烟草甲细胞色素P450还原酶基因的分子特征与表达模式分析

烟草甲Lasioderma serricorne是一种重要的仓储害虫,长期化学防治导致烟草甲已对多种传统熏蒸剂产生抗性,但其对新型熏蒸剂甲酸乙酯仍处于敏感水平。因此明确其体内细胞色素P450还原酶（cytochrome P450 reductase, CPR）对甲酸乙酯的代谢解毒功能,对开展该药剂的抗性监测及延缓抗性的发生发展具有重要意义。本研究旨在克隆烟草甲LsCPR基因,分析其分子特征和表达特性,为进一步明确其在烟草甲对甲酸乙酯解毒代谢过程中的作用奠定基础。利用RT-PCR技术克隆烟草甲LsCPR基因的开放阅读框（open reading frame, ORF）;利用生物信息学软件分析LsCPR编码蛋白的结构、特征和系统进化关系。采用实时定量PCR技术检测LsCPR在烟草甲不同发育阶段（低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫）、幼虫不同组织（表皮、肠道、脂肪体和马氏管）以及甲酸乙酯熏蒸胁迫后的表达模式。烟草甲LsCPR基因的ORF为2 046 bp（GenBank登录号：MZ423209）,编码681个氨基酸,具有典型的昆虫CPR基因FMN区域、NADPH区域和FAD等保守结构域。系统发育分析表明,烟草甲LsCPR与鞘翅目昆虫聚为一支,且与赤拟谷盗Tribolium castaneum CPR亲缘关系最近。LsCPR在烟草甲不同发育阶段均有表达,在高龄幼虫期的表达水平较高;在幼虫体内的表达部位主要在肠道,其次为脂肪体和马氏管,而在表皮的表达水平最低。LC10、LC30和LC50 3种浓度的甲酸乙酯处理24 h后,烟草甲LsCPR表达量随着胁迫浓度升高而上调且显著高于对照;甲酸乙酯LC50处理烟草甲不同时间（3、6、12、24和48 h）后,LsCPR基因上调表达,24 h时达到表达高峰。推测LsCPR是参与烟草甲代谢甲酸乙酯的候选基因。     

烟草甲谷胱甘肽S-转移酶基因LsGSTe1的表达及其与甲酸乙酯耐受性的关系

【目的】探究烟草甲Lasioderma serricorne谷胱甘肽S-转移酶(glutathione S-transferase, GST)基因LsGSTe1的分子特性和生物学功能。【方法】在烟草甲转录组数据的基础上,利用RT-PCR技术扩增LsGSTe1基因,并进行生物信息学分析;采用qPCR技术检测LsGSTe1在烟草甲不同发育阶段(低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫)及高龄幼虫不同组织(表皮、中肠、脂肪体、马氏管)中的表达水平,以及在甲酸乙酯熏蒸胁迫后的5龄幼虫中的表达变化。进一步采用RNAi技术沉默烟草甲5龄幼虫LsGSTe1基因,通过生物测定分析烟草甲对熏蒸剂甲酸乙酯的敏感性变化。【结果】获得LsGSTe1基因的全长cDNA序列(GenBank登录号:MN480468),开放阅读框长684 bp,编码227个氨基酸,N端和C端均存在催化保守位点。系统发育分析表明该基因属于GSTs的Epsilon家族。qPCR结果表明,LsGSTe1在烟草甲不同发育阶段均有表达,且在高龄幼虫期的表达量较高;表达部位主要在幼虫脂肪体,其次为中肠和表皮,而在马氏管中的表达量最低。LC     

Sterols in Laodelphax striatellus with special reference to the intracellular yeastlike symbiotes as a sterol source

Sterols were analysed to investigate the sterol source in Laodelphax striatellus and three other rice plant-sucking homopterous insects. In L. striatellus, cholesterol, 24-methylenecholesterol and β-sitosterol were detected. The host plant (rice) contained campesterol, stigmasterol and β-sitosterol. From the honeydew excreted by L. striatellus, cholesterol, β-sitosterol and negligible amounts of campesterol were recovered. Laodelphax striatellus possesses yeastlike symbiotes which can be destroyed by high temperature. Fifth instar nymphs, which have been exposed to 35°C for 3 days in their 1 st instar, showed lower cholesterol concentration and markedly reduced amounts of 24-methylenecholesterol. From the results it is concluded that L. striatellus has two sterol sources: one from the host plant and the other from the yeastlike symbiotes which appear to provide 24-methylenecholesterol.     

烟草甲clip丝氨酸蛋白酶基因的克隆及其对外源激素和免疫胁迫的表达响应(英文)

【目的】作为细胞外信号级联通路的重要组成部分,含有clip结构域的丝氨酸蛋白酶(clip-domain serine proteases, CLIPs)在昆虫发育和先天免疫过程中起着重要作用。本研究旨在克隆烟草甲Lasioderma serricorne CLIP基因,解析其在烟草甲不同发育阶段和幼虫不同组织中的表达模式,分析其在外源激素20-羟基蜕皮酮(20E)和免疫胁迫后的表达特征,为进一步研究其生理功能奠定基础。【方法】采用RT-PCR技术克隆获得烟草甲两个CLIPs基因(LsCLIP1和LsCLIP2)全长cDNA序列,并利用生物信息学软件预测其编码蛋白的结构和特征,利用MEGA 6.06构建昆虫CLIPs系统发育树;利用实时荧光定量PCR(quantitative real-time PCR, qPCR)研究这两个基因在不同发育阶段［低龄幼虫(卵孵化后24 h内)、高龄幼虫(4龄以上)、蛹(化蛹后48 h以上)、早期成虫(化蛹后24 h内)和晚期成虫(化蛹后7 d)］、5龄幼虫不同组织(表皮、脂肪体、肠道和剩余组织)中以及注射20E(120 ng/幼虫)和来源于大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus的肽聚糖(0.2 μL)后4龄幼虫中的表达模式。【结果】克隆获得烟草甲LsCLIP1和LsCLIP2基因的cDNA全序列,其开放阅读框长度均为1 194 bp,编码397个氨基酸。序列分析显示,其氨基酸序列各自具有一个clip结构域和胰蛋白酶结构域。系统发育分析表明,CLIP1和CLIP2都属于subfamily C CLIPs。qPCR结果表明,LsCLIP1和LsCLIP2基因在所检测的各发育阶段和幼虫各组织中均有表达,分别尤以蛹期和表皮中表达量最高;经20E和肽聚糖诱导后,烟草甲幼虫体内LsCLIP1和LsCLIP2基因的表达量明显提高。【结论】推测LsCLIP1和LsCLIP2可能参与了烟草甲的蜕皮发育和对免疫胁迫的应激响应。本研究将为后续研究昆虫CLIPs的分子调控提供参考。     

Complete mitochondrial genome of a carabid beetle,Damaster mirabilissimus mirabilissim (Coleoptera: Carabidae)

In the present study, we report the 16 823‐bp long complete mitochondrial genome (mitogenome) of a carabid beetle, Damaster mirabilissimus mirabilissim (Coleoptera: Carabidae), which is endangered in Korea. The gene arrangement of D. m. mirabilissim mitogenome is identical to the most common type found in insects. The start codon of the D. m. mirabilissim COI gene is a typical ATN codon. On the other hand, the initiation codon for ND1 gene is TTG, instead of ATN. All transfer RNAs (tRNAs) exhibit a stable canonical clover‐leaf structure, except for tRNA^Ser(AGN), the dihydrouridine arm of which forms a simple loop. The 1703‐bp long A+T‐rich region is the second longest among the complete adephagan mitogenome sequences, next to Macrogyrus oblongus belonging to Gyrinoidea. One of the unusual features of the genome is the presence of a tRNA^Leu(UUR)‐like sequence in the A+T‐rich region. This sequence displays the proper anticodon sequence and the potential to form secondary structures, but also harbors many mismatches in the stems.     

Freshwater population of Ulva flexuosa (Ulvaceae,Chlorophyta) as a food source for great pond snail: Lymnaea stagnalis (Mollusca,Lymnaeidae)

Cosmopolitan species of the genus Ulva (Ulvaceae; Chlorophyta) that populate the littoral zone of marine habitats constitute a staple diet for a variety of organisms, particularly snails, shellfish, polychaetes, and birds. Occurrence of Ulva species (e.g., U. flexuosa and U . prolifera) has also been observed in freshwater inland ecosystems that have no contact with saline water. However, the influence of the development of macroalgal mats of Ulva on indigenous organisms in limnic ecosystems has not been established. This study investigates the trophic relationships between Ulva flexuosa and one species of snail from freshwater habitats in central Europe. During the summer, the great pond snail (Lymnaea stagnalis) consumed Ulva as a source of nutrition even when other algae and plants were available. Lymnaea stagnalis consumed an average of 100 mg of Ulva thalli per day. This level of biomass exceeded the consumption of an alternative food source, the shoots of Elodea canadensis. Ulva thalli are more actively consumed by great pond snails than Elodea shoots, and this is expressed in terms of the differences of biomass consumption. It was also observed that the interior of the monostromatic tubular thalli of Ulva flexuosa serves as a protective shelter for juvenile great pond snails.     

Nonlethal sampling of DNA from critically endangered diving beetles (Coleoptera: Dytiscidae) using a single antenna

Nonlethal DNA sampling is highly desirable in molecular genetic studies of protected and endangered species. To develop a demonstrably nonlethal method of obtaining DNA from endangered diving beetles (Dytiscus sharpi sharpi Wehncke, Cybister lewisianus Sharp and Cybister brevis Aubé), we amputated the antennae of these endangered diving beetles and investigated the impact of the amputation on reproductive behaviors, egg‐laying and lifespan. Diving beetles with either one or no antennae copulated without delay and laid eggs, comparable to the pairs of intact beetles under breeding conditions. The lifespan of antennae‐amputated D. sharpi sharpi was the same as that of the intact beetles. A single antenna was sufficient to allow polymerase chain reaction (PCR) detection of a mitochondrial DNA gene, cytochrome‐c oxidase subunit I (COI), and the sequence of the COI gene could be determined directly. The PCR‐ready genomic DNA was available both in fresh antennae isolated from living beetles and in old antennae from whole beetles preserved for at least 5–6 years in pure ethanol. These results suggest that an antenna is a good sampling site for isolating genomic DNA from endangered diving beetles without sacrificing and disturbing reproductive behaviors such as mating and egg‐laying, or lifespan.     

Longhorn beetles and predatory clerid beetles attracted to a blend of longhorn beetle pheromone compounds in a Central European oak forest (Coleoptera: Cerambycidae,Cleridae)

1. A blend of longhorn beetle pheromones was tested as a generic attractant in a Central European oak forest. Overall, 20 cerambycid species totalling 1250 specimens were captured using two trap types.
2. More adults of Phymatodes testaceus and Leiopus nebulosus nebulosus were attracted to pheromone-baited traps compared to solvent controls. Significant numbers of four other species were caught by panel traps but not funnel traps. For the cerambycine Pyrrhidium sanguineum, significantly more beetles were caught in treatment traps than controls. For the cerambycine Anaglyptus mysticus, lepturines Cortodera humeralis and Rhagium sycophanta, the numbers of beetles caught in treatment or control traps were similar, indicating no attraction to the lure blend. Adults of a predatory clerid beetle Clerus mutillarius were caught in significantly larger numbers by both baited trap types in comparison to controls, totalling 1514 specimens.
3. Antennae of both sexes of C. mutillarius showed responses to 3-hydroxyhexan-2-one and 2-methylbutan-1-ol, and males also responded to syn-2,3-hexanediol.
4. Strong attraction of C. mutillarius suggests eavesdropping on the pheromones of cerambycids and that such attractant baits, without traps, might be used to manipulate the local population density of predators in a push-pull biological control program.

     

Ground beetles (Coleoptera,Carabidae) as indicators of change and pattern in the agroecosystem: Longer surveys improve understanding

Investigations concerned with ground beetle (Carabidae) dynamics in the agroecosystem have generally been limited to relatively short time periods in one crop, commonly wheat, whilst other studies have concentrated on the influence of landscape structure and non-crop habitat on beetle activity, again usually in one crop. Results of a seven-year survey at Nafferton in northern England, with recording from all crops in an organic rotation and from three types of field boundary, indicated that the lack of field boundary management had the greatest influence on ground beetle activity/density in this particular agroecosystem. However, within the crop rotation, grass/clover limited activity/density compared with that in cereal, potato and bean crops and there were also differences within cereal crops. Data from a 17-year survey at Drayton Environmental Change Network (ECN) site in midland England indicate that previous understanding of ground beetle activity and distribution in intensively managed landscapes may have been limited by temporal constraints. At Drayton, changes in surrounding crop cover, with the introduction of willow coppice and reduced area of arable land and agricultural inputs, considerably influenced ground beetle activity/density and assemblages in non-crop habitat. Small, active species dominating the assemblage in the first few years of the Drayton survey were gradually replaced by larger, non-flying species, especially after all the willow coppice had been planted. In both surveys, activity/density of ground beetles was related to disturbance, affecting vegetation cover and structure, but at the field scale at Nafferton and at the farm scale at Drayton. Longer surveys in a variety of landscapes such as those by the United Kingdom Environmental Change Network are likely to improve understanding of activity, diversity and distribution of invertebrates, which are fundamental requirements if predators such as ground beetles are required for ecosystem service provision.     

Cork-oak woodlands as key-habitats for biodiversity conservation in Mediterranean landscapes: a case study using rove and ground beetles (Coleoptera: Staphylinidae, Carabidae)

Land-use intensification in Mediterranean agro-forest systems became a pressure on biodiversity, concerning particularly the woodland sensitive species. In 2001, the effects of a land-use gradient from old-growth cork-oak forest to a homogeneous agricultural area were assessed using rove beetles as indicators in a Mediterranean landscape. The aim was to find which species were negatively affected by land-use intensification at the landscape level and whether they benefited from cork-oak patches occurring along the land-use gradient. A total of 3,196 rove beetles from 88 taxa were sampled from all landscape types. Agricultural area recorded significantly higher numbers of abundance and species richness in relation to the cork-oak mosaics, i.e. the old-growth forest and the managed agro-forest landscapes (montados). Moreover, 70% of rove beetle indicator species common enough to be tested by IndVal displayed their highest indicator value for agriculture, showing a lower number of woodland indicators in comparison to ground beetles. Nevertheless, one rove beetle taxon was considered a specialist of closed woodland mosaics while no specialist ground beetle was found for that landscape typology. Some rare rove beetle species were also important in typifying diversity patterns of old-growth cork-oak forests. Hence, future management in Mediterranean landscapes should take into account not only indicator species common enough to be tested by IndVal, but also rare and endemic species. Considering the added value of cork-oak woodland cover for sensitive rove and ground beetle diversity, the strengthening of cork-oak woodland connectivity seems to be a crucial management that is required in agricultural Mediterranean landscapes.     

Fungal community composition in the gut of rove beetles (Coleoptera: Staphylinidae) from the Canadian boreal forest reveals possible endosymbiotic interactions for dietary needs

The goal of this study was to investigate the fungal community composition in the gut of Staphylinidae from boreal forest in order to better understand the diversity and the complexity of fungus-insect relationships. DNA gut content analyses of nine abundant rove beetle species (Coleoptera, Staphylinidae) living in the boreal balsam fir forest ecosystem (Montmorency Forest, Quebec, Canada) were performed to identify the fungal taxa present either as endosymbiotic taxa or as a source of nutrition. A total of 42 fungal operational taxonomic units (OTUs) were recorded from the analysis of 441 fungal ITS rDNA sequences recovered from gut extracts. The OTU richness per species ranged between four in Tachinus quebecensis and 16 in Atheta ventricosa. The fungal mycobiota in posterior gut extracts was dominated by Saccharomycetales (12 OTUs), followed by Sordariomycetes (nine OTUs). No significant difference was observed between the OTU richness recorded within each of the three subfamilies of rove beetles investigated. The core mycobiome of the posterior gut extracts was dominated by three OTUs related to yeasts, with ITS sequences having pairwise similarities equal to or greater than 99% with Candida mesenterica, Debaryomyces spp. and Ophiostoma pluriannulatum. These results provide some evidence of the consumer-resource relationships of these beetles. Predominance of yeast and fungal spores in the posterior gut of rove beetles suggests that they may play an important role in their dietary requirements and as endosymbionts.     

A new genus and a new subgenus of cavernicolous beetles from Furong Jiang valley,southwestern China (Coleoptera: Carabidae: Trechinae)

A new genus and a new subgenus of semi-aphaenopsian trechine beetles are described from Furong Jiang valley in the bordering areas of Chongqing Shi (Municipality) and Guizhou Province, southwestern China: Deuveaphaenops Tian & Huang, n. gen. (type species: Deuveaphaenops qimenxicus Tian & Huang, n. sp. from Qimenxia Cave II, Wulong Xian, Chongqing) and Furongius Tian & Huang, n. subgen. (type species: Deuveaphaenops (Furongius) gelaophilus Tian & Huang, n. sp. from cave Xianmi Dong, Daozhen Xian, Guizhou). Deuveaphaenops probably belongs to the complex of Cathaiaphaenops, Qianotrechus, Guizhaphaenops and Sinotroglodytes.     

Patterns in the distribution, abundance and body size of carabid beetles (Coleoptera: Caraboidea) in relation to dispersal ability

The effects of dispersal ability, measured as two wing size categories (brachypterous vs. macropterous), on the distribution, abundance and body size, and on the relationships between these variables were examined in eighty-four species of carabid beetles over twenty-two sites in the northern Iberian peninsula. Geographic ranges of species (restricted to the northern Iberian peninsula vs. widespread—European or wider range) were also taken into account in the analyses because macropterous species significantly tended to exhibit wider geographic ranges than did brachypterous species. Regional distributions were wider in brachypterous-restricted and brachypterous-widespread species than in macropterous-widespread species. The three groups did not differ in abundance. Differences in regional distributions between groups may be explained by referring to a trade-off between dispersal ability and establishment ability indicated in the literature. Macropterous species would occupy relatively few sites due to a high frequency of unsuccessful colonizations. The relationships between regional distribution and abundance were positive for all the three groups, brachypterous-restricted, brachypterous-widespread and macropterous-widespread species. The regression line for the last group showed a lower elevation than those for brachypterous-restricted and brachypterous-widespread species. This fact was probably due to differences in regional distributions between groups. No relationship between abundance and body size was significant. Regressions of regional distribution on body size were positive in brachypterous-restricted and brachypterous-widespread carabids, but the relationship was not significant in macropterous-widespread carabids. These results were interpreted in terms of differences in body size–dependency of travelling velocities between flying and running carabids.     

印度加尔各答市区外围一地的露尾甲物种组成、多度和季节性发生(英文)

【目的】印度大部分露尾甲在腐烂的水果和蔬菜上大量发生,其种群在一年中表现出明显的季节性波动。据推测,露尾甲种群很大程度上依赖于温度、湿度和降雨之类的环境因子。【方法】本研究调查了2013-2015年印度加尔各答市区外围一地Garia的露尾甲物种组成、季节性发生和种群结构,记录了其活跃时期、季节性多度和影响其发生的因素。【结果】调查期间在调查地共发现数目不等的6个物种。其中最常见露尾甲为Urophorus humeralis,它是个体数量最多的物种且在一年中几乎所有月份均有发生;其他常见物种为Epuraea ocularis和E. luteola。不同物种在食物发酵的连续阶段进入诱捕器中。最初12 h被捕获的是Epuraea 属的种类,而在诱捕器中食物严重腐烂的后续阶段发现最多的是U. humeralis。在合适范围的气温(22~29℃)和相对湿度(82.5%~86%)下,物种丰富度最高,表明这些环境变量对露尾甲种群具有重要影响。【结论】加尔各答主要水果和蔬菜在季风后季节种植,在季风后季节取食这些作物的露尾甲发生量(物种丰富度和多度)最高。这一研究结果可能有助于制定针对这些甲虫的有效田间治理策略。     

Genetic variation in a phylogenetic context: Responses of two specialized leaf beetles (Coleoptera: Chrysomelidae) to host plants of their congeners

This work explores the possibility that constraints on genetic variation guide host shifts and are responsible for the evolutionary conservatism of host affiliation in phytophagous insects. To this end, we used full- and half-sib breeding designs to screen two species of the North American bettle genus Ophraella for genetic variation in larval and adult feeding responses to several host plants of other species of Ophraella. All the plants are in the family Asteraceae. In O. conferta, we observed effectively no feeding response, and hence no genetic variation in response, to three of five test plant species; only those plants related to the species' natural hosts evoked genetically variable responses. In O. artemisiae, adults displayed genetic variation in response to a congener of the natural host, but not to two distantly related plants. However, significant variation among full-sib broods in larval feeding suggests the existence of nonadditive genetic variance in feeding response to all five species of test plants—although survival was very low on most of them. The results suggest that patterns of presence versus apparent absence of detectable genetic variation may be related to the chemical similarity of plants to the insects' natural hosts, but not evidently to the phylogenetic history of host affiliation within the genus. Almost all genetic correlations in responses to host plants were not significantly different from zero; the few significant correlations were positive, and negative correlations that might explain host specificity were not found. Our data do not explain why exclusive shifts to new hosts should occur, but the apparent lack of genetic variation in responses to some plants suggests that the direction of host shifts is genetically constrained.     

Paroster extraordinarius sp. nov., a new groundwater diving beetle from the Flinders Ranges, with notes on other diving beetles from gravels in South Australia (Coleoptera: Dytiscidae)

The first groundwater (stygobitic) diving beetle is reported from South Australia. Paroster extraordinarius sp. nov. (Dytiscidae: Hydroporini) is described and figured. Its morphology and mitochondrial DNA sequences place it in the hydroporine genus Paroster Sharp. Phylogenetic analysis shows that the new species is imbedded in a clade of stygobitic species from the Yilgarn area of Western Australia. The evolution of this species is discussed and compared with observations on the behaviour and distribution of other dytiscid beetles found in subterranean habitats in South Australia.     

A survey of ground beetles (Coleoptera: Carabidae) in Ahuriri Scenic Reserve,Banks Peninsula,and comparisons with a previous survey performed 30 years earlier

Carabidae (Coleoptera) are commonly used as indicators of ecosystem health due to their high diversity, conservation value and relative ease of identification. Carabids were monitored at Ahuriri Scenic Reserve, Port Hills, Canterbury, from August 2007 to September 2008 using pitfall traps. Carabid captures, species richness and distribution across the reserve were compared with those found in a similar study in 1977–1978. Monthly carabid captures were highly correlated with average temperature in both studies. There was little evidence of carabids responding to habitat factors, such as leaf litter depth, pH or ground cover. However, Megadromus antarcticus was positively associated with canopy openness and ground cover, whereas Holcaspis angustula was more abundant where canopy cover was extensive and there was a relatively high incidence of bare rock. Thirteen species of carabid were recorded in 1977–1978, of which six were absent, four had decreased, one was stable and two species increased in abundance from collections made in 2007–2008. The loss of one Banks Peninsula and five New Zealand endemic beetle species from this reserve is of concern, and future work should aim to gain more detailed information on the habitat requirements of these species to assist conservation management strategies.     

Alphitobius diaperinus Panzer (Insecta,Coleoptera) in a single house of a broiler production facility as a potential source of pathogenic bacteria for broilers and humans

Pest infestation in any stage can lead to a quality reduction in the finished products. This study aimed to detect Escherichia coli, Salmonella spp., Campylobacter spp., and Staphylococcus aureus in Alphitobius diaperinus adults, and in samples from broiler swabs, administered water and feed collected in a single house from a broiler production facility in central Italy. Three samplings were carried out, each collecting ninety adult beetles for microbial detection in the external, faecal and internal content; ten cloacal swab samples; and one sample of both administered feed and water. Microbiological cultures and biochemical identification were performed on suspected cultures and confirmed by species-specific PCRs. A. diaperinus was abundantly found near the windows, under the manger and in the corners of the facility. Salmonella enterica serovar Cholerasuis was found at the external surface of the beetles, while Staphylococcus xylosus and E. coli were in the faecal content. The latter micro-organism together with Staphylococcus lentus, S. xylosus and other staphylococcal species were detected in the internal microbiota. E. coli and Campylobacter spp. were observed in cloacal swabs, and S. xylosus in one feed sample. The study findings support evidence for Salmonella spp. and E. coli, and remark that adherence to sanitation rules and biosecurity procedures are required.