中国松毛虫属(Dendrolimus Germar:Lasiocampidae)的研究及新种记述

松毛虫属变异较复杂, 过去文献上的记载比较紊乱, 本篇着重以各虫期的形态特征为主, 修订记载了我国严重为害松柏科的6种松毛虫, 计:西伯利亚松毛虫(Dendrolimus sibiricus Tschetv.), 赤松毛虫(D.spectabilis Butler), 马尾松毛虫(D.punctatus Walker), 铁杉毛虫(D.superansButler), 云南松毛虫(D.latipennis Walker), 和思茅松毛虫(D.kikuchii Mats.), 同时记述了2个新种, 计:油松毛虫 (D.tabulaeformis Tsai et Liu)和西昌松毛虫(D.xichangensis Tsai et Liu)及1个新亚种:赭色松毛虫(D.kikuchii ochraceus Tsai et Liu)。模式标本保存在中国科学院动物研究所。 新种油松毛虫和赤松毛虫、马尾松毛虫很近似, 但其♀成虫前翅较狭长, 中横线与外横线为白色, 亚外缘斑列内侧有白色斑;♂外生殖器小抱针长度约为大抱针的1/2;♀外生殖器侧前阴片接近圆形。卵壳上内层室和中层室壁很薄, 外层室中央凹下部分呈不规则条状。幼虫毛片束发达, 片状毛小, 先端极少有齿状突起, 无贴体倒伏鳞毛。 新种西昌松毛虫和云南松毛虫、思茅松毛虫比较近似, 但成虫前翅中横线与外横线之间明显形成褐色宽带;♀外生殖器前阴片愈合成一块。卵壳表面无花斑。幼虫胸部背面无明显毒毛带, 但体侧有黑丛毛。根据以上几点, 显然和其他两种松毛虫有区别。 新亚种赭色松毛虫和思茅松毛虫极近似, 只是体色显著呈深赭色, 地理分布、寄主植物亦与原种有别, 因此定为新亚种。 此外, 文中制有各虫期检索表。     

松毛虫雌性信息素的研究与应用

目前,在我国对松林危害最严重的害虫主要有马尾松毛虫Dendrolimnspunctatus(Walker)、落叶松毛虫D.superaus(Butler)、油松毛虫D.tabulaeformisTsaietLiu、赤松毛虫D.spectabilis(Butler)、思茅松毛虫D.kikuchiiMatsumura、云南松毛虫D.houiLajonqui埁re等。综述6种松毛虫的雌性信息素的成分鉴定、合成、EAG反应,人工合成雌性信息素的田间活性测定,性诱剂在林间的设置技术及种群监测等应用。     

Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species （Lepidoptera： Lasiocampidae）

Pine caterpillar moths, Dendrolimus spp. （Lepidoptera： Lasiocampidae）, are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins （OBPs） participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans （Butler）, D. punctatus （Walker）, D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. la＇kuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure.     

松毛虫的杂交遗传试验

本文报道了马尾松毛虫Dendrolimus punctatus Walker和油松毛虫Dendrolimus tabwlaeformis Tsai et Liu杂交遗传试验的部分结果.研究表明,不论从食性、形态特征、混合配对和单个配对试验的结果来看,油松毛虫和马尾松毛虫应为同一物种.其形态学特征,尤其是外生殖器解剖构造的差异,并未反映出种的特性和种间的生殖隔离.油松毛虫与马尾松毛虫杂交,无论正交或反交,都能产生有生育力的F₁代,F₁代自交产生F₂代,F₂代自交产生F₃代.但考虑到油松毛虫在我国的分布与油松的分布大体相一致,它与马尾松毛虫在形态学和生态学等方面多少存在着一些差异,因此可认为它是马尾松毛虫的一个亚种,可称为Dendrallmua punctatus tabulaeformis(Tsai et Liu).     

毒蛾绒茧蜂饲养繁殖技术

<正> 毒蛾绒茧蜂Apanteles liparidis(Bouche)是松毛虫Dendrolimus和舞毒蛾Lymantria dispar(L.)等害虫幼虫期的重要内寄生天敌,自然寄生率很高。1982年Schaefer等在北京、黑龙江、吉林等地考察发现,该蜂对舞毒蛾的寄生率为11.5～39.8％。1979年刘振恒等报道,在河北省丰宁县,该蜂对油松毛虫D.tabulaeformis Tsai et Liu越冬幼虫的平均寄生率为16.7％,对落叶松毛虫D.superans(Butler)的寄生率为15.6％。该蜂繁殖量大,寄主范围较广。为探讨室内大量繁殖此蜂的可能性,1990年作者利用人工饲料饲养的舞毒蛾幼虫     

松毛虫质型多角体病毒的宿主域与交叉感染

自1956年从赤松毛虫Dendrolimus spectabilis上首次发现赤松毛虫质型多角体病毒1型（D. spectabilis cytovirus 1,DsCPV-1）以来,先后从马尾松毛虫D. punctatus、油松毛虫D. tabulaeformis、赤松毛虫、德昌松毛虫D. p. tehchangensis、文山松毛虫D. p. Wenshangensis和落叶松毛虫D. superans上发现了质型多角体病毒(cytoplasmic polyhedrosis virus,CPV)。病毒基因组dsRNA电泳图谱分析表明,这些松毛虫CPV的不同分离株均属于质型多角体病毒1型（cytovirus 1）。这些松毛虫CPV病毒可以感染鳞翅目10科35种昆虫,其中对多种昆虫具有很高的感染力和良好的杀虫效果,可以从中筛选替代宿主生产松毛虫CPV杀虫剂,用于害虫生物防治。松毛虫CPV接种某些昆虫后病毒的基因组dsRNA电泳图谱发生了改变,可能是异源病毒诱发了宿主自身潜伏型病毒的感染复制。     

四种松毛虫不同地理种群遗传多样性的等位酶分析

【目的】采用等位酶电泳技术对中国松毛虫属Dendrolimus 4种共9个地理种群进行遗传多样性和遗传分化研究。【方法】对6种等位酶系统乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）、苹果酸酶（ME）、乙醇脱氢酶（ADH）、甲酸脱氢酶（FDH）、谷氨酸脱氢酶（GDH）进行聚丙烯酰胺凝胶电泳分析。【结果】在4种松毛虫9个地理居群中共检测到10个基因位点,其中4个位点为多态位点,检测到17个等位基因; 种群总体水平多态位点比率P=40%,平均有效基因数A=1.700,平均期望杂合度He=0.151,种群平均遗传距离为0.001～0.285; 其中马尾松毛虫指名亚种Dendrolimu punctatus Walker 6个居群的遗传分化度Fst=0.265,基因流Nm=0.692。4种松毛虫之间遗传关系最近的是落叶松毛虫D. superans Butler和马尾松毛虫的地理亚种赤松毛虫D. punctatus spectabilis Butler,遗传关系最远的是落叶松毛虫D. superans Butler和云南松毛虫D. houi Lajonquiere。【结论】 马尾松毛虫居群间遗传分化程度较大,基因交流较少,遗传漂变已经成为导致该物种种群分化的主要原因之一;遗传距离与地理距离存在一定相关性。     

松毛虫属（Dendrolimus) 部分种类亲缘关系与遗传分化的等位酶分析

采用等位酶聚丙烯酰胺凝胶电泳技术对松毛虫属5个种和亚种的野生种群进行了亲缘关系和遗传变异的研究.8种等位酶系统(乳酸脱氢酶LDH、苹果酸脱氢酶MDH、苹果酸酶ME、乙醇脱氢酶ADH、甲酸脱氢酶FDH、谷氨酸脱氢酶GDH、过氧化物酶POD、过氧化氢酶CAT)共检测到12个基因位点,其中6个位点为多态位点,检测到15个等位基因.松毛虫属5个种和亚种的总体水平多态位点比率P＝50%,平均有效基因数A = 1.917,平均期望杂合度He =0.267,平均遗传距离为0.0730～0.5701.遗传参数表明松毛虫属昆虫种间存在较高程度的遗传变异,聚类图和遗传距离数据表明赤松毛虫与马尾松毛虫亲缘关系最近,落叶松毛虫与思茅松毛虫亲缘关系最远.     

三种松毛虫不同地理种群遗传多样性

运用13个ISSR引物对赤松毛虫(Dendrolimus spectabilis)、油松毛虫(Dendrolimus tabulaeformis)、落叶松毛虫(Dendrolimus superans)的种群遗传分化进行分析.13 个引物共产生195条带,其中184条具多态性,总的多态位点百分率为94.36%,扩增谱带具有明显的种间多态性.Shannon 信息指数和Nei信息指数均表明落叶松毛虫群体内的遗传多样性最高,油松毛虫与赤松毛虫则相差不多.各种松毛虫的不同地理居群出现了遗传分化,由Nei指数估计的群体间的遗传分化系数分别为15.50%、32.57%和41.92%.用UPGMA法对Nei′s遗传距离作聚类分析.聚类结果表明:不同地域的油松毛虫遗传距离与地理距离呈一定程度的相关趋势.     

不同林分类型下油松毛虫（Dendrolimus tabulaeformis Tsai et Liu）种群遗传多样性

采用AFLP技术对平泉县的1个油松-落叶松混交林和2个油松纯林中油松毛虫(Dendrolimus tabulaeformis Tsai et Liu)的135个个体进行了遗传多样性和遗传结构的研究,并调查了3个油松林群落的各种环境条件.结果表明,纯林中油松毛虫种群的遗传多样性高于混交林中的油松毛虫种群;林木生长状况为影响不同油松纯林群落中油松毛虫种群遗传多样性的重要因素;混交林对油松毛虫种群之间的基因流有阻断作用,油松毛虫种群的基因流大小与油松林之间的物种多度呈反相关.     

云南松毛虫成虫的营养成份

本文从松毛虫资源化的角度,探讨云南松毛虫成虫的营养价值。该虫的蛋白质、脂肪和总糖含量分别为６８．３０％、６．５６％和１．３１％,含１５种氨基酸（色氨酸未测）,并含Ｖ－Ａ、Ｖ－Ｂ_１、Ｖ－Ｂ_２、Ｖ－Ｃ、Ｖ－Ｄ、Ｖ－Ｅ等多种维生素和卵磷脂以及丰富的微量元素。几丁质含量较高,为 １７． ８３％。     

Host range of Dendrolimus kikuchii nucleopolyhedrovirus

Dendrolimus kikuchii Matsumura nucleopolyhedrovirus (DekiNPV) has been considered as a biological control agent against D. kikuchii. We examined the infectivity and replication of DekiNPV in 11 other lepidopteran species, including Dendrolimus houi (Lajonquiere), Dendrolimus punctatus (Walker), D. punctatus wenshanensis (Tsai et Liu), Dendrolimus spectabilis (Butler), Hyphantria cunea (Drury), Lymantria dispar (L.), Ectropis grisescens (Warren), Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner), Plutella xylostella (L.) and Spodoptera litura (Fabricius) larvae to define the host range of DekiNPV and identify suitable alternate hosts. Our study showed that DekiNPV is highly specific to D. kikuchii, but triggers covert infections in H. cunea, D. houi, D. punctatus, D. punctatus wenshanensis and D. spectabilis larvae into overt symptoms, indicating that DekiNPV is suitable as an ideal biocontrol agent for D. kikuchii, D. houi, D. punctatus, D. punctatus wenshanensis, D. spectabilis and H. cunea.     

基于Cyt b基因序列分析的松毛虫种群遗传结构研究

为了揭示松毛虫种群的遗传结构,采用DNA序列测定的方法测定了松毛虫不同种群的线粒体细胞色素b (Cyt b)基因的部分序列,并利用分子生物学软件分析其核苷酸组成、转换和颠换、氨基酸组成、遗传距离及亲缘关系。结果显示:在获得的Cyt b 基因387bp的序列中碱基A,T,C,G平均含量分别为40.1%、33.5%、9.5%、16.9%,A+T含量明显高于G+C含量表现出强烈的A、T偏向性,密码子第3位点的A+T含量高达86.5%,这种偏向性在种群间无明显差异。碱基替换主要发生在密码子第三位,转换大于颠换,且种群内替换高于种群间。该序列片段中共有39个核甘酸位点发生变异,遗传距离为0.000-0.100,显示出较小的遗传变异。蛋白质氨基酸由除谷氨酸以外的19种氨基酸组成。聚类分析结果表明马尾松毛虫和油松毛虫亚种遗传距离较近,种群间的遗传分化与生态环境有关。     

Mitochondrial phylogenomics and genetic relationships of closely related pine moth (Lasiocampidae: Dendrolimus) species in China,using whole mitochondrial genomes

Background

Pine moths (Lepidoptera; Bombycoidea; Lasiocampidae: Dendrolimus spp.) are among the most serious insect pests of forests, especially in southern China. Although COI barcodes (a standardized portion of the mitochondrial cytochrome c oxidase subunit I gene) can distinguish some members of this genus, the evolutionary relationships of the three morphospecies Dendrolimus punctatus, D. tabulaeformis and D. spectabilis have remained largely unresolved. We sequenced whole mitochondrial genomes of eight specimens, including D. punctatuswenshanensis. This is an unambiguous subspecies of D. punctatus, and was used as a reference for inferring the relationships of the other two morphospecies of the D. punctatus complex. We constructed phylogenetic trees from this data, including twelve published mitochondrial genomes of other Bombycoidea species, and examined the relationships of the Dendrolimus taxa using these trees and the genomic features of the mitochondrial genome.

Results

The eight fully sequenced mitochondrial genomes from the three morphospecies displayed similar genome structures as other Bombycoidea species in terms of gene content, base composition, level of overall AT-bias and codon usage. However, the Dendrolimus genomes possess a unique feature in the large ribosomal 16S RNA subunits (rrnL), which are more than 60 bp longer than other members of the superfamily and have a higher AC proportion. The eight mitochondrial genomes of Dendrolimus were highly conservative in many aspects, for example with identical stop codons and overlapping regions. But there were many differences in start codons, intergenic spacers, and numbers of mismatched base pairs of tRNA (transfer RNA genes).Our results, based on phylogenetic trees, genetic distances, species delimitation and genomic features (such as intergenic spacers) of the mitochondrial genome, indicated that D. tabulaeformis is as close to D. punctatus as is D. punctatus wenshanensis, whereas D. spectabilis evolved independently from D. tabulaeformis and D. punctatus. Whole mitochondrial DNA phylogenies showed that D. spectabilis formed a well-supported monophyletic clade, with a clear species boundary separating it from the other congeners examined here. However, D. tabulaeformis often clustered with D. punctatus and with the subspecies D. punctatus wenshanensis. Genetic distance analyses showed that the distance between D. tabulaeformis and D. punctatus is generally less than the intraspecific distance of D. punctatus and its subspecies D. punctatus wenshanensis. In the species delimitation analysis of Poisson Tree Processes (PTP), D. tabulaeformis, D. punctatus and D. punctatus wenshanensis clustered into a putative species separated from D. spectabilis. In comparison with D. spectabilis, D. tabulaeformis and D. punctatus also exhibit a similar structure in intergenic spacer characterization. These different types of evidence suggest that D. tabulaeformis is very close to D. punctatus and its subspecies D. punctatus wenshanensis, and is likely to be another subspecies of D. punctatus.

Conclusions

Whole mitochondrial genomes possess relatively rich genetic information compared with the traditional use of single or multiple genes for phylogenetic purposes. They can be used to better infer phylogenetic relationships and degrees of relatedness of taxonomic groups, at least from the aspect of maternal lineage: caution should be taken due to the maternal-only inheritance of this genome. Our results indicate that D. spectabilis is an independent lineage, while D. tabulaeformis shows an extremely close relationship to D. punctatus.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1566-5) contains supplementary material, which is available to authorized users.     

油松毛虫受球孢白僵菌感染的组织病理学变化

为了研究昆虫病原真菌对松毛虫的致病机理, 提供北方地区松毛虫生物防治的科学依据, 本研究采用球孢白僵菌Beauveria bassiana (Bals.) Vuillemin 菌株1573感染油松毛虫Dendrolimus tabulaeformis Tsai et Liu, 通过扫描电镜和石蜡切片光学显微镜观察技术, 研究了菌株的感染过程和虫体的组织病理学变化。结果表明, 该病原真菌通过穿透表皮入侵油松毛虫, 染菌后24 h, 观察到分生孢子附着于头部的颅顶, 单眼、触角和口器的基部, 在胸、腹部附着于毛簇、毛瘤、棘状突和节间褶。染菌后36 h, 孢子萌发长出菌丝, 在入侵部位, 菌丝的端部特化成附着胞和侵入钉。染菌后48 h, 菌丝依靠机械力和胞外酶的作用穿透表皮, 虫体表皮出现了裂痕和黑化。染菌后72 h, 菌丝已进入体腔, 感染血淋巴、脂肪体、肌肉、消化道、丝腺和神经组织, 并利用血液和内部组织器官作为营养大量繁殖, 此时, 虫体发涨, 表皮变暗。染菌后96 h, 菌丝占据了松毛虫的血腔, 内部的组织结构被完全瓦解, 松毛虫死亡。最后, 菌丝冲破体壁, 在尸体表面长出新的分生孢子。这些结果说明, 球孢白僵菌B. bassiana菌株1573是一种油松毛虫的高致病性菌株, 可以引起虫体的一系列组织病理变化而致其死亡。