高温胁迫对筛豆龟蝽成虫抗氧化能力的影响

为探索筛豆龟蝽Megacopta cribraria (Fabricius)对高温的耐受性及其体内抗氧化系统对极端高温胁迫的响应机制.以25℃为对照温度,经高温37℃、40℃、43℃和46℃胁迫处理4h后,测定筛豆龟蝽成虫存活率及其体内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性以及总抗氧化(T-AOC)能力和丙二醛(MDA)含量的变化.在37℃～46℃高温胁迫下,筛豆龟蝽成虫存活率与对照无显著差异,其体内SOD和CAT活性较对照组均显著增加,其中,SOD活性在37℃最高,随着温度进一步升高,其活性有所下降,但仍显著高于对照.CAT一直保持较高活性,随着处理温度的升高CAT活性呈逐渐升高趋势,在极端高温46℃时其活性最高.POD活性在37℃～ 40℃显著高于对照,43℃和46℃处理与对照无显著差异.T-AOC活性在各处理温度下均显著高于对照,且随着处理温度的升高T-AOC活性呈先升高后降低趋势.各处理MDA含量与对照无显著差异.筛豆龟蝽体内3种抗氧化酶的活力、总抗氧化能力和MDA在适应高温胁迫过程中发挥了重要作用.     

Mouthpart morphology and feeding behavior of the invasive kudzu bug,Megacopta cribraria (Hemiptera: Plataspidae)

The invasive kudzu bug, Megacopta cribraria, was first reported in North America in 2009 and has subsequently spread through most of the southeastern United States, causing yield loss in soybean. Since detection in the USA, research has focused mainly on managing this newly established pest, but many important characteristics of the pest's mouthpart morphology and feeding behavior are unknown. Qualitative and quantitative comparisons of nymph and adult mouthparts and sensilla were made through scanning electron microscopy and light microscopy, and feeding behavior was examined using electropenetrography (EPG) and paraffin histology. Morphologies observed were similar to what has previously been reported for other piercing–sucking hemipterans. The relationship between rostrum length and body size (pronotum width and dorsal length) exhibited negative allometry. Rostrum length exhibited an isometric relationship with interocular width. Adult females (n=9) probed soybean stems 1.3±0.8 times in 9 h, with an average probe time of 2.3±1.3 h. EPG waveforms were characterized and correlated with behavior. Salivary sheaths were shown to terminate in the vascular tissue; four of five sheaths terminated in the phloem. This is the first time that the feeding behavior of a member of the Plataspidae has been recorded using EPG. Results add to our current limited knowledge of plataspid mouthpart morphology and provide a baseline for further research on the feeding behaviors of M. cribraria and other soybean‐feeding hemipterans.     

From Asian curiosity to eruptive American pest: Megacopta cribraria (Hemiptera: Plataspidae) and prospects for its biological control

The kudzu bug or bean plataspid, Megacopta cribraria (Fabricius), is native to Asia where it appears to be widely distributed (although the taxonomy is not entirely clear), but is infrequently a pest of legumes. This bug appeared in 2009 in the southeastern United States, where it is closely associated with kudzu, Pueraria montana Lour. [Merr.] variety lobata [Willd.] Maesen & S. Almeida. However, the insect has become a consistent economic pest of soybeans, Glycine max (L.) Merr., and some other leguminous crops in areas where large numbers can build in kudzu, in addition to being a considerable nuisance in urban landscapes where kudzu occurs. The insect has remarkable capacity for movement and has spread rapidly from nine Georgia counties in 2009 to seven states in 2012. Despite being a nuisance in urban areas and a crop pest, high populations of the bug also reduce the biomass of kudzu, which is itself a seriously problematic invasive weed, complicating the status of M. cribraria in its expanded range. Extant predators and a pathogen in the US have been observed attacking kudzu bugs in the laboratory and field, but no parasitism of eggs or nymphs has been observed to date. A single record of parasitism of an adult kudzu bug by a tachinid fly is known from the US, but no other adult parasitism has been observed in the US or elsewhere. Extant enemies may eventually significantly reduce the bug’s populations, but at present native enemies appear to be insufficient for the task, and exotic enemies from the kudzu bug’s native range may offer the best possibility for effective biological control in the US. Based on the available literature, the best option for an importation biological control program appears to be the platygastrid egg parasitoid Paratelenomus saccharalis (Dodd) because of its apparent host specificity, intimate biological linkages with M. cribraria, and wide geographic distribution in the Eastern Hemisphere. Other natural enemies may eventually emerge as good candidates for importation, but at present P. saccharalis appears to be the most promising.     

光照强度对筛豆龟蝽生长发育及繁殖的影响

【目的】为明确光照强度对筛豆龟蝽 Megacopta cribraria 种群生长发育及生殖力的影响,进一步探索筛豆龟蝽对光环境的适应规律。【方法】在温度24±1℃,相对湿度60%±10%,光周期 16L ∶8D 条件下,通过测定寄主植物大豆上筛豆龟蝽在不同光照强度(500, 2 500, 4 500, 6 500, 8 500 和10 500 lx)下的生长发育及繁殖指标,包括各虫态发育历期、成虫寿命、存活率、若虫期营养积累效率等;利用生命表方法进一步分析光照强度对种群趋势变化的影响。【结果】光照强度对筛豆龟蝽各虫态发育历期、存活率以及成虫生殖力等均产生显著影响。各阶段的发育历期有随光照强度增加而缩短的趋势,其发育速率和若虫期营养积累效率均与光照强度呈显著正相关。各阶段发育速率与光照强度的关系模型分别为:卵期 V 1= 0.129968exp(0.011310 L I)(R^2=0.610, P <0.0001),若虫期 V 2=0.000767LI^0.014182 ( R^2 =0.980, P <0.0001),雌虫产卵前期 V 3=0.019000 L 0.418000 I ( R^2 =0.837, P <0.0001);若虫期营养积累效率与光照强度之间符合三次函数模型 y =0.0004 x^3-0.0064 x^2+0.0361 x +0.0660 ( R^2 =0.983, P <0.0001);筛豆龟蝽种群趋势指数 I 随光照强度增加而显著增大,光照强度低于2 500 lx时,其种群不能完成生殖过程。【结论】光照强度可显著影响筛豆龟蝽种群生长发育及生殖力。较高的光照强度(10 500 lx)更有利于其种群数量增长,而较低的光照强度(≤2 500 lx)将导致种群逐渐消亡。     

The biology and preliminary host range of Megacopta cribraria (Heteroptera: Plataspidae) and its impact on kudzu growth

The bean plataspid, Megacopta cribraria (F.), recently was discovered in the United States feeding on kudzu, Pueraria montana Lour. (Merr.) variety lobata (Willd.), an economically important invasive vine. We studied its biology on kudzu and its impact on kudzu growth. We also tested its ability to use other common forest legumes for oviposition and development. Flight intercept traps operated from 17 May 2010 to 31 May 2011 in a kudzu field near Athens, GA showed three peaks of adult flight activity suggesting there are two generations per year on kudzu. Vine samples examined for eggs from April 2010 to April 2011 and June to October 2011 showed two periods of oviposition activity in 2010, which coincided with the peaks in adult activity. In 2011, the second period of oviposition began on or before 24 June and then egg abundance declined gradually thereafter until late August when we recovered <2 eggs/0.5 m of vine. Samples of the five nymphal instars and adults on vines did not show similar trends in abundance. Adults did not lay eggs on the various legume species tested in 2010 in a no-choice test possibly because the cages were too small. In the 2011 field host range experiments conducted in a kudzu field by using 12 legume species, M. cribraria preferentially oviposited on kudzu over soybean, Glycine max Merrill., but they still laid 320 eggs per plant on soybean. Lespedeza hirta (L.) Hornem. and Lespedeza cuneata (Dum. Cours.) G. Don had 122.2 and 108.4 eggs per plant, respectively. Kudzu and soybean were the only species M. cribraria completed development on. Plots protected from M. cribraria feeding by biweekly insecticide applications had 32.8% more kudzu biomass than unprotected plots. Our results show that M. cribraria has a significant impact on kudzu growth and could help suppress this pest weed.     

Mating aggregation of a stink bug,Megacopta punctissimum (Montandon) (Heteroptera: Plataspidae)

Summary Adults of a stink bug,Megacopta punctissimum, form aggregations on stems of the bush-clover,Lespedeza crytobotria, in spring. They do not oviposit there, but feed and mate within aggregations. Oviposition is made on other leguminous plants such as the kudzu-vine,Pueraria lobata. Mean size of aggregations (groups of two or more individuals which sit on a stem at distance less than 3 cm from their nearest neighbors) consisted of 4.4 individuals. The sex ratios within aggregations were similar to overall sex ratio of the population including solitary individuals (0.58). More than 50% of females found in aggregations were copulating. Percentage of femalesin copula in larger aggregations was higher than that in smaller aggregations, and this difference was considered to be caused by the higher chance of sexual enconters in larger aggregations. It was suggested that the aggregation of this species on the bush-clover is not a ‘harem’ (a male monopolizes a group of females reported in some other stink bugs) but is mating aggregation to raise the chance of mating. This work was partially supported by Grant-in-Aid (No. 56480039) from the Ministry of Education, Science and Culture.     

Formation and maintenance of mating aggregations in a stink bug,Megacopta punctissimum (Montandon) (Heteroptera: Plataspidae)

Adults of a stink bug,Megacopta punctissimum, formed mating aggregations on their host plants: a few pairs in copula and a few bachelor males (males not in copula) stayed in the aggregation and the bachelor males waited for arriving females to mate with. The processes of formation and maintenance of the aggregations were observed using the individual marking technique. Aggregations initiated by 2 males or by 1 male and 1 female were usually joined by 1 or more individuals and lasted for several days, but aggregations initiated by 2 females broke up within 1 h. Aggregations were not maintained by the same members. The residence time (time from joining an aggregation to leaving the aggregation without copulating) of males was longer than that of females. There was a negative correlation between the residence time of males and the number of bachelor males in an aggregation when bugs joined it, while the residence time of females was positively correlated with the number of bachelor males in the aggregation. When bugs copulated after joining an aggregation, the postcopulatory residence time (time from completing copulation to leaving the aggregation) was longer in males than in females. Thus, males had a stronger tendency to initiate and maintain aggregations than females.     

Female choice for male gregariousness in a Stink bug,Megacopta punctissimum (Montandon) (Heteroptera,Plataspidae)

Adults of a stink bug,Megacopta punctissimum, form mating aggregations on their host plants, based on the gregarious habit of males. A female was released on a stem on which there was an aggregation of 2 mating pairs and 1 bachelor male. Next, the bachelor male was released on a stem on which no bugs were present, and the same female was released there. Sequences of courtship behavior were compared between them. This experiement was repeated for 41 pairs of males and females, and about half (20 cases) of these experiments were made in the reverse order. Males were the active sex in courtship, whilst females either accepted the courtship, or escaped from courting males. Females accepted courtship with a higher probability when males courted in aggregations (73%) compared to solitary conditions (22%). This was because the escape behavior of females from the males was reduced if females detected the presence of other bugs near the males. It was concluded that female choice is a selective force for gregariousness in males.     

A revision of the genus Hemitrochostoma (Hemiptera,Heteroptera, Plataspidae)

The genus Hemitrochostoma Bergroth, 1913 (Hemiptera, Heteroptera, Plataspidae) is redescribed and reviewed. Two species are recognized: the type species Hemitrochostoma altilabris Bergroth, 1913 (Malaysia: Sarawak), and Hemitrochostoma rutabulum sp. n. (Malaysia: Perak). The following new subjective synonymies are proposed: Hemitrochostoma Bergroth, 1913 = Inflatilabrum Tomokuni, 2012, syn. n.; Hemitrochostoma altilabris Bergroth, 1913 = Inflatilabrum lambirense Tomokuni, 2012, syn. n.     

Ability of <Emphasis Type="Italic">Megacopta cribraria</Emphasis> (Hemiptera: Plataspidae) to Cling to Different Surfaces against Extreme Wind

The kudzu bug, Megacopta cribraria (Fabricius), is native to Asia but recently invaded the US and is expanding its distribution rapidly. To assess the probability of this bug traveling by attaching to the exteriors of fast-moving vehicles, we investigated the ability of M. cribraria adults to cling to stages with different surfaces (cloth, metal, or glass) against extreme airflows in a transparent acrylic tube connected to a vacuum cleaner. On the stages with cloth and metal, insects remained on the stages at 100 km/h wind speed. Estimated wind velocities required to blow 50 % of insects from the stages within 1 min were more than 100 km/h (cloth); 60 km/h (metal); and 40 km/h (glass) for males and 100 km/h (cloth); 50 km/h (metal); and 30 km/h (glass) for females. Together with frequent observations of attached M. cribraria on vehicle exteriors in the field, our results indicate a fairly high probability of range expansion of this species by attachment to vehicles.     

一株牙鲆源黏质沙雷氏菌YP1的分离鉴定及致病性分析

黏质沙雷氏菌(Serratia marcescens)是引起人类、动物及植物感染的重要条件致病菌,但其作为鱼类致病菌却鲜有报道。【目的】本研究以从患病牙鲆(Paralichthys olivaceus)病灶处分离的一株黏质沙雷氏菌YP1为研究对象,分析黏质沙雷氏菌对鱼类的致病性及对疾控的影响。【方法】利用形态学、分子生物学及生理生化实验综合鉴定菌株YP1;利用菌株YP1进行人工感染实验、组织病理实验及药敏试验,研究其感染症状、组织病理学、毒力和药物敏感性。【结果】分离自患病牙鲆体表溃疡病灶处的菌株YP1鉴定为黏质沙雷氏菌。感染实验结果显示,牙鲆和斑马鱼的半数致死量(LD₅₀)分别为3.44×10⁷CFU/g和6.28×10⁵CFU/g,除牙鲆外菌株YP1对其他鱼类也具有高致病性;菌株YP1主要导致牙鲆腹水,同时伴有呼吸急促、摄食减弱、脱肛、白便、鳃缺血及多脏器膨大出血等症状,并随着感染时间的延长对脏器损伤呈加重趋势。病理组织切片结果显示,菌株YP1对牙鲆鳃、肠、肝、脾、肾、心均造成损伤。药敏试验结果表明,YP1对左氧氟沙星、诺氟沙星等14种药物敏感;但对氨苄西林、头孢拉定等19种药物具有耐药性。【结论】本研究结果证实了黏质沙雷氏菌是能导致牙鲆腹水病的一种病原菌,同时对其他鱼类也具高致病性,为该菌感染鱼类导致疾病的检测、鉴别和防治提供科学依据。     

Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut (Reticulitermes santonensis)

Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55 °C. Its activity was increased by reducing agents and decreased by Cu(2+), some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.     

从松叶蜂分离的球孢白僵菌及其致病性

从东北伊通红松林采集的自然染病死亡的帕克阿扁松叶蜂Acantholyda 虫尸上分离获得一株球孢白僵菌,定名为FDB01。用该菌株孢子悬浮液感染油松阿扁叶蜂Acantholyda posticalis和落叶松叶蜂Pristiphora erichsonii幼虫,研究该菌株的致病情况。结果表明,用浓度为2×108孢子/mL的孢子悬浮液处理16d后,该菌株对油松阿扁叶蜂和落叶松叶蜂的致死率分别达到了94.4%和100%,说明该菌株对松叶蜂具有很强的致病力。     

Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect, Triatoma infestans (Hemiptera: Reduviidae)

Recently, we have cloned several Kazal-type serine protease inhibitors from the midgut of the Triatoma infestans bug. A single gene composed of multi Kazal-type domains, in tandem, encodes these inhibitors. In this work, we describe the purification and characterization of recombinant infestins 3-4 and 4, which are potent factor XIIa inhibitors (KI=67 pM and 128 pM, respectively). We also identified the first native factor XIIa inhibitor from a hematophagous insect. The factor XIIa inhibitory activity of infestin 4 demonstrates extremely efficient anticoagulant activity, prolonging activated partial thromboplastin time by approximately 3 times. Our results suggest that infestins perform a very important role in the T. infestans midgut during meal acquisition and digestion by controlling blood coagulation by means of inhibiting thrombin and factor XIIa.     

Identification and characterization of a novel endoglucanase (CMCase) isolated from the larval gut of Bombyx mori

While screening for cellulase-producing fungi from insect gut, a fungus with high endoglucanase (carboxymethyl cellulase; CMCase) activity was isolated from the larval gut of Bombyx mori. Based on morphological characteristics and using an 18S rRNA-based molecular phylogenetic approach, the fungus, strain BMC-2, was identified as a Mucor sp. expressing a novel alkalotolerant cellulase. The maximum production of cellulase by the BMC-2 strain was observed at 55 °C and pH 8.0. The CMCase activity was inhibited by Cu^2 + > Na⁺ > Zn^2 + > Mg^2 + > Ba^2 +, and induced by Ca^2 +, Mn^2 +, Fe^2 +, and K⁺.     

Identification and characterization of chitinolytic bacteria isolated from a freshwater lake

To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of chitinase of four selected strains was higher than that of a reference strain. The molecular size of one chitinase produced by Andreprevotia was greater than that of typical bacterial chitinases. The dialyzed culture supernatant containing chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents.     

Isolation,characterization and pathogenicity of bacteria from Rhynchites bacchus (Coleoptera: Rhynchitidae)

The leafroller weevil beetle (Rhynchites bacchus L., Coleoptera: Rhynchitidae) is one of the most serious pests of apple, plum, apricot, cherry and peach (nectarine) fruit worldwide. The aim of this study was to isolate and characterize pathogenic bacteria from this pest for possible use in biocontrol, and to determine their pathogenicity. R. bacchus were collected from the Eastern Black Sea Region of Turkey in 2007. Based on their morphological, physiological and biochemical features, five different bacterial isolates were obtained from adults and larvae. In addition, 16S rRNA gene was amplified and sequenced to confirm isolate identification. According to the numerical and molecular data, the culturable bacterial flora of R. bacchus was determined as Bacillus licheniformis (Rb1), Serratia marcescens (Rb2), Enterobacter hormaechei (Rb3), Paenibacillus sp. (Rb4), and Enterobacter sp. (Rb5). Isolate Rb2 produced the highest mortality (73%) against larvae within 10 days after inoculation (P<0.05). The others, Rb1, Rb3, Rb4 and Rb5, caused 20, 13, 26 and 13% mortality in the larvae within the same period. These results indicate that S. marcescens Rb2 seems to be the most promising biocontrol agent againts this pest.     

Identification and characterization of a baculovirus from Lonomia obliqua (Lepidoptera: Saturniidae)

A baculovirus has been isolated from larvae of Lonomia obliqua, a Saturniidae of medical importance due to a potent toxin found in their spines. Electron Microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra of approximately 1 microm in diameter containing multiple nucleocapsids per envelope. This baculovirus was thus named Lonomia obliqua multicapsid nucleopolyhedrovirus (LoobMNPV). Restriction endonuclease profiles of viral DNA digested with three restriction enzymes were obtained and the genome size was estimated to be 95.52 +/- 2.3 kbp. The polyhedrin gene of LoobMNPV was identified and its DNA sequence was determined. Phylogenetic analysis of the polyhedrin gene showed that the LoobMNPV polyhedrin belongs to group I NPV and that it is closely related to the polyhedrin of the NPV of Amsacta albistriga.     

Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Asiatic citrus psylla, Diaphorina citri, Kuwayama (Hemiptera: Liviidae) is an economic pest of citrus groves and a vector of the bacterium, Candidatus Liberibacter spp., one of the causative agents of citrus greening. In order to estimate the infectivity of six different isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea, fungal bioassay was performed on the adults of D. citri. Adults of D. citri were treated individually with 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, 2 × 10⁸ spores/mL fungal concentrations by the immersion method. Subsequent to fungal bioassay, treated D. citri were used to determine the levels of esterase and glutathione S‐transferases (GST) enzymes over a period of 3–7 days. The mortality results suggest that I. fumosorosea isolates (If‐02) caused 82.2% mortality on the seventh day of treatment. However, B. bassiana isolate (Bb‐08) with lowest LC₅₀ (1.4 × 10⁷ spores/mL) proved to be highest potential isolate against D. citri. Biochemical determination of esterase and GST activity assay showed significant differences in activities after infection of fungi. Significantly high activity of esterase was observed by Bb‐01 (27.0 unit per mg protein) on the seventh day, while Ma‐11.1 and If‐2.3 (16.9 and 36.3 unit per mg protein) on the third day post treatment. However, maximum GST's activity was showen by isolates Bb‐08,Ma‐M₂ and If‐2.3 (37.6, 1.40 and 10.9 unit per mg protein) on the third day. The current investigation will help to explore the relations between the insect defense system and entomopathogenic fungi. Moreover, the determination of enzymatic activities will be useful for selecting the most pathogenic isolates.