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.     

Coocclusion of Helicoverpa armigera Single Nucleopolyhedrovirus (HearSNPV) and Helicoverpa armigera Multiple Nucleopolyhedrovirus (HearMNPV): Pathogenicity and Stability in Homologous and Heterologous Hosts

Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) is a virulent pathogen of lepidopterans in the genera Heliothis and Helicoverpa, whereas Helicoverpa armigera multiple nucleopolyhedrovirus (HearSNPV) is a different virus species with a broader host range. This study aimed to examine the consequences of coocclusion of HearSNPV and HearMNPV on the pathogenicity, stability and host range of mixed-virus occlusion bodies (OBs). HearSNPV OBs were approximately 6-fold more pathogenic than HearMNPV OBs, showed faster killing by approximately 13 h, and were approximately 45% more productive in terms of OB production per larva. For coocclusion, H. armigera larvae were first inoculated with HearMNPV OBs and subsequently inoculated with HearSNPV OBs at intervals of 0–72 h after the initial inoculation. When the interval between inoculations was 12–24 h, OBs collected from virus-killed insects were found to comprise 41–57% of HearSNPV genomes, but the prevalence of HearSNPV genomes was greatly reduced (3–4%) at later time points. Quantitative PCR (qPCR) analysis revealed the presence of HearSNPV genomes in a small fraction of multinucleocapsid ODVs representing 0.47–0.88% of the genomes quantified in ODV samples, indicating that both viruses had replicated in coinfected host cells. End-point dilution assays on ODVs from cooccluded mixed-virus OBs confirmed the presence of both viruses in 41.9–55.6% of wells that were predicted to have been infected by a single ODV. A control experiment indicated that this result was unlikely to be due to the adhesion of HearSNPV ODVs to HearMNPV ODVs or accidental contamination during ODV band extraction. Therefore, the disparity between the qPCR and end-point dilution estimates of the prevalence of mixed-virus ODVs likely reflected virus-specific differences in replication efficiency in cell culture and the higher infectivity of pseudotyped ODVs that were produced in coinfected parental cells. Bioassays on H. armigera, Spodoptera frugiperda and Mamestra brassicae larvae revealed that mixed-virus OBs were capable of infecting heterologous hosts, but relative potency values largely reflected the proportion of HearMNPV present in each mixed-virus preparation. The cooccluded mixtures were unstable in serial passage; HearSNPV rapidly dominated during passage in H. armigera whereas HearMNPV rapidly dominated during passage in the heterologous hosts. We conclude that mixed-virus coocclusion technology may be useful for producing precise mixtures of viruses with host range properties suitable for the control of complexes of lepidopteran pests in particular crops, although this requires validation by field testing.     

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective: CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods: The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results: In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe3+ or hemin alone. Either co-addition of 5-ALA and Fe3+ or addition of 5-ALA or Fe3+ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). Conclusion: 5-ALA and Fe3+ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression.     

An odorant receptor from the common cutworm (Spodoptera litura) exclusively tuned to the important plant volatile cis‐3‐Hexenyl acetate

Olfaction plays an important role in insect behaviours. The odorant receptor (OR) repertoire, housed within the dendritic membrane of sensory neurons, is one of the primary determinants of odour recognition. ORs in moths could be classified into pheromone receptors (PRs) and non‐pheromone receptors (non‐PR ORs). Much research in the field of insect olfaction recently has been focused on PRs of the male moth, but few Lepidoptera studies have been done on the functional study of non‐PR ORs. In the present study, we identified and characterized four non‐PR ORs from Spodoptera litura (Lepidoptera: Noctuidae) antennae. The tissue expression pattern showed that the four ORs were mainly expressed in adult antennae and further in situ hybridization revealed SlituOR12 was expressed in both long and short sensilla trichodea and sensilla basiconica. A functional analysis of the four SlituORs was conducted in the heterologous expression system Xenopus oocytes. SlituOR12 was exclusively and sensitively tuned to cis‐3‐Hexenyl acetate and SlituOR19 slightly responded to 4′‐Ethylacetophenone; however, SlituOR44 and SlituOR51 did not respond to any chemicals tested in this study. It is proposed that SlituOR12 might partially account for some key behaviours of the female, such as detection of host location and oviposition site.     

The Complete Sequence of the First Spodoptera frugiperda Betabaculovirus Genome: A Natural Multiple Recombinant Virus

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.     

Effects of zinc exposure on the reproduction of Spodoptera litura Fabricius (Lepidoptera: Noctuidae)

Reproductive toxicity of Zn to insects was investigated in this study. By exposing phytophagous insect Spodoptera litura Fabricius to Zn in artificial diets of larvae, we investigated the effects of Zn on reproduction at ecological and molecular levels. A significantly shorter period of laying eggs was observed in S. litura exposed to 300–750 mg Zn/kg. The oviposition rate, fecundity and hatchability of female adults treated with 750 mg Zn/kg were significantly lower than those of the controls (31.43%, 20.95% and 52%, respectively, compared to the control). The Zn accumulation and vitellin (Vn) content in eggs were tested by inductively coupled plasma-atomic emission spectrometry and Bradford combining Western-blot, respectively. The results showed that Zn accumulated in the eggs, which has affected the weight and Vn content of eggs with significant negative correlations. The down-regulated expression levels of vitellogenin (Vg) mRNA were detected by real-time polymerase chain reaction (RT-PCR): the relative quantity of Vg mRNA was less than half of the controls at higher than 450 mg Zn/kg wet weight. These results indicated that excess Zn made expression of Vg gene down-regulated and caused poor accumulation of egg yolk, which led to a reduction in egg numbers and failure of eggs to hatch.