Functional analysis of two mitogen-activated protein kinases involved in thermal resistance of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae).

Predatory mites are important biological control agents used against phytophagous mites and small insects. They face various environmental pressures, especially fluctuating climate factors. Neoseiulus californicus, a commercially available phytoseiid mite, is adapted to a wide range of temperature conditions. We investigated the regulatory mechanisms governing the plastic response of N. californicus for coping with environmental temperature variations. The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved pathway of cell signal transduction that responds to environmental stress. We isolated two MAPKK genes (NcMAPKK4 and NcMAPKK6) from N. californicus and studied their functions. Developmental stage-specific expression level analysis showed that in adults, particularly females, NcMAPKK4 and NcMAPKK6 levels were higher than in other developmental stages. The expression level analysis at extremely high and low temperature conditions demonstrated that NcMAPKK4 could be induced significantly by adverse thermal stresses, whereas NcMAPKK6 distinctly responded to heat shock, indicating their different roles in thermal stress responses. After silencing of NcMAPKK4, both heat and cold resistance decreased significantly, whereas NcMAPKK6 knockdown had a greater influence on heat resistance. Knockdown of NcMAPKKs also reduced the activities of antioxidant enzymes, suggesting the regulation of NcMAPKKs was closely related to the antioxidant process in oxidative stress caused by external stimuli. These results indicate an important role of NcMAPKKs in the response to thermal stress and provide insight into the MAPK cascade pathway in the environmental adaptation mechanisms of phytoseiid mites.

Effects of UV-B radiation on the survival, egg hatchability and transcript expression of antioxidant enzymes in a high-temperature adapted strain of Neoseiulus barkeri.

Biological control of spider mites in hot and dry weather is a serious technical issue. A high-temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes was selected from its conventional strain (CS), via long-term heat acclimation and frequent heat hardenings in our previous studies. However, the environment of high temperature is usually associated with enhanced ultraviolet (UV) radiation. In the present study, the physiological effects of UV-B radiation on survival rate and egg damage of N. barkeri were investigated, as well as the activities and expression profiles of antioxidant enzymes to UV-B radiation stress. UV-B radiation had deleterious effects on egg hatchability and survival of N. barkeri. Adults of the HTAS strain were less UV-B resistant than those of the CS strain; they also had lower levels of enzymatic activity of superoxide dismutase (SOD) and catalase against oxidative damage and weaker upregulation of SOD genes. The mRNA expression of three SOD genes of CS adult females immediately increased whereas that of HTAS showed almost no difference under UV-B stress for 1 h. The results showed the HTAS of N. barkeri had lower fitness under UV-B stress compared with the CS of N. barkeri. These results suggested that long-term heat acclimation may exert a profound impact on the developmental physiology of N. barkeri.

Sustaining induced heat shock protein 70 confers biological thermotolerance in a high-temperature adapted predatory mite Neoseiulus barkeri (Hughes).

BACKGROUND: In fluctuating climatic environments, heat acclimation in predatory mites is a superior adaptation strategy for effective agricultural pest management and can be used to enhance the abilities critical in biological control efficiency. We investigated the regulatory mechanism governing the remarkable plastic response of thermotolerance in a high-temperature adapted strain (HTAS) and discerned the differences in the defensive reactions between the HTAS and the conventional strain (CS) in the predatory mite Neoseiulus barkeri. RESULTS: At 42 °C, the relative expression levels of four identified HSP70 genes increased rapidly in both N. barkeri strains; meanwhile the expression of NbHSP70-1 and NbHSP70-2 in CS sharply decreased after 4 h, displaying a distinct contrast with the remaining elevated expression in HTAS. Western blot analysis showed that the protein level of NbHSP70-1 in CS was dramatically elevated at 0.5 h and decreased at 6 h at 42 °C. Conversely, in HTAS, NbHSP70-1 was constantly induced and peaked at 6 h at 42 °C. Furthermore, HSP70 suppression by RNAi knockdown had a greater influence on the survival of HTAS, causing a higher mortality under high temperature than CS. Finally, the recombinant exogenous NbHSP70-1 protein enhanced the viability of E. coli BL21 under a lethal temperature of 50 °C. CONCLUSION: Sustained accumulation of HSP70 proteins results in predatory phytoseiid mites with the thermotolerance advantage that could promote their biological control function to pests. The divergent constitutive regulation of HSP70 to a thermal environment is conducive to the flexible adaptability of predators in the higher trophic level to trade off under extremely adversity stress.

Evaluation of Neoseiulus barkeri (Acari: Phytoseiidae) for Control of Eotetranychus kankitus (Acari: Tetranychidae).

Eotetranychus kankitus Ehara (Acari: Tetranychidae) is an important pest in Chinese citrus orchards. In the present study, we aimed to evaluate the potential of Neoseiulus barkeri (Hughes) (Acari: Phytoseiidae) for the biological control of E. kankitus. A two-sex life table of E. kankitus and N. barkeri was constructed to estimate development and fecundity. The functional response and stage-specific predation rate were analyzed to evaluate predation capacity. In addition, a timing model was used to project populations of E. kankitus with release of N. barkeri. Results showed that N. barkeri was able to develop and reproduce when fed on E. kankitus. The functional responses of N. barkeri on different stages of E. kankitus all fit the Holling II disc equation. When mixed stages of E. kankitus coexisted, N. barkeri mainly consumed larvae and nymphs. Based on the life tables and stage-specific predation rates, population projection revealed the stage structure and growth rate of N. barkeri on E. kankitus. Although E. kankitus had the higher growth rate, it was maintained at a low population level for several weeks after release of N. barkeri. The results highlighted the potential for utilizing N. barkeri as a biological control agent of E. kankitus.

Does Long-Term Feeding on Alternative Prey Affect the Biological Performance of Neoseiulus barkeri (Acari: Phytoseiidae) on the Target Spider Mites?

The predatory mite Neoseiulus barkeri (Hughes) is a good biological control agent for many small sucking pests. We aimed to determine whether rearing long term on alternative prey versus target prey species affected the performance of N. barkeri. Therefore, we investigated the prey preference, life tables, and population parameters of N. barkeri between alternative prey Tyrophagus putrescentiae (Schrank) and three species of spider mites, Tetranychus urticae Koch, Panonychus citri (McGregor), and Eotetranychus kankitus Ehara. We found that N. barkeri preferred the tetranychid mites to the alternative prey. Between the tetranychid mites, the predator consumed more P. citri and E. kankitus than T. urticae. When reared on T. urticae, the total developmental time and longevity of N. barkeri were the longest, whereas the intrinsic rate of increase was the lowest, indicating that the biotic fitness of predatory mite preyed on target of T. urticae was higher than on alternative prey of T. putrescentiae. However, total developmental time, longevity, and fecundity did not differ between N. barkeri reared on T. putrescentiae and P. citri, although these parameters were higher than those for mites reared on E. kankitus, indicating that the predatory mite reared on T. putrescentiae may not be affected to control P. citri, and that coexistence of P. citri and E. kankitus may enhance the control efficiency of N. barkeri. Altogether, our results demonstrated that long-term feeding on the alternative prey T. putrescentiae did not affect the performance of the predatory mite N. barkeri on various target spider mites.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction.

In insects, the accessory gland proteins (Acps) secreted by male accessory glands (MAGs) account for the majority of seminal fluids proteins. Mixed with sperm, they are transferred to the female at mating and so impact reproduction. In this project, we identified 2,927 proteins in the MAG secretions of the oriental fruit fly Bactrocera dorsalis, an important agricultural pest worldwide, using LC-MS analysis, and all sequences containing open reading frames were analyzed using signalP. In total, 90 Acps were identified. About one third (26) of these 90 Acps had a specific functional description, while the other two thirds (64) had no functional description including dozens of new classes of proteins. Hence, several of these novel Acps were abundant in the MAG secretions, and we confirmed their MAG-specific expression by qPCR. Finally and interestingly, one of these novel proteins was functionally predicted as juvenile hormone-binding protein, suggesting the impact of Acps with reproductive events in the female. Our results will aid in the development of an experimental method to identify Acps in insects, and in turn this information with new Acps in B. dorsalis will pave the way of further exploration their function in reproduction and potential development as new insecticide targets.

Comparative proteomic analysis of Bactrocera dorsalis (Hendel) in response to thermal stress.

Temperature is one of the most important environmental variables affecting growth, reproduction and distribution of insects. The rise of comparative proteomics provides a powerful tool to explore the response in proteins to thermal stress. As an important worldwide pest, the oriental fruit fly Bactrocera dorsalis causes severe economic losses to crops. To understand the response of B. dorsalis to thermal stress, we performed a comparative proteome analysis of this insect after exposure to extreme low and high temperatures using two-dimensional electrophoresis. Among the separated proteins, 51 diverse protein spots were present differently in response to extreme temperatures. Using tandem mass spectrometry sequencing analysis 39 proteins were successfully identified, which included 13 oxidoreductases, 10 binding proteins, 5 transferases, and 2 each of lyases, isomerases, ligases, and developmental proteins. Subsequently, the expression of these protein transcripts was studied by RT-qPCR to validate the proteomic results. In conclusion, this study provides a first look into the thermal stress response of B. dorsalis at the protein level, and thus it paves the way for further functional studies in the physiological mechanism related to thermal stress.