Selection of reference genes for RT-qPCR analysis in Trichogramma chilonis (Hymenoptera: Trichogrammatidae)

Trichogramma chilonis is an important natural enemy for control of various Lepidoperan crop pests. The biology of T. chilonis is well-studied, but the molecular mechanisms of this biology require further study. Screening suitable reference genes is a vital step for use of RT-qPCR to understand underlying molecular physiology. In the present study, nine candidate reference genes including elongation factor 2 (EF2), ribosomal proteins (RPS23, RPL13, and RPL44), malate dehydrogenase (MDH), eukaryotic translation initiation factor 3 subunit F (EIF3F), zinc finger protein 268 (ZFP268), muscle specific protein 20 (MP20), and ATP synthase subunit alpha (ATP5F1A) were evaluated at different conditions including development stage, diet, temperature, and insecticide treatments. Four common algorithms (the Delta Ct method, geNorm, BestKeeper, and NormFinder) and RefFinder were used to analyze gene expression stability. Our results indicated that two reference genes used for normalization were sufficient, and the optimal combinations were: RPS23 and EF2 for developmental stages, ZFP268 and EF2 for feeding with different diets, ZFP268 and RPL13 for temperature treatments, and EF2 and RPL44 for insecticide treatments. The results provide preliminary determination of suitable reference gene for standard RT-qPCR analyses in T. chilonis, which might establish the foundation for further molecular biology research.     

Larval feeding on Bt hybrid and non-Bt corn seedlings by Harmonia axyridis (Coleoptera: Coccinellidae) and Coleomegilla maculata (Coleoptera: Coccinellidae)

Zoophytophagy is an omnivorous activity that occurs when a primarily carnivorous species feeds on plant material. Plant feeding by beneficial predators may have negative consequences if the plant material has been chemically treated, contains toxins, or was transgenically altered. Although common in predaceous Hemiptera, zoophytophagy has been rarely studied in aphidophagous coccinellids. This study examined the likelihood of feeding on Bt and non-Bt corn seedlings by third- and fourth-instar coccinellid larvae, the regularity of feeding events by fourth instars, and the effect of leaf feeding on development time and adult size. Both third- and fourth-instar Harmonia axyridis Pallas and Coleomegilla maculata DeGeer consume leaf tissue, with fourth instars being significantly more likely to feed on corn seedlings. C. maculata larvae ingested leaf tissue more frequently than H. axyridis. Furthermore, when given access to corn seedlings daily, development time of fourth-instar C. maculata increased after Bt hybrid corn treatments compared with non-Bt corn treatments. Zoophytophagous feeding behavior is thought to sustain predators during times of low prey availability, and leaf tissue feeding by coccinellids has typically been attributed to their need for water. However, in this study, tissue feeding regularly occurred even though coccinellid larvae had constant access to water and a daily ad libitum supply of aphids. We suggest that, in addition to environmental conditions, the physiological state of the zoophytophagous species will influence the probability of plant feeding.     

异色瓢虫不同色斑型成虫的耐寒性研究

【目的】研究异色瓢虫Harmonia axyridi不同色斑型成虫的耐寒能力,以明确其色斑型比例所呈现的季节性变化的适应意义。【方法】本文测定了异色瓢虫实验种群和自然种群不同色斑型成虫的过冷却点(Supercooling point,SCP)及低温存活率。【结果】同一发育温度下异色瓢虫实验种群不同色斑型成虫的SCP和低温存活率均没有显著性差异(P>0.05),但是低温暴露中饲养在25℃下成虫的存活率下降更为剧烈。异色瓢虫自然种群黑底型与黄底型成虫的SCP呈现出明显的季节性变化,其SCP在7月份最高,分别为﹣8.2℃和﹣7.5℃;在1月份达到最低,分别为﹣16.8℃和﹣18.2℃。越冬开始(11月)黑底型成虫的SCP低于黄底型成虫的,整个越冬期间黄底型成虫的SCP一直低于黑底型成虫的,但是异色瓢虫黑底型与黄底型越冬成虫的低温存活率却没有显著性差异(P>0.05)。【结论】越冬期间异色瓢虫黄底型成虫的SCP低于黑底型的,这样可以通过体液过冷却的方式来避免结冰造成的伤害,以增强其越冬种群的耐寒能力,这也可能是越冬期间异色瓢虫黄底型成虫数量显著上升的一个原因。     

异色瓢虫生物生态学研究进展

原产于亚洲的异色瓢虫[Harmonia axyridis Pallas(Coleoptera:Coccinellidae)]是最重要的捕食性瓢虫种类之一,被广泛应用于农林业害虫的生物防治.本文综合分析了国内外近几十年对异色瓢虫的研究成果,对其重要的生物学及生态学特性进行了归纳,主要涉及生活史、繁殖行为及策略、捕食行为和自残行为等,分析了相关研究对其应用前景的影响.基于异色瓢虫的人工饲养、农药交互作用以及入侵生态影响等的总结,提出了预防异色瓢虫造成生态不平衡的具体措施.     

Ten years of invasion: Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in Britain

1. Harmonia axyridis was first recorded in Britain in 2004. Two subsequent earlier records were received from 2003. 2. The UK Ladybird Survey, a citizen science initiative involving online recording, was launched in 2005 to encourage people across Britain to track the spread of H. axyridis. Tens of thousands of people have provided records of H. axyridis and other species of ladybirds, creating an invaluable dataset for large‐scale and long‐term research. Declines in the distribution of seven (of eight assessed) native species of ladybird have been demonstrated, and correlated with the arrival of H. axyridis, using the records collated through the UK Ladybird Survey. 3. Experimental research and field surveys have also contributed to our understanding of the ecology of H. axyridis and particularly the process of invasion. Harmonia axyridis arrived in Britain through dispersal and introduction events from regions in which it was deliberately released as a biological control agent. The rapid spread of this species has been attributed to its high natural dispersal capability by means of both flight and anthropogenic transport. A number of factors have contributed to the successful establishment and indeed dominance of this polymorphic species within aphidophagous guilds, including high reproductive capacity, intra‐guild predation, eurytopic nature, high resistance to natural enemies within the invaded range, and potentially phenotypic plasticity. 4. The global invasion by H. axyridis and subsequent research on this species has contributed to the general understanding of biological invasions.     

异色瓢虫越冬聚集行为对其能量代谢的影响

异色瓢虫以成虫滞育越冬,越冬期间聚集行为尤为显著。为探讨异色瓢虫越冬聚集行为的适应意义,本研究利用便携式红外线CO2分析仪测定了6 种聚集度（分别用1头、2头、5头、10 头、20头和50头表示）下异色瓢虫越冬成虫的呼吸量,并通过呼吸量计算得到各聚集度下的呼吸速率（Respiration rate, Rr）和能量代谢速率（Metabolism rate, Rm）。结果表明: 不同聚集度间异色瓢虫越冬成虫的呼吸速率和能量代谢速率均存在显著性差异,相关性分析表明Rr和Rm值与聚集度之间呈显著负相关。即随着聚集度的增加,越冬成虫的呼吸速率和能量代谢速率呈逐渐降低的趋势。此外,利用“Y”形嗅觉仪测定了异色瓢虫实验种群和越冬种群成虫对不同味源的选择趋性,表明异色瓢虫均对各自的群体表现出趋性,而其越冬聚集行为的发生需要一种特殊的气味物质。本研究表明聚集行为可以有效降低异色瓢虫的新陈代谢速率,减少能量消耗,有利于其种群成功越冬,这对其越冬来说具有非常重要的意义。     

异色瓢虫气味结合蛋白基因HaxyOBP1和HaxyOBP6的克隆及表达谱分析

【目的】本研究旨在探索异色瓢虫Harmonia axyridis气味结合蛋白的结构和分布,更好地了解气味结合蛋白在异色瓢虫嗅觉系统中的作用。【方法】利用生物信息学方法克隆异色瓢虫2个气味结合蛋白基因序列并对其蛋白结构进行分析;通过实时荧光定量PCR分析克隆获得的这2个基因在异色瓢虫各个发育阶段和成虫不同组织中的表达水平。【结果】本研究成功克隆得到异色瓢虫两个气味结合蛋白基因HaxyOBP1和HaxyOBP6(GenBank登录号分别为MG757923和MG757927)。HaxyOBP1开放阅读框全长447 bp,编码148个氨基酸,具有6个保守的半胱氨酸,表明HaxyOBP1属于Classic OBPs。HaxyOBP1在异色瓢虫各发育阶段均有表达,在雄性成虫中表达量最高;组织表达谱分析表明HaxyOBP1在雄虫头部表达量最高。HaxyOBP6开放阅读框全长414 bp,编码137个氨基酸,具有4个保守的半胱氨酸,表明HaxyOBP6属于Minus-C OBPs。HaxyOBP6在成虫期的表达量明显高于幼虫阶段,并且在雄成虫中的表达量显著高于雌成虫;组织表达谱分析表明HaxyOBP6在雄成虫头部和雌成虫翅中表达量最高。【结论】本研究克隆得到异色瓢虫两个气味结合蛋白基因,表达谱分析表明HaxyOBP1和HaxyOBP6分别在异色瓢虫成虫头和翅中具有较高的表达水平,说明气味结合蛋白基因可能在异色瓢虫非嗅觉组织中同样具有重要作用。本研究结果为深入研究异色瓢虫气味结合蛋白结构和功能奠定了基础。     

多巴脱羧酶对异色瓢虫生殖力的调控机制

[目的]为了探究多巴脱羧酶(DOPA decarboxylase,DDC)对异色瓢虫Harmonia axyridis生殖力的影响及其调控机制.[方法]利用RNA干扰技术(RNA interference,RNAi),将异色瓢虫4龄幼虫的DDC基因(HaDDC)抑制表达,成虫羽化后第8天开始统计20 d内累计产卵量及产...     

异色瓢虫幼虫对卵的种内自残和种间捕食

自残现象的发生在异色瓢虫Harmonia axyridis中很是常见,为明确自残行为对其存活和生长发育的影响,在非选择性条件下我们对异色瓢虫幼虫对非姊妹卵的种内自残和对七星瓢虫Coccinella septempunctata卵的种间捕食进行了研究。结果表明,异色瓢虫取食同种卵和异种卵均可完成生长发育,与取食蚜虫相比：①其从卵到成虫及蛹期的发育历期明显缩短（P<0.05）,其中1龄幼虫发育历期缩短非常明显（P<0.01）;②自然条件下高死亡率的1龄幼虫的存活率明显提高（P<0.05）;③2-4龄幼虫、蛹和成虫的体重变轻（P<0.05）,且体长在4龄幼虫间也表现出显著差异（P<0.05）。这表明异色瓢虫种内自残和种间捕食对其在食物恶劣条件下延续种群至关重要,但对种群的繁衍却不是最理想的途径。     

Nitrogen fertiliser affects the functional response and prey consumption of Harmonia axyridis (Coleoptera: Coccinellidae) feeding on cereal aphids

Predator–prey interactions are influenced by nitrogen availability. Wheat (Triticum aestivum cv. Solstice) plants were provided with four levels of nitrogen and examined the responses of coccinellid predator, Harmonia axyridis to cereal aphids, Rhopalosiphum padi and Sitobion avenae. Increased nitrogen application improved nitrogen contents of the plants and also the body weight of cereal aphids feeding on them. In no‐choice feeding trials, H. axyridis consumed more aphids on low fertilised plants, suggesting a compensatory consumption to overcome reduced biomass (lower aphid size). Total biomass devoured by H. axyridis on all nitrogen fertiliser treatments was not statistically different. Logistic regression analysis of the proportion of prey consumed demonstrated that all developmental stages (larval and adult) of H. axyridis exhibited the type II functional response on all nitrogen fertiliser treatments. The rate of successful search (a′) of third and fourth instars and adults were the same across all fertiliser treatments suggesting that nitrogen fertilisation did not affect a′. Maximum handling time for the first instars of H. axyridis on R. padi (3.81 h^?1) and S. avenae (4.59 h^?1) was on the highest nitrogen treatment while minimum handling time was for the adults of H. axyridis on R. padi (0.20 h^?1) and S. avenae (0.20 h^?1) on the lowest nitrogen treatment. Handling time varied at varying fertiliser treatments within all instars and affected the predator's efficiency. The functional response curve, rate of successful search and handling time provide the information needed to understand the predator–prey interaction between H. axyridis and these cereals aphids. This could lead to the development of a better strategy for the biological control of R. padi and S. avenae at any particular level of nitrogen fertiliser regime in the field crops.     

Polymorphism of the harlequin ladybird Harmonia axyridis (Coleoptera, Coccinellidae) Baikal population

The phenotypic composition of the Baikal population of the harlequin ladybird Harmonia axyridis (Pallas, 1773) was analyzed based on the material collected in Irkutsk Province in 2009–2010. Significant microgeographic phenotypic variation was revealed on the Baikal coast and in the city of Irkutsk. The morph axyridis, widespread in Siberia, was dominant (80–90%) but the light-colored morph succinea was also present with high frequencies (43–59%) in some samples. Some individuals possessed an elytral ridge, their frequency in the Baikal population being low (4–6%). An increase in the number of the morph succinea in Irkutsk Province is not related to hybridization between the populations from the Far East and Siberia.     

海藻糖酶基因TRE2-like和TRE2在异色瓢虫羽化过程中的表达与功能

【目的】异色瓢虫Harmonia axyridis是一种重要的捕食性天敌昆虫,海藻糖在异色瓢虫的变态发育、羽化等整个生命过程都起着重要的作用。本研究以前期获得的类似膜结合型海藻糖酶(TRE2-like)与膜结合型海藻糖酶(TRE2)基因为基础,探讨在异色瓢虫羽化阶段这两个海藻糖酶的潜在功能,为阐明异色瓢虫从蛹发育到成虫时海藻糖代谢机制提供参考。【方法】根据TRE2-like和TRE2基因序列设计双链RNA(dsRNA)区域片段并合成对应的dsRNA,通过RNAi将其注射到异色瓢虫2日龄蛹中。采用实时荧光定量PCR(RT-qPCR)检测RNAi处理后羽化第1天的异色瓢虫成虫糖代谢相关基因的表达;同时采用蒽酮比色法、酶标法等分别测定RNAi处理后羽化第1天的异色瓢虫成虫主要糖类物质含量及TRE活性变化,并观察异色瓢虫羽化后的表型变化。【结果】结果表明,与对照组(dsGFP注射组)相比,异色瓢虫2日龄蛹被注射TRE2-like或TRE2 dsRNA后,其新羽化成虫体内TRE2-like和TRE2表达量均极显著下调,且少数个体出现了蜕皮与翅形成困难等畸形表型。可溶性海藻糖酶活性在注射dsTRE2-like后显著降低,膜结合型海藻糖酶活性在注射dsTRE2后显著降低;注射dsTRE2后糖原含量显著下降,注射dsTRE2-like后糖原和海藻糖含量显著下降,注射dsTRE2-like+dsTRE2后糖原和葡萄糖含量显著下降,且海藻糖含量极显著下降。注射dsTRE2-like, dsTRE2和dsTRE2-like+dsTRE2后可溶性海藻糖酶基因TRE1-1和TRE1-2表达下降或显著下降,而TRE1-5表达上升或显著上升,海藻糖合成酶(trealose-6-phosphate synthase, TPS)、糖原磷酸化酶(glycogen phosphorylase, GP)、糖原合成酶(glycogen synthase, GS)基因的表达均显著下调。【结论】TRE2-like和TRE2基因表达被抑制后,异色瓢虫海藻糖等代谢受到影响。研究结果为探究异色瓢虫体内膜结合型海藻糖酶的潜在功能和调控机制奠定了基础。     

Expansion of Harmonia axyridis Pallas (Coleoptera: Coccinellidae) to European Russia and adjacent regions

An invasive alien species, the harlequin ladybird Harmonia axyridis (Pallas, 1773), has quickly expanded its distribution in Eastern Europe. Records of H. axyridis from 31 localities in Lithuania, Latvia, the Ukraine, European Russia, and the Northern Caucasus are summarized and mapped. Within the last few years this species has established in south Latvia, on the Baltic Sea shore (Kaliningrad oblast and Lithuania), in the western and central Ukraine, Crimea, and in the Northern Caucasus. Besides that, individual specimens have been found in 4 more localities in European Russia. The species is recorded from Lipetsk oblast (European Russia), Crimea, and Nikolaev oblast (the Ukraine) for the first time.     

异色瓢虫对蚜虫性外激素的行为反应研究

蚜虫性外激素假荆芥内酯(Z,E-Nepetalactone,NEP)能够吸引多种捕食性和寄生性天敌昆虫,它专一性强,生物活性突出,对非靶标生物安全。异色瓢虫Harmonia axyridis(Pallas)作为一种重要的捕食性天敌,被广泛应用于农业生产中。为明确异色瓢虫对蚜虫性外激素的行为反应,本研究在室内用"Y"型嗅觉仪测定了异色瓢虫成虫对不同剂量NEP的嗅觉行为反应;并在田间,每7 d释放100μL浓度为1 mg/m L的NEP,测定了NEP对异色瓢虫种群数量变动的影响。室内嗅觉测定试验结果表明异色瓢虫雌虫对10μL剂量NEP有显著的选择性,而雄虫对1μL剂量NEP有显著的选择性;田间试验发现,释放NEP的处理区异色瓢虫数量显著高于对照区。本研究为蚜虫性外激素在害虫绿色防控中的应用提供了依据。     

Walking activity of flightless Harmonia axyridis (Coleoptera: Coccinellidae) as a biological control agent

The use of flightless strains of the multicolored Asian lady beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), established via artificial selection, can be highly effective as a biological control agent for aphids. However, flightless H. axyridis must depend on walking for dispersion. Therefore, data on the walking activity levels in flightless strains are important for the development of effective methods when releasing these agents in the field. Results of measurement of walking activity levels using an infrared actograph showed that walking activity levels during the daytime (but not nighttime) in both sexes of pure flightless strains tended to be lower than those of control strains. We also found that walking activity levels during the daytime for the F1 generation of hybrid strains, produced by reciprocal crossing between two pure flightless strains, were approximately equal to those of pure strains; the reduction in walking activity levels was not recovered by hybrid vigor. Our results indicate that the reduction in walking activity levels in the pure flightless strains was not caused merely by inbreeding depression stemming from the artificial selection process. Instead, potentially flight ability and walking activity levels in this species may be controlled by the pleiotropic effect of a gene.     

Record of the invasive alien ladybird Harmonia axyridis (Coleoptera, Coccinellidae) from Kenya

The biological control agent and alien invasive ladybird Harmonia axyridis (Pallas, 1773) was recorded for the first time in Kenya, and in equatorial Africa, in 2010.     

Cannibalizing Harmonia axyridis (Coleoptera: Coccinellidae) larvae use endogenous cues to avoid eating relatives

Cannibalism is widespread among many different organisms, and can have both negative and positive fitness consequences. Avoiding eating relatives can minimize negative fitness consequences of cannibalism. Such avoidance requires kin discrimination, but evidence for this ability among cannibals is limited with little data that address the cues used in such discrimination. We examined whether larvae of the ladybird beetle Harmonia axyridis avoid eating their relatives. We further manipulated environmental factors to create individuals that had similar or dissimilar rearing environments to begin to test for endogenous versus exogenous recognition cues. In our experiments, third-instar larvae were much less likely to cannibalize if they were interacting with a relative. Larvae that did cannibalize kin required more encounters, and significantly delayed cannibalism, compared to larvae cannibalizing unrelated individuals. Acquired cues were less important. Even though the different rearing environment resulted in significantly different phenotypic effects, similarity or dissimilarity of rearing environment had no effect on cannibalism, and there was no interaction between environments and relatedness in cannibalism. We suggest that H. axyridis has a well-developed kin discrimination system, and that kin recognition in this ladybird beetle is based on endogenous rather than exogenous cues. We also argue that these cues reflect either direct or indirect genetic effects on larval phenotypes.     

环境颜色对异色瓢虫生长发育及繁殖能力的影响

在室内利用不同颜色的饲养容器,对异色瓢虫Harmonia axyridis (Pallas) 生长发育、体长体重变化及繁殖能力进行了比较研究。结果显示：不同环境颜色条件对异色瓢虫幼虫各龄历期均有显著影响; 尽管蛹期受不同颜色影响显著,但其总胚后发育期在各处理间差异不显著。异色瓢虫1龄和2龄幼虫体长增量在不同颜色处理间无显著差异,但3龄幼虫在红色条件下体长增量显著小于其余各处理。不同环境颜色条件下异色瓢虫各虫期体重增量均有显著差异,而4龄幼虫体重增量在各处理中均显著大于其余各虫期,并占总胚后发育期体重增量的50%以上。异色瓢虫成虫的交配持续时间在各处理间无显著差异,但其产卵前期在黄色及绿色条件下显著小于其余各处理。其首堆产卵量在各处理间无显著差异,但在红色及自然光下的48 h累计产卵量均显著小于其余处理。     

冷驯化对异色瓢虫后代生长发育及适合度的影响

为明确冷驯化处理对异色瓢虫Harmonia axyridis后代生长发育及适合度的影响, 本研究通过两性生命表的制作, 在室内条件下调查了冷处理后其后代发育历期、 成虫产卵前期、 寿命和生殖力以及后代生命表参数。结果表明: 冷驯化使异色瓢虫后代卵的发育历期延长, 幼虫（1-4龄）和蛹的发育历期则缩短; 随着冷驯化时间的延长, 后代体长和体重增量均减小。且完成发育的后代成虫产卵前期延长, 寿命缩短, 生殖力下降。后代生命表参数内禀增长率（r）、 周限增长率（λ）、 净生殖率（R₀）和年龄特征存活率（l_x）均降低, 但是后代雌虫所占比例却升高。此外, 冷驯化类型对异色瓢虫后代生长发育的影响也不相同。经相同时间（5 d）的低温诱导, 变温诱导的后代成虫寿命比恒定低温诱导的长, 但是生殖力却低; 变温诱导的后代生命表参数（r, λ和R₀）均小于恒定低温诱导的, 但是l_x却高于恒定低温诱导的。结果说明, 异色瓢虫亲代经历冷驯化, 这种对低温的可塑性反应会延伸到下一代, 而且还能够影响后代的适合度, 这对其在低温下的存活和繁殖具有重要的意义。