ABSTRACT
Objective To compare the complete mitochondrial genome sequences of two phenotypes of Paragonimus westermani isolated from Fujian Province with different sizes of metacercariae, and perform a phylogenetic analysis of various geographical isolates of P. westermani from Asia, so as to identify the possible genetic characteristics associated with the P. westermani phenotypes. Methods P. westermani metacercariae with different sizes (large metacercariae, 380–420 μm in diameter; small metacercariae, 320–340 μm) isolated from freshwater crabs were used to infect dogs, and the eggs and adult worms of P. westermani were collected from the dog stool samples and lung tissues. Then, the egg size and morphology were compared. In addition, genomic DNA was extracted from the adult worms of the two phenotypes of P. westermani and used for the PCR amplification to yield the complete mitochondrial genome sequence. Sequence structure and phylogenetic analyses were performed based on the complete mitochondrial genome of P. westermani. Results Following infection with large and small P. westermani metacercariae, the adult worms recovered from the dog lung had a thick body, and had oral and ventral suckers. The ventral sucker was located slightly in front of the midline of the body, and testes, ovary and vitelline gland were seen in the adult worms. Following fixation, the adults appeared oval, with an approximately 1.7∶1 of the length-width ratio. The length and width of the eggs isolated from the fecal samples of dogs infected with large and small P. westermani metacercariae varied significantly, and the large metacercariae produced bigger eggs than the smaller metacercariae. Based on the morphological features of adults and eggs and the ITS2 sequences, both phenotypes were identified as P. westermani. The complete mitochondrial genome sequence analysis of adults showed almost consistent sequences in the protein-coding region of the mitochondrial genome of adult worms derived from large and small metacercariae, with a major variation seen in the former non-coding region. Sliding window analysis revealed the most polymorphic region within the ND4 gene across the mitochondrial genome from various geographical isolates of P. westermani, and phylogenetic analysis showed that both phenotypes were clustered into the Chinese branch of P. westermani, which was close to the Japanese branch and distinct from the South/Southeast Asian branch. Conclusions The genetic distance between the phenotypes of P. westermani isolated from Fujian Province is near at a mitochondrial genome level, with no remarkable genetic differentiation seen; however, the mutation and structural changes in the non-coding region may result in the phenotypic variations. In addition, there is a distinct variation of the evolutionary rate in the mitochondrial coding genes, suggesting the selection of appropriate molecular markers during the phylogenic researches.
ABSTRACT
OBJECTIVE: To identify a myeloid differentiation factor 88 (MyD88) in Oncomelania hupensis, and characterize the role of MyD88 against Schistosoma japonicum infection. METHODS: The complete cDNA of MyD88 in O. hupensis was obtained by using rapid amplification of cDNA ends (RACE), and homologues sequences and conserved domains were aligned and the structure of MyD88 was predicted either. A phylogenetic tree of MyD88 was further constructed with other species. In addition, the mRNA expression level of O. hupensis MyD88 before and after S. japonicum infection was investigated by real-time quantitative PCR (RT-qPCR). RESULTS: The cDNA of O. hupensis MyD88 consisted of 1 406 bp open reading frame (ORF), encoding 468 amino acid residues, which contained death domain and Toll/interlrukin-1 receptor (TIR) domain, the typical features of MyD88 family proteins. The predicted amino acid sequence of O. hupensis MyD88 shared 38%-52% identity with other mollusc. O. hupensis MyD88 was phylogenetically closeted to Biomphalaria glabrata MyD88. The O. hupensis MyD88 existed in all selected tissues and expressed highly in hemocyte, up-regulated after S. japonicum infection in all selected tissues except cephalopodium, especially higher in whole snail and hemocyte. CONCLUSIONS: MyD88-dependent signaling pathway is present in O. hupensis and plays an important role in innate immune response against S. japonicum infection.
