Identification,expression, and molecular evolution of microRNAs in the “living fossil” Triops cancriformis (tadpole shrimp)

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a “living fossil,” whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first–fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1–3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a “living fossil.” However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.     

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal‐to‐zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc‐miR‐205 and bovine bta‐miR‐10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc‐miR‐205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte‐to‐embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.     

Identification and comparative analysis of the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> hemocytes microRNAs in response to <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> infection

MicroRNAs (miRNAs) are a class of noncoding RNA molecules that function as negative regulators of gene expression and play important roles in a wide spectrum of biological processes, including in immune response. However, the physiological regulation function of Pinctada fucata miRNAs, specially their immunomodulation has not been explored yet. Here, two small RNA libraries from hemocytes of P. fucata with or without Vibrio alginolyticus infection were constructed and sequenced using the high-throughput Illumina deep sequencing technology. In total, 11,939,992 and 11,083,327 raw reads, corresponding to 10,993,546 and 9,988,179 clean reads, were respectively obtained in the control and infected libraries. A total of 276 miRNAs, including 225 known miRNAs and 51 putative novel miRNAs, were identified by bioinformatic analysis. By using pairwise comparison between two libraries, 93 miRNAs were found to be significantly differentially expressed, with 42 and 51 miRNAs exhibiting up-regulation and down-regulation, respectively. Thereinto, some known miRNAs were considered to be immune-related. Real-time PCR were implemented for 6 miRNAs co-expressed in the control and infected samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. After miRNA targets were predicted, GO and KEGG pathway enrichment analysis were performed, and the results indicated that ten of the differentially expressed miRNAs were involved in immune-related pathways, and might participate in the host immune response to V. alginolyticus. These results of identification and comparative analysis of miRNAs might deepen our understanding of host-pathogen interactions and immune defense mechanisms in P. fucata.     

Orientobilharzia turkestanicum is a member of Schistosoma genus based on phylogenetic analysis using ribosomal DNA sequences

In the present study, samples representing Orientobilharzia turkestanicum from cattle, sheep, cashmere goat and goat in Heilongjiang Province, China, were characterized and grouped genetically by sequences of internal transcribed spacer (ITS, including ITS-1 and ITS2) and 28S ribosomal DNA (28S rDNA). The ITS and 28S rDNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with that of other members of the Schistosomatidae available in GenBank™, and phylogenetic relationships between them were re-constructed using the neighbor-joining and maximum parsimony methods. The lengths of ITS-1, ITS-2 and 28S rDNA sequences for all O. turkestanicum samples from different hosts were 384 bp, 331 bp and 1304 bp, respectively. While the ITS-1 sequences of O. turkestanicum from each of the four different hosts, and ITS-2 of O. turkestanicum from cattle, sheep and cashmere goat were identical, respectively, the ITS-2 of O. turkestanicum from goat differed from that of O. turkestanicum from cattle, sheep and cashmere goat by one nucleotide. The 28S rDNA sequences of O. turkestanicum from sheep and cashmere goat were identical, but differed from that of O. turkestanicum from cattle and goat by two nucleotides, with the latter two also having identical 28S rDNA sequence. Phylogenetic analyses based on the combined sequences of the ITS-1 and ITS-2, or the 28S rDNA sequences placed O. turkestanicum within the genus Schistosoma, and it was phylogenetically closer to the African schistosome group than to the Asian schistosome group. These results should have implications for studying the origin and evolution of O. turkestanicum and other members of the Schistosomatidae.