Isolation of the Astacin-like metalloprotease coding gene (astl) and assessment of its insecticidal activity against Spodoptera littoralis and Sitophilus oryzae

Astacin- like metalloprotease (astl) is a multi-domain metallopeptidase that has protease activity against a number of organisms; including fish, frogs, birds and insects. In this present investigation, the full length of astl cDNA was cloned from spider species, Hasarius adansoni. Sequencing of the cloned astl cDNA proved that its full length including 802 bp with 714bp open reading frame encoding for 238 amino acids. The catalytic domain comprised of 489 nts was cloned and expressed by the yeast expression system Pichia pastoris and its insecticidal activity was determined against two species of agricultural insects Spodoptera littoralis (Lepidoptera:Noctuidae) and Sitophilus oryzae (Coleoptera:Curculionidae). Bioassay was performed using three concentrations (100,500 and 1000 ppm) for four days for S. littoralis and 14 days for S. oryzae. In addition, the astl was fused to the GNA snowdrop lectin in the same frame and expressed in P. pastoris. The synergistic effect of astl and GNA was examined on the S. littoralis larvae and S. oryzae adults. The mortality percentages of the fused protein (Ha-astl/GNA) “1000 ppm” after 4 days, were 78.6%± 4.16 and 71.66% ±3.51 for first and second spodpotera larval instars, respectively. While, lower mortality of the fused protein of the same concentration was observed on S. oryzae adults, 49.3±2.08 %.


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The spiders and predacious beetles are considered the most venomous 34 terrestrial animals (Windley et al., 2012). Spider venoms are a cocktail of 35 thousand peptide toxins, and most of these are likely to have insecticidal 36 activity against a wide range of insect orders (Escoubas et al., 2006;Windley et 37 al., 2012;and King et al., 2002). Many of the insecticidal peptide toxins have 38 been isolated from spider venoms and their activities have been demonstrated. 39 Three families of insect-specific peptide toxins (atracotoxins), from 40 Hadronyche versuta, are specific blockers of arthropods voltage gated calcium 41 channels (Mukherjee et al., 2006). Orally active venom (OAIP-1) was isolated 42 from the Australian tarantula spider, Selenotypus plumipes, (Hardy et al.,43 2013). Some of spider toxins show no adverse effect on economically 44 important insects such as Hv1a that is harmless to the pollinating insect honey 45 bee Apis mellifera (Nakasu et al. 2014). Proteins belong to family of astacin-3 46 like metalloprotease were described as toxins that exist in the venom of 47 different spider species of Loxosceles (Trevisan-Silva et al., 2010). 48 Astacin (astl) is a multi-domain metalloprotease that is distinguished by the 49 presence of zinc binding motif (HEXXHXXGXXH) and methionine-turn 50 (MXY) (Gomis-Rüth et al., 2012). They are either secreted or membrane-51 anchored as inactive zymogen which is activated by cleaving the inhibiting 52 pro-segment (Yiallouros et al., 2002 andGuevara et al., 2010). The astl gene is 53 expressed as hatching enzyme in the oocyte and in the developing embryo 54 (Gomis-Rüth et al., 2012). It is suggested that hatching enzyme is responsible 55 for degrading embryonic envelopes of crustaceans, fish, frogs, and birds 56 (Gomis-Rüth et al., 2012). It is also known as ovastacin in mammals (Quesada 57 et al., 2004) and plays a role in egg-sperm interaction (Sachdev et al., 2012). 58 The astacin gene copy number in the genome is different from organism to 59 another and it is suggested that its number determines its roles in the organism 60 (Gomis-Rüth et al., 2012). According to MEROPS database 61 (http://merops. sanger.ac.uk), Nematode genome (Caenorhabditis elegans) 62 contains the largest copy number, around 40 copies of astacin gene, because it 63 plays role in breaking down the host connective tissue (Möhrlen et al., 2003). 64 However in mammals and birds, the astacin gene is represented by a limited 65 copy number, six, compared to those of lower vertebrate and invertebrate 66 genomes (Stöcker et al., 2013). 67 Based on the fusion protein technology, a carrier protein such as lectin could be 68 used, thus allowing the spider venom toxins to act orally. Fitches et al. (2012) 4 69 pointed 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.

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The 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.,  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 polyhedrosis virus (Elnagar andEl-Sheikh, 1990 andJones et al., 1994 and91 Atia et al., 2016) and Bacillus thuringiensis and its derivative (Navon et al.,5 92 1983 and Moussa et al., 2016). However, S. littoralis and S. oryzae developed 93 resistant against biological control agents (Salama et al., 1989). New 94 biopesticides are always needed to be involved in control strategy. 95 In this article, the astacin like metalloprotease coding gene (astl) has been 96 isolated from spider Hasarius adansoni, expressed in yeast and the toxicity of 97 the astl protein from and astl/GNA fused protein were demonstrated against S.  The total RNA was extracted from the cephalothorax region of the spider by The 489bp fragment of astl catalytic region was amplified using specific 163 primer set "astlFHE/astRX" (Table 1) was amplified using astlFHE and GNARX primers (Table 1)  at 28°C for 2-3 days on YPDS medium plates containing100mg/ml Zeocin. 184 Then, the colonies were numbered and transferred to YPD plates, and 185 incubated for two more days and allowed to grow. A number of colonies was 186 screened by universal primers"Alfa factor/3'AOX1" followed by nested PCR 187 by specific primers (astlFHE/astlRX and astlFHE/GNARX) (Table 1).

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The grown yeast colonies were numbered and dissolved in 20mM NaOH then and T94) (Fig. 1).

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The deduced amino acid sequence was aligned using the BLASTp tool in the 264 NCBI website. The covered score was 88-99% and the identity ranged between  (Table 1) and the positive colonies were confirmed by sequencing. respectively, thus revealing a 319 bp fragment in the positive lanes (Fig. 5B).

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The positive colonies were confirmed by different combinations between the 296 universal and specific primers (Fig. 5C).    8) and (9).

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The results of the feeding experiment also showed that the larvae 329 survived in the different treatments expressed a significant retardation in their 330 growth (Fig. 10). Some of them developed to malformed pupae or failed to 331 pupate or retarded to pupate compared to the control (Fig. 11). After a recovery 332 period where larvae were transferred onto untreated castor leaves and allowed 333 to grow for 10 to 15 days, the larvae did not restore normal weight. The Bioinsecticides are being used as potential alternatives to chemical insecticides.

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The sources of biopesticides are natural organisms, or their metabolic products 359 including insecticidal toxins derived from insect predators and parasitoids. thioredoxin-ω-HXTX-Hv1a has also been shown to be lethal to these 435 caterpillar species (Khan et al., 2006 peptides to the larval haemolymph (Fitches et al., 2002;  Adams ME, Herold EE, Venema VJ. Two classes of channel-specific toxins 485 from funnel web spider venom. J. Comp. Physiol. 1989;164: 333-342. Management in Tropical and Subtropical Cropping Systems. 1990