Comparative Evolution of S7 Intron 1 and Ribosomal Internal Transcribed Spacer in Coilia nasus (Clupeiformes: Engraulidae)

Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.     

快速检测淋球菌porA和16S rRNA基因的环介导等温扩增技术的建立

目的建立快速检测淋球菌porA和16S rRNA基因的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,探讨其用于淋球菌感染早期诊断的可行性。方法利用Primer-ExplorerV3软件分别针对淋球菌porA及16S rRNA基因设计6条引物(2条内引物FIP、BIP,2条外引物F3、B3,2条环引物LF、LB),以淋球菌标准菌株基因组DNA为模板,进行LAMP扩增,同时,以外引物F3、B3为PCR引物,进行PCR扩增,并比较2种扩增方法的灵敏度和特异性。结果LAMP法与PCR法灵敏度相同,可检测到10个拷贝的目的基因;其针对淋球菌porA和16S rRNA基因的引物对脑膜炎奈瑟菌、肺炎链球菌和大肠埃希菌的DNA均不能扩增。结论已成功建立了检测淋球菌porA和16S rRNA基因的LAMP技术,为淋球菌的快速检测提供了新的手段,有望成为淋球菌常规检测的简便方法。     

Biology,Pest Status,Microbiome and Control of Kudzu Bug (Hemiptera: Heteroptera: Plataspidae): A New Invasive Pest in the U.S.

Soybean is an important food crop, and insect integrated pest management (IPM) is critical to the sustainability of this production system. In recent years, the introduction into the United States of the kudzu bug currently identified as Megacopta cribraria (F.), poses a threat to soybean production. The kudzu bug was first discovered in the state of Georgia, U.S. in 2009 and since then has spread to most of the southeastern states. Because it was not found in the North American subcontinent before this time, much of our knowledge of this insect comes from research done in its native habitat. However, since the U.S. introduction, studies have been undertaken to improve our understanding of the kudzu bug basic biology, microbiome, migration patterns, host selection and management in its expanding new range. Researchers are not only looking at developing IPM strategies for the kudzu bug in soybean, but also at its unique relationship with symbiotic bacteria. Adult females deposit bacterial packets with their eggs, and the neonates feed on these packets to acquire the bacteria, Candidatus Ishikawaella capsulata. The kudzu bug should be an informative model to study the co-evolution of insect function and behavior with that of a single bacteria species. We review kudzu bug trapping and survey methods, the development of bioassays for insecticide susceptibility, insecticide efficacy, host preferences, impact of the pest on urban environments, population expansion, and the occurrence of natural enemies. The identity of the kudzu bug in the U.S. is not clear. We propose that the kudzu bug currently accepted as M. cribraria in the U.S. is actually Megacoptapunctatissima, with more work needed to confirm this hypothesis.     

Single Nucleotide Polymorphisms in Growth Hormone Gene and Their Association with Growth Traits in Siniperca chuatsi (Basilewsky)

Growth hormone (GH) has been considered as a candidate gene for growth traits in fish. In this study, polymorphisms of the GH gene were evaluated for associations with growth traits in 282 Siniperca chuatsi individuals. Using directly sequencing, four single nucleotide polymorphisms (SNPs) were identified in GH gene, with two mutations in intron 4 (g.4940A>C, g.4948A>T), one mutation in exon 5 (g.5045T>C) and one in intron 5 (g.5234T>G). Notably, three of them were significantly associated with growth performance, particularly for g.4940A>C which was highly correlated with all the four growth traits. In conclusion, our results demonstrated that these SNPs in GH gene could influence growth performance of S.chuatsi and could be used for marker-assisted selection (MAS) in this species.     

Homogeneous Nature of Malaysian Marine Fish Epinephelus fuscoguttatus (Perciformes; Serranidae): Evidence Based on Molecular Markers,Morphology and Fourier Transform Infrared Analysis

Taxonomic confusion exists within the genus Epinephelus due to the lack of morphological specializations and the overwhelming number of species reported in several studies. The homogenous nature of the morphology has created confusion in the Malaysian Marine fish species Epinephelus fuscoguttatus and Epinephelus hexagonatus. In this study, the partial DNA sequence of the 16S gene and mitochondrial nucleotide sequences of two gene regions, Cytochrome Oxidase Subunit I and III were used to investigate the phylogenetic relationship between them. In the phylogenetic trees, E. fuscoguttatus was monophyletic with E. hexagonatus species and morphology examination shows that no significant differences were found in the morphometric features between these two taxa. This suggests that E. fuscoguttatus is not distinguishable from E. hexagonatus species, and that E. fuscoguttatus have been identified to be E. hexagonatus species is likely attributed to differences in environment and ability to camouflage themselves under certain conditions. Interestingly, this finding was also supported by Principal Component Analysis on Attenuated Total Reflectance–Fourier-transform Infrared (ATR-FTIR) data analysis. Molecular, morphological and meristic characteristics were combined with ATR-FTIR analysis used in this study offer new perspectives in fish species identification. To our knowledge, this is the first report of an extensive genetic population study of E. fuscoguttatus in Malaysia and this understanding will play an important role in informing genetic stock-specific strategies for the management and conservation of this highly valued fish.     

Ontogenic Expression Pattern and Genetic Polymorphisms of the Fatty Acid Transport Protein 4 (FATP4) Gene in Chinese Chicken Populations

In the current research, the polymorphism of FATP4 gene was analyzed in Erlang Mountainous chickens. A total of nine genetic variants were identified by FATP4 gene sequencing analysis across the chicken samples. Significant associations (p < 0.05) were observed for two SNPs (g.5608778C>T and g.5608814G>A in exon 6) with certain carcass traits (such as live weight, carcass weight, eviscerated weight) in S01 and S05 populations, respectively. Meanwhile, in S05 population, haplotype 3 (T-G) and haplotype 2 (C-A) were associated with higher and lower partial carcass traits such as live weight, carcass weight, eviscerated weight and semi-eviscerated weight, respectively. Moreover, we investigated the expression profile of this gene during ontogenesis in Mountainous black-boned chicken. Quantitative real-time PCR analysis showed that FATP4 mRNA had the highest expression level in small intestine tissue over all other tissues examined. The FATP4 mRNA levels presented remarkable developmental changes with age in the various tissues. These results suggested that the FATP4 gene might play an important role in controlling chicken carcass traits.     

The Complete Mitochondrial Genome of Mindarus keteleerifoliae (Insecta: Hemiptera: Aphididae) and Comparison with Other Aphididae Insects

The mitogenome of Mindarus keteleerifoliae Zhang (Hemiptera: Aphididae) is a 15,199 bp circular molecule. The gene order and orientation of M. keteleerifoliae is similarly arranged to that of the ancestral insect of other aphid mitogenomes, and, a tRNA isomerism event maybe identified in the mitogenome of M. keteleerifoliae. The tRNA-Trp gene is coded in the J-strand and the same sequence in the N-strand codes for the tRNA-Ser gene. A similar phenomenon was also found in the mitogenome of Eriosoma lanigerum. However, whether tRNA isomers in aphids exist requires further study. Phylogenetic analyses, using all available protein-coding genes, support Mindarinae as the basal position of Aphididae. Two tribes of Aphidinae were recovered with high statistical significance. Characteristics of the M. keteleerifoliae mitogenome revealed distinct mitogenome structures and provided abundant phylogenetic signals, thus advancing our understanding of insect mitogenomic architecture and evolution. But, because only eight complete aphid mitogenomes, including M. keteleerifoliae, were published, future studies with larger taxon sampling sizes are necessary.     

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.     

Antiangiogenesis,Loss of Cell Adhesion and Apoptosis Are Involved in the Antitumoral Activity of Proteases from V. cundinamarcensis (C. candamarcensis) in Murine Melanoma B16F1

The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.     

Associations of MTHFR C677T and MTRR A66G Gene Polymorphisms with Metabolic Syndrome: A Case-Control Study in Northern China

Prior evidence indicates that homocysteine plays a role in the development of metabolic syndrome (MetS). Methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G polymorphisms are common genetic determinants of homocysteine levels. To investigate the associations of the MTHFR C677T and MTRR A66G polymorphisms with MetS, 692 Chinese Han subjects with MetS and 878 controls were recruited. The component traits of MetS and the MTHFR C677T and MTRR A66G genotypes were determined. A significant association was observed between the MTHFR 677T allele and increased risk of MetS, high fasting blood glucose, high waist circumference, and increasing number of MetS components. The MTRR A66G polymorphism was associated with an increased risk of MetS when combined with the MTHFR 677TT genotype, although there was no association found between MetS and MTRR A66G alone. Furthermore, the MTRR 66GG genotype was associated with high fasting blood glucose and triglycerides. Our data suggest that the MTHFR 677T allele may contribute to an increased risk of MetS in the northern Chinese Han population. The MTRR A66G polymorphism is not associated with MetS. However, it may exacerbate the effect of the MTHFR C677T variant alone. Further large prospective population-based studies are required to confirm our findings.     

Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for amino acid production. In the present study, 543 genes showed a significant change in their mRNA expression levels in l-lysine-producing C. glutamicum ATCC21300 than that in the wild-type C. glutamicum ATCC13032. Among these 543 differentially expressed genes (DEGs), 28 genes were up- or downregulated. In addition, 454 DEGs were functionally enriched and categorized based on BLAST sequence homologies and gene ontology (GO) annotations using the Blast2GO software. Interestingly, NCgl0071 (bioB, encoding biotin synthase) was expressed at levels ~20-fold higher in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain. Five other genes involved in biotin metabolism or transport—NCgl2515 (bioA, encoding adenosylmethionine-8-amino-7-oxononanoate aminotransferase), NCgl2516 (bioD, encoding dithiobiotin synthetase), NCgl1883, NCgl1884, and NCgl1885—were also expressed at significantly higher levels in the l-lysine-producing ATCC21300 strain than that in the wild-type ATCC13032 strain, which we determined using both next-generation RNA sequencing and quantitative real-time PCR analysis. When we disrupted the bioB gene in C. glutamicum ATCC21300, l-lysine production decreased by approximately 76%, and the three genes involved in biotin transport (NCgl1883, NCgl1884, and NCgl1885) were significantly downregulated. These results will be helpful to improve our understanding of C. glutamicum for industrial amino acid production.     

Reducing Capacity,Chlorogenic Acid Content and Biological Activity in a Collection of Scarlet (Solanum aethiopicum) and Gboma (S. macrocarpon) Eggplants

Scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants are important vegetables in Sub-Saharan Africa. Few studies have been made on these crops regarding the diversity of phenolic content and their biological activity. We have studied the reducing activity, the chlorogenic acid and other phenolic acid contents in a collection of 56 accessions of scarlet eggplant, including the four cultivated groups (Aculeatum, Gilo, Kumba, Shum) and the weedy intermediate S. aethiopicum-S. anguivi types, as well as in eight accessions of gboma eggplant, including the cultivated S. macrocarpon and its wild ancestor, S. dasyphyllum. A sample of the accessions evaluated in this collection has been tested for inhibition of nitric oxide (NO) using macrophage cell cultures. The results show that there is a great diversity in both crops for reducing activity, chlorogenic acid content and chlorogenic acid peak area (% of total phenolic acids). Heritability (H²) for these traits was intermediate to high in both crops. In all samples, chlorogenic acid was the major phenolic acid and accounted for more than 50% of the chromatogram peak area. Considerable differences were found among and within groups for these traits, but the greatest values for total phenolics and chlorogenic acid content were found in S. dasyphyllum. In most groups, reducing activity was positively correlated (with values of up to 0.904 in the Aculeatum group) with chlorogenic acid content. Inhibition of NO was greatest in samples having a high chlorogenic acid content. The results show that both crops are a relevant source of chlorogenic acid and other phenolic acids. The high diversity found also indicates that there are good prospects for breeding new scarlet and gboma eggplant cultivars with improved content in phenolics and bioactive properties.