Life cycles,phenology and genetic structure of endangered Megacrania tsudai Shiraki (Phasmatodea: Phasmatidae): Male individuals from a geographic parthenogenesis species

Megacrania tsudai, a peripherally distributed member of Megacrania, requires conservation in Taiwan; it has limited distribution in Taiwan and its eastern offshore islands. It feeds on screw pines (Pandanus odoratissimus) in nature and has demonstrated a specific defensive mechanism involving actinidine secretion from the prothoracic gland. However, details of its distribution area, life cycle and developmental phenology remain largely unknown. In this study, a field survey and review of published works revealed M. tsudai distribution in coastal zones and along river shores near estuaries. At room temperature, the egg period was 128 days. The development of the first to sixth instars required 17, 26, 27, 26, 34 and 43 days, respectively, on average; and a generation cycle required approximately 204 days. The phenology of the mesonotal granules was recorded. Moreover, genetic analysis of the mitochondrial cytochrome oxidase I (COI), 16S ribosomal DNA (16S rDNA) and the nuclear ribosomal spacer indicated the occurrence of genetic drift. Therefore, the rearing procedures proposed in this study for the primary and last instars of M. tsudai can facilitate its conservation. Megacrania tsudai was previously recorded as parthenogenetic; however, two male individuals were fostered unexpectedly. The male body length was 91 mm, which is shorter than the female length (120 mm). During mating, the male climbs onto the female's back and protrudes its genitalia downward. Geographical parthenogenesis is likely the reproductive strategy among peripheral M. tsudai; however, the rarely found M. tsudai male could be an intermediate link of reproductive strategy in the transition from tychoparthenogenesis to parthenogenesis.     

Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo

Background

Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.

Methodology/Principal Findings

This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)^cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR⁺ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR⁺ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR⁺ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR⁺ fluorescent signaling.

Conclusion/Significance

The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.     

Chemical polymorphism and antifungal activity of essential oils from leaves of different provenances of indigenous cinnamon (Cinnamomum osmophloeum)

The essential oils isolated from nine geographical provenances of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) leaves were examined by GC-MS and their chemical constituents were compared. According to GC-MS and cluster analyses the leaf essential oils of the nine provenances and their relative contents were classified into six chemotypes-cinnamaldehyde type, cinnamaldehyde/cinnamyl acetate type, cinnamyl acetate type, linalool type, camphor type and mixed type. In addition, the antifungal activities of leaf essential oils and their constituents from six chemotypes of indigenous cinnamon were investigated in this study. Results from the antifungal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against white-rot fungi, Trametes versicolor and Lenzites betulina and brown-rot fungus Laetiporus sulphureus. The antifungal indices of leaf essential oils from these two chemotypes at the level of 200 micro/ml against T. versicolor, L. betulina and L. sulphureus were all 100%. Among them, the IC(50) (50% of inhibitory concentrations) value of the essential oil of cinnamaldehyde type leaf against L. sulphureus was 52-59microg/ml. Cinnamaldehyde possessed the strongest antifungal activities in comparison with other constituents of the essential oils from cinnamaldehyde type leaf, at the level of 100microg/ml its antifungal indices against T. versicolor, L. betulina and L. sulphureus were 100%. The IC50 values of cinnamaldehyde against T. versicolor, L. betulina and L. sulphureus were 73, 74 and 73microg/ml, respectively.     

The Susceptive Alendronate-Treatment Timing and Dosage for Osteogenesis Enhancement in Human Bone Marrow-Derived Stem Cells

Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on ostegenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM) according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic.     

Insecticidal activities of leaf essential oils from Cinnamomum osmophloeum against three mosquito species

The larvicidal activities of leaf essential oils and their constituents from six chemotypes of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) trees were evaluated against three mosquito species. Results of larvicidal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against Aedes albopictus larvae, and their LC(50) values in 24h were 40.8 microg/ml (LC(90)=81.7 microg/ml) and 46.5 microg/ml (LC(90)=83.3 microg/ml), respectively. Results of the 24-h mosquito larvicidal assays also showed that the effective constituents in leaf essential oils were trans-cinnamaldehyde and benzaldehyde and that the LC(50) values of these constituents against A. albopictus larvae were below 50 mug/ml. In addition, cinnamaldehyde type leaf essential oil and trans-cinnamaldehyde have also exhibited great larvicidal performance against Culex quinquefasciatus and Armigeres subalbatus larvae. Comparisons of mosquito larvicidal activity of trans-cinnamaldehyde congeners revealed that alpha-methyl cinnamaldehyde, benzaldehyde, and trans-cinnamaldehyde exhibited strong mosquito larvicidal activity.     

Analysis of landscape spatial pattern changes in urban fringe area: a case study of Hunhe Niaodao Area in Shenyang City

The urban fringe is areas where land use changes and develops rapidly, analyzing land use changes and driving forces in urban fringe area is of great significance for landscape pattern and ecological environment construction. This study takes the Hunhe Bird Island area of Shenyang City as the research object, uses Landsat remote sensing images from 2005 to 2019 as the basic data, and uses the land transfer matrix and landscape index method to analyze the overall landscape environment and six land types. Using government policies and statistical data to analyze the driving forces of landscape changes. The research results show that the landscape matrix had changed, the overall landscape structure had changed drastically, the ecological environment was unstable, and human interference factors had increased, and the fragmentation phenomenon had become more and more serious. Each land type has different environmental changes. Except for the relatively stable water environment, other land types have different degrees of fragmentation. The reason is that the driving force of landscape pattern changes mainly comes from the influence of human factors, which is closely related to population, economic development and government policies. Therefore, future development and protection strategies are proposed for each land type in the urban fringe area. For the overall development of urban fringe area, it is advocated to make full use of land resources, strengthen the correct judgment and forecast of urban population and economy, formulate the correct urban boundary strategy for sustainable development, avoid the ecological environment problems faced by excessive land development, and realize the “Rational expansion” of cities.

     

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H₂O₂ elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2-80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H₂O₂ amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H₂O₂, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H₂O₂ elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H₂O₂ homeostasis in leaves caused by developmental cues and environmental stimuli.     

Fishery policy when considering the future opportunity of harvesting

Free access to a common pool of resource in a country may lead to over-exploitation and sacrifice future opportunities of harvesting. As such, the protection of a common fishery resource is worth investigating. In this paper we develop a two-period model and a multi-period model to analyze the optimal inter-temporal utilization of a finite resource of stock and propose to impose a tax on the harvest rate as an efficient mechanism with an aim at economic sustainability by incorporating the future opportunity of harvesting into the models as a major component of social objectives. The sensitivity analysis of the two-period model shows that (1) labor inputs for harvesting in Period 1 should be reduced, the biomass of fishery stock will increase, but the harvesting in Period 2 should be amplified and the biomass of fishery stock in Period 2 will not be affected if the current generation owns a higher valuation on the future opportunity of harvesting; (2) a higher internal regeneration rate leads to higher harvesting in each period and a higher level of fishery stock in Period 1, but an uncertain level of fishery stock in Period 2; (3) with a higher discount rate the harvesting in Period 1 should increase, but the harvesting in Period 2 should fall and the level of fishery stock in each period will be reduced; (4) a higher fish price in Period 1 leads to higher harvesting in Period 1, but reduced harvesting in Period 2. As a consequence, the level of fishery stock in each period will be reduced; (5) the effect of a change in fish prices in Period 2 on the harvesting and the level of fishery stock in Period 1 is uncertain, but the change in fish prices in Period 2 gives a positive effect on harvesting in Period 2 and a negative effect on the level of fishery stock in Period 2; (6) higher labor wages in Period 1 lead to lower harvesting, but a higher level of fishery stock in Period 1. This encourages an increase in harvesting in Period 2 and leads to a higher level of fishery stock in Period 2; and (7) a change of the labor wage in Period 2 affects the harvesting and the level of fishery stock in Period 1 indecisively, but it gives negative effects on the harvesting in Period 2 and positive effects on the level of fishery stock in Period 2.     

Application of fractals: create an artificial habitat with several small (SS) strategy in marine environment

In 2006, Lan and Hsui applied perspectives on landscape ecology to propose a spatially explicit model, and suggested that the cluster type deployment of artificial habitat in a marine environment might be better than other configurations in maximizing habitat complexity with a fractals approach. The limitation is that, however, it could only design a single large island pattern subject to its solving algorithm. Hence, by applying more sophisticated algorithms, Evolution computational (EC) algorithm, we mimic more spatially explicit structural patterns to conduct several small (SS) clusters pattern in an artificial habitat such as those found in nature landscape; additionally, the deployment in a community follows the concept of DARCs model, which applied the cellular automata (CA) concept with Moore neighborhood rule. The results show that with all the new knowledge that has been gained through the appropriate application of fractal geometry to natural sciences, it is clear that understanding how landscape ecology influences population ecology has allowed population ecologists to gain new insights into their field.     

Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli.

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis. The mutant protein, OraSE-K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.