Study On Mechanisms Of Panonychus Citri (McGregor) In Response To Thermal Stress

The citrus red mite Panonychus citri (McGregor), belonging to Arachnida, Acarina, Tetranychidae, Panonychus, is an important pest that devastates both deciduous and evergreen fruit trees such as citrus, pear, peach, and holly. When the mites are rampant, they will lead to great loss of output. Recent population outbreaks of P. citri may be attributed to a disruption in the orchard ecosystem, caused mainly by the application of broad-spectrum pesticides targeting other insects, leading to decreases in the natural enemy pressure on P. citri. In addition, the citrus red mite is often difficult to manage because of their ability to rapidly develop resistance to various acaricides due to its short life cycle. Nowadays, the P. citri has become one of the most serious pests in citrus orchard. In China, citruses have been cultivted in subtropical zones, and the mite populations on citrus trees have two infestation peaks every year, one in early summer and one in the autumn; but maintain low densities during the hot summer and cold winter. In main citrus producing areas where summer temperatures often reach or exceed 40℃, while winter temperatures may decline below 0℃. The optimum temperature zone for the citrus red mite ranges from 20-30℃, and 35℃is the proximity of the upper thermal threshold. So the citrus red mites are often exposed to the thermal stress in hot summer and cold winter. In general, tolerance mechanisms for extreme temperature are complex and act by various biochemical molecules such as polyols, lipids, and proteins. The sub-lethal hyperthermia induces heat shock responses involving increased expression of a suite of heat shock proteins (Hsps) which results in acquired thermo-tolerance of organisms. Heat or cold shock may also disturb the redox balance in animals and lead to oxidative stress. The antioxidant systems have an important role in deal with the damage caused by oxidative stress.Based on the economic importance of the citrus red mite and the significance of thermal-stress responses, the current study was aiming at the adaption mechanisms of the mite to environment stress, and four new heat shock proteins (Hsps) genes were isolated, and their expression patterns were analyzed. In addtion, the response of antioxidant system of the citrus red mite under the thermal stress were investigated. Our results will not only provide great insights into exploring the developmental and physiological functions of the Hsps from the mite, but also provide evidence for clarifying the adaptive mechanisms of the mite to thermal stress. In the meantime, the current results enrich and develop the scientific theoretical study system for the antioxidant response in thermal stress. The study was supported in part by Special Fund for Agroscientific Research in the Public Interest (nyhyzx07-057,201103020), the Program for Innovative Research Team in University (IRT0976), Natural Science Foundation of Chongqing (CSTC,2009BA1042), and the Earmarked Fund for Modern Agro-industry (Citrus) Technology Research System.The main results are as follows:1 Effects of thermal stress on survival and fecundity of P. citriTemperature is one of the main factors influencing the arthropod population dynamics. The survival and fecundity of P. citri were studied following treated by 32, 35,38, and 41℃for short time, respectively. Temperature of 25℃served as a control. The results showed that the egg hatch rate decreased with the increasing of temperatures, and when temperature rose above 35℃, the hatch rate decreased significantly with comparison to control. The total average eggs laid per female was 53.7 eggs at 25℃, but it decreased significantly after stressed for 1 h under above temperatures, and the decrease tendency kept pace with the stress time. At 25℃, the average longevity of female adult was 12.1 d, but it was shortened after stressed for 1 h by high temperatures, and the longevity became shorter and shorter with stress time prolonged. In addition, high-temperature stress had negative effect on age-specific survival rage and age-specific fecundity of female adult, and the disadvantage became even worse with the length of stress time.2 Antioxidant response of P. citri to thermal stressThermal stress is one of the factors that have the possibility to generate oxidative stress products in the citrus red mites. It has been confirmed that heat or cold shock may disturb the redox balance in this species and lead to oxidative stress. Catalase (CAT) and peroxidase (POD) activity as well as total antioxidant capacity (T-AOC) changed slightly in removal of ROS when the citrus red mites exposed to thermal stress. Nevertheless, superoxide dismutase (SOD) and glutathione-S-transferases (GSTs) play important role in the process of antioxidant response to thermal stress. Although T-AOC changed slightly, SOD and GSTs could be induced by the accumulation of products from oxidative stress. As one important index of lipid peroxidation, malondialdehyde (MDA) kept at an almost normal level in a time-dependent manner, which suggest that the citrus red mites is capable of efficiently dealing with ROS induced by thermal stresses.3 Evaluation and validation of reference genes for RT-qPCR in P. citriReference genes used for the quantification of mRNA expression may be affected by the experimental condition, so the stabilities of seven candidate reference genes (Pc5.8SrRNA, PcActin, PcEf-1α, PcGapdh, PcRpⅡ, PcSdha, and Pcα-Tubulin) in P. citri were systematically analyzed at different developmental stages and after thermal stresses using geNorm and NormFinder software, respectively. The stage of egg, larva, nymph, and adult (male and female) was used for different developmental stages experiment, and thermal stresses included cold shock (0,5 and 10℃) and heat shock (35,38 and 41℃), respectively. The treated time was 1 h, and 25℃served as a control. The aim of this study was to evaluate housekeeping genes of P. citri for their suitability as reference genes in quantitative real-time PCR (RT-qPCR), and provide appropriate reference genes to explore the gene expression patterns of the heat shock proteins (Hsps) in P. citri. According to the analysis by geNorm and NormFinder, it was found that Pc5.8SrRNA, PcActin, and PcSdha were less stabe whether at developmental stages or under thermal stresses. Whereas, PcRpⅡwas the most stable gene at different developmental stages, PcGapdh and Pcα-Tubulin also kept stable during 1 h cold shock. If multiple reference genes needed for validation target genes mRNA expression under above condition, the most appropriate genes number was 2, and the combination of PcEf-1α+PcRpⅡwas recommended at different developmental stages; for cold shock experiment, the most appropriate genes number was also 2, the combination of PcGapdh+Pcα-Tubulin was recommended; whereas for heat shock experiment, the most appropriate genes number was 3, and the combination of PcEf-1α+PcGapdh+ PcRpⅡwas recommended.4 Molecular cloning and sequences analysis of Hsp90 and Hsp70 family genes in P. citri4.1 Hsp90The full-length cDNA of Hsp90 family gene PcHsp90 was cloned from P. citri using the combined techniques of reverse transcriptase-PCR (RT-PCR) with rapid amplification of cDNA ends (RACE). Its GenBank accession number was GQ495086. The complete cDNA of consisted of 2,763 nucleotides with an open reading frame (ORF) of 2,193 nucleotides which encode a protein of 730 amino acids residues. Molecular characterizations of the putative proteins have been predicted by Protparam software. The deduced amino acid sequence included 5 typical motifs of the Hsp90 family:NKEIFLRELISNSSDALDKIR, LGTIARS, IGQFGVGFYSAYLVAD, IKLYVRRVFI, and GVVDSEDLPLNISRE. The amino acid sequences ended with "MEEVD" motif which was the characteristic of cytosolic Hsp90. A phylogenetic tree was constructed based on the amino acid sequence deduced from PcHsp90 with other 12 Hsp90 amino sequences from GenBank using software MEGA 4.1 with Neighbor-joining method. The result showed that PcHsp90 clustered with Hsp90 from Tetranychus cinnabarinus firstly, next with Hsp90 of Ixodes scapularis, and kept relatively longer hereditary distance with Hsp90s from insects. In addition, its 3-D structure model was constructed by SWISSS-MODEL. Similar to other Hsp90, three domains were found:NBD (Nuleotide binding domain), MD (Middle domain), and CTD (C-terminus domain).4.2 Hsp70sThe full-length cDNA of three Hsp70 family genes PcHsp70-1, PcHsp70-2, and PcHsp70-3 were cloned from P. citri. GenBank accession numbers were GQ495083, GQ495084, and GQ495085, respectively. The complete cDNA of PcHsp70-1 consisted of 2,586 nucleotides with an ORF of 1,977 nucleotides which encoded a protein of 658 amino acids residues. The complete cDNA of PcHsp70-2 consists of 2.405 nucleotides with an ORF of 1,968 nucleotides, encoding a protein of 655 amino acids residues. The complete cDNA of PcHsp70-3 consists of 2,300 nucleotides with an ORF of 2,028 nucleotides, encoding a protein of 675 amino acids residues.Molecular characterizations of the putative proteins have been predicted by Protparam software. The amino acids sequences were encoded by PcHsp70-1, PcHsp70-2, and PcHsp70-3. respectively, included three typical motifs of the Hsp70 family genes. The motifs for PcHsp70-1 amino acids sequence were as follows: IDLGTTYS. IFDLGGGTFDVSLL, and IVLVGGSTRIPKIQK. The motifs for PcHsp70-2 were IDLGTTYS, IFDLGGGTFDVSIL, and IVLVGGSTRIPKIQK. The motifs for PcHsp70-3 were IDLGTTYS. VFDLGGGTFDVSLL, and IVLVGGSTRIPKIQQ. The C-terminus motifs of PcHsp70-1 and PcHsp70-2 amino acid sequences both were "EEVD", which was the characteristic of cytosolic Hsp70. So PcHsp70-1 and PcHsp70-2 were located in cytosol. However, the C-terminus motif of PcHsp70-3 amino acid sequence was "KDEL", which was the typical characteristic of ER (endoplasmic reticulum) protein at C-terminus. Accordingly PcHsp70-3 was attributed to ER Hsp70. The percentages of similarity among the three amino acids sequences were as follows:87% between PcHsp70-l and PcHsp70-2,64.7% between PcHsp70-l and PcHsp70-3, and 65.8% between PcHsp70-2 and PcHsp70-3. To analyze the sequence homology and phylogenetic relationships, the complete deduced protein sequences of the three Hsp70s, together with other Hsp70 sequences from 27 other organisms obtained from GenBank, were used for the phylogenetic tree construction. A Neighbor-joining phylogenetic tree was constructed by MEGA 4.1 The tree consisted of three branches, including the Hsp70 family members of the different intracellular compartments (cytosol, endoplasmic reticulum, and mitochondria). As expected, PcHsp70-l and PcHsp70-2 belonged to cytosolic Hsp70s and the PcHsp70-3 was clustered with the endoplasmic reticulum Hsp70 cluster from other species. In addition, their 3-D structure models were constructed by SWISSS-MODEL. All of them included 2 domains, NBD (Nucleotide binding domain) and SBD (Substrate binding domain).5 The mRNA expression profiles of Hsp90 and Hsp70 genes5.1 Developmental expression profiles of the four Hsp genesThe developmental expression profiles of Hsp90 and Hsp70 genes from P. citri were analyzed using PcRpII as an internal control gene. The developmental stages included egg, larvae, nymph, male adult, and female adult. The results indicated that expression of PcHsp90 in egg, nymph, and adult was high. Expressions of PcHsp70-l and PcHsp70-3 genes changed slightly at all stages. However, the expression level of PcHsp70-3 distinctly increased with development. Based on the above results, it was concluded that PcHsp90 and PcHsp70-2 possibly have connection with growth and development of P. citri. It was necessary for PcHsp70-l and PcHsp70-3 to maintain basal physiological function of the mite.5.2 Expression profiles under short-term low-temperature stressThe expression profiles of Hsp90 and Hsp70 genes from P. citri were analyzed with Pcα-Tubulin gene as an internal control gene under low-temperature (0,5, and 10℃) stress for 1 h. Temperature of 25℃served as a control. The mRNA expressions levels of the four genes were all decreased compared with the control. It may suggest that other Hsps or substances were synthesized during stress, thus resulting in the decrease of the 4 Hsps under short-term low-temperature stress.5.3 Expression profiles under short-term high-temperature stressThe expression patterns of Hsp90 and Hsp70 genes from P. citri were analyzed with PcRpII as an internal control gene under high-temperature (35,38, and 41℃) stress for 1 h. Temperature of 25℃served as a control. The results showed that expressions of the 4 genes were all up-regulate after heat shock, but only the expressions of PcHsp90 and PcHsp70-2 significantly enhanced at 41℃compared to control. At 41℃, the expression of PcHsp90 and PcHsp70-2 was up to 6.75- and 28.37-fold compared with the control. It was concluded that all the four genes all can be induced under short-term high-temperature, but PcHsp90 and PcHsp70-2 had more important role in deal with heat stress.In conclusion, PcHsp70-1 and PcHsp70-3 were necessary to maintain basal physiological function of P. citri, while wouldn't be induced under short-term thermal stress. PcHsp90 can be induced by heat shock and was also relative with basal physiological function; there was a close relationship between PcHsp70-2 and development of P. citri, especially it can be dramatically induced by heat shock.6 Heterologous expression of P. citri Hsp70s in Escherichia coliApplying the double digestion of FastDigest" BamHⅠand FastDigestR NotⅠrestriction enzymes with the DNA recombination technology, the expression vectors for PcHsp70-1,PcHsp70-2, and PcHsp70-3 based on pET 28a (+) vector in Escherichia coli was constructed. This provided the basis for Hsp70s expression in vitro, and made it possible for study the characteristics and physiological function of Hsp70s in future.In conclusion, the current study made it clear that short-term high-temperature had negative effect on the survival and fecundity of P. citri. The antioxidant response of P. citri to thermal stress was subsequently determined. To explore the molecular mechanisms of P. citri response to thermal stress, one Hsp90 and 3 Hsp70 genes were cloned from P. citri. To validate the stability of 7 housekeeping gene, the best stable reference genes at developmental stages and under thermal stress were selected using geNorm and NormFinder software. Then the mRNA expression profiles of the four genes at different developmental stages and under thermal stress were analyzed, and the role of the Hsp90 and Hsp70 genes in response to thermal stress were clarified. Besides, the expression vectors for 3 Hsp70 genes based on pET 28a (+) vector in Escherichia coli were constructed for studying the Hsp70 characteristics in future. These results not only help us understanding the mechanisms of P. citri in response to thermal stress at biological, physiological and molecular levels, but also provided the evidence to understand the population dynamics of the mite.