Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae.

In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Effect of sublethal concentrations of the bioinsecticide spinosyn treatment of Trichoplusia ni eggs on the caterpillar and its parasitoid, Trichogramma brassicae.

BACKGROUND: Integrated Pest Management (IPM) seeks to combine multiple management strategies for optimal pest control. One method that is successfully employed in IPM is the use of beneficial organisms. However, in severe circumstances when pest insects exceed threshold limits, insecticides may still need to be implemented. Thus, understanding the effects of insecticides on biocontrol agents, such as parasitoid wasps, is paramount to ensure sustainable agroecosystems. Sublethal effects of the bioinsecticide spinosyn, a mixture of the bacterial Saccharopolyspora spinosa (Mertz and Yao) fermentation products spinosyn A and D, on eggs of Trichoplusia ni (Hübner), a cruciferous crop pest, and its egg parasitoid Trichogramma brassicae (Bezdenko) was investigated. RESULTS: The LC50 for spinosyn A and D (dissolved in ethanol) on T. ni eggs is 54 ng mL-1. Transcriptomics on caterpillars (1st and 3rd instars) that hatched from eggs treated with sublethal concentrations of spinosyn identified the upregulation of several genes encoding proteins that may be involved in insecticide resistance including detoxification enzymes, such as cytochrome P450s, glutathione S-transferases and esterases. Sublethal T. ni egg treatments did not affect parasitoid emergence, however, there was a marked increase in the size of T. brassicae hind tibia and wings that emerged from spinosyn-treated eggs. CONCLUSIONS: For the caterpillar, treatment of eggs with sublethal concentrations of spinosyn may induce insecticide resistance mechanisms. For the parasitoids, their increased size when reared in spinosyn-treated eggs suggests that the emerged wasps may have higher performance. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Compatibility of the fungus Beauveria bassiana and Trichoplusia ni SNPV against the cabbage looper Trichoplusia ni: crop plant matters.

BACKGROUND: Microbial insecticides are an important weapon in insect pest management, but their use is still relatively limited. One approach for increasing their efficacy and use could be to combine different pathogens to increase pest mortality. However, little is known about whether increasing pathogen diversity will improve pest management. Here, we investigated the compatibility of two pathogens for the management of the cabbage looper, Trichoplusia ni, T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus Beauveria bassiana, on two crops, tomato and broccoli. The pathogens were applied to individual plants using ultra low volume sprays, alone or in combination, either synchronously or asynchronously. Healthy third-instar T. ni larvae were introduced to the plants before application and collected by destructive sampling 24 h after the last pathogen application. RESULTS: Combined applications did not result in an increase in larval mortality compared to TniSNPV alone, although mortality was generally high. B. bassiana was considerably less effective on broccoli compared to tomato. In both the combined treatments, virus-induced mortality was approximately 50% lower when applied together with the fungus, while fungus-induced mortality was not affected by the virus, even when the virus was introduced 24 h before the fungus. CONCLUSION: While our results suggest that applying this combination of entomopathogens would not be beneficial for pest management, this study illustrates the need to consider the target crop as an important driver of the efficacy of both single and mixed pathogen applications in the field. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cross-Regional Pollination Behavior of Trichoplusia ni between China and the Indo-China Peninsula.

Noctuid moths, a group of "non-bee" pollinators, are essential but frequently underappreciated. To elucidate their roles in cross-regional pollination, this study selected the agriculturally significant species, cabbage looper (CL) Trichoplusia ni, as a representative model. From 2017 to 2021, this study was conducted on Yongxing Island, situated at the center of the South China Sea. We investigated the flower-visiting activities of CL, including its occurrence, potential host species, and geographic distribution in the surrounding areas of the South China Sea. First, the potential transoceanic migratory behavior and regional distribution of CL were systematically monitored through a comprehensive integration of the data obtained from a searchlight trap. The transoceanic migratory behavior of CL was characterized by intermittent occurrence, with the major migratory periods and the peak outbreak yearly. Furthermore, trajectory analysis confirmed the ability of CL to engage in periodic, round-trip, migratory flights between Southeast Asian countries and China. More importantly, an observation of pollen on the body surface demonstrated that 95.59% (130/136) of the migrating individuals carried pollen. The proboscis and compound eyes were identified as the primary pollen-carrying parts, with no observable gender-based differences in pollen-carrying rates. Further, identifying the pollen carried by CL using morphological and molecular methods revealed a diverse range of pollen types from at least 17 plant families and 31 species. Notably, CL predominantly visited eudicot and herbaceous plants. In conclusion, this pioneering study has not only revealed the long-distance migration activities of these noctuid moths in the East Asian region but also provided direct evidence supporting their role as potential pollinators. These findings offer a critical theoretical basis to guide the development of scientific management strategies.

Characterization of the Antimicrobial Activities of Trichoplusia ni Cecropin A as a High-Potency Therapeutic against Colistin-Resistant Escherichia coli.

The spread of colistin-resistant bacteria is a serious threat to public health. As an alternative to traditional antibiotics, antimicrobial peptides (AMPs) show promise against multidrug resistance. In this study, we investigated the activity of the insect AMP Tricoplusia ni cecropin A (T. ni cecropin) against colistin-resistant bacteria. T. ni cecropin exhibited significant antibacterial and antibiofilm activities against colistin-resistant Escherichia coli (ColREC) with low cytotoxicity against mammalian cells in vitro. Results of permeabilization of the ColREC outer membrane as monitored through 1-N-phenylnaphthylamine uptake, scanning electron microscopy, lipopolysaccharide (LPS) neutralization, and LPS-binding interaction revealed that T. ni cecropin manifested antibacterial activity by targeting the outer membrane of E. coli with strong interaction with LPS. T. ni cecropin specifically targeted toll-like receptor 4 (TLR4) and showed anti-inflammatory activities with a significant reduction of inflammatory cytokines in macrophages stimulated with either LPS or ColREC via blockade of TLR4-mediated inflammatory signaling. Moreover, T. ni cecropin exhibited anti-septic effects in an LPS-induced endotoxemia mouse model, confirming its LPS-neutralizing activity, immunosuppressive effect, and recovery of organ damage in vivo. These findings demonstrate that T. ni cecropin exerts strong antimicrobial activities against ColREC and could serve as a foundation for the development of AMP therapeutics.

Trichomes mediate plant-herbivore interactions in two Cucurbitaceae species through pre- and post-ingestive ways.

Plant structural defenses such as trichomes exert a significant selection pressure on insect herbivores. However, whether variation in structural defense traits affects common herbivores in related plant species is less understood. Here, we examined the role of trichomes in plant-herbivore interactions in two commonly cultivated members in Cucurbitaceae: bottle gourd (Lagenaria siceraria) and cucumber (Cucumis sativa). In common garden experiments when the two species were grown together, we observed that they differed in their attractiveness to four major herbivore species (Trichoplusia ni, Acalymma vittatum, Diaphania indica, and Anasa tristis) and, consequently, their feeding behavior. We found that L. siceraria consistently harbored less herbivores, and the two lepidopteran herbivores (T. ni and D. indica) were found to take significantly longer time to commence feeding on them, a primary mode of pre-ingestive defense function of trichomes. To tease apart structural and chemical modes of defenses, we first used scanning electron microscopy to identify, quantify, and measure trichome traits including their morphology and density. We found that C. sativa has significantly lower number of trichomes compared to L. siceraria, regardless of trichome type and leaf surface. We then used artificial diet enriched with trichomes as caterpillar food and found that trichomes from these two species differentially affected growth and development of T. ni showing cascading effects of trichomes. Taken together, we show that trichomes, independent of chemical defenses, are an effective pre- and post-ingestive defense strategy against herbivores with negative consequences for their feeding, growth, and development. Supplementary Information: The online version contains supplementary material available at 10.1007/s10340-023-01611-x.

Resistance of Cabbage Loopers to Bacillus thuringiensis (Bt) Toxin Cry1F and to Dual-Bt Toxin WideStrike Cotton Plants.

The Cry proteins from Bacillus thuringiensis (Bt) are major insecticidal toxins in formulated Bt sprays and are expressed in genetically engineered Bt crops for insect pest control. However, the widespread application of Bt toxins in the field imposes strong selection pressure on target insects, leading to the evolution of insect resistance to the Bt toxins. Identification and understanding of mechanisms of insect resistance to Bt toxins are an important approach for dissecting the modes of action of Bt toxins and providing knowledge necessary for the development of resistance management technologies. In this study, cabbage looper (Trichoplusia ni) strains resistant to the transgenic dual-Bt toxin WideStrike cotton plants, which express Bt toxins Cry1Ac and Cry1F, were selected from T. ni strains resistant to the Bt formulation Bt-DiPel. The WideStrike-resistant T. ni larvae were confirmed to be resistant to both Bt toxins Cry1Ac and Cry1F. From the WideStrike-resistant T. ni, the Cry1F resistance trait was further isolated to establish a T. ni strain resistant to Cry1F only. The levels of Cry1F resistance in the WideStrike-resistant and the Cry1F-resistant strains were determined, and the inheritance of the Cry1F-resistant trait in the two strains was characterized. Genetic association analysis of the Cry1F resistance trait indicated that the Cry1F resistance in T. ni isolated in this study is not shared with the Cry1Ac resistance mechanism nor is it associated with a mutation in the ABCC2 gene, as has so far been reported in Cry1F-resistant insects. IMPORTANCE Insecticidal toxins from Bacillus thuringiensis (Bt) are highly effective for insect control in agriculture. However, the widespread application of Bt toxins exerts strong selection for Bt resistance in insect populations. The continuing success of Bt biotechnology for pest control requires the identification of resistance and understanding of the mechanisms of resistance to Bt toxins. Cry1F is an important Bt toxin used in transgenic cotton, maize, and soybean varieties adopted widely for insect control. To understand the mode of action of Cry1F and mechanisms of Cry1F resistance in insects, it is important to identify Cry1F-specific resistance and the resistance mechanisms. In this study, Trichoplusia ni strains resistant to commercial "WideStrike" cotton plants that express Bt toxins Cry1Ac and Cry1F were selected, and a Cry1F-specific resistant strain was isolated. The isolation of the novel Cry1F-specific resistance in the T. ni provided an invaluable biological system to discover a Cry1F-specific novel resistance mechanism.

Host Cytoskeleton Gene Expression Is Correlated with the Formation of Ascovirus Reproductive Viral Vesicles.

Ascoviruses are large DNA viruses that primarily infect lepidopteran larvae. They differ markedly from other plant or animal viruses by initiating replication in the nucleus, then inducing nuclear lysis followed by extensive cellular hypertrophy and subsequent cleavage of the entire enlarged cell into numerous viral vesicles. Most progeny virions are assembled in these vesicles as they circulate in the hemolymph. Here, we report transcriptome studies of host cytoskeletal genes in larvae infected with ascoviruses from 6 h to 21 days post-infection (dpi). We focused on the cabbage looper, Trichoplusia ni, infected with the Trichoplusia ni ascovirus (TnAV), along with supporting studies on the fall armyworm, Spodoptera frugiperda, infected with the Spodoptera frugiperda ascovirus (SfAV). In T. ni, many cytoskeleton genes were upregulated at 48 hours post-infection (hpi), including 29 tubulins, 21 actins, 21 dyneins, and 13 kinesins. Mitochondrial genes were upregulated as much as two-fold at 48 hpi and were expressed at levels comparable to controls in both T. ni and S. frugiperda, even after 21 dpi, when several cytoskeleton genes remained upregulated. Our studies suggest a temporal correlation between increases in the expression of certain host cytoskeletal genes and viral vesicle formation. However, these results need confirmation through functional genetic studies of proteins encoded by these genes.

Bt Cry1Ac resistance in Trichoplusia ni is conferred by multi-gene mutations.

The three-domain Cry toxin Cry1Ac from Bacillus thuringiensis （Bt） is an important insecticidal toxin in Bt sprays and has been used in transgenic Bt-crops to confer insect resistance. The cabbage looper, Trichoplusia ni, has developed resistance to Bt sprays in commercial greenhouses, and the resistance to Cry1Ac has been previously identified to be associated with altered expression of the APN1 and APN6 genes and be genetically linked to a locus on chromosome 15. In this study, the Cry1Ac resistance locus in T. ni was further finely mapped, and the specific Cry1Ac resistance-conferring mutation in the resistance locus was identified to be a 4 bp frameshift insertion in the ABCC2 gene by whole genome resequencing, midgut transcriptome analysis, candidate gene cDNA sequencing and mutation site genomic DNA sequencing. By CRISPR/Cas9 mutagenesis, a series of ABCC2 and ABCC3 mutant T. ni strains were generated, and the role of ABCC2 in the toxicity of Cry1Ac in T. ni was confirmed. The results from this study also showed that knockout of ABCC2 in T. ni conferred resistance to Cry1Ac at a level lower than that in the greenhouse-derived resistant T. ni strain and that the Cry1Ac resistance-associated alteration of APN1 and APN6 expression was independent of ABCC2 gene mutations, indicating that the altered expression of APN1 and APN6 was controlled by another gene mutation in Cry1Ac resistant T. ni. Furthermore, T. ni larval bioassays showed that the level of Cry1Ac resistance in F1 families from reciprocal crosses of the Cry1Ac resistant strain with an ABCC2 knockout CRISPR strain was significantly higher than that in ABCC2 knockout strain, indicating the presence of additional Cry1Ac resistance-conferring mutation(s) in the Cry1Ac resistant strain. Therefore, the resistance to Cry1Ac in T. ni is conferred by a mutation in ABCC2 and an additional mutation (or mutations) which leads to altered expression of APN1 and APN6. The additional Cry1Ac resistance mutation or mutations remain to be identified.

Chimeric VLPs Bearing VP60 from Two Serotypes of Rabbit Haemorrhagic Disease Virus Are Protective against Both Viruses.

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

Metabolic response to larval herbivory in three Physalis species.

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.

Temporal Occurrence of Plusiinae on Soybean in the Mississippi River Delta.

The subfamily Plusiinae of the moth family Noctuidae is made up 400 species worldwide. Two species of the subfamily, the soybean looper, Chrysodeixis includens (Walker), and the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), are important defoliating insect pests of various field crops and have been the subject of previous Plusiinae surveys in the Southern U.S. Soybean fields were sampled in the Mississippi Delta from 2010 to 2012 to determine the temporal occurrence of various Plusiinae species on soybean. As in previous surveys, C. includens was the most common Plusiinae species in soybean during the 3-yr survey, especially in late season collections (July-September). Rachiplusia ou (Guenée) (Lepidoptera: Noctuidae) was the predominant species observed in early season collections (May-early July). Populations of R. ou during the first sample dates during 2010 were much higher than those observed during the other years of the survey. Only three collected larvae successfully developed into T. ni adults, one each collected during May, June, and July. Although R. ou was not commonly reported in previous studies on soybean, it occurred in low numbers during June and July during the 3-yr study. The temporal occurrence and species composition followed a predictable pattern in all 3 yr of the study.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

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 Hübner (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.

Functional redundancy of structural proteins of the peritrophic membrane in Trichoplusia ni.

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.

Soil Microbiomes From Fallow Fields Have Species-Specific Effects on Crop Growth and Pest Resistance.

Communities of microorganisms in the soil can affect plants' growth and interactions with aboveground herbivores. Thus, there is growing interest in utilizing soil microbiomes to improve plant performance in agriculture (e.g., for pest control), but little is known about the phenotypic responses of various crop species to different microbiomes. In this study, we inoculated four crop species from different botanical families, maize (Zea mays, Poaceae), cucumber (Cucumis sativus, Cucurbitaceae), tomato (Solanum lycopersicum, Solanaceae), and lettuce (Lactuca sativa, Asteraceae), with diverse soil microbiomes originating from actively-managed agricultural fields or fallow fields under varying stages of succession (1, 3, and 16-years post-agriculture) sourced from a large-scale field experiment. We compared the crops' responses to these different microbiomes by assessing their growth and resistance to two generalist insect pests, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda). These different microbiomes affected both plant growth and resistance, but the effects were species-specific. For instance, lettuce produced the largest leaves when inoculated with a 3-year fallow microbiome, the microbiome in which cucumber performed worst. Plants were generally more resistant to T. ni when inoculated with the later succession microbiomes, particularly in contrast to those treated with agricultural microbiomes. However, for tomato plants, the opposite pattern was observed with regard to S. frugiperda resistance. Collectively, these results indicate that plant responses to microbiomes are species-specific and emphasize the need to characterize the responses of taxonomically diverse plant species to different microbiomes.

A new nodavirus-negative Trichoplusia ni cell line for baculovirus-mediated protein production.

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 agents. 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.

Leaf vibrations produced by chewing provide a consistent acoustic target for plant recognition of herbivores.

Plant defenses that respond to the threat of herbivory require accurate sensing of the presence of herbivores. Herbivory cues include mechanical damage, elicitors from insect saliva or eggs, and airborne volatiles emitted by wounded plants. Plants can also respond to the leaf vibrations produced by chewing herbivores. However, previous studies of the influence of feeding vibrations on plant defenses have been limited to single species pairs. In this study we test the hypothesis that chewing vibrations differ among herbivore species, both in their acoustic features and in their potential effect on plant defense responses. We first compare the acoustic traits of larval feeding vibrations in ten species from six families of Lepidoptera and one family of Hymenoptera. We then test responses of Arabidopsis thaliana plants to variation among feeding vibrations of different individuals of one species, and to feeding vibrations of two species, including a pierid butterfly and a noctuid moth. All feeding vibrations consisted of repetitive pulses of vibration associated with leaf tissue removal, although chewing rates varied between species and between large and small individuals within species. The frequency spectra of the vibrations generated by leaf feeding were similar across all ten species. Induced increases in anthocyanins in A. thaliana did not differ when plants were played vibrations from different individuals, or vibrations of two species of herbivores with different chewing rates, when amplitude was held constant. These results suggest that feeding vibrations provide a consistent set of cues for plant recognition of herbivores.

Ectomycorrhizal fungi induce systemic resistance against insects on a nonmycorrhizal plant in a CERK1-dependent manner.

Below-ground microbes can induce systemic resistance against foliar pests and pathogens on diverse plant hosts. The prevalence of induced systemic resistance (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 fungus Laccaria bicolor on the nonmycorrhizal plant Arabidopsis thaliana. We used the cabbage looper Trichoplusia ni and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto) as readouts for ISR on Arabidopsis. We found that root inoculation with L. bicolor triggered ISR against T. ni and induced systemic susceptibility (ISS) against the bacterial pathogen Pto. We found that L. bicolor-triggered ISR against T. ni was dependent on jasmonic acid signaling and salicylic acid biosynthesis and signaling. Heat-killed L. bicolor and chitin were sufficient to trigger ISR against T. ni and ISS against Pto. The chitin receptor CERK1 was necessary for L. bicolor-mediated effects on systemic immunity. Collectively our findings suggest that some ISR responses might not require intimate symbiotic association, but rather might be the result of root perception of conserved microbial signals.

Stimulation of Insect Herbivory by Elevated Temperature Outweighs Protection by the Jasmonate Pathway.

Rising global temperatures are associated with increases in the geographic range, population size, and feeding voracity of insect herbivores. Although it is well established that the plant hormone jasmonate (JA) promotes durable resistance to many ectothermic herbivores, little is known about how JA-mediated defense is influenced by rising temperatures. Here, we used the Arabidopsis-Trichoplusia ni (cabbage looper) interaction to investigate the relative contribution of JA and elevated temperature to host resistance. Video monitoring of T. ni larval behavior showed that elevated temperature greatly enhanced defoliation by increasing the bite rate and total time spent feeding, whereas loss of resistance in a JA-deficient mutant did not strongly affect these behaviors. The acceleration of insect feeding at elevated temperature was not attributed to decreases in wound-induced JA biosynthesis, expression of JA-responsive genes, or the accumulation of defensive glucosinolates prior to insect challenge. Quantitative proteomic analysis of insect frass, however, provided evidence for a temperature-dependent increase in the production of T. ni digestive enzymes. Our results demonstrate that temperature-driven stimulation of T. ni feeding outweighs the protective effects of JA-mediated resistance in Arabidopsis, thus highlighting a potential threat to plant resilience in a warming world.