Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Immune response of insect vectors to transmitted pathogens or insect hosts against parasites are well studied, whereas the mechanism of tripartite interactions remains elusive. In this study, we investigated the immune interactions of the vector beetle Monochamus alternatus ( Ma) to the devastating plant parasitic nematode Bursaphelenchus xylophilus ( Bx) and the insect parasitic nematode Howardula phyllotretae ( Hp). We report the unique immune mechanism by which the vector beetle tolerates many devastating Bx in its trachea, yet that immune tolerance is compromised by the parasitic nematode Hp. Contact with either nematode species triggers epithelial reactive oxygen species (ROS) production in Ma. Only the entry of Bx, not Hp, infection, induces increased expression of antioxidative genes, through which the ROS levels are balanced in the trachea of beetles. Furthermore, we found that up-regulation of antioxidative genes was induced by the interaction of Toll receptors. In contrast, beetles infected by Hp retain high levels of oxidative stress and melanization in trachea, and as a result, decrease Bx loading. This study highlights the role of Toll receptors in mediating the activation of antioxidative genes in immune tolerance to plant parasitic nematodes, and suggests the use of insect parasites as a biologic control.-Zhou, J., Zhao, L.-L., Yu, H.-Y., Wang, Y.-H., Zhang, W., Hu, S.-N., Zou, Z., Sun, J.-H. Immune tolerance of vector beetle to its partner plant parasitic nematode modulated by its insect parasitic nematode.

Comparative analysis of the Monochamus alternatus immune system.

The pine sawyer beetle, Monochamus alternatus, is regarded as a notorious forest pest in Asia, vectoring an invasive pathogenic nematode, Bursaphelenchus xylophilus, which is known to cause pine wilt disease. However, little sequence information is available for this vector beetle. This hampered the research on its immune system. Based on the transcriptome of M. alternatus, we have identified and characterized 194 immunity-related genes in M. alternatus, and compared them with homologues molecules from other species known to exhibit immune responses against invading microbes. The lower number of putative immunity-related genes in M. alternatus were attributed to fewer C-type lectin, serine protease (SP) and anti-microbial peptide (AMP) genes. Phylogenetic analysis revealed that M. alternatus had a unique recognition gene, galectin3, orthologues of which were not identified in Tribolium castaneum, Drosophila melanogastor, Anopheles gambiae and Apis mellifera. This suggested a lineage-specific gene evolution for coleopteran insects. Our study provides the comprehensive sequence resources of the immunity-related genes of M. alternatus, presenting valuable information for better understanding of the molecular mechanism of innate immunity processes in M. alternatus against B. xylophilus.

Reproductive effects of cadmium on sperm function and early embryonic development in vitro.

Cadmium is a major environmental toxicant that is released into the atmosphere, water and soil in the form of cadmium oxide, cadmium chloride, or cadmium sulfide via industrial activities, such as the manufacturing of batteries and pigments, metal smelting and refining and municipal waste incineration. In the present study, we investigated the effects of cadmium exposure on sperm quality parameters, fertilization capacity and early embryonic development. Our study showed that in vitro incubation of human or mouse sperms with cadmium for a long time (up to 24 hours) could significantly decreased sperm motility in a concentration- and time-dependent manner. Exposure to cadmium in the environment for a short term (30 min) did not affect sperm motility but significantly reduced in vitro fertilization rate. We also evaluated the effects of cadmium at concentrations of 0.625 µg/ml, and 1.25 µg/ml on early embryonic development in vitro and observed that the blastocyst formation rate dramatically decreased with increasing cadmium concentration. This finding emphasizes the hazardous effects of cadmium on sperm quality as well as on natural embryo development and raises greater concerns regarding cadmium pollution.

Population divergence of aggregation pheromone responses in Ips subelongatus in northeastern China.

The Asian larch bark beetle, Ips subelongatus, is considered to be the major pest of larch within its natural range. We investigated the electrophysiological and behavioral characteristics as well as mitochondrial DNA cytochrome oxidase subunit I sequences of I. subelongatus from 13 geographic populations throughout northeastern China in order to explore population divergence of aggregation pheromone responses and the extent of potential genetic divergence. Electrophysiological analyses showed that antennae of I. subelongatus from all the six tested populations responded strongly to (S)-(-)-ipsenol (100% detection; 0.35-0.73 mV) in gas chromatography (GC)-electroantennographic detection (EAD) analyses, while its antipode, (R)-(+)-ipsenol was antennally inactive. I. subelongatus populations varied in their responses to (R)-(-)- and (S)-(+)-ipsdienol in GC-EAD analyses. Behavioral bioassays demonstrated that (S)-(-)-ipsenol alone was significantly attractive at all the tested sites, supporting its status as a key pheromone component of I. subelongatus, whereas (S)-(+)-ipsdienol was inactive alone. Adding (S)-(+)-ipsdienol to (S)-(-)-ipsenol did not have any effect on the trap catches from some populations in Inner Mongolia. However, (S)-(+)-ipsdienol showed a strong synergistic effect on (S)-(-)-ipsenol from several populations in Jilin and Liaoning Provinces, and a weak synergistic effect from some transition populations in Heilongjiang Province. Furthermore, 27 mitochondrial haplotypes were found among the 13 populations (intraspecific nucleotide divergence, 0.1%-1.1%). Analyses of molecular variance and haplotype networks indicated that different geographic populations have developed some genetic variation but did not form completely independent groups. From an applied point of view, a universal synthetic binary blend of racemic ipsenol and (S)-(+)-ipsdienol might have a potential for monitoring or even mass-trapping of I. subelongatus across northeastern China, even though some populations only use (S)-(-)-ipsenol alone as their active pheromone component.

MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are considered to be very important in regulating the growth, development, behavior and stress response in animals and plants in post-transcriptional gene regulation. Pinewood nematode, Bursaphelenchus xylophilus, is an important invasive plant parasitic nematode in Asia. To have a comprehensive knowledge about miRNAs of the nematode is necessary for further in-depth study on roles of miRNAs in the ecological adaptation of the invasive species. METHODS AND FINDINGS: Five small RNA libraries were constructed and sequenced by Illumina/Solexa deep-sequencing technology. A total of 810 miRNA candidates (49 conserved and 761 novel) were predicted by a computational pipeline, of which 57 miRNAs (20 conserved and 37 novel) encoded by 53 miRNA precursors were identified by experimental methods. Ten novel miRNAs were considered to be species-specific miRNAs of B. xylophilus. Comparison of expression profiles of miRNAs in the five small RNA libraries showed that many miRNAs exhibited obviously different expression levels in the third-stage dispersal juvenile and at a cold-stressed status. Most of the miRNAs exhibited obviously down-regulated expression in the dispersal stage. But differences among the three geographic libraries were not prominent. A total of 979 genes were predicted to be targets of these authentic miRNAs. Among them, seven heat shock protein genes were targeted by 14 miRNAs, and six FMRFamide-like neuropeptides genes were targeted by 17 miRNAs. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression levels of target genes. CONCLUSIONS: Basing on the fact that a negative correlation existed between the expression profiles of miRNAs and the mRNA expression profiles of their target genes (hsp, flp) by comparing those of the nematodes at a cold stressed status and a normal status, we suggested that miRNAs might participate in ecological adaptation and behavior regulation of the nematode. This is the first description of miRNAs in plant parasitic nematodes. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs in plant parasitic nematodes.

[Recombinant adenovirus mediate RNA silencing of perforin in NK-92 cells and its function].

AIM: To adenovirus-mediated RNAi technology be used to down-regulate perforin expression in NK-92 cells. This study will provide convenience for further investigation of the mechanism of perforin. METHODS: First, we designed three perforin shRNAs (shPF) and identified their knockdown efficiency at mRNA levels. Then we constructed recombinant adenovirus with shPF. After that, we transfected NK-92 cells with Ad-shPF and detected the expression of perforin at mRNA and protein levels. RESULTS: We confirm that the design of shPF sequences are effective. Moreover, the mRNA level of NK-92 cells treated by Ad-shPFP1 drop to 32% compare to Ad-scramble treated control. Furthermore, using Western blot, we find Ad-shPF1 treatment can obviously down-regulate the expression of perforin protein. CONCLUSION: Recombinant adenovirus with perforin shRNA can knockdown the expression of perforin in NK-92 cells.

Chemotaxis of the pinewood nematode, Bursaphelenchus xylophilus, to volatiles associated with host pine, Pinus massoniana, and its vector Monochamus alternatus.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, the most important invasive species in pine forests of Asia, is transported to new pine hosts by beetles of the genus Monochamus. Third-stage dispersal juveniles (J(III)) aggregate in pupal chambers around the vector as it matures. We demonstrated that the ratio of three terpenes (alpha-pinene, beta-pinene, and longifolene at 1:2.7:1.1) released by larval Monochamus alternatus strongly attract J(III), whereas the different ratio (1:0.1:0.01) of these three terpenes found in healthy xylem of Pinus massoniana attracts only the propagative stage (J(n)) of the nematode. We suggest that the volatiles produced by the host plants could be the basis of a chemoecological relationship between plant parasitic nematodes and their vector insects. Capture of J(III) with terpene-baited trap tubes deployed for 2 hr in the field was demonstrated. This technique may lead to the development of rapid sampling methodologies for use at either ports-of-entry or in the field.

[Effects of Tagetes erecta extracts on glutathione S-transferase and protease activities and protein content in Tetranychus viennensis].

With in vivo and in vitro Tagetes erecta roots under light and dark as test materials, this paper studied the effects of their extracts on the glutathione S-transferase and protease activities and protein content in Tetranychus viennensis. The results showed that the chloroform extract of T. erecta roots had the highest light-activated activity, followed by water extract, and methanol extract. After treated with chloroform extract, the glutathione S-transferase and protease activities in T. viennensis increased markedly, while its protein content decreased obviously. The variation degree of T. viennensis protease activity and protein content was significantly higher when the chloroform extract came from the T. erecta roots under light, suggesting that there existed active matters in the extract, which could promote the activation of protease, and thus, the decomposition of protein in T. viennensis. The bioactivity of T. erecta metabolites was mainly of light-activated one.