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Volume 31 Issue 1
Jul.  2019
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Distribution and Function of the Ovarian Nematode Contortylenchus genitalicola (Tylenchida: Allantonematidae) on Monochamus alternatus (Coleoptera: Cerambycidae) in China

  • Corresponding author: YANG Zhong-qi, yangzhqi@126.com
  • Received Date: 2017-10-10
  • Objective Japanese scholars found that the pine sawyer Monochamus alternatus carried not only pine wood nematode (Bursaphelenchus xylophilus), but also an another nematode, which was identified as a new specie and named as ovarian nematode, Contortylenchus genitalicola Kosaka et Ogura (Tylenchida:Allantonematidae). It was recognized that the nematode was parasitic in the ovary of M. alternatus. This new ovarian nematode has not been reported in China till now. This study is to determine whether this nematode existing in China and its function in the cerambycid. Method Capturing emergent M. alternatus in batches and groups, dissecting the beetle, the dead larvae of M. alternatus, and the infected pine wood to find the nematode. Result It was found that M. alternatus adults in China carried the ovarian nematode indeed. The nematode has 3 niches. It existed in the cerambycid adult and larva of M. alternatus, as well as in the xylem of the infected pine trees. The 4th instar larvae of the ovarian nematode inoculated into the xylem of pine tree when the female M. alternatus laying eggs. Then some of the 4th instar nematode in the xylem parasitized the larvae of M. alternatus as host, while some still lived in the xylem. While the cerambycid developed from egg to larvae and to pupa, the ovarian nematode in xylem could move to the body of the cerambycid adult, finishing and restarting the life cycles among the niches. It was found in the investigation that 44.4% of the cerambycid adults carried this ovarian nematode, in which 43.8% of female adults and 45.0% male adults with the nematode respectively and the data showed no significant difference. The average quantity of nematodes carried by one cerambycid adult was 574, in which each female carried 816 and each male carried 308 in average, and the data showed significant differences. The preliminary study on the ovarian nematode life history indicated that the nematode could only develop stages from egg to 1st-4th instar larva, but did not complete the whole life history in the adult body of the cerambycid. However, in the cerambycid larva and the xylem of infested tree, it was able to finish the whole life history. Conclusion The ovarian nematode is unable to kill the adults of M. alternatus although it could parasitic in the adult body, however it could kill the cerambycid larvae as a parasitic nematode. It is also found that the ovarian nematode often coexists with pine wood nematode and has a similar way with the pine wood nematode living in the infested pine trees. It is necessary to confirm whether the ovarian nematode has a pathological effect to infested pine tree similar as the pine wood nematode.
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Distribution and Function of the Ovarian Nematode Contortylenchus genitalicola (Tylenchida: Allantonematidae) on Monochamus alternatus (Coleoptera: Cerambycidae) in China

    Corresponding author: YANG Zhong-qi, yangzhqi@126.com
  • 1. Ningbo Forest Pest Control and Quarantine Station, Ningbo 315000, Zhejiang, China
  • 2. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry/Key Laboratory of Forest Protection, China, State Forestry Administration, Beijing 100091, China

Abstract:  Objective Japanese scholars found that the pine sawyer Monochamus alternatus carried not only pine wood nematode (Bursaphelenchus xylophilus), but also an another nematode, which was identified as a new specie and named as ovarian nematode, Contortylenchus genitalicola Kosaka et Ogura (Tylenchida:Allantonematidae). It was recognized that the nematode was parasitic in the ovary of M. alternatus. This new ovarian nematode has not been reported in China till now. This study is to determine whether this nematode existing in China and its function in the cerambycid. Method Capturing emergent M. alternatus in batches and groups, dissecting the beetle, the dead larvae of M. alternatus, and the infected pine wood to find the nematode. Result It was found that M. alternatus adults in China carried the ovarian nematode indeed. The nematode has 3 niches. It existed in the cerambycid adult and larva of M. alternatus, as well as in the xylem of the infected pine trees. The 4th instar larvae of the ovarian nematode inoculated into the xylem of pine tree when the female M. alternatus laying eggs. Then some of the 4th instar nematode in the xylem parasitized the larvae of M. alternatus as host, while some still lived in the xylem. While the cerambycid developed from egg to larvae and to pupa, the ovarian nematode in xylem could move to the body of the cerambycid adult, finishing and restarting the life cycles among the niches. It was found in the investigation that 44.4% of the cerambycid adults carried this ovarian nematode, in which 43.8% of female adults and 45.0% male adults with the nematode respectively and the data showed no significant difference. The average quantity of nematodes carried by one cerambycid adult was 574, in which each female carried 816 and each male carried 308 in average, and the data showed significant differences. The preliminary study on the ovarian nematode life history indicated that the nematode could only develop stages from egg to 1st-4th instar larva, but did not complete the whole life history in the adult body of the cerambycid. However, in the cerambycid larva and the xylem of infested tree, it was able to finish the whole life history. Conclusion The ovarian nematode is unable to kill the adults of M. alternatus although it could parasitic in the adult body, however it could kill the cerambycid larvae as a parasitic nematode. It is also found that the ovarian nematode often coexists with pine wood nematode and has a similar way with the pine wood nematode living in the infested pine trees. It is necessary to confirm whether the ovarian nematode has a pathological effect to infested pine tree similar as the pine wood nematode.

  • 日本学者在上世纪70年代初证实,松褐天牛(Monochamus alternatus Hope)成虫是松林毁灭性病害的病原——松材线虫 (Bursaphelenchus xylophilus Nickle) [1-2]最主要的传播媒介昆虫[3]。随后,许多学者对松褐天牛成虫携带松材线虫的部位、分布、数量作了广泛而深入的研究[4-8]。我国发生松材线虫病后,许多研究者也证实松褐天牛成虫是引起我国松材线虫病流行的最重要的媒介昆虫[9-11],并作为防治对象,通过控制松褐天牛来达到控制松材线虫病的目的[12-14]。防治松褐天牛多用化学和物理的方法进行[15-20]。近年来对开发利用天敌防治松褐天牛技术取得了重要进展[21-22]。斯氏线虫(Steinernema spp.)和异小杆线虫(Heterorhabditis spp.)是昆虫的重要天敌[23-25], 但除少量的人工寄生试验外[26],至今未发现有自然寄生松褐天牛的斯氏线虫或异小杆线虫。日本学者在上世纪90年代初报道了松褐天牛雌成虫生殖器官内携带有一种线虫,并定名为卵巢线虫(Contortylenchus genitalicola Kosoka et Ogura),认为是松褐天牛成虫的重要天敌[27-29]。我国迄今尚未见关于卵巢线虫的研究报道。笔者经过研究,发现我国的松褐天牛成虫体上也分布有该卵巢线虫,并对该线虫的形态、分布、寄生性和生态作用作了初步研究,现将结果报道如下。

1.   材料与方法
  • 在宁波北仑区、慈溪市、余姚市等松疫木堆场选取当年受松材线虫病危害枯死的带有较多松褐天牛蛀入孔的松树,锯取长1 m、直径6~9 cm的松木段备用。这些松木段带有松褐天牛幼虫蛀入孔,无其成虫羽化孔,放在实验室铁丝笼中饲养,让木段中的松褐天牛幼虫继续发育,待次年松褐天牛成虫羽化出后采集供试验用。这些松木段的选取原则是要保证有足够数量的松褐天牛幼虫在木质部蛹室中越冬。松木段预置工作分别在松材线虫发生区选取:2001年11月在北仑区选取7段;在2006年10月在余姚市选取7段;2012年12月在慈溪市选取7段,置入铁丝笼备用;2002年、2007年、2013年5月后,分别在饲养笼中采集饲养出的松褐天牛成虫36、35和37头供解剖试验用。同时,不定期在林间锯伐、劈松木段,采集树干中死亡的松褐天牛幼虫或蛹供试验和分析用。

  • ① 成虫体表携带的线虫观察。采集在铁丝笼内羽化出孔的松褐天牛成虫,在NTX-2B型体视镜下观察体表、气门孔口、尾孔口是否有线虫活动,并作记录;

    ② 成虫体内携带的线虫观察。把经体表观察有线虫的松褐天牛置于直径9 cm的培养皿中,用医用剪,剪去头、足、翅,再剪去胸腹部的腹板和背板缝接边沿,再用昆虫针挑去腹部背板,将气管系统、生殖系统、腹腔略作分离后,移至解剖镜下观察是否有线虫活动,并作记录;

    ③ 分离线虫。把经②观察到有线虫的松褐天牛成虫加蒸馏水20 mL, 用手术刀切碎成虫残体,分离线虫,每头天牛成虫放置在1培养皿内,静置12 h,以使线虫充分逸出;

    ④ 镜检线虫。在解剖镜下,挑线虫置载玻片上,盖上盖玻片后在XSP-103B型显微镜下鉴定线虫种类(活体观察),并用卡西欧EX-P505型数码相机,通过自设制的连接装置随时从显微镜目镜中摄取显微照片;

    ⑤ 线虫数量统计。在解剖镜下对培养皿内的线虫进行人工计数。数量少的,直接在镜下计数;数量多的,对培养皿分成8区,在设定的体视镜视野面积下逐区计数,最后将8个分区线虫数合计,取平均数;再用公式计算线虫数量:线虫数量=分区线虫平均数×培养皿面积/体视镜视野面积。

    ⑥ 线虫行为观察。在上述松褐天牛解剖实验、外形观察、分离计数等过程中,同时在镜下观察线虫离开或进入松褐天牛成虫的部位和行为及在松褐天牛幼虫中的活动情况。

    ⑦ 线虫形态观察。对松褐天牛成虫解剖所得的线虫,按Ogura氏描述的虫态、虫龄和形态的方法和标准[29]识别出卵巢线虫。

  • 松褐天牛成虫的卵巢线虫携带率按雌雄分别统计,并作频率差异显著性检验;成虫的卵巢线虫携带量,只统计携带卵巢线虫的松褐天牛个体,雌雄分别统计;携带量数据作平均数差异显著性t检验;用Excel软件作图。

2.   结果与分析
  • 解剖成虫时镜下最常见的是3龄卵巢线虫,但卵、孕胎卵、1龄、2龄、3龄、4龄和侵染型4龄线虫都能见到。按Ogura氏方法和标准[15]对卵巢线虫的虫态和虫龄作形态描述:

    卵:短圆柱形,两端圆,长约54 μm,直径约定27 μm(图 1:a);

    Figure 1.  The morphology of Contortylenchus genitalicola

    孕胎卵:能见幼虫胚胎卷曲在卵壳内的卵(图 1:b);

    1龄线虫:线形,长约150 μm,直径30 μm(图 1:c);

    2龄线虫:由1龄虫蜕皮而成,虫体圆柱形,略有香蕉状弯曲,头部有唇区缩缢明显,圆锥形,口针不明显;体内充满脂肪质颗粒物,体长约230~300 μm,直径约35 μm;尾圆柱形,顶端圆(图 1:d);

    3龄线虫:线形,长约400~600 μm,直径约40 μm(图 1:e);头部特化为圆球形,能见口针,长约8 μm(图 1:f);尾部圆柱形,末端圆(图 1:g);

    4龄线虫:线形,长约600 ~700 μm,直径约45 μm,头部唇区平,圆盘形缩缢不明显;口针基结明显,长约11 μm;尾部圆柱形,末端圆。4龄幼虫可分为耐久型和扩散型二虫态:耐久型4龄幼虫特点是4龄虫态形成后不破3龄虫表皮,而是长时间包裹在3龄虫的表皮下做伸缩活动,当4龄虫头部向后缩时能见到球形的3龄虫头壳与柱形的4龄虫头壳的区别(图 1:h),而尾形与3龄蜕相同(图 1:i);分散型4龄虫即离开了3龄虫蜕的4龄虫,头部不见球形的3龄蜕(图 1:j),虫尾圆柱形(图 1:k);

    成虫:雌成虫尾形圆锥形,顶端圆(图 1:l),阴门无盖(图 1:m);雄虫未确定。

  • 卵巢线虫的3龄幼虫由于头部呈球形,最易识别,并区别于松褐天牛成虫体上的其它线虫,所以在镜下以找到3龄幼虫为标准,做了卵巢线虫在成虫体上分布及年度变化(表 1)。由表 1可见,卵巢线虫在成虫体上分布率为44.4%,在雌成虫上分布率为43.8%,在雄成虫上分布率为45.0%,经频率差异显著性检验,u=1.058<uα=0.05=1.96, 没有显著差异(表 1)。在松褐天牛成虫体内,卵巢线虫很易检出;松褐天牛体表只能检到有极少数线虫,这可能是体内线虫向外蠕动而出的个体。因此,在松褐天牛体外能检到线虫,其体内也必能检到,没有发现体外检到线虫,体内检不到线虫的情况。体内线虫分布率为44.4%,体外线虫分布率为4.6%,经差异检验,u=6.805 7>uα=0.05=1.96达极显著水平(表 1)。从年度分析,卵巢线虫携带率有逐年下降趋势,2002年携带率52.8%,2007年为43.2%,2013年降到37.1%,经差异显著检验,u02-07=0.821,u02-13=1.328,u07-13=0.528<uα=0.05=1.96均未达到显著差异(表 1)。

    年度
    Year
    检查头数
    Number adults examined
    性别
    Sex
    数量
    Number
    体表镜检
    Checking nematodes on body surface of the adults
    解剖镜检
    Checking nematodes in inside of the adults
    线虫携带率
    Rate with nematodes/%
    有线虫头数
    The adults with nematodes
    无线虫头数
    The adults without nematodes
    有线虫头数
    The adults with nematodes
    无线虫头数
    The adults without nematodes
    体表
    Outside of the adults
    体内
    Inside of the adults
    2002 36 15 1 14 8 7 6.7 53.3
    21 0 21 11 10 0 52.4
    sum 36 1 35 19 17 2.8b 52.8a
    2007 37 16 1 17 7 9 6.3 43.8
    21 1 22 9 12 4.8 42.9
    sum 37 2 35 16 19 5.4b 43.2a
    2013 35 17 1 16 6 11 5.9 35.3
    18 1 17 7 11 5.6 38.9
    sum 35 2 33 13 22 5.7b 37.1a
    Average 4.6b 44.4a
    注:a, b不同小写字母表示在P<0.05水平上差异显著。
    Note: Different small letters mean significant difference at P<0.05 level.

    Table 1.  Percentage of M. alternatus adult carried ovarian nematode

  • 线虫在松褐天牛体内以卵、1龄、2龄、3龄、4龄虫态存在,由于总数量统计有困难,均以2龄以上的虫态计数。对21头带卵巢线虫的松褐天牛计数,最少带80条,最多带2 405条,每头松褐天牛平均带574条。按年度计量,卵巢线虫数量有逐渐下降趋势。其中2002年每头松褐天牛成虫平均带679.14±301.69条卵巢线虫,2007年每头松褐天牛成虫平均带600.14±161.87条,2013年每头松褐天牛成虫平均带441.86±120.73条,经方差分析,3年度所带线虫量, df=2、20,F=0.337, P=0.722, 均无显著差异。按松褐天牛雌雄性别计数,雌松褐天牛平均卵巢线虫816±188.58条,雄天牛平均带308±59.85条,经方差分析,F=6.066,df=1、21,P=0.031 5,差异显著,这可能与雌虫生殖系统空间大、能容纳较多线虫有关。2002年每头松褐天牛雌成虫平均带916±518.18条,雄成虫平均带362±120.27条;2007年每头松褐天牛雌成虫平均带794±229.01条,雄成虫平均带346±149.33条;2013年每头松褐天牛雌成虫平均带711±162.55条,雄成虫平均带240±78.21条。对这3年度雌、雄携带线虫量作方差分析,2002年,df=1、6,F=0.797, P=0.413, 无显著差异;2007年,df=1、6,F=2.210, P=0.197, 无显著差异;2013年,df=1、6,F=8.198, P=0.035, 有显著差异(图 2)。

    Figure 2.  The numbers of the Contortylenchus genitalicola nematodes carried by the Monochamus alternatus beetle

  • 卵巢线虫有3个生态位。松褐天牛成虫或蛹是卵巢线虫的第一个生态位,在松褐天牛尾部和雌雄生殖器内都能检到生活着的卵巢线虫(图 3a);松树木质部是卵巢线虫第二个生态位,但在松树上未能检到卵巢线虫的卵,幼虫及成虫与其它线虫混杂在一起,不易分离;卵巢线虫第三个生态位是松褐天牛的幼虫,偶然在蛹室内劈到1头黑色死亡松褐天牛幼虫,镜检发现在这头死幼虫体内有活着的卵巢线虫。这一发现,初步证明卵巢线虫的生态位变换过程是:松树木质部内的卵巢线虫成虫在4月中下旬起逐步进入松褐天牛的蛹或直接进入在羽化的松褐天牛雌雄成虫的生殖系统,完成从第2生态位进入第1生态位过程。在松褐天牛生殖器内,卵巢线虫交尾产卵;然后卵孵化,发育到4龄幼虫;此时松褐天牛咬穿松木,携带着卵巢线虫出孔;松褐天牛成虫飞到健康松树上补充营养,然后交尾;交尾时雄松褐天牛所带的卵巢线虫转移到雌松褐天牛生殖器内;交尾后雌松褐天牛在树干上产卵,同时把所携带的卵巢线虫也一同注入松树的韧皮部,完成从第1生态位进入第2生态位过程(图 3b, c)。进入韧皮部的4龄卵巢线虫开始蜕皮为成虫,接着交尾产卵在韧皮部增殖,扩散,深达木质部;与此同时松褐天牛卵也孵化发育为幼虫,在松树韧皮部取食,并逐渐长大;此时在松褐天牛幼虫周边的部分卵巢线虫就会侵入寄生松褐天牛幼虫并致死幼虫,寄生致死的幼虫黑色,解剖可见大量线虫活动,完成从第2生态位进入第3生态位过程。松褐天牛幼虫死亡后,卵巢线虫离开,再进入松木质部,完成从第3生态位到第2生态位过程。第2生态位的卵巢线虫继续寻找寄主,重复第2生态位到第3生态位,再从第3生态位进入第2生态位的过程。这是卵巢线虫生态位变换和寄生过程,只是初步的了解,寄生率及具体的寄生过程尚需作进一步的调查研究。此时,未被寄生的松褐天幼虫逐渐发育为4龄虫;4龄幼虫在木质部咬坑道,筑成蛹室,次年4月下旬开始化蛹,卵巢线虫再次从第2生态位进入第1生态位;当松褐天牛羽化成虫,咬出羽化孔后,通过补充营养、交尾产卵,卵巢线虫又从第1生态位进入第2生态位,完成一个大循环(图 4)。

    Figure 3.  The ovarian nematode distribution in the anal part of beetle of M. alternatus

    Figure 4.  The alternation and cycle of environment site of ovarian nematode

  • 卵巢线虫在松褐天牛成虫体内,产的卵经约4 d后胚胎开始发育,变为孕胎卵;孕胎卵约1d就出卵壳为1龄幼虫;1龄幼虫经1~2 d蜕皮为2龄幼虫;2龄幼虫约2~3 d蜕皮为3龄幼虫;3龄幼虫约2~3 d蜕皮为4龄幼虫。4龄幼虫的2种虫态:A仍然生活在3龄幼虫的蜕内,时间可长达10~15 d;B直接由3龄幼虫蜕皮而成或由A离开3龄幼虫蜕而成。4龄幼虫具有耐久性和扩散性,生活时间较长;4龄幼虫聚集在松褐天牛的卵巢,待松褐天牛产卵时一同进入松树皮内。进入松树木质部的4龄卵巢线虫蜕皮为成虫,随即交尾、产卵、幼虫孵化,直到再形成成虫。随着卵巢线虫在树内生长、增殖,此时,产入松树皮下的松褐天牛卵也孵化为幼虫,开始取食松树形成层组织,逐渐发育;当松褐天牛幼虫2龄后,其周围的卵巢线虫会侵入部分松褐天牛幼虫体内,致死寄主;随后,卵巢线虫离开死亡的松褐天牛幼虫,回到松树木质部。卵巢线虫在松树木质部生活和对松褐天牛幼虫的寄生阶段,刚发现,加之未见到雄成虫,对其生活史及生物学特性只是初步观察和分析,研究尚是空白。

    Figure 5.  Showing the life history cycle of the ovarian nematode in the inner of the M. alternatus and pine wood

3.   讨论
  • 松材线虫病已成为对我国森林生态系统危害最大的有害生物,而松褐天牛成虫由于能携带、传播松材线虫,因而成为防治松材线虫病的标靶害虫。但实际上松褐天牛能携带多种线虫[20],卵巢线虫就是松褐天牛所携带的另一种线虫。日本学者认为卵巢线虫是松褐天牛成虫的寄生线虫[27-29]。我们经研究,发现我国的松褐天牛上也携带有卵巢线虫,而且在松褐天牛的雌雄成虫体内都检测到了卵巢线虫,表明这种线虫并非仅由雌虫卵巢所携带;而且该线虫的数量在松褐天牛的雌、雄成虫中基本相同。卵巢线虫在松褐天牛体内生活时间大约为30 d,能在松褐天牛生殖器内繁殖,但只能完成从成虫产卵到4龄扩散型幼虫,不能完全完成其完整的生活史。当松褐天牛成虫产卵时,卵巢线虫也随同松褐天牛的卵一起被接种到寄主松树韧皮部和形成层。卵巢线虫也能在松树中生活、繁殖,在松树中的生活时间长达9个月,但其在松树内生活的过程尚不明暸。卵巢线虫常与松褐天牛体内的真菌绕成一团,这两者有何关系也尚不清楚,日本学者研究证明,该线虫可在真菌培养剂基上完成生活史[27-28],说明卵巢线虫可能以真菌为其食物源。

    日本学者认为,卵巢线虫是寄生松褐天牛卵巢的寄生线虫[29]。这个结论有待于进一步研究证实。第一, 寄生于卵巢的寄生线虫应该对松褐天牛的卵巢有特别的趋性,而实际上卵巢线虫在雄成虫生殖器内也有分布,这表明卵巢线虫对卵巢没有特别的趋性,不是雌性松褐天牛卵巢特有的线虫;第二,寄生线虫的寄生结果必然要引起寄主感病甚至导致寄主死亡,但实际情况是,卵巢线虫能在雌雄松褐天牛成虫体内生活,未发现携带大量卵巢线虫的松褐天牛致病或死亡,表明寄生依据不足。松褐天牛成虫只是卵巢线虫的搭载和运输工具,卵巢线虫必须依靠松褐天牛成虫才能实现在松树与松树之间的传播循环。当然,对这方面的研究还是空白,疑点很多,需要进一步研究来证实。但我们发现,卵巢线虫对松褐天牛幼虫可能有寄生作用,能在黑变腐烂的松褐天牛幼虫体上找到卵巢线虫。如能证实这一点,表明卵巢线虫参与了对松褐天牛幼虫种群数量的调节作用,同时也表明卵巢线虫在松树体内完成另一部分的生活史过程。卵巢线虫在松树体内完成部分生活史,在松褐天牛成虫体内完成一部分生活史,形成一个完整的循环体系。

    有一个问题值得注意:卵巢线虫形态多变复杂,不同的生活阶段就有不同的形态相适应[29]。日本学者用真菌培养的卵巢线虫形态与松褐天牛体内的卵巢线虫形态有极大的差异[29]。本文只报道在松褐天牛体内的卵线虫的形态,这些线虫形态也很复杂,为何其2龄幼虫有2种形态“胖形”与“线形”,其具有什么功能,现在所知很少。更值得注意的是卵巢线虫与松材线虫能同时存在于松褐天牛体上,同样在松树与松褐天牛之间循环,是否对松树也有致病作用,是值得深入研究的问题。从卵巢线虫的生活史和在松褐天牛与松树的循环过程分析可见,卵巢线虫也可能与松材线虫具有同样的病原作用[30-31]。现在研究报道认为,松材线虫的病原作用是其携带的细菌引起的[9, 32-39],松材线虫只是起到了传播媒介作用,即把细菌传带到松树体内的组织中。卵巢线虫同松材线虫一样,体上也能带大量的细菌,甚至携带大量的真菌。这些传送到松树体内组织中去的微生物很可能有病原物,导致松树感病枯死。当然这些仅是假设,还有待于进一步研究证实。

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