Abstract:In their natural environment, plants are often attacked simultaneously by many insect species. The specificity of induced plant responses that is reported after single herbivore attacks may be compromised under double herbivory and this may influence later arriving herbivores. The present study focuses on the dynamics of induced plant responses induced by single and double herbivory, and their effects on successive herbivores. Morphological (leaf length, area and trichome density) and chemical changes (leaf alkenyl and indole glucosinolates) in Brassica juncea were evaluated four, ten, fourteen and twenty days after damage by the specialist Plutella xylostella alone, or together with the generalist Spodoptera litura. To assess the biological effect of the plant's responses, the preference and performance of both herbivores on previously induced plants were measured. We found that alkenyl glucosinolates were induced twenty days after damage by P. xylostella alone, whereas their levels were elevated as early as four days after double herbivory. Trichome density was increased in both treatments, but was higher after double herbivory. Interestingly, there was an overall decrease in indole glucosinolates and an increase in leaf size due to damage by P. xylostella, which was not observed during double damage. S. litura preferred and performed better on undamaged plants, whereas P. xylostella preferred damaged plants and performed better on plants damaged fourteen and ten days after single and double herbivory, respectively. Our results suggest that temporal studies involving single-versus multiple-attacker situations are necessary to comprehend the role of induced plant responses in plant-herbivore interactions.Response to Reviewers: Reviewer #1: Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation1. The paper is well written, but a bit lengthy, particular in the Introduction.We have shortened the introduction from the earlier 1228 words to 927 words by deleting sentences from page 3 (lines 6-9; 13-14; 17; 19-21) Reviewer #2: 3. Concerning the presentation of the data I have difficulties with the fact that the graphs in the figures start only at day 4.As mentioned on Page 7 line 2-4, we started the period of our studies from day 4 because it is already well established that glucosinolate induction starts from third days onwards following herbivore damage (Hopkins et al., 2009; Mathur et al., 2011).4. In figure 1b the authors interpret the data that the glucosinolate content is induced during the complete study period. But the levels remain constant after day 4.Indeed, we meant to say that the levels in damaged plants remained higher during the entire study. We have changed the wording accordingly on page 10.5. Even more problems arise from figure 2. The authors state that the trichome density is increasing due to herbivory. This conclusion is just based on the observation that already on day 4 the number of trichomes between treated and untreated leaves is significantly different...

Studies on the susceptibility of F 1 neonates of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) collected from chickpea in Delhi and cotton in Punjab, Haryana and Rajasthan in northern India, to Bacillus thuringiensis ssp. kurstaki HD-73, and the impact of host crop diets on insect susceptibility, were carried out by diet incorporation bioassays. The susceptibility of F 1 neonates of H. armigera to Bacillus thuringiensis ssp. kurstaki HD-73 ranged from twofold (LC 50 96 h, 84.5 -164.2 µ g (ai) l − 1 ) for chickpea to about fivefold (LC 50 96 h, 51.1-247.7 µ g (ai) l − 1 ) for cotton. The F 1 neonates of insects collected from pearl millet were twice as tolerant as those collected from cotton and sunflower at Sirsa to B. thuringiensis ssp. kurstaki HD-73, suggesting that there was an influence of host crops on insect susceptibility. Insects originally collected from cotton fields at Bhatinda and reared for four generations on a chickpea-based meridic diet were used to initiate host-specific colonies of H. armigera. These host-specific colonies were allowed to complete one generation on meridic diets prepared with different hosts, viz., cabbage, cauliflower, chickpea, green pea, pearl millet, and pigeon pea. Larvae of H. armigera were heaviest on the 15th day, and had a higher growth rate on a pigeon pea-based diet than all other host diets. The larval period was shorter on chickpea and pigeon pea, with higher percentage pupation than all other host-diets. The pupal weight of H. armigera was greater on chickpea and pigeon pea diets than on other host diets. The growth and development of larvae was significantly poorer on pearl millet diet than on other host diets. The F 1 neonates of H. armigera belonging to cabbage, cauliflower, and pearl millet host-specific colonies were more susceptible than those belonging to chickpea, green pea, and pigeon pea host-specific colonies to B. thuringiensis ssp. kurstaki HD-73, suggesting the importance of proteinaceous nutrients in tolerance. The F 1 neonates of the pearl millet colony of H. armigera grown on a chickpea-diet for 4 days were significantly more tolerant to B. thuringiensis ssp. kurstaki HD-73 than those reared on the pearl millet-based diet. These studies show the impact of the host diet of H. armigera on tolerance to B. thuringiensis .

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Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hübner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values <1.3 ppm. Furthermore, one or both toxins were effective against the other major pest Lepidoptera, except for S. litura or H. armigera which were less susceptible. No cross-resistance has been found between Cry1Ba2 and Cry1Ca4, suggesting cry1Ba2+cry1Ca4 cauliflower and cabbage could be an effective and sustainable tool to control, P. xylostella, the key lepidopteran pest on cruciferous vegetable crops, as well as most other Lepidoptera. As the CIMBAA plants are being developed, further tests are needed to determine whether they will express these proteins at sufficient levels to control all the Lepidoptera. Sustainable use of the dual-gene plants also is discussed.

BACKGROUND The fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith), native to the Americas, is a new invasive pest that was reported in India for the first time in May 2018. Being polyphagous, FAW can infest several different hosts and increase its population all year round. In this context, the present study was conducted under laboratory conditions to evaluate the biological parameters of FAW on four different hosts, Zea mays (maize), Gossypium hirsutum (cotton), Ricinus communis (castor) and Brassica oleracea var. botrytis (cauliflower), and a semi‐synthetic diet. RESULTS The shortest life cycle of 32.8 ± 0.52 days in males and 34.1 ± 0.43 days in females was observed on maize. Semi‐synthetic diet was superior in terms of higher mean fecundity (1324.6 ± 61.21 eggs), larval weight (503 ± 0.02 mg), pupal weight (263 ± 0.01 mg) and adult female weight (128 ± 0.0 mg) compared with natural hosts. Cotton was the least preferred host with a longer life cycle of 49.5 ± 0.50 days. Head capsule width and length were measured and the growth rate was validated using Dyar's rule. The mean width and length of the head capsule of first‐instar larvae of FAW on different hosts was 0.35 ± 0.00 mm. The maximum width (2.76 ± 0.03 mm) and length (2.31 ± 0.03 mm) were observed in sixth‐instar larvae grown on diet. CONCLUSION The results of this study will be instrumental in understanding and formulating management strategies for FAW.

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.