Pipermotuoense, a new species of Piperaceae from Xizang, China.

Pipermotuoense X.W.Qin, F.Su & C.Y.Hao, a new species of Piperaceae from Xizang, China, is described and illustrated in this paper. The new species resembles P.yinkiangense and P.anisotis, but it can be readily distinguished from the compared species by several characteristics. Gonophyll leaves are chartaceous and the leaf secondary vein count is 7-9, with the outermost pair being very weak when there are nine veins. Additionally, the apical pair arises 2-4 cm above the base and the leaf base is asymmetrical, with bilateral petioles that cling and heal together. Pistillate floral bracts are sessile, with 3, 4 or 5 stigmas. The description of the new species includes photographs, detailed descriptions, notes on etymology, distribution and habitat, as well as comparisons with morphologically similar species.

Staurantherafloribunda, a new species of Gesneriaceae from Yunnan, China.

Staurantherafloribunda F.Su, C.Y.Hao & K.Tan, a new species of Gesneriaceae from Yunnan, China, is described and illustrated here. It is morphologically similar to S.grandifolia Benth. in the shape of corolla, stigma, leaves and the number of stamens. However, it can be readily distinguished from the compared species by its dense cymes, leaf indumentum, lack of a corolla spur, calyx colour and stamen shape. The description of the new species, photographs, detailed descriptions, notes on etymology, distribution and habitat, as well as comparisons with morphologically similar species, are provided.

The Interaction of Mandarin Fish DDX41 with STING Evokes type I Interferon Responses Inhibiting Ranavirus Replication.

DDX41 is an intracellular DNA sensor that evokes type I interferon (IFN-I) production via the adaptor stimulator of interferon gene (STING), triggering innate immune responses against viral infection. However, the regulatory mechanism of the DDX41-STING pathway in teleost fish remains unclear. The mandarin fish (Siniperca chuatsi) is a cultured freshwater fish species that is popular in China because of its high market value. With the development of a high-density cultural mode in mandarin fish, viral diseases have increased and seriously restricted the development of aquaculture, such as ranavirus and rhabdovirus. Herein, the role of mandarin fish DDX41 (scDDX41) and its DEAD and HELIC domains in the antiviral innate immune response were investigated. The level of scDDX41 expression was up-regulated following treatment with poly(dA:dT) or Mandarin fish ranavirus (MRV), suggesting that scDDX41 might be involved in fish innate immunity. The overexpression of scDDX41 significantly increased the expression levels of IFN-I, ISGs, and pro-inflammatory cytokine genes. Co-immunoprecipitation and pull-down assays showed that the DEAD domain of scDDX41 recognized the IFN stimulatory DNA and interacted with STING to activate IFN-I signaling pathway. Interestingly, the HELIC domain of scDDX41 could directly interact with the N-terminal of STING to induce the expression levels of IFN-I and ISGs genes. Furthermore, the scDDX41 could enhance the scSTING-induced IFN-I immune response and significantly inhibit MRV replication. Our work would be beneficial to understand the roles of teleost fish DDX41 in the antiviral innate immune response.

Roles of extracellular matrix components in Tiger frog virus attachment to fathead minnow (Pimephales promelas) cells.

Tiger frog virus (TFV) belongs to the genus Ranavirus (family Iridoviridae) and causes significant harm in cultured frogs, resulting in substantial losses in ecological and economic field in Southern China. Attachment is the first step in viral life cycle, which is dependent on the interactions of virions with extracellular matrix (ECM) components. Studying this process will help in understanding virus infection and controlling viral diseases. In this study, the roles of primary ECM components in TFV attachment were investigated. The results on the kinetics of virus attachment showed TFV successful attachment to the cell surface as a relatively rapid process after TFV was used to inoculate cells for 10 min at 4 °C. Western blot and quantitative PCR analyses results showed that soluble fibronectin, collagen IV, laminin, or hyaluronic acid treatment with TFV caused no significant effect on virus attachment. Soluble heparin, heparan sulfate and chondroitin sulfate A/B could inhibit TFV attachment in a dose-dependent manner. Enzymic digestion by cell surface heparin/heparan sulfate using heparinase I, II, and III could significantly prevent TFV attachment, suggesting that heparan sulfate plays an important role in TFV attachment. Furthermore, the binding assays of heparin-agarose beads and virion showed that TFV virions specifically bound with heparin in a dose-dependent manner. Given that heparin is a structural analogue of heparan sulfate, the above results suggest that heparan sulfate might serve as an attachment factor of TFV infection. Our work would be beneficial to understand the mechanisms of TFV attachment and the interactions of TFV with cellular receptor(s).

The roles of mandarin fish STING in innate immune defense against Infectious spleen and kidney necrosis virus infections.

The mandarin fish Siniperca chuatsi is a cultured freshwater fish species that is popular in China because of its high market value. With the development of high-density cultural mode in mandarin fish, viral diseases such as Infectious spleen and kidney necrosis virus (ISKNV) are becoming increasingly serious. Stimulator of interferon genes (STING) is a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the roles of STING in innate immune response of mandarin fish remain unknown. In the present study, S. chuatsi STING (scSTING)-mediated host immune response against ISKNV infection was investigated. ScSTING transcription level increased remarkably in response to ISKNV infection, LPS, PMA, or poly (I:C) stimulation in mandarin fish fry (MFF-1) cells. Immunofluorescence results showed that scSTING localized majorly in the endoplasmic reticulum. scSTING overexpression remarkably increased the expression levels of scIFN-h, scMx, scISG15, scPKR, scViperin, scIL-1ß, scIL-18, and scTNF-α genes. IFN-ß-luciferase report assay results showed that the relative expressions of luciferin were remarkably increased in MFF-1 cells. Site mutation of serine (S) on C-terminus of scSTING showed that both S388 and S396 were important for mediated signaling. Furthermore, scSTING overexpression inhibited ISKNV infection, and knockdown of scSTING promoted ISKNV infection, indicating that scSTING could suppress ISKNV infection in MFF-1 cells. These observations suggested that the scSTING played an important role in innate immune against ISKNV infection. Our work would help elucidate the roles of teleost fish STING in innate immunity.

The microbiota-gut-brain interaction in regulating host metabolic adaptation to cold in male Brandt's voles (Lasiopodomys brandtii).

Gut microbiota play a critical role in orchestrating metabolic homeostasis of the host. However, the crosstalk between host and microbial symbionts in small mammals are rarely illustrated. We used male Brandt's voles (Lasiopodomys brandtii) to test the hypothesis that gut microbiota and host neurotransmitters, such as norepinephrine (NE), interact to regulate energetics and thermogenesis during cold acclimation. We found that increases in food intake and thermogenesis were associated with increased monoamine neurotransmitters, ghrelin, short-chain fatty acids, and altered cecal microbiota during cold acclimation. Further, our pair-fed study showed that cold temperature can alter the cecal microbiota independently of overfeeding. Using cecal microbiota transplant along with ß3-adrenoceptor antagonism and PKA inhibition, we confirmed that transplant of cold-acclimated microbiota increased thermogenesis through activation of cAMP-PKA-pCREB signaling. In addition, NE manipulation induced a long-term alteration in gut microbiota structure. These data demonstrate that gut microbiota-NE crosstalk via cAMP signaling regulates energetics and thermogenesis during cold acclimation in male Brandt's voles.

Tiger frog virus ORF104R interacts with cellular VDAC2 to inhibit cell apoptosis.

Ranaviruses belong to the family Iridoviridae, and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa, and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo.

Identification and functional analysis of the Mandarin fish (Siniperca chuatsi) hypoxia-inducible factor-1α involved in the immune response.

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha, scvegf, and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (scHIF-1α) was also characterized. Multiple sequence alignments showed that scHIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that scHIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of scHIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of scHIF-1α could induce the HIF signaling pathway, whereas knockdown of scHIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of scHIF-1α was closely related to the circulatory system. Furthermore, scHIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that scHIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of scHIF-1α could inhibit MRV and SCRV infections, whereas knockdown of scHIF-1α could promote such infections. Those results suggested that scHIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease.

Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway.

The cellular endosomal sorting complex required for transport (ESCRT) pathway is a multifunctional pathway involved in cell physiological activities. While the majority of RNA viruses bearing L-domains are known to hijack the ESCRT pathway to complete the budding process, the budding of large and complex enveloped DNA viruses, especially iridoviruses, has been rarely investigated. In the present study, we use the tiger frog virus (TFV) as a model to investigate whether iridoviruses are released from host cells through the ESCRT pathway. Inhibition of class E proteins and auxiliary proteins (VPS4A, VPS4B, Tsg101, Alix, and Nedd4.1) reduces extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in TFV release. The respective interactions of TFV VP031L, VP065L, VP093L with Alix, Tsg101, Nedd4 suggest the underlying molecular mechanism by which TFV gets access to the ESCRT pathway. Co-depletion of Alix, Tsg101, and Nedd4.1 induces a significant reduction in extracellular virion production, which implies the functional redundancy of host factors in TFV budding. Those results are first observation that iridovirus gains access to ESCRT pathway through three ways of interactions between viral proteins and host proteins. Our study provides a better understanding of the budding mechanism of enveloped DNA viruses.

Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation.

Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles.

Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-ß-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.

Volatile organic compound emissions from different stages of Cananga odorata flower development.

Headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used to identify the volatile organic compounds (VOCs) of the different flower development stages of Cananga odorata for the evaluation of floral volatile polymorphism as a basis to determine the best time of harvest. Electronic nose results, coupled with discriminant factor analysis, suggested that emitted odors varied in different C. odorata flower development stages, including the bud, display-petal, initial-flowering, full-flowering, end-flowering, wilted-flower, and dried flower stages. The first two discriminant factors explained 97.52% of total system variance. Ninety-two compounds were detected over the flower life, and the mean Bray-Curtis similarity value was 52.45% among different flower development stages. A high level of volatile polymorphism was observed during flower development. The VOCs were largely grouped as hydrocarbons, esters, alcohols, aldehydes, phenols, acids, ketones, and ethers, and the main compound was ß-caryophyllene (15.05%-33.30%). Other identified compounds were ß-cubebene, D-germacrene, benzyl benzoate, and α-cubebene. Moreover, large numbers of VOCs were detected at intermediate times of flower development, and more hydrocarbons, esters, and alcohols were identified in the full-flowering stage. The full-flowering stage may be the most suitable period for C. odorata flower harvest.

[Effects of forest gap on herbaceous plant diversity in mixed birch-fir forest of Taibai Mountain].

To better understand the effects of forest gap on the herbaceous species community in a mixed birch-fir forest of Taibai Mountain in Qinling, CCA ordination and random permutation test were employed to analyze the distribution pattern of the species composition across a gradient of gap size, and the relationships between the distribution of 55 herbaceous species with > or = 5 individuals and the habitat variables (convexity, slope, and soil total N, total P, available N, available P, pH, and organic matter). In this forest, gap area occupied 19.8% of the total land area, gap density was 20.7 per hm2, and gap size varied from 25.6 to 279.1 m2, with a mean of 93.7 m2. The species richness in herbaceous layer in gaps was significantly positively correlated with gap size, but of the 69 herbaceous species identified in the gaps, most species were found across all gap sizes, and only eight species were found in larger gaps (>120 m2). No successional change was observed in the herbaceous species distribution with gap size. The CCA ordination and random permutation test also showed that 27.3% of the 55 species with abundance > or = 5 had significant association with the eight habitat variables. It was concluded that gap size contributed to the species richness, but determined the diversity constitution in random.

Effect of needle damage on the release rate of Masson pine (Pinus massoniana) volatiles.

The effect of needle damage on the release rate of Masson pine (Pinus massoniana Lamb.) volatiles was examined. Needles were continuously damaged by mechanical damage (MDP) or by feeding of pine caterpillar (Dendrolimus punctatus) larvae (LFP); undamaged pine was used as a control (UDP). Volatiles were collected before damage, and at 16, 24, 40, 48, 64, 72, 88 and 96 h post-damage, and analyzed. The analyses revealed that 19 compounds identified as constitutive volatiles from UDP were terpenes and green leaf odors. The release rate of volatiles from MDP or LFP was higher than that from UDP. At 96 h post-damage, emission from MDP or LFP returned to the same level as that of UDP. Some volatiles, including sabinene, ocimene, limonene-1,2-epoxide, linalool, linalool acetate, germacrene D: -4-ol, farnesol, and (E)-4,8-dimethyl-1,3,7-nonatriene were induced by mechanical damage and/or larval attack. Furthermore, the release rate of linalool acetate, farnesol, or (E)-4,8-dimethyl-1,3,7-nonatriene from LFP was higher than that from MDP. Based on an exact estimation of the proportion of damaged pine needles, a significant linear correlation between the release rate of total volatiles identified and the proportion of damaged needles was found in the case of LFP but not MDP.

Putative chemical signals about sex, individuality, and genetic background in the preputial gland and urine of the house mouse (Mus musculus).

To explore whether preputial gland secretions and/or urine from the house mouse (Mus musculus) can be used for coding information about sex, individuality, and/or the genetic background of strain [ICR/albino, Kunming (KM), and C57BL/6], we compared the volatile compositions of mouse preputial glands and urine using a combination of dichloromethane extraction and gas chromatography coupled with mass spectrometry (GC-MS). Of the 40 identified compounds in preputial gland secretions, 31 were esters, 2 sesquiterpens, and 7 alcohols. We failed to find any compound unique to a specific sex, individual, or strain. However, many low molecular weight compounds between the sexes, most compounds among individuals, and several compounds among the 3 strains varied significantly in relative ratios. These quantitative differences in preputial gland volatiles (analog coding) are likely to convey information about sex, individual, and the genetic background of mouse strain. We identified 2 new main and male-elevated compounds, 1-hexadecanol (Z=3.676, P=0.000, N=19 in ICR; Z=3.576, P=0.000, N=18) and 1-hexadecanol acetate (Z=3.429, P=0.000, N=19 in ICR; Z=3.225, P=0.001, N=18), which were eluted in GC chromatogram after the 2 sesquiterpens. They might also be potential male pheromones, in addition to the well-known E-beta-farnesene and E,E-alpha-farnesene. Additionally, a few compounds including 1-hexadecanol also varied with strains and might also code for genetic information. Of the 9 identified volatile compounds in male urine, (s)-2-sec-butyl-4,5-dihydrothiazole and R,R-3,4-dehydro-exo-brevicomin are known urine-originated male pheromones from previous studies. We also detected 6-hydroxy-6-methyl-3-heptanone, a male urinary pheromonal compound, which had not been directly detected by GC-MS previously. Chemical analysis shows that the genetically more closely related ICR and KM strains had a higher similarity in the volatile compositions of preputial glands and urine than that between ICR or KM and C57BL/6. R,R-3,4-dehydro-exo-brevicomin, in particular, was sensitive to genetic shifts and differed in relative abundance among the 3 strains, whereas (s)-2-sec-butyl-4,5-dihydrothiazole differed between ICR or Km and C57BL/6. Hence, these 2 compounds might code for information about their genetic background.