Effect of Different Wheat-Based Diets and Corn Meal Addition on Development Parameters of Ephestia kuehniella (Zeller, 1879) (Lepidoptera: Pyralidae).

The expansion of Integrated Pest Management (IPM), including biological control, has had several positive consequences for the agricultural environment and participants in the production chain. To enable successful operation and applications of biological control, production of insects used for rearing natural enemies (parasitoids and predators) must be optimized to reduce time and costs and improve production both qualitatively and quantitatively. The present study evaluated the effect of wheat varieties, the main component of artificial diets for Ephestia kuehniella, on the reproductive performance and biological parameters of this flour moth, which is used for mass production of Trichogramma spp. (Hymenoptera: Trichogrammatidae) and other parasitoids and predators. Four varieties of wheat were compared: BRS 327, BRS Marcante, BRS Parrudo, and KBR, with and without the addition of corn E. kuehniella reared on 97% BRS 327 wheat flour + 3% nutritional yeast had the best biological parameters and substitution of corn for about half of the wheat increased the number of eggs per female.

Selection and Suitability of an Artificial Diet for Tuta absoluta (Lepidoptera: Gelechiidae) Based on Physical and Chemical Characteristics.

Tuta absoluta (Meyrick, 1917) is a key tomato pest in South America and, recently, in Europe and Africa. To develop efficient control methods for this pest, adequate rearing protocols are desirable. As an alternative to tomato leaves (natural diet), we evaluated four artificial diets. Biological traits including larval and pupal viability and development time, pupal weight and deformations were assessed. Additionally, the optimum container size and larval density were evaluated. The diet based on casein, wheat germ and cellulose allowed the best development of T. absoluta, showing higher viability and no negative effects on larval instars and pupal weight. The best container was a glass tube measuring Ø 1 × h 6 cm, topped with waterproof cotton, with a density of three larvae. To evaluate the suitability of this diet, T. absoluta was reared during eight generations and life-table parameters were estimated for the F1, F3, F6, and F8 generations. The total viability (egg-adult) increased over the generations, reaching 75% in the eighth generation. Based on life-table estimations no differences among generations were found. The net reproductive rate (Ro) was higher than 40, the intrinsic rate of increase (rm) ranged between 0.08 and 0.11, the finite rate of increase (λ) was 1.1, the mean generation time (T) have a maximum of 44 d and doubling time ranged from 5.89-8.32 generations. These results indicated that a diet based on casein, wheat germ and cellulose was suitable for T. absoluta rearing in laboratory conditions.

Relative Fitness of Helicoverpa armigera (Lepidoptera: Noctuidae) on Seven Host Plants: A Perspective for IPM in Brazil.

The cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) is a widespread pest of many cultivated and wild plants in Europe, Africa, Asia, and Australia. In 2013, this species was reported in Brazil, attacking various host crops in the midwestern and northeastern regions of the country and is now found countrywide. Aiming to understand the effects of different host plants on the life cycle of H. armigera, we selected seven species of host plants that mature in different seasons and are commonly grown in these regions: cotton (Gossypium hirsutum, "FM993"), corn (Zea mays, "2B587"), soybean (Glycine max, "99R01"), rattlepods (Crotalaria spectabilis), millet (Pennisetum glaucum, "ADR300"), sorghum (Sorghum bicolor, "AGROMEN70G35"), and cowpea (Vigna unguiculata, "SEMPRE VERDE"). The development time of immatures, body weight, survivorship, and fecundity of H. armigera were evaluated on each host plant under laboratory conditions. The bollworms did not survive on corn, millet, or sorghum and showed very low survival rates on rattlepods. Survival rates were highest on soybean, followed by cotton and cowpea. The values for relative fitness found on soybean, cotton, cowpea, and rattlepods were 1, 0.5, 0.43, and 0.03, respectively. Survivorship, faster development time, and fecundity on soybean, cotton, and cowpea were positively correlated. Larger pupae and greater fecundity were found on soybean and cotton. The results indicated that soybean, cotton, and cowpea are the most suitable plants to support the reproduction of H. armigera in the field.

Lethal and Sublethal Impacts of Acaricides on Tamarixia radiata (Hemiptera: Eulophidae), an Important Ectoparasitoid of Diaphorina citri (Hemiptera: Liviidae).

The use of synthetic acaricides for management of pest mites may alter the efficacy of the ectoparasitoid Tamarixia radiata (Waterston) in biological control of Diaphorina citri Kuwayama, the vector of the bacteria associated with huanglongbing (HLB) in citrus orchards. We evaluated the toxicity of 16 acaricides that are recommended for the control of citrus-pest mites to T. radiata. Acrinathrin, bifenthrin, carbosulfan, and fenpropathrin caused high acute toxicity and were considered harmful (mortality >77%) to T. radiata. Abamectin, diflubenzuron, etoxazole, fenbutatin oxide, fenpyroximate, flufenoxuron, hexythiazox, propargite, spirodiclofen, and sulfur caused low acute toxicity and affected the parasitism rate and emergence rate of adults (F1 generation), and were considered slightly harmful to T. radiata. Dicofol and pyridaben did not affect the survival and action of the ectoparasitoid, and were considered harmless. In addition to its acute toxicity, carbosulfan caused mortality higher than 25% for >30 d after application, and was considered persistent. Acrinathrin, bifenthrin, fenpropathrin, fenpyroximate, propargite, and sulfur caused mortalities over 25% until 24 d after application and were considered moderately persistent; abamectin was slightly persistent, and fenbutatin oxide was short lived. Our results suggest that most acaricides used to control pest mites in citrus affect the density and efficacy of T. radiata in the biological control of D. citri. However, further evaluations are needed in order to determine the effect of these products on this ectoparasitoid under field conditions.

Control of Diatraea saccharalis by the endophytic Pantoea agglomerans 33.1 expressing cry1Ac7.

Despite the fact that Bacillus thuringiensis (Bt) is found in more than 90 % of the products used against insects, it has some difficulty reaching the internal regions where the larvae feed. To solve this problem, many genetically modified microorganisms that colonize the same pests have been developed. Thus, the endophytic bacterium Pantoea agglomerans (33.1), which has been recently described as a promising sugarcane growth promoter, was genetically modified with the pJTT vector (which carries the gene cry1Ac7) to control the sugarcane borer, Diatraea saccharalis. Firstly, the bioassays for D. saccharalis control by 33.1:pJTT were conducted with an artificial diet. A new in vivo methodology was also developed, which confirmed the partial control of larvae by 33.1:pJTT. The 33.1:pJTT strain was inoculated into sugarcane stalks containing the D. saccharalis larvae. In the sugarcane stalks, 33.1:pJTT was able to increase the mortality of D. saccharalis larvae, impair larval development and decrease larval weight. Sugarcane seedlings were inoculated with 33.1:pJTT, and re-isolation confirmed the capacity of 33.1:pJTT to continuously colonize the sugarcane. These results prove that P. agglomerans (33.1), a sugarcane growth promoter, can be improved by expressing the Cry protein, and the resulting strain is able to control the sugarcane borer.

Biology of the Huanglongbing vector Diaphorina citri (Hemiptera: Liviidae) on different host plants.

Although many studies have been conducted on the development and reproductive potential of Diaphorina citri Kuwayama, 1908 (Hemiptera: Liviidae) in different host species, few have evaluated these parameters on different varieties of the same host species. This study evaluated the influence of five commercial varieties of citrus (Citrus spp. L.)--Hamlin, Natal, Pêra, Ponkan, and Valencia-and orange jasmine [Murraya exotica (L.) Jack] on the development of D. citri. Survival rates for the egg stage were highest on orange jasmine (85.7%) and on Valencia (83.3%). The lowest viability of the nymphal stage was also observed on Hamlin, averaging 57.4%. Values for total viability ranged from 65.9 to 32.6%, and were highest on Valencia. The longest egg-adult development time was on Natal, with a mean of 18.4 d; the shortest total development time was on orange jasmine, with a mean of 17.3 d. Based on the fertility life table, the net reproductive rate (Ro) of D. citri was 2.5 times higher when reared on Valencia than on Hamlin. The other parameters (duration of each generation [T], finite rate of increase [lambda], and innate capacity to increase in number [r(m)]) also demonstrated that Valencia is best suited to this insect. The results obtained for the biological parameters and the fertility life table indicate that Valencia and orange jasmine were the most suitable hosts, whereas Hamlin was least suitable for the development of D. citri. These results provide information for the installation of new citrus groves, especially in the choice of varieties to be planted and the location of different varieties within the groves, with a view toward the management of Huanglongbing or HLB.

Trichogramma pretiosum parasitism and dispersal capacity: a basis for developing biological control programs for soybean caterpillars.

In order to succeed in biological control programs, not only is it crucial to understand the number of natural enemies to be released but also on how many sites per area this releasing must be performed. These variables might differ deeply among egg parasitoid species and crops worked. Therefore, these trials were carried out to evaluate the parasitism (%) in eggs of Anticarsia gemmatalis and Pseudoplusia includens after the release of different densities of the egg parasitoid Trichogramma pretiosum. Field dispersal was also studied, in order to determine appropriate recommendations for the release of this parasitoid in soybean fields. The regression analysis between parasitism (%) and densities of the parasitoid indicated a quadratic effect for both A. gemmatalis and P. includens. The maximum parasitism within 24 h after the release was reached with densities of 25.6 and 51.2 parasitoids per host egg, respectively, for the two pests. Parasitism of T. pretiosum in eggs of P. includens decreased linearly as the distance of the pest eggs from the parasitoid release sites increased. For P. includens, the mean radius of T. pretiosum action and the area of parasitoid dispersal in the soybean crop were 8.01 m and 85.18 m2, respectively. We conclude that for a successful biological control program of lepidopteran pests using T. pretiosum in soybean fields, a density of 25.6 parasitoids per host egg, divided into 117 sites per hectare, should be used.

Practical and Simple Systems for Food Intake Studies on Phloem-Sucking Insects.

Studies of insect feeding behavior are useful in different areas of entomology such as plant resistance, biology, and insecticide efficacy. For chewing insects, this kind of technique is well established, but for sap-sucking insects, especially tiny ones such as whiteflies, aphids, and psyllids, these tests can be laborious. Manipulation is difficult and can damage the plant, affecting the results. We describe here three types of cages for tests with small insects, one for seedlings in pots, a second for larger plants or plants in the field, and a third for caging insects on part of a leaf. These cages have been useful for different types of studies in addition to feeding behavior and can facilitate research with small phytophagous sucking insects.

The Enemy is Outside: Releasing the Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in External Sources of HLB Inocula to Control the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Liviidae).

Huanglongbing (HLB), the most destructive citrus disease worldwide, was first recorded in Brazil in 2004, and since then, more than 50 million trees identified with this disease have been eliminated. The disease is managed mainly by controlling the psyllid vector Diaphorina citri Kuwayama, 1908 (Hemiptera: Liviidae). Although the presence of the insect in commercial citrus groves is low, HLB infection rates increase in areas bordering the groves. The disease is transmitted by psyllids from host citrus plants in areas outside the managed groves, such as abandoned or organic groves and residential trees, and from orange jasmine plants in urban settings. In order to provide information to support HLB control, this study evaluated the biotic and abiotic variables that affect the dynamics of D. citri populations after releases of the parasitoid wasp Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae) in external sources of HLB inocula. The study was divided into two parts. After releasing the parasitoids in non-commercial areas, we determined the following: (a) the variables that significantly affected the number of nymphs collected on shoots in the same non-commercial area; (b) the variables that significantly affected the number of adult psyllids collected in a neighboring commercial citrus area. Our results indicated that the number of nymphs in external areas was affected only by the host plant and rainfall. However, periodic parasitoid releases significantly reduced the number of adult psyllids collected in the commercial area. The results indicate that the release of parasitoids in external sources of inocula has the potential to maximize actions for D. citri control, contributing to the reduction of psyllid populations in commercial areas. Consequently, this strategy may help to manage the disease infection without an increase in insecticide use.

Temperature Thresholds and Thermal Requirements for Development and Survival of Dysmicoccus brevipes (Hemiptera: Pseudococcidae) on Table Grapes.

Temperature is an important climate factor that has a direct influence on insect biology and consequently a crucial role in forecasting and integrated pest management. The mealybug Dysmicoccus brevipes (Cockerell) (Hemiptera: Pseudococcidae) is one of the most common species in Brazilian vineyards. Here, development and survival of D. brevipes on leaves of table grapes (Vitis vinifera cv. Itália) were studied at five constant temperatures (15, 20, 25, 28, and 30 ± 1°C) under laboratory conditions. We investigated the developmental time and nymphal survival, temperature thresholds, and the degree-day requirements for each life stage of D. brevipes. The developmental time for the nymphal stage ranged from 32 to 130 days with decreasing the temperature, with the temperatures of 28 and 30°C providing the shorter developmental time. Survival of the entire nymphal stage was affected by the temperature, ranging from 81% at 20°C to 1% at 15°C. The predicted upper lethal temperature was 34.5°C, while the optimal temperature was 28.6°C. The minimum threshold for total development of D. brevipes occurred at 8.2°C. Dysmicoccus brevipes required 678.4 degree-days to complete development from first-instar nymph to adult. The temperature of 30°C was the most suitable for the development of D. brevipes. The thermal thresholds estimated for D. brevipes life cycle suggest that this species can develop in different table grape-producing regions of Brazil, in a temperature range between 8 and 35°C.

Comparing Potential as Biocontrol Agents of Two Neotropical Parasitoids of Liriomyza sativae.

We evaluated the potential of two parasitoids as biocontrol agents of Liriomyza sativae Blanchard in northeastern Brazil. The two species were the koinobiont larval-pupal endoparasitoid Phaedrotoma scabriventris Nixon (Braconidae) and the idiobiont larval endoparasitoid Chrysocharis vonones (Walker) (Eulophidae). The biological parameters evaluated were survivorship, parasitism, and host-feeding, at 25 and 30°C. Differences between the species were observed at 25°C, but not at 30°C. At 25°C, the total parasitism for P. scabriventris (196.1 ± 17.7) and C. vonones (176.6 ± 7.24) was similar and with higher values compared to 30°C, 102.5 ± 8.81 and 89.1 ± 5 66 parasitized larvae, respectively. However, C. vonones showed a 3.97 lower survivorship as well as higher daily parasitism (1.4-fold) and host-feeding means (1.9-fold) than the braconid at 25°C. The results indicate a potential for both natural enemies to be used as biocontrol agents of L. sativae. The differences between species detected at 25°C suggest the best conditions for the application of each parasitoid.

Potential of Two Trichogrammatidae species for Helicoverpa armigera control.

Helicoverpa armigera (Hübner) has attacked several economically important crops since it was first reported in Brazil in 2013. Because insecticides have been used intensively to control H. armigera, resistant populations have been detected more frequently. One alternative to insecticides is the use of biological control with egg parasitoids from the family Trichogrammatidae (Hymenoptera). The present study evaluated the use of different species and strains of these parasitoids to control H. armigera in soybean. Five strains of Trichogramma pretiosum (Riley): RV (Rio Verde, Goiás, Brazil), SO (Sorriso, Mato Grosso, Brazil), 47A (Irvine, CA, USA), PR (Colombo, Paraná, Brazil), and AN (Anhumas, São Paulo, Brazil), and Trichogrammatoidea annulata (De Santis) denominated TR, from Anhumas, were used. The following parasitoid biological parameters were evaluated: parasitism rates (%), offspring viability (%) number of parasitoids emerged per egg, egg-adult duration (days), female longevity, and offspring sex ratio. Strain RV of T. pretiosum from central Brazil performed best.

Lethal and Sublethal Toxicity of Insecticides to the Lacewing Ceraeochrysa Cubana.

The lethal and sublethal effects of 11 insecticides on the predator Ceraeochrysa cubana (Hagen) were assessed under laboratory conditions. First-instar larvae and adults ≤ 48 h old were sprayed with the highest insecticides doses allowed to control Diaphorina citri Kuwayama in the citrus crop. The survival and duration rates of the different development stages, sex ratio, pre-oviposition period, fecundity, and fertility of the insects were evaluated. In the larval bioassay, chlorpyrifos and malathion had lethal effect which none larvae survived. Azadirachtin, lambda-cyhalothrin + chlorantraniliprole, lambda-cyhalothrin + thiamethoxam, and thiamethoxam had lethal and sublethal effects that did not allow to estimate the life table parameters because the low number of couples formed. Esfenvalerate, imidacloprid WG and SC, phosmet, and pyriproxyfen had sublethal effects which were reflected in the net reproductive rate and in the intrinsic rate of natural increase. In bioassay using adults, none of the individuals survived in the chlorpyrifos, lambda-cyhalothrin + chlorantraniliprole, lambda-cyhalothrin + thiamethoxam, malathion, or thiamethoxam treatments, and the azadirachtin, esfenvalerate, imidacloprid WG and SC, phosmet, and pyriproxyfen treatments were significantly lower compared to the control. None of the insecticides was harmless to first-instar larvae and adults of C. cubana under laboratory conditions showing their potential to reduce the efficiency of this predator.

Selection of an Artificial Diet for Laboratory Rearing of Opogona sacchari (Lepidoptera: Tineidae) (Bojer, 1856).

The banana moth Opogona sacchari (Bojer) (Lepidoptera: Tineidae) is a polyphagous pest that can cause serious damage, in particular to banana crops in southern Brazil. The insect is a quarantine pest in several countries, including Argentina, the main consumer market for bananas from southern Brazil. Little information is available about the biology and ecology of this moth, such as a suitable diet for laboratory rearing. In order to provide support for integrated pest management of the pest, this study furnished data for selecting two diets suitable for continuous laboratory rearing of O. sacchari, one based on dried beans, wheat germ, soy bran, brewer's yeast, and casein and another diet with wheat germ and casein as protein sources. With both diets, the viability of the egg-adult period exceeded 68%, with fertility over 338 eggs per female. A corrected biotic potential analysis gave similar values for the two diets.

Platynota rostrana (Walker) (Tortricidae) AND Phidotricha erigens Raganot (Pyralidae): artificial diet effects on biological cycle.

The lepidopterans Platynota rostrana (Walker) (Tortricidae) and Phidotricha erigens Raganot (Pyralidae) have been found frequently in citrus groves in São Paulo State in recent years. Since in Brazil, the fertility cycle of these two species is largely unknown, as are details of the damage wrought by them in crops, this research studied these aspects of the two species, which were kept under laboratory conditions (temperature 25 +/- 2 degrees C, 70 +/- 10% RH, 14 h photophase) and on an artificial diet. The duration of the biological cycle (egg-adult) for P. rostrana was 38.3 days and total viability was 44.0%; for P. erigens these values were 32.5 days and 63.6%, respectively. Both species showed five larval instars. Females of P. rostrana laid an average of 308 eggs, whereas those of P. erigens laid an average of 106 eggs. In both species, female pupae were heavier than males. Male and female longevity for both species was nearly 10 days. Based on the data obtained, the artificial diet produced better results in P. rostrana than in P. erigens. If these species, which have the potential to reach pest status in the citrus groves of São Paulo State, could be reared on an artificial diet, research on their control by alternative methods would be easier.

Effects of Self-Superparasitism and Temperature on Biological Traits of Two Neotropical Trichogramma (Hymenoptera: Trichogrammatidae) Species.

It is common for a female trichogrammatid to lay more than one egg per host, a phenomenon known as self-superparasitism, which exposes her offspring to intraspecific, intrinsic competition (IIC) with its own siblings. Information about how often self-superparasitism occurs and how IIC interacts with abiotic factors is rare, especially regarding the Neotropical Trichogramma species. Here we determined the frequency of self-superparasitism in Trichogramma atopovirilia Oatman & Platner (Ta) and T. pretiosum Riley (Tp), and the effects of IIC and temperature on the sex ratio, egg-to-adulthood period, and survivorship of both species' offspring. Individual females were offered eggs of Spodoptera frugiperda (J.E. Smith) for 30 min. A group of parasitized hosts was then dissected for determination of the self-superparasitism frequency, while another group was incubated at 15, 18, 21, 24, 27, 30, and 33°C. High rates of self-superparasitism were found in both Ta (0.55 ± 0.07) and Tp (0.62 ± 0.06). IIC interacted with temperature decreasing Ta and Tp's survivorship, lengthening the egg-to-adulthood period in Tp and shortening it in Ta, and balancing Ta's sex ratio. Based on survivorship rate, Ta and Tp could not be differentiated if their immatures develop in absence of IIC. However, in its presence, Tp was 3 × more likely to survive than Ta at 33°C, while at 15°C Ta survived 2× better than Tp These results show that self-superparasitism can be very common in both Ta and Tp, and that its effects on the species' biological traits and competitiveness strongly depend on the IIC-temperature interaction.

Adult Citrus Leafminers (Phyllocnistis citrella) Are Not Efficient Vectors for Xanthomonas axonopodis pv. citri.

Interest in the interaction between the citrus leafminer (Phyllocnistis citrella) and citrus bacterial canker, caused by Xanthomonas axonopodis pv. citri, has increased as a greater incidence and severity of canker-diseased plants was observed in groves infested with the citrus leafminer. To determine whether adults of the citrus leafminer could act as vectors of citrus canker, we investigated two potential mechanisms for direct spread by leafminer adults using experimental microcosms. First, adult leafminers were raised on canker-infected foliage and were allowed to mate and lay eggs on healthy plants. These plants then were observed for development of citrus canker symptoms. In a second set of experiments, adults raised on healthy plants were given free access to canker-diseased plants during the period in which they mated and laid eggs on healthy plants. In all, 3,119 mines were produced by developing larvae on a total of 2,384 leaves examined for citrus canker symptoms. No symptoms of citrus bacterial canker disease were observed on any of the healthy test plants in 37 independent experimental trials conducted to test these two potential mechanisms of spread of citrus canker, and the pathogen was not recovered from insects exposed to symptomatic Rangpur lime plants. The upper limit on the rate of transmission was estimated to be less than 0.2% per oviposition event based on the binomial probability distribution. However, when adult P. citrella insects were artificially contaminated with high levels of X. axonopodis pv. citri, transmission to Rangpur lime plants with the induction of citrus canker was observed. This suggests that the ability of P. citrella to transmit X. axonopodis pv. citri is limited by the rate at which it can acquire inoculum from infected plants. The results support the conclusion that adult citrus leafminers are not efficient vectors for citrus canker bacteria, and the disease is unlikely to be spread this way.

Behavior of Trichogramma atopovirilia Oatman & Platner and T. pretiosum Riley (Hymenoptera: Trichogrammatidae) on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) egg masses.

In this study we analyzed the impact of physical barriers of Spodoptera frugiperda (J. E. Smith) egg-masses on the behavior of Trichogramma atopovirilia Oatman & Platner and Trichogramma pretiosum Riley. The duration of drumming, drilling, oviposition, period spent over the egg-mass, and interval between parasitized eggs were timed, and the number of parasitized eggs were recorded. The presence of scales on the egg-masses caused a significant increase in the time spent by both parasitoids on each process and a decrease in the residence time over the egg-mass and in the number of parasitized eggs, with an increase in the number of egg layers. There was a significant decrease in the number of parasitized eggs in relation to egg-masses with one layer and no scales. We observed that the physical barriers in fall armyworm egg-masses changed the behavior of T. atopovirilia and T. pretiosum, affecting their parasitization capacity.

Sensilla on the antenna and ovipositor of the parasitic wasps trichogramma galloi Zucchi and T. pretiosum Riley (Hym., Trichogrammatidae).

The understanding of the stimuli perceived by these parasitoids to accept and exploit a potential host may support the development of artificial hosts for their in vitro rearing. The most common structures involved in the host selection process in parasitoids are the antenna and the ovipositor. Sensilla present on these structures are involved in host recognition and acceptance, and the identification of which kind of stimulus (physical or chemical) is perceived by the sensilla could provide data about the host characteristics that elicit the parasitism behavior. The antenna and ovipositor of the wasps Trichogramma galloi and T. pretiosum were studied to identify their sensillar structure and their possible functions in the host selection process, as a support to the development of an artificial host for the in vitro rearing of these parasitoids. Seven sensilla and one setiform structure were present on the antenna of both parasitoid species, with a mixed, chemo-, or mechanoreception function. The ovipositor has four different sensilla and their role in the host evaluation is suggested.

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases.

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.