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The South American soybean pest, Rachiplusia nu (Guenée), is naturally infected by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Rachiplusia nu nucleopolyhedrovirus (RanuNPV). We compared their pathogenicity to fourth-instar R. nu larvae, by evaluating time to death and virus spread throughout the tissues in single and mixed infections. Bioassays showed that generalist AcMNPV had a faster speed of kill than specific RanuNPV, while the mixed-virus treatment did not statistically differ from AcMNPV alone. Histopathology evidenced similar tissue tropism for both viruses, but co-inoculation resulted in mostly AcMNPV-infected cells. In sequential inoculations, however, the first virus administered predominated over the second one. Implications on baculovirus interactions and biocontrol potential are discussed. Graphical Abstract

Entomopathogenic fungi are widely recognized as agents of biological control worldwide. Their use in agriculture for the regulation of pest populations is a promising alternative to conventional insecticides. Furthermore, recent studies have shown that entomopathogenic fungi fulfill an additional role in plants as growth promoters. The purpose of this investigation was to assess the growth and yield of corn plants colonized with Beauveria bassiana and its effect on the lepidopteran pest Rachiplusia nu. Effects of the fungus on plant growth, crop yield, and vertical transmission were evaluated in the field. Feeding preferences of R. nu larvae were assessed in the laboratory using a “choice test”. Corn plants inoculated with B. bassiana showed an increase in height, number of leaves, grain weight, yield, and percentage of seed germination compared to control plants. Consumption of B. bassiana-colonized corn plants by R. nu larvae was reduced compared to feeding levels observed on non-inoculated plants. This study showed that endophytic B. bassiana can provide multiple benefits to Zea mays and can play an important role in future integrated pest management programs.

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.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Hydrophobin-fused domain III of dengue envelope proteins serotypes 1 and 2 were expressed in Rachiplusia nu larvae and purified by aqueous two-phase system. This biotechnological approach of hydrophobin-fused proteins, which allowed obtaining 97.7 µg/larva of fusion protein DomIII serotype 1 and 61.4 µg/larva of fusion protein DomIII serotype 2, represents an integrated strategy for simple production of recombinant antigens. Purified fusion proteins induced serotype-specific neutralizing antibodies without cross-reaction against other serotypes and arboviruses after mouse immunization. hydrophobin-fused domain III of dengue envelope protein could be a promising strategy for easy and low-cost production of components of a tetravalent sub-unit vaccine against dengue.

Six trap designs were assessed for capturing noctuid moths in field trials in the United Kingdom and Argentina. The traps were baited with either a sex pheromone for Autographa gamma in the UK trials and Helicoverpa gelotopoeon (Dyar) (Lepidoptera: Noctuidae) in Argentina, or a floral odor blend. In the UK trials the Universal Trap and a funnel sleeve trap were compared; in Argentina the funnel sleeve trap, a homemade bucket trap, and (L.) (Lepidoptera: Noctuidae) three sticky traps: LepTrap, wing trap, and delta trap were compared. Comparisons were made between the traps and attractants and captures of noctuid moths and nontarget insects. Traps baited with the floral attractant caught a lower number but a wider range of noctuid species including Helicoverpa, Spodoptera, Rachiplusia, Dargida, Mythimna, Chrysodeixis, Agrotis, and Autographa spp., and nontarget insects. In the UK trials, the funnel sleeve trap caught significantly more A. gamma than the Universal Trap. The addition of an insecticide to the Universal Trap marginally increased trap catches. In the Argentinian trials, the homemade bucket and the sleeve traps outperformed all sticky traps in most situations regardless of crop environment and attractant type. The homemade bucket and the funnel sleeve traps are also considerably cheaper than the other trap designs. Of the sticky traps the LepTrap caught more noctuids than the wing and delta traps. The results suggest that the bucket trap and the sleeve trap have a much greater maximum capacity and possibly a greater retention efficiency for noctuid moths compared to the sticky traps.

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 cross-attracted 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. Graphical Abstract

The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering.

Lepidoptera, stink bugs, and weevils are important pests in soybean. For lepidopteran control, insecticides and seed treatments are used. As an alternative, Bt soybean was developed to control primary pests of Lepidoptera such as Rachiplusia nu (Guenée) (Noctuidae), Chrysodeixisincludens (Walker) (Noctuidae), Anticarsia gemmatalis Hübner (Erebidae), Helicoverpa gelotopoeon (Dyar) (Noctuidae), and Crocidosema aporema (Walsingham) (Tortricidae). However, the use of transgenic plants, and the resulting reduction of insecticide against target pests, may allow other pest species to become more prevalent in agricultural environments. Soybean expressing Cry1Ac against different lepidopteran nontarget and target insect pests was evaluated, and its performance was compared with non-Bt soybean with seed treatment. The treatments were Bt soybean, non-Bt soybean with seed treatment (Fortenza® diamide insecticide, Syngenta, Buenos Aires, Argentina), and non-Bt soybean without seed treatment. Larvae of H. gelotopoeon, Spodoptera albula (Walker) (Noctuidae), Spodoptera cosmiodes (Walker) (Noctuidae), Spodoptera eridania (Stoll) (Noctuidae), and Spodoptera frugiperda (J. E. Smith) (Lep.: Noctuidae) were used. The plants of each treatment were infested with larvae of each species, and the percentage of leaf damage produced by each species was recorded. The results showed that Bt soybean provided control of H. gelotopoeon and had a suppressive effect on S. frugiperda and S. albula. However, S. eridania and S. cosmiodes were not susceptible to the Cry1Ac protein in MON 87701 × MON 89788 soybean when evaluated by greenhouse infestation. Considering the performance of each species using non-Bt soybean without seed treatment, S. eridania would represent a potential risk in soybean crops.

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers.