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Side effects of Bacillus thuringiensis on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae)
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Side effects of Bacillus thuringiensis on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae) Gabriela da Silva Rolima, Angelica Plata-Ruedab, Luis Carlos Martínezc,∗, Genésio Tâmara Ribeirod, José Eduardo Serrãoc, José Cola Zanuncioe a
Departamento de Fitotecnia, Universidade Federal de Viçosa, 36570-900, Viçosa, Minas Gerais, Brazil Instituto de Ciências Agrárias, Universidade Federal de Viçosa, 38810-000, Viçosa, Minas Gerais, Brazil c Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais, Brazil d Universidade Federal de Sergipe – Departamento de Ciências Florestais, 49100-000, São Cristóvão, Sergipe, Brazil e Departamento de Entomologia, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parasitoid wasp Production of immatures Repellency Reproductive performance Survival Toxicity
The endoparasitoid wasp Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is used to control defoliating lepidopteran pests. Chemical insecticides are not compatible with natural enemies, but bioinsecticides, such as Bacillus thuringiensis Berliner (Bt), have great potential for use in integrated pest management. However, interactions between Bt and P. elaeisis still need to be investigated. This study aimed to evaluate the effects of Bt on parental and first-generation P. elaeisis parasitizing Bt-susceptible and -resistant Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). An additional aim was to determine the toxicity of Bt to susceptible third-instar S. frugiperda larvae. Larvae were exposed to lethal concentrations (LC50 and LC90) of Bt and then allowed to be parasitized by P. elaeisis. Parasitoid longevity, immature production, reproductive performance, and behavioral responses were evaluated. Bt repelled P. elaeisis and reduced immature production. Parental and first filial generation parasitoids of both sexes emerged from Bt-treated larvae showed lower survivorship than controls. Parasitoids had poorer reproductive performance in Bt-susceptible and -resistant pupae than in untreated pupae. Palmistichus elaeisis emerged from Bt-susceptible and -resistant S. frugiperda showed altered host-searching behavior and reproductive parameters, which indicates low compatibility between the bioinsecticide agent and the parasitoid wasp.
1. Introduction Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is a gregarious endoparasitoid wasp that develops in coleopteran and lepidopteran species (Zanuncio et al., 2008; Barbosa et al., 2016). Females of P. elaeisis lay eggs on host pupae, and, after emergence, their parasitic larvae feed on host organs and tissues (Soares et al., 2009). The parasitoid is native to the Neotropics and has polyphagous habits, feeding on lepidopterans of economic importance, such as Anticarsia gemmatalis Hübner (Noctuidae) (Pereira et al., 2013), Diaphania hyalinata Linnaeus (Crambidae) (Pratissoli et al., 2007), Psorocampa denticulata Schaus (Notodontidae) (Zanuncio et al., 2015), Spodoptera frugiperda (J.E. Smith) (Noctuidae) (Bittencourt and Berti-Filho, 1999), and Thyrinteina arnobia (Stoll) (Geometridae) (Barbosa et al., 2016). The species shows great potential as a biological control agent. For use in integrated pest management (IPM), natural enemies must
∗
be compatible with other control methods (Zanuncio et al., 2016; Alcántara-de La Cruz et al., 2017; Martínez et al., 2018a). Chemical control is currently the most widely used method, and several studies have been aimed at developing more selective insecticides to minimize their impact on natural enemies (Desneux et al., 2007). However, the side effects of chemical agents on humans, non-target organisms, and the environment as a whole motivate the search for more sustainable pest control methods (Desneux et al., 2007; Nicholson, 2007; Pedlowski et al., 2012). Natural enemies of pests as parasitoids are susceptible to insecticides, which may affect development, longevity, fecundity, and parasitism rate (Alcántara-de La Cruz et al., 2017), while bacteria-based bioinsecticides to pest control can be compatible with parasitoids (Desneux et al., 2007). Bacillus thuringiensis Berliner (Bt) is a rod-shaped, Gram-positive bacterium that produces insect-toxic crystal (Cry) proteins during the sporulation phase (Bravo et al., 2011). When dissolved and activated by
Corresponding author. E-mail address: [email protected] (L.C. Martínez).
https://doi.org/10.1016/j.ecoenv.2019.109978 Received 23 May 2019; Received in revised form 12 November 2019; Accepted 14 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Gabriela da Silva Rolim, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109978
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to evaluate the toxicity of the bioinsecticide to Bt-susceptible S. frugiperda larvae, construct concentration–mortality curves, and estimate lethal concentrations (LC). Distilled water was used as negative control. Three replicates of 30 third-instar S. frugiperda larvae were placed individually in Petri dishes (90 × 1.5 mm) and fed with Bt suspensions (2 μL) in 1 g of artificial diet. The number of dead larvae was recorded for 8 days.
insect proteases upon ingestion, Cry proteins form pores in the midgut membrane, causing septicemia and insect death (Oestergaard et al., 2007; Castro et al., 2019). Specific Bt strains developed for target insect groups are less toxic to vertebrates and therefore have lower environmental impact (Bravo et al., 2011). For instance, Bt subsp. kurstaki can be used to control lepidopterans (Sanahuja et al., 2011). Bt strains and toxins have been used for the production of bioinsecticides, and cry genes for the development of transgenic plants (Romeis et al., 2006). Transgenic plants expressing Cry proteins have revolutionized the IPM of different cropping systems (Sanahuja et al., 2011; Flagel et al., 2018). Plants expressing a single Cry protein may favor outbreaks of resistant pests ( HYBaranek et al., 2017), but expression of two or more active Bt toxins attenuates this problem (Santos-Amaya et al., 2015). On the other hand, natural enemies exposure to Bt proteins by feeding on Bt-resistant prey or hosts can compromise their quality (Romeis et al., 2008). In fact, the existing debate about the effect of Bt proteins on natural enemies has focused on whether any purported negative effects are due to the Bt protein or quality of the host or prey on which the natural enemy fed (Shelton et al., 2002). The combined use of Bt and parasitoids is a promising IPM strategy because of its low cost, high efficiency, and minimal environmental risk (Barrat et al., 2010). However, the effects of Bt on P. elaeisis are still unknown. This study aimed to evaluate the reproductive parameters, progeny size, and development time of P. elaeisis parasitizing Bt-susceptible and Bt-resistant S. frugiperda pupae exposed to the bacterium.
2.3. Time–mortality test Bt-susceptible third-instar S. frugiperda larvae were placed one per Petri dish and exposed to the lethal concentrations (LC25, LC50, LC75, and LC90) of Bt determined in the concentration–mortality test. A control was performed using distilled water. Exposure procedures and conditions were the same as described above for the concentration–mortality test (section 2.2). The number of live insects was recorded every 12 h for 8 days. Four replicates of 30 insects were used for each Bt concentration following a completely randomized design. 2.4. Production of immature parasitoids Bt-susceptible and -resistant third-instar S. frugiperda larvae were treated with Bt at the estimated LC50 and LC90 or with distilled water as control. After pupation, S. frugiperda were exposed to parasitism by P. elaeisis for 24 h. The experiment was arranged in a completely randomized design, with three replicates of 10 pupae per treatment. Pupae parasitized by P. elaeisis were placed individually in glass vials (2.5 × 8 cm) and examined using an MX-20 specimen radiography system (Faxitron X-Ray Corp, Wheeling, IL, USA) equipped with a 14bit digital camera. The location of each parasitoid within each S. frugiperda pupa was recorded at 5, 10, 15, and 20 days after parasitization. The number of live larvae and pupae of P. elaeisis per S. frugiperda pupa was also recorded.
2. Material and methods 2.1. Insects Palmistichus elaeisis and S. frugiperda (only Bt-susceptible population) individuals were mass reared at the Laboratory of Insect Biological Control of the Federal University of Viçosa (Viçosa, Minas Gerais, Brazil). Bt-resistant individuals were collected from Cry1F-expressing corn plants (30F35H DuPont Pioneer®, Santa Cruz do Sul, Rio Grande do Sul, Brazil). Pupae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) were used to mass rearing of P. elaeisis. Tenebrio molitor individuals were kept in plastic boxes (60 × 40 × 12 cm) at 25 ± 1 °C and 70 ± 10% relative humidity (RH) under a 12:12 h light/dark (L/D) photoperiod regime. Larvae and adults were fed ad libitum with wheat bran (75% carbohydrates, 12% protein, 11% minerals, and 2% lipids), pieces of sugarcane [Saccharum officinarum Linnaeus (Poaceae)] stems, and pieces of chayote [Sechium edule Swartz (Cucurbitaceae)] until pupation. P. elaeisis adults were mated for 24 h and placed in glass tubes (14 × 2.2 cm) covered with a fine net and containing T. molitor pupae. After parasitization, newly emerged (24 h old) P. elaeisis individuals were placed in glass tubes (14 × 2.2 cm) in a controlled environment room (25 ± 2 °C, 70 ± 10% RH, and 12:12 h L/D photoperiod) and fed with pure honey ad libitum. Bt-susceptible and -resistant S. frugiperda larvae were grown separately in polyethylene pots (15 × 9 cm) in a controlled room (25 ± 2 °C, 75 ± 5% RH, and 12:12 h L/D photoperiod). Larvae were fed with an artificial diet consisting of 6.2 g of agar, 15.2 g of brewer's yeast, 23.7 g of wheat germ, 50 g of bean, 15.3 g of ascorbic acid, 0.5 g of sorbic acid, 1 g of methylparaben, and 1.2 mL of mold inhibitor solution (41.8% propionic acid and 4.2% phosphoric acid) (Isenhour et al., 1985).
2.5. Behavioral responses Adult P. elaeisis were placed individually in Petri dishes (90 × 15 mm) lined with filter paper disc (9 cm diameter, 3 μm pore size, 0.5% ash content, and 80 g/m2 density) (Nalgon Scientific Equipment, Itupeva, São Paulo, Brazil), which was fixed onto the bottom of the plate with synthetic glue. Half of the arena was treated with 1 mL of Bt suspension at the LC50 or LC90, and the other half with distilled water. A P. elaeisis adult was released in the center of the Petri dish and monitored for 10 min. Each treatment consisted of 20 insects arranged in a completely randomized design. Locomotor activities were recorded using a digital camcorder (XL1 3CCD NTSC, Canon, Lake Success, NY, USA) equipped with a 16 × video lens (ZoomXL 5.5–88 mm, Canon, Lake Success, NY, USA). A video tracking system (ViewPoint Life Sciences, Montreal, Quebec, Canada) was used to analyze the videos and measure the distance traveled and the amount of time spent on each half of the arena. Insects that spent less than 1 s on the Bt-treated half were considered repelled, those that spent less than 5 min on the Bt-treated surface were considered irritated, and insects that spent more than 5 min were considered unaffected (Fiaz et al., 2018; Plata-Rueda et al., 2019a). 2.6. Survivorship in parental and first-generation parasitoids Twenty-five S. frugiperda pupae per line (resistant/susceptible) were treated with the LC50 and LC90 of Bt. After 24 h, pupae were individually exposed, for 96 h, to parasitism by two 72 h old mated P. elaeisis in glass vials (14 × 2.2 cm) capped with cotton plugs and containing a drop of honey. During this time, insects were maintained at 25 ± 2 °C and 75 ± 5% RH under a 12:12 h L/D photoperiod regime. Pupae were kept in the glass tubes until emergence of the parasitoids. The number of live male and female adult parasitoids was
2.2. Concentration–mortality test Bt kurstaki strain HD-1 (Dipel®, Abbott Laboratories, North Chicago, IL, USA) was diluted in distilled water to obtain a stock suspension (100 g L−1), from which dilutions were prepared as needed. Six concentrations (1.562, 3.125, 6.25, 12.5, 25, and 50 mg mL−1) were used 2
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counted daily for two generations.
2.7. Reproductive performance of the parasitoid Bt-susceptible and -resistant S. frugiperda pupae treated with the estimated LC50 and LC90 of Bt were exposed to parasitism by two 72 h old P. elaeisis couples for 96 h in glass tubes (14 × 2.2 cm) containing a drop of honey, capped with cotton plug, and kept at 25 ± 2 °C and 75 ± 5% RH under a 12:12 h L/D photoperiod. The insects were maintained under these conditions until emergence of the parasitoid or the host. Fifteen replicates were used per treatment. Number of parasitoids emerged, egg-to-adult development time, and sex ratio [number of females/(males + females)] were determined in two generations.
2.8. Statistical analysis Concentration–mortality data were subjected to probit analysis to generate concentration–mortality curves (Finney, 1964). Time–mortality data were submitted to survival analysis by the Kaplan–Meier log-rank test using Origin Pro v. 9.1. (OriginLab Corporation, 2013). Parasitoids that survived until the end of the experiment were treated as censored. For the emerged parasitoids, development time, and sex ratio data between the S. frugiperda pupae (susceptible/resistant) were used general linear models (GLMs) assuming a binomial distribution and logit transformation (Proc Genmod) with emerged parasitoids (number of adult parasioids) or sex ratio as the dependent variable, and time longevity or (female/male proportion) as predictor variables. Logit transformation was performed to stabilize the variance and meet the assumptions of normality for analysis. Behavioral response data was subjected to analysis of variance (one-way ANOVA) and compared by Tukey's honestly significant difference (HSD) test at a significance level of 0.05. Behavioral response data was arcsine–transformed to satisfy normality and homoscedasticity assumptions. Data were analyzed using SAS for Windows v. 9.0. (SASInstitute, 2002).
Fig. 1. Survivorship of Bacillus thuringiensis (Bt)-susceptible Spodoptera frugiperda larvae exposed to different lethal concentrations of Bt. Values were determined by the Kaplan–Meier method and compared using the log-rank test (χ2 = 20.39, P < 0.001).
3.3. Production of immature parasitoids After 5 and 20 days of inoculation, no live P. elaeisis larvae were observed in susceptible and resistant S. frugiperda pupae exposed to Bt (Fig. 2A). The number of live P. elaeisis larvae did not differ between S. frugiperda pupae lines (susceptible/resistant) and treatments after 10 days of parasitoid inoculation (generalized linear model; χ2 = 2.44, P = 0.115). However, after 15 days, there were significant differences between the S. frugiperda pupae lines and the treatments (generalized linear model; χ2 = 15.62, P < 0.001). No pupae of P. elaeisis were observed in susceptible and resistant S. frugiperda 5 or 10 days after parasitoid exposure. The number of P. elaeisis pupae was significantly different between S. frugiperda pupae lines. Spodoptera frugiperda resistant had more parasitoid pupae after 15 (generalized linear model; χ2 = 32.81, P < 0.001) or 20 days (generalized linear model; χ2 = 13.38, P < 0.001) (Fig. 2B).
3. Results 3.1. Concentration–mortality responses of Bt-susceptible larvae
3.4. Behavioral response
The goodness-of-fit of the concentration–mortality model was adequate (P > 0.05) for estimating toxicological parameters (Table 1). The results confirmed the toxicity of Bt to susceptible S. frugiperda. Mortality in the control group was less than 1%.
Representative walking trails of P. elaeisis on half-treated arenas are shown in Fig. 3A. The distance walked was higher for the control (323.9 ± 46.6 cm) than for parasitoids placed in arenas containing the LC50 (182.3 ± 16.4 cm) and LC90 of Bt (142.7 ± 20.7 cm) (F2,41 = 12.06, P < 0.001) (Fig. 3B). The resting time did not differ between control (255.4 ± 25.3 s) and parasitoids kept in arenas half treated with LC50 (245.6 ± 24.5 s) and LC90 (220.7 ± 18.3 s) (F2,41 = 0.91, P = 0.563) (Fig. 3C).
3.2. Time–mortality responses of bt-susceptible larvae Survival analysis of susceptible S. frugiperda larvae exposed to Bt revealed significant differences between lethal concentrations (χ2 = 20.39, DF = 4, P < 0.001) (Fig. 1). After 200 h, survival was 100% in non-Bt-exposed (control) larvae, declining to 70%, 6.25%, 0%, and 0% with LC25, LC50, LC75, and LC90, respectively.
3.5. Survival of parental and first-generation adult parasitoids P. elaeisis emerged from Bt-treated S. frugiperda pupae (susceptible and resistant) showed reduced survival compared with the control, especially in the filial generation (Fig. 4). Survival time was reduced by 12.2 days for females (χ2 = 380.39, DF = 4, P < 0.001) and 12.4 days for males (χ2 = 148.47, DF = 4, P < 0.001) developing in susceptible pupae treated with LC50. Parasitoids emerged from Bt-resistant S. frugiperda exposed to LC50 showed a mean survival reduction of 18.4 days for females (χ2 = 673.4, DF = 4, P < 0.001) and 19.2 days for males (log-rank test: χ2 = 157.46, DF = 4, P < 0.001).
Table 1 Lethal concentrations of Bacillus thuringiensis against susceptible Spodoptera frugiperda larvae, according to probit analysis (DF = 5, slope ± SE = 8.175 ± 1.09, intercept = 5.506). Lethal concentration
Estimated concentration (mg mL−1)
Confidence interval 95%
χ2 (Pvalue)
LC25 LC50 LC75 LC90
5.67 6.39 7.12 7.84
5.22–5.99 6.10–6.68 6.83–7.53 7.45–8.48
0.87 (0.97)
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Fig. 2. Immature production of Palmistichus elaeisis from Spodoptera frugiperda pupae (susceptible/resistant) treated with Bacillus thuringiensis (Bt) at the estimated LC50 and LC90 or distilled water (control). A) Temporal sequence of X-ray images showing the number of live larvae (L) and pupae (P) of P. elaeisis in Bt-susceptible and -resistant S. frugiperda pupae treated with the LC50 and LC90 of Bt or distilled water. B) Proportion of number of larvae and pupae (mean ± SD) of P. elaeisis found in Bt-susceptible and -resistant S. frugiperda pupae to Bt (estimated LC50 and LC90 values besides control) for 10, 15 and 20 days.
Fig. 3. Behavior response of Palmistichus elaeisis caused by Bacillus thuringiensis (Bt). A) Representative tracks showing the locomotor activity of P. elaeisis over a 10 min period in arenas half treated with Bt (upper half of each arena). Red tracks indicate high walking speed; green tracks indicate low (initial) walking speed. Distance walked (B) and resting time (C) (mean ± SEM) of P. elaeisis kept for 10 min in arenas half treated with the LC50 and LC90 of Bt or distilled water (control). Letters indicate significant differences between treatments by Tukey's HSD test (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. Survivorship of Palmistichus elaeisis parental (F1) and first-generation (F2) in Bacillus thuringiensis (Bt)-susceptible and-resistant Spodoptera frugiperda pupae treated with the LC50 and LC90 of Bt or distilled water (control). Values were determined by the Kaplan–Meier method and compared using the log-rank test. Females × susceptible pupa (A), males × susceptible pupae (B), females × resistant pupae (C), and males × resistant pupae (D).
insect mortality, as observed in other Lepidoptera species, such as Tuta absoluta (Meyrick) (Gelechiidae) ( Gonzalez-Cabrera et al., 2011), Helicoverpa armigera (Hübner) (Noctuidae) (Regode et al., 2016), Plutella xylostella (Linnaeus) (Plutellidae) (Stemele, 2016), and Spodoptera litura (Fabricius) (Noctuidae) (Vineela et al., 2017). Bt caused mortality in S. frugiperda in a concentration-dependent manner, as reported for other insects (Oestergaard et al., 2007; Bravo et al., 2011). In this case, symptoms in S. frugiperda were consistent with the known effects of Bt protoxins and cell lysis (Tabashnik et al., 2015; Castro et al., 2019). S. frugiperda mortality was observed long after Bt exposure, confirming the slow action of the bioinsecticide via ingestion ( Gómez et al., 2014). Low survivorship of susceptible S. frugiperda was also observed in larvae exposed to transgenic plants, such as Bt cotton (Armstrong et al., 2011) and Bt corn expressing the Vip3Aa20 protein (Burtet et al., 2017). Laboratory bioassays revealed that Bt had negative effects on the parasitoid wasp P. elaeisis. The number of P. elaeisis larvae and pupae found in Bt-susceptible and Bt-resistant S. frugiperda varied according to Bt concentration. Results showed that 15 days after parasitism, the number of parasitoid larvae in Bt-susceptible S. frugiperda was lower than in Bt-resistant S. frugiperda. Similar results were reported for immature Trichogramma bourarachae Pintureau & Babault (Hymenoptera: Trichogrammatidae) (Ksentini et al., 2010) and Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) (Ebrahimi et al., 2012). The insecticidal action of Bt affects insect population growth rate, longevity, reproduction, and survivorship at different stages (Liu et al., 2005; Mohan et al., 2008), compromising parasitoid larvae and their progeny. Bt exposure altered the behavior and locomotor activity of P. elaeisis. Exposure to toxic compounds can affect the walking patterns of
3.6. Reproductive performance There was a reduction in the egg-to-adult development time between parental and first-generation P. elaeisis developed from Bt-susceptible and -resistant S. frugiperda pupae. The development time of adult parasitoids emerged from susceptible and resistant S. frugiperda pupae exposed to Bt did not differ in the parental generation (generalized linear model; χ2 = 3.44, P = 0.035). The development time of P. elaeisis developed in susceptible and resistant pupae exposed to Bt was different in first filial generation (generalized linear model; χ2 = 6.84, P < 0.001) (Fig. 5A). The number of adult parasitoids emerged from susceptible and resistant S. frugiperda pupae exposed to Bt did not differ in the parental generation (generalized linear model; χ2 = 1.55, P = 0.221). However, when hosts were exposed to Bt, there was a reduction in the number of first filial generation adult parasitoids emerged from susceptible and resistant pupae (generalized linear model; χ2 = 12.97, P < 0.001) in the control and LC50 groups (Fig. 5B). The proportion of females of P. elaeisis developed in susceptible and resistant pupae exposed to Bt was different in the parental generation (generalized linear model; χ2 = 6.44, P < 0.001) but no significant differences in the first filial generation developed from resistant Bttreated hosts (generalized linear model; χ2 = 1.84, P = 0.111) (Fig. 5C). 4. Discussion Exposure of S. frugiperda larvae to Bt via ingestion resulted in high 5
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-resistant hosts decreased over generations and can dramatically reduce parasitoid wasp populations in the field. Previous studies reported the detrimental effects of Bt on parasitoids (Chilcutt and Tabashnik, 1999) following ingestion of the Bt-toxins via artificial diet (Salama et al., 1996) and transgenic plants (Bernal et al., 2002; Baur and Boethel, 2003). Negative effects caused by Bt on parasitoids have been reported: reduced survival rate (Blumberg et al., 1997), lower emergence rates (Atwood et al., 1997), increased larval development time, and reduced longevity and fecundity (Baur and Boethel, 2003). Parasitoids complete their development on a host and are likely to be adversely affected if their Bt susceptible hosts are treated with Bt toxin and are weakened or killed (Bernal et al., 2002), a phenomenon usually referred to as hostquality mediated effects (Baur and Boethel, 2003). In this study, the negative impact on P. elaeisis survival was host-quality mediated when it developed inside susceptible or resistant S. frugiperda pupae, compromising the parasitism potential of this biological control agent in the field. The development time, proportion of females, and number of parasitoids emerged from Bt-treated (susceptible and resistant) S. frugiperda pupae decreased compared with the control. Parasitoids may complete larval development and emerge before their Bt-infected host dies, provided that the level of Bt toxin does not cause host death. Nevertheless, S. frugiperda nutrition is affected by the presence of Bt toxins in the intestinal membrane, resulting in host malnutrition (Groot and Dicke, 2003) and, consequently, poor parasitoid performance. Direct exposure of adult parasitoids to Bt is unlikely because plant nectar does not contain Bt toxins (Groot and Dicke, 2003) and because Bt toxins bind to receptors in the midgut epithelium of host larvae and lose their toxicity to the parasitoids. However, parasitoids at immature stages may be indirectly affected by lethal or sublethal effects on host health and development (Bernal et al., 2002). The effects caused by Bt are indirect, and affect the quality of the host, compromising the performance of generations of P. elaeisis. Hymenoptera species can reproduce by arrhenotokous parthenogenesis. Therefore, the reduction in the proportion of females of P. elaeisis emerged from Bt-susceptible or -resistant hosts may be due to lower sperm production or male sterility, as already observed in P. elaeisis exposed to other insecticides (Rabeling and Kronauer, 2013). Female-biased sex ratios in parasitoid wasps with spatially structured populations have been studied (Flanagan et al., 1998). Sex allocation theory explains the evolution of how organisms allocate investment into male or female offspring (Shuker et al., 2005). In this study, exposure to the Bt disrupts the ability P. elaeisis females or males to facultatively allocate sex. Effects in the proportion of females suggest that exposure to Bt-susceptible at different concentrations alters the machinery females use to allocate sex adaptively. This disruption imposes a significant cost to P. elaeisis females, considering the mortality cost by susceptible or Bt-resistant host selection, and a cost by the reduction in fecundity seen here and in other parasitoids (Cook et al., 2016). In general, Bt had side effects on P. elaeisis. Exposure to the bacteria reduced immature production and reproductive performance over the generations and changed the parasitoid's host-searching behavior. Thus, the use of Bt as a bioinsecticide may affect the establishment and performance of P. elaeisis. The results of this study provide useful information for the development of effective and compatible IPM strategies.
Fig. 5. Reproductive performance (mean ± SD) of Palmistichus elaeisis emerged from Bacillus thuringiensis (Bt)-susceptible and -resistant Spodoptera frugiperda pupae exposed to the LC50 and LC90 of Bt. A) Egg-to-adult development time. B) Number of adults/pupa. C) Proportion of females (sex ratio).
insects as a result of damage to the nervous system, which may lead to stimulation or reduction in mobility (Martínez et al., 2018b; PlataRueda et al., 2018). The short distances traveled by P. elaeisis after exposure indicate Bt repellency (Dias-Pini et al., 2014). Bt also repelled Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae) (Wright and Cornelius, 2012). The parasitoid Tranosema rostrale (Brishke) (Hymenoptera: Ichneumonidae) avoided an area treated with Bt in a choice test (Schoenmaker et al., 2001). Studies have shown that biological and chemical insecticides can disrupt important insect activities, such as substrate foraging and food recognition (Plata-Rueda et al., 2019b). In parasitoids, side effects on olfactory orientation and walking can disrupt host recognition, limiting the parasitism rate (Erb et al., 2001). The survivorship of P. elaeisis emerged from Bt-susceptible and
Author Contribution Statement Gabriela da Silva Rolim: Methodology, Investigation, WritingOriginal draft preparation. Angelica Plata-Rueda: Conceptualization, Visualization, Investigation. Luis Carlos Martínez: Supervision, Validation, Writing- Reviewing and Editing. Genésio Tâmara Ribeiro: Conceptualization, Visualization, Investigation. José Eduardo Serrão: Writing- Reviewing and Editing. José Cola Zanuncio: WritingReviewing and Editing. 6
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Side effects of Bacillus thuringiensis on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae) Gabriela da Silva Rolima, Angelica Plata-Ruedab, Luis Carlos Martínezc,∗, Genésio Tâmara Ribeirod, José Eduardo Serrãoc, José Cola Zanuncioe a
Departamento de Fitotecnia, Universidade Federal de Viçosa, 36570-900, Viçosa, Minas Gerais, Brazil Instituto de Ciências Agrárias, Universidade Federal de Viçosa, 38810-000, Viçosa, Minas Gerais, Brazil c Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais, Brazil d Universidade Federal de Sergipe – Departamento de Ciências Florestais, 49100-000, São Cristóvão, Sergipe, Brazil e Departamento de Entomologia, Universidade Federal de Viçosa, 36570-000, Viçosa, Minas Gerais, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Parasitoid wasp Production of immatures Repellency Reproductive performance Survival Toxicity
The endoparasitoid wasp Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is used to control defoliating lepidopteran pests. Chemical insecticides are not compatible with natural enemies, but bioinsecticides, such as Bacillus thuringiensis Berliner (Bt), have great potential for use in integrated pest management. However, interactions between Bt and P. elaeisis still need to be investigated. This study aimed to evaluate the effects of Bt on parental and first-generation P. elaeisis parasitizing Bt-susceptible and -resistant Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). An additional aim was to determine the toxicity of Bt to susceptible third-instar S. frugiperda larvae. Larvae were exposed to lethal concentrations (LC50 and LC90) of Bt and then allowed to be parasitized by P. elaeisis. Parasitoid longevity, immature production, reproductive performance, and behavioral responses were evaluated. Bt repelled P. elaeisis and reduced immature production. Parental and first filial generation parasitoids of both sexes emerged from Bt-treated larvae showed lower survivorship than controls. Parasitoids had poorer reproductive performance in Bt-susceptible and -resistant pupae than in untreated pupae. Palmistichus elaeisis emerged from Bt-susceptible and -resistant S. frugiperda showed altered host-searching behavior and reproductive parameters, which indicates low compatibility between the bioinsecticide agent and the parasitoid wasp.
1. Introduction Palmistichus elaeisis Delvare & LaSalle (Hymenoptera: Eulophidae) is a gregarious endoparasitoid wasp that develops in coleopteran and lepidopteran species (Zanuncio et al., 2008; Barbosa et al., 2016). Females of P. elaeisis lay eggs on host pupae, and, after emergence, their parasitic larvae feed on host organs and tissues (Soares et al., 2009). The parasitoid is native to the Neotropics and has polyphagous habits, feeding on lepidopterans of economic importance, such as Anticarsia gemmatalis Hübner (Noctuidae) (Pereira et al., 2013), Diaphania hyalinata Linnaeus (Crambidae) (Pratissoli et al., 2007), Psorocampa denticulata Schaus (Notodontidae) (Zanuncio et al., 2015), Spodoptera frugiperda (J.E. Smith) (Noctuidae) (Bittencourt and Berti-Filho, 1999), and Thyrinteina arnobia (Stoll) (Geometridae) (Barbosa et al., 2016). The species shows great potential as a biological control agent. For use in integrated pest management (IPM), natural enemies must
∗
be compatible with other control methods (Zanuncio et al., 2016; Alcántara-de La Cruz et al., 2017; Martínez et al., 2018a). Chemical control is currently the most widely used method, and several studies have been aimed at developing more selective insecticides to minimize their impact on natural enemies (Desneux et al., 2007). However, the side effects of chemical agents on humans, non-target organisms, and the environment as a whole motivate the search for more sustainable pest control methods (Desneux et al., 2007; Nicholson, 2007; Pedlowski et al., 2012). Natural enemies of pests as parasitoids are susceptible to insecticides, which may affect development, longevity, fecundity, and parasitism rate (Alcántara-de La Cruz et al., 2017), while bacteria-based bioinsecticides to pest control can be compatible with parasitoids (Desneux et al., 2007). Bacillus thuringiensis Berliner (Bt) is a rod-shaped, Gram-positive bacterium that produces insect-toxic crystal (Cry) proteins during the sporulation phase (Bravo et al., 2011). When dissolved and activated by
Corresponding author. E-mail address: [email protected] (L.C. Martínez).
https://doi.org/10.1016/j.ecoenv.2019.109978 Received 23 May 2019; Received in revised form 12 November 2019; Accepted 14 November 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Gabriela da Silva Rolim, et al., Ecotoxicology and Environmental Safety, https://doi.org/10.1016/j.ecoenv.2019.109978
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to evaluate the toxicity of the bioinsecticide to Bt-susceptible S. frugiperda larvae, construct concentration–mortality curves, and estimate lethal concentrations (LC). Distilled water was used as negative control. Three replicates of 30 third-instar S. frugiperda larvae were placed individually in Petri dishes (90 × 1.5 mm) and fed with Bt suspensions (2 μL) in 1 g of artificial diet. The number of dead larvae was recorded for 8 days.
insect proteases upon ingestion, Cry proteins form pores in the midgut membrane, causing septicemia and insect death (Oestergaard et al., 2007; Castro et al., 2019). Specific Bt strains developed for target insect groups are less toxic to vertebrates and therefore have lower environmental impact (Bravo et al., 2011). For instance, Bt subsp. kurstaki can be used to control lepidopterans (Sanahuja et al., 2011). Bt strains and toxins have been used for the production of bioinsecticides, and cry genes for the development of transgenic plants (Romeis et al., 2006). Transgenic plants expressing Cry proteins have revolutionized the IPM of different cropping systems (Sanahuja et al., 2011; Flagel et al., 2018). Plants expressing a single Cry protein may favor outbreaks of resistant pests ( HYBaranek et al., 2017), but expression of two or more active Bt toxins attenuates this problem (Santos-Amaya et al., 2015). On the other hand, natural enemies exposure to Bt proteins by feeding on Bt-resistant prey or hosts can compromise their quality (Romeis et al., 2008). In fact, the existing debate about the effect of Bt proteins on natural enemies has focused on whether any purported negative effects are due to the Bt protein or quality of the host or prey on which the natural enemy fed (Shelton et al., 2002). The combined use of Bt and parasitoids is a promising IPM strategy because of its low cost, high efficiency, and minimal environmental risk (Barrat et al., 2010). However, the effects of Bt on P. elaeisis are still unknown. This study aimed to evaluate the reproductive parameters, progeny size, and development time of P. elaeisis parasitizing Bt-susceptible and Bt-resistant S. frugiperda pupae exposed to the bacterium.
2.3. Time–mortality test Bt-susceptible third-instar S. frugiperda larvae were placed one per Petri dish and exposed to the lethal concentrations (LC25, LC50, LC75, and LC90) of Bt determined in the concentration–mortality test. A control was performed using distilled water. Exposure procedures and conditions were the same as described above for the concentration–mortality test (section 2.2). The number of live insects was recorded every 12 h for 8 days. Four replicates of 30 insects were used for each Bt concentration following a completely randomized design. 2.4. Production of immature parasitoids Bt-susceptible and -resistant third-instar S. frugiperda larvae were treated with Bt at the estimated LC50 and LC90 or with distilled water as control. After pupation, S. frugiperda were exposed to parasitism by P. elaeisis for 24 h. The experiment was arranged in a completely randomized design, with three replicates of 10 pupae per treatment. Pupae parasitized by P. elaeisis were placed individually in glass vials (2.5 × 8 cm) and examined using an MX-20 specimen radiography system (Faxitron X-Ray Corp, Wheeling, IL, USA) equipped with a 14bit digital camera. The location of each parasitoid within each S. frugiperda pupa was recorded at 5, 10, 15, and 20 days after parasitization. The number of live larvae and pupae of P. elaeisis per S. frugiperda pupa was also recorded.
2. Material and methods 2.1. Insects Palmistichus elaeisis and S. frugiperda (only Bt-susceptible population) individuals were mass reared at the Laboratory of Insect Biological Control of the Federal University of Viçosa (Viçosa, Minas Gerais, Brazil). Bt-resistant individuals were collected from Cry1F-expressing corn plants (30F35H DuPont Pioneer®, Santa Cruz do Sul, Rio Grande do Sul, Brazil). Pupae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) were used to mass rearing of P. elaeisis. Tenebrio molitor individuals were kept in plastic boxes (60 × 40 × 12 cm) at 25 ± 1 °C and 70 ± 10% relative humidity (RH) under a 12:12 h light/dark (L/D) photoperiod regime. Larvae and adults were fed ad libitum with wheat bran (75% carbohydrates, 12% protein, 11% minerals, and 2% lipids), pieces of sugarcane [Saccharum officinarum Linnaeus (Poaceae)] stems, and pieces of chayote [Sechium edule Swartz (Cucurbitaceae)] until pupation. P. elaeisis adults were mated for 24 h and placed in glass tubes (14 × 2.2 cm) covered with a fine net and containing T. molitor pupae. After parasitization, newly emerged (24 h old) P. elaeisis individuals were placed in glass tubes (14 × 2.2 cm) in a controlled environment room (25 ± 2 °C, 70 ± 10% RH, and 12:12 h L/D photoperiod) and fed with pure honey ad libitum. Bt-susceptible and -resistant S. frugiperda larvae were grown separately in polyethylene pots (15 × 9 cm) in a controlled room (25 ± 2 °C, 75 ± 5% RH, and 12:12 h L/D photoperiod). Larvae were fed with an artificial diet consisting of 6.2 g of agar, 15.2 g of brewer's yeast, 23.7 g of wheat germ, 50 g of bean, 15.3 g of ascorbic acid, 0.5 g of sorbic acid, 1 g of methylparaben, and 1.2 mL of mold inhibitor solution (41.8% propionic acid and 4.2% phosphoric acid) (Isenhour et al., 1985).
2.5. Behavioral responses Adult P. elaeisis were placed individually in Petri dishes (90 × 15 mm) lined with filter paper disc (9 cm diameter, 3 μm pore size, 0.5% ash content, and 80 g/m2 density) (Nalgon Scientific Equipment, Itupeva, São Paulo, Brazil), which was fixed onto the bottom of the plate with synthetic glue. Half of the arena was treated with 1 mL of Bt suspension at the LC50 or LC90, and the other half with distilled water. A P. elaeisis adult was released in the center of the Petri dish and monitored for 10 min. Each treatment consisted of 20 insects arranged in a completely randomized design. Locomotor activities were recorded using a digital camcorder (XL1 3CCD NTSC, Canon, Lake Success, NY, USA) equipped with a 16 × video lens (ZoomXL 5.5–88 mm, Canon, Lake Success, NY, USA). A video tracking system (ViewPoint Life Sciences, Montreal, Quebec, Canada) was used to analyze the videos and measure the distance traveled and the amount of time spent on each half of the arena. Insects that spent less than 1 s on the Bt-treated half were considered repelled, those that spent less than 5 min on the Bt-treated surface were considered irritated, and insects that spent more than 5 min were considered unaffected (Fiaz et al., 2018; Plata-Rueda et al., 2019a). 2.6. Survivorship in parental and first-generation parasitoids Twenty-five S. frugiperda pupae per line (resistant/susceptible) were treated with the LC50 and LC90 of Bt. After 24 h, pupae were individually exposed, for 96 h, to parasitism by two 72 h old mated P. elaeisis in glass vials (14 × 2.2 cm) capped with cotton plugs and containing a drop of honey. During this time, insects were maintained at 25 ± 2 °C and 75 ± 5% RH under a 12:12 h L/D photoperiod regime. Pupae were kept in the glass tubes until emergence of the parasitoids. The number of live male and female adult parasitoids was
2.2. Concentration–mortality test Bt kurstaki strain HD-1 (Dipel®, Abbott Laboratories, North Chicago, IL, USA) was diluted in distilled water to obtain a stock suspension (100 g L−1), from which dilutions were prepared as needed. Six concentrations (1.562, 3.125, 6.25, 12.5, 25, and 50 mg mL−1) were used 2
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counted daily for two generations.
2.7. Reproductive performance of the parasitoid Bt-susceptible and -resistant S. frugiperda pupae treated with the estimated LC50 and LC90 of Bt were exposed to parasitism by two 72 h old P. elaeisis couples for 96 h in glass tubes (14 × 2.2 cm) containing a drop of honey, capped with cotton plug, and kept at 25 ± 2 °C and 75 ± 5% RH under a 12:12 h L/D photoperiod. The insects were maintained under these conditions until emergence of the parasitoid or the host. Fifteen replicates were used per treatment. Number of parasitoids emerged, egg-to-adult development time, and sex ratio [number of females/(males + females)] were determined in two generations.
2.8. Statistical analysis Concentration–mortality data were subjected to probit analysis to generate concentration–mortality curves (Finney, 1964). Time–mortality data were submitted to survival analysis by the Kaplan–Meier log-rank test using Origin Pro v. 9.1. (OriginLab Corporation, 2013). Parasitoids that survived until the end of the experiment were treated as censored. For the emerged parasitoids, development time, and sex ratio data between the S. frugiperda pupae (susceptible/resistant) were used general linear models (GLMs) assuming a binomial distribution and logit transformation (Proc Genmod) with emerged parasitoids (number of adult parasioids) or sex ratio as the dependent variable, and time longevity or (female/male proportion) as predictor variables. Logit transformation was performed to stabilize the variance and meet the assumptions of normality for analysis. Behavioral response data was subjected to analysis of variance (one-way ANOVA) and compared by Tukey's honestly significant difference (HSD) test at a significance level of 0.05. Behavioral response data was arcsine–transformed to satisfy normality and homoscedasticity assumptions. Data were analyzed using SAS for Windows v. 9.0. (SASInstitute, 2002).
Fig. 1. Survivorship of Bacillus thuringiensis (Bt)-susceptible Spodoptera frugiperda larvae exposed to different lethal concentrations of Bt. Values were determined by the Kaplan–Meier method and compared using the log-rank test (χ2 = 20.39, P < 0.001).
3.3. Production of immature parasitoids After 5 and 20 days of inoculation, no live P. elaeisis larvae were observed in susceptible and resistant S. frugiperda pupae exposed to Bt (Fig. 2A). The number of live P. elaeisis larvae did not differ between S. frugiperda pupae lines (susceptible/resistant) and treatments after 10 days of parasitoid inoculation (generalized linear model; χ2 = 2.44, P = 0.115). However, after 15 days, there were significant differences between the S. frugiperda pupae lines and the treatments (generalized linear model; χ2 = 15.62, P < 0.001). No pupae of P. elaeisis were observed in susceptible and resistant S. frugiperda 5 or 10 days after parasitoid exposure. The number of P. elaeisis pupae was significantly different between S. frugiperda pupae lines. Spodoptera frugiperda resistant had more parasitoid pupae after 15 (generalized linear model; χ2 = 32.81, P < 0.001) or 20 days (generalized linear model; χ2 = 13.38, P < 0.001) (Fig. 2B).
3. Results 3.1. Concentration–mortality responses of Bt-susceptible larvae
3.4. Behavioral response
The goodness-of-fit of the concentration–mortality model was adequate (P > 0.05) for estimating toxicological parameters (Table 1). The results confirmed the toxicity of Bt to susceptible S. frugiperda. Mortality in the control group was less than 1%.
Representative walking trails of P. elaeisis on half-treated arenas are shown in Fig. 3A. The distance walked was higher for the control (323.9 ± 46.6 cm) than for parasitoids placed in arenas containing the LC50 (182.3 ± 16.4 cm) and LC90 of Bt (142.7 ± 20.7 cm) (F2,41 = 12.06, P < 0.001) (Fig. 3B). The resting time did not differ between control (255.4 ± 25.3 s) and parasitoids kept in arenas half treated with LC50 (245.6 ± 24.5 s) and LC90 (220.7 ± 18.3 s) (F2,41 = 0.91, P = 0.563) (Fig. 3C).
3.2. Time–mortality responses of bt-susceptible larvae Survival analysis of susceptible S. frugiperda larvae exposed to Bt revealed significant differences between lethal concentrations (χ2 = 20.39, DF = 4, P < 0.001) (Fig. 1). After 200 h, survival was 100% in non-Bt-exposed (control) larvae, declining to 70%, 6.25%, 0%, and 0% with LC25, LC50, LC75, and LC90, respectively.
3.5. Survival of parental and first-generation adult parasitoids P. elaeisis emerged from Bt-treated S. frugiperda pupae (susceptible and resistant) showed reduced survival compared with the control, especially in the filial generation (Fig. 4). Survival time was reduced by 12.2 days for females (χ2 = 380.39, DF = 4, P < 0.001) and 12.4 days for males (χ2 = 148.47, DF = 4, P < 0.001) developing in susceptible pupae treated with LC50. Parasitoids emerged from Bt-resistant S. frugiperda exposed to LC50 showed a mean survival reduction of 18.4 days for females (χ2 = 673.4, DF = 4, P < 0.001) and 19.2 days for males (log-rank test: χ2 = 157.46, DF = 4, P < 0.001).
Table 1 Lethal concentrations of Bacillus thuringiensis against susceptible Spodoptera frugiperda larvae, according to probit analysis (DF = 5, slope ± SE = 8.175 ± 1.09, intercept = 5.506). Lethal concentration
Estimated concentration (mg mL−1)
Confidence interval 95%
χ2 (Pvalue)
LC25 LC50 LC75 LC90
5.67 6.39 7.12 7.84
5.22–5.99 6.10–6.68 6.83–7.53 7.45–8.48
0.87 (0.97)
3
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Fig. 2. Immature production of Palmistichus elaeisis from Spodoptera frugiperda pupae (susceptible/resistant) treated with Bacillus thuringiensis (Bt) at the estimated LC50 and LC90 or distilled water (control). A) Temporal sequence of X-ray images showing the number of live larvae (L) and pupae (P) of P. elaeisis in Bt-susceptible and -resistant S. frugiperda pupae treated with the LC50 and LC90 of Bt or distilled water. B) Proportion of number of larvae and pupae (mean ± SD) of P. elaeisis found in Bt-susceptible and -resistant S. frugiperda pupae to Bt (estimated LC50 and LC90 values besides control) for 10, 15 and 20 days.
Fig. 3. Behavior response of Palmistichus elaeisis caused by Bacillus thuringiensis (Bt). A) Representative tracks showing the locomotor activity of P. elaeisis over a 10 min period in arenas half treated with Bt (upper half of each arena). Red tracks indicate high walking speed; green tracks indicate low (initial) walking speed. Distance walked (B) and resting time (C) (mean ± SEM) of P. elaeisis kept for 10 min in arenas half treated with the LC50 and LC90 of Bt or distilled water (control). Letters indicate significant differences between treatments by Tukey's HSD test (P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. Survivorship of Palmistichus elaeisis parental (F1) and first-generation (F2) in Bacillus thuringiensis (Bt)-susceptible and-resistant Spodoptera frugiperda pupae treated with the LC50 and LC90 of Bt or distilled water (control). Values were determined by the Kaplan–Meier method and compared using the log-rank test. Females × susceptible pupa (A), males × susceptible pupae (B), females × resistant pupae (C), and males × resistant pupae (D).
insect mortality, as observed in other Lepidoptera species, such as Tuta absoluta (Meyrick) (Gelechiidae) ( Gonzalez-Cabrera et al., 2011), Helicoverpa armigera (Hübner) (Noctuidae) (Regode et al., 2016), Plutella xylostella (Linnaeus) (Plutellidae) (Stemele, 2016), and Spodoptera litura (Fabricius) (Noctuidae) (Vineela et al., 2017). Bt caused mortality in S. frugiperda in a concentration-dependent manner, as reported for other insects (Oestergaard et al., 2007; Bravo et al., 2011). In this case, symptoms in S. frugiperda were consistent with the known effects of Bt protoxins and cell lysis (Tabashnik et al., 2015; Castro et al., 2019). S. frugiperda mortality was observed long after Bt exposure, confirming the slow action of the bioinsecticide via ingestion ( Gómez et al., 2014). Low survivorship of susceptible S. frugiperda was also observed in larvae exposed to transgenic plants, such as Bt cotton (Armstrong et al., 2011) and Bt corn expressing the Vip3Aa20 protein (Burtet et al., 2017). Laboratory bioassays revealed that Bt had negative effects on the parasitoid wasp P. elaeisis. The number of P. elaeisis larvae and pupae found in Bt-susceptible and Bt-resistant S. frugiperda varied according to Bt concentration. Results showed that 15 days after parasitism, the number of parasitoid larvae in Bt-susceptible S. frugiperda was lower than in Bt-resistant S. frugiperda. Similar results were reported for immature Trichogramma bourarachae Pintureau & Babault (Hymenoptera: Trichogrammatidae) (Ksentini et al., 2010) and Diadegma insulare (Cresson) (Hymenoptera: Ichneumonidae) (Ebrahimi et al., 2012). The insecticidal action of Bt affects insect population growth rate, longevity, reproduction, and survivorship at different stages (Liu et al., 2005; Mohan et al., 2008), compromising parasitoid larvae and their progeny. Bt exposure altered the behavior and locomotor activity of P. elaeisis. Exposure to toxic compounds can affect the walking patterns of
3.6. Reproductive performance There was a reduction in the egg-to-adult development time between parental and first-generation P. elaeisis developed from Bt-susceptible and -resistant S. frugiperda pupae. The development time of adult parasitoids emerged from susceptible and resistant S. frugiperda pupae exposed to Bt did not differ in the parental generation (generalized linear model; χ2 = 3.44, P = 0.035). The development time of P. elaeisis developed in susceptible and resistant pupae exposed to Bt was different in first filial generation (generalized linear model; χ2 = 6.84, P < 0.001) (Fig. 5A). The number of adult parasitoids emerged from susceptible and resistant S. frugiperda pupae exposed to Bt did not differ in the parental generation (generalized linear model; χ2 = 1.55, P = 0.221). However, when hosts were exposed to Bt, there was a reduction in the number of first filial generation adult parasitoids emerged from susceptible and resistant pupae (generalized linear model; χ2 = 12.97, P < 0.001) in the control and LC50 groups (Fig. 5B). The proportion of females of P. elaeisis developed in susceptible and resistant pupae exposed to Bt was different in the parental generation (generalized linear model; χ2 = 6.44, P < 0.001) but no significant differences in the first filial generation developed from resistant Bttreated hosts (generalized linear model; χ2 = 1.84, P = 0.111) (Fig. 5C). 4. Discussion Exposure of S. frugiperda larvae to Bt via ingestion resulted in high 5
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-resistant hosts decreased over generations and can dramatically reduce parasitoid wasp populations in the field. Previous studies reported the detrimental effects of Bt on parasitoids (Chilcutt and Tabashnik, 1999) following ingestion of the Bt-toxins via artificial diet (Salama et al., 1996) and transgenic plants (Bernal et al., 2002; Baur and Boethel, 2003). Negative effects caused by Bt on parasitoids have been reported: reduced survival rate (Blumberg et al., 1997), lower emergence rates (Atwood et al., 1997), increased larval development time, and reduced longevity and fecundity (Baur and Boethel, 2003). Parasitoids complete their development on a host and are likely to be adversely affected if their Bt susceptible hosts are treated with Bt toxin and are weakened or killed (Bernal et al., 2002), a phenomenon usually referred to as hostquality mediated effects (Baur and Boethel, 2003). In this study, the negative impact on P. elaeisis survival was host-quality mediated when it developed inside susceptible or resistant S. frugiperda pupae, compromising the parasitism potential of this biological control agent in the field. The development time, proportion of females, and number of parasitoids emerged from Bt-treated (susceptible and resistant) S. frugiperda pupae decreased compared with the control. Parasitoids may complete larval development and emerge before their Bt-infected host dies, provided that the level of Bt toxin does not cause host death. Nevertheless, S. frugiperda nutrition is affected by the presence of Bt toxins in the intestinal membrane, resulting in host malnutrition (Groot and Dicke, 2003) and, consequently, poor parasitoid performance. Direct exposure of adult parasitoids to Bt is unlikely because plant nectar does not contain Bt toxins (Groot and Dicke, 2003) and because Bt toxins bind to receptors in the midgut epithelium of host larvae and lose their toxicity to the parasitoids. However, parasitoids at immature stages may be indirectly affected by lethal or sublethal effects on host health and development (Bernal et al., 2002). The effects caused by Bt are indirect, and affect the quality of the host, compromising the performance of generations of P. elaeisis. Hymenoptera species can reproduce by arrhenotokous parthenogenesis. Therefore, the reduction in the proportion of females of P. elaeisis emerged from Bt-susceptible or -resistant hosts may be due to lower sperm production or male sterility, as already observed in P. elaeisis exposed to other insecticides (Rabeling and Kronauer, 2013). Female-biased sex ratios in parasitoid wasps with spatially structured populations have been studied (Flanagan et al., 1998). Sex allocation theory explains the evolution of how organisms allocate investment into male or female offspring (Shuker et al., 2005). In this study, exposure to the Bt disrupts the ability P. elaeisis females or males to facultatively allocate sex. Effects in the proportion of females suggest that exposure to Bt-susceptible at different concentrations alters the machinery females use to allocate sex adaptively. This disruption imposes a significant cost to P. elaeisis females, considering the mortality cost by susceptible or Bt-resistant host selection, and a cost by the reduction in fecundity seen here and in other parasitoids (Cook et al., 2016). In general, Bt had side effects on P. elaeisis. Exposure to the bacteria reduced immature production and reproductive performance over the generations and changed the parasitoid's host-searching behavior. Thus, the use of Bt as a bioinsecticide may affect the establishment and performance of P. elaeisis. The results of this study provide useful information for the development of effective and compatible IPM strategies.
Fig. 5. Reproductive performance (mean ± SD) of Palmistichus elaeisis emerged from Bacillus thuringiensis (Bt)-susceptible and -resistant Spodoptera frugiperda pupae exposed to the LC50 and LC90 of Bt. A) Egg-to-adult development time. B) Number of adults/pupa. C) Proportion of females (sex ratio).
insects as a result of damage to the nervous system, which may lead to stimulation or reduction in mobility (Martínez et al., 2018b; PlataRueda et al., 2018). The short distances traveled by P. elaeisis after exposure indicate Bt repellency (Dias-Pini et al., 2014). Bt also repelled Coptotermes formosanus Shiraki (Blattodea: Rhinotermitidae) (Wright and Cornelius, 2012). The parasitoid Tranosema rostrale (Brishke) (Hymenoptera: Ichneumonidae) avoided an area treated with Bt in a choice test (Schoenmaker et al., 2001). Studies have shown that biological and chemical insecticides can disrupt important insect activities, such as substrate foraging and food recognition (Plata-Rueda et al., 2019b). In parasitoids, side effects on olfactory orientation and walking can disrupt host recognition, limiting the parasitism rate (Erb et al., 2001). The survivorship of P. elaeisis emerged from Bt-susceptible and
Author Contribution Statement Gabriela da Silva Rolim: Methodology, Investigation, WritingOriginal draft preparation. Angelica Plata-Rueda: Conceptualization, Visualization, Investigation. Luis Carlos Martínez: Supervision, Validation, Writing- Reviewing and Editing. Genésio Tâmara Ribeiro: Conceptualization, Visualization, Investigation. José Eduardo Serrão: Writing- Reviewing and Editing. José Cola Zanuncio: WritingReviewing and Editing. 6
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Acknowledgments
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