Heliothis Virescens Research Articles

Parasitic Wasp Mediates Plant Perception of Insect Herbivores.

Microplitis croceipes is a solitary parasitoid that specializes on noctuid larvae of Helicoverpa zea and Heliothis virescens. Both the parasitoid and its hosts are naturally distributed across a large part of North America. When parasitoids deposit their eggs into hosts, venom and polydnaviruses (PDVs) are also injected into the caterpillars, which can suppress host immune responses, thus allowing parasitoid larvae to develop. In addition, PDVs can regulate host oral cues, such as glucose oxidase (GOX). The purpose of this study was to determine if parasitized caterpillars differentially induce plant defenses compared to non-parasitized caterpillars using two different caterpillar host/plant systems. Heliothis virescens caterpillars parasitized by M. croceipes had significantly lower salivary GOX activity than non-parasitized caterpillars, resulting in lower levels of tomato defense responses, which benefited parasitoid performance by increasing the growth rate of parasitized caterpillars. In tobacco plants, parasitized Helicoverpa zea caterpillars had lower GOX activity but induced higher plant defense responses. The higher tobacco defense responses negatively affected parasitoid performance by reducing the growth rate of parasitized caterpillars, causing longer developmental periods, and reduced cocoon mass and survival of parasitoids. These studies demonstrate a species-specific effect in different plant-insect systems. Based on these results, plant perception of insect herbivores can be affected by parasitoids and lead to positive or negative consequences to higher trophic levels depending upon the particular host-plant system.

Diversity of Insect Pests Damaging Quality of “Huitlacoche” (Corn Smut) at Saltillo, Coahuila, Mexico.

Huitlacoche or corn smut, Ustilago maydis (DC.) Corda, is a biotrophic fungal pathogenic. The scales that grow in ears of maize, Zea mays L., are used as fresh or raw vegetables. In some regions of Mexico, maize is planted to harvest “huitlacoche”, a crop also susceptible to attack by insect pests that affect its quality. The purpose of this study was to recognize entomofauna in maize ears infected with huitlacoche (corn smut) and identify pest species that attack huitlacoche in five maize hybrids. We collected a total of 515 specimens in four orders, seven families, and 11 species. The orders Coleoptera and Lepidoptera comprised 71.4% of the total number of families, 81.8% of the species, and 99.6% of individuals. The percentage of damaged ears ranged from 47.5 to 65.0%. Species best represented were Cotinis mutabilis (Gory and Percheron), Spodoptera frugiperda (J.E. Smith), Heliothis virescens (Fabricius), and Carpophilus lugubris Murray considered primary pest of huitlacoche because they were present in all maize studied. Shannon-Wiener diversity (0.592 bits per individual) and Simpson (0.302 bits per individual) indices showed less diversity of individuals and moderate species dominance. Greatest richness of species was in hybrids ‘AN388’ and ‘H-318’, each with seven.

The expression patterns of SNMP1 and SNMP2 underline distinct functions of two CD36-related proteins in the olfactory system of the tobacco budworm Heliothis virescens.

In insects, male and female pheromone signals are detected by olfactory sensory neurons (OSNs) expressing the "sensory neuron membrane protein type 1". SNMP1 is supposed to function as a co-receptor involved in the transfer of pheromones to adjacent pheromone receptors. In the moth Heliothis virescens, we previously found OSNs that project their dendrites into pheromone-responsive trichoid sensilla and are associated with cells containing transcripts for the HvirSNMP1-related protein HvirSNMP2. Like HvirSNMP1, HvirSNMP2 belongs to the CD36-family of two-transmembrane domain receptors and transporters for lipophilic compounds, but its role in the olfactory system is unknown. Here, we generated polyclonal anti-peptide antibodies against HvirSNMP2 as well as HvirSNMP1 and conducted an in-depth immunohistochemical analysis of their subcellular localization in the antenna of both sexes. In line with a function in pheromone detection, HvirSNMP1 was immunodetected in the somata and the dendrites of distinct OSNs in subsets of trichoid sensilla. These trichoid sensilla contained only one α-SNMP1-positive OSN in males and clusters of 2-3 labeled cells in females. In contrast, experiments with α-SNMP2-antibodies revealed a broad labeling of non-neuronal support cells (SCs) that are associated with OSNs in likely all trichoid and basiconic sensilla of the antenna with no differences between sexes. Detailed confocal microscope examinations of olfactory sensilla revealed SNMP2-like immunoreactivity close to the apical membrane of SCs and interestingly inside the sensillum. Together, these findings indicate a potential function of SNMP2 in pheromone- as well as general odorant-responsive sensilla and a role fundamentally different from SNMP1.

Improving Risk Assessment of Noctuid Pests at North American Ports and Farms by Differentiating Egg Morphology

Abstract Heliothine eggs are commonly found on agricultural commodities and ornamental plants transported through domestic and international commerce. Tobacco budworm [Chloridea (Heliothis) virescens (F.)], Chloridea subflexa (Guenée), and the corn earworm [Helicoverpa zea (Boddie)] are indigenous pests of the American continent. Interceptions of the Old World bollworm [Helicoverpa armigera (Hübner)] at various ports of entry into the United States have been detected due to the invasion of this pest in South America, adding to the complexity and importance of decision making at plant inspection stations. The ability to distinguish the eggs of C. virescens from C. subflexa and Helicoverpa species is a critical component for conducting risk assessments by quarantine authorities, taxonomists, and crop consultants. We developed a simple, rapid (~60 min), inexpensive ($0.06 per sample), and accurate (100% reliability) technique to distinguish C. virescens eggs from the possibility of being H. armigera based on the presence, number, and/or size of aeropyle holes on the primary ribs of eggs, near the micropylar rosette. In this location, aeropyles were easily visible at 40× magnification in 213 fresh and ethanol-preserved C. virescens eggs once treated with Hoyer’s solution. A small number of C. subflexa had one or two aeropyles on a few of the primary ribs, whereas no aeropyles were found on 411 H. zea and 269 H. armigera eggs analyzed using optical, electron, and confocal microscopy. We conclude that in most cases multiple aeropyle holes positively identify H. virescens eggs from H. zea or H. armigera, and C. subflexa. This technique potentially will reduce the number of specimens that need to be sent for molecular identification thereby saving program time and resources.

3H-117, a structural protein of Heliothis virescens ascovirus 3h (HvAV-3h).

The open reading frame 117 (3h-117) of Heliothis virescens ascovirus 3h (HvAV-3h), which is a conserved coding region present in all completely sequenced ascovirus members, was characterized in this study. By RT-PCR detection, 3h-117 transcription began at 6-h post-infection (hpi) and remained stable until 168 hpi in HvAV-3h-infected Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. In addition, 3h-117 putatively encodes a 21.5-kDa protein (3H-117) predicted to be a CTD-like phosphatase. Western blot analysis using a prepared rabbit polyclonal antibody specific to 3H-117 showed that the product could be detected at 24 hpi, which remained stably detectable until 168 hpi. The same analysis also demonstrated that the 3H-117 protein localized in the virions of HvAV-3h. Immunofluorescence analysis showed that at 24 hpi, 3H-117 was mainly located in the nuclei of H. armigera larval fat body cells and later spread into the cytoplasm. In summary, our results indicate that 3H-117 is a structural protein of HvAV-3h.

Efficacy of biological control agents, synthetic insecticides, and their combinations to control tobacco budworm [Heliothis virescens (Lepidoptera: Noctuidae)] in pigeon pea

Tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is an important pest in pigeon pea [Cajanus cajan (L.) Mill.] in Puerto Rico and the tropics. The use of chemicals is the most effective strategy to control this pest. Our objective was to assess the efficacy of two biological control agents, five synthetic insecticides, and four combinations to control tobacco budworm larvae. Pigeon pea plots were sprayed with 11 insecticide treatments and water as control in 2017 and 2018. Tobacco budworm incidence and severity, and data of dry-pod/seed yield were evaluated at harvest. Plots sprayed with water, the biological control agents [Steinernema carpocapsae (Weiser) (SC), and nucleopolyhedrovirus (NP)], and chlorpyrifos had significantly higher incidences (>24%) compared with plots applied with spinetoram alone and combined with SC and NP (12–18%) in both planting years. However, there were no significant differences (P ≥ 0.05) between treatments for tobacco budworm severity (mean scores of 4.00 to 4.83 in 2017 and 2.90 to 3.60 in 2018). In contrast, significantly lower incidence (<2.00) and severity (≤2.50) were recorded with applications of chlorantraniliprole and its combination with NP and SC. However, the use of low dosages of chlorantraniliprole + NP (0.20 + 0.10 l/ha), and chlorantraniliprole + SC + oil (0.20 l + 550 g + 1.25 l/ha) increased significantly dry-pod (2.18 and 2.20 t/ha) and seed-yield (1.27 and 1.23 t/ha) only in 2017; while high dosages of β-cyfluthrin (0.20 l/ha) and chlorantraniliprole (0.30 l/ha) increased yield significantly (2.04 and 2.44; 1.72 and 1.69 t/ha) compared to the control (1.25 and 0.76 for dry-pod; and 0.78 and 0.26 t/ha for seed in 2017 and 2018, respectively). We concluded that timely applications of β-cyfluthrin and chlorantraniliprole may increase net incomes over $4000/ha and should be incorporated in insecticide rotations to control tobacco budworm in pigeon pea.

Ecdysteroidogenesis in Heliothis virescens (Lepidoptera: Noctuidae): Recombinant Prothoracicotropic Hormone and Brain Extract Show Comparable Effects.

Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.

Population genetics of lepidopteran (noctuidae) collected on transgenic and non-transgenic maize in Mexico

Allele frequency of the resistance of the gene Onb3GalT5 to Cry proteins was analyzed in insect populations (Helicoverpa zea and Heliothis virescens), collected from maize transgenic and non-transgenic plants. The genetic parameters estimated in these populations were allele and genotype frequency, genetic balance and Wright statistics (Fis, Fit and Fst). Statistical analyses were performed using the package Genepop (4.0.10). Results showed that from the DNA of H. virescens, collected on non-transgenic maize, a higher number of bands were amplified in a range from 250 bp to 600 bp. In contrast, the bands amplified from insects collected on transgenic maize were in the range of 250 bp-500 bp, being the 400 bp band in both cases the most frequent, while in the DNA of Helicoverpa zea, collected on transgenic maize, three bands (350 bp, 400 bp and 450 bp) were amplified, and in H. zea, collected on non-transgenic maize, only two bands (350 bp and 400 bp) were observed. Genetic diversity in insect populations was higher than within these same populations.

In Vitro Infectious Risk Assessment of Heliothis virescens ascovirus 3j (HvAV-3j) toward Non-target Vertebrate Cells.

As specific pathogens of noctuid pests, including Spodoptera exigua, S. litura, Helicoverpa armigera, and Mythimna separata, ascoviruses are suitable for the development of bioinsecticides. In this study, the infectivity of Heliothis virescens ascovirus 3j (HvAV-3j) on insect and mammalian cells was evaluated. HvAV-3j infection induced drastic morphological changes in Sf9, HzAM1, SeFB, and HaFB cells, including swelling and detachment. Notably, the latter phenomena did not occur in HvAV-3j-inoculated mammalian cells (HEK293, 7402, HePG2, PK15, ST, and TM3). MTT assays indicated that HvAV-3j inhibited the growth of host insect cells from the 6th hpi, but no effects were detected in the HvAV-3j-inoculated mammalian cells. Furthermore, viral DNA replication, gene transcription, and protein expression were investigated, and the results consistently suggested that HvAV-3j viruses were not able to replicate their genomic DNA, transcribe, or express their proteins in the non-target vertebrate cells. The HvAV-3j genes were only transcribed and expressed in the four insect cell lines. These results indicated that HvAV-3j was infectious to cells derived from S. frugiperda, S. exigua, H. armigera, and H. zea but not to cells derived from human, pig, and mouse, suggesting that ascoviruses are safe to non-target vertebrate cells.

Genome Size Reversely Correlates With Host Plant Range in Helicoverpa Species.

In organisms with very low percentages of transposable elements (TEs), genome size may positively or negatively correlate with host range, depending on whether host adaptation or host modification is the main route to host generalism. To test if this holds true for insect herbivores with greater percentages of TEs, we conducted flow cytometry to measure the endopolyploidy levels and C-values of the host modification (salivary gland and mandibular gland in head), host adaptation (midgut), and host use-independent tissues (male gonad, hemolymph, and Malpighian tubules) of the generalist Helicoverpa armigera and the head of its older specialist sister H. assulta. Larval salivary gland displayed a consecutive chain of endopolyploidy particles from 8Cx to higher than 32Cx and larval head and midgut had endopolyploidy nuclei clusters of 16Cx and 32Cx, whereas larval male gonad, hemolymph, and Malpighian tubules possessed no endopolyploidy nuclei of higher than 8Cx. The estimated genome size of the Solanaceae plant specialist H. assulta is 430 Mb, significantly larger than that of its older generalist sister Heliothis virescens (408 Mb) and those of its two generalist descendants H. armigera (394 Mb) and H. zea (363 Mb). These data not only reveal a negative correlation between host plant range and genome size in this terminal lineage, but also imply that Helicoverpa species appear to depend more on host modification than on host adaptation to achieve polyphagy.

Imidacloprid is compatible with control provided by the predator Jalysus wickhami Van Duzee (Hemiptera: Berytidae) in flue-cured tobacco (Nicotiana tabacum L.)

Jalysus wickhami Van Duzee is the most abundant predator in North Carolina flue-cured tobacco production but information on the effect of contemporary pest management practices and interactions with other arthropods is lacking. We measured the effect of systemic imidacloprid on J. wickhami in field experiments during 2015 and 2016 by surveying its abundance, the abundance of its prey; the pests Heliothis virescens (Fabricus), Manduca sexta L., and Manduca quinquemaculata (Haworth, 1803); and other predatory arthropods in the agro-ecosystem. Systemic imidacloprid applications did not reduce J. wickhami abundance nor increase the abundance of H. virescens, M. sexta, and M. quinquemaculata, indicating natural control was not affected. J. wickhami abundance was positively correlated with the abundance of prey and predators from another feeding guild, suggesting species interactions have significant implications for the predators.

Do age and mating status affect olfactory response of the parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) to host-related plant odors?

Background: Parasitic wasps (parasitoids) use volatile organic compounds released by herbivore-infested plants to locate their hosts. Response of parasitoids to plant odors may be plastic and dependent on their physiological state. Using Microplitis croceipes (Hymenoptera: Braconidae), a relatively specialized larval endoparasitoid of Heliothis virescens (Lepidoptera: Noctuidae), we asked whether age and mating status of parasitoids affect their olfactory response to host-related odors. Methods: Four odor stimuli of varying complexity were selected based on previous reports of parasitoid response to cotton volatiles: cis-3-hexenol (a green leaf volatile), α-pinene (a constitutive monoterpene), a 50/50 v/v binary mixture ( cis-3-hexenol + α-pinene), and H. virescens-infested cotton odors. Female M. croceipes used in Y-tube olfactometer bioassays were either mated or unmated, and grouped 1-3, 4-6, and 7-9 d-old. Female parasitoids used in electroantennogram (EAG) recording were mated and grouped 1-3, 4-6, 7-9 and 10-12 d-old. Results: In Y-tube olfactometer bioassays, neither age nor mating status played a major role in the attraction of parasitoids to test odor stimuli, with two exceptions: 4-6 d-old mated parasitoids showed attraction to the binary mixture, and 1-3 d-old mated parasitoids showed attraction to H. virescens-infested cotton. Age did not affect EAG response of parasitoids to test stimuli. Conclusions: The present results suggest that age and mating status do not play a major role in modulating olfactory responses of M. croceipes to host-related plant odors. Instead, plasticity of olfactory response may be limited in M. croceipes due to strong innate sensitivity to host-related odor cues.

Do age and mating status affect olfactory response of the parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) to host-related plant odors?

Background: Parasitic wasps (parasitoids) use volatile organic compounds released by herbivore-infested plants to locate their hosts. Response of parasitoids to plant odors may be plastic and dependent on their physiological state. Using Microplitis croceipes (Hymenoptera: Braconidae), a relatively specialized larval endoparasitoid of Heliothis virescens (Lepidoptera: Noctuidae), we asked whether age and mating status of parasitoids affect their olfactory response to host-related odors. Methods: Four odor stimuli of varying complexity were selected based on previous reports of parasitoid response to cotton volatiles: cis-3-hexenol (a green leaf volatile), α-pinene (a constitutive monoterpene), a 50/50 v/v binary mixture ( cis-3-hexenol + α-pinene), and H. virescens-infested cotton odors. Female M. croceipes used in Y-tube olfactometer bioassays were either mated or unmated, and grouped 1–3, 4–6, and 7–9 d-old. Female parasitoids used in electroantennogram (EAG) recording were mated and grouped 1–3, 4–6, 7–9 and 10–12 d-old. Results: In Y-tube olfactometer bioassays, neither age nor mating status played a major role in the attraction of parasitoids to test odor stimuli, with two exceptions: 4–6 d-old mated parasitoids showed attraction to the binary mixture, and 1–3 d-old mated parasitoids showed attraction to H. virescens-infested cotton. Age did not affect EAG response of parasitoids to test stimuli. Conclusions: The present results suggest that age and mating status do not play a major role in modulating olfactory responses of M. croceipes to host-related plant odors. Instead, plasticity of olfactory response may be limited in M. croceipes due to strong innate sensitivity to host-related odor cues.

Proteomic analysis of the Heliothis virescens ascovirus 3i (HvAV-3i) virion.

Ascoviruses are enveloped, circular, double-stranded DNA viruses that can effectively control the appetite of lepidopteran larvae, thereby reducing the consequent damage and economic losses to crops. In this study, the virion of a sequenced Heliothis virescens ascovirus 3i (HvAV-3i) strain was used to perform proteomic analysis using both in-gel and in-solution digestion. A total of 81 viral proteins, of which 67 were associated with the virions, were identified in the proteome of HvAV-3i virions. Among these proteins, 23 with annotated functions were associated with DNA/RNA metabolism/transcription, virion assembly, sugar and lipid metabolism, signalling, cellular homoeostasis and cell lysis. Twenty-one viral membrane proteins were also identified. Some of the minor 'virion' proteins identified may be non-virion contaminants of viral proteins synthesized during replication, identified by more recent and highly sensitive methods. The extensive identification of the ascoviral proteome will establish a foundation for further investigation of ascoviral replication and infection.

Production and Distribution of Aldehyde and Alcohol Sex Pheromone Components in the Pheromone Gland of Females of the Moth Chloridea virescens.

Aldehydes are components of many moth sex pheromones, and are thought to be produced from analogous alcohols by oxidase(s) in the cell membrane or the gland cuticle. This implies that the two types of components are produced and/or stored in different parts of the gland: alcohols in cells and aldehydes in cuticle. Few studies have investigated the distribution of components in moth pheromone glands. Using rinse/extract sampling, stable isotope tracer/tracee methods, and decapitation/ pheromone biosynthesis activating neuropeptide stimulation, we studied production and distribution of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-hexadecenol (Z11-16:OH) in the gland of Chloridea virescens (formerly Heliothis virescens). The rinse, which likely sampled the surface and outer cuticle, contained large amounts of aldehyde and small amounts of alcohol. By contrast, the residual extract, which likely sampled cells and less solvent-accessible (inner) cuticle, had large amounts of alcohol and small amounts of aldehyde. When a tracer (U-13C-glucose) was fed to females, the aldehyde had higher isotopic enrichment than the alcohol in the rinse, but not in the residual extract, showing that in the rinse pool, Z11-16:Ald was, on average, synthesized before Z11-16:OH. This is consistent with greater aldehyde than alcohol flux through the cuticle. While our results are consistent with cell/cuticle synthesis sites for alcohol/aldehyde components, we cannot rule out both being synthesized in gland cells. We propose two alternative conceptual models for how site of production, cuticular transport and catabolism/metabolism might explain the relative masses of Z11-16:Ald and Z11-16:OH translocated to the pheromone gland surface in female C. virescens.

Novel Factors of Viral Origin Inhibit TOR Pathway Gene Expression.

Polydnaviruses (PDVs) are obligate symbionts of endoparasitoid wasps, which exclusively attack the larval stages of their lepidopteran hosts. The Polydnavirus is injected by the parasitoid female during oviposition to selectively infect host tissues by the expression of viral genes without undergoing replication. Toxoneuron nigriceps bracovirus (TnBV) is associated with Toxoneuron nigriceps (Hymenoptera: Braconidae) wasp, an endoparasitoid of the tobacco budworm larval stages, Heliothis virescens (Lepidoptera: Noctuidae). Previous studies showed that TnBV is responsible for alterations in host physiology. The arrest of ecdysteroidogenesis is the main alteration which occurs in last (fifth) instar larvae and, as a consequence, prevents pupation. TnBV induces the functional inactivation of H. virescens prothoracic glands (PGs), resulting in decreased protein synthesis and phosphorylation. Previous work showed the involvement of the PI3K/Akt/TOR pathway in H. virescens PG ecdysteroidogenesis. Here, we demonstrate that this cellular signaling is one of the targets of TnBV infection. Western blot analysis and enzyme immunoassay (EIA) showed that parasitism inhibits ecdysteroidogenesis and the phosphorylation of the two targets of TOR (4E-BP and S6K), despite the stimulation of PTTH contained in the brain extract. Using a transcriptomic approach, we identified viral genes selectively expressed in last instar H. virescens PGs, 48 h after parasitization, and evaluated expression levels of PI3K/Akt/TOR pathway genes in these tissues. The relative expression of selected genes belonging to the TOR pathway (tor, 4e-bp, and s6k) in PGs of parasitized larvae was further confirmed by qRT-PCR. The down-regulation of these genes in PGs of parasitized larvae supports the hypothesis of TnBV involvement in blocking ecdysteroidogenesis, through alterations of the PI3K/Akt/TOR pathway at the transcriptional level.

Do age and mating status affect olfactory response of the parasitoid, Microplitis croceipes (Hymenoptera: Braconidae) to host-related plant odors?

Background: Parasitic wasps (parasitoids) use volatile organic compounds released by herbivore-infested plants to locate their hosts. Response of parasitoids to plant odors may be plastic and dependent on their physiological state. Using Microplitis croceipes (Hymenoptera: Braconidae), a relatively specialized larval endoparasitoid of Heliothis virescens (Lepidoptera: Noctuidae), we asked whether age and mating status of parasitoids affect their olfactory response to host-related odors. Methods: Four odor stimuli of varying complexity were selected based on previous reports of parasitoid response to cotton volatiles: cis-3-hexenol (a green leaf volatile), α-pinene (a constitutive monoterpene), a 50/50 v/v binary mixture (cis-3-hexenol + α-pinene), and H. virescens-infested cotton odors. Female M. croceipes used in Y-tube olfactometer bioassays were either mated or unmated, and grouped 1–3, 4–6, and 7–9 d-old. Female parasitoids used in electroantennogram (EAG) recording were mated and grouped 1–3, 4–6, 7–9 and 10–12 d-old. Results: In Y-tube olfactometer bioassays, neither age nor mating status played a major role in the attraction of parasitoids to test odor stimuli, with two exceptions: 4–6 d-old mated parasitoids showed attraction to the binary mixture, and 1–3 d-old mated parasitoids showed attraction to H. virescens-infested cotton. Age did not affect EAG response of parasitoids to test stimuli. Conclusions: The present results suggest that age and mating status do not play a major role in modulating olfactory responses of M. croceipes to host-related plant odors. Instead, plasticity of olfactory response may be limited in M. croceipes due to strong innate sensitivity to host-related odor cues.

Chemical-mediated counter defense: attraction of two parasitoid species to the defensive secretion of host larvae

Carnivorous animals put selection pressure on their herbivorous preys, leading to the development of defensive traits in prey species and counter defensive traits in carnivores—an evolutionary arms race for survival. For instance, larvae of the moth Heliothis virescens use defensive oral secretions to deter attacking parasitic wasps (parasitoids). However, it is not clear whether parasitoids also use the same secretion for host location. If carnivores follow the herbivore model, evolution should favor specialist over generalist parasitoid species in their ability to manipulate host defensive chemicals. To test this prediction, Microplitis croceipes and Cotesia marginiventris, both larval endoparasitoids of H. virescens, were used as a study system. Microplitis croceipes is relatively specialized on Heliothis/Helicoverpa species while C. marginiventris is a generalist parasitoid on several noctuid host species. Innate behavioral responses of parasitoids to host defensive secretion were tested in Petri dish arena and Y-tube olfactometer bioassays. Heliothis virescens defensive secretion was manually applied to a spot in a sector (secretion zone) of the arena. Tracking data suggests that both parasitoid species are likely to locate the secretion spot once they enter the secretion zone. Comparing the two species, however, M. croceipes (specialist) spent significantly more time in the secretion zone and spot than C. marginiventris (generalist). In Y-tube olfactometer bioassays, the secretion elicited a strong attraction in M. croceipes but not in C. marginiventris, supporting our prediction. We discussed chemical-mediated counter defense in natural enemies of pest insects and the significance of the results to parasitoid-host coevolution.

Differential heliothine susceptibility to Cry1Ac associated with gut proteolytic activity

The Cry1Ac protein is the most active insecticidal toxin from the bacterium Bacillus thuringiensis (Bt) to members of the heliothinae subfamily in Lepidoptera, which includes some of the most devastating pests of corn and cotton worldwide. However, there are wide discrepancies in susceptibility among members of this subfamily in the US. Specifically, susceptibility to Cry1Ac in Helicoverpa zea (Hz) is >100-fold lower when compared to Heliothis virescens (Hv) larvae. The biochemical properties and Cry1Ac protoxin processing activity of gut digestive fluids from larvae of Hz and Hv were compared to test their role in differential susceptibility to Cry1Ac. Comparatively lower protease activity, associated with slower Cry1Ac proteolytic processing, was detected in digestive fluids of Hz compared to Hv. Moreover, Cry1Ac toxin processed by Hz digestive fluids displayed significantly lower toxicity in vitro against cultured insect cells compared to toxin activated by Hv proteases. These data support a contributing role for gut proteases in differential susceptibility to Cry1Ac in heliothine larvae.

Simple Methods to Devitalize Eggs and Larvae ofHeliothis virescensandHelicoverpa zeaunder Laboratory Conditions

Heliothine moths are economically damaging insect pests, significantly affecting world crop production each year. Several species in the subfamily are commonly used in agricultural research laboratories. Research protocols should include methods to ensure that excess individuals are completely devitalized before being discarded, preventing further spread of the pests. Processes such as ultracold temperature freezing, autoclaving, or holding insects under adverse conditions for a long period of time are commonly used devitalization methods. Each method adds significant amounts of time and cost; also increasing competition for space in already crowded laboratories. While these frequently used methods might be necessary for some very resilient species, other species can succumb under less elaborate methods of devitalization. One hour of exposure to undiluted household bleach, brief exposure (>20 seconds) of wetted individuals to microwave radiation, storage in a household freezer for 48 hours, and contact with 70% isopropyl alcohol for 24 hours completely devitalized eggs and L1 larvae of corn earworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (Fab.). These very effective methods might also be applicable for other noctuid species.