• A common HPLC-PDA method for amino acid analysis in insects and plants

    A common method for analysis of 17 amino acids from various insect species and plant parts was standardized using HPLC-PDA. Prior to hydrolysis, lyophilization of test samples was found indispensible to remove excess moisture, which interferes in hydrolysis and separation of amino acids. For the hydrolysis of plant and insect samples, 500 and 100 µL of boiling HCl, respectively, and 20 µL of hydrolyzed samples used for derivatization, provided best results. Gradient profile of mobile phase and run time up to 65 min were standardized to (i) encompass the problems related to eluting underivatized sample part, (ii) optimize the use of mobile phase and run time, and (iii) get better separation of different amino acids. Analysis of Chilo partellus larvae reared on sorghum seedling powder based artificial diet indicated that arginine and histidine quantities were on par in both samples. However, methionine was higher, and leucine, isoleucine, lysine, phenylalanine, threonine and valine were lower in sorghum seedlings than in C. partellus larvae, suggesting compensation of these amino acids by the insect through voracious feeding, as is being expected from artificial diet. This method was found highly sensitive, reproducible and useful for the analysis of amino acids for better understanding of insect-plant interactions.

  • Inheritance of resistance in Pectinophora gossypiella

    The mode of inheritance of resistance to Bt toxins Cry1Ac and Cry2Ab was studied in the Pectinophora gossypiella (Saunders). Both strains, Jalg-R showing 605-fold resistance to Cry1Ac and another Jal-Sc showing 37.7-fold resistance to Cry2Ab were mated with susceptible insects to study inheritance of Cry1Ac and Cry2Ab resistance. The resistance in both cases was semi-dominant and autosomal in nature.  Further, association of alkaline phosphatase with Cry2Ab resistance was studied. ALP ranged from 60-65 to 115-120 kDa in resistant strains and 60 to 105 kDa in susceptible strains. ALP activity was more in resistant insects (26.35 µM/min/µg) as compared to susceptible insects (6.45 µM/min/µg) and untreated control (9.95 µM/min/µg).

  • Silencing of insect molecular targets through RNA interference

    Chitin has to be degraded during the development of the insects periodically and hence chitinase plays an important role during the insect growth and development. Chitinase expression therefore coincides with the molting process and is developmentally regulated in insects. Putative chitinase genes from S. litura and H. armigera respectively were identified through genome-wide search. siRNAs were designed and synthesized from the EST region of chitinase gene of H. armigera as well as S. litura. Feeding and injection assay of SiRNA as well as dsRNA against neonates and 10 day old larvae respectively showed inter-molt mortality. This provides proof of applying SiRNA to knockdown of chitinase gene.

    The effect of chitinase specific dsRNA was studied on larval growth and development of H. armigera and S. litura with feeding of neonates and last instar larvae @ 0.50 µg/g and 0.50 µg/insect, respectively. The mortality of larvae occurred during molting due to interference with chitinase that plays an important role prior to deposition of new cuticle. The phenotypic effects involved larval-larval, larval-pupal, pupal-adult deformities. Further studies also showed interference with fecundity and fertility in eggs of next generation. RT-PCR studies showed inhibition of chitinase. Expression profiling of chitinase gene on 1.2% agarose gel (Fig.3) showed an enormous reduction in levels of chitinase transcripts in dsRNA treated samples as compared to chitinase transcripts in untreated control, internal  andnon-target siRNA control (ApSuc2). Besides that decrease in titres was also detected in malformed pupae, malformed adults as well as in next generation (1st instar larvae) showing later on persistent effect of siRNA and dsRNA treatment after feeding for four days in neonates and one time single dose feeding in last instar of both the test insects.

  • Insect gut microbes

    Generic identities of about 150 gut bacterial isolates from three lepidopteran insects H. armigera, Plutella xylostella and Antherarea assamensis were established by using 16s rRNA probes. The analysis revealed the presence of diverse group of gut bacterial genera including Delftia, Stenotrophomonasp, Pseudomonas, Bacillus, Acetobacter, Acinetobacter, Gamma proteobacterium, Lysinibacillus and Enterobacter which have potential role in insecticide detoxification, nitrification, amide metabolism and other biological activity in insects. Metagenomic analysis of gut homogenates of these lepidopteran insect species led to the identification of about 5846 Operational Taxonomic Units (OTUs) in the three species. Alpha and beta diversities of gut bacterial strains were found to be significantly higher in P. xylostella in comparison to other two species.

  • Insect-Plant Interactions

    The Gas Chromatography (GC) profiling of selected maize genotypes viz., CPM 2, CPM 8, CPM 13, CPM 15, and CPM 18 along with resistant check CML 334 vis-à-vis the C. partellus larvae fed on these genotypes for different fatty acids revealed that the contents of myristic acid, palmitic acid, margaric acid, linoleic acid, stearic acid, methyl 11-eicosenoate, and eicosanoic acid were comparatively lower in the test maize genotypes as compared to the C. partellus larvae fed on them. However reverse was the trend for methyl 3-methoxy tetradecanoate. Furthermore, the fatty acids viz., cinnamic, linolenic, bhenic, and lignoceric acids were present in the maize seedlings, while absent from the C. partellus larvae fed on them. Conversely, the fatty acids viz., palmitoleic acid, methyl 14-methyl hexadecanoate, oleic acid and erucic acid in spite of absence from the maize seedlings were found present in the C. partellus larvae.

  • Biosystematics
    • Mapping of species distribution of  Melolonthinae and Rutelinae (Scarabaeidae: Coleoptera) in four north Indian states  viz., Uttar Pradesh, Uttarakhand, Himachal Pradesh and Rajasthan was done based on surveys.  Thirty one species belonging to 12 genera with 3 new records and one new species were documented in Himachal Pradesh. Uttar Pradesh and Uttarakhand recorded 23 and 28 species under 8 and 11 genera, respectively with melolonthines dominating in the former and rutelines in the latter. Species diversity was comparatively less in Rajasthan with 16 species under 8 genera. Melolonthine species dominated in all states except Uttarakhand where rutelines were predominant. Spatial resource partitioning was observed among the different species. The dominating species in Uttar Pradesh was Holotrichia nagpurensis followed by H. consanguinea whereas it was Anomala dimidiata and A. cantori in Uttarakhand. In Himachal Pradesh and Rajasthan, the dominating species were Brahmina coriacea, H. longipennis and H. consanguinea and Maladera insanabilis.
    • Revisionary studies of tribe Coelidiini was completed with 15 new species from Indian subcontinent under 4 genera, Calodia, Olidiana, Mahellus and Taharana..
    • Taxonomic studies  conducted on family Halictidae resulted in several new records from India viz., Nomia (Acunomia) strigata and   Nomoides  fortunatus     from Tamil Nadu and Halictus  fimbriatus from Jammu & Kashmir and Shimla and numerous new distribution records within India; cataloging of all the known 198 species under 13 genera in this subfamily from India with their synonyms, revisionary details and zoogeographic distribution; taxonomic accounts of a total of 27 species under 8 genera have been standardized to an uniform format with additional characters, morphometric ratios, coloured photographs and line diagrams.
    • Four new species of genus Sycobia viz., S. nigra, S. benjamina, S. pasighatensis and S. madani  have been described as new to science. Further a key for the identification of all the six Indian species including the new ones has been formulated.
    • A new leafhopper species Cofana trilobata sp. nov. was described from Barapani, Meghalaya. This species can be distinguished by the following features: pygofer moderately produced with acutely rounded caudo-dorsal margin; aedeagal shaft, in lateral view, uniformly broad throughout length with round membranous apex and serrated dorsal and ventral margins, with minute spine-like spicules throughout length which are more numerous at apex laterally and triangular membranous region midventrally; hind margin of female seventh sternite convex with caudal projection feebly trilobed.

  • IPM and pest forecasting models
    • Key-mortality factor analysis in BPH population: Life table of BPH was studied under glasshouse conditions and subsequently adapted to field situation with inclusion of pertinent biotic mortality factors. The life table analysis of six cohorts showed that around 70% of BPH mortality occurred during 2nd nymphal instar, which resulted in type A survivorship curve. Key factor analysis by graphical and regression method revealed 2nd nymphal instar to be the key-mortality stage in BPH life cycle. Regression coefficients (b) of egg (KE) and 1st to 5th nymphal instar (KN1 to KN5), were found to be 0.061, 0.034, 0.826, 0.027, 0.0002 and 0.165, respectively. The highest b value (0.826) for 2nd nymphal instar indicated it to be the major contributor to BPH mortality. Mortality factors of BPH population were accounted to be egg infertility, egg and nymphal predation, mechanical injury to nymphs during exit from hatching site, dispersal, wrong selection of feeding site by nymphs, and unfavourable humidity and temperature. Type A survivorship curves suggested that in order to take advantage of natural mortality factors, pest management against BPH need not be applied before completion of 3rd nymphal instar especially in first generation during the crop season. Afterwards, timely management interventions can be ensured through continuous monitoring of BPH population on the crop.

    • Forewarning of BPH outbreak: Following BPH outbreak during kharif 2008, analysis of 10-year weather data (1999-2008) had indicated likely contributory factors to be i) early onset of  summer rainfall, and ii) intermittent rainfall with more number of rainy days. The BPH outbreak recurred in NCR during kharif 2013. Weather parameters during 2008-2013 were thus analysed to validate role of rainfall pattern on pest outbreak. Data for different years indicated June rainfall, rainfall frequency and mean daily humidity were maximum in 2013 followed by 2008, while total rainfall was highest in 2013 followed by 2010 and 2008.  Early onset of rainfall followed by more frequent rains that led to higher humidity and moderate temperature favoured fast multiplication of BPH playing a role in its outbreak.
    • Development of leaf folder population-weather model: Peaks of leaf folder light trap catches (LFTC) during 5 years (2008-2012) at Ludhiana (Punjab) showed significant relationship with maximum temperature (Tmax), morning relative humidity (RH1), evening relative humidity (RH2) and sunshine hours (SSH) of 28th standard meteorological week (SMW). Weather-based prediction model was developed by regressing square root-transformed peak leaf folder trap catch observed during different years against mean values of aforesaid weather parameters of 28th SMW.
      √LFTC = -72.559+2.007Tmax28smw + 0.019RH128smw+ 0.042RH228smw+0.362SSH28smw  (R2=0.99, P<0.0001). The model was validated with weather and peak leaf folder trap catch observed during 2007.
    • In an attempt at validation of eco-friendly insect pest management schedule, baseline data on use of insecticides in okra was gathered from village Paladi. Farmers applied 6-8 sprays for management of pests. Insect pest management schedule with focus on conservation of natural enemies developed at experimental plots in IARI farm was validated in collaboration with NCIPM. Maximum natural enemy population was observed in plots where maize was sown as an intercrop in IPM trial conducted in farmer’s field with okra while population of leafhoppers (3.25/ 3leaves) and white fly (2.20/ 3 leaves) was low. Seed treatment with imidacloprid @ 5 g/kg seed, intercropping with maize and one spray each of spinosad @ 75g a.i./ha and chlorantraniliprole @ 20 g a.i./ha thirty days after sowing was best. Spiders and coccinellids conserved by intercropping with maize were found feeding on leaf hoppers in main crop okra.
    • In an evaluation trial of insecticide mixtures in okra, leafhoppers population was minimum in the treatments of spraying by triazophos + deltamethrin @ 360 g a.i./ha (12.33/15 leaves) followed by treatment with profenophos + cypermethrin @ 440 g a.i./ha (15.33/ 15 leaves) as against control (28.67/ 15 leaves) at 7th day after spray.
    • In the trial on brinjal novel insecticides i.e. fipronil (80% WG), thiacloprid (21.7% SC), diafenthiuron  (50% WP) and chlorantraniliprole (18.5% SC) were evaluated against brinjal shoot and fruit borer and were compared with acephate, chlorfenapyr, abamectin,  alpha cypermethrin with 3 sprays of each insecticide at fortnightly interval. Results indicated that fipronil (50 g a.i./ha) was most effective followed by thiacloprid (20 g a.i./ha), diafenthiuron (40 g a.i./ha), chlorantraniliprole (10 g a.i./ha) with 9.61, 14.91, 17.12 and 18.43%  infestation on weight basis against 39.18 % in control.
    • The soybean lines evaluated against stem fly and yellow mosaic virus (YMV) disease revealed that varieties DS 2705, DS 2706, DS 2806, SL 958, SL 979 and SL 982 were highly resistant to YMV with incidence rating up to 1 and also to stem fly, and hence considered promising sources of resistance to major pests of soybean.
    • Weather based population model for legume pod borer, Maruca vitrata on pigeon pea was developed based on the data for crop seasons of 2011-12 and 2012-13 by regressing the incidence of larval population on mean values of different weather parameters recording at different weeks. The resultant model y = -31.6 + 0.52Tmax – 0.58Tmin + 0.10RF + 0.31RH + 1.09SSH was validated for crop seasons of 2013-14 (r2 = 0.83).
  • Insect Toxicology
    • Laboratory studies on the relative toxicity of some new insecticides to larvae by topical bioassay revealed differences among the field populations of S. litura collected from Delhi, Sonepat and Varanasi. All the three populations were found to be most susceptible to chlorantraniliprole, followed by emamectin benzoate and indoxacarb compared to other insecticides. However, no definite trend of resistance development was observed. Varanasi population was most resistant to spinosad than Sonepat and Delhi populations.
    • The effect of four different larval diets (cauliflower, soybean, castor and artificial diet) on insecticide (profenophos and cypermethrin) susceptibility to S. litura was studied. The larvae reared on castor and artificial diets were found to be more susceptible to both the insecticides than those reared on cauliflower and soybean. Cypermethrin was 4.4 fold less toxic than profenofos for the larvae fed on artificial diet.  Profenofos was equally toxic to the larvae fed on different diets while cypermethrin was 6.6 fold less toxic to the larvae fed on cauliflower as compared to those fed on artificial diet.
    • Pesticide-Risk-Analysis (PRA) of various termiticides used in the rabi crops (2012-13) was done for residues in the harvested commodities viz., wheat, barley, gram and lentil. Residues were found below detectable level, implying the safety of termiticides recommended viz., imidacloprid 17.6%SL, chlorpyriphos 20% EC, and fipronil 0.3% G.
  • Biological control of insect pests
    • Corcyra egg cards treated with tomato leaf extracts from reproductive, vegetative and seedling stage of the crop when exposed to Trichogramma brasiliensis and T. japonicum for parasitisation showed up to 83.33% parasitisation in case of tomato extracts from reproductive phase as compared to vegetative and seedling stage by both the species.  In Y-tube olfactometer studies, T. brasiliensis and T. japonicum were attracted more towards flowering plants than the other two stages. The leaf volatile profiles of the tomato seedling, vegetative and reproductive phase revealed the presence of 19 different hydrocarbons at varying levels. The level of heptadecane was more in reproductive phase than vegetative and seedling stage. The whole body washes of H. armigera attracted T. brasiliensis and T. japonicum. The egg cards treated with body washes of H. armigera showed 76 and 60% by T. brasiliensis and T. japonicum, respectively. The antennae of T. brasilensis showed significantly better response to the extracts of reproductive phase of tomato and body wash of H. armigera in EAG.
    • In another Y-tube olfactometer study mustard flowers infested with aphids attracted 76.6% of the Coccinella septumpunctata  population. The response of alate forms of mustard aphid was 62.2% towards leaves with flower than only leaf (29.0 %) or flower (7.2 %).
    • Bioassay of imidacloprid, profenofos and thiodicarb against the adults of Aenasius bambawalei, a parasitoid ofmealy bugs indicated that the Gujarat (Kalawad) population was more tolerant to profenofos and Tamil Nadu (Tholuthur) population was more tolerant to thiodicarb than five other populations. All six populations of A. bambawalei were equally susceptible to imidacloprid. Dharwad population of A. bambawalei showed higher fecundity and longevity at high temperature of 38 oC.
    • Eight isolates of Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metsch.) Sorokin, obtained from ITCC and NCIPM were screened against adults of Bactrocera dorsalis, larvae of C. cephalonica  and Spodoptera litura by exposing the insects to two week old culture. Three isolates ITCC No.6628, ITCC No. 6645 and B. NCIPM were found pathogenic to fruit flies causing 100 % mortality within 6 days. However, first two isolates were less toxic to C. cephalonica at and C. septumpunctata at 1 x 109 conidia/ml over three weeks.