Isolation of the Astacin-like metalloprotease coding gene (astl) and assessment of its insecticidal activity againstSpodoptera littoralisandSitophilus oryzae

Abstract

14Astacin-like metalloprotease (astl) is a multi-domain metallopeptidase that has 15 protease activity against a number of organisms; including fish, frogs, birds and 16 insects. In this present investigation, the full length of astl cDNA was cloned 17 from spider species, Hasarius adansoni. Sequencing of the cloned astl cDNA 18 proved that its full length including 802 bp with 714bp open reading frame 19 encoding for 238 amino acids. The catalytic domain comprised of 489 nts was20 cloned and expressed by the yeast expression system Pichia pastoris and its 21 insecticidal activity was determined against two species of agricultural insects 22 Spodoptera littoralis (Lepidoptera:Noctuidae) and Sitophilus oryzae 23 (Coleoptera:Curculionidae). Bioassay was performed using three 2 24 concentrations (100,500 and 1000 ppm) for four days for S. littoralis and 14 25 days for S. oryzae. In addition, the astl was fused to the GNA snowdrop lectin 26 in the same frame and expressed in P. pastoris. The synergistic effect of astl 27 and GNA was examined on the S. littoralis larvae and S. oryzae adults. The 28 mortality percentages of the fused protein (Ha-astl/GNA) "1000 ppm " after 4 29 days, were 78.6%± 4.16 and 71.66% ±3.51 for first and second spodpotera 30 larval instars, respectively. While, lower mortality of the fused protein of the 31 same concentration was observed on S. oryzae adults, 49.3±2.08 %. 32 4 69pointed out that the neurotoxin protein from the Australian funnel web spider 70 Hadronyche versuta, Ѡ-hexatoxin-Hv1a was lethal to many insect species 71 when injected, while it is non-toxic through feeding. However, the Ѡ-72 hexatoxin-Hv1a/GNA fusion protein showed oral insecticidal activity against 73 insects from different orders. 74The agricultural and horticultural pest insects cause 40% loss of the crop yield 75 worldwide (Oerke et al., 1994) that is estimated by 17.7 billions dollars 76 annually (Oliveira et al., 2014). Spodoptera littoralisis one of the most 77 destructive agricultural lepidopteron insect pests that can damage many 78 economical crops such as cotton, maize, tomato and vegetables (Salama et al., 79 1970). The larvae of S. littoralis feed on the leaves, fruits, flowers, buds and 80 bolls of cotton making them useless (Bishari, 1934). Sitophilus oryzae is one of 81 the important insect pests of stored grains such as rice, wheat and their products 82 (Baloch, 1992). S. oryzae causes loss of weight, reduction in the nutritional 83 value and increasing the infection with other mites and fungi, therefore the 84 commercial value of the stored grains decreased (Madrid et al., 1990). These 85 agriculture pests are controlled by different methods such as chemical 86 insecticides which are extensively used in Egypt (Issa et al., 1984a; Issa et al., 87 1984b; and Abo-El-Ghar et al. , 1986). Therefore, resistant strains of insect 88 pests appeared in the field (Sawicki, 1986). An alternative control method that 89 used in control strategy is biological agents such as natural enemies, nuclear 90 polyhedrosi...