Identification and Physicochemical Properties of the Novel Hemolysin(s) From Oral Secretions of Helicoverpa armigera (Lepidoptera: Noctuidae)

Hemolysins cause the lysis of invading organisms, representing major humoral immunity used by invertebrates. Hemolysins have been discovered in hemolymph of Helicoverpa armigera larvae as immune factors. As oral immunity is great important to clear general pathogens, we presumed that hemolysins may be present in oral secretions (OS). To confirm this hypothesis, we conducted four testing methods to identify hemolysin(s) in larval OS of H. armigera, and analyzed physicochemical properties of the hemolysin in comparison with hemolytic melittin of Apis mellifera (L.) (Hymenoptera: Apidae) venom. We found hemolysin(s) from OS of H. armigera for the first time, and further identified in other lepidopteran herbivores. It could be precipitated by ammonium sulfate, which demonstrates that the hemolytic factor is proteinaceous. Labial gland showed significantly higher hemolytic activity than gut tissues, suggesting that hemolysin of OS is mainly derived from saliva secreted by labial glands. Physicochemical properties of hemolysin in caterpillar’s OS were different from bee venom. It was noteworthy that hemolytic activity of OS was only partially inhibited even at 100°C. Hemolytic activity of OS was not inhibited by nine tested carbohydrates contrary to bee venom melittin. Moreover, effects of metal ions on hemolytic activity were different between OS and bee venom. We conclude that there is at least a novel hemolysin in OS of herbivorous insects with proposed antibacterial function, and its hemolytic mechanism may be different from melittin. Our study enriches understanding of the potential role of hemolysins in insect immunity and provides useful data to the field of herbivorous insect-pathogen research.     

Na+/K+-ATPase Activation by cAMP in the Midgut of Lutzomyia longipalpis (Lutz & Neiva, 1912; Diptera: Psychodidae)

Lutzomyia longipalpis (Lutz & Neiva, 1912) females have been intensively studied regarding the regulation of midgut pH. The mechanisms involved in pH regulation are complex, and some aspects remain to be clarified. Here, we investigated the role of the Na⁺/K⁺-ATPase pump as an electrochemical potential generator and its modulation by the second messenger cAMP in the midgut of female L. longipalpis. Our results suggest that not only may Na⁺/K⁺-ATPase be the main generator of an electrochemical potential across membranes in the midgut of female L. longipalpis, but also its activity is positively regulated by cAMP. cAMP-mediated Na⁺/K⁺-ATPase pump activity might be necessary to maintain the transport of the nutrients produced during blood digestion.     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

Feeding behavior of a potential insect pest, Lygus hesperus, on four new industrial crops for the arid southwestern USA

Camelina (Camelina sativa), guayule (Parthenium argentatum), lesquerella (Physaria fendleri), and vernonia (Centrapalus pauciflorus [formerly Vernonia galamensis]) are either under limited commercial production or being developed for production in the southwestern USA. Insect pests are a potential economic threat to all these new crops, with Lygus hesperus, the western tarnished plant bug, among the most prominent due to its regional abundance and propensity to feed on reproductive plant tissue. The objectives of this study were to establish baseline data on the feeding behavior and potential impact of L. hesperus on camelina, guayule, lesquerella and vernonia. Behavioral observations of adult females and males, and nymphs of this insect were made in the laboratory. Insects spent ≈35% of their time either probing (=tasting) or feeding on various reproductive and vegetative tissues of guayule, lesquerella or vernonia, but only 20% on camelina. When insects did probe and feed they preferred reproductive tissues, primarily flowers and siliques/achenes, and there were differences in these behaviors relative to crop but not generally to insect stage or sex. Insects probed and fed more on flower tissue of guayule and vernonia compared with camelina and lesquerella, and more on siliques of lesquerella compared with achenes of vernonia. When probing and feeding on vegetative tissue, there was generally a preference for stems compared with leaves in all crops except guayule. Results show that L. hesperus will readily feed on the economically important tissues of all crops, and although research has shown that this feeding did not consistently affect lesquerella yield, further work will be needed to determine if such feeding poses a risk to commercial production of camelina, guayule or vernonia.     

Insect pests and associated root pathogens of sainfoin in western USA

Sainfoin, Onobrychis viciifolia, Scop., is a perennial forage legume that was introduced into North America as an alternative to alfalfa, Medicago sativa L. Sainfoin does not cause bloating in livestock, is comparatively drought-resistant, and is not attacked by several economically important alfalfa insect pests. Plant survival in long-term sainfoin fields is reduced by root pathogens that invade through scars caused by root-feeding insects. Sitona scissifrons Say (Coleoptera: Curculionidae) weevils feed in sainfoin foliage and their larvae attack roots. Weevil populations were suppressed by application of carbofuran for two years in a newly established field. During the third year, there were fewer feeding scars and a lower incidence of tap-root disease in treated plots, indicating larval activity was associated with incidence of disease. In a seven-year trial near Bozeman, MT, alfalfa weevils. Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), and pea aphids, Acrythosiphon pisum (Harris) (Homoptera: Aphididae) attacked alfalfa but not sainfoin. Both crops were attacked by S. scissifrons, however, as well as by a complex group of plant bugs, including Lygus elisus VanDuzee, L. hesperus Knight, and Adelphocoris lineolatus (Geoze) (Hemiptera: Miridae). A seed-infesting insect, Bruchophagous spp. (Coleoptera: Curculionidae), was found in only sainfoin.     

Feeding disruption tests for monitoring the frequency of larval lepidopteran resistance to Cry1Ac, Cry1F and Cry1Ab

An alternative to traditional larval lepidopteran resistance-monitoring bioassays was developed. Feeding disruption tests were developed for detecting insects resistant to three Bacillus thuringiensis (Bt) proteins: Cry1Ac, Cry1F and Cry1Ab. The assays rely on a diagnostic dose of Bt toxin in 100-μl hydratable meal pads of artificial diet containing blue indicator dye. The assay was formatted as a portable (palm-sized) plastic plate containing an array of 16 test wells, each containing a single hydratable meal pad with one insect added per well. The diagnostic dose was the concentration of Bt in meal pad rehydration solution that reduced 24 h dyed fecal production of Bt-susceptible neonates to ≤2 fecal pellets per larva. Bt-resistant neonates were able to consume the diagnostic dose of the insecticidal protein and produce >2 blue fecal pellets. The feces were distinctly visible on the white background of the feeding disruption test plate. Diagnostic doses were determined with lab-strain Bt-susceptible Heliothis virescens and Helicoverpa zea. For H. virescens, the diagnostic doses were 10, 20 and 15 μg/ml for Cry1Ac, Cry1F and Cry1Ab, respectively. For H. zea, the diagnostic doses were 40, 200 and 500 μg/ml, respectively. The assays were validated against a lab-strain of Bt-resistant H. virescens and with susceptible larval H. virescens collected as eggs from field-grown tobacco in North Carolina.     

Crop hosts and genotypic resistance influence the biological activity of Bacillus thuringiensis towards Helicoverpa armigera

Studies on the influence of genotypic resistance on biological activity of a commercial formulation of Bacillus thuringiensis (Bt) and pure Bt toxin Cry1Ac were carried out to develop appropriate strategies for pod borer, Helicoverpa armigera management in chickpea, sorghum, pigeonpea and cotton. The interaction effects of host plant resistance and biological activity of commercial Bt/Cry1Ac were studied by incorporating the lyophilized tissues of chickpea leaves, milk stage sorghum grain, pigeonpea pods and cotton squares into the artificial diet with and without Bt formulation or Cry1Ac. The H. armigera larval weights were significantly lower in insects reared on diets with square powder of the insect - resistant Bt-cotton RCH 2 + Bt/Cry1Ac and pod powder of insect - resistant pigeonpea genotype, ICPL 332WR + Bt/Cry1Ac as compared to the larvae reared on diets with leaf powder of H. armigera susceptible chickpea genotype, ICCC 37 and the standard artificial diet. Pupation and adult emergence were significantly lower in insects reared on diets with tissues of pod borer-resistant genotypes + Bt/Cry1Ac as compared to insects reared on diets with tissues of the insect susceptible genotypes + Bt/Cry1Ac. Insects reared on diets containing insect-resistant and -susceptible genotypes of sorghum, pigeonpea and cotton and pod borer-resistant genotype of chickpea (ICC 506EB) + Bt/Cry1Ac did not lay any eggs. However, eggs were laid by the insects reared on diets containing pod borer-susceptible genotype of chickpea, ICCC 37 and on the standard artificial diet + Bt/Cry1Ac. The insects reared on diets with sorghum genotype, ICSV 745, and Bt-cotton, RCH 2 without Bt/Cry1Ac also did not lay eggs. The results suggested that Bt/Cry1Ac is more effective for management of H. armigera when deployed in combination with insect-resistant genotypes of cotton, chickpea, pigeonpea and sorghum.     

Southwestern corn borer damage and aflatoxin accumulation in conventional and transgenic corn hybrids

Southwestern corn borer (Diatraea grandiosella Dyar) is a major pest of corn (Zea mays L.) in the southern United States. In addition to the direct yield losses caused by southwestern corn borer, larval feeding on developing ears provides a site for fungi to enter the ear. Aspergillus flavus Link: Fries infection and the subsequent accumulation of aflatoxin in corn grain are major limitations to profitable corn production in the southern United States. This investigation was conducted to determine the effectiveness of transgenic corn hybrids expressing the δ-endotoxin insecticidal (CryIAb) proteins isolated from Bacillus thuringiensis (Bt) in reducing southwestern corn borer damage and aflatoxin accumulation. Ear damage and aflatoxin accumulation were compared among 10 pairs of conventional nonBt and transgenic Bt corn hybrids following infestation with southwestern corn borer and inoculation with A. flavus using kernel-wounding and nonwounding techniques. Both nonBt and Bt hybrids exhibited high levels of aflatoxin accumulation when inoculated with a kernel-wounding technique. When inoculated with a non-wounding technique and infested with southwestern corn borer, aflatoxin accumulation was significantly higher in nonBt than Bt hybrids. Aflatoxin accumulation was also significantly higher for nonBt hybrids inoculated with A. flavus and infested with southwestern corn borer than for hybrids that were only inoculated with A. flavus. Southwestern corn borer larval establishment was significantly higher on nonBt hybrids than on Bt hybrids. Larval survival was extremely low on the Bt hybrids. The results of this investigation indicate that these Bt hybrids should be effective in reducing aflatoxin contamination in areas where high southwestern corn borer infestations occur. The reduced levels of aflatoxin accumulation associated with Bt hybrids are likely a consequence of reduced insect damage rather than resistance to A. flavus infection or aflatoxin accumulation per se.     

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus.     

Do Sesamia nonagrioides (Lepidoptera; Noctuidae) Gravid Females Discriminate Between Bt or Multivitamin Corn Varieties? Role of Olfactory and Visual Cues

The Mediterranean corn borer, Sesamia nonagrioides Lefèbvre, is a key pest of corn and a main target of Bacillus thuringiensis (Bt) corn in Northeast Spain. Trends for future biotechnology crops indicate that Bt, non-Bt, and stacked corn varieties with metabolic pathways for vitamin-increased traits could coexist in same region. Knowledge of the oviposition response of gravid females of S. nonagrioides to these different varieties could be extremely important for managing strategies aimed for delaying resistance development. In dual-choice assays, we examined the host preference of gravid females of S. nonagrioides for four corn varieties: a new transgenic corn with increased vitamin levels, its near isogenic counterpart (M37W), a Bt corn plant, and its near isogenic counterpart. Olfactory cues were the predominant ones when gravid females looked for a suitable host to lay eggs, and no synergistic effects were observed when both visual and olfactory cues were present. When the plant was visible, the females preferred the odors emitted by the nontransgenic to its multivitamin transgenic counterpart and when they only could detect the volatiles they also preferred the nontransgenic M37W variety to the Bt corn variety. If gravid females are less attracted to corn with an increased level of vitamins, this could impact insect resistance management and the value of refuge plants, if such traits are stacked with an insect resistance trait.     

Interaction between host plant resistance and biological activity of Bacillus thuringiensis in managing the pod borer Helicoverpa armigera in chickpea

The legume pod borer Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) has developed high levels of resistance to conventional insecticides, and therefore, efforts are being made to develop transgenic chickpea expressing toxin genes from the bacterium Bacillus thuringiensis (Bt) for controlling this pest. However, there is an apprehension that acid exudates in chickpea might interfere with the biological activity of Bt. Therefore, we studied the biological activity of Bt (Biolep^R) on four chickpea genotypes with different levels of resistance to H. armigera under field conditions, and by incorporating lyophilized leaf and pod tissue into the artificial diet with and without Bt. The pH of the acid exudates varied from 2.1 to 2.9, and malic and oxalic acids were the major components of the acid exudates in different chickpea genotypes. There was no survival of H. armigera larvae in chickpea plants sprayed with 0.1, 0.2 and 0.5% Bt. There was a significant reduction in larval survival, larval and pupal weights and fecundity, and prolongation of larval and pupal periods in chickpea plots sprayed with Bt (0.05%) as compared to the unsprayed plots. Biological activity of Bt was lower on artificial diets with leaf or pod powder of chickpea genotypes, which might be because of a low intake of Bt toxins due to the antifeedant effects of acid exudates in the chickpea or reduction in biological activity of Bt due to the interaction of biochemical constituents in chickpea with the Bt toxins. Larval survival, larval and pupal weights, pupation and adult emergence were significantly lower on diets with leaf or pod powder of the H. armigera-resistant genotypes than on the susceptible check. Chickpea genotypes with resistance to H. armigera acted in concert with Bt to cause adverse effects on the survival and development of this insect. The results suggested that development of transgenic chickpeas expressing toxin genes form Bt will be quite effective for controlling of the pod borer, H. armigera.     

No fitness costs associated with resistance of Busseola fusca (Lepidoptera: Noctuidae) to genetically modified Bt maize

A concern regarding planting of Bt crops is that their widespread cultivation could lead to evolution of insect resistance to Bt toxins. In South Africa, the noctuid maize stem borer (Busseola fusca [Fuller]), is resistant to Bt maize (Zea mays L.; MON810) which produces Cry1Ab protein. The presence of fitness costs in resistant populations could be a valuable component of resistance management since the non-Bt maize refuge may select against resistance. The aim of the study was to determine if there are fitness costs associated with Bt resistance of B. fusca. Life history parameters were compared between individuals of a Bt maize resistant B. fusca population when feeding on Bt or non-Bt maize. Similar comparisons were done using a control population. Field collected larvae as well as their F₁-generation were used in the study. The following parameters were compared: pupal mass, moth longevity, fecundity, fertility, larval mass and survival, and sex ratio. Except for LT50-values, no fitness costs were associated with the resistance trait in the highly resistant B. fusca population. The absence of fitness costs and presence of resistant populations may promote the use of a multi-gene strategy which would be expected to impact negatively on fitness.     

Effect of Teflubenzuron Ingestion on Larval Performance and Chitin Content in <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis>

The potential of teflubenzuron was assessed in a series of laboratory studies in order to achieve consistent, long-term, integrated management of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Teflubenzuron exhibited excellent stomach toxicity to the larvae. Its larvicidal activity was comparable with those of cyhalothrin, chlorantraniliprole and spinosad. Moreover, the teflubenzuron-fed larvae consumed less foliage, grew slower, and needed a longer period to develop, in a dose dependent manner. Most of these larvae died during larval-larval molting, larval-pupal ecdysis or adult emergence. Furthermore, chitin contents in body carcass (without midgut) and integument specimen of the teflubenzuron-fed larvae significantly decreased, whereas the chitin amount in the midgut peritrophic matrix was not affected. In addition, uridine diphosphate-N-acetylglucosamine-pyrophosphorylase gene (LdUAP1), which was mainly responsible for chitin biosynthesis in ectodermally-derived tissues, was suppressed after teflubenzuron ingestion, in contrast to its partner LdUAP2 for chitin formation in the midgut peritrophic matrix. In a word, by inhibition of chitin production in ectodermally-derived tissues, teflubenzuron is an effective benzoylurea insecticide to L. decemlineata larvae. It can be a valuable tool in effective integrated pest management and insecticide resistance management programs against L. decemlineata.     

Galleria mellonella (Lepidoptera: Pyralidae) Hemocytes Release Extracellular Traps That Confer Protection Against Bacterial Infection in the Hemocoel

Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.     

Herbivory tolerance of cotton expressing insecticidal proteins from Bacillus thuringiensis: responses to damage caused by Helicoverpa spp. and to manual bud removal

Transgenic cotton varieties expressing Cry IA(c) insecticidal proteins from Bacillus thuringiensis (`Bt cotton') remain vulnerable to non-lepidopteran insects. In addition, they are susceptible to lepidopteran pests when the efficacy of Bt toxins falls because of ontogenetic and/or environmental factors. Hence the importance of knowing to what extent Bt cotton is able to tolerate damage. The degree of tolerance of Bt cotton to actual and simulated insect damage was assessed in three field experiments. Exp. 1 compared the effects of Helicoverpa spp. (Lepidoptera: Noctuidae) on the growth, development and yield of Bt cotton with those of its near isogenic non-Bt counterpart in two genetic backgrounds (Siokra V15, Sicala V2) under two regimes of chemical control of insects (S1: nine insecticide applications during the growing cycle, S2: six insecticide applications). Exp. 2 compared insecticide-protected Bt crops with crops manually damaged to simulate (a) early-season loss of vegetative buds, (b) loss of flowerbuds, and (c) loss of both vegetative and reproductive buds. Also using manual damage, Exp. 3 evaluated the effect of timing of flowerbud loss on the yield and maturity time of insecticide-protected Bt crops. In Exp. 1, well-protected Bt crops (S1) yielded 24% more than their less-protected counterparts (S2). The less protected crops had, however, substantially more immature fruit at the end of the season highlighting a considerable potential for recovery. Poor soil conditions, interacting with season length accounted for the difference between potential and actual compensation in crops that were exposed to almost continuous damage by Helicoverpa spp. Under more favourable growing conditions, maturity was delayed but yield of damaged Bt crops was unaffected by discrete episodes of simulated herbivory (Exps. 2 and 3). The introduction of B. thuringiensis genes into cotton does not seem to have reduced the considerable capacity of the crop to tolerate insect damage, and this attribute should be considered in the development of pest management strategies for Bt crops.     

Monte Carlo simulation method for forecasting the timing of pest insect attacks

The timing of attack by pest insects can vary greatly both from region to region and from year to year because the rates at which insects complete their life cycles depend mainly on temperature. In addition, variation between individual insects in their rate of development can lead to a spread of activity. A simulation method, based on rates of insect development, has been produced for forecasting the timing of attack by pest insects. Variability is incorporated using the ‘same-shape property’ which implies that the coefficient of variation of the rate of insect development is independent of temperature. The method is feasible because it uses a fixed number of individuals from one generation to the next and simulates the timing of events rather than the population dynamics of the insects. The method is sufficiently flexible to allow insect resting phases to occur at the appropriate instant for each individual. Its effectiveness in predicting the behaviour of the cabbage root fly (Delia radicum), the carrot fly (Psila rosae), the bronzed blossom beetle (Meligethes aeneus) and the large narcissus fly (Merodon equestris) is demonstrated for one site in one season. To be of practical use, the method has been designed to use standard meteorological data. Estimation of soil temperatures at 6 cm and the derivation of equations for estimating diurnal variation are described in two appendices. The basis of the method is the integration of a temperature-dependent rate function over time. A third appendix describes a quicker and more accurate method of integration than that usually employed in pest forecasting work.     

Comparison of the potential of organophosphorous insecticides and soaps in conifer aphid control

A comparison of the toxicity to two aphids, Elatobium abietinum and Adelges cooleyi, and a coccinellid predator, A. obliterata, of dimethoate, fenitrothion and malathion with Safer's insecticidal soap (SIS) and saponified corn oil and sunflower oil confirmed that the organophosphates were more toxic. This did not detract from the effectiveness of the soaps under field conditions provided that appropriately higher concentrations were applied. SIS was not more toxic to the insects than the saponified oils with corn oil soap, which is rich in oleic and linoleic acid, frequently producing higher insect mortality levels. In E. abietinum a linear relationship was demonstrated between insecticide susceptibility and aphid instar, with late instars apparently becoming progressively more susceptible. This relationship disappeared when LC₅₀ values were corrected for size differences. Larval Aphidecta obliterata were more susceptible than adults and field applications of these chemicals should be timed to avoid the larval feeding period. Phytotoxic symptoms appeared on Sitka spruce needles after application of sunflower oil soap, current year needles being particularly susceptible to damage. Corn oil soap or SIS could be substituted during this susceptible period.     

Microbial control of the European skipper, Thymelicus lineola Ochs.

A research programme was initiated to develop biological control methods for the European skipper, Thymelicus lineola Ochs. (Lepidoptera: Hesperiidae) after its appearance in the Province of Quebec. A baculovirus and Bacillus thuringiensis could both be used to control T. lineola in the field. With B. thuringiensis a dose of 2·5 × 10⁹ International Units/ha resulted in 100% mortality of second- to fourth-instar larvae in 6–15 days; dosages of 5 and 10 × 10⁹ I.U./ha caused 100% mortality within 3–6 days. Field application of the nuclear polyhedrosis virus caused 50, 80 and 100% larval mortality after 15, 19 and 27 days respectively. B. thuringiensis provided a curative method of control, whereas the virus can be used either as a curative or as a preventive method of controlling T. lineola because it is transmitted to the progeny. It is suggested that the best way to control T. lineola is by the combined use of both micro-organisms.