A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow

Abstract

A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

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“…The first group includes the nuclear polyhedrosis (NPV) and granulosis viruses from the Baculoviridae family of doublestranded DNA viruses that infect insects from the orders of Lepidoptera, Hymenoptera, and Diptera (Van Regenmortel et al, 2000). They serve as the basis of microbial formulations against Lepidoptera pests, including the cotton bollworm, which are widely used worldwide (Chen et al, 2000;Shapiro et al, 2002;Yu et al, 2015;Kolosov et al, 2017;Eroğlu et al, 2019). NPV populations can grow rapidly, increasing it number at billion-fold rate per insect.…”

Effect of Different Light Spectrum in Helicoverpa armigera Larvae during HearNPV Induced Tree-Top Disease

“…Larvae of Helicoverpa armigera (Hübner) were reared on a wheat bran rich artificial diet at temperature 28 ± 1 °C, 50% relative humidity and the photoperiod of 12 L: 12 D (Light: Dark) [32]. HearNPV viral occlusion bodies (OB) were amplified in H. armigera 4th instar larvae; collected, purified, quantified and stored at 4 °C as described previously [33].…”

“…Newly molted third instar larvae were starved for 24 h and fed with the viral dose of LD90 (2800 OBs/larvae) [33]. Infection was done following a modified droplet feeding method [14].…”

A novel method for construction of baculovirus bacmids

“…One form is occluded form, which is responsible for transmission between different insect individuals, and the other form, budded virus, which spreads from cell to cell within a susceptible insect. Baculoviruses possess a variety of unique features and have been used in multiple elds including over-expression of foreign genes [4][5][6], bio-control of pests [7][8][9], surface display [10][11][12] and gene delivery [13,14]. Despite these applications, functions of many baculovirus genes are not fully understood and the mechanisms underlying viral replication, assembly and release remain to be elucidated.…”