Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.
Dendrolimus kikuchii Matsumura, 1927 is a serious forest pest causing great damage to coniferous trees in China. Despite its economic importance, the population genetics of this pest are poorly known. We used three mitochondrial genes (COI, COII and Cytb) to investigate the genetic diversity and genetic differentiation of 15 populations collected from the main distribution regions of D. kikuchii in China. Populations show high haplotype and nucleotide diversity. Haplotype network and phylogenetic analysis divides the populations into three major clades, the central and southeastern China (CC+SEC) clade, the eastern China (EC) clade, and the southwestern China (SWC) clade. Populations collected from adjacent localities share the same clade, which is consistent with the strong relationship of isolation by distance (r = 0.74824, P = 0.00001). AMOVA analysis indicated that the major portion of this molecular genetic variation is found among the three groups of CC+SEC, EC and SWC (61.26%). Of 105 pairwise FST comparisons, 93 show high genetic differentiation. Populations of Puer (PE), Yangshuo (YS) and Leishan (LS) are separated from other populations by a larger genetic distance. Distributions of pairwise differences obtained with single and combined gene data from the overall populations are multimodal, suggesting these populations had no prior population expansion in southern China. The nonsignificant neutral test on the basis of Tajima’ D and Fu’s Fs, and the lack of a star-shaped haplotype network together with the multiple haplotypes support this hypothesis. Pleistocene climatic fluctuations, combined with the host specificity to Pinus species, made these regions of south China into a refuge for D. kikuchii. The high level of population genetic structuring is related to their weak flight capacity, their variations of life history and the geographic distance among populations.
Cydia pomonella is a serious invasive insect pest in China, and has caused severe damage to the production of apple and pear in its invaded areas. This species is distributing in the northwest and northeast of China, but no occurrence of it has been recorded in the large areas (about 3000-5000 km away) between the invaded northwestern and northeastern regions despite continuous monitoring. As yet the genetic diversity and invasion origin of the C. pomonella populations in Northwestern and Northeastern China is obscure. In this study, we investigate the genetic diversity of 14 populations of C. pomonella sampled throughout the main distribution regions in Northwestern (Xinjiang and Gansu Provinces) and Northeastern (Heilongjiang Province) China and compared them with nine populations from Europe and other continents using the mitochondrial COI, COII and Cytb genes. Both the populations from Northeastern and Northwestern China shared some haplotypes with populations from other countries. Haplotypes of the three mitochondrial genes had a different distribution in Northeastern and Northwestern China. The northeastern populations had more private haplotypes than the northwestern populations. A large number of the individuals from northwestern populations shared a few haplotypes of each of the three genes. The haplotype numbers and haplotype diversities of the northeastern populations were similar to those of field populations in other countries, but were higher than those of the northwestern populations. Populations from the Northwestern China showed similar haplotype number and haplotype diversity. We conclude that the population genetic background of C. pomonella populations in Northeastern and Northwestern China varies due to different invasion sources and that this should be considered before the application of new pest control tactics.
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