萘降解质粒pND6的分离和鉴定

从工业废水中分离的假单胞菌 (Pseudomonassp .)ND6菌株 ,能以萘为唯一碳源生长 ,使无机盐培养基(MM)中 2g/L的萘在 48h内降解 98% .该菌株含有一个 115kb的大质粒pND6 .DNA杂交实验表明 ,pND6质粒含有与恶臭假单胞菌 (P .putida)G7菌株的NAH7质粒同源的萘降解基因 .图 3表 1参 14     

石油降解菌株G5 16S rDNA全序列的系统学分析

用PCR方法扩增了一株石油降解菌株G5的16S rRNA基因全序列，并对其进行了克隆和测序．对该序列在GenBank中的BLAST结果表明，所有与该序列高度同源的序列都是假单胞菌的16S rRNA基因．其中假单胞菌的代表菌株Pseudomonas aeruginosa，P．fluoroscens，P .putida，P.syringae的16S rRNA基因序列与G5的16S rRNA基因序列同源性分别为93．4％，98．4％，96．3％，97．5％．对G5和其他39株假单胞菌的16S rRNA基因序列进行聚类分析，获得的系统发育树与RDP(Ribosomal Database Project)报道的系统发育树基本一致，其中菌株G5与5株P．chlororaphis聚类在一起．图2参7     

假单胞菌反式氯双烯内酯异构酶基因克隆和序列分析

从土壤中分离得到一株降解2,4-二氯酚能力较强的假单胞菌菌株GT241-1,从该菌株中克隆出参与降解2,4-二氯酚的反式氯双烯内酯异构酶基因(dcpE).克隆策略是采用Southern杂交对其邻近基因进行定位后构建基因组文库,再用斑点杂交从基因文库中筛选目的转化子.经序列测定得知,dcpE基因编码区1062bp.核苷酸和推测的编码氨基酸序列分析表明,dcpE与已在GenBank登记的相关基因有一定的差异.图5表1参12     

萘降解菌NAP_A的分离、降解性能和分子系统学研究

该文研究降解多环芳烃类环境污染物的微生物资源、降解活性和分子系统特征.利用萘-无机盐选择性培养基分离萘降解菌,用培养技术和气相色谱法检测菌株对底物的利用和降解情况,用分子克隆技术获得菌株的16S rRNA基因并测序,用DNAMAN软件对菌株的16S rRNA基因序列进行比对和系统发育分析.从淄博张店污水处理厂的活性污泥中分离到一株能降解萘的菌株NAP_A.此菌株在30 ～35 ℃和pH 7条件下较快的降解底物萘,其中30 ℃下,10 d内可以将初始质量浓度为320 mg·L-1的萘降解90％± 4.5％.对菌株NAP_A的16S rRNA基因进行了克隆和测序(EU142847),基于菌株NAP_A和相关菌株的16S rRNA基因进行系统发育分析,结果表明菌株NAP_A位于苍白杆菌属的分枝中,其中与假中间苍白杆菌种的同源性最高,可达99％,因此推断NAP_A菌是一株假中间苍白杆菌.此前并无苍白杆菌属成员降解多环芳烃的报道.这是首例苍白杆菌属成员降解多环芳烃的报道,对今后在环境污染防治中开发利用此类细菌具有指导意义.     

菲降解菌的筛选鉴定及其降解酶基因的研究

经过富集从活性污泥中筛选出两株菲降解菌WSCII和WSCIII,经16SrDNA鉴定,它们分别属于鞘氨醇单胞菌属(Sphingomonassp. )和假单胞菌属(Pseudomonassp. ),在28℃下4d内对菲( 0. 6g/L)的降解率分别达到了70. 9%和78. 1%;在LB、萘和菲为碳源的培养条件下,测得菌体的双加氧酶比活力不同.在菲的诱导下,两株菌的双加氧酶比活力最高,而以LB和萘为碳源时酶的比活力较低,其中WSCII在萘中不能生长;这表明该酶在两菌中皆为底物诱导表达.以其它菌的萘双加氧酶基因大亚基片段做模板制备异源探针,与两菌总DNA进行斑点杂交;结果表明,这两株菌可能含有不同的菲双加氧酶基因.利用简并引物进行PCR扩增菲双加氧酶,结果仅能从WSCIII的总DNA中扩增得到目的片段,序列分析证明,该基因与已报道的(P. putidaNCIB9816 -4, AF491307 )双加氧酶同源性最高为98%. 图5参10     

假单胞菌表达载体pYMB03的构建与性能分析

恶臭假单胞菌(Pseudomonas putida)是具有强抗逆性能的环境优势菌,构建其质粒表达载体有着明显的应用潜力.将恶臭假单胞菌AB92019菌株中肽聚糖相关脂蛋白编码基因的启动子PoprL和质粒载体pTrcHis-B的多克隆位点片段插入到质粒载体pUCPl8的EcoRL/HInIII位点,获得了重组载体pYMB03.用绿色荧光基因gfp作为标记进行外源蛋白表达的结果表明,该载体能分别在恶臭假单胞菌AB92019菌株和大肠杆菌DH5a菌株中,由启动子PoprL启动组成型表达GFP蛋白并使细胞产生可见荧光.经SDS-PAGE验证,所产生的GFP蛋白分别占细胞总蛋白的12.5%和5.O%.重组菌株YMB001中GFP表达量与菌体培养时间有关,在稳定期后期其相对荧光强度达到最大值(D600nm=l.0),但与培养温度未见相父性.对携带该载体的2株重组恶臭假单胞菌7次168 h继代培养测定,载体pYMB03的稳定性均为100%.     

苏云金芽胞杆菌菌株WB9编码活性因子的基因分析

采用PCR—RFLP及PCR技术对苏云金芽胞杆菌新分离菌株WB9的ICP、VIP、几丁质酶和肠毒素4种活性因子的编码基因进行了分析．结果表明，该菌株含有编码ICP的cry1Aa、cry1Ab、cry1Cb、cry1Fa和cry1GaS个cry1基因型，编码Vip3A蛋白的vip3A基因以及编码3种肠毒素的hblA、bceT和entS基因；不含编码几丁质酶的基因．将vip3A、hblA、bceT和entS基因PCR产物回收纯化后直接测序，经在线的BLAST软件进行同源性分析，前两个基因片段与Bt已公布基因序列的相应片段同源性分别为99％和96％～98％，bceT基因片段与蜡质芽孢杆菌bceT相应片段同源性为97％．     

34株假单胞菌的泛基因组分析

假单胞菌属在各种环境广泛分布且具有重要的生态应用价值,然而当前研究对其环境适应性遗传基础的解析尚不充分.选择33株有效发表且具有全基因组序列以及一株新分离的假单胞菌的全基因组进行泛基因组学分析.结果表明,选择的假单胞菌属菌株泛基因组含有173 271条蛋白序列,共有30 847个同源基因家族,包括1 505个核心基因,基因组呈开放型.通过分析不同基因组的基因组演化特征,发现通过基因水平转移获得的基因占整个基因组的比例为32%-48%,其平均基因岛数量为53个.发现假单胞菌属泛基因组的开发性与其频繁的基因水平转移事件和快速的基因进化有关.进一步分析表明,不同基因组中的功能基因家族按照菌株的环境分布进行聚类,表明对特殊环境的适应决定了假单胞菌的遗传特征.本研究初步揭示了假单胞菌属细菌遗传特征的共性与特性,结果可为解析假单胞菌属环境适应性的遗传基础与功能多样性的演化发生机制提供研究线索.(图8表1参37)     

质粒pACYC184电转化松材线虫携带的荧光假单胞菌GcM5－1A

以质粒pACYC184为材料,研究了松材线虫携带的荧光假单胞菌的最适电转化条件.结果表明,荧光假单胞菌GcM5-1A培养至D600nm为0.5时制备感受态细胞,以300 mmol/L蔗糖溶液为电击缓冲液,质粒DNA终浓度为0.01 ng/μL,在25 μF电容、9 kV/cm电场强度、200 Ω电阻的电击条件下电击一次,可得最大的电转化效率,即5.5×106/μg(DNA).SDS-PAGE分析显示,转化质粒pACYC184的氯霉素抗性基因在受体细胞内得到了表达.本方法的建立为松材线虫携带的荧光假单胞菌的基因导入提供技术支持.为进一步通过基因敲除的方法,选择性地失活特定功能基因,进而确定这些基因在松材线虫病病理进程中的作用奠定基础.图2参16     

紫花苜蓿根际氢氧化细菌的分离与鉴定

利用气体循环培养体系从陕西乾县HUP-豆科植物紫花苜蓿(Medicago sativa)根际土壤中分离获得37株细菌.菌株氧化氢能力测定结果表明,8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌类群;根据其形态特征、培养特征和生理生化特性,鉴定为7个不同属:假单胞菌属(Pseudomonas)、邻单胞菌属(Plesiomonas)、脂肪杆菌属(Pimelobacter)、黄色杆菌属(Xanthobacter)、勒米诺氏菌属(Leminorella)、地杆菌属(Terrabacter)和稀有杆菌属(Rarobacter);其中氧化氢能力最强的优势菌株WMQ-7 16S rDNA序列(GenBank登录号为EU807744)长度为1 451bp,GC含量为53.8%,其核苷酸序列与假单胞菌属同源性高于99%,在系统发育树上位于同一分支,将WMQ-7菌株鉴定为假单胞菌属(Pseudomonas).图2表5参16     

Rapid,recurring, structured survey versus bioblitz for generating biodiversity data and analysis with a multispecies abundance model

A bioblitz inexpensively and quickly generates biodiversity data, but bioblitzes are often conducted with haphazard, unreplicated sampling. Results tend to be taxonomically, geographically, or temporally biased, lack metadata, and consist of lists of observed taxa that do not enable further analyses or correction for imperfect detection. A rapid, recurring, structured survey (RRSS) uses a structured sampling design and temporal and spatial replication to survey randomly selected sites on a conservation property. We participated in a loosely structured bioblitz and a subsequent RRSS at Big Canoe Creek Nature Preserve in Springville (St. Clair County), Alabama (USA) to compare observed richness derived from the 2 survey approaches. The RRSS data structure enabled us to fit models that accounted for imperfect detection to estimate abundances, occupancy probabilities, and habitat associations. The loosely structured bioblitz data could not be used in such models. We present a new integrated multispecies abundance model that we applied to avian RRSS data. Our model extension enables estimation for the community, employs data augmentation to estimate the number of undetected species, and incorporates covariates. The RRSS generated a more comprehensive and less biased list of observed taxonomic richness than the loosely structured bioblitz (e.g., 73 vs. 45 bird species and 104 vs. 63 insect families from the RRSS vs. loosely structured bioblitz, respectively). Models fit to the RRSS data identified seasonal patterns in avian community composition and allowed for estimation of habitat–occupancy relationships for insect taxa. The RRSS protocol has potential for broad transferability as a standardized, quick, and inexpensive way to inventory biodiversity and estimate ecological parameters while providing an outreach opportunity.     

Compliance with and ecosystem function of biodiversity offsets in North American and European freshwaters

Land-use change via human development is a major driver of biodiversity loss. To reduce these impacts, billions of dollars are spent on biodiversity offsets. However, studies evaluating offset project effectiveness that examine components such as the overall compliance and function of projects remain rare. We reviewed 577 offsetting projects in freshwater ecosystems that included the metrics project size, type of aquatic system (e.g., wetland and creek), offsetting measure (e.g., enhancement, restoration, and creation), and an assessment of the projects’ compliance and functional success. Project information was obtained from scientific and government databases and gray literature. Despite considerable investment in offsetting projects, crucial problems persisted. Although compliance and function were related to each other, a high level of compliance did not guarantee a high degree of function. However, large projects relative to area had better function than small projects. Function improved when projects targeted productivity or specific ecosystem features and when multiple complementary management targets were in place. Restorative measures were more likely to achieve targets than creating entirely new ecosystems. Altogether the relationships we found highlight specific ecological processes that may help improve offsetting outcomes.     

A palaeo-hydrogeological model for arsenic contamination in southern and south-east Asia

An argument is presented in which areas of natural arsenic contamination of modern groundwaters throughout Asia have a common origin. Arsenic originally accumulated in oceanic ferro-manganoan sediments of the eastern Palaeo-Tethys. This was further concentrated through oceanic crustal extinction in what later became the south-east Chinese accreted mineralised terrain. Proto-Himalayan uplift of this area created the palaeo-drainage systems of the Ganges – Brahmaputra, Irrawaddy, Mekong, and Red Rivers, with consequent headwater erosion of arsenic-rich sediments. Their downstream deposition as immature and easily redistributed Neogene sandstones, silts, and iron-rich clays has created secondary and tertiary reservoirs of adsorbed and authigenic arsenic, from which the current arsenic-rich groundwaters have evolved. Considering river basins within the above palaeo-hydrogeological framework provides a basis for assessing the risk of arsenic in groundwater basins of south and south-eastern Asia.     

Cost‐effective conservation of an endangered frog under uncertainty

How should managers choose among conservation options when resources are scarce and there is uncertainty regarding the effectiveness of actions? Well‐developed tools exist for prioritizing areas for one‐time and binary actions (e.g., protect vs. not protect), but methods for prioritizing incremental or ongoing actions (such as habitat creation and maintenance) remain uncommon. We devised an approach that combines metapopulation viability and cost‐effectiveness analyses to select among alternative conservation actions while accounting for uncertainty. In our study, cost‐effectiveness is the ratio between the benefit of an action and its economic cost, where benefit is the change in metapopulation viability. We applied the approach to the case of the endangered growling grass frog (Litoria raniformis), which is threatened by urban development. We extended a Bayesian model to predict metapopulation viability under 9 urbanization and management scenarios and incorporated the full probability distribution of possible outcomes for each scenario into the cost‐effectiveness analysis. This allowed us to discern between cost‐effective alternatives that were robust to uncertainty and those with a relatively high risk of failure. We found a relatively high risk of extinction following urbanization if the only action was reservation of core habitat; habitat creation actions performed better than enhancement actions; and cost‐effectiveness ranking changed depending on the consideration of uncertainty. Our results suggest that creation and maintenance of wetlands dedicated to L. raniformis is the only cost‐effective action likely to result in a sufficiently low risk of extinction. To our knowledge we are the first study to use Bayesian metapopulation viability analysis to explicitly incorporate parametric and demographic uncertainty into a cost‐effective evaluation of conservation actions. The approach offers guidance to decision makers aiming to achieve cost‐effective conservation under uncertainty.     

Parasitic wasps orient to green leaf volatiles

Summary Undamaged plants emit low levels of green leaf volatiles (GLVs), while caterpillar-damaged and artificially damaged plants emit relatively higher levels of certain GLVs. Female braconid parasitoids,Microplitis croceipes, oriented to both damaged plants and to individual GLVs in no-choice tests in a wind tunnel, but seldom oriented to undamaged plants. Female ichneumonid parasitoids,Netelia heroica, also oriented to individual GLVs in a wind tunnel. Males of both wasp species failed to orient to the GLVs. These data show that leaf-feeding caterpillars can cause the release of GLVs, and that parasitic wasps can respond to these odors by flying upwind (chemoanemotactic response), which brings the wasps to their caterpillar hosts. This supports the hypothesis that plants communicate with members of the third trophic level,i.e., plants under herbivore attack emit chemical signals that guide natural enemies of herbivores to sites of plant damage. In this interaction, the GLVs serve as tritrophic plant-to-parasitoid synomones. That parasitoids from two different wasp families oriented to GLVs suggests that the response may be widespread among the Hymenoptera.Mention of a commercial or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture     

Potential for Electropositive Metal to Reduce the Interactions of Atlantic Sturgeon with Fishing Gear

Atlantic sturgeon (Acipenser oxyrhynchus) populations have been declared either endangered or threatened under the U.S. Endangered Species Act. Effective measures to repel sturgeon from fishing gear would be beneficial to both fish and fishers because they could reduce both fishery‐associated mortality and the need for seasonal and area closures of specific fisheries. Some chondrostean fishes (e.g., sturgeons and paddlefishes) can detect weak electric field gradients (possibly as low as 5 Μv/cm) due to arrays of electroreceptors (ampullae of Lorenzini) on their snout and gill covers. Weak electric fields, such as those produced by electropositive metals (typically mixtures of the lanthanide elements), could therefore potentially be used as a deterrent. To test this idea, we recorded the behavioral responses of juvenile Atlantic sturgeon (31–43 cm fork length) to electropositive metal (primarily a mixture of the lanthanide elements neodymium and praseodymium) both in the presence and absence of food stimuli. Trials were conducted in an approximately 2.5 m diameter × 0.3 m deep tank, and fish behaviors were recorded with an overhead digital video camera. Video records were subsequently digitized (x, y coordinate system), the distance between the fish and the electropositive metal calculated, and data summarized by compiling frequency distributions with 5‐cm bins. Juvenile sturgeon showed clear avoidance of electropositive metal but only when food was present. On the basis of our results, we conclude that the electropositive metals, or other sources of weak electric fields, may eventually be used to reduce the interactions of Atlantic sturgeon with fishing gear, but further investigation is needed. El Potencial del Metal Electropositivo para Reducir las Interacciones del Esturión Atlántico con Instrumentos de Pesca Bouyoucos, Bushnell & Brill 13–003     

The role of time and species identities in spatial patterns of species richness and conservation

Many conservation actions are justified on the basis of managing biodiversity. Biodiversity, in terms of species richness, is largely the product of rare species. This is problematic because the intensity of sampling needed to characterize communities and patterns of rarity or to justify the use of surrogates has biased sampling in favor of space over time. However, environmental fluctuations interacting with community dynamics lead to temporal variations in where and when species occur, potentially affecting conservation planning by generating uncertainty about results of species distribution modeling (including range determinations), selection of surrogates for biodiversity, and the proportion of biodiversity composed of rare species. To have confidence in the evidence base for conservation actions, one must consider whether temporal replication is necessary to produce broad inferences. Using approximately 20 years of macrofaunal data from tidal flats in 2 harbors, we explored variation in the identity of rare, common, restricted range, and widespread species over time and space. Over time, rare taxa were more likely to increase in abundance or occurrence than to remain rare or disappear and to exhibit temporal patterns in their occurrence. Space–time congruency in ranges (i.e., spatially widespread taxa were also temporally widespread) was observed only where samples were collected across an environmental gradient. Fifteen percent of the taxa in both harbors changed over time from having spatially restricted ranges to having widespread ranges. Our findings suggest that rare species can provide stability against environmental change, because the majority of species were not random transients, but that selection of biodiversity surrogates requires temporal validation. Rarity needs to be considered both spatially and temporally, as species that occur randomly over time are likely to play a different role in ecosystem functioning than those exhibiting temporal structure (e.g., seasonality). Moreover, temporal structure offers the opportunity to place management and conservation activities within windows of maximum opportunity.     

Main nutrient compounds in food bodies of Mexican <Emphasis Type="Italic">Acacia</Emphasis> ant-plants

Summary. Myrmecophytic plants use obligate ant mutualists as a constitutive indirect defence mechanism. These plants often produce cellular food bodies (FBs) to nourish their resident ants. Lipids, proteins, and even highly specialised compounds such as glycogen have been reported from FBs, but detailed chemical analyses of FB composition have so far been presented only for Southeast Asian Macaranga and Central American Piper myrmecophytes. Here we report the chemical composition of FBs of five myrmecophytic Acacia (Fabaceae) species from Mexico using HPLC (carbohydrates and proteins) and GC-MS (lipids). Feeding experiments revealed no hints on any use of external food sources by the inhabiting Pseudomyrmex ants. These ants obviously rely completely on FBs and extrafloral nectar provided by their hosts. The total content of nutrients in Acacia FBs was 15-25% of FB dry mass, being much lower than in Macaranga or Piper FBs. Proteins were dominating (8-14 % dm) in Acacia FBs and thus were present in higher amounts than in Macaranga FBs, yet in lower amounts than in Piper. Lipids contributed 1-9 % of dry mass, showing a lower proportion than in FBs of Macaranga or Piper. Carbohydrates made up 3-11 % dm, reaching in most Acacia species the same range as observed in Macaranga and in Piper FBs. Water content was 18-24 % of FB fresh mass, and structural tissue obviously made up a much higher proportion in Acacia FBs than in Macaranga or Piper FBs. Both characters might represent an adaptation to producing FBs unprotected at the leaf tips under dry conditions. Acacia FBs contain all amino acids and all fatty acids that are considered essential for insects, and their contents of lipids and proteins are higher than in the leaves from which they are ontogenetically derived. This indicates a putatively adaptive enrichment of nutritionally valuable compounds in structures functioning as ant-food.     

Examining the uncertain origin and management role of martens on Prince of Wales Island,Alaska

Conservation biologists are generally united in efforts to curtail the spread of non‐native species globally. However, the colonization history of a species is not always certain, and whether a species is considered non‐native or native depends on the conservation benchmark. Such ambiguities have led to inconsistent management. Within the Tongass National Forest of Alaska, the status of American marten (Martes americana) on the largest, most biologically diverse and deforested island, Prince of Wales (POW), is unclear. Ten martens were released to POW in the early 1930s, and it was generally believed to be the founding event, although this has been questioned. The uncertainty surrounding when and how martens colonized POW complicates management, especially because martens were selected as a design species for the Tongass. To explore the history of martens of POW we reviewed other plausible routes of colonization; genetically and isotopically analyzed putative marten fossils deposited in the late Pleistocene and early Holocene to verify marten occupancy of POW; and used contemporary genetic data from martens on POW and the mainland in coalescent simulations to identify the probable source of the present‐day marten population on POW. We found evidence for multiple routes of colonization by forest‐associated mammals beginning in the Holocene, which were likely used by American martens to naturally colonize POW. Although we cannot rule out human‐assisted movement of martens by Alaskan Natives or fur trappers, we suggest that martens be managed for persistence on POW. More generally, our findings illustrate the difficulty of labeling species as non‐native or native, even when genetic and paleo‐ecological data are available, and support the notion that community resilience or species invasiveness should be prioritized when making management decisions rather than more subjective and less certain conservation benchmarks.