Development and characterization of microsatellite markers in the Colorado potato beetle,Leptinotarsa decemlineata

The characterization of 11 Leptinotarsa decemlineata microsatellite loci isolated using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol is reported. Three to 15 alleles per locus were detected in 54 beetles collected from four populations of L. decemlineata. The mean number of alleles was 7.4 ± 3.2 and the level of expected heterozygosity ranged from 0.451 to 0.798. The total exclusionary probabilities using these loci for the first and the second parent were 0.982 and 0.999, respectively. These are the first microsatellite loci characterized from the Colorado potato beetles that can be used for estimating genetic diversity, population structure and parentage analysis.     

Biological control potential of Turkish entomopathogenic nematodes against the Colorado potato beetle,Leptinotarsa decemlineata

Three Turkish isolates of the entomopathogenic nematodes Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora were evaluated under laboratory conditions (in different temperatures and doses) for their biocontrol efficiency against last instar Leptinotarsa decemlineata. Herein, the effects of infective juveniles in aqueous suspension against L. decemlineata were evaluated. S. feltiae appeared to be the most pathogenic nematode among the tested species.     

Ecdysteroids and adult diapause in the Colorado potato beetle, Leptinotarsa decemlineata

ABSTRACT. The difference in ecdysteroid activity in short-day (10-h photo-phase) and long-day (16-h photophase) Leptinotarsa decemlineata Say adults was characterized by high performance liquid chromatography, which revealed that for short-day beetles ecdysterone and ecdysone are the major constituents of the free ecdysteroids, whereas in long-day beetles products co-migrating with 2-deoxyecdysone and 2-deoxyecdysterone are just as abundant. Ecdysteroids were detected (15 ng/ml) only in the haemolymph of females. Ligation experiments showed that the induction of the diapause syndrome by inactivation of the corpora allata or the brain not only resulted in the formation of the typical diapause proteins but also in an increase of the ecdysteroid activity. It is suggested that adult diapause in L.decemlineata is regulated by a multifactorial system.     

Abdominal movements, heartbeats and gas exchange in pupae of the Colorado potato beetle, Leptinotarsa decemlineata

The rhythms of abdominal movements, heartbeats and gas exchange in the pupae of Leptiontarsa decemlineata (Say) were recorded simultaneously using an electrolytic respirometer and infrared gas analyser, both combined with contact thermography. Abdominal pulsations and heartbeat occurred periodically with little variance among individuals. The abdominal pulsations and heartbeat pauses varied individually within large limits, with the frequency of abdominal pulsations being six to seven times lower than that of the heart pulses. A proportion of the pupae (20%) showed discontinuous gas exchange with large, actively ventilated CO₂ bursts, whereas others (≈ 25%) exhibited continuous regular microcycles (flutter) with abrupt intake of air into the tracheae before discrete microbursts of carbon dioxide. The abdominal pulsations exerted only a minor influence on ventilation during the microcycles. More than 90% of the bursts of abdominal movement coincided with a series of forward directed heartbeats, but interspersed between the bouts of abdominal movement commonly two to three heartbeat pulses were observed that were not associated with abdominal movements. A period of abdominal movement associated with a heartbeat pulse was commonly initiated by one or two vigorous strokes of abdominal rotation.     

Effects and modes of action of citrus limonoids against Leptinotarsa decemlineata

ABSTRACT Limonin and its five derivatives were evaluated in feeding bioassays for depression effects on feeding, growth and development of the fourth stadium Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Epilimonol and limonin diosphenol were most effective in depression of feeding and growth, with ED₅₀s 10 and 8 μg/cm². At a dosage of 31.6 μg/cm², epilimonol, limonin diosphenol and limonin caused both significant feeding reduction and developmental delay, while deoxylimonol, tetrahydrolimonin and limonol were not effective. Mouth-part coating and antifeedant bioassays show that feeding inhibition at host acceptance level seems to be the primary mode of action of epilimonol and limonin diosphenol.     

新疆石河子和博州地区马铃薯甲虫抗药性检测

马铃薯甲虫Leptinotarsa decemlineata(Say)在新疆北部的危害逐年加重。本文采用点滴法检测了石河子、博州和昌吉3个地区4个马铃薯甲虫田间种群对有机磷、氨基甲酸酯、拟除虫菊酯、新烟碱类等15种杀虫药剂的抗性水平。结果表明,所检测的沙湾、玛纳斯、博乐和奇台4个马铃薯田间种群对拟除虫菊酯类高效氯氟氰菊酯和溴氰菊酯产生了11～155倍的抗性,博乐种群对高效氯氰菊酯抗性为27倍;4个种群对氨基酸酯类的丁硫克百威也产生了33～59倍的抗性。另外,对硫丹和阿维菌素也分别产生了11～23倍和4～7倍的抗性。但对水胺硫磷等4种有机磷和吡虫啉等3种新烟碱类杀虫药剂仍然比较敏感。研究结果为上述地区马铃薯甲虫的有效控制提供重要依据。     

Effects of four Bacillus spp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say)

Four species of Bacillus were isolated from soil in an effort to find safe, effective and alternative biological control agents against plant pests. These bacteria were identified as Bacillus pumilus, Bacillus sphaericus, Bacillus megaterium and Bacillus cereus on the basis of fatty acid methyl ester analysis and carbon utilization profiles by using Microbial Identification and Biolog Microplate Systems. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that B. pumilus caused 95.7 and 26.7% mortality and B. sphaericus caused 74.5 and 23.3% mortality of Leptinotarsa decemlineata larvae and adults, respectively. B. cereus and B. megaterium showed 51.1 and 29.7%, respectively, of L. decemlineata larvae. This study presents at least two Turkish isolates from the genus Bacillus showing high insecticidal activity against L. decemlineata.     

Cry3A毒素和氯菊酯对马铃薯甲虫神经-肌肉系统的电生理作用比较

对Cry3A毒素和氯菊酯杀虫剂经口注射处理的马铃薯甲虫的前肠、中肠和腿节样本的神经肌肉自发动作电位发射进行了记录。两种化合物均可呈现很典型的神经电生理症状：在初期阶段,它们均引起连放动作电位的发放程度大幅增加,而发放间隔期延长,且随着中毒加深而拉长。氯菊酯可在腿节样本引起典型高频爆排,表现出间隔非常短的静息期特征,但Cry3A毒素只在肠道样本中表现上述特征。而且Cry3A毒素可使处理甲虫的呕吐物大幅增加,而取食减少。这些结果显示处理虫的肠道神经肌肉系统较腿部神经肌肉系统对Cry3A毒素更敏感,Cry3A毒素最初的神经毒性或细胞毒性作用引起肠道活动的紊乱是其昆虫毒性作用的重要机制。     

Genetic relationships of introduced Colorado potato beetle Leptinotarsa decemlineata populations in Xinjiang,China

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an infamous invasive species worldwide that aggressively attacks potato and other Solanaceae crops. CPB was first found in China in 1993 and has since spread across 2.77 × 10⁵ km² in Xinjiang Uygur Autonomous Region. To better understand genetic variation and migration patterns, we used seven polymorphic microsatellite loci to elucidate the genetic relationships and gene flow among 10 CPB populations across Xinjiang. (i) Overall low levels of genetic diversity were detected on the entire population in Xinjiang but most of the diversity was retained among populations during invasion. (ii) The mean pairwise F_ST was low (0.071 ± 0.043) among populations. The genetic differentiation was little (pairwise F_ST 0.038 ± 0.016) between the five interior populations (Wusu, Urumqi, Jimsar, Qitai and Mulei) and Tacheng population. The six populations might come from the same genetic group via Bayesian clustering and were closely related on a neighbor‐joining tree. Combining the history data, the five interior populations may have originated from Tacheng. (iii) Gene flow was frequent, especially among the five interior populations. Individuals from the interior populations could be assigned to Tacheng at higher probabilities (means 0.518 ± 0.127) than vice versa (means 0.328 ± 0.074), suggesting that the beetle population has spread from the border to the interior in Xinjiang.     

Host-plant discrimination and evolution of feeding preference in the Colorado potato beetle Leptinotarsa decemlineata

ABSTRACT. The behaviour of newly emerged adult Colorado potato beetles on preferred hosts follows a stereotyped pattern of sampling, feeding, grooming and rest. Reduced meal sizes on less-preferred hosts is accompanied by increased sampling and frequent interruptions in feeding. A systematic increase in pre-ingestive sampling on less-preferred foodplants indicates that beetles discriminate among closely related species within the Solanaceae. This ability may depend primarily on stimuli perceived at, and near, the leaf surface. Three geographic populations of beetles have adapted to different local host plants, but have not lost their preference for feeding on an ancestral host species. Host shifts by oligophagous insects to related plant species may evolve through selection for feeding generalists in isolated populations, and may not require genetic changes affecting the perception of a particular novel host.     

Effect of diet and feeding history on flight of Colorado potato beetle, Leptinotarsa decemlineata

We evaluated the hypothesis that Colorado potato beetle (Leptinotarsa decemlineata Say) (CPB) flight frequency is related to diet, and that it changes with duration of food unavailability or exposure to poor quality food by exposing adult overwintered and summer CPB populations to an acceptable host plant (conventional foliage), a poor host (insect resistant transgenic foliage expressing Bacillus thuringiensis tenebrionis[Btt] Cry3a toxin) and no host. Exposure to poor host and no host treatments (with or without water) decreased mean daily flight frequencies and the overall number of overwintered CPB flying, but increased the mean daily flight frequency and number of summer population CPB that flew. Overwintered CPB did not react to an absence of plants at emergence whereas summer CPB increased mean daily flight frequencies when plants and water were not available. The flight response to insect resistant foliage was similar to that for starvation treatments in both populations indicating that flight may not be triggered by Btt toxins but by starvation brought on by feeding on poor quality food. Flight was observed in all treatments for the duration of the test with two exceptions; overwintered beetles fed insect resistant foliage ceased flying after day 17 and summer beetles starved without water ceased after day 8 of a 29‐day study.     

Sequencing and characterization of a cDNA encoding a ferritin subunit of Colorado potato beetle, Leptinotarsa decemlineata

A differentially expressed cDNA fragment (P311) from Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), was identified by restriction fragment differential display-polymerase chain reaction (RFDD-PCR) technique, and showed a strong similarity to ferritin heavy chain subunits of other organisms. Based on P311, we constructed specific primers and obtained a 840-bp cDNA fragment spanning the open reading frame of CPB ferritin subunit using the rapid amplification of cDNA ends (RACE) technique. The sequence encodes 213 amino acid residues, including a 19 amino acid signal peptide. The sequence has a conserved cysteine in the N-terminus and has the seven conserved residues that comprise the ferroxidase center, which is the feature of heavy chain ferritins of vertebrates. The CPB ferritin subunit has high amino acid sequence identity with the Apriona germari (69.3%), Galleria mellonela (54.5%), Manduca sexta (54.0%), Drosophila melanogaster (53.2%), Calpodes ethlius (51.4%), and Nilaparvata lugens (47.6%) but lower identity with the Anopheles gambiae (38.7%) and Aedes aegypti (37.8%). Using Northern blot analysis, the subunit mRNA was identified from fat body and midgut of 4th instars with much higher mRNA levels found in midgut than that in fat body (2.5-fold). Nevertheless, only the levels of mRNA in fat body was induced by dexamethasone (1.5-fold).     

Effect of vermicompost on nutrition and intermediary metabolism of Colorado potato beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Colorado potato beetle (CPB) is the important pest of potato throughout the world. The study showed the effects of vermicompost on nutritional indices, digestive enzyme activities and intermediary metabolism of the larvae and the adults of CPB. Vermicompost affected significantly the efficiency of conversion of ingested food (ECI) and efficiency of conversion of digested food (ECD) in addition to activities of carbohydrases and proteases in both larvae and adults. Amount of total phenol compounds increased in the leaves of the potatoes grown on the soil containing 30% of vermicompost compared to control and it were amended by 15% of vermicompost. In case of intermediary metabolism, activities of aspartate aminotransferase and γ-glutamyl transferase showed no significant differences in the control and the treated larvae, but those were fluctuated upper and lower when 15% of vermicompost was added into growth plots. The amount of low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in the larvae of CPB showed no significant differences among treatments. However, the amounts of LDL, HDL, glycogen and protein of the adults significantly increased using 30% of vermicompost. Results of the current study clearly revealed significant effects on some physiological processes in CPB fed on the plants grown in the different vermicompost treatments.     

Body movements and their role as triggers of heartbeats in pupae of the Colorado potato beetle Leptinotarsa decemlineata

Rhythmic body movements and their role as triggers of intermittent heartbeats were studied in pupae of Leptinotarsa decemlineata Say. Heartbeats and body movements were recorded simultaneously by means of an optical method (infra‐red cardiography) combined with respirometry. IR‐cardiography allows heartbeats and body movements to be distinguished on the basis of their different rates (40–80 min^?1 and 4–8 min^?1, respectively) and amplitude. In the mid‐interecdysial period, abdominal movements in the pupae were always accompanied by heart activity beginning after the first 5–12 abdominal strokes. Simultaneous periods of abdominal movements and heartbeats lasted 2–5 min, while the intervening pause ranged from 40 to 72 min at 24°C. Experiments of forced heart activity showed that a slight external tactile stimulus (prodding once with a single hair), applied prior to an expected heartbeat bout, evoked abdominal movements followed soon afterwards by contractions of the dorsal vessel. Repeated prodding with a hair evoked body rotating movements (1–3 strokes) at any time with heartbeats starting at the first movement. We surmise that rhythmic body movements play an active role in the triggering of heart activity in pupae according to the principle of cardiac reflex response. This is a possible mechanism for synchronizing periods of heart activity with periods of rhythmic body movements. Haemolymph circulation in pupae is favoured when heartbeat bouts are accompanied by active body movements.     

短时低温对马铃薯甲虫种群增长的影响

[目的]马铃薯甲虫Leptinotarsa decemlineata是我国重要的检疫性害虫,对茄科植物危害严重.本研究旨在明确出现倒春寒短时低温对马铃薯甲虫种群增长的影响.[方法]马铃薯甲虫卵在8℃下分别处理1,3和5d,以27℃下饲养的卵作为对照,调查卵孵化率及孵化后幼虫的生长发育和成虫繁殖情况,用种群参数评估短时低...     

Adult diapause in the Colorado potato beetle, Leptinotarsa decemlineata: effects of external factors on maintenance, termination and post-diapause development

ABSTRACT. This study tests the effects of temperature, water, food and photoperiod on breaking diapause and resumption of activity (emergence from the soil) in Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) diapausing under natural winter conditions or at constant temperatures in the laboratory. Behavioural and physiological criteria were used to characterize the various phases of diapause. Three successive phases are distinguished during hibernation: 'diapause development' or true diapause, a facultative 'post-diapause quiescence' and, finally, a transient phase of post-diapause development leading to emergence from the soil. Diapause development is completed within 3 months in the field and its duration depends on temperature. Although they are buried in the soil during this phase, beetles remain sensitive to photoperiod when artificially exposed to it. They do not emerge from the soil when exposed to higher temperatures. Thereafter, they stay in a quiescent state maintained by low temperature, low humidity or lack of food. The response to temperature changes during hibernation. In the soil, activity begins when soil temperature reaches 4–5C, but this temperature is too low to permit postdiapause development. The transient phase has a temperature threshold between 8 and 10C, whereas emergence from the soil occurs only when the temperature exceeds 11C. Post-diapause development is influenced strongly by temperature and humidity. After emergence, post-diapause development leads eventually to reproduction. Food is essential for reproduction after diapause whereas photoperiod plays no further role.     

Effect of photoperiod and allatectomy on reproduction and (second) diapause induction in post-diapause adult females of the Colorado potato beetle,Leptinotarsa decemlineata Say

Summary

The effect of photoperiod and allatectomy on the rates of oviposition and juvenile hormone (JH) synthesis was studied in female beetles after the break of adult diapause. The results indicate that after diapause, photoperiod has no effect on oviposition rates. However, the photoperiodic effect on diapause induction is restored within a few days after the break of the first diapause. Allatectomy performed within one day after diapause did not induce a second diapause, whereas about 40% of the beetles allatectomized after 3 or more days responded by entering a second diapause. Oogenesis in the operated animals was never completely prevented under long and short day conditions. These preliminary results suggest a change in the sensitivity of post-diapause beetles to photoperiodic and endocrine manipulation as compared with pre-diapause insects.     

Differential responses of galeal gustatory sensilla of the adult Colorado potato beetle, Leptinotarsa decemlineata (Say), to leaf saps from host and non-host plants

ABSTRACT Responses of galeal sensilla of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), to leaf sap from Solanum tuberosum and a number of other solanaceous and non-solanaceous plants are described. A single cell responds vigorously to expressed sap from S.tuberosum , -and even to ten-fold dilutions of expressed sap. The response is characterized by a high spike frequency, low phasic activity and a tonic phase which can last for tens of seconds. At 8 s following stimulus application the primary response is typically 50% of its original activity, indicating slow adaptation to the sap stimulus. The primary response varies little among multiple stimulations of single sensilla and among sensilla on the same animal. There is a secondary response to potato leaf sap from one or more cells which have low spike amplitudes. This is usually of much lower frequency than the response from the primary cell, and it is highly variable.
The prominent and reproducible single cell response to potato sap may be an important component in signalling the presence of a host plant. This hypothesis is tested using saps from two other species of Solanum which are hosts, and several solanaceous and non-solanaceous non-hosts. Responses of galeal sensilla to the three host-plants tested were qualitatively similar and reproducible while responses to the non-hosts were highly variable and qualitatively different from responses to host saps. These results are interpreted and discussed in the context of a possible mechanism for chemosensory coding of complex stimuli in the insect/plant relationship.
Analysis of the raw electrophysiological data required extensive use of a microcomputer and several programs were developed during the course of this study. The computer analyses are briefly described in the paper and additional information and copies of the programs are available from the authors.     

马铃薯甲虫气味结合蛋白的序列和基因表达谱分析

【目的】昆虫气味结合蛋白(OBPs)在昆虫嗅觉行为中发挥着重要作用。马铃薯甲虫Leptinotarsa decemlineata是马铃薯上一种最主要的毁灭性害虫。为阐明该虫嗅觉识别分子机制,本研究对马铃薯甲虫26个OBP基因序列特征及组织表达谱进行研究。【方法】基于马铃薯甲虫触角转录组测序数据,利用生物信息学方法及qRT-PCR技术,分别对马铃薯甲虫26个LdecOBPs (LdecOBP1-LdecOBP26)的系统进化及基因的组织表达谱进行分析。【结果】除LdecOBP26基因外,其余25个LdecOBPs基因序列均具有完整的开放阅读框,编码120~255个氨基酸残基,预测的蛋白分子量为13.66~29.38 kD,等电点为4.12~8.42,它们属于两个亚家族,其中13个为Classical-C OBPs, 12个为Minus-C OBPs。除LdecOBP3和LdecOBP26外,其他24个LdecOBPs的N端均由16~23个氨基酸组成的信号肽序列。不同的OBPs亚家族均具有各自典型保守的Cys残基。LdecOBPs之间高度分化,氨基酸序列一致性在3.20%~41.91%。系统进化树分析表明,LdecOBPs与沙葱萤叶甲Galeruca daurica的GdauOBPs亲缘关系最近。基因表达谱分析显示,26个LdecOBPs基因在马铃薯甲虫的不同组织中表达,其中有12个LdecOBPs基因(LdecOBP2, LdecOBP4, LdecOBP6, LdecOBP9, LdecOBP10, LdecOBP12, LdecOBP13, LdecOBP16, LdecOBP20-22和LdecOBP24)在触角中高表达,2个LdecOBPs基因(LdecOBP5和LdecOBP17)在足中高表达,其他12个LdecOBPs基因(LdecOBP1, LdecOBP3, LdecOBP7, LdecOBP8, LdecOBP11, LdecOBP14, LdecOBP15, LdecOBP18, LdecOBP19, LdecOBP23, LdecOBP25和LdecOBP26)在触角、头(去除触角)、胸、腹、足和翅这些组织中均表达。【结论】本研究结果为进一步研究马铃薯甲虫嗅觉识别分子机制奠定了基础。