Rachiplusia Nu Research Articles

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.

Efecto de las arañas (Arachnida: Araneae) como depredadoras de insectos plaga en cultivos de alfalfa (Medicago sativa) (Fabaceae) en Argentina

Spiders are predators that reduce insect pest populations in agroecosystems. Trials were conducted to measure the selectivity against different insect preys, the daily consumption, effect of predators alone and together with a known number of preys, and the indirect effect of predators on vegetation. For this, experimental units (1 x 1m) were used covered with a fine plastic mesh. Misumenops pallidus, Oxyopes salticus and Araneus sp. were used as generalist predators, and aphids, weevils, locusts, chrysomelids and Lepidoptera larvae as their potential preys. Among the preys offered, the spiders preferred Lepidoptera larvae compared to the other two pests groups (weevils and aphids). The maximum consumption rate was of 93.33% for Lepidoptera larvae, 25.33% for aphids and 11.67% for weevils. The Q Index values for the three species of spiders showed a positive selectivity only for defoliating larvae. O. salticus showed the highest values of consumption rates while Rachiplusia nu was the most consumed. The maximum value of consumption in 24 hours was showed by O. salticus on R. nu (C) = 2.8. The association of several species of predatory spiders increased the total number of insects captured, and also showed that the addition of spiders caused a decrease in the number of leaves damaged by the effect of lepidopterous larvae.

Recombinant peroxidase production in species of lepidoptera frequently found in Argentina

Horseradish peroxidase isozyme C (HRPC) is an important commercial biocatalyst. In this study, a screening of different lepidopteran species frequently found in Argentina to produce this protein was carried out. Two recombinant viruses were constructed: AcMNPV HRPC polyhedrin-minus (occ-), an intrahemocelical infective virus; and AcMNPV HRPC polyhedrin-plus (occ+), to achieve an oral infective baculovirus. Each lepidopteran species was infected either with AcMNPV HRPC occ- or AcMNPV HRPC occ+ and the harvesting days post-infection (dpi) were optimized. All species were susceptible to AcMNPV HRPC occ- infection, giving Spodoptera frugiperda the best yield: 41 μg per larva. Rachiplusia nu was highly susceptible to oral infection, reaching 22 μg per larva at 4 dpi. HRPC was purified by IMAC from S. frugiperda extracts with a yield of 86% and a purification factor of 29.

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.

Potential Use of the Hemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus Expressed in Rachiplusia nu Larvae as an Immunogen for Chickens

The hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus (NDV) was obtained as a recombinant antigen in Rachiplusia nu larvae. When it was used as an immunogen in chickens, a solid immune response, including neutralizing antibodies, was detected, demonstrating the potential use of this simple and economic strategy in the design of recombinant anti-NDV vaccines.

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.

Ocorrência de Rachiplusia nu (Guenée) (Lepidoptera: Noctuidae) em fumo (Nicotiana tabacum L.) no Rio Grande do Sul

The occurrence of Rachiplusia nu (Guenée) is registered for the first time in tobacco [Nicotiana tabacum L. (Solanaceae)]. The specimens were collected in a Virginia tobacco field, in Venâncio Aires, State of Rio Grande do Sul. Caterpillars occurred in large infestations, causing important damage to tobacco leaves, and demanding chemical control to minimize losses. This occurrence indicates that this species can feed on Virginia tobacco, which corresponds to 80% of the tobacco area of Rio Grande do Sul, and so presents high risk to become an important pest in tobacco fields.

Laboratory and Field Evaluations of Transgenic Soybean Exhibiting High-Dose Expression of a Synthetic Bacillus thuringiensis cry1A Gene for Control of Lepidoptera

Transgenic lines of soybean, Glycine max (L.) Merrill, expressing a synthetic cry1A gene (tic107) from Bacillus thuringiensis (Bt), were evaluated in screenhouse and conventional field trials for efficacy against lepidopteran pests. In screenhouse trials, Bt soybean and negative checks (isogenic segregants and parental lines) were evaluated against Anticarsia gemmatalis Hübner and Pseudoplusia includens (Walker) in the United States and against A. gemmatalis, Epinotia aporema (Walsingham), Rachiplusia nu (Guenée), and Spilosoma virginica (F.) in Argentina. Bt soybean exhibited virtually complete efficacy against each of these pests, whereas negative checks suffered significant damage. Bt soybean and negative checks also were evaluated in conventional trials against native populations of A. gemmatalis and P. includens in the southeastern United States. Each of these insects caused significant damage to negative checks in one or more locations, whereas Bt soybean exhibited virtually complete efficacy against these pests. In the laboratory, lyophilized leaf tissues from Bt soybean incorporated in artificial diet at a concentration representing a 25-fold dilution of fresh tissue caused complete mortality of A. gemmatalis and near complete mortality of P. includens neonates after 11 d, whereas mortality on negative checks did not exceed 10% for either insect. Average TIC107 expression approached or exceeded 50 microg/g fresh weight at V3 stage of growth and 200 microg/g by R6 stage of growth. These results demonstrate that expression of TIC107 in soybean can not only achieve highly efficacious control of several lepidopterans under field conditions but also provide a high dose for effective insect resistance management.

High-level expression of recombinant 3AB1 non-structural protein from FMDV in insect larvae

For its potential usefulness in diagnosis, the non-structural protein 3AB1 from foot-and-mouth disease virus was expressed as a soluble protein by using Autographa californica nuclear polyhedrosis virus as a vector. The 3AB1 coding sequence was introduced into AcNPV genome via pBAcPAK3AB1 transfer vector to originate Ac3AB1 recombinant baculovirus of phenotype occ −. Rachiplusia nu larvae were injected with supernatants of Sf9 cells infected with Ac3AB1 and 5 days post-infection total protein extracts were obtained. An intense band of approximately 21.5 kDa was observed when total larvae extracts were SDS-PAGE resolved and the recombinant protein detected by an FMDV-infected guinea pig serum. ELISA tests and Western blot experiments were carried out using sera both from FMDV-infected cattle and from vaccinated animals. The recombinant protein was only recognized by sera from infected animals, suggesting that this method of production in insect larvae could be applied to an efficient mass production of proteins of diagnostic interest.

Placement of Noctuid Eggs (Lepidoptera) on Soybean Plants

The distribution of eggs of Noctuidae on the leaflets of soybean plants was analyzed during 2 seasons. Four species were found in the 1st yr; Anticarsia gemmatalis Hubner was the most frequent, followed by Rachiplusia nu Guenee, Spodoptera latifascia Walker, and Chrysodeixis (=Pseudoplusia) includens Walker. However, in the 2nd yr, R. nu, instead of C. includens, was the least frequent species. A. gemmatalis and S. latifascia oviposited mainly on medium-height plant regions but also on leaflets with different average sizes. R. nu and C. includens laid their eggs in the upper and lower plant regions, respectively. All species oviposited preferentially on the ventral surface. These results suggest that spatial parameters of the plant are important for Noctuidae in choosing where to oviposit on soybean.

Characterization and Biological Properties of Embryonic Stages of Four Noctuidae (Lepidoptera)

The oviposition rate, the spatial oviposition pattern, and common morphological and temporal embryonic characteristics of each stage, and the caterpillar eclosion patterns of Anticarsia gemmatalis (Hueb.), Rachiplusia nu (Guen.), Spodoptera latifascia (Walk.), and Chrysodeixis (=Pseudoplusia) includens (Walk.) were observed under laboratory conditions. The average daily oviposition rate differed among species. The egg-larva developmental time lag did not show a normal distribution. Development was fastest in A. gemmatalis, followed by R. nu and S. latifascia, C. includens being the slowest. The species showed an overlap in different embryonic time stages. The mean overlap time was lower and the same in A. gemmatalis and R. nu than in S. latifascia and C. includens . Egg viability was the same in all species. The morphological characterization of the embryos of the 4 species allowed the description of 8 stages. Independently of species, 46% of the caterpillars ate the whole chorionic layer and the other 54% ate it partially.

Eye Attached Hemipollinaria in the Hawkmoth and Settling Moth Pollination of Habenaria (Orchidaceae): A Study on Functional Morphology in 5 Species from Subtropical South America

Abstract:Morphological adaptations to sphingophily and pollination by moths was studied in 5 South American Habenaria species. For H. gourlieana and H. hieronymi direct evidence of hawkmoth (Agrius cingulatus and Manduca sexta) and settling moth (Rachiplusia nu) pollination, respectively, by hemipollinaria attachment on the eyes, is presented. In two other species (H. paucofilia, H. rupicola) pollination by settling moths and eye attachment of the hemipollinaria can be inferred by indirect evidence (placement of scales and massulae on the flowers) and by flower structure. For the fifth species (H. montevidensis) pollination by small moths or mosquitoes with hemipollinaria attachment on the proboscis is postulated. A comparative study in floral features revealed clear morphological divergence between sphingophilous and phalaenophilous species. In addition to deeper spurs the former have slender, exerted, and upturned petal lobes (acting as mechanical guide to the hovering visitors), a much developed median rostellar lobe (acting as deflecting surface of the hawkmoths towards the viscidia), flexible and sinuous hemipollinarium caudicles (appropriate for frontal strikes against the stigma when hemipollinaria are brought by the hawkmoths dangling against the flower). Male efficiency was compared between 4 species with overlapping flowering time in the same area. Male efficiency factors were unexpectedly low in all species. Only in one species (H. hieronymi) each pollen donation accounted for more than one pollination. H. gourlieana is part of a more or less rich sphingophilous flora interacting with the same two long‐tongued hawkmoth species. Interspecific competition for pollen placement on the pollinator's body surface is probably low on account of different pollination mechanisms.

Laboratory and field studies in Chile on the control of Epinotia aporema (Lepidoptera: Olethreutidae) and Rachiplusia nu (Lepidoptera: Noctuidae) on Phaseolus vulgaris beans with growth regulators, Bacillus thuringiensis, and avermectin

Laboratory and field trials were conducted to evaluate the effectiveness of control of the larvae of Rachiplusia nu and Epinotia aporema (Lepidoptera: Noctuidae, and Olethreutidae, respectively), on Phaseolus vulgaris beans, by avermectin, Bacillus thuringiensis, diflubenzuron, triflumuron, and endosulfan. Two days after application in the laboratory, endosulfan resulted in 90% mortality of R. nu; avermectin, B. thuringiensis, triflumuron and diflubenzuron caused 48, 36, 28, and 16% mortality, respectively, on R. nu larvae. Triflumuron and diflubenzuron had a low initial activity against the larvae of E. aporema (10 and 4% mortality 2 days after application). All treatments obtained high final mortality levels (84–98%) of R. nu. Diflubenzuron and triflumuron caused slow initial effects on R. nu and E. aporema, but a high final cumulative mortality, in part attributable to malformation of pupae. In the field trial, all treatments had generally fewer larvae than the control. Fewer pods were damaged by E. aporema with endosulfan and avermectin 12 days after application than in the other treatments. The percentage of damaged pods increased largely thereafter in the controls, reaching 16.3% 26 days after the application date. Triflumuron was the only treatment that did not differ from the control 15 days after application, corroborating its slow insecticide action on E. aporema in the laboratory.