Nu Larvae Research Articles

Life table study of Rachiplusia nu (Lepidoptera: Noctuidae) on different food sources and artificial diet.

Rachiplusia nu Guenée is a polyphagous species able to develop on several cultivated and non-cultivated host plants. However, basic life history information about this pest on hosts is scarce. In this study, R. nu larvae did not survive on leaves of non-Bt corn, wheat, Bt cotton that expresses proteins Cry1Ac+Cry2Ab2 or on Intacta2 Xtend soybean that expresses the Cry1A.105/Cry2Ab2/Cry1Ac proteins. Rachiplusia nu showed a viable egg-to-adult biological cycle (54%-66.3%) on non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip hosts, similar to larvae raised on the artificial diet. In addition, R. nu was unable to complete larval development on non-Bt cotton, and only 45.2% of R. nu larvae reached the pupal stage when fed leaves of intacta RR2 PRO soybean that expresses the Cry1Ac protein. Larval and pupal mass of surviving insects on Cry1Ac soybean leaves were also lower (larval: 0.104g; pupal: 0.099g) in relation to other food sources (larval: 0.165-0.189g; pupal: 0.173-0.192g). The total fecundity of R. nu on Cry1Ac soybean leaves was ≈65% lower in relation to other food sources. This fact caused ≈60% the net reproductive rate (Ro) and intrinsic rate of increase (rm) when compared to other food sources. Our findings indicate that the Cry1Ac soybean negatively affects the biological parameters of R. nu. Non-Bt soybean, sunflower, canola, vetch, Persian clover, alfalfa, bean, and forage turnip are viable food sources for the survival and development of R. nu.

Genetic diversity of the polyphagous pest Rachiplusia nu across crops and locations in the Argentine Pampas

AbstractRachiplusia nu (Guenée) (Lepidoptera: Noctuidae, Plusiinae) is a defoliator of soybean (Glycine max L., Fabaceae), sunflower (Helianthus annuus L., Asteraceae), alfalfa (Medicago sativa L., Fabaceae), and horticultural crops in temperate southern South America; however, there is little genetic information available about this pest insect. Recently, concerns about this insect have increased because of its suspected resistance to Bt soybean and its spread to tropical regions. To better understand pest dynamics, and therefore implement suitable management strategies, a thorough knowledge of the genetic variability and spatial structuring of populations is required. In this paper, R. nu larvae were sampled from various crops and locations in central Argentina (where the pest is endemic) to assess diversity and population structure through mtDNA sequence analysis. In addition to the analysis of the standard cytochrome c oxidase subunit I (COI) ‘barcode’ fragment, a new set of primers was developed to explore genetic variation in the cytochrome b (Cytb) gene. COI and Cytb concatenated sequences revealed an overall high haplotype diversity (mean ± SD = 0.798 ± 0.037) and low nucleotide diversity (0.00137 ± 0.00013). The haplotype network showed a total of 19 haplotypes, separated by at most five mutational steps. A main central haplotype and four additional frequent haplotypes (accounting for 80% of the specimens examined) were distributed across populations with no obvious pattern related to host plant or geographic origin. Analysis of molecular variance further supported a weak population structure, where genetic variation within populations was the main source of total variation (ca. 96%). Pairwise comparisons showed only minor genetic differentiation among some of the populations studied. Implications of these findings in the context of R. nu control are discussed.

Ten years of Cry1Ac Bt soybean use in Argentina: Historical shifts in the community of target and non-target pest insects

Long-term changes in the insect communities in genetically modified (GM) crops expressing target-specific pesticidal proteins can occur and matter for optimized integrated pest management. Using monitoring data of commercial soybean fields from 2012 to 2022, we documented shifts in the abundance of target and non-target insects of the GM Cry1Ac Bacillus thuringiensis (Bt) soybean in two provinces of northwestern Argentina. Before adoption of Bt soybean, lepidopterans prevailed in the crop-associated insect-pest community. They were 65% more abundant than stink bugs and weevils, and the highest population levels (a mean of 169 individuals/plot) were recorded during the vegetative and early reproductive stages. After introducing Bt soybean, the abundance of target lepidopterans (i.e., erebids, heliothines, and plusiines) was the lowest in the 2017–2018 crop season. In the 2020–2021 and 2021–2022 seasons, Rachiplusia nu larvae (Lepidoptera: Noctuidae: Plusiinae) were recorded in Bt soybean fields. The numbers of non-target lepidopterans (Spodoptera spp.) increased year by year, and they were the prevailing lepidopteran species on Bt soybean in 2020–2021 and the R1-R5 (reproductive) soybean growth stages. Weevil abundance was higher on Bt soybean than non-Bt soybean. Stink bugs were more frequent in Tucumán than Catamarca, and their abundances were higher during R1-R5 than during vegetative and late-reproductive stages. The temperature, precipitation, and solar radiation conditioned the abundance of some of the pest complexes. Efforts to monitor and manage secondary or Bt-resistant populations of insect pests are necessary and should be continued and complemented with studies of pest-susceptibility shifts to inform sound locally adapted integrated pest management programs.

Effect of Chlorantraniliprole on Beneficial Fauna Associated with Soybean Cultivation

Using insecticides on soybean (Glycine max, L.) may induce lethal and sublethal effects on beneficial fauna associated with soybean cultivation. In Argentina, the application of pesticides in extensive and intensive crops for weed and insect control has increased by more than 400 % over the last 20 years. This study aimed to determine the effect of chlorantraniliprole applied under field conditions on predators and parasitoids associated with Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae). Phytophagous and predatory organisms were recorded in chlorantraniliprole-treated and untreated field plots during the 2014-2015 crop season. R. nu larvae were collected, individually placed in Petri dishes, and reared in a conditioned growth chamber until adult Lepidoptera or parasitoids appeared. Data were analyzed with the generalized linear mixed model. In untreated plots, the parasitoid Copidosoma sp. reduced the R. nu population growth curve below the economic injury level. In chlorantraniliprole-treated fields, differences before and after application were significant (p > 0.05), indicating that the insecticide affected predators (Araneidae, Syrphidae, Hemiptera) and parasitoids (Hymenoptera). Additionally, the Berger-Parker dominance index showed parasitoids were more sensitive to chlorantraniliprole than predators. Future research on selective insecticides with other action modes would be needed to evaluate sublethal effects on natural enemies.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.

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.

Comparative Pathogenesis of Generalist AcMNPV and Specific RanuNPV in Larvae of Rachiplusia nu (Lepidoptera: Noctuidae) Following Single and Mixed Inoculations.

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.

Simple production of hydrophobin-fused domain III of dengue envelope protein and induction of neutralizing antibodies against the homotypic serotype of dengue virus.

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 97.7µg/larva of fusion protein DomIII serotype 1 and 61.4µg/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.

Endophytic Effects of Beauveria bassiana on Corn (Zea mays) and Its Herbivore, Rachiplusia nu (Lepidoptera: Noctuidae).

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.

Recombinant protein purification in baculovirus-infected Rachiplusia nu larvae: An approach towards a rational design of downstream processing strategies based on chromatographic behavior of proteins

Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones. Two-dimensional (2D) gel electrophoresis showed differences in the protein patterns of infected and non-infected larvae. Hydrophobic interaction matrices adsorbed the bulk of larval proteins, thus suggesting that such matrices are inappropriate for this system. Only 0.03% and 2.9% of the total soluble protein from the infected larval extract was adsorbed to CM-Sepharose and SP-Sepharose matrices, respectively. Immobilized metal ion affinity chromatography represented a solid alternative because it bound only 1.4% of the total protein, but would increase the cost of the purification process. We concluded that cation-exchange chromatography is the best choice for easy purification of high-isoelectric-point proteins and proteins with arginine tags, since very few contaminating proteins co-eluted with our target protein.

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.

Biological and molecular features of Nosema rachiplusiae sp. n., a microsporidium isolated from the neotropical moth Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae).

Light, electron microscopy and DNA analyses were performed to characterize a microsporidium infecting Rachiplusia nu larvae from a laboratory rearing in Argentina. Diplokaryotic spores were oval and measured 3.61 ± 0.29 × 1.61 ± 0.14μM (fresh). The spore wall was composed of an electron-dense exospore and an electron-lucent endospore, ca. 30nm and 100-120nm thick, respectively. The polar filament was arranged in a single rank of 10-12 coils (typically 11). Microsporidian cells were found in the cytoplasm, next to the endoplasmic reticulum (especially the prespore stages) and generally surrounded by electron-lucent spaces. The infection was polyorganotropic; the fat body appeared as the most heavily invaded tissue, followed by tracheal matrix and epidermis. A molecular phylogeny based on the small (SSU) and large subunit (LSU) ribosomal RNA genes clearly placed the new isolate within the "Nosema bombycis clade". Considering both SSU and LSU concatenated partial sequences, the microsporidium from R. nu showed 99.5% nucleotide similarity with N. bombycis and 99.8% with its closest relative, a microsporidium isolated from Philosamia cynthia. According to its genetic and biological features, the R. nu isolate is proposed as the new species Nosema rachiplusiae sp. n., expanding the limited knowledge on microsporidia associated to endemic South-American moths.

Compatibility of chemical insecticides and entomopathogenic fungi for control of soybean defoliating pest, Rachiplusia nu

Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) is one of the major lepidopteran pests defoliating soybeans (Glycine max Merrill) in Argentina. The combined use of chemical insecticides and entomopathogenic fungi is a promising pest-control option to minimize adverse chemical effects. In this work, we evaluated the interactions between five insecticides—two being considered biorational—and five fungal entomopathogenic strains under laboratory conditions in order to determine the possible usefulness of combinations of these agents against R. nu. The insecticides were tested for compatibility at four doses by in vitro bioassay and for the lethality of R. nu by inoculations at three doses. Fungal strains were applied at 1×108, 1×106, and 1×104conidia/ml. The combinations of those insecticides with Beauveria bassiana (LPSc 1067, LPSc 1082, LPSc 1098), Metarhizium anisopliae (LPSc 907), and Metarhizium robertsii (LPSc 963) caused higher R. nu–larval mortalities than any of the individual agents alone. We observed significant differences in the in vitro conidial viability, vegetative growth, and conidia production of the five strains of entomopathogenic fungi exposed to different doses of the chemical insecticides. The combination gamma-cyhalothrin–LPSc-1067 caused the highest percent mortality of R. nu larvae, with synergism occurring between the two agents at 50% and 25% of the maximum field doses.

Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus. The antigen was expressed as a fusion to hydrophobin I (DomIIIHFBI) to easily purify it by an aqueous two-phase system (ATPS) and to efficiently immobilize it in immunoassay plates. A high level of recombinant DomIIIHFBI was obtained in R. nu, where yields reached 4.5 mg per g of larva. Also, we were able to purify DomIIIHFBI by an ATPS with 2% of Triton X-114, reaching a yield of 73% and purity higher than 80% in a single purification step. The recombinant DomIIIHFBI was efficiently immobilized in hydrophobic surface plates. The immunoassay we developed with the immobilized antigen was able to detect IgG specific for dengue virus type 2 in serum samples and not for other serotypes.

High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae

Wheat germ agglutinin (WGA) is a homodimeric lectin stabilized by non-covalent interactions. Each monomer of 171 residues, which has a complex structure with 16 disulfide bridges, determines two sites for the specific binding of N-acetyl-d-glucosamine and one for the specific binding of N-acetylneuraminic acid. Because of these folding requirements, the production of high yields of recombinant WGA is still not possible and its extraction from wheat germ is the only source for commercial purposes. This work reports for the first time the expression of WGA isolectin A (WGA A), using a baculovirus expression system in Sf9 cells and Rachiplusia nu larvae. High levels of recombinant WGA A were obtained in both cases, especially in R. nu, where yields reached 346.6±88.5μg/g of larvae. Also, an integrated purification method was developed based on aqueous two-phase separation coupled to affinity chromatography using chitosan mini-spheres. The recombinant WGA A was able to recognize ovoalbumin sugar moieties, cross-react with anti-WGA serum and agglutinate human red blood cells, and showed the same behavior as that of commercial WGA in SDS-PAGE and RP-HPLC analyses.

Expression and purification of recombinant feline interferon in the baculovirus-insect larvae system

Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and insect larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28±0.15)×106Uml−1 and (1.3±0.2)×106Uml−1 respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7±0.2)×106Uml−1 and (3.5±0.4)×106Uml−1 at 2dpi in Rachiplusia nu larvae and (1.1±0.2)×106Uml−1 and (1.0±0.15)×106Uml−1 at 5dpi in Spodoptera frugiperda larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots.

Description of a novel single mutation in the AcMNPV polyhedrin gene that results in abnormally large cubic polyhedra

We describe a point mutation in the AcMNPV polyhedrin gene that produces abnormally large cubic polyhedra in packaging cell lines. A polyhedrin mutant baculovirus in which the single change E44G was introduced confirmed that this mutation and no other alterations in the AcMNPV genome was responsible for the abnormal phenotype. Although baculoviral VP39 protein was detected inside mutant polyhedra, electron microscopy demonstrated that only a proportion of the large crystals allow occlusion of virions. When compared with wild-type polyhedra, the mutant inoculum showed reduced oral infectivity for Rachiplusia nu larvae. Hence, the amino acid 44 substitution in the AcMNPV polyhedrin protein alters polyhedrin assembly and affects viral occlusion and infectivity.

Trans-complementation of polyhedrin by a stably transformed Sf9 insect cell line allows occ− baculovirus occlusion and larval per os infectivity

Oral infection of insect larvae with baculovirus is an advantageous methodology for producing high levels of recombinant proteins and for achieving plague control. However, many recombinant baculoviruses express a foreign protein in lieu of the polyhedrin and hence do not form occlusion bodies (occ−), resulting in extremely reduced per os infectivity in larvae.To overcome this limitation, stably transformed insect cell lines expressing polyhedrin capable of occluding occ− recombinant baculovirus by trans-complementation were developed to obtain oral inoculum for insect larvae infection.First, the optimum regulatory region of polyhedrin promoter was determined utilizing chloramphenicol acetyl transferase (CAT) as the reporter gene. After infection with occ− baculovirus, the higher expression levels of CAT were achieved when a region of 2735bp that contained sequences known to have transcriptional enhancer functions were present upstream the polyhedrin coding sequence. This regulatory region was selected to drive polyhedrin expression in insect cell lines.Transfection of Sf9 cells with plasmid carrying polyhedrin gene and stable cell lines established by selection with blasticidin showed polyhedrin expression and, moreover, crystalline polyhedron-like structures were visualized by optic microscopy. Oral infectivity was demonstrated by fluorescence detection in Rachiplusia nu larvae infected with occluded AcGFPpolh− baculovirus obtained using the system presented here.

Expression and purification of horseradish peroxidase in insect larvae

A strategy for obtaining a high-level expression of horseradish peroxidase isozyme C (HRPC) in insect larvae is herein described. The HRPC-6xHis coding sequence was inserted into the AcNPV genome via the pAcGP67HRPC-6xHis transfer vector to originate an AcHRPC-6xHis recombinant baculovirus of phenotype occ −. Rachiplusia nu larvae were injected with a viral stock derived from culture supernatants of Sf9 cells infected with AcHRPC-6xHis. Enzyme concentration at day 3 post-infection was 230 ± 10 mg/kg haemolymph and 100 ± 14 mg/kg larvae, an expression level never attained by other expression systems. HRPC-6xHis was purified from the crude larval extract or haemolymph by immobilised metal ion affinity chromatography with yields of 88.8% and 89.0% and purification factors of 18.9 and 14.0, respectively, in a single step. The purity of the final product was 90%.

Induction of virus-neutralizing antibodies by immunization with Rachiplusia nu per os infected with a recombinant baculovirus expressing the E2 glycoprotein of bovine viral diarrhea virus

This work describes the development of a novel protein expression system based on Rachiplusia nu larvae for the production of the recombinant E2 protein to be used as a vaccine candidate against bovine viral diarrhea virus (BVDV). A recombinant baculovirus (Ac-E2pol+) bearing the E2 glycoprotein coding sequence of BVDV was obtained. Fourth-instar R. nu larvae were infected orally with recombinant polyhedra and the expression of E2 protein was confirmed by immunoblot. In order to test the recombinant product as a vaccine candidate, an immunization assay was performed and the neutralizing humoral immune response against BVDV NADL strain was evaluated. Mice vaccinated with Ac-E2pol+ extracts of per os infected larvae developed a neutralizing antibody titer of 3.16 after the administration of three doses of the immunogen. This report demonstrates the efficacy of per os infected larval extracts as a BVDV recombinant immunogen, which constitutes an easier and economic approach for producing recombinant antigens.