A new method for loading Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on adult Monochamus alternatus (Coleoptera: Cerambycidae)

A new method was developed for loading the pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, on the beetle Monochamus alternatus Hope. Postdiapause beetle larvae were sterilized with 70 and 99.9% aqueous ethanol and placed singly in flasks where B. xylophilus reproduced on the fungus Ophiostoma minus (Hedgcock) H. et P. Sydow that had been grown on autoclaved barley grain and Pinus densifiora Sieb. et Zucc. wood chips. The fungus produced a large nematode population that developed to a high proportion of third-stage dispersal juveniles that molted to the fourth-stage dispersal juveniles. The survival rate was 80%, and the mean nematode load was 10,096. It took a mean of 5 wk to obtain the nematode-infested beetles after the initiation of nematode rearing.     

Canopy-related adult density and sex-related flight activity of Monochamus alternatus (Coleoptera: Cerambycidae) in pine stands

Monochamus alternatus Hope is an important vector of the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, which causes pine wilt disease. To determine canopy-related differences in the activity density of M. alternatus adults in pine stands, sticky screen traps with no lure were suspended under and in the canopy at 3.8 and 10 m above the ground of a Pinus densiflora Siebold et Zuccarini stand during a flight season. The five upper and five lower traps caught 338 and 54 adults, respectively, during a flight season from 9 June to 12 October 2011, indicating that activity density was six times as high in the canopy as under it. Male-biased sex ratio was observed for trap catches during a season: The sex ratio was male-biased in the early half of the flight season and 1:1 in the late half. There was no difference in sex ratio between the inside and outside of the canopy. To determine the sex-related difference in flight activity, the adults were captured by two different measures in two other mixed stands of P. densiflora and P. thunbergii Parlatore. Kicking tree trunks to cause beetles to drop revealed a 1:1 sex ratio of catches, whereas sticky screen traps tended to show a male-biased sex ratio in a 4-year study, suggesting that males were more active fliers than females.     

温度对松褐天牛传递松材线虫数量的影响

松材线虫病是一种重要的检疫性病害,松褐天牛是传播松材线虫的主要媒介昆虫.本实验研究了温度对松褐天牛传递松材线虫数量的影响.设置22℃、28℃和34℃ 3个温度梯度,结果表明松褐天牛成虫的寿命随温度升高而缩短,其中雌虫寿命短于雄虫,且羽化进度不太一致,多数松褐天牛携带的松材线虫量在1000～9999条之间.通过对松褐天牛取食传入松枝的松材线虫量的追踪记录发现,随着温度升高,传播线虫的高峰时段随之提前.34℃高温下松褐天牛的传递率最低,表明高温对松材线虫病的发生具有抑制作用.     

Deterred oviposition response of Monochamus alternatus (Coleoptera: Cerambycidae) to oviposition scars occupied by eggs

1 The oviposition behaviour and response of Monochamus alternatus females to oviposition scars were investigated in the laboratory. 2 Prior to oviposition, females gnawed at the bark surface of Pinus densiflora bolts to make a wound. Then females turned their bodies 180° to position their ovipositors over the wounds and inserted them under the bark through the wounds. After an oviposition, a jelly was deposited while the ovipositor was still inserted. The females then withdrew their ovipositors and rubbed the oviposition scars with the tips of their abdomens. 3 When searching females encountered oviposition scars, they stopped walking and drummed the surface and inside of the oviposition scars with their maxillary and labial palpi. 4 Eighty-six percent of females left oviposition scars containing single eggs after the palpation. By contrast, when females encountered oviposition scars containing no eggs, 76% of them began to gnaw at the scars and 64% deposited single eggs. The response to artificial oviposition scars was similar to that to vacant oviposition scars made by the females. 5 The results of various observations and experiments showed that the females could recognize oviposition scars and discriminate the scars occupied by single eggs from vacant ones, and suggested that the palpation of oviposition scars was the critical discrimination behaviour, indicating mediation by chemical cues.     

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae)

The Japanese pine sawyer, Monochamus alternatus , is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point (SCP) and cumulative probability of individuals freezing (CPIF). The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle's death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or < 0 °C, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.     

Functional response of the parasitoid Sclerodermus sp.(Hymenoptera:Bethylidae) to the third instar larvae of host Monochamus alternatus ( Coleoptera: Cerambycidae)

为评估松褐天牛肿腿蜂Sclerodermus sp.(膜翅目:肿腿蜂科)对其寄主松褐天牛Monochamus alternatus 3龄幼虫的控制作用,通过功能反应试验,研究了松褐天牛肿腿蜂对松褐天牛3龄幼虫补充营养时期的捕食功能反应和补充营养后的寄生功能反应以及肿腿蜂的密度效应.结果表明:当松褐天牛肿腿蜂密度固定为1头/缸时,肿腿蜂因补充营养对松褐天牛3龄幼虫的捕食功能反应符合Holling Ⅱ型方程,1头松褐大牛肿腿蜂雌蜂对松褐天牛3龄幼虫的最大致死量为9.48头,平均为3.75头;寄生作用的功能反应符合Holling Ⅰ型方程,可知肿腿蜂的寄主密度阈限为4头就能满足补充营养和寄生的需要;在捕食和寄生两个阶段其对松褐天牛幼虫的寻找效应随着寄主密度的增加呈线性减弱.当松褐天牛幼虫密度固定为30头/缸时,随着肿腿蜂的密度增加,其补充营养阶段的捕食作用和之后的寄生作用均呈线性升高,寻找效应则呈线性减弱;由蜂虫比和致死总量的模型可得肿腿蜂与天牛幼虫比例为0.9805(近1∶1)时,可使松褐天牛幼虫死亡总数最大.这些结果表明,松褐天牛肿腿蜂是松褐天牛幼虫期的有效天敌.本研究为评价松褐天牛肿腿蜂对寄主的控制能力提供了基础数据和方法,并为其在野外的释放量提供了依据.     

Prevalence of laterally transferred Wolbachia genes in Japanese pine sawyer,Monochamus alternatus (Coleoptera: Cerambycidae)

Lateral gene transfer is any process in which an organism incorporates genetic material from a phylogenetically distant and reproductively isolated organism. A previous survey demonstrated that two Japanese populations of Monochamus alternatus Hope carried many Wolbachia genes that had been acquired by lateral gene transfer in their own genomes, but were not infected with Wolbachia. To understand the prevalence of Wolbachia infection and laterally transferred Wolbachia genes in this beetle, we performed a broader survey of natural populations covering two subspecies of M. alternatus from Japan (M. alternatus endai) and Taiwan (M. alternatus alternatus). We detected laterally transferred Wolbachia genes in all Japanese and Taiwanese populations of M. alternatus, but no Wolbachia infection in any population. In addition, we confirmed the absence of Wolbachia infection and of transferred Wolbachia genes in two Japanese populations of Monochamus saltuarius Gebler, which is a congeneric relative of M. alternatus. Our findings suggest that the Wolbachia endosymbiont as gene donor has disappeared from M. alternatus, and that the transfer of part of its genome to M. alternatus occurred in the ancestor of M. alternatus before the subspeciation event.     

Inhibitory Effect of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on B. xylophilus Boarding Adult Monochamus alternatus (Coleoptera: Cerambycidae)

Inhibitory effects of Bursaphelenchus mucronatus on the number of B. xylophilus carried by an adult Monochamus alternatus were investigated using artificial pupal chambers. When pupal chambers were infested with either B. xylophilus or B. mucronatus, the load of B. xylophilus onto the beetle was greater (P < 0.001) than that of B. mucronatus. However, within the pupal chamber there was no difference in the abundance of the third-stage dispersal juveniles, which would molt to the fourth-stage dispersal juveniles to board beetles. The nematode load on beetles that emerged from pupal chambers infested with both Bursaphelenchus species was smaller (P = 0.015) than that of beetles with B. xylophilus alone but greater (P < 0.001) than that of beetles with B. mucronatus alone, suggesting an inhibitory effect of B. mucronatus. As a result of this study, the rate of inhibition of B. mucronatus on molting of third-stage dispersal juveniles of B. xylophilus to fourth-stage dispersal juveniles was 0.65, which resulted in great inhibition on boarding beetles at a rate of 0.7.     

A male-produced aggregation pheromone of Monochamus alternatus (Coleoptera: Cerambycidae), a major vector of pine wood nematode

The beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an efficient vector of pine wood nematode, the causal pathogen of pine wilt disease, that has resulted in devastating losses of pines in much of Asia. We assessed the response of adult M. alternatus to 2-(undecyloxy)-ethanol, the male-produced pheromone of the congeneric M. galloprovincialis Dejean, in field experiments in Fujian Province, People's Republic of China. Both sexes of M. alternatus were attracted to lures consisting of 2-(undecyloxy)-ethanol combined with the host plant volatiles alpha-pinene and ethanol. A follow-up experiment showed that 2-(undecyloxy)-ethanol was synergized by both ethanol and alpha-pinene. Coupled gas-chromatography mass-spectrometry analyses of volatiles sampled from field-collected beetles of both sexes revealed that 2-(undecyloxy)-ethanol was a sex-specific pheromone component produced only by males. The combination of 2- (undecyloxy) -ethanol with ethanol and/or alpha-pinene will provide a valuable and badly needed tool for quarantine detection, monitoring, and management of M. alternatus.     

Role of host volatiles in mate location by the Japanese pine sawyer, Monochamus alternatus hope (Coleoptera: Cerambycidae)

We evaluated the responses of male and female Monochamus alternatus Hope (Coleoptera: Cerambycidae) to various terpenes commonly associated with host trees. Electroantennogram (EAG) tests were conducted with 12 plant volatile compounds and ethanol. Antennae of both sexes were highly sensitive to (R)-(+)-alpha-pinene, (+)-3-carene, (-)-beta-pinene, and terpinolene. Both sexes of M. alternatus were attracted by traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene. Our results support the first of the three-stage hypothesis posed by Ginzel and Hanks that suggests that location of stressed trees by cerambycids involves three stages: (1) both sexes locate larval hosts by using plant volatiles as kairomones; (2) males produces sex pheromones to attract females after both sexes land on the larval hosts; (3) males and female recognize each other by contract pheromones in their epicuticular wax layer. Males and females showed differences in their EAG responses to several compounds, including (R)-(+)-alpha-pinene, (-)-beta-pinene, myrcene, (+)-3-carene, (R)-(+)-limonene, terpinolene, and trans-caryophyllene. In all cases, males exhibited greater sensitivity than females. In laboratory assays, male M. alternatus showed strong preference for 1% (+)-alpha-pinene and 1% (-)-beta-pinene over other compounds. In field assays, traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene caught more beetles than control traps. We found strong male bias in beetle catches in baited traps and those captured on the stem of stressed trees despite a strong female bias in emerging beetles in 2004. We hypothesize that male M. alternatus are more responsive than females to plant volatiles and that males have more capacity than females in finding mating locations.     

聚集性信息素和植物源信息素对松墨天牛的联合诱捕作用

我们评价了聚集性信息素和植物源信息素对松墨天牛Monochamus alternatus Hope(Coleoptera:Cerambycidae)在林间的联合诱捕作用,以期研制出更优的引诱剂控制松墨天牛成虫的危害。结果显示,樟子松墨天牛M.galloprovincialis雄虫挥发出来的聚集性信息素2-undecyloxy-1-ethanol对松墨天牛雌雄成虫均有引诱效果,而且樟子松墨天牛性信息素与植物源信息素之间相互增效作用十分显著,比性信息素或植物源信息素单独使用效果提高4⁵倍。松墨天牛雌虫卵巢发育情况显示,成虫期在浙江地区大约持续4个月,松墨天牛成虫期可以分为两个阶段,第一个阶段为取食期(卵巢未成熟),从5月9日到7月9日;第2个阶段为产卵期(卵巢成熟),从6月3日到9月18日,两个阶段在一段时期有重叠。聚集性信息素和植物源信息素联合应用将在防控松墨天牛危害,遏制松材线虫病传播方面发挥重要作用。     

Microarray gene expression analysis of Monochamus alternatus (Coleoptera: Cerambycidae) after treatment with a sublethal dose of chloramine phosphorus

Monochamus alternatus Hope (Coleoptera: cerambycidae), the Japanese pine sawyer beetle, is a serious pest management concern in pine stands. A new organophosphorus insecticide, chloramine phosphorus (CP), has been applied as an insecticide in China to control M. alternatus. In this study, we investigated gene expression changes in M. alternatus after 4 h of exposure to CP using a 60-mer oligonucleotide microarray. The results showed that 356 genes were differentially expressed, of which 76 were upregulated and 280 were downregulated. GO enrichment analysis of the differentially expressed genes indicated that those involved in structural molecule activity and transporter activity were among the most highly represented. Within the cellular component category of GO, cell and cell part were the most represented GO terms in upregulated genes. “Cellular process” was the most represented GO term in the biological process category among the downregulated genes. Molecular pathway analysis showed that a large portion of the differentially expressed genes were associated with metabolic pathways. This study sought to identify new biomarkers as nonacetylcholinesterase targets to assess the secondary effects of CP on M. alternatus and will provide a valuable resource for the scientific community to study the molecular toxicology of CP.     

Fluorescein-5 isothiocyanate conjugated-chitin-binding domain probe (FITC-CBD)-coupled detection of chitin in the peritrophic membrane of Monochamus alternatus (Coleoptera: Cerambycidae)

This study investigated the potential of a fluorescein-5 isothiocyanate conjugated-chitin-binding domain (FITC-CBD) probe to detect chitinous materials in the peritrophic membrane (PM) structure of the Japanese pine sawyer beetle, Monochamus alternatus. Results through direct observations indicated that the fluorescent probe specifically bound to chitin, and specifically labeled the chitinous materials in the PM. Applying the probe was easy, as fluorescence was stable, and the excitation maximum of the structures stained with the probe was within a range covered by most existing fluorescence microscopes. These advantages make fluorescence staining an easy method of choice for specifically visualizing the chitin-rich internal structures of insects. Additionally, this simple approach might have the added advantage of revealing chitinolysis events in vivo and in vitro.     

松墨天牛化学感受组织荧光定量PCR内参基因的鉴定与筛选

【目的】本研究拟选择适合用于分析松墨天牛Monochamus alternatus化学感受组织中基因表达的内参基因。【方法】依据转录组测序结果进行内参基因鉴定,利用RT-q PCR技术分析内参基因在松墨天牛不同发育阶段和不同性别化学感受组织间的表达差异,并利用软件ge Norm,Norm Finder和Best Keeper比较其表达的稳定性。【结果】松墨天牛转录组中鉴定出9个候选内参基因(Actin,TUB,18S rRNA,RPS27A,RPS3,RPL10,AK,GAPDH和EF1A),其中后7个候选内参基因在松墨天牛中被首次鉴定,松墨天牛候选内参基因和其他昆虫相应基因的同源性很高。9个候选内参基因引物均具有良好的扩增效率,18S rRNA的表达水平最高,EF1A的表达水平最低;18S rRNA和Actin在不同样品间的表达水平差异最大,GAPDH和TUB表达水平在不同样品间差异最小。ge Norm和Norm Finder软件分析认为,GAPDH是最稳定的内参基因,TUB是较为稳定的内参基因,18S rRNA和Actin是最不稳定的内参基因;Best Keeper软件分析认为,GAPDH和TUB是合适的内参基因,18S rRNA和Actin是不适合的内参基因。最适合校正松墨天牛化学感受组织中基因表达数据的内参基因数量为2个,即GAPDH和TUB,并且这样的内参基因组合可以用于不同发育阶段和不同性别的不同化学感受组织。【结论】本研究结果为利用RT-q PCR技术准确分析松墨天牛和其他天牛基因包括化学感受组织基因相对表达量的内参基因选择提供了重要参考。     

Reproductive traits of Monochamus galloprovincialis (Coleoptera: Cerambycidae) under laboratory conditions

The pine sawyer Monochamus galloprovincialis (Olivier) is the vector of the introduced pine wood nematode Bursaphelenchus xylophilus (Steiner & Bührer) Nickle in Portugal, and until recently was considered a secondary forest insect. Under laboratory conditions, a study of biological and reproductive traits of 37 insect pairs was conducted. The longevity of both sexes was similar, being 61.2+/-6.5 days for males and 64.0+/-6.3 days for females (mean+/-SE). Sixteen small-sized insects (22% of the population) died within 20 days and before starting to reproduce. The sexual maturation period (without egg laying) was 20.4+/-0.7 days (mean+/-SE), while the oviposition period lasted 54.0+/-4.2 days (mean+/-SE). The oviposition rate increased very quickly during the first weeks of life, peaking to almost two eggs per day during days 30-44, and gradually dropping in the following weeks. The females laid an average of 67.0+/-5.96 (mean+/-SE) eggs through their lives. The hatch rate was 92.6+/-1.0% (mean+/-SE). There were large individual variations in longevity and fecundity parameters, and principal component analysis based on 16 morphological and biological parameters separated the breeding insects into four distinct groups. Almost half of the reproducing beetles were large-sized insects, with high longevity and fecundity traits. Overall, the reproductive potential of the Portuguese population of M. galloprovincialis seems to be smaller than that described for other Monochamus vectors of the pine wood nematode both in North America and Japan.     

Sensilla on the antennal flagellum of the sawyer beetles Monochamus notatus (Drury) and Monochamus scutellatus (Say) (Coleoptera: Cerambycidae)

The antennae of the sawyer beetles Monochamus notatus and M. scutellatus were examined with the light and scanning electron microscopes to determine the types, number, distribution and innervation of the sense organs. Nine types of sensilla are described. Both short, thin-walled pegs (sensilla basiconica) and reversely curved thick-walled hairs (sensilla trichodea) are chemoreceptors. There are three types of long, thick-walled hairs (sensilla chaetica) which may be mechanoreceptors. One of these is modified in males to form unique snail-shaped pegs. A few dome-shaped organs, probably campaniform sensilla, were found. In addition to sense organs, many glands occur in association with the sensilla, and the antennae are well supplied with dermal glands connected by canals to small pores on the surface.     

Influence of larval frass extracts on the oviposition behaviour of Monochamus alternatus (Col., Cerambycidae)

Abstract:  The oviposition behaviour and response of adult females of Monochamus alternatus to short lengths (bolts) of Pinus massoniana trunks treated with hexane extracts of larval frass of M. alternatus was investigated in the laboratory. Females gnawed significantly fewer oviposition scars, and deposited significantly fewer eggs, on extract-treated bolts than on control bolts. These results suggest the presence of a possible oviposition deterrent in larval frass of M. alternatus . Hexane extracts of larval frass were analysed by gas chromatography–mass spectrometry, and α -pinene, β -pinene, 3-carene, limonene, longifolene and butylated hydroxytoluene were identified. Experiments with synthetic mixtures indicated that a mixture of monoterpenes and butylated hydroxytoluene exhibited oviposition-deterring activity.     

Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes)

We examined the amount of maturation feeding and transmission of pinewood nematodes, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Nematoda: Parasitaphelenchidae), to healthy pine (Pinus spp.) trees by pine sawyer Monochamus alternatus Hope (Coleoptera: Cerambycidae) adults infected with Beauveria bassiana (Balsamo) Vuill. (Deuteromycotina: Hyphomycetes). Inoculated beetles fed less than noninoculated beetles, probably because feeding by inoculated beetles began to decrease at about 4 d postinoculation and inoculated beetles ceased to feed for several days before their death. In inoculated beetles carrying >1,000 nematodes, some beetles died before nematode departure. The remaining heavily nematode-infested beetles lived until the beginning of nematode departure, but they had stopped feeding, preventing the nematodes from entering pine twigs. We suggest that microbial control of pine sawyer adults by B. bassiana may be effective in preventing transmission of pine wilt disease to healthy pine trees.     

Pine wilt disease in Yunnan,China: Evidence of non-local pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae) populations revealed by mitochondrial DNA

Abstract Monochamus alternatus (Hope) specimens were collected from nine geographical populations in China, where the pinewood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) was present. There were seven populations in southwestern China in Yunnan Province (Ruili, Wanding, Lianghe, Pu’er, Huaning, Stone Forest and Yongsheng), one in central China in Hubei Province (Wuhan), and one in eastern China in Zhejiang Province (Hangzhou). Twenty-two polymorphic sites were recognized and 18 haplotypes were established by analyzing a 565 bp gene fragment of mitochondrial cytochrome oxidase subunit II (CO II). Kimura two-parameter distances demonstrated that M. alternatus populations in Ruili, Wanding and Lianghe (in southwestern Yunnan) differed from the other four Yunnan populations but were similar to the Zhejiang population. No close relationship was found between the M. alternatus populations in Yunnan and Hubei. Phylogenetic reconstruction established a neighbor-joining (NJ) tree, which divided haplotypes of southwestern Yunnan and the rest of Yunnan into different clades with considerable bootstrapping values. Analysis of molecular variance and spatial analysis of molecular variance also suggested significant genetic differentiation between M. alternatus populations in southwestern Yunnan and the rest of Yunnan. Our research suggests that non-local populations of M. alternates, possibly from eastern China, have become established in southwestern Yunnan.