The bimodal adult activity of Monochamus alternatus (Coleoptera: Cerambycidae) caught in pheromone traps in Jeju can be explained by the competitive attractiveness of dying pine trees

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is known to be the primary vector of pinewood nematode Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle that causes pine wilt disease. Adult activity of M. alternatus caught in a pheromone trap on Jeju Island of Korea showed a bimodal form with the first peak in mid to late June and the second peak in mid to late September. The two peaks were separated between mid and late August, showing a valley. Accumulated degree‐days predicted that the emergence of the second generation adults could be possible just before the second peak. But actually no adults of the second generation occurred in the field development experiments of M. alternatus in 2016 and 2017. Pine trees without oleoresin flow (namely dying trees by the infection of pinewood nematode) were abundant during early July to early August. The bimodal adult activity pattern of M. alternatus could be partially explained by the competitive attractiveness of dying trees against pheromone traps, when we accepted the assumption that dying pine trees attract strongly M. alternatus.     

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.     

Oviposition deterrence associated with larval frass of the Japanese pine sawyer,Monochamus alternatus (Coleoptera: Cerambycidae)

To determine the effect of larval frass of Monochamus alternatus on oviposition preference of the female adults, three tests were performed in the laboratory. Individual females were provided with a frass-coated, Pinus densiflora bolt and an untreated bolt simultaneously and were allowed to oviposit for 24 h. They deposited a significantly smaller number of eggs on frass-coated bolts. The females supplied with frass-coated bolts deposited no eggs on them, indicating that the larval frass deterred the females from oviposition. When individual females were provided simultaneously with a pine bolt applied with a methanol extract of larval frass and another bolt applied with methanol alone, they deposited a significantly smaller number of eggs on frass extract-applied bolts for 24 h. These results suggest the presence of a putative oviposition deterrent in larval frass of M. alternatus. Each test was replicated 19-20 times using different females.     

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.     

Genetic differentiation of the pine wilt disease vector Monochamus alternatus (Coleoptera: Cerambycidae) over a mountain range - revealed from microsatellite DNA markers

To study the dispersal process of the pine sawyer Monochamus alternatus (Hope) in frontier populations, a microsatellite marker-based genetic analysis was performed on expanding populations at the northern limit of its range in Japan. In Asian countries, M. alternatus is the main vector of pine wilt disease, the most serious forest disease in Japan. Sawyers were collected from nine sites near the frontier of the pine wilt disease damage area. A mountain range divides the population into western and eastern sides. Five microsatellite loci were examined and a total of 188 individuals was genotyped from each locus with the number of alleles ranged from two to nine. The mean observed heterozygosity for all loci varied from 0.282 to 0.480 in the nine sites, with an overall mean of 0.364. None of the populations have experienced a significant bottleneck. Significant differentiation was found across the mountain range, but the genetic composition was similar amongst populations of each side. It is believed that the mountain range acts as a geographical barrier to dispersal and that gene flow without a geographical barrier is high. On the west side of the mountain range, a pattern of isolation by distance was detected. This was likely to be caused by secondary contact of different colonizing routes on a small spatial scale. Based on these data, a process linking genetic structure at local (kilometres) and regional spatial scales (hundreds of kilometres) was proposed.     

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.     

Smoke,pheromone and kairomone olfactory receptor neurons in males and females of the pine sawyer Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae)

The response of antennal olfactory receptor neurons (ORNs) of Monochamus galloprovincialis to several odourants was tested using single sensillum electrophysiology. Behaviourally active pheromone, and kairomone (host and sympatric bark beetle pheromone) odours were tested alongside smoke compounds released by burnt wood that are potentially attractive to the insect. The antennae bore several types of sensilla. Two plate areas in the proximal and distal ends of each antennal segment were covered with basiconic sensilla that responded to the odour stimuli. Sensilla basiconica contained one or two cells of different spike amplitude. The 32 male and 38 female ORNs tested responded with excitations or inhibitions to the different plant odours. In general the response of male and female receptors was very similar so they were pooled to perform a cluster analysis on ORN responses. Six ORNs were clearly specialised for pheromone reception. Responses to kairomone and smoke odours were less specific than those of pheromone, but a group of 9 cells was clearly excited by smoke compounds (mainly eugenol and 4-methyl 2-methoxyphenol), a group of 8 cells was very responsive to α-pinene, β-pinene and cis-verbenol, and a group of 14 cells responded to a wider range of compounds. The rest of the cells (47%) were either non-responsive or slightly inhibited by smoke compounds. Dose–response curves were obtained for several compounds. Different compounds induced significantly different latencies and these appeared to be unrelated to their boiling point.     

Application of harmonic radar to analyze dispersal behavior of the Japanese pine sawyer beetle,Monochamus alternatus (Coleoptera: Cerambycidae)

Monochamus alternatus (Hope) is a severe wood‐boring pest in coniferous forests and a major vector of pine wilt disease in East Asia. Harmonic radar is a powerful tool for studying the dispersal behavior of insects and it could be applied to control pine wilt disease. In this study, we validated the application of harmonic radar for analyzing the dispersal behavior of M. alternatus beetles in a field environment. We determined the wing capacities of the beetles and the effects of electronic tagging and marking on their movement, flight ability, survivorship, and food consumption in the laboratory to confirm the suitability of this technique. The detection rate and recovery rate for beetles were analyzed separately using radar on caged pine stands and in the field environment. The results showed that the minimum wing capacity of the Japanese pine sawyer was 24.9 mg, which was seven times the weight of the electronic tag (3.5 mg). Marking and tagging the beetles had no significant adverse effects on their movement, flight capacity, food consumption, and survivorship. The detection rate using the radar system and recovery rate based on visual observations of the beetles in caged pines were both 95.6%. However, in the field environment, the detection and recovery rates were only 55.6% and 37.8% after one week, respectively, and 33.3% and 7.8% after two weeks. Harmonic radar is a promising technique for studying the dispersal behavior of the Japanese pine sawyer, but its performance is not satisfactory and major improvements are required for both the radar system and electronic tags.     

松墨天牛对寄主树木的产卵选择

在室内采用选择行为方法,研究了松墨天牛Monochamus alternatus对几种松属植物的产卵选择行为。结果表明,松墨天牛对黑松Pinus thunbergii的选择性最强,对马尾松P.massoniana的选择性最弱,二者间差异显著,它们与火炬松P. taeda和湿地松P. elliottii间无明显差异。在黑松和马尾松的被松材线虫危害木与健康木之间,松墨天牛明显选择被害木产卵;具产卵痕枝段和虫粪处理过的枝段,对松墨天牛的产卵有显著抑制作用,松墨天牛明显地选择对照枝段产卵。     

中国松材线虫病空间分布格局

应用地统计学和克里格插值方法,分析了松材线虫病在中国的空间分布结构和空间相关性.研究表明,松材线虫病在中国呈聚集分布.在全国和江苏、安徽、广东、浙江5个不同空间尺度下,半变异函数均为球形,空间依赖性距离分别可达10 1888°、3.4464°、2.1581°、3.08°和2.4376°.松材线虫病在中国有着2个明显的聚集分布区域,一个位于(30.5～32.5°N,117.7～120.5°E),以南京为中心,包括江苏、安徽大部和浙江西北部,另一个位于(22.5～24°N,113～114.5°E ),在广东境内.松材线虫病在中国早期空间分布自然扩散模型为R = 5.4743e0.4139t(R2 = 0.9204).     

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。     

Temperature Effects on the Transmission of Bursaphelenchus xylophilus (Nemata: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae)

The object of this research was to investigate the effects of ambient temperature on the transmission of the pinewood nematode, Bursaphelenchus xylophilus, by its vector, Monochamus alternatus. Ninety M. alternatus were reared individually at one of three constant temperatures (16, 20, and 25 °C). As the ambient temperature decreased from 25 to 16 °C, longevity of vectors decreased, nematode transmission efficiency decreased, and the peak period of nematode transmission was delayed and its peak height decreased. Low temperature may inhibit the transmission process, and this inhibition could be partially responsible for preventing pine wilt disease from devastating pine forests in cool regions.     

Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera, Cerambycidae)

A mitochondrial DNA (mtDNA) study, based on 43 European populations (33 of them sampled in France) of Monochamus galloprovincialis , vector of the pinewood nematode, and 14 populations of its sister species Monochamus sutor was realized. Sequencing of 792 bp of the cytochrome oxidase I (COI) and 521 bp of the COII genes revealed numerous ambiguities on multiple nucleotide sites for half of M. galloprovincialis specimens studied (44.8%). Hypotheses of heteroplasmy and pseudogenes ( Numts ) were examined. The mtDNA isolation by alkaline lysis and cloning (for three successfully used individuals) both support the hypothesis that the ambiguous sequences amplified were not of mtDNA nature and validate the presence of Numts in the nuclear genome of M. galloprovincialis . Multiple copies of mtDNA-like sequences were found paralogous to COI, tRNA leucin and COII regions. Phenetic analysis placed different recently diverged mtDNA-like sequences as a close relative of mtDNA sequences, and grouped 10 closely related mtDNA-like sequences as a more basal clade, closer to ancestral states and to M. sutor . This result supports that this nuclear family of pseudogenes arose independently of the other events and may represent mitochondrial haplotypes sampled from M. galloprovincialis ancestral populations. This is the first time that Numts are proved for a longhorned beetle, whereas no Numts were found within its sister species M. sutor. The incorporation mechanism of Numts in unknown for M. galloprovincialis , however, excess of ambiguous sites corresponding to synonymous mutations placed on third codon position as well as the absence of Numts in M. sutor , conducted to the hypothesis of a recent transfer of these Numts in the nuclear genome of M. galloprovincialis .     

Synergistic attraction of pine sawyer Monochamus saltuarius (Coleoptera: Cerambycidae) to monochamol and α‐pinene

Monochamus (Coleoptera: Cerambycidae) species are longhorn pine sawyers that serve as insect vectors of the pinewood nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae), which are responsible for debilitating pine wilt disease. An aggregation pheromone, 2‐(1‐undecyloxy)‐1‐ethanol (hereafter referred to as monochamol), was shown to be effective at attracting Monochamus species. However, attraction of the pine sawyers to aggregation pheromones varied depending on semiochemicals, including host plant volatiles and kairomones. In this study, we investigated the abilities of monochamol and the host‐plant volatiles α‐pinene and ethanol to attract M. saltuarius in a pine forest in Cheongsong, Gyeongsangbuk‐do, Korea. A total of 91 M. saltuarius (28 males and 63 females) were captured. The combination of monochamol (700 mg) with α‐pinene and ethanol exhibited a synergistic effect on attracting M. saltuarius (11.0 beetles per trap), whereas monochamol alone and a mixture of α‐pinene and ethanol resulted in the capture of 3.2 beetles and 3.6 beetles per trap, respectively. Our results suggest that multi‐funnel traps baited with a blend of monochamol, α‐pinene and ethanol are highly effective for monitoring M. saltuarius and M. alternatus in pine forests.     

松褐天牛的交配行为

采用室内试验和野外观察相结合的方法,对松材线虫病的主要媒介昆虫松褐天牛Monochamus alternatus Hope的交配行为进行了研究。结果表明： 松褐天牛一次完整的交配包括相遇抱对、插入输精和配后保护3个阶段,在交配过程中雄虫有多次插入输精现象发生。室内试验中共观察到松褐天牛的交配123次,松褐天牛一次完整的交配过程平均需时63.49 min,其中输精前的抱对时间平均为1.68 min,交配过程中每次输精插入时间平均为57.60 s,配后保护时间为15.18 min。松褐天牛在开始交配的4天内平均交配5.15次,不同雄性个体所获得的交配机会差异很大。松褐天牛的交配行为表现出强烈的雄性竞争现象,雄虫能根据雌虫或自身的交配经历调整交配投入,当雌虫或者雄虫是初次交配时,总输精时间和插入输精的次数显著大于与有交配经历的雌虫或雄虫交配时的输精时间和插入输精次数。田间松褐天牛的交配行为与室内观察结果基本一致。     

Transmission of Pinewood Nematode Through Feeding Wounds of Monochamus carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode, Bursaphelenchus xylophilus, to mature, field grown Scots pines through feeding wounds of Monochamus carolinensis was investigated by caging nematode-infested beetles on pine branches for 24 hours. Nematodes were transmitted to 31 of 64 branches. Frequency of successful transmission was independent of the sex of the beetle but dependent upon beetle age. Transmission frequencies were highest for beetles 2, 3, and 4 weeks after emergence as adults. The number of nematodes transmitted per branch was low and did not differ between beetle sexes or among beetle age categories. The number of nematodes extracted per branch was correlated with the number of nematodes carried per beetle but was not correlated with the feeding area on the branch.     

Attractants for management of the pine sawyer beetle Monochamus sutor,a potential vector of Bursaphelenchus xylophilus

Monochamus sutor (Linnaeus) (Coleoptera: Cerambycidae) is a secondary wood borer that has been hypothesized as capable of transmitting Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD). This fact supposes a risk of spread of PWD over Europe and has created an urgent need for effective tools to detect and monitor both the nematode and the insect species that vectors it. Recent reporting of 2‐undecyloxy‐1‐ethanol as the M. sutor male‐produced aggregation pheromone has opened the possibility of developing an efficient lure for this species. It is known that some European bark beetle pheromone compounds and host volatiles kairomonally attract this species. Besides, smoke volatiles from burnt trees might play a role in M. sutor host location. In this work, field trapping experiments during 3 years in three countries (Spain, Sweden and Austria), aimed to develop an efficient pheromone‐kairomone lure operative for M. sutor management were carried out. Electroantennographic responses by M. sutor to Ips pheromones and to the Pityogenes chalcographus pheromone chalcogran were also studied. GC‐EAG recording showed that M. sutor males and females clearly responded to ipsenol and ipsdienol, and females also responded to 2‐methyl‐3‐buten‐2‐ol. Chalcogran elicited a response to M. sutor female antennae. In field tests, ipsenol was the most attractive kairomone to both sexes of M. sutor, whereas ipsdienol, cis‐verbenol and 2‐methyl‐3‐buten‐2‐ol were attractive and chalcogran was unattractive. When combined with the pheromone, most bark beetle kairomones increased catches of both sexes although chalcogran was completely ineffective. Thus, ipsenol was the strongest individual kairomone for M. sutor and the best single kairomone to be combined with the pheromone. Smoke volatile blends tested in Spain and Austria did not elicit responses, suggesting that these compounds are likely not involved in host finding by this species.     

Evolutionary relationships within European Monochamus (Coleoptera: Cerambycidae) highlight the role of altitude in species delineation

Phylogenetic relationships within the European Monochamus (Coleoptera: Cerambycidae) remain understudied despite their increasing importance in the Pine Wood Nematode spread in Europe. To clarify the delimitation and the evolutionary history of the two main European Monochamus species, Monochamus galloprovincialis and Monochamus sutor, as well as their sub‐species, a comparative study using morphological, molecular, and biogeographical criterions was conducted. Four morphological characters, including a newly‐described morphological character on the male genitalia, separated the two species. Additionally, molecular data revealed twelve and two single nucleotide polymorphisms in cytochrome oxidase c subunit I and 28S, respectively, supporting species segregation. By contrast, incongruence between morphological and genetic results did not allow discriminating the sub‐species of M. galloprovincialis and M. sutor, even though mitochondrial DNA revealed intraspecific differentiation, mostly consenting to a multiple refugia origin. Within‐species variability was explained to a large extent by biogeography (i.e. altitude, climate). These different ecological adaptations within beetle species, together with potential climate change impact, increase the risk of spreading the nematode across Europe to novel conifer hosts and challenge the European biosecurity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 354–376.     

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.     

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.