麦蛾柔茧蜂规模化人工饲养的关键技术及其冷胁迫的反应机制
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摘要
麦蛾柔茧蜂(Habrobracon hebetor)是一种分布和寄主都非常广泛的体外寄生蜂。前人的研究结果表明麦蛾柔茧蜂寄生能力强,繁殖速度快,因此具有很好的应用前景。为了推动麦蛾柔茧蜂应用于生物防治的进程,本论文研究了以印度谷螟作为中间寄主规模化饲养麦蛾柔茧蜂,优化印度谷螟(Plodia interpunctella)的饲料配方,寻找麦蛾柔茧蜂的嗜好幼虫龄期；在低温条件下(5℃)冷藏刚羽化的麦蛾柔茧蜂成虫；在不同温光条件下诱导麦蛾柔茧蜂滞育,滞育后长时间冷藏麦蛾柔茧蜂；克隆滞育相关的类似热激蛋白70基因,并测定在不同温光条件下饲养的麦蛾柔茧蜂体内类似热激蛋白70的相对表达量。主要研究结果如下：
用正交实验优化了印度谷螟的饲料配方。用小麦,甘油,蜂蜜,红枣,酵母5种配方组分,根据正交实验设计原理,按不同比例配制饲料用以饲养幼虫。分析饲料中各组成成分对印度谷螟的发育历期、化蛹率、羽化率以及后代产卵量影响。得到了各指标下的最优方案及各组分对该指标影响的主次顺序。饲养麦蛾柔茧蜂需要重量大的老熟幼虫,因此选用蛹重最大的配方：A3B4C2D3。
通过研究寄主龄期对麦蛾柔茧蜂寄生能力和繁殖的影响,明确了麦蛾柔茧蜂的嗜好幼虫龄期。通过在密闭空间里调查麦蛾柔茧蜂对印度谷螟幼虫各龄期的寄生率以及后代的羽化数,评价了麦蛾柔茧蜂最喜好寄主龄期及各龄期对麦蛾柔茧蜂后代羽化数的影响。结果显示,麦蛾柔茧蜂能寄生所有龄期的印度谷螟幼虫,但不管在有选择还是无选择条件下,三龄、四龄幼虫出蜂数都显著多于低龄幼虫。在有选择的条件下,麦蛾柔茧蜂更喜欢老龄幼虫。一般来说,在无选择的条件下低龄幼虫的被寄生率要高于在有选择的条件下。因而,印度谷螟低龄幼虫被寄生率较低,三龄、四龄幼虫更适合饲养麦蛾柔茧蜂。
通过冷藏麦蛾柔茧蜂成虫来评价冷藏对其冷藏代成虫及后代的影响。刚羽化的麦蛾柔茧蜂雌虫在5±1℃的条件下冷藏10、20、30、40、50和70d。记录数个生理指标用以确定评价冷藏代和F1代是否受冷藏的影响。冷藏时间长于30d时,冷藏代的寄生能力有所下降,但对F1代没有显著性影响。冷藏代的寿命和产卵量在冷藏20d以上显著下降,但冷藏时间长于50d才会对F1代有影响。不同冷藏时间对发育历期有显著性影响,但差异在2d以内。冷藏时间长短对F1和F2代的性比没有显著性影响。
在不同温光条件下诱导麦蛾柔茧蜂滞育。通过观察在低温和不同光照条件下饲养的麦蛾柔茧蜂,记录麦蛾柔茧蜂生理指标,结果发现麦蛾柔茧蜂的发育历期不仅受到温度的影响,而且受到光周期的影响。雌蜂羽化后在低温下的寿命显著长于羽化后放到高温下的寿命,但产卵量并非如此。解剖结果显示低温和短光照能抑制卵巢的发育。只有饲养和产卵条件均为低温短光照,雌蜂才具有较长的产卵前期,其余条件下雌蜂的产卵前期均很短。在20℃,10：14 L：D条件下饲养的麦蛾柔茧蜂雌蜂在20℃时的呼吸率显著低于在20℃,L：D=16：8条件下饲养的雌蜂。但20℃,长短光照条件下饲养的麦蛾柔茧蜂雌蜂在27.5℃时的呼吸率没有显著差异。另外,结果还显示在17.5和20℃,短光照条件下,麦蛾柔茧蜂能被诱导进入生殖滞育(reproductive diapauses)。
在明确滞育条件的基础上,研究了长时间冷藏对滞育麦蛾柔茧蜂的影响。冷藏滞育和非滞育麦蛾柔茧蜂,发现死亡率随着冷藏时间的延长而增高,但滞育麦蛾柔茧蜂的死亡率在冷藏8、12和16周后显著低于冷藏同时间段的非滞育麦蛾柔茧蜂。麦蛾柔茧蜂的寿命和产卵量随着在5℃中冷藏时间的延长而显著的下降。但滞育的麦蛾柔茧蜂,寿命和产卵量在8周以内和对照没有显著差异,并显著超过非滞育麦蛾柔茧蜂。无论冷藏后的雌蜂是否与雄蜂配对,后代雌蜂的百分比始终低于对照后代雌蜂的百分比,但不同冷藏时间段之间没有显著性差异。
最后本文克隆了滞育相关的类似热激蛋白70基因,并测定了在不同温光条件下饲养的麦蛾柔茧蜂体内类似热激蛋白70的相对表达量。通过设计简并性引物获得了麦蛾柔茧蜂体内三个类似热激蛋白70基因片段,分别命名为hsp70A6、hsp70B4和hsp70B6。运用3’和5’RACE技术获得这三个基因的cDNA全长序列。根据全长预测的氨基酸序列和其它昆虫的热激蛋白70做比对发现同源性很高。通过实时定量PCR检测了不同饲养条件下麦蛾柔茧蜂体内三个类似热激蛋白70的相对表达量。结果显示类似热激蛋白70A6和类似热激蛋白70B6的相对表达量在不同饲养环境中并没有显著性差异,但在高温饲养条件下,麦蛾柔茧蜂体内类似热激蛋白70B4的表达量要显著的高于低温下的表达水平。
Habrobracon hebetor (Say) (=Bracon hebetor)(Hymenoptera:Braconidae), a cosmopolitan ectoparasitoid, was considered as a biological control agents of various lepidopteran pests in worldwide due to its rapid growth and development rates. In order to promote the process of H. hebetor used in biological control, this paper study the indianmeal moth as the host for mass rearing H. hebeotr, and optimized the artificial diet recipe for indianmeal moth; find the best larva stage for rearing H. hebetor; evaluated the performance of newly emerge H. hebetor adult after different stogage duration at 5℃; try to induce diapauses under different conditions, and long term cold storage the adults enter diapauses; clone the heat shpck protein 70 genes which may have relationship with diapauses, and determine the expression patterns of hsp70sin adults which rearing from different conditions. The main results are as follows:
Wheat, glycerine, honey, dried jujube and yeast were used as the components of artificial diet for indianmeal moth. According to the orthogonal experimental design, different artificial diet recipes were made up to test the influence of each components to different parameters. The parameters include the development time of larvae stage, pupae rate, eclosion rate and adult fecundity. Analyze the influence of each components in artificial diet to different parameters and got the best artificial diet recipes for each parameters.
The interactions between trophic levels during parasitism and host location by H. hebetor Say (Hymenoptera:Braconidae) within a closed environment by carrying out the tests to evaluate the role host instar on the mortality of Plodia interpunctella and on the emergence of H. hebetor were examined. Results showed that H. hebetor was able to parasitize all instars (first through fourth) of P. interpunctella, but significantly fewer early instars (first through fourth) were parasitized. Parasitized third and fourth instars were more profitable to H. hebetor, irrespective of choice factor, as significantly more adult parasitoids emerged from third and fourth instars. H. hebetor females consistently showed a preference for fourth instars of P. interpunctella when they were offered a choice between early and late host instars in arenas. Generally, parasitization of early instars was higher in no-choice than in choice tests. The behavior of H. hebetor in relation to host choice and its influence on the pest mortality are discussed.
The effects of low temperature storage on the quality of adult parasitoids and their progeny were investigated. Newly emerged females were stored for 10,20,30,40,50, and 70 d at 5±1℃. Several reproductive and developmental parameters were then assessed to determine the quality of the adult parasitoids and their progeny. After more than 30 d of storage, there was a decrease in parental parasitism, but low temperature storage of parents had no effect on parasitism of the F1 generation. Parental longevity and fecundity decreased after more than 20 d of storage, but there was no effect of storage duration on the fecundity and longevity of the F1 generation until a storage duration of 50 d. Development time varied with storage duration but differences were within 2 d. Storage duration had no effect on the sex ratio of F1 and F2 generations. Our data show that H. hebetor can be cold stored for up to 20 d without adversely affecting the performance of the parasitoid. Therefore, short-term storage of H. hebetor adults could be used for Hmaintaining and accumulating large numbers of parasitoids in mass rearing programs.
Several physiological parameters of H. hebetor rearing in different photoperiod at low temperature were investigated. Found that the development time of H. hebetor not only significant effect by temperature, but also the photoperiod. The longevity of female leaving at low temperature after emergence was significant longer than the female move to high temperature, but the fecundity was not. The performance of H. hebetor adult females which rearing from different photoperiod at low temperature were studied. Dissecting result showed that short photoperiod and low temperature would suppress the development of ovary. The female have long preoviposition period when the conditions for rearing and laying eggs are always short photoperiod and low temperature, otherwise, the preoviposition period is very short. When the test temperature set at 20℃, the respiration rate for the female rearing from 20℃,10:14 L:D was significant lower than the female rearing from 20℃, L:D=16:8. But there have no significant difference when the test temperature set at 27.5℃. Our data showed that at 17.5 and 20℃, the wasp can induction into reproductive diapauses at short photoperiod.
The effects of long-term cold storage on the performance of diapausing H. hebetor were investigated. We found that the mortality during storage increased with increasing duration and the mortality for diapausing females were lower than nondiapausing females after 8,12, and 16 weeks of storage. Longevity and egg laying of H, hebetor reared at nondiapause condition decreases rapidly with increasing storage duration at 5℃. But for the female rearing from diapauses conditions, the longevity and egg laying have no significant difference with culture when the storage duration are short than 8 weeks, and always significantly higher than nondiapausing female. The female percentage of F1 offspring for the female after storage is always lower than culture regardless pair with male or not, but there have no difference for the female after different storage durations.
Three partial genes sequence of heat shock protein 70's in H. hebetor were amplified by degenerate primer, and they were tentatively named as hsp70A6, hsp70B4 and hsp70B6. 3'and 5'RACE were followed to develop the full length clone. Deduced the amino acid sequence and comparison with other insects hsp70s shows high similarity. The theoretical molecular weight and isoelectric point were estimated. Expression patterns of the hsp70s were investigated in adults which rearing from different conditions. Real time quantitative PCR analyse were carried out to determine the different expression on mRNA levels. The expression of hsp70A6 and hsp70B6 mRNA were similar among the different rearing conditions, hsp70B4 mRNA was increase expression in high temperature rearing condition compare to low temperature rearing condition. Results suggest that the expression hsp70A6 and hsp70B6 are constitutive and hsp70B4 is relation to the temperature of rearing condition.
用正交实验优化了印度谷螟的饲料配方。用小麦,甘油,蜂蜜,红枣,酵母5种配方组分,根据正交实验设计原理,按不同比例配制饲料用以饲养幼虫。分析饲料中各组成成分对印度谷螟的发育历期、化蛹率、羽化率以及后代产卵量影响。得到了各指标下的最优方案及各组分对该指标影响的主次顺序。饲养麦蛾柔茧蜂需要重量大的老熟幼虫,因此选用蛹重最大的配方：A3B4C2D3。
通过研究寄主龄期对麦蛾柔茧蜂寄生能力和繁殖的影响,明确了麦蛾柔茧蜂的嗜好幼虫龄期。通过在密闭空间里调查麦蛾柔茧蜂对印度谷螟幼虫各龄期的寄生率以及后代的羽化数,评价了麦蛾柔茧蜂最喜好寄主龄期及各龄期对麦蛾柔茧蜂后代羽化数的影响。结果显示,麦蛾柔茧蜂能寄生所有龄期的印度谷螟幼虫,但不管在有选择还是无选择条件下,三龄、四龄幼虫出蜂数都显著多于低龄幼虫。在有选择的条件下,麦蛾柔茧蜂更喜欢老龄幼虫。一般来说,在无选择的条件下低龄幼虫的被寄生率要高于在有选择的条件下。因而,印度谷螟低龄幼虫被寄生率较低,三龄、四龄幼虫更适合饲养麦蛾柔茧蜂。
通过冷藏麦蛾柔茧蜂成虫来评价冷藏对其冷藏代成虫及后代的影响。刚羽化的麦蛾柔茧蜂雌虫在5±1℃的条件下冷藏10、20、30、40、50和70d。记录数个生理指标用以确定评价冷藏代和F1代是否受冷藏的影响。冷藏时间长于30d时,冷藏代的寄生能力有所下降,但对F1代没有显著性影响。冷藏代的寿命和产卵量在冷藏20d以上显著下降,但冷藏时间长于50d才会对F1代有影响。不同冷藏时间对发育历期有显著性影响,但差异在2d以内。冷藏时间长短对F1和F2代的性比没有显著性影响。
在不同温光条件下诱导麦蛾柔茧蜂滞育。通过观察在低温和不同光照条件下饲养的麦蛾柔茧蜂,记录麦蛾柔茧蜂生理指标,结果发现麦蛾柔茧蜂的发育历期不仅受到温度的影响,而且受到光周期的影响。雌蜂羽化后在低温下的寿命显著长于羽化后放到高温下的寿命,但产卵量并非如此。解剖结果显示低温和短光照能抑制卵巢的发育。只有饲养和产卵条件均为低温短光照,雌蜂才具有较长的产卵前期,其余条件下雌蜂的产卵前期均很短。在20℃,10：14 L：D条件下饲养的麦蛾柔茧蜂雌蜂在20℃时的呼吸率显著低于在20℃,L：D=16：8条件下饲养的雌蜂。但20℃,长短光照条件下饲养的麦蛾柔茧蜂雌蜂在27.5℃时的呼吸率没有显著差异。另外,结果还显示在17.5和20℃,短光照条件下,麦蛾柔茧蜂能被诱导进入生殖滞育(reproductive diapauses)。
在明确滞育条件的基础上,研究了长时间冷藏对滞育麦蛾柔茧蜂的影响。冷藏滞育和非滞育麦蛾柔茧蜂,发现死亡率随着冷藏时间的延长而增高,但滞育麦蛾柔茧蜂的死亡率在冷藏8、12和16周后显著低于冷藏同时间段的非滞育麦蛾柔茧蜂。麦蛾柔茧蜂的寿命和产卵量随着在5℃中冷藏时间的延长而显著的下降。但滞育的麦蛾柔茧蜂,寿命和产卵量在8周以内和对照没有显著差异,并显著超过非滞育麦蛾柔茧蜂。无论冷藏后的雌蜂是否与雄蜂配对,后代雌蜂的百分比始终低于对照后代雌蜂的百分比,但不同冷藏时间段之间没有显著性差异。
最后本文克隆了滞育相关的类似热激蛋白70基因,并测定了在不同温光条件下饲养的麦蛾柔茧蜂体内类似热激蛋白70的相对表达量。通过设计简并性引物获得了麦蛾柔茧蜂体内三个类似热激蛋白70基因片段,分别命名为hsp70A6、hsp70B4和hsp70B6。运用3’和5’RACE技术获得这三个基因的cDNA全长序列。根据全长预测的氨基酸序列和其它昆虫的热激蛋白70做比对发现同源性很高。通过实时定量PCR检测了不同饲养条件下麦蛾柔茧蜂体内三个类似热激蛋白70的相对表达量。结果显示类似热激蛋白70A6和类似热激蛋白70B6的相对表达量在不同饲养环境中并没有显著性差异,但在高温饲养条件下,麦蛾柔茧蜂体内类似热激蛋白70B4的表达量要显著的高于低温下的表达水平。
Habrobracon hebetor (Say) (=Bracon hebetor)(Hymenoptera:Braconidae), a cosmopolitan ectoparasitoid, was considered as a biological control agents of various lepidopteran pests in worldwide due to its rapid growth and development rates. In order to promote the process of H. hebetor used in biological control, this paper study the indianmeal moth as the host for mass rearing H. hebeotr, and optimized the artificial diet recipe for indianmeal moth; find the best larva stage for rearing H. hebetor; evaluated the performance of newly emerge H. hebetor adult after different stogage duration at 5℃; try to induce diapauses under different conditions, and long term cold storage the adults enter diapauses; clone the heat shpck protein 70 genes which may have relationship with diapauses, and determine the expression patterns of hsp70sin adults which rearing from different conditions. The main results are as follows:
Wheat, glycerine, honey, dried jujube and yeast were used as the components of artificial diet for indianmeal moth. According to the orthogonal experimental design, different artificial diet recipes were made up to test the influence of each components to different parameters. The parameters include the development time of larvae stage, pupae rate, eclosion rate and adult fecundity. Analyze the influence of each components in artificial diet to different parameters and got the best artificial diet recipes for each parameters.
The interactions between trophic levels during parasitism and host location by H. hebetor Say (Hymenoptera:Braconidae) within a closed environment by carrying out the tests to evaluate the role host instar on the mortality of Plodia interpunctella and on the emergence of H. hebetor were examined. Results showed that H. hebetor was able to parasitize all instars (first through fourth) of P. interpunctella, but significantly fewer early instars (first through fourth) were parasitized. Parasitized third and fourth instars were more profitable to H. hebetor, irrespective of choice factor, as significantly more adult parasitoids emerged from third and fourth instars. H. hebetor females consistently showed a preference for fourth instars of P. interpunctella when they were offered a choice between early and late host instars in arenas. Generally, parasitization of early instars was higher in no-choice than in choice tests. The behavior of H. hebetor in relation to host choice and its influence on the pest mortality are discussed.
The effects of low temperature storage on the quality of adult parasitoids and their progeny were investigated. Newly emerged females were stored for 10,20,30,40,50, and 70 d at 5±1℃. Several reproductive and developmental parameters were then assessed to determine the quality of the adult parasitoids and their progeny. After more than 30 d of storage, there was a decrease in parental parasitism, but low temperature storage of parents had no effect on parasitism of the F1 generation. Parental longevity and fecundity decreased after more than 20 d of storage, but there was no effect of storage duration on the fecundity and longevity of the F1 generation until a storage duration of 50 d. Development time varied with storage duration but differences were within 2 d. Storage duration had no effect on the sex ratio of F1 and F2 generations. Our data show that H. hebetor can be cold stored for up to 20 d without adversely affecting the performance of the parasitoid. Therefore, short-term storage of H. hebetor adults could be used for Hmaintaining and accumulating large numbers of parasitoids in mass rearing programs.
Several physiological parameters of H. hebetor rearing in different photoperiod at low temperature were investigated. Found that the development time of H. hebetor not only significant effect by temperature, but also the photoperiod. The longevity of female leaving at low temperature after emergence was significant longer than the female move to high temperature, but the fecundity was not. The performance of H. hebetor adult females which rearing from different photoperiod at low temperature were studied. Dissecting result showed that short photoperiod and low temperature would suppress the development of ovary. The female have long preoviposition period when the conditions for rearing and laying eggs are always short photoperiod and low temperature, otherwise, the preoviposition period is very short. When the test temperature set at 20℃, the respiration rate for the female rearing from 20℃,10:14 L:D was significant lower than the female rearing from 20℃, L:D=16:8. But there have no significant difference when the test temperature set at 27.5℃. Our data showed that at 17.5 and 20℃, the wasp can induction into reproductive diapauses at short photoperiod.
The effects of long-term cold storage on the performance of diapausing H. hebetor were investigated. We found that the mortality during storage increased with increasing duration and the mortality for diapausing females were lower than nondiapausing females after 8,12, and 16 weeks of storage. Longevity and egg laying of H, hebetor reared at nondiapause condition decreases rapidly with increasing storage duration at 5℃. But for the female rearing from diapauses conditions, the longevity and egg laying have no significant difference with culture when the storage duration are short than 8 weeks, and always significantly higher than nondiapausing female. The female percentage of F1 offspring for the female after storage is always lower than culture regardless pair with male or not, but there have no difference for the female after different storage durations.
Three partial genes sequence of heat shock protein 70's in H. hebetor were amplified by degenerate primer, and they were tentatively named as hsp70A6, hsp70B4 and hsp70B6. 3'and 5'RACE were followed to develop the full length clone. Deduced the amino acid sequence and comparison with other insects hsp70s shows high similarity. The theoretical molecular weight and isoelectric point were estimated. Expression patterns of the hsp70s were investigated in adults which rearing from different conditions. Real time quantitative PCR analyse were carried out to determine the different expression on mRNA levels. The expression of hsp70A6 and hsp70B6 mRNA were similar among the different rearing conditions, hsp70B4 mRNA was increase expression in high temperature rearing condition compare to low temperature rearing condition. Results suggest that the expression hsp70A6 and hsp70B6 are constitutive and hsp70B4 is relation to the temperature of rearing condition.
引文
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