Preliminary Research On The Adaptive Mechanism Of Kaburagia Rhusicola In Tannin Environment

The du-ensiform gall aphid, Kaburagia rhusicola Takagi (Hemiptera:Pemphigidae) is an important economic insect whose non-migratory and migratory life cycle are coexist in, the du-ensiform gallunts are rich in tannin concentration which is maintained at approximately 70% of dry weight and induced by K. rhusicola in the summer host. In this study, to clarify the mechanism for K. rhusicola to adapt to high tannin in gall environment, basic works about physiological and biochemical response and hydrolyzed protein were done. Main results were summarized as following:1. Observation on du-ensiform gallnuts slice and research on tanninconcentrationAfter making the slice of the gallnut and detecting the tannin concentration in different parts of the gallnut, we analyse the position which the galling aphid feeding in and the tannin concentration that aphid intake in. It showed that there were significent difference among the outer epideral, intraepidermal and middle layer of gallnut from microscope, the outer epideral covered with non-glandular hairs; the cells of middle layer were large than others and were closely linked; the cells in intraepidermal were loosely arranged. There also existed significent difference in tannin concentration among different parts of the gallnuts, the tannin concentration in middle layer of gallnut is about 30.0%, intraepidermal is 13.2% and the juice is 7.2%, the results indicated that the tannin concentrations from different parts of gallnuts were not at the same level, the aphids may intake lower concentration from the juice which is a adaptation to high tannin environment.2. Research on tannase in K. rhusicolaIn our study, we did not find any ingredient about tannase in the aphids'supernatant in the range of 3.8-6.2 pH condition. Afetr purifying and separating the aphids' supernatant protein, we detected tannase in the three separation proteins and did not find any activity about the hydrolase yet.3. Response of the antioxidant system of K. rhusicola in gall environmentTo test the hypothesis that the effect of high tannin environment is to increase oxidative stress on the aphids. We treat spring migrant as contrast and measured the total antioxidant capacity and antioxidant enzymes activities of SOD, CAT, POD and GST in adult K rhusicola of different generation and analyse the response of the antioxidant system of K. rhusicola in the high tannin environment. We found that the activities of SOD and GST in aphid generations living within galls were enhanced except in AM generation when compared to control, T-AOC, CAT and POD activities increased over the course of the gall time. Our results show that the high tannin environment increase the level of oxidative stress in K. rhusicola.4. Cloning and sequence of cDNA of laccase-1 from K. rhusicolaThe cDNA of Lac-1 gene was cloned from K. rhusicola by means of reverse transcriptase and polymerase chain (RT-PCR) and 5'and 3'-rapid amplification of cDNA ends (RACE) analyses. The cDNA is 2,327 bp in length and encodes a putative protein of 591 amino acids (m.w.67.3583 kDa, pI 6.31). Multiple sequence alignments indicate that the deduced amino acid sequences of K. rhusicola show very high homology to their corresponding sequences in other species.The phylogenetic analysis of laccase sequences was consistent with well known classification of insects.5. Transcriptional analysis of antioxidant and detoxification genes in K. rhusicolaThe cDNA ofβ-Actin, Cu-Sod, Mn-Sod, Cat, Tpx and Gst genes were cloned from K. rhusicola by means of reverse transcriptase and polymerase chain (RT-PCR). In this study, we used quantitative real-time PCR method to describe the transcriptional and analysis of antioxidant genes in the aphids'midgut of different generations. The results showed that copper zinc superoxide dismutase (SOD-1), manganese superoxide dismutase (SOD-2), catalase (CAT), thioredoxin peroxidase (TPx), Glutathione-S-transferase (GST) and laccase-1 (LAC-1) transcripts were expressed differently in the midgut of different generations living in gall environment. Sod-1, Sod-2, Cat, Tpx and Gst established an antioxidant defense system and expressed at high levels in 1st and 2nd fundatrices, Lac-1 gene expressed at a high level in 3rd fundatrices when the antioxidant enzymes genes were low.Base upon these results, we postulate that K. rhusicola utilize antioxidant and phenol detoxification mechanisms together in the midgut to overcome the high phenolic environment.