Microfracture technique combined with mesenchymal stem cells inducer represses miR-708-5p to target special at-rich sequence-binding protein 2 to drive cartilage repair and regeneration in rabbit knee osteoarthritis.

Knee osteoarthritis (KOA) is a degenerative joint illness which leads to knee pain and functional limitation. In this study, we combined microfracture surgery with kartogenin (KGN), a small bioactive molecule used to promote the differentiation of mesenchymal stem cells (MSCs), and explored its impact on cartilage repair and possible latent mechanisms of action. The research offers a brand-new idea for the clinical cure of KOA. The microfracture technique in combination with KNG treatment was performed on a rabbit model of KOA. Animal behaviour was evaluated after the intra-articular injection of miR-708-5p and Special AT-rich sequence binding protein 2 (SATB2) lentiviruses. Later, the expression of the tumour necrosis factor α (TNF-α) and interleukin- 1 (IL-1), the pathology of synovial tissue and cartilage tissue, and the positive cartilage type II collagen, MMP-1, MMP-3 and TIMP-1 were detected. Finally, a luciferase assay was conducted to verify the interaction of miR-708-5p and SATB2. Our results showed that miR-708-5p was elevated in the rabbit KOA model; however, the expression of SATB2 was reduced. Meanwhile, the microfracture technology combined with MSCs inducer KGN drove cartilage repair and regeneration in rabbit KOA by repressing the miR-708-5p expression. We also found that miR-708-5p directly targeted the SATB2 mRNA to regulate its expression. Furthermore, our data urged that elevating miR-708-5p or restraining SATB2 may reverse the therapeutic effect of the microfracture technique combined with MSCs inducer on rabbit KOA. Microfracture technique combined with MSCs inducer represses miR-708-5p to target SATB2 to drive cartilage repair and regeneration in rabbit KOA. This indicates that the microfracture technique combined with MSCs inducers is supposed to be an effective latent method for osteoarthritis cure.

An Intestinal Symbiotic Bacterial Strain of Oscheius chongmingensis Modulates Host Viability at Both Global and Post-Transcriptional Levels.

A rhabditid entomopathogenic nematode (EPN), Oscheius chongmingensis, has a stable symbiotic relationship with the bacterial strain Serratia nematodiphila S1 harbored in its intestines and drastically reduced viability when associated with a non-native strain (186) of the same bacterial species. This nematode is thus a good model for understanding the molecular mechanisms and interactions involved between a nematode host and a member of its intestinal microbiome. Transcriptome analysis and RNA-seq data indicated that expression levels of the majority (8797, 87.59%) of mRNAs in the non-native combination of O. chongmingensis and S. nematodiphila 186 were downregulated compared with the native combination, including strain S1. Accordingly, 88.84% of the total uniq-sRNAs mapped in the O. chongmingensis transcriptome were specific between the two combinations. Six DEGs, including two transcription factors (oc-daf-16 and oc-goa-1) and four kinases (oc-pdk-1, oc-akt-1, oc-rtk, and oc-fak), as well as an up-regulated micro-RNA, oc-miR-71, were found to demonstrate the regulatory mechanisms underlying diminished host viability induced by a non-native bacterial strain. Oc-rtk and oc-fak play key roles in the viability regulation of O. chongmingensis by positively mediating the expression of oc-daf-16 to indirectly impact its longevity and stress tolerances and by negatively regulating the expression of oc-goa-1 to affect the olfactory chemotaxis and fecundity. In response to the stress of invasion by the non-native strain, the expression of oc-miR-71 in the non-native combination was upregulated to downregulate the expression of its targeting oc-pdk-1, which might improve the localization and activation of the transcription factor DAF-16 in the nucleus to induce longevity extension and stress resistance enhancement to some extent. Our findings provide novel insight into comprehension of how nematodes deal with the stress of encountering novel potential bacterial symbionts at the physiological and molecular genetic levels and contribute to improved understanding of host-symbiont relationships generally.

Differentiating between scavengers and entomopathogenic nematodes: Which is Oscheius chongmingensis?

Entomopathogenic nematodes (EPNs) continue to be explored for their potential usefulness in biological control and pest management programs. As more insect-associated species of nematodes are discovered and described, it is possible that scavengers and kleptoparasites may be mischaracterized as EPNs. If a nematode species is truly an entomopathogen it should display similar infectivity, as well as behaviors and preferences, to those of established EPN species, such as Steinernema carpocapsae. In this study we evaluated dauers of the putative EPN species Oscheius chongmingensis. We examined virulence, odor preferences as a measure of host-seeking behavior, and features of its bacterial symbiont Serratia nematodiphila. We determined that O. chongmingensis behaves more like a scavenger than an EPN. Not only did O. chongmingensis exhibit very poor pathogenicity in Galleria mellonella (wax moth larvae), it also displayed odor (host-seeking) preferences that are contrary to the well-known EPN S. carpocapsae. We also found that the bacterial symbiont of O. chongmingensis was antagonistic to S. carpocapsae; S. carpocapsae IJs were unable to develop when S. nematodiphila was a primary food source. We conclude that there is insufficient evidence to support the characterization of O. chongmingensis as an EPN; and based on the attributes of its preferences for already-infected or deceased hosts, suggest that this nematode is a scavenger, which may be on an evolutionary trajectory leading to an entomopathogenic lifestyle.

Oscheius microvilli n. sp. (Nematoda: Rhabditidae): A Facultatively Pathogenic Nematode from Chongming Island, China.

A new species, Oscheius microvilli n. sp., was found on Chongming Island (Shanghai, China). The new species is morphologically similar to the type strain of Oscheius myriophilus, but can be distinguished from it and other species of Oscheius on the basis of unique morphological characteristics of the bursa as well as male papillae. In this new species, the male bursal papillar formula is 2, 1, 3, 3 with everted tips in the first, fifth, and seventh pairs. The bursal rim is jagged, joins together anterior to the spicules, and is partially extended and decorated with microvilli. The spicules are incompletely separated, and the tail does not extend beyond the bursa. Phylogenetic trees of 18S rDNA and internal transcribed spacer indicate that the new species belongs to the insectivora group of the genus Oscheius; it is most closely related to O. myriophilus, and the two species can be distinguished on the basis of their different body length, morphological features of the bursa, and molecular data. The new species is facultatively associated with a bacterial strain of Serratia. The LC50 of this novel nematode against Galleria mellonella was 69.1 dauer juveniles per milliliter after 48 hr of infection.

Genome-wide profiling of chicken dendritic cell response to infectious bursal disease.

BACKGROUND: Avian infectious bursal disease virus (IBDV) is a highly contagious, immunosuppressive disease of young chickens, which causes high mortality rates and large economic losses in the poultry industry. Dendritic cells (DCs), which are antigen-presenting cells, have the unique ability to induce both innate and acquired immune responses and may significantly influence virus pathogenicity. To understand the interaction between IBDV and DCs, a microarray was used to analyse the response of DCs infected by IBDV. RESULTS: IBDV infection induced 479 upregulated and 466 downregulated mRNAs in chicken DCs. Analysis of Gene Ontology suggested that transcription from the RNA polymerase II promoter and the RNA biosynthetic process were enriched, and pathway analyses suggested that oxidative phosphorylation, as well as the T cell receptor and Interleukin-17 (IL-17) signalling pathways might be activated by IBDV infection. Moreover, microRNA (miRNA) and long non-coding RNA (lncRNA) alterations in IBDV-infected chicken DCs were observed. A total of 18 significantly upregulated or downregulated miRNAs and 441 significantly upregulated or downregulated lncRNAs were identified in IBDV-stimulated DCs. We constructed 42 transcription factor (TF)-miRNA-mRNA interactions involving 1 TF, 3 miRNAs, and 42 mRNAs in IBDV-stimulated DCs. Finally, we predicted the target genes of differentially expressed lncRNAs, and constructed lncRNA-mRNA regulatory networks. CONCLUSIONS: The results of this study suggest a mechanism to explain how IBDV infection triggers an effective immune response in chicken DCs.

Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.).

BACKGROUND: Salt stress is one of the most representative abiotic stresses that severely affect plant growth and development. MicroRNAs (miRNAs) are well known for their significant involvement in plant responses to abiotic stresses. Although miRNAs implicated in salt stress response have been widely reported in numerous plant species, their regulatory roles in the adaptive response to salt stress in radish (Raphanus sativus L.), an important root vegetable crop worldwide, remain largely unknown. RESULTS: Solexa sequencing of two sRNA libraries from NaCl-free (CK) and NaCl-treated (Na200) radish roots were performed for systematical identification of salt-responsive miRNAs and their expression profiling in radish. Totally, 136 known miRNAs (representing 43 miRNA families) and 68 potential novel miRNAs (belonging to 51 miRNA families) were identified. Of these miRNAs, 49 known and 22 novel miRNAs were differentially expressed under salt stress. Target prediction and annotation indicated that these miRNAs exerted a role by regulating specific stress-responsive genes, such as squamosa promoter binding-like proteins (SPLs), auxin response factors (ARFs), nuclear transcription factor Y (NF-Y) and superoxide dismutase [Cu-Zn] (CSD1). Further functional analysis suggested that these target genes were mainly implicated in signal perception and transduction, regulation of ion homeostasis, basic metabolic processes, secondary stress responses, as well as modulation of attenuated plant growth and development under salt stress. Additionally, the expression patterns of ten miRNAs and five corresponding target genes were validated by reverse-transcription quantitative PCR (RT-qPCR). CONCLUSIONS: With the sRNA sequencing, salt-responsive miRNAs and their target genes in radish were comprehensively identified. The results provide novel insight into complex miRNA-mediated regulatory network of salt stress response in radish, and facilitate further dissection of molecular mechanism underlying plant adaptive response to salt stress in root vegetable crops.

A New Species of Pristionchus (Rhabditida: Diplogastridae) and Its Bacterial Symbiont from Yixing, China.

A new nematode species, Pristionchus entomophilus n. sp., was collected during a soil sample survey in Yixing of Jiangsu province, eastern China. P. entomophilus n. sp. is distinguished by its unique characteristics. This new species is mainly hermaphroditic, with males seldom found. The new nematode has a similar body length but has much narrower body width compared with P. pacificus. Its body is covered with longitudinal ridges: 12 ridges on head, 13 or 14 ridges in the middle, 11 and 7 ridges in front and rear of the anus, respectively. The eurystomatous form mouth includes a triangular dorsal tooth, a large claw-like right subventral tooth, and a row of five ventral denticles placed opposite the dorsal tooth. Only eight pairs of genital papillae and a pair of phasmids are present in the tail of the male as the sixth pair of papillae having seemingly been degenerated and lost. Molecular phylogenetic trees based on 18S rDNA confirmed that the new species belongs to the genus Pristionchus and is most closely related to P. pacificus. Moreover, the new species was found to be occasionally associated with the entomopathogenic bacterial strain 09FLYB1 of Serratia nematodophila and be able to stably transfer the bacterial strain for several generations.

Exome sequencing identifies mutations in ABCD1 and DACH2 in two brothers with a distinct phenotype.

BACKGROUND: We report on two brothers with a distinct syndromic phenotype and explore the potential pathogenic cause. METHODS: Cytogenetic tests and exome sequencing were performed on the two brothers and their parents. Variants detected by exome sequencing were validated by Sanger sequencing. RESULTS: The main phenotype of the two brothers included congenital language disorder, growth retardation, intellectual disability, difficulty in standing and walking, and urinary and fecal incontinence. To the best of our knowledge, no similar phenotype has been reported previously. No abnormalities were detected by G-banding chromosome analysis or array comparative genomic hybridization. However, exome sequencing revealed novel mutations in the ATP-binding cassette, sub-family D member 1 (ABCD1) and Dachshund homolog 2 (DACH2) genes in both brothers. The ABCD1 mutation was a missense mutation c.1126G > C in exon 3 leading to a p.E376Q substitution. The DACH2 mutation was also a missense mutation c.1069A > T in exon 6, leading to a p.S357C substitution. The mother was an asymptomatic heterozygous carrier. Plasma levels of very-long-chain fatty acids were increased in both brothers, suggesting a diagnosis of adrenoleukodystrophy (ALD); however, their phenotype was not compatible with any reported forms of ALD. DACH2 plays an important role in the regulation of brain and limb development, suggesting that this mutation may be involved in the phenotype of the two brothers. CONCLUSION: The distinct phenotype demonstrated by these two brothers might represent a new form of ALD or a new syndrome. The combination of mutations in ABCD1 and DACH2 provides a plausible mechanism for this phenotype.

Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1.

OBJECTIVES: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. METHODS: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of circUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. RESULTS: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. CONCLUSION: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.

Five novel antimicrobial peptides from the Kuhl's wart frog skin secretions, Limnonectes kuhlii.

Five novel antimicrobial peptides (temporin-LK1, rugosin-LK1, rugosin-LK2, gaegurin-LK1, and gaegurin-LK2) are purified and characterized from Kuhl's wart frog skin secretions, Limnonectes kuhlii. They share obvious similarity to temporin, rugosin, and gaegurin antimicrobial peptide family, respectively. Their amino acid sequences were determined by Edman degradation and mass spectrometry, and further confirmed by cDNA cloning. Nine cDNA sequences encoding precursors of these five purified antimicrobial peptides and other four hypothetical antimicrobial peptides were cloned from the skin cDNA library of L. kuhlii. The deduced precursors are composed of a predicted signal peptide, an acidic spacer peptide, and a mature antimicrobial peptide. Most of them showed strong antimicrobial activities against Gram-positive and Gram-negative bacteria and fungi. The current work identified and characterized three families of antimicrobial peptides from L. kuhlii skins and confirmed that the genus of Limnonectes amphibians share similar antimicrobial peptide families with the genus of Rana amphibians. In addition, a unique antimicrobial peptide (temporin-LK1) with 17 residues including four phenylalanines, which is significantly different from other temporins (16 residues, one or two phenylalanines), was identified in this work. Such unique structure might provide novel template or leading structure to design antimicrobial agents.

4­Methoxydalbergione inhibits esophageal carcinoma cell proliferation and migration by inactivating NF­κB.

4­Methoxydalbergione (4­MD) can inhibit the progression of certain types of cancer; however, its effects on esophageal cancer (EC) remain unclear. The present study aimed to investigate the inhibitory effect of 4­MD on EC and its molecular mechanism. ECA­109 and KYSE­105 cells were treated with or without lipopolysaccharide (LPS) and 4­MD. Cell Counting Kit­8 and colony formation assays were used to analyze cell proliferation. Wound healing assay was performed to evaluate cell migration. ELISA and western blotting were performed to measure the expression levels of NF­κB and inflammatory cytokines. In cells treated with 4­MD, proliferation and migration were significantly inhibited, the levels of inflammatory cytokines were downregulated and the NF­κB signaling pathway was inactivated. Notably, proliferation, migration, inflammation and NF­κB were promoted by LPS, whereas 4­MD reversed the increases induced by LPS in EC cells. In conclusion, 4­MD may attenuate the proliferation and migration of EC cells by inactivating the NF­κB signaling pathway.

Two novel antimicrobial peptides from skin secretions of the frog, Rana nigrovittata.

Two novel antimicrobial peptides with similarity to brevinin-2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Their amino acid sequences were determined as GAFGNFLKGVAKKAGLKILSIAQCKLSGTC (brevinin-2-RN1) and GAFGNFLKGVAKKAGLKILSIAQCKLFGTC (brevinin-2-RN2), respectively, by Edman degradation. Different from brevinin-2, which is composed of 33 amino acid residues (aa), both brevinin-2-RN1 and -RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465-9) encoding precursors of brevinin-2-RN1 and -RN2 were screened from the skin cDNA library of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin-2-RN. Both brevinin-2-RN1 and -RN2 showed strong antimicrobial activities against gram-positive and gram-negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure.

Two antimicrobial and nematicidal peptides derived from sequences encoded Picea sitchensis.

Two antimicrobial peptides (piceain 1 and 2) derived from sequences encoded Picea sitchensis are identified. Their amino acid sequences are KSLRPRCWIKIKFRCKSLKF and RPRCWIKIKFRCKSLKF, respectively. One intra-molecular disulfide bridge is formed by these two half-cysteines in both piceain 1 and 2. Antimicrobial activities of synthesized piceains against several kinds of microorganisms were tested. They showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and fungus Candida albicans but little antimicrobial activity against Bacillus subtilis. The results of nematicidal test showed they exerted strong nematicidal activities against Caenorhabditis elegans, following exposure for 5 h at concentrations as low as 10 µg/ml. They had weak hemolytic abilities against human and rabbit red cells. At the concentration of 250 µg/ml, they induced red cell hemolysis of less than 5%. Circular dichroism spectra of the two antimicrobial peptides were investigated in several solutions. Their main secondary structure components are ß-sheet and random. The current work provides a novel family of antimicrobial and nematicidal peptides with unique disulfided loop containing nine amino acid residues.

Progressive osseous heteroplasia: a case report and literature review.

OBJECTIVE: To investigate the clinical features and pathogenesis of progressive osseous heteroplasia (POH) in children. METHODS: The clinical features and imaging findings of a child with POH are described, and family investigations and gene comparisons were performed, followed by a literature review. RESULTS: A 9-year-old female with no relevant family medical history initially presented with ectopic ossification of the skin and subcutaneous tissue of the right face that developed slowly. The ossification area extended to the right waist, back, and right knee. The unilateral body (limbs) was gradually invaded. The patient exhibited limited movement of the head, neck, and left shoulder joint, and experienced difficulty in opening her mouth. She also exhibited deformity of the toe, delayed development, insufficient language skills and behavioral ability, and difficulty in communicating with others, but had no apparent endocrine disorders. Blood calcium, phosphorus, and alkaline phosphatase levels were normal, and DNA sequencing did not yield a positive result. CONCLUSION: The clinical manifestations of POH include hard plaques, which can develop deep into the bone; however, there are currently no effective preventive or treatment measures.

Rhizobium kunmingense sp. nov., isolated from rhizosphere soil of Camptotheca acuminata Decne.

Strain LXD30(T) was isolated from rhizosphere soil of a plant of the species Camptotheca acuminata Decne which is native to warm, humid stream banks in southern China. Analysis of the 16S rRNA gene sequence revealed that the Gram-negative, rod-shaped bacterium fell within the realm of the genus Rhizobium and was most closely related to Rhizobium huautlense SO2(T) (96.4% sequence similarity) and Rhizobium cellulosilyticum LMG 23642(T) (96.4%). The isolate grew optimally at pH7.0 and 25-28 degrees C in the presence of 0-1% (w/v) NaCl. Major fatty acids were C16:0 (17.5%) and summed feature 7 (C18:1omega7c/omega9t/omega12t, 58.3%). Unequivocally low 16S rRNA (<97%), recA (<92%) and atpD (<90%) gene sequence similarities to all existing species of the genus and phenotypic characteristics all suggested that strain LXD30(T) (=KCTC 22609(T)=CGMCC 1.8903(T)) represents a novel Rhizobium species, for which the name Rhizobium kunmingense sp. nov. is proposed.

[Analysis of risk factors for cerebral infarction after hip arthroplasty].

OBJECTIVE: To investigate the independent risk factors for cerebral infarction so as to provide a theoretical basis for the prevention of cerebral infarction after total hip arthroplasty. METHODS: A retrospective analysis of 571 patients for hip arthroplasty was conducted from January 2003 to September 2008. Twenty-three patients were found with cerebral infarction postoperatively. Single-factor and multi-factor correlation analyses were tested for the patients with cerebral infarction after hip arthroplasty. RESULTS: The single-factor analysis for hip arthroplasty revealed that age (P = 0.001) and femoral neck fracture (P = 0.008) were the main factors for cerebral infarction. Furthermore, age was considered a risk factor for cerebral infarction after hip arthroplasty in multi-factor analysis (P = 0.029, OR = 1.054, 95%CI: 1.005 - 1.105). CONCLUSION: Advanced age (> 70 yr) and femoral neck fracture are the main independent risk factors for cerebral infarction after hip arthroplasty.

The first antimicrobial peptide from sea amphibian.

The crab-eating frog, Rana cancrivora, is one of only a handful of amphibians worldwide that tolerates saline waters. It typically inhabits brackish water of mangrove forests of Southeast Asia. A large amount of antimicrobial peptides belonging to different families have been identified from skins of amphibians inhabiting freshwater. No antimicrobial peptide from sea amphibians has been reported. In this paper, we firstly reported the antimicrobial peptide and its cDNA cloning from skin secretions of the crab-eating frog R. cancrivora. The antimicrobial peptide was named cancrin with an amino acid sequence of GSAQPYKQLHKVVNWDPYG. By BLAST search, cancrin had no significant similarity to any known peptides. The cDNA encoding cancrin was cloned from the cDNA library of the skin of R. cancrivora. The cancrin precursor is composed of 68 amino acid residues including a signal peptide, acidic spacer peptide, which are similar to other antimicrobial peptide precursors from Ranid amphibians and mature cancrin. The overall structure is similar to other amphibian antimicrobial peptide precursors although mature cancrin is different from known peptides. The current results reported a new family of amphibian antimicrobial peptide and the first antimicrobial peptide from sea amphibian.

A phospholipase A1 platelet activator from the wasp venom of Vespa magnifica (Smith).

Wasp is an important venomous animal that can induce human fatalities. Aortic thrombosis and cerebral infarction are major clinical symptoms after massive wasp stings but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnifin, contains phospholipase-like activity and induces platelet aggregation. The cDNA encoding magnifin is cloned from the venom sac cDNA library of the wasp. The predicted protein was deduced from the cDNA with a sequence composed of 337 amino acid residues. Magnifin is very similar to other phospholipase A(1) (PLA(1)), especially to other wasp allergen PLA(1). Magnifin can activate platelet aggregation and induce thrombosis in vivo. The current results proved that PLA(1) in wasp venom could be contributable to aortic thrombosis after massive wasp stings.

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes.

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP=90 (80-105)microm, ES=104 (92-120)microm, tail length=111 (89-159)microm, and a=19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide.

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xylophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xylophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those from the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal, Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xylophilus originated in North America and that the B. xylophilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.