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Chysodeixis includens (Walker) is a polyphagous economic pest in agricultural landscapes. To detect the occurrence of this pest in the field, trapping using sex pheromone lures is often implemented. However, other plusiine species are cross-attracted to these lures and may be misidentified as C. includens due to their morphological similarities. The objectives of this study were to provide region-specific information on the abundance of C. includens throughout the year as well as document the occurrence of related plusiines crossattracted to C. includens sex pheromone traps in the Florida Panhandle. Twelve commercial fields of peanut and twelve commercial fields of cotton located across Escambia, Santa Rosa, and Jackson counties were monitored with Trécé delta traps baited with C. includens sex pheromone lures (Alpha Scents, Inc.; West Linn, OR) from June 2017 to June 2019. There was no difference in C. includens flight across dryland or irrigated fields. Identifications revealed that in addition to C. includens, the following species of the subfamily Plusiinae were crossed-attracted: Argyrogramma verruca (Fabricius) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), Ctenoplusia oxygramma (Geyer) (Lepidoptera: Noctuidae), and Rachiplusia ou (Guenée) (Lepidoptera: Noctuidae). The occurrence of each species in the region and their flight phenology are documented. Chysodeixis includens abundance was greatest in September and decreased through December. Due to the high abundance of C. oxygramma and similar flight phenology to C. includens, this is the likeliest species to skew estimations and influence management decisions of C. includens, especially early in the crop season, when C. includens abundance is low. Made available in DSpace on 2022-05-24T05:01:37Z (GMT). No. of bitstreams: 1 Alexandre-Seasonal-flight-patterns.pdf: 30387592 bytes, checksum: 403e5b227747fa7fc1a3e4cad2be3161 (MD5) Previous issue date: 2021

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.

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.

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.

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.

Many species of moths have a common control mechanism for synthesizing sex pheromone: the circadian release of pheromone biosynthesis-activation neuropeptide (PBAN) that switches pheromone synthesis on/off during the day. One apparent exception to this is the noctuid moth Trichoplusia ni (Hubner), in which pheromone synthesis appears continuous through the photoperiod, with circadian release of PBAN controlling emission rate of pheromone during calling. Sex pheromone biosynthesis was reinvestigated in T. ni using stable isotope tracer-tracee and gland sampling techniques to ascertain how pheromone quantities in gland cells and on the gland cuticular surface varied and were controlled. It was found that (i) carbohydrate from adult female feeding is used to synthesize sex pheromone, (ii) most of the stored acetate ester pheromone component(s) is contained in gland cells, (iii) a large pool of pheromone is synthesized and stored through the photoperiod with a slow turnover rate, (iv) although pheromone is synthesized throughout the photoperiod, its rate can vary, influenced by release of PBAN and possibly by an unidentified head factor, with both affecting carbohydrate uptake into the acetyl CoA pheromone precursor pool, and (v) as suggested previously, PBAN also influences translocation of pheromone out of the cell to the cuticular surface, possibly by causing breakdown of intracellular lipid droplets storing pheromone molecules. This work suggests that the quantitative synthesis and emission of pheromone in T. ni, and possibly other moths, is regulated by multiple complementary biochemical mechanisms.

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.

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.

Baculoviruses have been used for the expression of heterologous proteins of biotechnological interest. However, most of these proteins are obtained by homologous co-transfection recombination in cell lines, limiting their use. Recently, the CRISPR/Cas9 system has excelled in its high efficiency in editing specific sequences without the need for insect cell lines. In this work, the CRISPR/Cas9 system was used to edit the genome of Trichopusia ni granulovirus (TnGV) and transformation of insects by the PIG bombardment method. A homologous repair vector (pTnGV101) was designed with regions orf5 and orf7, as well as sgRNA flanking TnGV P10 of this virus. The bombardment transformation was carried out at 175 psi with 40% of infected T. ni larvae, of which 38% expressed the reporter protein EGFP. These results demonstrate that the CRISPR/Cas9 system and PIG bombardment can be used for genetic modification of baculovirus in vivo.

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.

Tyrosinases are melanocyte-specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra-melanosomal domains of tyrosinase, TYRtr (19 – 469), and tyrosinase-related protein 1, TYRP1tr (25 – 472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero-oligomeric complexes to maintain the protective function.

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

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.

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.

Nucleocapsid (N) protein is the most highly expressed of all avian metapneumovirus (aMPV) viral proteins and stimulates a substantial immune response in infected animals. Codon optimized recombinant N (rec-N) protein from aMPV subtypes A, B, and C were expressed using the baculoviral expression system in Trichoplusia ni (Tni) insect cells. A mixture of purified rec-N antigens from each subtype was used as a coating antigen and was evaluated in indirect ELISA (iELISA) to assess antibody response in serum samples collected from experimentally infected chickens and turkeys with different aMPV subtypes. Also, archived field serum samples that were collected from different poultry submissions were used. Receiver operating characteristic (ROC) analysis was performed using chicken and turkey serum samples that were confirmed by indirect fluorescent antibody (IFA) test for serostatus (positive n = 270, negative n = 610). The ROC analysis showed sensitivity and specificity of 97 % at a cut-off value of 0.25. The rec-N iELISA was compared with a commercial whole virus-based APV kit. The rec-N iELISA showed comparable results in detecting antibody response in aMPV infected chicken sera but was more sensitive in detecting early antibody response in aMPV infected turkey serum samples. Our results further confirm the presence of aMPV antibodies in Canadian domestic poultry populations. The developed aMPV-rec N iELISA offers a safe and valuable alternative to whole virus-based iELISA for serodiagnosis and seroepidemiological surveillance of the disease in domestic poultry.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hubner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

Cell lines derived from Trichoplusia ni (Tn) are widely used as hosts in the baculovirus-insect cell system (BICS). One advantage of Tn cell lines is they can produce recombinant proteins at higher levels than cell lines derived from other insects. However, Tn cell lines are persistently infected with an alphanodavirus, Tn5 cell line virus (TnCLV), which reduces their utility as a host for the BICS. Several groups have isolated TnCLV-negative Tn cell lines, but none were thoroughly characterized and shown to be free of other adventitious viruses. Thus, we isolated and extensively characterized a new TnCLV-negative line, Tn-Nodavirus Negative (Tn-NVN). Tn-NVN cells have no detectable TnCLV, no other previously identified viral contaminants of lepidopteran insect cell lines, and no sequences associated with any replicating virus or other viral adventitious agent. Tn-NVN cells tested negative for >60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma. Finally, Tn-NVN cells grow well as a single cell suspension culture in serum-free medium, produce recombinant proteins at levels similar to High Five(™) cells, and do not produce recombinant glycoproteins with immunogenic core α1,3-fucosylation. Thus, Tn-NVN is a new, well-characterized TnCLV-negative cell line with several other features enhancing its utility as a host for the BICS.

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.