Efficacy of biorational insecticides against Bemisia tabaci (Genn.) and their selectivity for its parasitoid Encarsia formosa Gahan on Bt cotton

The toxicity of seven biorational insecticides [five insect growth regulators (Buprofezin, Fenoxycarb, Pyriproxyfen, Methoxyfenozide, and Tebufenozide) and two oil-extracts of neem and bitter gourd seeds] against Bemisia tabaci and their selectivity for its parasitoid, Encarsia formosa were evaluated in laboratory and field conditions for 2 years (2018–2019) in Pakistan. Toxicity results demonstrate that Pyriproxyfen, Buprofezin, and Fenoxycarb proved to be effective (80–91% mortality and 66.3–84.2% population-reduction) against B. tabaci followed by Methoxyfenozide, Tebufenozide (50–75% mortality and 47.8–52.4% population-reduction), and then oil-extracts of neem and bitter gourd (25–50% mortality and 36.5–39.8% population-reduction) in the laboratory [72 h post-application exposure interval (PAEI)] and field trails (168 h PAEI), respectively. All tested biorationals, except Methoxyfenozide [(slightly-harmful/Class-II), i.e., causing mortality of parasitoids between a range of 25–50%] and Tebufenozide [(moderately-harmful/Class-III), i.e., causing mortality of parasitoids between the ranges of 51–75%], proved harmless/Class-I biorationals at PAEI of 7-days in the field (parasitism-reduction < 25%) and 3-days in the lab (effect < 30%). In laboratory bioassays, exposure of parasitized-pseudopupae and adult-parasitoids to neem and bitter gourd oils demonstrated that these compounds proved harmless/Class-I biorationals (< 30% mortality). Alternatively, Pyriproxyfen, Buprofezin, Fenoxycarb, Methoxyfenozide, and Tebufenozide were slightly-harmful biorationals (30–79% mortality) against the respective stages of E. formosa. We conclude that most of the tested biorationals proved harmless or slightly harmful to E. formosa, except tebufenozide after PAEI of 7-days (168 h) in the field and, therefore, may be used strategically in Integrated Pest Management (IPM) of B. tabaci.


Scientific Reports
| (2021) 11:2101 | https://doi.org/10.1038/s41598-021-81585-x www.nature.com/scientificreports/ and thiamethoxam), insecticide-mixtures [NOVASTAR (bifenthrin + abamectin) and DELTAPHOS (deltamethrin + triazophos)], sulfoximines (sulfoxaflor), insect growth regulators [(IGRs) (buprofezin, pyriproxyfen)] and Tetronic/Tetramic acid derivatives (Spirotetramat, spiromesifen) 4,[9][10][11][12][13][14][15][16][17][18][19][20] . However, indiscriminate use of insecticides has resulted in the rapid development of resistance to sucking insects, including whitefly populations and adverse effects on their natural enemies 3,8,14,[17][18][19][20] . In plants, conventional and systemic IGRs insecticides are biotransformed by nonmicrosomal (IGRs) or microsomal biotransformation reactions (hydrolysis) and associated enzymes, which greatly vary among types, species, and varieties of plants (crops) 21,22 ; hence, their impacts on non-target arthropods greatly vary on a variety of plant species 4,21 . Bt crops have great potential to augment the role of biocontrol in integrated pest management systems. Conventional and Bt cotton cultivars have different production technology [different sowing-time, fertilizer (nutrient) and water requirements, etc.] and influence various biotic stresses and exogenic molecules differently due to differences in their genetic background and physiology/metabolism 22 . Variation in the agronomic aspects of their production technologies (nutrients, irrigation, etc.) affects the quantity and quality of specialized-metabolite biosynthesis differently in Bt and non-Bt crops 23,24 . Some reports also advocate that Bt crops' physiology and metabolism affect the toxicity of systemic insecticides against pest and its selectivity against natural enemies through metabolic interaction of Bt-toxin (proteins) or its metabolites (protoxins) with insecticide molecules within the plant tissues 25 . A few in-vivo studies have indicated that a combination of synthetic and microbial insecticides (including entomopathogenic fungi and bacteria) can have additive, antagonistic or synergistic toxicity effects against target insects [26][27][28] . However, research on the aforementioned lines has not been reported on Bt cotton. The use of synthetic biorationals, including IGRs and botanical extracts for controlling whiteflies has increased in Pakistan, but their efficacy against B. tabaci and selectivity for E. formosa in Bt cotton has yet to be explored. IGRs are highly selective insecticides having biorational impacts on pests 8,[29][30][31][32] . As biorational insecticides, they disrupt hormonal regulation and ultimately result in abnormal molting, growth, and development in several insects. They are used for the control of many insect species acting as ecdysone agonists (tebufenozide and halofenozide), juvenile hormone mimic (pyriproxyfen) and chitin synthesis inhibitors (benzoylphenylureas and buprofezin). Buprofezin (2-ter-butylimino-3-isopropyl-5-phenylperhydro-1, 3, 5-thiadiazin-4-one) possesses strong contact and stomach toxicity and has been reported as less toxic and highly selective for natural enemies (predators and parasitoids), fish, birds, and silkworms. Buprofezin inhibits chitin synthesis, interrupts metabolism, and causes mortality without molting or due to deformed molting 4,33 . It has been evaluated against whiteflies on cotton 4,34 , planthoppers on rice 35 , jassids on brinjal 36 , and mealybugs on grapes 37,38 and cotton 39 . Fenoxycarb acts as a juvenile hormone mimic and has been documented as an effective molecule for controlling various insect pests infesting economic crops, ornamentals, and fruits. It interrupts the metamorphosis, growth, and development of all insect life stages and inhibits oogenesis and the development of ovaries 40 . Pyriproxyfen also possesses juvenile hormone mimic properties but has no indirect lethal effects on female fertility, egg viability, sex ratios of different predators, and parasitism capacity of various parasitoids 41,42 . Tebufenozide and methoxyfenozide as IGRs act both as stomach and contact poisons with a 3-week residual activity period. Both result in mortality by a failure of feeding in target insects or by disrupting the normal process of molting, specifically in lepidopteran pests 43,44 . These IGRs have been documented safe for honeybees and most of the other natural enemies 45 .
IGRs have been reported as highly compatible insecticides with certain species of parasitoids of B. tabaci; hence, biocontrol efficacy of E. formosa can be complemented with strategic application of IGRs in greenhouse and field IPM program of crops 4,46 . However, before devising IGRs and parasitoid-based IPM strategy for B. tabaci, it is essential to investigate the non-target effects of IGRs on various biological parameters of E. formosa 46 . Some studies have shown that different development stages of Hymenopterous parasitoids respond differently when exposed to IGR treatments 41,42,46,47 . For example, the results of a leaf and glass-vial residue bioassays confirmed that pyriproxyfen, fenoxycarb, and buprofezin caused no considerable effects on the survival of adults of E. formosa. The residues of pyriproxyfen and fenoxycarb resulted in a slight reduction in parasitism compared with buprofezin. However, pyriproxyfen, fenoxycarb, and buprofez caused a significant decrease in the rates of parasitoids' adult emergence 46 . Encarsia formosa females emerging from the buprofezin-treated pupae exhibited shorter lifespan (0.7-1.7 days), lower parasitism rate (11.7%), and less adult emergence (11.6-17.8%) than control treatment 47 48 . Based on the mortality scale specified by the International Organization for Biological Control (IOBC), pyriproxyfen proved harmful, whereas buprofezin and fenoxycarb proved slightly-moderately harmful to larvae and harmless to pupae of E. formosa when assessed through leaf and glass-vial residues bioassays 46 .
Similarly, botanicals have been reported as environmentally safe compounds with lower toxic effects on mammals, natural enemies and pollinators. These compounds are considered important components of IPM programs of any crop. Extracts of neem (Azadirachta indica A.) and bitter gourd (Momordica chrantia L.) were effective for the management of sucking insect pests of cotton 49 . The most prominent constituent of the seedkernels, leaves, and bark of neem is azadirachtin, which has a strong insecticidal property. It not only induces some behavioral effects like antifeedency, repellency, etc. in insects by interfering with chemoreceptory mechanisms, but also disrupts the growth mechanism of insects through interfering with the neuroendocrine control of molting and edysis 50 . Momordica charantia, is a tropical to sub-tropical vine in the family Cucurbitaceae. The plant produces triterpenoid compounds in the leaves, fruits and seeds, which have been reported to have antifeedant and oviposition-inhibition properties on arthropods [51][52][53][54] . However, the impact of these biorational insecticides is not well understood on the whitefly and its parasitoid in Bt cotton.
A comprehensive understanding of the influence of environmental factors (temperature, relative humidity, photoperiod, etc.) on the interaction between pest and its bioagent is of utmost importance for successful and  56 . Biological behavior and response of biocontrol agents toward their host/prey under insecticides' stress is also influenced by several environmental factors (humidity, temperature, rainfall, wind-speed, etc.) which are responsible for the postapplication degradation and dissipation of insecticides 4,57 . The results reviewed in some research reports show that a steady increase in temperature (beyond 30 °C) and decrease in relative humidity (below 70%) cause gradual degradation of IGRs' residues on plants or in other media, reduction in their half-life and resultantly reduction in their toxicity against parasitoids/predators and its host/prey 4,57,58 . Based on the aforementioned facts, it is concluded that B. tabaci is a major cotton pest parasitized by E. formosa and Eretmocerus species and foliar application of insecticides is the most important pest management technique for B. tabaci and/or sucking pest complex in Pakistan. The conventional insecticides used for the management of B. tabaci and/or sucking pest complex are not target specific and suppress the population of their respective predators and parasitoids in the agro-ecosystem. Synthetic-biorationals, including IGRs and botanicalextracts are currently being researched for the management of B. tabaci and/or sucking pest complex. However, most of the published work highlights the research studies conducted on various insecticides in other countries on fruit-crops and crops other than Bt-cotton, where there are differences in environmental conditions and technology. It has been reported that a biorational tool is effective on some crops, but not on others because of the variation in several factors [like plant-species (crop types), plant development-stages, physiochemical properties of chemicals, formulation of chemicals, application methods and climatic conditions (humidity, temperature, rainfall, wind-speed, etc.)] which are responsible for the degradation and dissipation of biorational insecticides after their spray on plants 4,57 . For example, foliar application of ASSAIL (acetamiprid) 20% SL) @ 300 mL/ha demonstrated a significant variation in B. tabaci population reduction on five cotton varieties including super-98 (65% reduction), BH-163 (98% reduction), VH-159 (73% reduction), NIAB-884 (83% reduction) and NIAB-101 (68% reduction) at 72 h post-application interval 9 .
Whiteflies currently threaten cotton production in Pakistan; therefore, it is important to identify biorational tools that are readily available for the management of whitefly populations. Again, management programs that are developed for other countries in other crops may not relate to the subtropical and relatively arid environmental conditions in Pakistan where cotton is such an important crop to the country and the citizens of Pakistan. There is a need for an extensive investigation on the efficacy of such biorationals against whitefly and their selectivity against natural enemies. The current study evaluated seven biorational insecticides including two oil-extracts (Neem-extract, bitter gourd-extract) and five IGRs (buprofezin, fenoxycarb, pyriproxyfen, methoxyfenozide, and tebufenozide) for their efficacy against B. tabaci and selectivity for its parasitoid E. formosa on Bt cotton in laboratory and field conditions. The findings would have applications in developing countries where the goal is to use fewer toxic pesticides for protecting humans, natural enemies, pollinators, and the environment.

Materials and methods
Collection and preparation of botanical extracts. For the preparation of botanical extracts of neem (A. indica L.) and bitter gourd (M. charantia L.), one kilogram each of fresh neem and bitter gourd seeds was purchased from a local market and rinsed thoroughly in distilled water. The seeds were then dehulled, put in paper bags, and subsequently dried inside an oven at 50 °C until a constant weight was obtained. The dried seeds of both neem and bitter gourd were cooled down to room temperature and ground into a powder in a grinding mill separately. After grinding, the powder of each was stored in separate air-tight containers and labeled. For extraction of oil, 50 g of neem seed powder was processed through soxhelt extraction using n-Hexane as the solvent. The miscella of n-hexane and neem oil obtained by soxhelt extraction was purified at 68 °C (boiling point of n-hexane) by evaporation of n-hexane inside the rotary evaporator. Neem oil dissolves easily in water and does not need a surfactant. The same procedure was followed for the extraction of oil from bitter gourd seeds. For laboratory and field experiments, 1000 ppm of each extract was prepared by dissolving 1.0 mL of extract in 1L of water.
Insect growth regulators. Five IGRs, buprofezin (chitin synthesis inhibitor), fenoxycarb, pyriproxyfen (juvenile hormone mimics), methoxyfenozide, and tebufenozide (molting hormone agonists) were purchased from a local market and evaluated at their field recommended doses by using a 3.5-gallon knapsack sprayer with a Teejet hollow cone spray cores D3 disk (Spraying systems Co., Keystone Heights, FL USA) spraying 1380-L of water/ha for each application in the field and by an atomizer in laboratory studies. The field recommended dose rate for buprofezin (Agrochemicals, Karachi, Pakistan), fenoxycarb (Syngenta, Karachi, Pakistan), pyriproxyfen methoxyfenozide (Agrochemicals, Karachi, Pakistan) and tebufenozide (Valent Biosciences, Lahore, Pakistan) were 500, 200, 120, 200 and 160 mL/0.4 ha, respectively. Laboratory evaluation. The leaves infested with pseudopupae of whitefly were clipped from untreated cotton plants potted in earthen-pots kept in the field for natural whitefly infestation and its parasitism by E. formosa. The leaves were placed on moistened filter paper in Petri dishes and maintained at 25 ± 2 °C and 75 ± 5% relative humidity in an incubator until the adult parasitoids emerged. The emerged parasitoids were aspirated and kept in glass jars provided with sponge rubber foam strips soaked with adult diet (honey, distilled water, and yeast-powder in 1:9:1 ratio) for two days. After two days, ten parasitoids (5 males and 5 females) were aspirated www.nature.com/scientificreports/ and released into parasitizing-chambers. Each parasitizing-chamber consisted of potted cotton (Bt-cotton var. Lalazar) infested with unparasitized pseudopupae, larval instars, and twenty whitefly adults. The experimental layout was a Completely Randomized Design (CRD) with three replications. The total number of pseudopupae and other larval instars present on the leaves inside the chamber were also counted. The cotton plant inside the chamber was sprayed with biorational insecticides using the atomizer. Neem was applied at the manufactured labeled rate (60 mL to 3. 8 L) without a surfactant. Parasitoids were released into the chamber 24 h after treatment. The numbers of dead whitefly adults, larval instars, and male/female adult parasitoids were counted after the three-day treatment and data obtained were transformed into percent corrected mortality over control using Henderson-Tilton's formula.
Observations were made 2 weeks after treatment to count dark-colored parasitized whitefly pseudopupae to determine the rate of parasitism, and percent parasitism was calculated. The black-colored parasitized pseudopupae were also kept under observation for 1 week and the number of E. formosa adults that emerged from parasitized pseudopupae were counted and the data obtained were transformed into percent emergence of adult E. formosa from parasitized pseudopupae. The rate of parasitism was calculated as the percent emergence of adult E. formosa from the parasitized pseudopupae. The parasitoid mortality percentage inside whitefly pseudopupae was computed by subtracting the adult emergence percentage from 100 and then transformed into percent corrected parasitoid mortality over control using Henderson-Tilton's formula.
The laboratory experiment was duplicated in 2018 and 2019. Since there was no significant difference in parasitization rate or success rate of parasitism between the two years, data from the two experiments were pooled in statistical analysis.
The mortality scale specified by the IOBC to describe the effects of pesticides on beneficial species for lab studies (< 30% = harmless/Class-I; 30-79% = slightly harmful/Class-II; 80-99% = moderately harmful/Class-III; > 99% = Harmful/Class-IV) 59 was used to ascertain the degree of toxicity or safety of tested insecticides for E. formosa.
Field evaluation. Bt-cotton var. Lalalzar was sown in Randomized Complete Block Design (RCBD) with three replications per treatment plot measuring 34 × 54 m 2 . Sowing was done with a plant to plant distance of 45 cm and row to row distance of 90 cm. The treatments were applied at their field recommended doses when the whiteflies reached their economic threshold level (ETL). The rest of the agronomic practices were carried out uniformly in all treatments. Whitefly population was recorded 24 h before and 24, 48, 72 and 168 h postapplication exposure interval (PAEI). The data were collected according to Frank and Liburd's method of pest scouting 60 . In total, 10 plants were selected randomly from every replication and the population (both adults and nymphs) of whiteflies per leaf was recorded visually with the help of hand-lens from the upper, middle, and lower leaf of the first, second and third plants respectively. The collected whitefly population data were transformed into percent population reduction in whitefly samples collected at 24, 48, 72-and-168 h PAEI as compared to the whitefly population recorded 24 h before treatments' application. The survival rate percentage was calculated.
In the parasitization experiment, five plants were tagged in each replication of each treatment and the numbers of parasitized and unparasitized whitefly pseudopupae were counted 24 h PAEI. The parasitized pupae were marked with a permanent marker and percent parasitism 24 h before the application was calculated. Then the number of newly parasitized pseudopupae of whitefly were counted and marked with permanent marker at 24, 48, 72, and 168 h PAEI. The percent reduction in parasitism due to biorational insecticides was computed for each PAEI using Sun-Shepard's formula 61 .
The mortality/parasitism-reduction scale specified by the IOBC to describe effects of pesticides on the survival/performance of beneficial species for semi-field and field studies (< 25% = harmless/Class-I; 25-50% = slightly harmful/Class-II; 51-75% = moderately harmful/Class-III; > 75% = harmful/Class-IV) 59 was used to describe the tested biorational insecticides as harmful or safe.
Five leaves with parasitized whitefly pseudopupae were removed from each treated and control plants. The parasitized pseudopupae present on each leaf were counted. The clipped leaves were placed on water-soaked and tissue-paper wrapped sponge adjusted in Petri dishes which were kept covered with perforated lid tightened with a rubber band. The Petri dishes were then placed inside the growth chamber maintained at 30 ± 2 °C and 65 ± 5% RH until the adult parasitoid emerged. This method was repeated for each PAEI. The emerging parasitoid adults from parasitized whitefly pseudopupae for each biorational insecticide and the control for each PAEI were counted and the data obtained were transformed into percent mortality and adult emergence of the parasitoid.
The field experiment was duplicated in 2018 and 2019. Since there was no significant difference in dependent variables between the 2 years, data from the two duplicated experiments were pooled in statistical analysis. Ethics approval and consent to participate. The authors agree to all the concerned regulations.
Consent for publication. The authors agree to publish this scientific paper at the Scientific Reports.  (Tables 3, 4).
The results explained an exposure-interval dependent reduction in the whitefly population. The percent population reduction increased with an increase in PAEI of each biorational insecticide up to 72 h. Alternatively, a decreasing trend in percent whitefly population reduction was observed for PAEI of 168 h. Specifically, at all PAEI, pyriproxyfen demonstrated the highest percent whitefly population reduction and proved to be a more effective biorational insecticide than the other tested compounds, followed by buprofezin and fenoxycarb. Methoxyfenozide and tebufenozide were less effective than pyriproxyfen and buprofezin in reducing the whitefly population. Neem-extract and bitter gourd-extract were the least effective products, demonstrating a 26.6-41.6% and 21.5-36.5% reduction in whitefly populations, respectively; however, Neem-extract was more effective than bitter gourd-extract (Table 1).   11,11,20, and 19 times, fenoxycarb 10, 14, 18, and 15 times, methoxyfenozide 9, 13, 14 and 11 times, tebufenozide 7, 12, 13 and 10 times, Neem-extract 6, 11, 11 and 7 times and bitter gourd-extract 5, 10, 9 and 5 times more reduction in whitefly populations than the control at PAEI of 24, 48, 72 and 168 h, respectively (Table 1). A substantial increase in whitefly parasitism by E. formosa over time was recorded in all biorational insecticides treated plots. Table 3. Percent emergence and mortality of adult E. formosa when parasitized pseudopupae were exposed to different insecticides at different post-application exposure intervals on field cultivated Bt cotton. Means with the same lowercase letters are not significantly different at α = 0.05 (Tukey's test). Values in parenthesis indicate the corrected percent mortality of E. formosa inside the parasitized whitefly pseudopupae over control at different PAEI. HL, SH, MH and HF abbreviations inside the parenthesis indicate Harmless/Class-I, Slightlyharmful/Class-II, Moderately-harmful/Class-III and Harmful/Class-IV insecticides, respectively. df Degree of freedom. P, F and Df values = the respective times listed above. a Degree of freedom of treatment. b Error degree of freedom. **Highly significant at probability level of 5%.  www.nature.com/scientificreports/ Pyriproxyfen, buprofezin, fenoxycarb, methoxyfenozide, tebufenozide, Neem-extract, and bitter gourd-extract treated plots exhibited significantly lower whitefly parasitism at shorter PAEI (24 h) compared with higher whitefly parasitism at longer PAEI (168 h). The percent reduction in whitefly parasitism by E. formosa in all biorational insecticides treated plots was higher at 24 h PAEI; then, a considerable decline in parasitism reduction was recorded with increasing PAEI. Neem-extract, bitter gourd-extract, and pyriproxyfen proved moderately harmful to E. formosa, reducing parasitism to 51-75%, while buprofezin, fenoxycarb, methoxyfenozide, and tebufenozide were harmful and reduced parasitism by more than 75% at the PAEI of 24 h (Table 2).
At PAEI of 48 h, bitter gourd-extract was recorded as slightly harmful; Neem-extract, pyriproxyfen, and buprofezin were moderately harmful, whereas fenoxycarb, methoxyfenozide, and tebufenozide were attributed as harmful biorational insecticides. A comparison of IOBC toxicity scale with the calculated percent reduction in parasitism at PAEI of 72 h revealed that Neem-extract, bitter gourd-extract, pyriproxyfen, buprofezin, and fenoxycarb demonstrated less than 25% reduction in parasitism and were recognized as harmless/safe for E. formosa; while methoxyfenozide and tebufenozide attributing parasitism reduction between the range of 50-75% and > 75% were assorted as moderately-harmful and harmful insecticides, respectively. An increase in whitefly parasitism by E. formosa was observed in all plots treated with biorational insecticides (− 6.6 to − 15.4%) except in those plots which were treated with methoxyfenozide (43.3% parasitism reduction) and tebufenozide (63.3% parasitism reduction) at PAEI of 168 h. At this PAEI, pyriproxyfen, buprofezin, fenoxycarb, Neem-extract, and bitter gourd-extract were classified as harmless insecticides, while methoxyfenozide and tebufenozide were assorted as slightly harmful and moderately harmful insecticides, respectively ( Table 2).
Percent emergence of adult E. formosa from field-collected parasitized whitefly pseudopupae varied significantly for different treatments at various PAEI (p < 0.05) ( Table 3). An increasing trend in percent emergence and a decreasing trend in mortality of adult E. formosa was observed from those parasitized whitefly pseudopupae, which were collected at 24, 48, 72, and 168 h PAEI from cotton plots treated with all biorational insecticides. Neem-extract, pyriproxyfen, and buprofezin were found to be moderately harmful at 24  Impact of biorationals under laboratory conditions. The treatments had a highly significant effect on variation in mortality of adults and nymphs of whitefly, mortality of parasitoid (E. formosa) males and females, and percent adult emergence of E. formosa from parasitized pseudopupae (p < 0.05) ( Table 4). Pyriproxyfen (90.4% adults and 88.8% nymphs), buprofezin (90.5% adults and 84.4% nymphs) and fenoxycarb (85.7% adults and 80.2% nymphs) had mortality higher than 80% in whitefly adults and nymphs. In comparison, methoxyfenozide and tebufenozide had a 72.6 and 65.1% mortality in adults and 66.5 and 59.9% mortality in nymphs. Plant extracts had less than 50% mortality rates in whitefly adults and nymphs ( Table 4).
The male and female parasitoids (E. formosa) released 24 h after the treatment application exhibited less than 50% mortality when exposed to pyriproxyfen (35.6% in females and 41.1% in males), buprofezin (40.4% in females and 46.6% in males), neem (30.6% in females and 36.3% in males) and bitter gourd (20.4% in females and 26.5% in males). Fenoxycarb, methoxyfenozide and tebufenozide demonstrated mortality in male and female E. formosa in the range of 50.8-76.3% (Table 4).
According to the mortality scale specified by IOBC to describe the effects of pesticides on beneficial species for laboratory, bitter gourd-extract proved harmless. In contrast, pyriproxyfen, buprofezin, fenoxycarb, methoxyfenozide, tebufenozide, and neem-extract proved slightly harmful for males and females individuals of E. formosa. Under laboratory conditions, more than 50% parasitism of whitefly pseudopupae by E. formosa (54.7-67.5%) was observed when pyriproxyfen, buprofezin, bitter gourd-extract, and neem-extract were applied 24 h before the release of the parasitoid. Statistically, similar parasitism was also recorded in the control treatment (69.4%). Fenoxycarb and methoxyfenozide demonstrated 44.4 and 37.5% parasitism, respectively, while tebufenozide resulted in the lowest amount of parasitism (25.6%) when applied 24 h before the release of the parasitoid. The data regarding the emergence of adult E. formosa from parasitized pseudopupae treated with insecticides indicate that bitter gourd and neem extract proved harmless (mortality < 30%) while pyriproxyfen, buprofezin, fenoxycarb, methoxyfenozide, and tebufenozide were slightly harmful (morality 30-79%) insecticides at parasitized pseudopupal stages (Table 4).

Discussion
Only a few studies have focused on the importance of buprofezin, fenoxycarb, pyriproxyfen, methoxyfenozide, and tebufenozide (IGRs), Neem-extract and bitter gourd-extract in integrated management of B. tabaci in Bt-cotton. This is the first detailed study comparing the efficacy of these biorational insecticides against B. tabaci and evaluating their selectivity for its parasitoid, E. formosa on Bt-cotton. Whitefly, B. tabaci and its two potential parasitoid E. formosa and Eretmocerus species 1-4 are targeted directly and/or indirectly through foliar application of conventional insecticides. These pesticides are intended to suppress B. tabaci but inadvertently www.nature.com/scientificreports/ reduce these key natural enemies' populations in the agro-ecosystem 63 . The laboratory and field studies in the present study demonstrate that adults and nymphs of B. tabaci show increased mortality in the laboratory and higher population reduction in field when exposed to pyriproxyfen followed by buprofezin, fenoxycarb, methoxyfenozide, tebufenozide neem-extract, and bitter gourd-extract. Similar findings were reported where buprofezin effectively controlled and significantly reduced the whitefly population in the field 4,63 . The present research results regarding the performance of pyriproxyfen and buprofezin against whiteflies were also consistent with the results that demonstrate the lower B. tabaci population in plots treated with buprofezin, pyriproxyfin, buprofezin + fenpropethrin, and pyriproxyfin + fenpropethrin (non-Bt cotton studies) 38,64 . However, the toxicity of tebufenozide, methoxyfenozide, fenoxycarb, and bitter gourd-oil cannot be compared or contradicted, as very little information is available in the literature reviewed. Under field conditions, the reduction in the whitefly population increased with an increase in PAEI up to 72 h PAEI; however, a non-significant decreasing trend in population reduction in all treatments was observed in the field study at PAEI of 168 h. These results indicate that the efficacy of the tested biorationals started to decrease while the whitefly population flare-up, which may be attributed either to a reduction in the natural enemies' population due to biorational stress or the emergence of a new population from pseudopupae in the treated plots. The results of commercial-scale field studies confirm that IGRs have very slow lethal impacts during the initial few hours of exposure. These lethal effects gradually increase and become significant within 1-3 weeks of post-application intervals because IGRs cause mortality in immature stages of B. tabaci only during ecdysis not before it 4,65,66 . The lethal effects of IGRs only on ecdysis of B. tabaci immatures are the possible reason behind the higher reduction in pest population during the longer post-exposure interval. This study also confirms that the tested IGRs proved effective against B. tabaci, causing a significantly higher reduction in its population (36.92-73.21%) up to 168 days (1 week). The observation on population reduction of B. tabaci in the field continued only for 168 h; hence, significant efficacy of IGRs reported for many weeks (2-5 weeks) by researchers 65,66 cannot be discussed. The reduction in the whitefly population in the field up to 72 h PAEI may be attributed to a decrease in fecundity and egg-hatching as well as the mortality of nymphal stages during ecdysis due to IGRs 4,65,66 . The reduction in the efficacy of tested IGRs against B. tabaci at 168 days (1 week) in the present field study is attributed to prolonged exposure (168 days) of residues of applied IGRs to adverse field-experimental conditions (> 40 °C, < 55% RH), which result in steady photo-degradation (photolysis) of IGRs residues on the crop. Several factors like plant-species, plant development-stages, physiochemical properties of chemicals, formulation of chemicals, application methods, and climatic conditions (humidity, temperature, rainfall, windspeed etc.) are responsible for the degradation and dissipation of agrochemicals (insecticides, fertilizers etc.) after their spray on plants 4,57 . It has been documented that a steady increase in temperature (beyond 30 °C) and a decrease in relative humidity (below 70%) result in the gradual degradation of residues of IGRs, reduction in their half-life, and resultantly reduction in their efficacy 4,57,58 . A variation in the toxicity/performance of tested biorationals (five IGRs and two plant-extracts) in present laboratory and field studies may be attributed to differences in experimental conditions experienced by the target insect (B. tabaci) and biorational molecules in the laboratory (30 ± 2 °C, 65 ± 5% RH) and field (> 40 °C and relative humidity fluctuating between 50 and 70% in cotton crop). Higher mortality in laboratory study may be due to the fact that the B. tabaci population under laboratory conditions was exposed to treated plants where they were forced to perform their life activities under no-choice bioassays, unlike the field study, where the population was exposed to abiotic conditions (temperature, humidity, and rainfall), exhibiting lower population reduction.
The results regarding the effects of tested biorationals to B. tabaci parasitoid (E. formosa) indicate that percent parasitism increased, percent mortality of E. formosa inside host-pseudopupae decreased, the percent reduction in parasitism declined, and the toxicity class of tested insecticides changed from harmful to harmless with an increase in PAEI from 24 to 168 h. An increase in parasitism and a decline in the reduction of parasitism of B. tabaci pseudopupae by adult E. formosa exhibited in the present study may be due to insecticides losing their contact toxicity with an increasing exposure interval of 168 h. This is confirmed by the declining trend in population-reduction and population flare-up of whiteflies at this exposure interval (168 h) in the present study. This steady reduction in contact toxicity of tested chemicals and flaring up of host (whitefly) population (as observed in the present study) enhance the survival rate of parasitoids, encourage the availability of sufficient host and ultimately result in a higher parasitism rate. The tested IGRs are recommended against insect pests on crops where these IGRs absorb into and form a reservoir of their toxic residues within the leaf-tissues (local-systemic/translaminar action). This type of absorption of IGRs residues increases with post-application interval and hence minimizes their residual toxicity on the treated leaf-surface 4,57 . Such types of post-application behavior of IGRs molecules ensure a gradual increase in toxicity against sucking pests like whiteflies (but not longer than 1-2 weeks) and a steady decrease in contact toxicity against predators, parasitoids 57 and other nontarget arthropod fauna 4 . The selectivity of the tested biorational insecticides to the parasitoid, E. formosa, may be related to the difference in their dose rate, formulations and mode of action, the parasitoid's exposed stage, and post-application exposure interval 4,64,67 .
At the maximum PAEI in this study (168 h), methoxyfenozide was slightly harmful, and Class-II insecticide (parasitism reduction between 25 and 50%) and tebufenozide was moderately harmful and Class-III insecticide (parasitism reduction between 50 and 75%). Alternatively, three IGRs (buprofezin, fenoxycarb and pyriproxyfen,) and two oil-extracts of neem and bitter gourd seeds, were harmless and Class-I insecticides as these biorationals demonstrated less than 25% reduction in parasitism in the field study. This reduction in parasitism would not be considered significant based on IOBC standards specified for field studies 59 . There is little information available in the literature regarding the categorization of insecticides based on parasitism reduction percentage of E. formosa. However, in another study, it was reported that methoxyfenozide ( www.nature.com/scientificreports/ of methoxyfenozide at its field recommended dose (39.8 g a.i./100 L H 2 O) proved non-toxic to Trichogramma nr. brassicae (egg parasitoid of Helicoverpa species) in both laboratory (filter paper bioassay conducted at 25 °C in plastic Petri dish under Potter Spray Tower against female parasitoid chilled at 4 °C for 3 min) and field trials (foliar spray with by Silvan battery powered backpack sprayer with T-jet cone nozzle on tomato beds), as it demonstrated no harmful effects on adult emergence from sprayed parasitized eggs of T. nr. brassicae 69 . These results are not in agreement with our findings about methoxyfenozide, which was categorized as a slightly-harmful biorational insecticide against E. formosa because methoxyfenozide caused mortality of parasitoids between a range of 25-50% in field-study and 30-79% in lab studies. These mortality ranges fall in mortality scale specified by the IOBC for slightly-harmful/Class-II insecticides against beneficial species for field and lab studies 59 . These variations in results may exist due to difference in the concentration/dose-rates of methoxyfenozide (200 mL/0.4 ha), materials and methods used in the field (foliar spraying by 3.5-gallon knapsack sprayer with a Teejet hollow cone spray cores D3 disk using 1380-L of water/ha) and lab (foliar application by an atomizer on potted cotton maintained at 25 ± 2 °C and 75 ± 5% RH) studies, choice of test parasitoid (E. formosa), and the life stage (adult of E. formosa and unparasitized pseudopupae and larval instars of B. tabaci) used in our experiments. Some studies demonstrate that different development stages of Hymenopterous parasitoids respond differently when exposed to IGRs' treatments 47 . There is a variable response to different stages of parasitoids to insecticides and IGRs. The effects of these compounds on predators/parasitoids are contradictory and inconsistent 46,47 . The results of the present study show that at the maximum PAEI (168 h), pyriproxyfen, buprofezin, fenoxycarb, and methoxyfenozide were in Class-II biorationals (slightly harmful, 25-50% mortality), neem and bitter gourd oils were Class-I biorationals (harmless, < 25% mortality), and tebufenozide was in Class-III biorationals (moderately harmful, 50-75% mortality) when emergence and mortality of E. formosa were recorded inside the treated host pseudopupae in lab bioassay. In parasitized pseudopupae-exposure bioassay (field), neem and bitter gourd oil were demonstrated as harmless/Class-I biorationals (< 30% mortality), while pyriproxyfen, buprofezin, fenoxycarb, methoxyfenozide, and tebufenozide were confirmed as slightly-harmful biorationals (30-79% mortality) against E. formosa stages developing inside host pseudopupae. Our results regarding pyriproxyfen are not in agreement with other findings, which confirm pyriproxyfen as highly harmful to larvae but moderately harmful to pupae of E. formosa 46,48 . Similarly, present results regarding buprofezin are contradictory to the results which demonstrate buprofezin as harmless to E. formosa pupae 46 . However, our findings regarding fenoxycarb corroborate other studies indicating that fenoxycarb is slightly harmful IGR to larvae and pupae of E. formosa 46 and Eretmocerus eremicus 70 . Our findings regarding the toxicity of methoxyfenozide and tebufenozide to E. formosa stages developing inside host pseudopupae cannot be compared or contradicted as no information is available in reviewed literature in this line of study. The adult-exposure lab bioassay demonstrated that all tested biorationals except bitter gourd oil (harmless/Class-I) were classed as slightly-harmful biorationals (30-79% mortality) against male and female E. formosa adults; however, female-adults exhibited lower mortality than the male-adults. Our lab bioassay results regarding pyriproxyfen, buprofezin and fenoxycarb are highly in accordance with the results that demonstrate these three IGRs as slightly harmful to E. formosa adults in glass-vial bioassay 46 . The reasons behind the higher mortality of E. formosa male-adults than its female-adults has not been investigated in the present study. Azadirachtin-A, azadirachtin-B, nimbina and salamina at 9.6 ppm were in general safe (Class-1) to both adults and pupae of Trichogramma pretiosum 71 . These botanicals did not affect T. pretiosum and proved highly compatible with this parasitoid. NEEMSETO proved safe for T. pretiosum in the first 24 h 72 . These results are highly consistent with our findings regarding neem oil extract (harmless). Azadirachtin, buprofezin, tebufenozide, and pyriproxyfen are harmless to parasitoids and slightly detrimental to parasitoid emergence 64,67,73 . The present field experiment results indicate that pyriproxyfen, buprofezin and fenoxycarb are harmless to parasitoids and slightly detrimental to the emergence of adult E. formosa from parasitized pseudopupae at 72 h PAEI. These results demonstrate that these IGRs, especially pyriproxyfen, can be used in integration with E. formosa. Our field bioassay findings regarding pyriproxyfen, buprofezin and fenoxycarb are highly in accordance with the results that demonstrate these three IGRs as harmless to E. formosa adults on tomato leaves in field bioassay 46 . Similarly, different researchers' findings on buprofezin and E. formosa compatibility confirm that leaf residues of buprofezin are not detrimental to B. tabaci parasitoids (Encarsia and Eretmocerus species) as it does not affect the foraging of adults parasitoids of these species on treated leaves [74][75][76][77] . These findings 74-77 also confirm our results concerning buprofezin as harmless IGR to E. formosa adults in field bioassay.
Bt-cotton has a different production technology regarding pesticide, fertilizer (nutrient), and water requirements. These factors influence various biotic stresses and exogenic molecules differently due to differences in their genetic background and physiology/metabolism 22 . Some of the differences in the efficacy of the insecticides tested may be attributed to differences in these ecological requirements for Bt-crops. These variations in the agronomic technologies may affect the quantity and quality of specialized-metabolite biosynthesis differently in Bt and non-Bt crops 23,24 , which may affect a pesticide's performance on a sucking pest such as B. tabaci 25 .
The present study results affirm that most of the tested biorationals (compounds) are moderately harmful to harmful within the first 48 h PAEI. Afterward, these molecules are classed as slightly harmful to harmless www.nature.com/scientificreports/ biorationals. These results highlight the most appropriate PAEI of these compounds before and after which these molecules are compatible with E. formosa. It is evident from these results that effective integration of pyriproxyfen, buprofezin, and fenoxycarb with E. formosa can be achieved if either these IGRs are sprayed at least 72 h before augmentation of E. formosa or E. formosa is augmented after 72 h of sprays of these IGRs. However, there is a need to investigate and reconfirm it in an IGR and E. formosa based IPM experiment with different PAEI ranging from 3 to 15 days. In the present study, compatibility effects of these biorationals were assessed only on one whitefly parasitoid i.e., E. formosa, which is one component of bioagents-complex of B. tabaci in Bt cotton. So, it is vitally imperative and essential to validate the compatibility effects of these biorationals against other predator and parasitoid species of whitefly under lab, semi-field and field conditions to come up with a sound and effective biorational-bioagent based IPM program for whitefly.

Conclusions
The present study results affirm that most of the tested biorationals (compounds) are relatively more toxic within the first 48 h PAEI and afterward prove comparatively safer to E. formosa. These results also define an effective PAEI (at least 72 h) for comparatively safer biorational molecules. Most of the tested biorationals proved harmless or slightly harmful to E. formosa, except tebufenozide after PAEI of 7 days in the field. Hence, they can be exploited strategically in B. tabaci management program. Overall, the compatibility ranking of tested biorationals to E. formosa was bitter gourd-extract > neem-extract > pyriproxyfen > buprofezin > fenoxycarb > methoxyfenozide > tebufenozide; while their order of toxicity to B. tabaci was pyriproxyfen > buprofezin > fenoxycarb > methoxyfenozide > tebufenozide > neem-extract > bitter gourd-extract. These two rankings indicate that pyriproxyfen, buprofezin, and fenoxycarb proved effective against B. tabaci and are relatively more compatible with E. formosa. Hence, effective integration of these three molecules with E. formosa, and an efficient E. formosa supplemented augmentative biocontrol system can be established either by devising a spray-schedule strategy of these three IGRs at least 72 h before augmentation of E. formosa or by planning E. formosa augmentation after 72 h of sprays of these IGRs. Nonetheless, it is emphasized that an IGR and E. formosa-based IPM experiments should be conducted at PAEI, ranging between 3 and 15 days to define IGR and E. formosa-based efficient strategy for effectual management of B. tabaci.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.