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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。
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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);
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);
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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)。
表 1 松褐天牛成虫携带卵巢线虫百分数
Table 1. Percentage of M. alternatus adult carried ovarian nematode
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 adults2002 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. -
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)。
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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)。
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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。
卵巢线虫(Contortylenchus genitalicola)在中国松褐天牛上的分布与作用研究
Distribution and Function of the Ovarian Nematode Contortylenchus genitalicola (Tylenchida: Allantonematidae) on Monochamus alternatus (Coleoptera: Cerambycidae) in China
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摘要:
目的 日本学者Kosaka与Ogura发现松褐天牛成虫除携带松材线虫外,雌成虫卵巢内还携带有另一种线虫,他们将其命名为卵巢线虫,并认为这种线虫是松褐天牛成虫的寄生性线虫。我国对该种线虫的研究迄今未见报道。为了证明这种线虫在我国是否存在和分布,开展了本项研究。 方法 分期分批捕获刚羽化的松褐天牛雌雄成虫,采用解剖松褐天牛成虫松树木质部及感病的松褐天牛幼虫等方法,调查卵巢线虫的存在与分布。 结果 通过调查研究,发现我国的松褐天牛成虫体内有该卵巢线虫存在,分布于松褐天牛成虫、幼虫体内和松树木质部3个部位;在松褐天牛成虫体内的卵巢线虫通过松褐天牛雌成虫产卵而接种、进入寄主树木木质部中。同时,木质部的卵巢线虫也有一部分进入松褐天牛幼虫体内寄生,另一部分仍在木质部生活,当松褐天牛幼虫再次发育为成虫时,在木质部中的卵巢线虫和已被寄生的松褐天牛幼虫体内的卵巢线虫再次进入松褐天牛成虫体内,完成循环。卵巢线虫在松褐天牛雌、雄虫体内均有分布,携带率为44.4%,其中松褐天牛雌成虫携带率为43.8%,松褐天牛雄成虫携带率为45.0%,两者间无显著差异;每头松褐天牛成虫平均携带卵巢线虫574条,其中雌成虫平均携带816条,雄成虫平均携带308条,具显著差异。初步研究表明,卵巢线虫在松褐天牛成虫体内只能完成产卵到1~4龄的幼虫阶段,不能完成一个完整的世代;在松褐天牛幼虫体内寄生和在木质部生活的卵巢线虫能完成一个完整世代,但具体过程尚不清楚;调查中未观察到卵巢线虫对松褐天牛成虫有寄生致病或致死的现象,但对松褐天牛幼虫有寄生致死的能力;卵巢线虫常与松材线虫同时存在,而且侵入松树及离开松树的方式与松材线虫相同。 结论 我国松褐天牛体内也发现有卵巢线虫存在;目前尚不能证明该线虫对松褐天牛成虫具有寄生致死性,但对松褐天牛幼虫具有一定的寄生致死性;卵巢线虫的生活史与松材线虫相似,是否与松材线虫一样对松树具有危害性以及其病理作用还有待于进一步研究。 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. -
表 1 松褐天牛成虫携带卵巢线虫百分数
Table 1. Percentage of M. alternatus adult carried ovarian nematode
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 adults2002 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. -
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