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An experiment was conducted to study and document the copulation duration and effect of mating on female calling behaviour of Indian population of an invasive pest, Spodoptera frugiperda (J. E. Smith). Results showed that 58.39% of the females in absence of males and 88.18% of females in presence of males exhibited calling on first scotophase and onset time of calling was early in females in presence of males. Calling length and calling bouts varied significantly with increase in age in virgin females, while in multiple mated females it showed declining trend. The copulation duration of S. frugiperda varied between 78.00±7.35 min to 197.14±11.06 min with maximum copulation duration and percentage of females mated was observed on second scotophase. The fecundity of multiple mated females ranged between 841 to 1849 with mean of 1176±113 while in single mated females it ranged between 476 to 1368 with mean of 878±175. Practically our results indicated that the calling and mating takes place on first scotophase and it was on peak in second scotophase. This information can be used in formulating biorational molecules, which may modulate calling behaviour and improve mating disruption in S. frugiperda for its management.

Purpose: The impact of gamma radiation on the reproductive biology of the spotted pod borer, Maruca vitrata (Fabr.) (Lepidoptera: Crambidae) was ascertained to explore the potential of the radio-ge...

Circadian rhythms are endogenous, and synchronize biological functions at the most suited time of the day. Insects like other organism display a wide array of circadian functions, which are controlled by a central timing system, in coordination with the peripheral clocks found in many tissues. Many insect behaviours including locomotion, courtship, mating, egg laying and photoperiodism, are influenced by the circadian systems. In this mini-review, we briefly describe the involvement of circadian clocks in various physiological processes and correlate their functions in insect life, focusing on lepidopteran pests (major group of Indian crop pests) and discuss their role(s) in the development of effective pest management strategy.

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating. (C) 2017 The Author(s). Published by Elsevier Ltd.

In this study we identified a number of moth (Lepidoptera) species that are potential targets for the sterile insect technique (SIT), and we assessed the feasibility of using stable isotope signatures as markers to distinguish mass-reared from wild moth species. Large natural differences in the isotopic signatures of commercially available sugars render them novel markers for mass-reared insects. Sugar beet (Beta vulgaris L.; Caryophyllales: Amaranthaceae), a C3 plant, has a stable isotopic signature (a measure of the ratio of the stable isotopes 13C:12C) of around −27‰ relative to Vienna Pee Dee Belemnite (VPDB; the international C isotope standard for the stable isotopes, 13C and 12C), and sugarcane (Saccharum spp.; Poales: Poaceae), a C4 plant, has an isotopic signature of around −11‰. Thus by means of such a distinct isotope ratio in the sugar in the diet, mass-reared insects can be easily distinguished from wild insects with a high degree of certainty. It was shown that the method could be extended using a multiple isotope approach, with 15N or a full suite of C, N, S and O isotopes. Intrinsic isotope marking of mass-reared moths proved to be an accurate means of distinguishing wild from mass-reared populations, based on isotopic differences between the wild host plant species and the diets used in mass-rearing, which where possible, had been manipulated to contain the isotopically divergent sugar type. This intrinsic labeling using stable isotopes could be useful in the assessment of the quality of mass-reared moths, because a stable isotope is a marker that does not affect the insect in any detrimental manner. ResumenEn este estudio se identificaron una serie de especies de polillas (Lepidoptera) como objetivos potenciales para la tecnica del insecto esteril (TIE), y se evaluo la viabilidad del uso de firmas isotopicas estables como marcadores para distinguir especies de polillas salvajes de las polillas criadas en masa. Las diferencias naturales mayores en las firmas isotopicas de los azucares comerciales disponibles los hacen nuevos marcadores para los insectos criados en masa. La remolacha azucarera (Beta vulgaris L .; Caryophyllales: Amaranthaceae), una planta C3, tiene una firma isotopica estable (una medida de la proporcion de los isotopos estables: 13C:12C) de alrededor de −27 ‰ relativa a Viena Pee Dee Belemnite (VPDB; el estandar internacional para el isotopo C de los isotopos estables, 13C y 12C), y la cana de azucar (Saccharum spp .; Poales: Poaceae), una planta C4, que tiene una firma isotopica de alrededor de −11 ‰. Por lo tanto, por medio de esta relacion de isotopos distinta en el azucar en la dieta, los insectos criados en masa se pueden distinguir facilmente de los insectos salvajes con un alto grado de certeza. Se demostro que el metodo podria ampliarse mediante un enfoque multiple de isotopos, con 15N o un grupo completo de isotopos C, N, S y O. El marcado de los isotopos intrinsecos de las polillas criadas en masa demostro ser un medio preciso para distinguir entre las poblaciones salvajes y las poblaciones criadas en masa, basado en las diferencias isotopicas entre las especies de plantas hospederas silvestres y de las dietas usadas en la cria en masa, que cuando sea posible, habian sido manipuladas para contener el tipo de azucar isotopicamente divergente. Este marcador intrinseco utilizando isotopos estables podria ser util en la evaluacion de la calidad de las polillas criadas en masa, porque un isotopo estable es un marcador que no afecta el insecto de cualquier manera perjudicial. View this article in BioOne

Sperm behavior represents one of the attributes in the radiogenetic technique called inherited sterility crucial for its effectiveness to suppress populations of Spodoptera litura (F.) (Lepidoptera: Noctuidae). Quantitative and qualitative assessments of S. litura sperm behavior (production, descent, activation, movement and transfer to females) were made in parental (P) males that received sub-sterilizing irradiation doses of either 100 or 130 Gy and in their F1 generation males. Age dependent production of eupyrene and apyrene spermatozoa in the testes were not affected by such irradiation in the P males, nonetheless a slight but significant effect occurred in the F1 generation. During the rhythmic cycles of sperm descent in the photophase and scotophase, the profile and proportion of sperm descent from the testes to the upper vas deferens (UVD), seminal vesicle (SV) and duplex were not significantly influenced by irradiation in P and F1 males. Sperm activation—assessed as percent active apyren...

Measurement of natural stable isotope signatures of insects has proven to be a useful method in determining their natal origin, feeding strategies and mating behavior, and has spawned a new discipline of insect isotope forensics. However the complexity of measuring isotope signatures using elemental analysis-isotope ratio mass-spectrometry (EA-IRMS) has encountered significant technical barriers, which together with costs, have hindered the advancement and wide spread adoption of this potentially useful approach. In the last decade simpler technologies to measure stable isotope ratios in samples based on laser spectroscopy have been developed. We foresee that these instruments will fuel the advancement and adoption of insect isotope forensics into pest management programs because they are easy to use, more affordable, and complement existing marking tools. However prior to the adoption of these new technologies it is necessary to test their fitness for purpose and explore their limitations. In this paper we set out to test the suitability of using combustion module-cavity ring-down spectroscopy (CM-CRDS) for measuring isotope ratios in an entomological context, covering marking of insects, mating behavior and dietary assessment. We carried out a series of experiments to compare the isotope measurements made with the CM-CRDS with the traditional method of EA-IRMS. Initially we compared measurements of carbon isotope values of a variety of common diet components used in insect rearing, which covered a wide range of carbon isotope values. There was good agreement in the isotope values obtained, using the CM-CRDS and the EA-IRMS with similar accuracy and precision. Secondly we compared the 2 methods to measure carbon isotope values of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) samples, and again there was good agreement in the values obtained. Thirdly we tested whether the CM-CRDS could be used to determine the paternity of spermataphores, based on the differential isotope signatures of the paternal and maternal moths, as a result of differences in their rearing diets. The CM-CRDS proved to be fit for the purpose of measuring isotope values in the spermataphores of common cutworm and suggested that the CM-CRDS technology would be suitable for these isotope based mating studies in moths if the spermatophores were of sufficient mass. Finally we explored a number of the issues surrounding CM-CRDS and isotope technologies in general, e.g., logistical considerations, economics of operation, sample size, etc. We conclude that CM-CRDS is a suitable instrument for measuring stable isotope carbon signatures in moths, and most probably other sufficiently large insects and that CM-CRDS could be easily used in both operational and research contexts.

The dose of radiation applied to the Oriental leafworm (also known as the common cutworm), Spodoptera litura (F.) (Lepidoptera: Noctuidae) for use in inherited sterility (IS) sterility programs must not materially reduce the quality and competitiveness of the released males. To assess the quality of sub-sterilized male moths and their F1 progeny, their flight and mating behaviors were investigated. Thus, parental (P) generation S. litura males that had been irradiated with either 100 or 130 Gy and their F1 male descendants were subjected to flight assay tests to assess flight ability, orientation towards a pheromone source and mating behavior, i.e., mating performance using various sex ratios, sequential matings, and effect of female age. The orientation behavior of either P or F1 males towards pheromone-baited traps was not different from that of non-irradiated males. Mating abilities of irradiated P generation males and their F1 male progeny with non-irradiated females were studied using severa...

The effects of gamma radiation were observed on the metamorphosis and reproduction as key target features to be intercepted for disinfestation of the three major pest bruchid species, Callosobruchus maculatus (Fabricius), C. analis (Fabricius) and C. chinensis (Linnaeus) irradiated in the various ontogenic stages. The present study was aimed to determine an efficacious generic dose of phytosanitary irradiation (PI) for bruchid disinfestation in pulses. Callosobruchus chinensis was the most radio-resistant species among the present tested species, and male bruchids were more radio-resistant than the females in this species complex. In this complex of tested bruchid species, the gamma dose required to cause 100% metamorphic disruption in the most radio-resistant pre-imaginal stage (pharate adult) was 20–46% higher than the dose required to induce complete sterility in the most radio-resistant imaginal stage (4–5 day old). The effective dose inducing 99.9% impact (ED99.9) was 105 Gy for sterility induction but ~181 Gy to induce metamorphic disruption in the most radio-resistant pre-imaginal pharate adult in C. chinensis, whereas the most radio-resistant imaginal stage (4–5 day old adult) of this species required 124 Gy for complete sterilization. Based on these results, 160–200 Gy as phytosanitary irradiation dose range was evaluated on a semi-large scale testing on the pigeonpea pulse seeds having mixed infestation of Callosobruchus species complex, which indicated that 200 Gy might be considered as a feasible generic dose of phytosanitary irradiation for bruchid disinfestation in pulses.

The tobacco cutworm, Spodoptera litura, is among the most widespread and destructive agricultural pests, feeding on over 100 crops throughout tropical and subtropical Asia. By genome sequencing, physical mapping and transcriptome analysis, we found that the gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione-S-transferase, were massively expanded in this polyphagous species, enabling its extraordinary ability to detect and detoxify many plant secondary compounds. Larval exposure to insecticidal toxins induced expression of detoxification genes, and knockdown of representative genes using short interfering RNA (siRNA) reduced larval survival, consistent with their contribution to the insect’s natural pesticide tolerance. A population genetics study indicated that this species expanded throughout southeast Asia by migrating along a South India–South China–Japan axis, adapting to wide-ranging ecological conditions with diverse host plants and insecticides, surviving and adapting with the aid of its expanded detoxification systems. The findings of this study will enable the development of new pest management strategies for the control of major agricultural pests such as S. litura.

To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells.Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit.A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism.Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Lepidopteran insects/insect cells display 50–100 times higher radioresistance than humans, and are evolutionarily closest to mammals amongst all radioresistant organisms known. Compared to mammalian cells, Lepidopteran cells (TN-368, Sf9) display more efficient antioxidant system and DNA repair and suffer considerably less radiation-induced DNA/cytogenetic damage and apoptosis. Recent studies indicate that a considerably lower radiation-induced oxidative stress may significantly reduce macromolecular damage in Lepidopteran cells. Since nitrosative stress contributes in radiation-induced cellular damage, we investigated its nature in the γ-irradiated Sf9 cells (derived from Spodoptera frugiperda ; order Lepidoptera; family Noctuidae) and compared with BMG-1 human cell line having significant NOS expression. Radiation induced considerably less ROS/RNS in Sf9 cells, which remained unchanged on treatment with NOS inhibitor l -NMMA. Surprisingly, growth of Sf9 cultures or irradiation could not induce NO or its metabolites, indicating negligible basal/radiation-induced NOS activity that remained unchanged even after supplementation with arginine. Cytosolic calcium release following high-dose (1000–2000 Gy at 61.1 cGy s −1 ) γ-irradiation or H 2 O 2 (250 μM) treatment also failed to generate NO in Sf9 cells having high constitutive levels of calmodulin, whereas BMG-1 cells displayed considerable calcium-dependent NO generation even following 10 Gy dose. These results strongly imply the lack of calcium-mediated NOS activity in Sf9 cells. Addition of exogenous NO from GSH-NO caused considerable increase in radiation-induced apoptosis, indicating significant contribution of constitutively attenuated nitrosative stress response into the radioresistance of Lepidopteran cells. Our study demonstrates for the first time that a calcium-insensitive, attenuated nitrosative stress response may contribute significantly in the unusual radioresistance displayed by Lepidopteran insect cells.

Effect of temperature on the physiological activity and resistance status of Culex quinquefasciatus was studied. The LT50 (lethal time) of deltamethrin, lambdacyhalothrin resistant and susceptible 4th-instar larvae decreased with increasing temperature. The LT50 of deltamethrin resistant strains was significantly lower than the susceptible strains but not so in lambdacyhalothrin resistant strains. The developmental period of the thermal exposed and non-exposed larvae of resistant strains were nearly equal, however a significant difference was observed in case of susceptible strain. The thermal exposed susceptible larvae developed 3.3 days faster than the thermal un-exposed population. The percentage of survival of thermal exposed and un-exposed resistant strains were nearly equal. However, the thermal exposed population survived 12.9% less than the thermal un-exposed susceptible population. Longevity of thermal exposed deltamethrin and lambdacyhalothrin resistant females was significantly higher than the un-exposed population. The longevity of thermal exposed susceptible population was lower than the un-exposed population. The resistance of both the deltamethrin and lambdacyhalothrin resistant strains increased when exposed to high temperature for 3 h. The difference in longevity between thermal un-exposed and exposed resistant male population was not significantly different. Among the un-exposed population, highest pupation was recorded in the susceptible strains, and lowest in the deltamethrin resistant strain. The fact that the thermal exposed resistant strains were more successful than the un-exposed population could be related to the presence of resistant gene.

The infective behaviour of entomopathogenic nematodes (EPNs), Steinernema glaseri (Steiner), reared within radiosterilized host larvae of the tropical pest, Spodoptera litura (Fabricius), was ascertained towards the unirradiated same host (S. litura) for successive generations and compared with the infectivity of controls (EPNs derived from unirradiated host larvae). A primary goal was to establish a safe mode of transport and dispersal of EPNs without concern that uninfected, reproductively competent hosts would be inadvertently released. Based on prior studies on radiation-mediated effects, two gamma doses (40 and 70 Gy), were used for radiosterilization of last instar S. litura larvae. Tests were performed using the following parameters: Regimen I (Control) with normal infective juveniles (N-IJs) vs. normal (N) hosts; Regimen II with N-IJs vs. Irradiated hosts; Regimen III with F1 IJs (harvested from Regimen II) vs. N-hosts; and Regimen IV with F2 IJs (harvested from Regimen III) vs. N-hosts. ...

Efficacy of the entomopathogenic nematode (EPN), Steinernema glaseri, (Steiner) cultured in radio-sterilized host, was studied vis-a-vis radiation-induced F1 sterility on a tropical lepidopteran pest, Spodoptera litura (Fabr.). To ensure safe transport of S. glaseri EPNs in vivo, host radio-sterilization was done; and the parasitising performance of S. glaseri infective juveniles (IJs), cultured in irradiated last instar S. litura larvae (with either 40 or 70 Gy of gamma rays) was evaluated on F1 sterile insects (progeny of male moths treated with 100 Gy, 130 Gy). S. glaseri EPNs cultured in radio-sterilized larvae at 40 Gy, had better infective potential than those cultured in sterilized host larvae at 70 Gy. F1 sterile larval hosts (progeny from 100 or 130 Gy treated parents) were equally acceptable to the EPNs cultured in radio-sterilized hosts, although the nematode harvest was reduced on F1 sterile hosts at 130 Gy. Infectivity of IJs derived from F1 sterile host was almost similar on F1 ster...

Role of cytochrome-c in insect cell apoptosis is highly controversial, with many earlier reports suggesting lack of involvement of mitochondrial factors in Drosophila while more recent studies have indicated otherwise, thus warranting more in-depth studies of insect cell apoptosis. In the present study, we investigated mitochondrial involvement during actinomycin-D induced apoptosis in Sf9 Lepidopteran cells. Cytochrome-c was released from mitochondria very early during apoptosis, and was preceded quickly by ROS generation and cardiolipin peroxidation. Albeit cytochrome-c release and apoptosis induction were inhibited by bongkrkicacid (BKA) it appears that the release is independent of permeability transition pore (PTP) as it preceded mitochondrial Ca(2+) buildup and mitochondrial membrane potential (MMP) loss. Further, the release was found to be unaffected by PTP inhibitor cyclosporin-A. Bax inhibitory peptide BiP-P5 could effectively block both cytochrome-c release and apoptosis induction indicating dependence on Bax-channel formation. Inhibition of apoptosis by FSBA, a nucleotide analog that inhibits apoptosome formation through Apaf1 binding, suggested activity of apoptosome similar to mammalian cells. Mitochondria isolated from treated cells activated caspases in the cytosolic fraction of untreated cells while mitochondrial lysates of treated or untreated cells had similar effect. Sequestering cytochrome-c in mitochondrial lysates inhibited DEVDase activity, and addition of purified cytochrome-c and dATP to Sf9 cytosolic fraction induced DEVDase activity, suggesting that cytochrome-c may be exclusively required for Lepidopteran apoptosis. This is the first detailed study demonstrating mitochondrial regulation of Lepidopteran insect cell apoptosis, and reiterates its homology with mammalian cell apoptosis while showing distinctive differences from earlier reports in Drosophila.

When given in a critical dietary dose range, the insecticidal bisacylhydrazine ecdysteroid agonists RH-5849 or tebufenozide (RH-5992) cause fifth stage Manduca sexta (L.) larvae to moult to a supernumerary sixth-stage giant larva. The effect is dependent on exposure to the chemicals immediately after the previous ecdysis. Previous removal of the corpora allata does not interfere with the induction of premature moulting by RH-5849 but completely prevents the formation of supernumerary larvae. The juvenilizing effect is therefore due to the interaction of the moult-promoting effect of the ecdysteroid agonists with the high titre of endogenous Juvenile Hormone that is present just after ecdysis to the fifth stage in this insect. The ecdysteroid agonists themselves appear to have no intrinsic Juvenile Hormone-agonist properties. Sixth-stage larvae resulting from exposure to critical dietary concentrations of RH-5849 are morphologically completely larval in character. When transferred to diet without the ecdysteroid agonist, they feed normally and gain weight, growing much larger than control fifth stage insects. At the end of the supernumerary stage, they cease to feed, wander in the usual way, and form a normal pupal cuticle but then die as pharate pupae without shedding the sixth-stage larval cuticle.

Previous studies on various insect cell lines have displayed very high radioresistance in Lepidoptera (butterflies and moths) as compared to mammals as well as other orders of Insecta including Diptera. Since NOS is known to modulate cellular radiation sensitivity, we carried out in silico analysis of Lepidopteran NOS and compared its structural and functional features including the sequence homology, predicted tertiary structure, post-translational phosphorylation and intracellular localization with the other species. Our study demonstrates that Lepidopteran NOS, while carrying significant sequence homology with mammalian nNOS, has structural/ functional features that may enhance resistance to radiation and other stress agents. A higher phosphorylation score of Lepidopteran NOS (0.885+/-0.02 as against 0.694+/-0.094 of mammalian NOS; predicted using Net Phos 2.0) was observed at many well-conserved phosphorylation sites, which may reduce NOS activation by stress agents including radiation. Further, the primarily cytoplasmic localization of Lepidopteran NOS (score 23 against 10 of mammalian NOS, derived using WoLFPSORT), aided by higher phosphorylation scores as well as sequence-driven cytoplasmic localizing signals, may significantly reduce amplification of extraneous oxidative damage. Based on these findings, we hypothesize that a primarily cytosolic and less responsive NOS could significantly contribute to radioresistance of Lepidopteran insects as well as their cultured cell lines.

Density of vectors and development of resistance against insecticides are two important aspects in the control of vector-borne diseases. Here, effect of temperature on different aspects of development of malathion-resistant and susceptible strains of Culex quinquefasciatus was evaluated in the laboratory. Fourth-instar larvae of C. quinquefasciatus were exposed to 37 degrees C, 39 degrees C, 40 degrees C, and 41 degrees C, and their LT(50) values calculated. The fourth-instar larvae were subjected to heat for 4 h at 39 degrees C and live larvae were reared until the completion of life cycle. The larvae of malathion-resistant strain were more tolerant to heat than the larvae of malathion-susceptible ones. The difference in mortality between 37 degrees C, 39 degrees C, 40 degrees C, and 41 degrees C of both the strains of C. quinquefasciatus were highly significant. Pupation percentage of heat-exposed larvae of both the strains was higher and delayed than the control ones. The percentage of adult emergence from the heat-exposed larvae was significantly lower than the control. The adult female of malathion-resistant strain showed increased longevity when exposed to heat at larval stage but reverse was true in case of malathion-susceptible. The highlights of the study would help in the management of resistance of mosquito vectors in the tropics where variable climatic condition is observed.

To investigate the effect of gamma radiation-induced alterations in antioxidant defence of radioresistant Sf9 insect cells.Sf9 cells were irradiated at doses ranging from 0.5-200 Gy. Lipid peroxidation and protein carbonylation levels were observed at 4 h post-exposure along with reduced glutathione/oxidized glutathione (GSH/GSSG) profile as well as specific activities of redox active enzymes superoxide dismutase (SOD), catalase, ascorbate peroxidase (APOx), and glutathione reductase (GR). Human brain malignant glioma (BMG-1) cells were used for comparing radiation response of mammalian cells.Sf9 cells displayed significantly less radiation-induced reactive oxygen/nitrogen species (ROS/RNS) generation, protein carbonylation and growth inhibition as compared to mammalian cells. Sf9 cells have higher basal APOx (∼4-fold), catalase (∼1.7-fold), SOD (∼1.3-fold) activity and GSH level (∼2.2-fold) compared to mammalian cells. A radiation dose-dependent increase in SOD, Catalase and APOx activity was found in Sf9 cells at least up to 100 Gy dose, while maximum activity in mammalian cells was achieved by 10 Gy.The present study suggests that Lepidopteran insect cells carry a stronger antioxidant system that protects against radiation-induced macromolecular damage, growth inhibition and cell death.

Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45-60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30-45 rain after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating, with little mixing. The proportion of eggs fertilized by sperm from the second mating (P-2) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12h:12h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.

To investigate age-correlated radiosensitivity in highly radioresistant lepidopteran pest, Helicoverpa armigera, upon exposure to ionizing radiation and to examine the irradiation impact on stress-molecular responses in F1 (first-filial) progeny of irradiated (100 Gy) male moths in relation to its reproductive behavior.Efficacy of sub-lethal gamma radiation was evaluated on two markedly apart ontogenic stages, neonates and adult moths. Differential growth, reproductive behavior and stress-indicating molecular responses were examined upto F1 progeny of sub-sterilized moths. Free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and Phenoloxidase cascade enzymes, pro-phenoloxidase (PPO), its activating enzyme (PPAE) were studied in irradiated and irradiated plus microbial challenge regimen (dual-stress) by Real-time RT-PCR (reverse-transcription-polymerase-chain-reaction).An inverse correlation of radiosensitivity with developmental age of insect was observed. F1 sterility was higher than parent sterility. F1 progeny exhibited protraction in development and decreased survival upon irradiation. Sex ratio in F1 progeny was skewed towards males. PPO, PPAE, SOD and CAT transcripts were downregulated upon neonate irradiation resulting in enhanced vulnerability of larvae to incidental microbial challenge. These transcripts were upregulated in F1 progeny of sub-sterilized male moths (100 Gy) upon dual-stress.Irradiation impact on stress-indicating molecular responses in F1 progeny is correlated with its reproductive performance. These observations will permit defining regimen having pragmatic viability of 'F1 sterility technique' for pest suppression. Gamma dose of 100 Gy would ensure balance between induced sterility of males and their field competitiveness. These parameters would facilitate integration of biocontrol strategy with parabiological 'Sterile Insect Release Technique'.

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis . Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda , and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura . Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

Purpose: To investigate homology and stress response of p53 (a 53 kDa tumor suppressor protein) orthologue in Sf9 Lepidopteran insect cell line that exhibits very high radioresistance. Materials and methods: Western immunoblotting, immunoprecipitation, degenerate RT-PCR (reverse transcriptionpolymerase chain reaction), electrophoretic gel mobility shift assay, flow cytometry and immuno-fluorescence microscopy were used for characterizing structural and functional features of Sfp53 (Spodoptera frugiperda p53) in g-irradiated or etoposide-treated Sf9 insect and BMG-1 (brain malignant glioma) human cells. Cells were pre-treated with caffeine for inhibiting ATM/ATR (ataxia-telangiectasia mutated protein/ATM and Rad-3-related protein) activation, wherever required. Results: A 47‐49 kDa protein band was observed with antibodies against three different epitopes, demonstrating conservation of respective domains in Sfp53. Immunoprecipitation also yielded similar-sized protein. Degenerate RT-PCR resulted in product of same size in both cell lines. Similar gel mobility shift of p53-binding oligonucleotide with BMG-1 and Sf9 cell lysates indicated analogous transcriptional activity of Sfp53. Constitutive Sfp53 level was higher than hp53 (human p53) and showed primarily cytoplasmic localization. Radiation-induced accumulation was considerably less in Sf9 even as an analogous ATM/ATR-dependent nuclear translocation was observed following g-irradiation and etoposide. Conclusions: A smaller-sized Sfp53 orthologue shows highly conserved native structure with DNA-binding, N-terminus and C-terminus domains, and has analogous p53 transcriptional activity. While its nuclear translocation and ATM/ATR dependence were similar to hp53, the cytoplasmic localization and subdued accumulation following g-irradiation indicate functional differences from human cells.

Biochemical mechanisms of insecticide resistance of thermal exposed and unexposed Culex quinquefasciatus strains are evaluated, which were not studied earlier. The activity of alpha- and beta-carboxylesterases and acetylcholinesterase of malathion susceptible and resistant strains were compared after thermal treatment. Three-day-old adult females were used for the malathion susceptibility test and biochemical assays, and males were used only for the susceptibility test. Thermal exposure brought about increase in resistance levels from 85% to 90% in males and 91% to 96.6% in females of resistant strain. The resistance status of the susceptibility strain was unchanged after thermal exposure. The activities of alpha- and beta-carboxylesterase of susceptible mosquitoes were within 800 and 700 U/mg protein, respectively. The alpha-carboxylesterase activity of the thermal exposed malathion-resistant population was significantly (t test, P0.05) higher than the unexposed resistant population, and the reverse was recorded in beta-carboxylesterase. The alpha-carboxylesterase activity of susceptible population was lower than the resistant population. The activity of alpha-carboxylesterase was higher than the beta-carboxylesterase in both the strains. Among the malathion resistant C. quinquefasciatus population, 2.3% population exhibited 30-40% inhibition which increased to 5.8% after the thermal exposure. Thermal exposure of mosquitoes increased the activity of both alpha-carboxylesterases and acetylcholinesterase but decreased the activity of beta-carboxylesterase.

Publisher Summary This chapter focuses on the dichotomous spermatogenesis in Lepidoptera. Research into how apyrene sperm development occurs and is regulated plays important dividends in understanding of normal spermatogenesis. The existence of apyrene spermatocytes that continue developing in a predictable way to produce spermatozoa after their nuclei become apoptotic presents a very convenient system to study differentiation of eukaryotic cell in the absence of newly synthesized informative RNA. Furthermore, the dichotomous eupyrene–apyrene dichotomy shows cells having the same genetical background but differentiating separately through a series of well characterized morphological markers that are easy to correlate with stages on molecular experimentation. Genetic techniques are poorly developed in the Lepidoptera, but molecular genetic techniques are now very powerful. The chapter gives some indications of where it might be profitable to look for genes controlling apyrene spermatogenesis and look forward to advances in this research area. Lepidopteran insects are important subjects of research in many areas of reproductive ecology, especially those of sperm allocation, sperm competition, and post copulatory sexual selection. The existence of two types of sperm in this group of insects is of fundamental importance to their reproductive biology, and there is no doubt that the evolutionary biology of sperm dichotomy will continue to be of great interest.

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.

Spodoptera litura reared on host plants and on synthetic diet were irradiated with two substerilizing doses of gamma radiation, 100 Gy and 130 Gy, and examined for inherited sterility. Irradiation affected mating success in the parental (P) and F1 generations. F1 sterility was higher than P sterility, and F1 males inherited more sterility than did F1 females. F1 progeny developed at a slower rate compared with controls. F1 survival to adulthood decreased with increasing dose of radiation. Sex ratio in F1 moths was skewed towards male. Life tables were constructed for S. litura reared on host plant and synthetic diets, and the impact of radiation on population characteristics was ascertained. Reproductive rate (R0) was significantly decreased as a consequence of irradiation, and the effect was more severe in F1 crosses than in P crosses. There was a negative correlation between the dose of radiation and the percent embryo formation in P crosses. Whereas in F1 crosses, radiation dose (given to male parents) was positively correlated with the percent unhatched embryonated eggs. Early mortality of eggs prevailed in unhatched eggs derived from P crosses, and late embryonic lethality was the major cause of F1 sterility. Effects of irradiation are discussed with an emphasis on assessing the potential of the inherited sterility principle for pest control.