Toxic and sublethal effects of buprofezin on the whitefly parasitoid Encarsia formosa Gahan

SUMMARY Acute toxicity of a buprofezin-based product (commercial product Elisa 440 SC) to pupae of the whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), and its effects on life history traits and population growth in F 1 generation of a commercial strain (“Dutch” strain, D) and two local populations from Serbia (Bujanovac, B; Negotin, N) were examined in laboratory bioassays. All trials were carried out at 27±1°C temperature and 60±10% relative humidity, and under 16/8 h daylight/darkness photoperiod in four replications. In an acute toxicity bioassay, tobacco leaves carrying parasitoid pupae (20 pupae per replicate) were treated with a series of buprofezin concentrations covering a 10-90% mortality range, and mortality was calculated based on the number of emerging adults 9 days after treatment. The following LC 50 (mg/l) estimates were obtained: 244.2, 281.5 and 199.5 (for B, N and D, respectively). The product based on buprofezin, applied to parasitoid pupae at concentrations within the LC 50 s and 95% confidence limits (264 mg/l for B and N; 220 mg/l for D), significantly prolonged the duration of juvenile development (2, 1.7 and 2.2 days for B, N and D, respectively, compared to control data). Females from all tested populations that emerged from the treated pupae and were exposed to the residual action of buprofezin lived shorter than control females (B, N and D by 1.5, 0.7 and 1.7 days, respectively). Also, females that emerged from the treated pupae achieved a significantly reduced level of parasitism (B, N and D by 11.7, 17.7 and 17.6 %, respectively), total adult emergence (B, N and D by 11.6, 17.8 and 17.8 %, respectively) and instantaneous rate of increase (B, N and D by 8.2, 6.8 and 12.5 %, respectively), compared to control. More precise determination of risks involved in the use of buprofezin requires its more detailed field testing.


INTRODUCTION
The greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), a cosmopolitan and polyphagous species, has been widespread in Serbia since the 1970s and it is frequently found in greenhouses as a serious pest of vegetables and ornamentals (Prijović et al., 2014). The parasitic wasp Encarsia formosa Gahan (Hymenoptera: Aphelinidae) has been used for many years for biological control of T. vaporariorum and has been one of the most successful biological agents in greenhouse crops around the world (van Lenteren & Martin, 1999;Enkegaard & Brødsgaard, 2006), but it is rarely used for that purpose in Serbia. E. formosa is uniparental, its females are primary endoparasitoids of the greenhouse whitefly, and males occur only rarely (Hoddle at al., 1998). Pesticide treatments are necessary when natural enemies fail to keep a pest population below its economic threshold (Albajes et al.,1999). The widespread use of chemical insecticides has caused whitefly resistance to compounds with various modes of action (Whalon et al., 2020).
Commercial field studies have shown that strategies of plant protection from pests based on initial use of insect growth regulators (IGR), such as buprofezin and pyriproxyfen, protect populations of natural enemies, unlike conventional insecticides (Naranjo et al., 2003). Buprofezin has a relatively small spectrum of activity against Hemiptera insects, including whiteflies (Cahill et al., 1996). This IGR has demonstrated efficacy in controlling T. vaporariorum (Masuda & Miyata, 2006), and has been effective in controlling B. tabaci in cotton (Natwick, 1993), even though less effective against B. tabaci biotype B (Masuda & Miyata, 2006) and B. tabaci biotype Q (Kobayashi, 2007). Buprofezin is not active against adults but reduces the viability of eggs of treated females, and it causes molting at younger larval stages, thus reducing the adult pest population only seven to 10 days after treatment (Horowitz & Ishaaya, 1996). Buprofezin, which inhibits chitin synthesis, is active during insect molting; it disturbs molting from larval stages to adulthood (interferes with embriogenesis in adults), affecting reproduction (Toscano et al., 2001;Yu, 2008).
Generally speaking, buprofezin is considered to be less harmful to parasitoids than to predators (Jones et al., 1998). Many studies have shown that different development stages of Hymenoptera parasitoids react in various ways to IGR treatments, showing a tendency of younger development stages to be more susceptible than the later and more mature stages (Gerling & Sinai, 1994;Darvas & Polgar, 1998). Parasitoids of the family Aphelinidae that survived the activity of buprofezin may suffer from harmful sublethal effects manifesting as shorter life span, lower fecundity, and wing deformation occurring after eclosion of parasitoid adults from the pupal stage (Stansly & Liu, 1997;Jones et al.,1998). Any indirect effect on natural enemies connected with buprofezin activity may occur as a consequence of its vaporability (De Cock et al., 1990).
In some cases, buprofezin performed poorly as regards its selectivity, which depends on the test species of insects (Stansly & Liu, 1997;Jones et al., 1998). Buprofezin classification based on quantification of life parameters and population growth of E. formosa was at the focus of research by Heydari et al. (2006) and Southwood and Handerson (2000). The present study focused on the effects of a buprofezin-based insecticide on life history traits and population growth of F 1 generation following treatment of pupae as the least susceptible development stage of the parasitoid. Data obtained in this study are discussed in terms of potentials for improving T. vaporariorum integrated management strategy.

MATERIALS AND METHODS
Two local populations of E. formosa were started from pupae collected in tunnel greenhouses of vegetables and ornamentals in locations without a history of using commercial parasitoid strains for biological control of greenhouse whiteflies: population B, collected in Bujanovac (GPS: 42°30'27'' N, 21°48'30'' E) on Solanum nigrum L, and population N, collected in Negotin (GPS: 44°13'00'' N, 22°31'00'' E) on Hibiscus sp. The emerged female wasps of each population were identified as E. formosa using the key given by Polaszek et al. (1992 The commercial insecticide product Elisa 440 SC (manufactured by Galenika Fitofarmacija, Serbia) is formulated as a suspension concentrate (SC). Its content of buprofezin, as the leading product ingredient, is standardised to 440 g/l.
All bioassays were performed in a climate chamber at 27 ± 1°C and 60 ± 10% R.H. with a 16/8 h light/ dark photoperiod and in four replicates. The bioassays were performed in Petri dishes (12 cm diameter), each having four (1 cm diameter) lid openings with muslin covers on top to provide ventilation and prevent internal condensation, and containing 1% agar layer upon which a tobacco leaf was set. The insecticide was diluted in distilled water and applied by spraying onto the entire area of each Petri dish (i.e. upturned lid and lower dish with a tobacco leaf placed on top of the agar layer). The insecticide was applied using a Potter spray tower (2 ml of spray liquid, 100 kPa air pressure, aqueous deposit 2.7 ± 0.2 mg/cm2).
In the acute toxicity bioassay with pupae, tobacco leaves with parasitised whitefly pupae were fixed to tin foil with Traganth-kit. After drying, the leaves were cut to pieces that carried about 20 parasitoid pupae (4 days old, i.e. 12 days after egg laying) and then placed on filter paper in plastic Petri dishes (filter paper was moistened with water to fix leaves in place during exposure). The pieces of tobacco leaves were then treated with a series of insecticide concentrations (8800, 4400, 440, 220, 110, 80, 55 and 27.5 mg a.i./l). Two hours after treatment, tobacco leaves were transferred to new Petri dishes, and they remained there until adults emerged from the pupae. Mortality assessment was based on the counts of emerged adults 9 days after treatment, compared to the number of treated pupae (EPPO, 2004). Concentrationmortality data were subjected to probit analysis using the POLO Plus software (LeOra Software, Berkeley, CA). A pairwise comparison of the LC 50 s was performed using the lethal dose ratio test: when 95% confidence limits (CLs) for LC ratios included 1, the LCs were not significantly different (Robertson et al., 2007).
A parasitism bioassay with F 1 generation wasps was carried out in which 20 pupae (4 days old, i.e. 12 days after egg laying) were treated in Petri dishes with the following buprofezin concentrations (mg/l): B and N with 264 mg/l, and population D with 220 mg/l (concentrations were within 95% confidence limits for the LC 50 calculated in acute toxicity bioassays, Table  1). All surviving female adults that emerged from the 20 treated pupae were transferred to Petri dishes containing third and fourth instar larvae/nymphs of the pest whiteflies that were offered for parasitizing at two day intervals until the death of the last female. The development time, longevity, parasitism/48 h and total parasitism, total emergence of adults and instantaneous rate of increase in F 1 generation of the parasitoid from all three populations were noted (Gholamzadeh et al., 2012). To determine the development time of surviving juveniles, when parasitoid adults were just before emergence from pupae, the number of eclosed adults was noted at 12 h intervals (Enkegaard, 1993). To determine the longevity of survived adults, the number of surviving parasitoid females was checked every other day and the longevity of females was calculated as the total number of days a female was alive assuming that its ultimate death occurred at the midpoint of 48 h. Parasitism was determined based on the number of parasitized host nymphs (black pupae) in each inspection interval (parasitism/48 h period) and the total number of parasitized host nymphs (total, lifetime parasitism). Adult emergence was calculated as the number of wasps that emerged from parasitized nymphs and reached adult stage.
In the parasitism bioassay, data on parasitism and survival of treated pupae were used to calculate the instantaneous rates of increase (r i ) using the equation: where N 0 is the initial number of individuals (i.e. 20 pupae per replicate), N f is the final number of individuals, i.e. black (parasitised) pupae and adults emerged, and Δt is the number of days elapsed between the start and the end of the bioassay. Positive r i values indicate a growing population, negative r i values indicate a population in decline and r i = 0 indicates a stable population (Walthall & Stark, 1997;Stark & Banks, 2003). The N f was determined at the end of the 14 th day of oviposition, the time interval that corresponds to the shortest oviposition period (period during which wasps oviposited over their lifetime). In parasitism bioassays, population N wasps had the shortest oviposition period. Kaplan-Meier analysis (SPSS for Windows, Version 17) was used to calculate the average female longevity, and survival curves were constructed (Enkegaard, 1993), which were analyzed by Log-rank test. Development time, longevity, parasitism/48 h period, total parasitism and adult emergence and instantaneous rate of increase data were analysed by two-way ANOVA (buprofezin treatment and population were the factors) with means separated by Fisher's LSD test (p < 0.05). Means of all parameters for treatment and control, for each population individually, were separated by Student's t-test (p < 0.05), using the software Statsoft Statistica 7.0.

RESULTS
The insecticide Elisa 440 SC caused similar levels of mortality to pupae of all three populations of E. formosa and no significant differences were noted among their LC 50 values (Table 1).
Juvenile developmental time of the parasitoid in nymphs treated with the insesticide was prolonged by 1.71-2.15 days, compared to the control. A two-way analysis of variance showed that the development period depended on treatment (F 1,18 =84.18, p<0.001), while population (F 2,18 =1.37, p=0.280) and interaction of the two (F 2,18 =0.33, p=0.721) had no significant effect ( Table 2).
The surviving parasitoid wasps that emerged from pupae exposed to the insecticide LC 50 (obtained from acute toxicity biossays) lived significantly shorter than control females. The relevant two-way analysis showed that the insecticide treatment of parasitised pupae (F 1,18 =28.   The survival curves for female wasps of the examined populations are shown in Figure 1. Females that emerged from pupae treated with buprofezin had lower survivorship than females which emerged from pupae treated only with distilled water ( -11.23, p=0.993).
Repetitive ANOVA analysis showed that parasitism/48 h periods of the females that emerged from pupae treated with buprofezin in all test populations were significantly affected by the observation period (F 7,126 =67.15, p<0.001). Between observation periods, all main effects and their associated interactions were significant, except the interaction of treatment and population (F 2,18 =0.16, p=0.851). Within each observation period, all main effects and their associated interactions were significant, except the interaction of treatment and observation period (F 7,126 =0.38, p=0.910), the 3-way interaction of treatment, population and observation period, which did not show a significant influence (F 7,126 =0.70, p=0.770) (Table 3).
The relevant two-way analysis of variance showed that 14 days after oviposition began (duration of the shortest oviposition period observed in wasps of population N), insecticide treatment (F 1,18 =254.8, p<0.001) and population (F 2,18 =20.7, p<0,001) as the main factors, and interaction of the two (F 2,18 =10.6, p<0.001), caused significantly different data of the instantaneous rate of increase (r i ) of wasp populations treated at the pupal stage. The insecticide Elisa 440 SC resulted in significant reductions in r i values of wasps from all test populations: females B by 8.17 % (F 1,6 =75.93, p<0.001), females N by 6.82 % (F 1,6 =30.18, p<0.01), and females D by 12.46 % (F 1,6 =241.01, p<0.001), compared to control wasps (Table 2).

DISCUSSION
After the recommended dose of buprofezin-based product (Elisa 440 SC) was applied, around 60 % of wasp adults failed to eclode from directly treated pupae of the parasitoid E. formosa. The LC 50 s ranged from 199.55 mg a.i./l (D) to 281.46 mg a.i./l (N), which is somewhat lower and higher, respectively, than the recommended buprofezin concentration for field treatment (264 mg a.i./l).
Buprofezin selectivity to beneficial insects was indicated more than three decades ago (Mullin & Croft, 1985;Hassan et al. 1994). In a similar study of buprofezin effects (formulation NNI-750; concentrations 125 and 250 ppm) on immatures of E. formosa and C. noacki, based on IOBC criteria (impact on survival, reproduction and parasitoid capacity) Garrido et al. (1985) graded buprofezin as harmless, noting no sterilization effects on either test parasitoid. Jones et al. (1998) reported different levels of mortality caused by a buprofezin test product, depending on parasitoid development stage (i.e. young larva or pupa), as it caused mortality among the early larval stages of E. luteola Howard, Eretmocerus eremicus and E. tejanus Rose and Zolnerowich. Similar studies have noted that parasitoids treated with buprofezin at later stages of larval development were less affected than those treated at earlier stages (Gerling & Sinai, 1994;Hoddle et al., 2001). In our own research, the buprofezin product that was applied directly onto parasitoid pupae at concentrations of 264 mg/l (B and N) and 220 mg/l (D), prolonged juvenile development significantly (1.96, 1.71 and 2.15 days in B, N and D wasps, respectively), and reduced significantly the longevity (1.52, 0.74 and 1.71 days in B, N and D wasps, respectively), total parasitism (11.66, 17.72 and 17.59 % in B, N and D wasps, respectively), total adult emergence (11.63, 17.79 and 17.56 % in B, N and D wasps, respectively) and instantaneous rate of increase of all test populations (8.17, 6.82 and 12.46 % in B, N and D wasps, respectively). In the present study, treated B females achieved better results than D females regarding nearly all observed parameters.
In another study, Gholamzadeh et al. (2012) treated E. formosa pupae directly with buprofezin (Applaud 40 SC, 1000 µg a.m./ml) and classified the insecticide into group I as harmless, based on its total effect (10.9 % reduction in beneficial capacity), and also based on its toxicity (23.14 % reduction in beneficial capacity). Besides, buprofezin showed no significant impact on parasitoid fecundity and longevity. Gerling and Sinai (1994) reported consistent data as they detected no harmful effects of buprofezin on one-day fecundity of E. luteola and Eretmocerus sp. females when the insecticide was applied to parasitised tobacco whitefly nymphs. Heydari (2004) also noted no detrimental effects of buprofezin on the longevity or fecundity of E. formosa.
Other studies, however, have reported results similar to ours regarding buprofezin selectivity below expectation. In a demographic study by Heydari et al. (2006), direct treatment of E. formosa pupae with the recommended concentration of buprofezin did not affect significantly the parasitoid population parameters, apart from net reproductive rate (R 0 ), which was significantly lower than control data. In another study by Southwood & Handerson (2000), life table data indicated a minor negative influence on the intrinsic rate of increase, which was reduced by 7.03 %. In contrast, Prabhaker et al. (2007) found buprofezin treatment (Applaud 40 SC, 400 g a.i./l; application by soaking leaves with parasitized larvae into buprofezin solution) to have significantly affected population stability parameters (R 0; intrinsic rate of increase -r m ; final rate of increase -l, and mean generation time -T) of the parasitoid wasp E. inaron. Sohrabi et al. (2013) reported data consistent with our present results, showing an adult eclosion rate of E. mundus pupae that was significantly reduced by the recommended buprofezin concentration and not dependent on the parasitoid development stage (larva or pupa).
It is possible to infer from the acquired data that the test product based on buprofezin (Elisa 440 SC) demonstrated a certain level of toxicity to E. formosa pupae, and that besides its lethality, buprofezin may also have sublethal effects on the parasitoid's physiology. Application of this growth regulator in early stages of whitefly development and parasitoid wasp release at a later stage of pest development (or in its next generation) may allow for biological and chemical control measures to coexist in practice. More precise determination of risks involved in the use of buprofezin (especially the toxicity of its gas phase) requires its more detailed field testing.