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.     

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.     

Influences of host volatiles on feeding behaviour of the Japanese pine sawyer, Monochamus alternatus

Abstract: Choice and no‐choice feeding assays on the twigs of three host species demonstrated the following feeding preference sequence by Monochamus alternatus: Pinus massoniana > Cedrus deodara > Pinus thunbergii. There were significant differences in the concentrations of α‐pinene, camphene, d ‐limonene, β‐phellandrene, longifolene and β‐caryophyllene in volatiles emitted by twigs among the three species. We tested the effects of six monoterpenes (α‐pinene, β‐pinene, 3‐carene, myrcene, limonene and β‐caryophyllene) added to an artificial diet consisting of bark from P. thunbergii on consumption rates by M. alternatus. The addition of α‐pinene at all four concentrations 0.4, 1.2, 3.6 and 10.8 μl/ml resulted in increases of a twofold greater consumption rate than the control at a concentration of 3.6 μl/ml. Limonene inhibited diet consumption at concentrations >0.4 μl/ml. The concentration of α‐pinene in volatiles emitted by twigs was significantly higher for P. massoniana than for P. thunbergii, whereas the reverse was true for limonene. There were no differences for any of the other host components, suggesting that α‐pinene and limonene may play an important role in the adult's selection and acceptance of suitable and unsuitable feed host. Mixed compounds promoted the consumption of artificial diet at a concentration of 0.4 μl/ml, whereas consumption was inhibited at a concentration of 10.8 μl/ml. There were significant linear correlations (β‐pinene: r² = 0.930, P < 0.05; myrcene: r² = 0.933, P < 0.05) between the amount of diets consumed and diet concentrations of β‐pinene and myrcene. In conclusion, host volatile terpenes may stimulate or repel M. alternatus depending on terpene concentrations they encounter during initial feeding and then possibly inhibit further feeding activity once concentrations increase to threshold levels.     

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.     

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。     

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.     

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer,Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.     

Heterologous expression and characterization of endocellulase from the Japanese pine sawyer Monochamus saltuarius (MsGHF5)

In this study, the endocellulase gene from Monochamus saltuarius (MsGHF5) was transformed into Escherichia coli (RosettaBlue(DE3)pLysS strain), and induced by IPTG. The molecular weight of recombinant MsGHF5 (rMsGHF5) was 78 kDa and was expressed as a fusion protein with maltose binding protein in pMAL‐c2 expression vector. Native‐PAGE was conducted with 0.1% carboxymethyl cellulose as a substrate, and the zymogenic bands were observed. The Michaelis constant and maximum velocity of rMsGHF5 were 0.199 mg/mL and 0.034 μmol/min/mL, respectively. The optimal condition for rMsGHF5 occurred at pH 5 and 30°C. Fe²⁺ and Mn²⁺ stimulated the activity of rMsGHF5 by 167 and 114% respectively, whereas Cu²⁺, Hg²⁺ and Zn²⁺ inhibited its activity.     

A demographic analysis of mortality caused by the pine wood nematode (Bursaphelenchus xylophilus) and pine sawyer beetles (Monochamus alternatus) in pine forests in the Seto Inland Sea-side,Japan

In northern Sweden breeding males of Tengmalm's owls (Aegolius funereus (L.)) were site tenacious during and between the peaks of the vole (staple food) cycles, but females only during the peaks. Most of these adults shifted nest boxes between successive years. They selected nest boxes randomly in a radius of 3 km. Juveniles, in contrast to site tenacious adults, dispersed outside their natal area. The females moved longer than the males prior to their first breeding. Five adult females were found to be nomadic. One of these nomadic females previously bred site tenaciously as long as food was abundant. Juveniles and adult males were not found to be nomadic. Emigration of adult females and juveniles occurred most frequently when vole populations declined. The breeding population increased sharply and received immigrants suggesting that nomadism may be essential in the population dynamics. Site tenacity and nomadism are discussed in terms of costbenefit to males and females, respectively. Emphasis is on the main functional roles of males (feeding femle and young) and females (incubation).     

Winter dormancy of the pine sawyer Monochamus galloprovincialis (Col., Cerambycidae) in Portugal

Abstract:  The pine sawyer Monochamus galloprovincialis is the vector of the introduced pine wood nematode Bursaphelenchus xylophilus in Portugal and has a fixed univoltine life cycle, overwintering as larvae inside the wood. We investigated the possibility of the existence of a phase of winter dormancy affecting the larvae, its induction and termination under controlled conditions and its incidence and termination in the field. In the laboratory, the majority of the newly hatched larvae which were maintained at a constant temperature of 23°C and different photoperiods became dormant in <70 days. Part of the population completed dormancy and reassumed development until adult emergence at the constant temperature, without the need for a chilling period. Although no critical photoperiod for dormancy maintenance or termination could be determined, dormancy termination was more frequent for larvae experiencing changes on daylength duration. In the field, dormancy incidence grew progressively from a minimum of 26% in October to a maximum of 100% in April, and dormancy appeared to be completed only during late winter. The results suggest that M. galloprovincialis larvae underwent an obligatory dormancy associated with the last larval instar, which conforms to the general definition of diapause, although presenting some affinities with oligopause. The role of this dormancy on the regulation of the pine sawyer's seasonal development and the number of generations is discussed.     

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.     

昆虫来源的几丁质酶的分离纯化及酶学性质

几丁质酶在真菌和昆虫的生理和发育过程中起着关键作用,该酶本身及其酶抑制剂是获取生物农药的重要途径。本研究从蚕蛹体内提取几丁质粗酶,经硫酸铵分级沉淀和Sephadex G-150分离得到几丁质酶。用SDS-PAGE测得该酶的分子量为88kDa。水解胶体几丁质的Km值为22.3μmol/L。酶反应的最适温度为45℃,最适pH值为6.0,金属离子和有机试剂对几丁质酶活性都有影响,其中高浓度的Mn2+对酶有较强的激活作用,而Cu2+、SDS则有较强的抑制作用。研究结果为基于几丁质酶的生物农药筛选研究奠定了基础。     

An operative kairomonal lure for managing pine sawyer beetle Monochamus galloprovincialis (Coleoptera: Cerymbycidae)

Abstract:  Monochamus galloprovincialis Olivier (Col., Cerambycidae) is a vector of the pine wood nematode, Bursaphelenchus xylophilus , causing the destructive pine wilt disease. An effective lure for monitoring and/or mass-trapping would be of great interest in the management of this pine sawyer. Males and females of this species show an attractive kairomonal response to blends composed of four pheromone compounds used by Ips spp. bark beetles and two host volatiles from pines. This six-component lure is highly attractive but may to be too complex and costly for practical use as each component is released from a separate lure. The role of each component, ipsdienol, ipsenol, cis -verbenol, methyl butenol, α -pinene and ethanol as attractants for M. galloprovincialis was field tested in Spain to obtain a simpler but equally effective bait. Ipsenol was confirmed as the strongest kairomonal signal to M. galloprovincialis synergizing response to α -pinene by 95 times. The addition of methyl butenol to this blend doubled the number of males and females trapped. On the other hand, neither ipsdienol, cis -verbenol nor ethanol improved the results when incorporated into the above three-component blend. A lure consisting of ipsenol, methyl butenol and α -pinene may be very cost-efficient in operational monitoring or mass trapping of M. galloprovincialis . Three potentially repellent candidates, (−)verbenone, methyl cyclohexenone and trans -conophthorin, were also tested against the attractive three-component bait. trans -Conophthorin significantly reduced male catches of M. galloprovincialis ; methyl cyclohexenone had no effect. Verbenone significantly enhanced the response of females to the attractive combination of α -pinene, ipsenol and methyl butenol.     

室内条件下莱氏猛叩甲对松墨天牛和黄粉虫的捕食能力

【目的】本研究旨在明确莱氏猛叩甲Tetrigus lewisi老熟幼虫分别对黄粉虫Tenebrio molitor和松墨天牛Monochamus alternatus末龄幼虫和蛹的捕食量的差异,评估莱氏猛叩甲的捕食能力,为松墨天牛M.alteratus的有效治理找到潜在的高效生物防治因子。【方法】于2014年11月在浙江富阳昌东村马鞍山砍下松墨天牛虫害木,通过林间野外调查,明确松墨天牛和莱氏猛叩甲的发生情况;将野外采集到的莱氏猛叩甲幼虫带回室内,分别饲喂黄粉虫和松墨天牛的末龄幼虫和蛹,每天记录捕食量,确定莱氏猛叩甲的捕食能力。【结果】野外调查中,我们在松墨天牛幼虫蛀食的坑道中发现了莱氏猛叩甲幼虫和松墨天牛幼虫被取食后的残体;在采集到1 094头松墨天牛幼虫的危害木内,共采得莱氏猛叩甲末龄幼虫36头,约为松墨天牛幼虫数量的3%。室内捕食实验表明,5 d内莱氏猛叩甲捕食黄粉虫幼虫总量为3.2头,平均每天捕食约0.6头,而捕食松墨天牛幼虫总量为8.0头,平均每天捕食约1.6头,莱氏猛叩甲对松墨天牛的总捕食量显著高于对黄粉虫幼虫的总捕食量(P0.0001);4 d内莱氏猛叩甲捕食黄粉虫蛹总量为5.6头,平均每天捕食约1.4头,而捕食松墨天牛蛹总量为7.8头,平均每天捕食约2.0头,莱氏猛叩甲对松墨天牛蛹的捕食量显著高于黄粉虫蛹(P=0.028)。回归分析还表明,莱氏猛叩甲幼虫的捕食量不受叩甲个体大小的影响。【结论】莱氏猛叩甲普遍分布在松墨天牛发生区,同时发现松墨天牛被取食后的残体,表明莱氏猛叩甲是松墨天牛的捕食性天敌。莱氏猛叩甲均能捕食黄粉虫和松墨天牛的幼虫和蛹;相比于捕食黄粉虫,莱氏猛叩甲对松墨天牛的幼虫和蛹具有更好的捕食效率,具有很好的生物防治前景,是未来松墨天牛治理潜在高效生物防治因子。     

松墨天牛成虫雌雄体型差异及蛹和成虫大小与越冬幼虫体重的关系

【目的】本研究旨在明确松墨天牛 Monochamus alteratus 成虫及越冬幼虫体型大小的差异,探讨该虫越冬后成虫体型大小和越冬幼虫体重大小关系及原因。【方法】于2014年5月1日至10月31日在浙江富阳野外诱捕松墨天牛,通过测量野外诱捕到的松墨天牛成虫体长、体宽,确定林间松墨天牛成虫体型的差异;松墨天牛越冬幼虫的采集和称重测量明确越冬幼虫的组成和体重大小差异;进而通过对越冬幼虫单头跟踪饲养至化蛹、羽化,确定越冬幼虫体重大小与发育所得蛹和成虫大小的关系,阐明林间松墨天牛成虫体型差异的原因。【结果】浙江富阳野外诱捕发现,松墨天牛的活动期间很长,从5月中旬到10月初一直能诱捕到松墨天牛成虫,高峰期在6和7月份。松墨天牛雌雄成虫体型差异很大,雌虫平均体长和体宽分别为20.59±0.19和6.59±0.06 mm;雄虫平均体长和体宽分别为19.90±0.26 和6.44±0.08 mm; 雌虫的平均体长明显高于雄虫,但二者体宽没有显著差异;并且雌雄成虫体长和体宽呈显著正相关。越冬幼虫的头宽测定表明头宽的变化很大,最小为2.20 mm,最大为4.24 mm,经比对越冬幼虫由4龄和5龄幼虫组成;幼虫体重差异大,平均体重为304.2 mg, 介于71.6~858.0 mg之间,其中5龄越冬幼虫显著重于4龄越冬幼虫。进一步将越冬幼虫单管饲养跟踪研究发现,越冬幼虫体重大小决定蛹和羽化后的成虫的大小,二者存在显著的正相关,并且由4龄越冬幼虫发育所得的蛹和成虫显著轻于由5龄幼虫发育所得的蛹和成虫。【结论】松墨天牛成虫体型差异很大,这与越冬幼虫体重差异相关;越冬幼虫的体重大小决定了其化蛹后的蛹重和羽化后成虫的体型和体重的大小;造成越冬幼虫体重差异的可能原因包括松墨天牛成虫扬飞周期长导致的产卵期长而使得天牛发育进度不一致以及寄主不同部位营养的差异。     

Acaricidal activity of the aggregation pheromone of Japanese pine sawyer against two-spotted spider mite

Japanese pine sawyer (Monochamus alternatus) (Coleoptera: Cerambycidae), a vector of pine wilt disease, produces male-produced aggregation pheromone called monochamol, 2-(1-undecyloxy)-1-ethanol (C₁₁OEtOH). After the identification of unique nematicidal activity of the synthetic monochamol and its homologues (ROEtOH: R = C₇-C₁₁) against Bursaphelenchus xylophilus nematode, their acaricidal activities were evaluated against two spotted spider mite (TSSM, Tetranychus urticae Koch) (Acari: Tetranychidae). In a leaf disc bioassay method, 100 mgL⁻¹ of monochamol and 2-(1-decyloxy)-1-ethanol (C₁₀OEtOH) exhibited 100% adult mortality and zero fecundity. Along with C₁₁OEtOH and C₁₀OEtOH, 2-(1-octyloxy)-1-ethanol (C₈OEtOH) and 2-(1-nonyloxy)-1-ethanol (C₉OEtOH) were also 100% acaricidal with complete oviposition inhibition at increased concentration of 1000 mgL⁻¹. When the TSSM eggs on leaf discs were immersed in 1000 mgL⁻¹ solutions, none of the components were ovicidal. Egg hatching was completely inhibited by three components (C₉OEtOH, C₁₀OEtOH and C₁₁OEtOH) at 6000 mgL⁻¹ and by four components (C₈OEtOH, C₉OEtOH, C₁₀OEtOH and C₁₁OEtOH) at 8000 mgL⁻¹. However, none of the compounds showed acaricidal effects in 0.5 h and 24 h residual toxicity tests at 1000 mgL⁻¹. This study highlights the potential acaricidal activity of monochamol and its homologues. Notably, the monochamol and its C₁₀-homologue could be listed as efficient acaricidal agents.     

金龟子绿僵菌MaYTTR-04菌株对松墨天牛成虫的致病力

松墨天牛Monochamus alternatus Hope是重大森林植物检疫性病害——松材线虫病的主要媒介昆虫。采用跗节接种法测定金龟子绿僵菌Metarhizium anisopliae MaYTTR-04菌株对松墨天牛成虫的致病力。结果表明： 在恒温条件下,松墨天牛第6天开始死亡,第18天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10⁶±0.2×10⁶时成虫的死亡率第18天达85%,第21天达95%; 时间效应指标值LT₅₀为14.7天。室内自然变温条件下,松墨天牛第3天出现死亡,第15天～21天为死亡高峰期,每头成虫接种孢子量为2.3×10.6±0.2×10⁶时的死亡率在第15天达85%,第21天达100%;时间效应指标值LT₅₀为12.9天。林间套笼21天后,菌液处理的成虫平均死亡率为60%,僵虫率为48.9%;而通过无纺布菌条的成虫死亡率为86.7%,僵虫率为75.6%。表明MaYTTR-04菌株对松墨天牛成虫有强的致病力,且在较高变温环境温度下致病力更强。结果说明该菌株可作为生产性菌株应用于林间防治松墨天牛。     

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757–767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A–500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS–PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc₅), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc_n, n=2–6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the β anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with K_i values depending on the pH of the reaction mixture (K_i=0.013−0.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin (k_cat/K_m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/K_m) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as K_m, k_cat and k_cat/K_m successively.