could choose between this species and to be at least L5, since the average duration of the larval stage in S. maize (t = −2.33, P = 0.031) (Table 2). These results are supported nonagrioides larvae reared at 25 °C and a 16:8 photoperiod was by the values of the feeding preference index (C), which indicated estimated in 32.5 days.43 However, only in maize all the larvae that S. nonagrioides larvae preferred maize to rice in rice-maize

Table 1 Performance of S. nonagrioides fed on plant parts (leaves and stems) of different plant species (mean ± SE)

Host Larval development (days) Pupal weight (mg) SGI¶ (mg day−1) Adult longevity (days) Fecundity Fertility (%)

Maizea 30.9 ± 0.4 188 ± 5 4.6 ± 0.1 8.7 ± 0.3 539 ± 32 88.2 ± 1.8 Sorghumb 48.3 ± 1.2* 128 ± 3* 2.7 ± 0.1* 5.1 ± 0.2* 125 ± 31 42.1 ± 14.0 Johnsongrassc 46.1 ± 1.7* 113 ± 5* 2.0 ± 0.1* 5.1 ± 0.5* –– F/t(p) 162.22 (<0.001) 56.19 (<0.001) 66.89 (<0.001) 10.40 (<0.001) 108.13 (<0.001) 3.04 (0.011)

Only those species in which S. nonagrioides completed its life cycle are shown. *Signiﬁcant differences from values obtained in maize (one-way ANOVA followed by Dunnett's t-test, P < 0.05). aSixty-ﬁve adults emerged from larvae fed on maize; 16 couples were set up for mating and oviposition. bThirty-four adults emerged from larvae fed on sorghum; 10 couples were set up for mating and oviposition. cEleven adults emerged from larvae fed on Johnsongrass. Female and male adults emerged at different times, so no couples could be set up. ¶SGI, standardized growth index. 5

100

Pupa 80 L6 60 L5 L4 40 L3 20 L2

0 Maize (10) Sorghum (22) Rice (22) Johnsongrass (21)

a Common reed and cattail have been excluded because the recovery rate of larvae at the end of the bioassay was below 10%. No larvae were recovered from giant reed. Figure 2 Frequency (%) of the different stages of the life cycle in S. nonagrioides recovered from infested plants. Common reed and cattail have been excluded because the recovery rate of larvae at the end of the bioassay was below 10%. No larvae were recovered from giant reed. Number of replicates per plant species is shown in brackets.

400

350

300

250 Maize * 200 Sorghum * Rice

Weight (mg) 150 * Johnsongrass 100 * 50

0 L5 L6 Pupa

a Common reed and cattail have been excluded because the recovery rate of larvae at the end of the bioassay was below 10%. No larvae were recovered from giant reed. * Significantly different from values obtained in maize (one-way ANOVA followed by Dunnett t test, p < 0.05). Figure 3 Larval and pupal weight per host species a (mean ± SE) in S. nonagrioides recovered from infested plants. Common reed and cattail have been excluded because the recovery rate of larvae at the end of the bioassay was below 10%. No larvae were recovered from giant reed. *Signiﬁcantly different from values obtained in maize (one-way ANOVA followed by Dunnett's t-test, P < 0.05).

Table 2 Relative consumption rate (mean ± SE) of S. nonagrioides on the three cultivated plant species using two-choice assays

Choice assay (n) RCR maize (mg mg−1 day−1) RCR rice (mg mg−1 day−1) RCR sorghum (mg mg−1 day−1) t (p)

Maize-rice (19) 3.83 ± 0.46 2.02 ± 0.25 – 3.78 (0.001)* Maize-sorghum (20) 2.74 ± 0.26 – 4.17 ± 0.47 –2.33 (0.031)*

*RCR differs signiﬁcantly in the two species tested in each two-choice assay (paired Student's t test, P < 0.05).

assays (0.80 ± 0.09), whereas sorghum was the preferred foliar tis- sorghum (51.6 ± 8.3%) in comparison with maize (18.7 ± 1.0%). sue in maize-sorghum (1.20 ± 0.10) assays. On the other hand, both RGR and ECI were higher in maize in comparison with rice (Table 3). 3.2.2 No-choice assays The RCR of leaf disks was significantly higher in maize compared 3.3 Oviposition preference on cultivated hosts with sorghum and rice. However, the RGR of larvae fed on sor- Females of S. nonagrioides showed preference for ovipositing on ghum was higher than in those fed on maize. This indicates that maize plants in comparison with sorghum or rice, so that the aver- larvae convert the ingested sorghum to biomass more efficiently age fecundity on maize was significantly higher than that 6 than in the case of maize, resulting in a significantly higher ECI in recorded in sorghum or rice (15- and 14-fold, respectively).

Table 3 Nutritional indices (mean ± SE) of S. nonagrioides in no-choice assays on the three cultivated hosts

Host (n) RCRa (mg mg−1 day−1) RGRb (mg mg−1 day−1) ECIc (%)

Maize (17) 4.85 ± 0.37 0.88 ± 0.06 18.7 ± 1.0 Sorghum (16) 2.87 ± 0.36* 1.19 ± 0.08* 51.6 ± 8.3* Rice (8) 2.98 ± 0.40* 0.13 ± 0.02* 4.7 ± 0.8* F (p) 10.4 (<0.001) 46.0 (<0.001) 94.6 (<0.001)

aRelative consumption rate. bRelative growth rate. cEfﬁciency of conversion index. *Signiﬁcant differences with regards to the values recorded on maize (one-way ANOVA followed by Dunnett's or T3 tests).

Table 4 Fecundity and fertility (mean ± SE) of S. nonagrioides in choice assays between maize-sorghum and maize-rice

Host pair n Fecundity t (p) Fertility (%) t (p)

Maize 387 ± 43 96.4 ± 1.1 18 6.90 (<0.001)* −0.85 (0.486) Sorghum 25 ± 16 99.4 ± 0.4

Maize 449 ± 50 88.7 ± 6.8 15 7.35 (<0.001)* 0.45 (0.671) Rice 32 ± 15 96.2 ± 1.6

*Signiﬁcant differences from values obtained in maize (Student's t-test, P < 0.05).

However, no significant differences were observed between hosts Common reed, giant reed and cattail were low-quality hosts for lar- regarding fertility in maize-sorghum or maize-rice assays (Table 4). val development. Even though S. nonagrioides has been reported to The average adult recovery rate per replicate was 80 ± 7% in feed on these species, all the studies reporting this behavior com- maize-sorghum assays and 93 ± 3% in maize-rice assays. prised African and Macaronesic populations,45–48 so the differences observed between them and the Spanish one might be due to host 3.4 Free amino acid and free sugar content of the specialization in S. nonagrioides populations from different areas in cultivated hosts which host availability differs. Free sugar content was significantly higher in rice leaf tissue com- Different studies in noctuids have reported the important role of pared to maize, whereas no differences were observed between sensory and physical cues, such as surface texture or stem thick- maize and sorghum. On the other hand, the total free amino acid ness, in the acceptability of a host species or plant part for ovipo- content was significantly higher in both sorghum and rice plants sition.49,50 In this vein, two other noctuid pests of maize, Busseola with regard to maize (Table 5). fusca and Mythimna unipuncta, have been observed to lay eggs on man-made structures that resemble the narrow slit used for ovipositor insertion in maize plants, emphasizing the important 4 DISCUSSION role of this kind of cues in eliciting oviposition in both species.51,52 Sesamia nonagrioides was able to lay eggs on all seven plants stud- Similarly, under laboratory conditions females of S. nonagrioides ied under no-choice conditions. However, based on the results of have been observed to lay viable eggs in artificial structures that the bioassays in which larvae were reared either on pieces of the mimic the tight gap between the stem and the leaf sheath (per- plants or on whole plants, its larval cycle could only be completed sonal observation), which is used by the females of this species – – in the three cultivated species tested maize, sorghum and rice to insert the ovipositor.53 This suggests that, in the absence of as well as on the weed Johnsongrass, which shares similarities with 44 its main host, S. nonagrioides females will lay eggs on a wide range sorghum, given that it is a hybrid of S. bicolor and S. propinquum. of plant species, even when they are unsuitable for larval development, as already observed in this and other noctuid species.54–56 Our results agree with the narrower larval feeding range often Table 5 Percentage of free sugars and free amino acid content observed in lepidopteran species in comparison with wider host (mean ± SE) of maize, sorghum and rice leaf tissue acceptance for oviposition.57 When females could choose between the primary cultivated Host Free sugars (%) Free amino acids (%) host (maize) and rice or sorghum in two-choice bioassays, they Maize 3.63 ± 0.32 0.256 ± 0.008 showed a strong oviposition preference for maize, as evidenced Sorghum 3.86 ± 0.30 0.631 ± 0.030 * by the more than 10 times higher fecundity recorded in this host Rice 5.14 ± 0.28 * 0.475 ± 0.001 * with regard to the other two species. Even though sorghum and F (p) 6.86 (0.028) 90.28 (<0.001) maize plants are phylogenetically very close and they share remarkable similarities in their architecture,58 the preference of * fi Signi cant differences from values obtained in maize (one-way S. nonagrioides females for laying eggs in maize rather than sor- ANOVA followed by Dunnett's t-test, P < 0.05). ghum has been previously reported by Dimotsiou et al.56 This is 7

in line with the better performance of S. nonagrioides observed on these larvae were developmentally delayed and their growth had maize, since larvae generally showed higher mortality, delayed been restricted in comparison with larvae recovered from maize developmental time, reduced growth, smaller larval and pupal plants. size, shorter adult life span and reduced fecundity and fertility in Altogether, our results indicate that only three out of the six the alternative hosts in comparison with maize in both larval per- tested species were suitable hosts for S. nonagrioides, apart from formance assays. These results are consistent with the ‘prefer- maize. Nevertheless, none of the potential alternative hosts tested ence-performance’ hypothesis that has been observed in several here should be used as natural unstructured refuges for Bt maize lepidopteran species,59,60 which predicts that, generally, adults and considered within the HDR strategy. This is because even if lay their eggs preferentially in hosts that are optimal for the devel- these plant species are present and abundant near or within (i.e. opment of their offspring.61 The differential development Johnsongrass) Bt maize fields in Spain and they coincide with observed depending on the host could lead to asynchronies in maize in both space and time, they do not comply with other the biological cycles, which in turn would result in different times requirements that must be met by unstructured refuges, that is, of emergence of the adults from different plant species in the that they are able to host a large population of high-quality field. This could have important implications in terms of resistance moths, and that no asynchrony exists between these moths and management, as the main function of the refuges is to provide those emerging from Bt fields.70–72 For some polyphagous target susceptible adults that will emerge at the same time as the resis- pests, natural refuges can be very effective, if the host plants in tant adults that may emerge in Bt maize fields. Therefore, devel- the refuge can produce sufficient numbers of high-quality opmental delay together with poorer quality adults produced in moths.71 Thus, natural hosts have been proved to contribute to alternative hosts would make them not a good option as refuges delay resistance development to Bt cotton in China and the USA for use within the HDR strategy. in the polyphagous and highly dispersive pests Helicoverpa armi- Sugars and amino acids detected by chemoreceptors have been gera, H. zea and Heliothis virescens.18,19,73 However, they have not reported to play a major role in discrimination between plants for been considered as effective refuges for Bt maize for Ostrinia nubi- oviposition in some lepidopteran species.21,62,63 However, results lalis in France and the USA, and for several stem-boring pests of are inconclusive when it comes to noctuid species.22,64 Our study maize in Africa.72,74,75 Based on our findings, the tested hosts does not reveal an oviposition preference of S. nonagrioides for would also not be suitable for S. nonagrioides, which in Spain is any of the plants based on their free sugars content, since, even considered an oligophagous or even facultatively monophagous though no differences in this parameter were found between species on Z. mays. Nonetheless, it must be kept in mind that S. maize and sorghum leaves, adults laid significantly more eggs nonagriodes may use these plants as refuges for short periods of on maize. However, maize had a significantly lower value in the time under adverse circumstances, e.g. to escape from Bt maize.76 total content of free amino acids in comparison with sorghum The results reported in this study confirm the premises assumed and rice, which could partially explain the differential oviposition in the resistance evolution model of S. nonagrioides to Bt maize in preference between maize and these species. the Ebro Valley regarding the low number of wild or cultivated The preference for laying eggs on maize shown by S. nona- alternative hosts plants for this noctuid pest.4 Furthermore, they grioides did not match the feeding preference for sorghum suggest that unstructured refuges composed of the tested plant compared to maize shown by second instar larvae in two- species would not help delay the development of resistance to choice bioassays, as expressed by RCR values. This contradic- Bt maize. Therefore, refuges for susceptible individuals should tion has also been observed in S. exigua, which preferred maize continue to be composed of non-Bt maize plants, and it is desir- for oviposition and other plant species for larval feeding,65 but able that compliance with refuge requirements increases from it differs from results reported in other species, where female the 92% reported in 201777 in maize-based agro-ecosystems. oviposition preference for different host plants was positively correlated with larval feeding preference.21,66,67 Asignificantly higher content of free amino acids, which have been proved to ACKNOWLEDGEMENTS stimulate feeding in other noctuid pests,38,68 was detected in This work was supported by grants AGL2012-34289 and AGL2015- sorghum leaf tissue compared to maize. However, this would 64825-R of the Ministerio de Economía y Competitividad notbeamajordriverofS. nonagrioides larval preference and (MINECO), and two grants received by M. Arias-Martín (BES- performance, since, even though rice contained higher levels 2010-037303, Ministerio de Ciencia e Innovación) and Ana M. of free amino acids than maize, larvae consumed more maize Camargo (BES-2013-064987, MINECO). We are grateful to Dr. Cris- in maize-rice choice assays and showed higher rates of relative tina Chueca (Instituto Nacional de Tecnología Agraria y Alimen- consumption, growth and efficiency of conversion in maize taria, Madrid, Spain) for providing the giant reed plants and to than in rice under no-choice conditions. In the same line, the Dr. Pedro L. Aguado (, Spain) and María del Mar Catalá (IRTA Cen- results of choice and non-choice assays show that the higher tro Experimental del Ebro, Tarragona, Spain) for providing the contentoffreesugarsrecordedinriceleavesincomparison seeds of sorghum and rice, respectively. We also thank N. Arranz, with maize or sorghum did not stimulate feeding in S. nona- G. Fernández-Mata and C. 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