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Insects are known plant pests, and some of them such as Trichoplusia ni feed on a variety of crops. In this study, Trichoplusia ni was fed distinct diets of leaves of Arabidopsis thaliana or Solanum lycopersicum as well as an artificial diet. After four generations, the microbial composition of the insect gut was evaluated to determine if the diet influenced the structure and function of the microbial communities. The population fed with A. thaliana had higher proportions of Shinella, Terribacillus and Propionibacterium, and these genera are known to have tolerance to glucosinolate activity, which is produced by A. thaliana to deter insects. The population fed with S. lycopersicum expressed increased relative abundances of the Agrobacterium and Rhizobium genera. These microbial members can degrade alkaloids, which are produced by S. lycopersicum. All five of these genera were also present in the respective leaves of either A. thaliana or S. lycopersicum, suggesting that these microbes are acquired by the insects from the diet itself. This study describes a potential mechanism used by generalist insects to become habituated to their available diet based on acquisition of phytochemical degrading gut bacteria.

The effects of deleting the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egt gene on speed of kill and virus productivity were compared in second and fourth instar Trichoplusia ni (Hubner) larvae. Time to death was significantly reduced in larvae infected with an egt deletion mutant compared to insects infected with the wild-type virus. Moreover, time to death was reduced by the same proportion (11%) in second and fourth instar larvae. Virus yield was also significantly lower in fourth instar larvae infected with the deletion mutant but no difference was apparent in second instar larvae. A comparison of cadaver weights showed that insects infected with the deletion mutant were lighter than those infected with the wild-type virus, suggesting that the decrease in virus yield resulted from a reduction in larval growth. An analysis of yield per unit body weight showed no evidence for differences in replication rate in the two viruses. To determine whether differences in larval growth rate were related to differences in feeding activity, frass production was monitored in fourth instar larvae. Larvae infected with the deletion mutant produced less frass than larvae infected with the wild-type virus. Whereas the average rate of feeding for fourth instars did not differ between virus treatments, the rate peaked and declined earlier for larvae infected with the deletion mutant than for those infected with wild-type virus, suggesting enhanced early feeding in the absence of egt expression.

Background The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest. Results Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides. Conclusion We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry.

We evaluated the host suitability and related traits of Trichoplusia ni Hübner (Lepidoptera: Noctuidae) and Chrysodeixis chalcites Esper (Lepidoptera: Noctuidae), which is nonnative in North America, for the native parasitoids Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) and Copidosoma floridanum Ashmead (Hymenoptera: Encyrtidae), and the nonnative parasitoid Cotesia vanessae Reinhard (Hymenoptera: Braconidae). For the larval parasitoid C. sonorensis and C. vanessae trials, three-day-old larvae of both hosts were used, whereas one-day-old eggs of both hosts were used for the egg–larval parasitoid C. floridanum trial. For suitability parameters on each host exposed separately to each of the three parasitoid species, we measured parasitoid emergence (parasitoid success), parasitoids that did not emerge (parasitoid cocoon mortality), the proportion of male offspring (parasitoid sex ratio), hosts that developed into moths (host success), hosts that died without developing into moths or producing a parasitoid (host mortality), parasitoids emerging from cocoon masses (brood size), and the developmental times of parasitoids and hosts. For C. sonorensis, the native host and the nonnative host were found to be similarly suitable. For C. vanessae, the native host was more suitable than the nonnative host. For C. floridanum, the native host was suitable, whereas the nonnative host was not; however, sublethal effects on both the native and nonnative hosts were observed. The differential suitability of the hosts observed in this study contributes to the understanding of this measure as a dynamic factor in the expansion of parasitoids into novel host species.

Four crop plants known to be hosts for the lepidopteran Trichoplusia ni (soybean, green bean, cotton, and cabbage) were treated with the biopesticide AfMNPV baculovirus in a dosage response assay. Treated soybean had, on average, a 6-fold increase in virus activity compared with the other crops. Leaf trichomes on soybeans were not found to be responsible for the observed increase of insecticidal activity. Three flavonoid compounds (daidzein, genistein, and kaempferol) were uniquely found only in the soybean crop, and were not detected in cotton, cabbage, or green bean plant matter. The individual flavonoid compounds did not cause T ni. mortality in no-virus assays when incorporated into artificial insect diet. The combination of the three flavonoid compounds at leaf level concentrations significantly increased baculovirus activity in diet incorporation assays. When the daidzein, genistein, and kaempferol were added to artificial diet, at 3.5–6.5 × leaf level concentrations, virus activity increased 1.5, 2.3, and 4.2-fold for each respective flavonoid. The soybean flavonoid compounds were found to synergistically improve baculovirus activity against T. ni.

Many factors affect the host range of baculoviruses. Chrysodeixis includens and Trichoplusia ni are two closely related members of the lepidopteran subfamily Plusiinae that are important insect pests of different crops worldwide. In Brazil, C. includens has recently become a major defoliating pest of soybean. In this study, we compared the susceptibility of C. includens and T. ni to an alphabaculovirus isolated from a C. includens population in Brazil named Chrysodeixis includens nucleopolyhedrovirus isolate GO (ChinNPV-GO). Our results showed that ChinNPV-GO infects and kills C. includens at low oral doses, whereas T. ni larvae exhibited much lower levels of susceptibility to oral infection. In contrast, ChinNPV-GO very similarly infected and killed C. includens and T. ni larvae after budded viruses were injected into the hemocoel. Altogether, we conclude that T. ni is much less susceptible to ChinNPV-GO due to a reduced ability of this virus to establish a primary infection in the midgut. Our results further support that ChinNPV-GO and other plusiinae-associated SNPVs studied to date exhibit restricted host ranges even though known hosts are phylogenetically closely related.

Most studies of stress-induced transposable element (TE) expression have so far focused on abiotic sources of stress. Here, we analyzed the impact of an infection by the AcMNPV baculovirus on TE expression in a cell line (Tnms42) and midgut tissues of the cabbage looper moth (Trichoplusia ni). We find that a large fraction of TE families (576/636 in Tnms42 cells and 503/612 in midgut) is lowly expressed or not expressed at all [≤ 4 transcripts per million (TPM)] in the uninfected condition (median TPM of 0.37 in Tnms42 and 0.46 in midgut cells). In the infected condition, a total of 62 and 187 TE families were differentially expressed (DE) in midgut and Tnms42 cells, respectively, with more up- (46) than downregulated (16) TE families in the former and as many up- (91) as downregulated (96) TE families in the latter. Expression log2 fold changes of DE TE families varied from −4.95 to 9.11 in Tnms42 cells and from −4.28 to 7.66 in midgut. Large variations in expression profiles of DE TEs were observed depending on the type of cells and on time after infection. Overall, the impact of AcMNPV on TE expression in T. ni is moderate but potentially sufficient to affect TE activity and genome architecture. Interestingly, one host-derived TE integrated into AcMNPV genomes is highly expressed in infected Tnms42 cells. This result shows that virus-borne TEs can be expressed, further suggesting that they may be able to transpose and that viruses may act as vectors of horizontal transfer of TEs in insects.

Effect of physical and nutritional conditions such as culture temperature, pH, light, and culture and substrate medium with supplements were assessed for vegetative growth and production of conidia, in addition to virulence against Lepidoptera and Coleoptera pests by strains of Beauveria sp. Dextrose, peptone, and yeast agar with wheat bran or amaranth flour, 23 to 25°C, 7.5-8.5 pH, and obscure light were optimum for radial growth. Light and pH for most production of conidia depended on the fungal strain. Most conidia per gram were produced with 4% amaranth added to oat grain and culture in dextrose, peptone, and yeast broth with 2% wheat bran. Both fungal strains killed 91.1-96.0% of Phyllophaga sp. (Coleoptera: Scarabaeidae) and Spodoptera exigua Hubner and Trichoplusia ni Hubner (Lepidoptera: Noctuidae). Results confirmed the possibility of increased production of B. bassiana conidia by protein supplements and physical growing conditions to maintain pathogenicity.

BACKGROUND Phytosanitary irradiation is a sustainable alternative to chemical fumigants for disinfesting fresh commodities from insect pests. However, irradiating insects in modified atmospheres with very low oxygen (

This study was focused on the detection, characterization and virulence of a new baculovirus isolate from the larvae of cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). T. ni is a polyphagous pest, and it has cosmopolitan distribution. An infected T. ni larvae was collected from a cabbage field in Turkey. Scanning electron microscopy studies showed the presence of typical occlusion bodies (OBs) with average size of 0.76 to 1.46 µm in the collected larvae. Since the virions have single nucleocapsid within the envelopes, the isolate was named as Trichoplusia ni single nucleopolyhedrovirus Turkey isolate (TrniSNPV- TR). The total genome of the TrniSNPV-TR was determined as 122.9 kb in size. Sequence analysis of the amplified late expression factor 8 (lef8), late expression factor 9 (lef9) and polyhedrin (polh) genes showed that the virus is a new isolate of nucleopolyhedroviruses and close to Trichoplusia ni SNPV isolates mentioned in the literature. However, this is the first study for the detection and characterization of a baculovirus from T. ni in Eurasian region. Insecticidal activities of the TrniSNPV-TR isolate (106 OBs/ml-1) against neonate, 3rd and 5th instar larvae of T. ni and Helicoverpa armigera showed 98%-91%, 91%-87% and 65%-60% mortalities, respectively, within 14 days. LC50 of TrniSNPV-TR was determined as 2×103-9×103, 3×104-7×104 and 1×105-2×105 OBs/ml on neonate, 3rd and 5th instar larvae, respectively. All these results showed that, TrniSNPV-TR has good potential to be utilized as a bio-pesticide against T. ni larvae in the future. Keywords: baculovirus; nucleopolyhedrovirus; Trichoplusia ni; TrniSNPV-TR; biological control.

In herbivorous insects, the choice that females make for a suitable host plant is crucial for survival of its offspring because the neonate larvae are generally not capable of moving great distances. The preference‐performance hypothesis states that herbivore females will choose to oviposit on hosts on which their offspring will have better performance. In this study, we investigated whether Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae) females are able to discriminate among a weedy race, a landrace, and a commercial cultivar of tomato plants, Solanum lycopersicum L. (Solanaceae), and how their choice affects the offspring performance. Additionally, we identified the volatile compounds and recorded the density of glandular trichomes of the tomato plants. Females did not show a preference for any of the three types of tomato plants. Females oviposited more on the adaxial surface of leaves of commercial cultivar plants than on (any surface of) leaves of weedy‐race plants. The relative abundance of volatiles varied quantitatively among the three types of tomato plants. Commercial cultivar plants released a higher abundance of volatiles than weedy race and landrace plants. Weedy‐race plants had a higher density of glandular trichomes types I and VI than the commercial cultivar. More neonate larvae died if fed on the weedy race and landrace plants than when reared on commercial cultivar plants. Results suggested that the higher mortality of T. ni larvae may be related to a higher density of glandular trichomes on landrace and weedy‐race plants than on commercial cultivar plants, although other constitutive and induced defenses may be involved. Our results do not support the preference‐performance hypothesis.

Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.

BACKGROUND: A chemical scaffold‐hopping approach from known 3‐hydroxyl‐3‐methylglutaryl‐CoA (HMG‐CoA) reductase inhibitors identified (E/Z)‐2‐arylstilbenes as novel insecticidal hits against two lepidopteran species, Spodoptera exigua and Trichoplusia ni. A structure–activity relationship (SAR) study of the aryl substituents and the E/Z conformations was carried out in an effort to improve insecticidal potency. RESULTS: A series of (E/Z)‐2‐arylstilbenes was prepared and separated to evaluate their insecticidal potency against lepidopterous species in diet‐feeding assays. The results showed that the (Z)‐2‐arylstilbenes were more active than their corresponding (E)‐isomers, and a stereoselective synthesis was utilized to expand the SAR of the (Z)‐2‐arylstilbenes. (Z)‐4'‐Fluoro‐3′‐methyl‐2‐(2,4‐difluorostyryl)‐4‐fluoro‐5‐methoxy‐1,1′‐biphenyl was the most potent analog in this study with strong activity against S. exigua, T. ni, Helicoverpa zea, Plutella xylostella and Pseudoplusia includens. CONCLUSION: The (Z)‐2‐arylstilbenes were found to have strong insecticidal potency against five lepidopteran species. Ultimately, synthetic efforts could not improve insecticidal potency to commercial levels, and a lack of UV stability led to efforts being discontinued. © 2019 Society of Chemical Industry

The Physalis genus includes species of commercial importance due to their ornamental, edible and medicinal properties. These qualities stem from their variety of biologically active compounds. We performed a metabolomic analysis of three Physalis species, i.e., P. angulata, P. grisea, and P. philadelphica, differing in domestication stage and cultivation practices, to determine the degree of inter-species metabolite variation and to test the hypothesis that these related species mount a common metabolomic response to foliar damage caused by Trichoplusia ni larvae. The results indicated that the metabolomic differences detected in the leaves of these species were species-specific and remained even after T. ni herbivory. They also show that each Physalis species displayed a unique response to insect herbivory. This study highlighted the metabolite variation present in Physalis spp. and the persistence of this variability when faced with biotic stressors. Furthermore, it sets an experimental precedent from which highly species-specific metabolites could be identified and subsequently used for plant breeding programs designed to increase insect resistance in Physalis and related plant species.

Simple Summary The insect family, Noctuidae, contains some of the most damaging pests of agriculture, including bollworms, budworms, and armyworms. Transgenic cotton and maize expressing Cry-type insecticidal proteins from Bacillus thuringiensis (Bt) are protected from such pests and greatly reduce the need for chemical insecticides. However, evolution of Bt resistance in the insects threatens the sustainability of this environmentally beneficial pest control strategy. Understanding the interaction between Bt toxins and their targets in the insect midgut is necessary to evaluate the risk of resistance evolution. ABC transporters, which in eukaryotes typically expel small molecules from cells, have recently been proposed as a target for the pore-forming Cry toxins. Here we review the literature surrounding this hypothesis in noctuids and other insects. Appreciation of the critical role of ABC transporters will be useful in discovering counterstrategies to resistance, which is already evolving in some field populations of noctuids and other insects. Abstract In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from Bacillus thuringiensis (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.

Incluye material complementario Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy. This study received financial support from the Spanish Ministry project AGL2017-83498-C2-1-R. Bioinsectis SL funded the activities of ABF.

Herbivore populations are regulated by plant defenses and natural enemies. While plant defense can suppress herbivore populations, these defenses adversely affect natural enemies thereby releasing herbivores from top-down control. Over their lifespans, herbivores and their natural enemies may experience substantial variation in plant defense. Defense variability can suppress the growth of herbivores, but the impacts of defense variability on natural enemies and top-down control of herbivores are unknown. We independently manipulated the mean and variation of a plant toxin experienced by individual Trichoplusia ni caterpillars and its parasitoid Copidosoma floridanum. Increases in the mean toxin concentration, but not its variance, experienced by individual T. ni and C. floridanum decreased the fitness of C. floridanum, whereas both mean and variance impacted T. ni fitness. Thus, increased defense variability for individual herbivores suppressed herbivore fitness with no perceptible cost to top-down control. However, impacts of variability depend heavily on scale of variability.

The polyhedral inclusion bodies (PIBs) of Spodoptera littoralis nucleopolyhedrosis virus (SlNPV) were extracted from droppings of both the house sparrow Passer domesticus Raf. and the cattle egret Bubulcus ibis Bon. Due to the wide host range of the SlNPV, the extracted PIBs were bioassayed versus newly hatched larvae (L1) of S. littoralis, S. exigua, Trichoplusia ni, and Autographa circumflexa belonging to the insect fauna of the Egyptian clover Trifolium alexandrinum L. and the greater wax moth Galleria mellonella. Mortality of treated neonates of the 5 tested insect species ranged between 80 and 100 % after 7 days from ingesting the PIB-contaminated diet. Feeding nymphs and adults of the earwig Labidura riparia Pallas on S. littoralis larvae infected with the nuclear polyhedrosis virus (SlNPV) passed viable PIBs in feces which were extracted and bioassayed versus lepidopteran larvae of L1. All of the virus-treated 5 lepidopteran species became infected with the virus and showed mortality of 44–96% at the 7th day post ingestion of the extracted PIBs. On the other hand, feces extract of the 3rd larval instar (L3) of the lacewing Parachrysopa pallens (R.) fed on the virus-diseased larvae of S. littoralis caused no mortality when assayed versus the 5 tested lepidopteran larvae proving absence of viable PIB polyhedra that might be due to the mode of larval feeding by external digestion of its host contents including the polyhedra. Results explain the potential role played by insectivorous birds in spreading the nucleopolyhedrosis viruses in the agroecosystem as well as certain predatory insects.

The baculovirus vector expression system (BEVS) combines cultured insect cells and genetically modified Autographa californica nuclear polyhedrosis virus (AcMNPV)-derived baculovirus vectors. This expression system has been widely used for the expression of hundred of proteins for more than 30 years, existing commercial products manufactured at large scale by this methodology, mainly subunit vaccines. At an industrial scale, insect cells, as any other cultured cells, require artificial media and a strict control of environmental sterile conditions in the complex and expensive bioreactors. Here we describe an efficient alternative to produce recombinant biologics using the versatile and productive baculovirus vectors. It consists in natural biocapsules (pupae from Trichoplusia ni (Hübner) Lepidoptera), containing millions of insect cells in perfect physiological conditions, ready to be programmed by a genetically modified AcMNPV-derived baculovirus vector to produce large quantities of any recombinant protein. This technology, denominated CrisBio, has been tested to produce dozens of proteins, reaching productivities on the range of milligrams per infected pupa, that can be translated into dozens of vaccine doses, for example. The biologics production by CrisBio was industrialized with the design of both insect rearing and pupae storage single-use plastic devices, compatible with machines specifically designed for the automation of pupae manipulation and inoculation. These devices and machines reduce manual operations, increase batches consistency and facilitate the scaled production of any recombinant protein. As a mode of examples, the productivity in CrisBio technology platform of two virus-like particle (VLP) vaccine antigens is described in this work.

The peritrophic membrane (or peritrophic matrix) (PM) in insects is formed by binding of PM proteins with multiple chitin binding domains (CBDs) to chitin fibrils. Multi-CBD chitin binding proteins (CBPs) and the insect intestinal mucin (IIM) are major PM structural proteins. To understand the biochemical and physiological role of IIM in structural formation and physiological function of the PM, Trichoplusia ni mutant strains lacking IIM were generated by CRISPR/Cas9 mutagenesis. The mutant T. ni larvae were confirmed to lack IIM, but PM formation was observed as in wild type larvae and lacking IIM in the PM did not result in changes of protease activities in the larval midgut. Larval growth and development of the mutant strains were similar to the wild type strain on artificial diet and cabbage leaves, but had a decreased survival in the 5th instar. The larvae of the mutant strains with the PM formed without IIM did not have a change of susceptibility to the infection of the baculovirus AcMNPV and the Bacillus thuringiensis (Bt) formulation Dipel, to the toxicity of the Bt toxins Cry1Ac and Cry2Ab and the chemical insecticide sodium aluminofluoride. Treatment of the mutant T. ni larvae with Calcofluor reduced the larval susceptibility to the toxicity of Bt Cry1Ac, as similarly observed in the wild type larvae. Overall, in the mutant T. ni larvae, the PM was formed without IIM and the lacking of IIM in the PM did not drastically impact the performance of larvae on diet or cabbage leaves under the laboratory conditions.

Most plants form mutualistic associations with arbuscular mycorrhizal (AM) fungi that are ubiquitous in soils. Through this symbiosis, plants can withstand abiotic and biotic stresses. The underlying molecular mechanisms involved in mediating mycorrhiza-induced resistance against insects needs further research, and this is particularly true for potato (Solanum tuberosum L. (Solanales: Solanaceae)), which is the fourth most important crop worldwide. In this study, the tripartite interaction between potato, the AM fungus Rhizophagus irregularis (Glomerales: Glomeraceae), and cabbage looper (Trichoplusia ni Hübner) (Lepidoptera: Noctuidae) was examined to determine whether potato exhibits mycorrhiza-induced resistance against this insect. Plant growth, insect fitness, AM fungal colonization of roots, and transcript levels of defense-related genes were measured in shoots and roots after 5 and 8 d of herbivory on mycorrhizal and nonmycorrhizal plants. AM fungal colonization of roots did not have an effect on potato growth, but root colonization levels increased by herbivory. Larval weight gain was reduced after 8 d of feeding on mycorrhizal plants compared with nonmycorrhizal plants. Systemic upregulation of Allene Oxide Synthase 1 (AOS1), 12-Oxo-Phytodienoate Reductase 3 (OPR3) (jasmonic acid pathway), Protease Inhibitor Type I (PI-I) (anti-herbivore defense), and Phenylalanine Ammonia Lyase (PAL) transcripts (phenylpropanoid pathway) was found during the tripartite interaction. Together, these findings suggest that potato may exhibit mycorrhiza-induced resistance to cabbage looper by priming anti-herbivore defenses aboveground. This study illustrates how mycorrhizal potato responds to herbivory by a generalist-chewing insect and serves as the basis for future studies involving tripartite interactions with other pests.

Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.

Octopamine, a trace amine in mammals, is a major neurotransmitter linked to important biological processes in insects. Interestingly, one of the molecular entities responsible for octopamine availability, the octopamine transporter (OAT), has not been identified in certain insect species. For instance, no OAT has been reported in the fly Drosophila melanogaster (Dm), and the molecule involved in octopamine reuptake in Drosophila is not known. Here, we used molecular modeling methodologies to obtain three-dimensional insights for the dopamine transporter (DAT) and OAT in a common agricultural pest insect, Trichoplusia ni (Tni). Our results show several similarities but also significant differences in the general structures of the proteins of Dm and Tni. One important difference is observed in the ligand binding cavity, where a negatively charged amino acid present in both dopamine transporters is replaced by a polar neutral residue in the Trichoplusia OAT. This modification could influence both the binding mode and the driving force involved in the transport mechanism of these amines into neurons of these species. We also obtained data that support the idea that octopamine could bind and possibly be transported by DmDAT. The structural characterization of macromolecules from different insect species is fundamental in the agricultural field to gain insights into the design of new compounds for controlling pests.

Host range expansion by parasitoids into a non-native host is a common ecological process in nature mediated by multiple factors. Here, we addressed whether foraging behaviour responses of parasitoids to the abundance of the different host species to which they are exposed is one mediating factor. We investigate the parasitism levels, the functional response and the switching behaviour of two strains of the larval parasitoid Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) exposed to different patch densities and host ratios of the native host Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) and the non-native Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae). With a few exceptions, the parasitism levels were similar on both hosts. Both strains of the parasitoid demonstrated a Type III functional response with parasitism level positively related to increasing host abundance. The switching response was positive for both strains of C. sonorensis when both species of hosts are present in the same host patch. Yet a switching response was demonstrated whereby the parasitised proportion of each host at each patch changed depending on host ratio, mediated by their preference for C. chalcites. The results of this study indicate that the foraging behaviour responses of C. sonorensis to the abundance of the host T. ni are factors mediating its host range expansion from the native T. ni to the non-native C. chalcites.

Elevated CO2 alters C3 plant tolerance to insect herbivory, as well as the induction kinetics of defense hormones salicylic acid (SA) and jasmonic acid (JA), but the underlying physiological mechanism causing this response is not well understood. In principle, SA could be induced under elevated CO2 by reactive oxygen signals generated in photosynthesis, ultimately influencing chemical defense. To test whether the effects of elevated CO2 on C3 plant chemical defense against herbivorous insects are modulated by photosynthesis, Arabidopsis thaliana var. Col-0 plants were grown in two 2 × 2 × 2 nested factorial combinations of ambient (400 ppm) and elevated (800 ppm) CO2, and two dimensions of light regimes comprising intensity (‘mild’ 150 μmol E m−2 s−1 vs. ‘low’ light, 75 μmol E m−2 s−1) and periodicity (‘continuous’, 150 μmol E m−2 s−1 vs. ‘dynamic’, in which lights were turned off, then on, for 15 min every 2 h). Plants were challenged with herbivore damage from third instar Trichoplusia ni (cabbage looper). Consistent with experimental predictions, elevated CO2 interacted with light as well as herbivory to induce foliar concentration of SA, while JA was suppressed. Under dynamic light, foliar content of total glucosinolates was reduced. Under combination of elevated CO2 and dynamic light, T. ni removed significantly more leaf tissue relative to control plants. The observations that CO2 and light interactively modulate defense against T. ni in A. thaliana provide an empirical argument for a role of photosynthesis in C3 plant chemical defense.

Adoption of biotech crops engineered to express insecticidal toxins from Bacillus thuringiensis (Bt) has revolutionized insect pest control in agriculture. For continuing effective application and development of the environmentally friendly Bt biotechnology, it is fundamental to understand pathways of toxicity of Bt toxins in insects. In this study, mutations were introduced in the midgut cadherin gene in the cabbage looper, Trichoplusia ni, by CRISPR/Cas9 mutagenesis. T. ni strains with mutations in the genes of two major receptors for Bt toxins, the midgut cadherin and ABCC2, and three Cry1A toxins with shared and differential midgut binding sites were used as an experimental system to dissect the roles of the cadherin and ABCC2 in the pathways of toxicity of Bt toxins. Results from assays of responses of the T. ni strains to different Bt toxins revealed that the cadherin and ABCC2 play independent roles in the mode of action of Cry1A toxins and that Bt toxins exert insecticidal activity through multiple redundant pathways of toxicity in insects. Besides the cadherin and ABCC2 pathways, there exists an additional major pathway of toxicity to be identified for Cry1Aa. The results also confirmed that the toxicity of Cry2Ab involves neither the cadherin nor the ABCC2 protein. The multiple pathway model for Bt toxins clarified from this study provided new insights into the molecular modes of action of Bt toxins and mechanisms of insect resistance to Bt toxins.

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed high biochemical and genetic similarities with the HD-73 standard strain, besides expressing only a Cry1Ac protein: Cry1Ac39. However, LBIT-1200 was 6.4 and 9.5 times more toxic against Manduca sexta and Trichoplusia ni larvae, respectively, than HD-73, which expresses the Cry1Ac1 protein. Sequencing of Cry1Ac1 and Cry1Ac39 proteins showed only four amino acid differences, including two amino acid replacements in domain III, as compared to the rest of Cry1Ac proteins reported to date: N547Y and R602G. These two amino acid substitutions in domain III were introduced in the Cry1Ac1 protein from HD-73 through site directed mutagenesis. Three mutants were obtained: two single and one double mutant, which were expressed in an acrystaliferous B. thuringiensis strain, as well as the native Cry1Ac1 and Cry1Ac39 toxins. Bipyramidal crystals produced by the single mutant strains were larger than the original Cry1Ac1 protein; however, the high toxicity levels previously observed in the strain LBIT-1200 were restored in the three mutants, when tested against M. sexta and T. ni. The substitution N547Y seems to have a higher contribution to LBIT-1200 toxicity.

Summary Plants can re‐programme their transcriptome, proteome and metabolome to deal with environmental and biotic stress. It has been shown that the rhizosphere microbiome has influence on the plant metabolome and on herbivore behaviour. In the present study, Trichoderma gamsii was isolated from Arabidopsis thaliana rhizosphere soil. The inoculation of roots of Arabidopsis thaliana with T. gamsii significantly inhibited the feeding behaviour of Trichoplusia ni and affected the metabolome as well as the content of phytohormones in Arabidopsis leaves. T. gamsii‐treated plant leaves had higher levels of amino acids and lower concentrations of sugars. In addition, T. gamsii‐treated plant leaves had more abscisic acid (ABA) and lower levels of salicylic acid (SA) and indole‐3‐acetic acid (IAA) in comparison with the untreated plants. Furthermore, the inoculation with T. gamsii on different signalling mutants showed that the induction of defences were SA‐dependent. These findings indicate that T. gamsii has potential as a new type of biocontrol agent to promote plant repellence to insect attacks.

Classical studies have described in detail the complex and regionalized morphology of the Malpighian tubule (MT) in larval Lepidoptera. Recent studies revealed unusual aspects of ion transport in the Malpighian tubules of the larva of the cabbage looper, Trichoplusia ni. These included: cation reabsorption via secondary cells (SC); coupling of SCs to neighbouring PCs via gap junctions to enable reabsorption; and a reversal from cation secretion to reabsorption by the principal cells in the distal ileac plexus region of the in situ tubule in response to dietary ion loading. The current paper aimed to identify molecular components of ion transport in the MTs of T. ni and to describe their role in the recently reported reversal of ion transport in ion-loaded animals. Using a combination of molecular, immunohistochemical and electrophysiological techniques, we assigned roles to Na+/K+-ATPase (NKA), V-type H+-ATPase (VA), Na+/K+/Cl− co-transporter (NKCC), K+/Cl− co-transporter (KCC), inward-rectifying K+ channel (Kir), and Na+/H+ exchangers (NHE)-7 and -8 in the transport of Na+ and K+ by the distal ileac plexus of T. ni. The yellow region of the tubule lacked all of the above except VA, and the white region lacked all of the above transporters but expressed an amiloride-sensitive Na+ channel (NaC). Overall, the ion transport machinery in the distal ileac plexus of the T. ni tubule shows remarkable similarity to that in tubules of other groups of insects, yet this region transports ions very differently. Shutdown of secretory ATPases and utilisation of the same molecular machinery in the face of changing ion gradients may enable ion transport reversal in lepidopteran MTs. We propose that gap junction-based coupling of the two cell types likely aids in toggling between ion secretion and ion reabsorption in this segment.

Cabbage looper (Trichoplusia ni) larvae are generalist herbivores that feed on numerous cultivated plants and weeds including crucifers, other vegetables, flowers, and field crops. Consuming plant material from a wide range of plant species exposes these larvae to a considerable variety of plant secondary metabolites involved in chemical defense against herbivory. The ability of the cabbage looper larvae to detoxify plant secondary metabolites, such as nicotine, has been attributed to the rapid induction of excretion via the Malpighian tubules. However, the role of metabolism in the detoxification of plant secondary metabolites in cabbage looper larvae is not well studied. We investigated nicotine metabolism in 4th larval instar cabbage looper using UPLC-MS/MS analysis to resolve the time course of nicotine metabolism, the kinetic distribution of nicotine, and the presence or absence of major metabolites of nicotine in larval tissue and excretions. The major metabolite found in our analysis was cotinine, with trace amounts of cotinine N-oxide and nicotine N-oxide. The nicotine metabolites detected are similar to those of the nicotine-tolerant Lepidopteran tobacco hornworm (Manduca sexta). The results of our study demonstrate that the 5′C-oxidation of nicotine to cotinine is the primary pathway for nicotine metabolism in cabbage looper larvae. This study showed that metabolism of nicotine and subsequent excretion of nicotine and its metabolites occurs in the larvae of the cabbage looper. Our results suggest that 5′C-oxidation in lepidopteran insects is a conserved metabolic pathway for the detoxification of plant secondary metabolites.

Characterization of new Bacillus thuringiensis strains is a valuable tool to discover novel insecticidal toxins and to manage resistance problems. In this study, seven Iranian Bt strains were selected according to their toxicity against Plodia interpunctella, to be thoroughly characterized based on their toxicity, protein profiling, proteomic analysis, gene content and β-exotoxin production. The toxicity was assessed by insect bioassays and cell viability assays (a less cost, time and material consuming technique), using four lepidopteran pests and four lepidopteran cell lines from Trichoplusia ni (Hi5), Helicoverpa zea (HzGUT), Spodoptera exigua (UCR-SE) and Spodoptera frugiperda (Sf21). The selected Bt strains showed similar protein electrophoretic profiles, but differed in toxicity. LC–MS/MS analysis of solubilized crystal proteins and gene content analyses (PCR screening) were compared and correlated with the toxicity results. Based on our data, three Bt strains could be considered as candidates for development of future bioinsecticides.

Bacillus thuringiensis Vip3 proteins are synthesized and secreted during the vegetative growth phase. They are activated by gut proteases, recognize and bind to midgut receptors, form pores and lyse cells. We tested the susceptibility to Vip3Aa and Vip3Ca of Cry1A-, Cry2A-, Dipel- and Vip3-resistant insect colonies from different species to determine whether resistance to other insecticidal proteins confers cross-resistance to Vip3 proteins. As expected, the colonies resistant to Cry1A proteins, Dipel (Helicoverpa armigera, Trichoplusia ni, Ostrinia furnacalis and Plodia interpunctella) or Cry2Ab (H. armigera and T. ni) were not cross-resistant to Vip3 proteins. In contrast, H. armigera colonies resistant to Vip3Aa or Vip3Aa/Cry2Ab showed cross-resistance to the Vip3Ca protein. Moreover, the Vip3Ca protein was highly toxic to O. furnacalis (LC50 not significantly different from that of Cry1Ab), whereas the Vip3Aa protein only showed moderate growth inhibition at the highest concentration tested (100 µg/g of diet). These results extend the cross-resistance studies between Vip3 and Cry proteins, show for the first time cross-resistance between proteins within the Vip3 subfamily, and points to O. furnacalis as a target for the Vip3Ca protein.

Insect saliva is one of the first secretions to come in contact with plants during feeding. The composition and role of caterpillar saliva has not been as thoroughly studied as that of sucking insects. This study focuses on characterizing the proteome of the cabbage looper (Trichoplusia ni) saliva using iTRAQ labeling and LC-MS/MS. We also measured how the saliva proteome changed when larvae were reared on different diets - cabbage, tomato, and an artificial pinto bean diet. We identified 254 proteins in the saliva out of which 63 were differentially expressed. A large percentage (56%) of the proteins identified function in protein metabolism, followed by proteins involved in vesicle transport (6%) and oxidoreductase activity (5%), among other categories. Several proteins identified are antioxidants or reactive oxygen species (ROS) scavengers. Among these ROS scavengers, we identified a catalase and further analyzed its gene expression and enzymatic activity. We also applied commercial, purified catalase on tomato and measured the activity of defensive proteins - trypsin proteinase inhibitor, polyphenol oxidase and peroxidase. Catalase gene expression was significantly higher in the salivary glands of larvae fed on tomato. Also, catalase suppressed the induction of tomato trypsin proteinase inhibitor levels, but not the induction of polyphenol oxidase or peroxidase. These results add to our understanding of proteomic plasticity in saliva and its role in herbivore offense against plant defenses.

Reactive oxygen species (ROS) can be elicited by many forms of stress, including pathogen attack, abiotic stress, damage and insect infestation. Perception of microbe- or damage-associated elicitors triggers an ROS burst in many plant species; however, the impact of herbivore fatty-acid amides on ROS elicitation remains largely unexplored. In this study we show that the lepidopteran-derived fatty-acid amide elicitor N-linolenoyl-L-glutamine (GLN18:3) can induce a ROS burst in multiple plant species. Furthermore, in Arabidopsis this ROS burst is partially dependent on the plasma membrane localized NADPH oxidases RBOHD and RBOHF, and an Arabidopsis rbohD/F double mutant produces enhanced GLN18:3-induced jasmonic acid. Quantification of GLN18:3-induced ROS in phytohormone-deficient lines revealed that in Arabidopsis reduced levels of jasmonic acid resulted in a larger elicitor-induced ROS burst, while in tomato reduction of either jasmonic acid or salicylic acid led to higher induced ROS production. These data indicate that GLN18:3-induced ROS is antagonistic to jasmonic acid production in these species. In biological assays, rbohD/F mutant plants were more resistant to the generalist herbivores Spodoptera exigua and Trichoplusia ni but not to the specialist Plutella xylostella. Collectively, these results demonstrate that in Arabidopsis herbivore-induced ROS may negatively regulate plant defense responses to herbivory.

We investigated the female oviposition preference and larval performance and behavior of Trichoplusia ni (Hubner) on cabbage, tomato, soybean (host plants), and wormseed (nonhost plant) in laboratory experiments. In addition, we investigated the role of trichomes in the oviposition of females. Females oviposited more often on cabbage than on tomato, soybean, or wormseed plants. Tomato was the second most preferred plant, followed by soybean and wormseed. Neonate larvae gained more weight on cabbage and tomato than on soybean or wormseed, while second-instar larvae grew better on cabbage than on tomato, soybean, or wormseed. The least growth of neonate larvae occurred on wormseed plants. The orientation of neonate and second-instar larvae to cabbage, tomato, soybean, and wormseed did not differ significantly. Neonate larvae settled equally on leaf discs of cabbage, tomato, soybean, and wormseed, while most second-instar larvae settled on leaf discs of cabbage in comparison with the other plants after 24 h of release. The foliar area consumed by neonate larvae was quite similar among plants, but second-instar larvae consumed more cabbage than tomato, soybean, or wormseed. Comparing different types of leaves, females oviposited more often on mature than young leaves of tomato, soybean, and wormseed. In contrast, females did not show any preference for ovipositing on young or mature leaves of cabbage. In general, we found that the density or length of nonglandular and glandular trichomes of tomato, soybean, and wormseed plants negatively affected oviposition of T. ni females. Fil: Coapio, G. G.. El Colegio de la Frontera Sur; México Fil: Cruz López, L.. El Colegio de la Frontera Sur; México Fil: Guerenstein, Pablo Gustavo. Provincia de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Universidad Autónoma de Entre Ríos. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones Científicas y Transferencia de Tecnología a la Producción; Argentina Fil: Malo, Edi A.. El Colegio de la Frontera Sur; México Fil: Rojas, J. C.. El Colegio de la Frontera Sur; México

The nucleotide and amino acid sequence of the ecdysteroid UDP-glucosyltransferase (egt), polyhedrin (pol), inhibitor of apoptosis (iap2 and iap3), late expression factor (lef) 9 and vp1054 genes of a South African isolate of Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) was compared to a Canadian isolate of TnSNPV. Nucleotide and amino acid sequence identity for the compared genes ranged from 89%to 98%and 95%to 99%, respectively. The deduced amino acid sequences of the TnSNPV egt, pol, iap3, lef-9 and vp1054 genes were used to infer phylogenetic trees and these were compared to the tree inferred from combined data sets consisting of the amino acid sequences of polyhedrin/granulin, lef-8 and lef-9 genes of 48 completely sequenced baculoviruses. The topologies of trees for the baculovirus core genes pol, lef-9 and vp1054 were better resolved than that of the auxiliary genes iap2, iap3 and egt when compared to the tree inferred from complete genome sequences. Sequence and phylogenetic analysis confirm...

Summary Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt‐resistant insects on Bt and non‐Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt‐resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt‐resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non‐Bt cotton and on transgenic Bt cotton leaves expressing a single‐toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R 0) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4‐day asynchrony of adult emergence between the susceptible T. ni grown on non‐Bt cotton leaves and the dual‐toxin‐resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.

Background Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability. Methods Using an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar ) as well as of the commonly-used T. ni High Five cell line. Results We detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar , N-glycans with glycolipid-like antennae containing α-linked N- acetylgalactosamine were also revealed. Conclusion The lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species. Significance The occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.

A biopesticide with a mixture of entomopathogenic microbial agents was studied for improvements in efficacy. Recently developed liquid fermentation techniques were used to produce blastospores of Beauveria bassiana (Balsamo) Vuillemin strain GHA (Bb), which were mixed with traditional fermentation of Bacillus thuringiensis Berliner kurstaki (Bt) spores and crystals. Based on a dosage response bioassay using Trichoplusia ni (Hubner) neonates exposed to treated leaf disks, the LC50 values were determined to be 3.85 × 107 spores mL−1 for the unformulated Bt and 3.58 × 107 blastospores mL−1 for the unformulated Bb. Using the same bioassay conditions, mixtures of Bt:Bb at ratios of 1:0, 0.75:0.25. 0.5:0.5, 0.25:0.75, and 0:1 indicated that the greatest mortality was caused by the 0.5:0.5 mixture ratio of the microbial agents. Based on these results, the 0.5:0.5 mixture was formulated as a dry powder by spray drying and applied to field-grown cabbage for comparison with commercial Bt and Bb products. Ov...

Trichoplusia ni is a polyphagous insect that feeds on many crops plants of economic importance. In this paper, we presented the complete mitochondrial genome of T. ni. The circular mitochondrial ge...

In recent years, several studies have examined the gut microbiome of lepidopteran larvae and how factors such as host plant affect it, and in turn, how gut bacteria affect host plant responses to herbivory. In addition, other studies have detailed how secretions of the labial (salivary) glands can alter host plant defense responses. We examined the gut microbiome of the cabbage looper (Trichoplusia ni) feeding on collards (Brassica oleracea) and separately analyzed the microbiomes of various organs that open directly into the alimentary canal, including the labial glands, mandibular glands, and the Malpighian tubules. In this study, the gut microbiome of T. ni was found to be generally consistent with those of other lepidopteran larvae in prior studies. The greatest diversity of bacteria appeared in the Firmicutes, Actinobacteria, Proteobacteria, and Bacteriodetes. Well‐represented genera included Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Diaphorobacter, Methylobacterium, Flavobacterium, and Cloacibacterium. Across all organs, two amplicon sequence variants (ASVs) associated with the genera Diaphorobacter and Cloacibacterium appeared to be most abundant. In terms of the most prevalent ASVs, the alimentary canal, Malpighian tubules, and mandibular glands appeared to have similar complements of bacteria, with relatively few significant differences evident. However, aside from the Diaphorobacter and Cloacibacterium ASVs common to all the organs, the labial glands appeared to possess a distinctive complement of bacteria which was absent or poorly represented in the other organs. Among these were representatives of the Pseudomonas, Flavobacterium, Caulobacterium, Anaerococcus, and Methylobacterium. These results suggest that the labial glands present bacteria with different selective pressures than those occurring in the mandibular gland, Malpighian tubules and the alimentary canal. Given the documented effects that labial gland secretions and the gut microbiome can exert on host plant defenses, the effects exerted by the bacteria inhabiting the labial glands themselves deserve further study., Labial gland secretions and the gut microbiota of insects affect defense responses of plants to herbivory. However, the microbiome of the labial gland has not been investigated. This work using cabbage looper, a serious pest of cole crops, demonstrates that the labial glands possess a community of bacteria that is distinct from the gut and other associated organs. This discovery warrants additional research to determine what role the labial gland microbiota has on the plant response to insect pests.

Below-ground microbes can induce systemic resistance (ISR) against foliar pests and pathogens on diverse plant hosts. The prevalence of ISR among plant-microbe-pest systems raises the question of host specificity in microbial induction of ISR. To test whether ISR is limited by plant host range, we tested the ISR-inducing ectomycorrhizal (ECM) fungus Laccaria bicolor on the non-mycorrhizal plant Arabidopsis. We found that root inoculation with L. bicolor triggered ISR against the insect herbivore Trichoplusia ni and induced systemic susceptibility (ISS) against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). We found that L. bicolor-triggered ISR against T. ni was dependent on jasmonic acid (JA) signaling and salicylic acid (SA) biosynthesis and signaling. We found that heat killed L. bicolor and chitin are sufficient to trigger ISR against T. ni and ISS against Pto and that the chitin receptor CERK1 is necessary for L. bicolor-mediated effects on systemic immunity. Collectively our findings suggest that some ISR responses might not require intimate co-evolution of host and microbe, but rather might be the result of root perception of conserved microbial signals.

Baculoviruses are large double-stranded DNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th-instar T. ni larvae were orally infected with AcMNPV occlusion bodies and the transcriptional responses of the T. ni midgut were analyzed at various times from 0 to 72 h postinfection, using transcriptome sequencing analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut and will be important for understanding how baculoviruses establish productive infections in the organism. IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni, infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.

Made available in DSpace on 2019-10-06T16:25:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-01 Fundação de Apoio à Pesquisa do Distrito Federal Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) We describe an unexpected feature observed for the heterologous expression of the Thyrinteina arnobia cypovirus polyhedrin from a recombinant baculovirus infection in different insect cell lines. The in cellulo-formed crystals varied in size and shape depending on the cell line. Crystals formed in Trichoplusia ni-derived cells were cubic (0.1-2 μm) and localized in both the nucleus and cytoplasm, whereas those formed in Spodoptera frugiperda-derived cells were ovate and ellipsoidal (0.1-3 μm) and also localized in both the nucleus and cytoplasm. The molecular basis for differences in the morphology, size, and location of cypovirus occlusion bodies is unclear, and cellular proteins might play a role in their formation and location. Cell Biology Department UnB-University of Brasília Laboratory of Insect Virology Department of Biochemistry and Molecular Biology Federal University of Santa Maria Plant Protection Department FCA/UNESP São Paulo State University, Campus of Botucatu Applied Biology Department FCAV/UNESP São Paulo State University, Campus of Jaboticabal Plant Protection Department FCA/UNESP São Paulo State University, Campus of Botucatu Applied Biology Department FCAV/UNESP São Paulo State University, Campus of Jaboticabal Fundação de Apoio à Pesquisa do Distrito Federal: 193.001532/2016 CNPq: 407908/2013-7 CNPq: 428799/2018-3 CAPES: CAPES/PROEX 23038.005848/2018-31

Understanding how hosts respond to pathogen attack is crucial to disease management. The response of a host can be particularly important if hosts have to defend against multiple pathogens which could either benefit from or be suppressed by prior pathogen exposure. Insect defence against viruses is less well understood than responses to other entomopathogens and much of the information available relates to in vitro studies and model systems. Baculoviruses are natural pathogens of insects, particularly Lepidoptera, and have been well-studied in terms of their ecology, pest control potential and molecular biology. In order to examine how an insect reacts to baculovirus challenge, we measured components of the cellular and humoral immune response of the cabbage looper Trichoplusia ni to Trichoplusia ni SNPV, a narrow-host range nucleopolyhedrovirus (NPV), over four doses and three times after pathogen challenge (18, 42 and 90 h). We found that total haemocyte numbers peaked at 42 h post-exposure at all doses, and declined linearly with increasing dose after the 18 h time point. Two immune-related enzymes, phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD), showed very different responses. PO levels were lowest at the 42 h time point and were not influenced by virus dose when each time point was examined separately. GLD levels declined over time but they interacted with virus dose in a non-linear manner, such that there was an increase in levels at intermediate virus doses after 18 h, no effect at 42 h, and then declined as infection progressed at 90 h post-infection. These data suggest that baculoviruses can rapidly infect haemocytes (or cause a reduction in their numbers) in a dose-dependent manner once the infection is systemic, likely reducing the ability of the host to counter subsequent infections. However, the data do not support a direct role for PO in defence against baculoviruses. Whether GLD plays a role in virus defence is still unclear.

Carotenoids are fundamental precursors for hormones and antioxidants, and insects must acquire carotenoids from their diet. Previous research has shown that insects can selectively absorb dietary carotenoids, often modifying them qualitatively or quantitatively, and quantities may be proportional to those found in the diet. Trichoplusia ni Hubner is a generalist herbivore with host plants varying greatly in carotenoid profiles and concentrations. Larvae sequester carotenoids in their hemolymph, and carotenoid sequestration contributes to their cryptic green coloration. Our objectives were to compare the types of carotenoids found in T. ni and their host plants to determine whether qualitative changes occurred, and compare the amounts of sequestered carotenoids in T. ni reared upon different host plants to determine whether quantitative variation influences sequestration. To fulfill these objectives, larvae were fed romaine lettuce (Lactuca sativa L. [Asterales: Asteraceae] var. longifolia) or kale (Brassica oleracea L. [Brassicales: Brassicaceae] var. sabellica) for a period of 5 d, and sequestered carotenoids from the entire insect were resolved with thin-layer chromatography and measured with spectrophotometer. All carotenoids resolved from plants were also resolved from larvae, and although the carotenoids of plants differed quantitatively, the sequestered carotenoids did not differ between host plants. Regardless of host plant species, T. ni sequestered carotenoids at concentrations up to 20 times higher than the concentrations found in the plants. Future research may be able to explicitly identify enzyme systems involved in the transport and modification of carotenoids in T. ni and other animals.

Klebsiella pneumoniae was isolated from infected pupae of Galleria mellonella and Pseudomonas aeruginosa was isolated from the entomopathogenic nematode Heterorhabditis bacteriophora hosted within the pupae of G. mellonella. Insect consumption and surface application of P. aeruginosa resulted in 83.33% and 81.66% mortality of Trichoplusia ni larvae, respectively. In contrast, 50% mortality was shown when T. ni larvae were fed with K. pneumoniae, and no larvae were killed when applying the bacterium to the larval cuticle. This report shows that two opportunistic human pathogens found in the insect-nematode ecosystem could kill insect pests.

Transporting epithelia are tissues that specialize in the directional movements of ions and water and are typically either secretory or reabsorptive. Recent work on the Malpighian tubule of larval lepidopterans (caterpillars) demonstrated that the distal ileac plexus segment of this epithelium is capable of rapidly switching between ion secretion and reabsorption. Subsequent transcriptomic studies suggested expression of voltage-gated ion channels in the lepidopteran MTs (which are not contractile and not innervated). The present study shows that isolated MTs of larval Trichoplusia ni express α1, β2, and α2δ4 subunits of voltage-gated Ca2+ channel CaV1 and that pan-CaVα immunoreactivity is present in the apical and basolateral membranes of the principal cells. Basolateral membrane potential (Vbl) in isolated MTs of larval Trichoplusia ni was influenced by CaV1 functioning; pharmacological inhibition of CaV1 reversed Vbl from inside-negative to inside-positive, and also reduced transepithelial potential (Vte), lowered [Ca2+]i and reversed the direction of K+ transport from secretion to reabsorption. Thus, our findings indicate that a functional CaV1 channel is necessary for constitutive K+ secretion observed in isolated preparations of lepidopteran MTs. Lastly, Vte and Vbl of isolated MTs were influenced by changes in bathing saline [K+]. Our findings suggest that epithelia may rely on CaV channels to enable robust ion secretion and downregulation of CaV channels, together with other transcriptional changes, enables ion reabsorption.

Since plant essential oils are composed with vast numbers of constituents, they often show complex interactions among the components. Although the research interest on the insecticidal activity of plant essential oils has been gaining more attention recently, most of the studies still focus on simple screening of active plant source or identification of active compounds. In the present study, insecticidal activity and synergistic interactions among the four major constituents of lemongrass (Cymbopogon citratus) essential oil were examined via topical application against the third instar larvae of the cabbage looper, Trichoplusia ni. Two synergistic binary combinations—citral + limonene and citral + geranyl acetate—were identified at the equivalent mixing ratios. The former was especially synergistic in larvae and additionally with respect to cytotoxicity in the ovarian cell line of the cabbage looper. Morphological observations indicated different cytotoxic modes of action of citral and limonene. GC–MS analysis of larval extracts in vivo revealed several metabolites of citral and limonene, with geranic acid and neric acid (from citral), and limonene-1,2-diol (from limonene) as the major ones. The insecticidal activity of geranic acid was very similar to that of the parent compound, citral, but limonene-1,2-diol failed to show any toxicity, indicating that inhibition of the metabolism could be a good strategy to enhance toxicity. Further, larval extracts following topical administration of a binary mixture of the two compounds revealed higher internal concentrations of both compared to their individual application, suggesting the possibility of enhanced cuticular penetration as the mechanism of synergy.

Glycosphingolipids (GSLs) play important roles in the cellular biology of vertebrate and invertebrate organisms, such as cell differentiation, tumor metastasis, and cell coordination. GSLs also serve as receptors for different bacterial toxins . For example, in the nematode Caenorhabditis elegans , GSLs function as receptors of the insecticidal Cry toxins produced by Bacillus thuringiensis (Bt), and mutations in bre genes involved in GSLs synthesis resulted in resistance to Cry5 toxin in this organism. However, the information of GSLs function in insects is still limited. In this study, three genes for glycosyltransferases, bre 2, bre 3, and bre 4, from Helicoverpa armigera were identified and cloned. The previously reported bre5 gene from H. armigera was also analyzed. Protein sequence alignments revealed that proteins codified by H. armigera Bre shared high identity with homologous proteins from other organisms. Expression profile analysis revealed that the expressions of bre genes varied in the different tissues and also in the different developmental stages of H. armigera. Finally, the heterologous expression of bre genes in Trichoplusia ni Hi5 cell line showed that the corresponding translated proteins were localized in the cytoplasm of Hi5 cells. These results provide the bases for further functional studies of bre genes and analyzing potential roles of GSLs in mode of action of Cry1A toxin in H. armigera.