全 文 ：　＊ This work w as supported by the S tate Key Laboratory of Integrated Management of Pest Insect &Rodents Opening
Research Foundat ion.
＊＊ To w hom correspondence should be addressed.
EFFECTSOF SUBLETHAL ESERINE ON THE CHEMICAL
COMMUNICATION SYSTEM OF ASIAN CORN BORER ,
OSTRINIA FURNACALIS (GUEN E)＊
YANG Zhi-hua＊＊ , DU Jia-wei , ZHOU Hong-chun and XU Shao-fu
Institute of Entomology , Chinese Academy of Science , Shanghai 200025 , China
(Received Dec.17 , 1999;accepted May 17 , 2000)
Abstract　　 Topically applied sublethal doses of eserine may interrupt chemical communication between the two
sexes of A sian corn borer , Ostrinia furnacalis (Guené e), by affecting calling and sex pheromone titre released
by the females.(1)Studies on the effects of sublethal eserine on the chemical communication system of O.f ur-
nacalis indicated tha t there was a decreasing probability of females calling and sex pheromone titre as the eserine
dosage increased from 0.27 to 2 700 ng , with no effect on the periodicity.How ever , at 2 700 ng , the E/ Z ra tio
of the sex pheromone components w as affected.The sexual chemical communica tion system could not control
w ithin narrow level.(2)The recovery test o f 27ng-treated female indicated that the calling percentages of the
1st , 2nd and 4th day post- treatment were 50%, 78% and 84% respectiv ely.Sex pheromone titre w as 40%,
40% and 80% of control female repectively.There w as a trend tow ard recovery.The sexual chemical communi-
cation sy stem could control within nar row level from the 2nd day post-treatment.
Key words　　Ostrinia furnacalis , sex pheromone , calling behaviour , eserine , sublethal , recovery
1　INTRODUCTION
The behavior o f insects , and all animals , is governed by the interactions among neu-
rons w ithin their nervous systems.Insecticides have been selected and sometimes designed
for their remarkable abili ty to kill insects.Most attack specific sites w ithin the insect s ner-
vous system (Matsumura 1985).Insecticidal effects on insects may be considered in two
categories:first , the mortality resulting from the direct toxic effect of the compound;sec-
ond , the behavioral effect of sublethal doses of insecticides on insect pests.Sublethal effects
occurred when insufficient molecules to cause death reached the sites of insecticidal action.
In recent years , there have been few detailed studies concerning the potential behav-
io ral effects of sublethal doses of insecticides.For example , sublethal doses of insecticides
have been shown to adversely affect calling in Pectinophora gossypiella (Haynes and Baker
1985), the wing fanning response of male Pectinophora gossypiella (Saunders) to
pheromones(Floyd and Crowder 1981), and the pheromone-mediated flight behavior of
Grapholitha molesta(Busck)(Linn and Roelofs 1984).The potential for control of behav-
io r with sublethal neurotoxicants is just beginning to be realized , but these early studies
have already revealed that further studies such as the recovery of calling behavior and
pheromone production affected by the sublethal neurotoxicants , the mechanism of sublethal
effects on chemical communication system , are specially necessary today .
The production of sex pheromone in a number of moth species follows a diel rhythm
250　 ENTOMOLOGIA SINICA　Vol.7 , No.3 , September 2000 ,pp.250 —256
which may be regulated by neural(Tang et al.1987), neuroendocrine (Raina and Klun
1984), or hormonal factors(Cusson and McNeil 1989).The production of sex pheromone
in Asian corn borer , O .f urnacalis , appear to be under the control of a neuroendocrine
regulation factor(Qiu and Cao 1989).It is not clear whether and how the sublethal neuro-
toxicants affect this process.Therefore , the aim of the present study was to elucidate the ef-
fects of sublethal neurotoxicants on the calling behavior and the sex pheromone production in
O.f urnacalis.
2　METHODS AND MATERIALS
2.1　Insect
Larval O .furnacalis were reared on artificial diet modified from that described by
Qiu et al.(1989).Pupae were separated by sex .Male and female moths were held in
25cm×15cm×35cm screened cages housed within separate environmental chambers(14L∶
10D , (26±1)℃).Moths had access to 10%sugar water.
2.2　Chemicals and sublethal dose
Technical grade eserine was diluted in distilled acetone.Stock solutions of eserine were
prepared at 0.27 , 27 , 2700 ng/μL.Adult females were dosed topically with 0.27 ng(n =
50), 27ng(n =50), 2 700 ng(n =50)eserine solutions on the ventral portion of the ab-
domen , and to determine sublethal doses.The control females were dosed topically w ith
1μL acetone(n=50).After 24 h and 100 h , the mortali ties for each group were noted.
Solutions were prepared within 1 week of use.The sublethal dose that causes litt le mortali ty
(<5%)by 24h after treatment and the mortality <30%occurred by 100h was selected for
the further research.
2.3　Calling behavior observation
Calling behavior by normal or eserine-treated virg in female , O .f urnacalis , was ob-
served in an environmental chamber (26℃, 14 L∶10D)with dim incandescent red back-
lighting in an otherwise dark room.Calling behavior was defined as gland protrusion.At
30min intervals throughout the 10h scotophase , females were noted as calling or not calling.
2.4　Analytical methods
The sublethal neurotoxicants that affected sex pheromone biosynthesis o f O .fur-
nacalis was verified by sampling sex pheromone gland extracts at the peak period of calling
(8 or 8.5 h after the initiation of scotophase).Twenty to thirty female moths in their sec-
ond scotophase were used at the various intervals.Gas chromatographic(GC)analyses were
performed on a Hewlett Packard 5880 capillary gas chromatograph equipped with a flame
ionization detector (100-200℃, 10℃/min.).A 25 m-0.2 mm-ID HP-17 column was
used for the quantitative determination.
3　RESULTS
3.1　Lethal effects
Table 1 gives the result of the lethal effects by 24 h and 100h after the treatment w ith
dose eserine.0.27 ng/ female and 27 ng/ female of eserine cause lit tle mortality (<5%)by
24 h after treatment and the mortali ty occurred by 100 h after treatment is less than 30%.
We chose these dosages of eserine in our experiment to determine the sublethal effects on the
251Yang Z.H.et al .:Effects of sublethal eserine on chemical communication system 　
chemical communication system of O .furnacalis.We also chose 27 ng/ female of eserine in
the recovery test to determine the recovery of the chemical communication system affected
by eserine (Table 1).
Table 1　Mortalities occurred by 24h and 100h after sublethal eserine treatment.
T ime after
treatment
Mor tality
Control
(n=50)
Eserine
0.27 ng(n=50) 27 ng(n=50) 2 700 ng(n =5)
24 h 0 0 0 0
100 h 2% 14% 28% 40%
3.2　The effects of sublethal eserine on calling behavior of O.furnacalis
One-day-old moths were treated with eserine at 1h before the beginning of scotophase.
Moths were cold-anesthetized on ice during the dosing procedure.The calling behavior of
the virgin female which treated by sublethal eserine was observed in an environmental cham-
ber(14L∶10D , (26±1)℃)with dim red backlighting in an otherwise dark room.In half
an hour of observation , each female was noted as either calling or not calling (Fig .1).
Fig.1　Sublethal effects of eserine on calling behavior by female O.furnacalis.Control
mo ths were dosed with 1μL of acetone , and other moths w ere dosed with the designated
　　　　　quantity of eserine in 1μL of acetone 1h before the beg inning of scotophase.
From Fig .1 , the calling behavior by females was significantly affected by sublethal
doses of eserine.Generally , there was a decreasing probability that a female performed call-
ing behavior as the dose of eserine increased.At the peak of calling (8 or 8.5 h after the ini-
tiation of scotophase), there was a significant difference between the probabilities of calling
by the normal female and sublethal eserine-treated female.These sublethal doses of eserine
apparently affected the probabili ty of calling , but not its periodicity .
3.3　Recovery of calling behavior by females treated by sublethal eserine
In this study , we need to know if the calling behavior by the females treated with the
sublethal eserine was recovered.The experimental results showed that the percentages of fe-
male calling w ithin the 1st , 2nd and 4th day after treated with eserine in the dosage of 27
ng/ female were 50%, 78%and 84% respectively (Fig.2).
The recovery of calling behavior by females was incomplete 4 days after treatment.
However , there was a trend toward recovery .This result indicated that sublethal eserine in-
terfered with female calling in a relatively long period.In the field , the decreasing probabili-
252 　 ENTOMOLOGIA SINICA　Vol.7 , No.3 , September 2000
Fig.2　Recovery duration of suble thal effects o f eserine on(27 ng/ female)calling behavio r of female
O.furnacalis.Control females and 1st-day post-treatment females were dosed 1 h before the begin-
ning of sco tophase.F rom 1st to 4th day , the calling percents of control females w ere the same 100%.
ty of calling w ill certainly cause the decreasing of mating rate , as a result , the offspring pop-
ulation density would certainly decrease.
3.4　Sublethal effects of eserine on sex pheromone biosynthesis of O.furnacalis
From the above results , we know that eserine treatment will significantly affect the
calling behavior of O .f urnacalis females , but we do not know whether it will affect sex
pheromone biosynthesis of O .f urnacalis females.Therefore , we have determined the sex
pheromone titre and the E/ Z ratio o f pheromone components by GC analysis.
The results of analyses indicated that there was a decreasing probability of sex pheromone titre
as the dose increased from 0.27 to 2700 ng.The periodicity was not affected in this case.Howev-
er , at 2 700 ng-treated group , E/Z was also affected.Its variation coefficient(C.V.%)is more
than 10%.The result indicated that the sexual chemical communication system could not control
within narrow level.At 27 ng-treated group , no significant effect was found on E/ Z , but the
C.V.%could not control under 10%(Table 2、3).
Table 2　The sex pheromone titre biosynthesized by female O.furnacalis treated by the sublethal dosages o f es-
erine(n=30).
Dosages
(ng/ female)
E-12-14:Ac＊
(ng)
Z-12-14:Ac＊＊
(ng)
14:Ac＊＊＊
(ng)
Average
(ng)
control 2.91±1.92 3.63±1.48 2.26±1.68 8.80±2.48
0.27 1.56±1.72 2.08±1.93 0.92±0.67 4.56±4.27
27 1.20±1.27 1.58±1.46 0.97±1.00 3.75±3.58
2700 0.92±0.87 1.53±1.52 0.69±0.53 3.14±2.84
＊(E)-12-te tradecenyl acetates;＊＊(Z)-12-tetradeceny l acetates;＊＊＊ tetradecenyl aceta tes.
　　Since the serious effects of sublethal eserine on the calling behaviors and sex pheromone
biosynthesis , it was more important to study if the sex pheromone biosynthesis affected by
eserine could be recovered.The results of recovery test were shown as Table 4.
253Yang Z.H.et al .:Effects of sublethal eserine on chemical communication system 　
Table 3　The E/Z ratio s of sex pheromone
components of female O.f urnacalis treated
by the sublethal dosages of eserine(n =30).
Dosages(ng/ female) E/(E +Z)＊ C.V.(%)
Control 0.44a 8.8
0.27 0.41ab 7.2
27 0.41ab 13.0
2 700 0.39b 13.0
＊ Values followed by the same letter were
determined not to be different
by DUNCAN s test ,
　　The recovery test of 27ng-treated group indi-
cated that sex pheromone titre o f the 1st , 2nd , 4th
day post-treatment was 40%, 40%, 80% of the
control group.There was a trend toward recovery.
T he sexual chemical communication system could
control w ithin narrow level(C.V.%<10%)at the
2nd day post-treatment.
Table 4　Recovery of chemical communication sy stem of O.f urnacalis(Guenée)dosed at 27ng/ female of
eserine.
Days
after
treatment
Chemical
communication
system
Control
(n=25)
27
ng-treatment
(n =25)
Percent of
recovery
P-value
(t-tes)
1 Sex pheromone titre
E/(E +Z)
C.V.(%)
9.10±3.42
0.43
4.1
3.75±3.58
0.41
13
～ 40%
　
　
P>0.05
2 Sex pheromone titre
E/(E +Z)
C.V.(%)
7.99±3.92
0.44
5.5
3.02±2.37
0.45
7.7
～ 40%
　
　
P>0.05
4 Sex pheromone titre
E/(E +Z)
C.V.(%)
4.79±2.74
0.43
9.6
3.83±3.29
0.45
10
～ 80%
　
　
P>0.05
4　DISCUSSION
The release of pheromone in O .furnacalis and most other species of moths depends
on an active behavioral process of everting the pheromone gland.We found that sublethal
doses of eserine substantially decreased the probability that females would call , without af-
fecting the periodicity of this activity (Fig.1).Similar effect were found in Glossina
austeni Newst and Pectinopora gossypiella (Haynes and Baker 1985).The effect of eserine
on the behavior of calling in female O .furnacalis would be expected to curtail mating since
the males are dependent on the volatile pheromone released by the females.
Pheromone emission by O .furnacalis is controlled by interrelated physiolog ical and
behavioral factors.Hunt and Haynes(1990)suggested that calling behavior and transport
o f pheromone to the gland s surface are synchronized in time and occur only during the sco-
tophase.These activities are under hormonal and neuronal controls.Therefore , neurotoxi-
cants , which at sublethal doses , might affect females calling and sex pheromone production
by disrupting hormonal or neuronal activity .
An effect of sublethal eserine on sex pheromone biosynthesis by females has not been
documented before this study.Sex pheromone biosynthesis in insects involves a complex co-
ordination of physiological activit ies.Our data suggest that sublethal neurotoxicants could
interfere w ith this process.Sublethal doses of neuroactive insecticides may cause latent toxi-
254 　 ENTOMOLOGIA SINICA　Vol.7 , No.3 , September 2000
city , enzyme induction , inhibitory effects on mating behavior and insect physiological pro-
cess.
The ratios between Z and E isomers are generally believed to be more important for re-
productive isolation than other ratios in the pheromone blend(see for instance of Carde and
Baker 1984).The pheromone components(E)- and (Z)-12-tetradecenyl acetates of O .
f urnacalis , are biosynthesized from palmitic acid by Δ14 desaturation , followed by chain
shortening(β-oxidation), reduction and acety lation(Zhao 1995).Since the regulation mod-
el is not very clear , it is not clear how sublethal eserine affect the ratios between Z and E
isomers.Detailed study is necessary in future.
S tudies of the behavioral effects of sublethal neurotoxicants are important.It is clear
that almost all insecticides interfere w ith the normally well orchestrated behavioral patterns
of insects-pests and thus may be a new applied technique of insecticides.An important relat-
ed point is that the sublethal dosage of the insecticides not only favors the beneficial insects
but also slows down the development of resistance of the insect pests against the insecti-
cides.
Perhaps the most important aspect of our research , and of all previous studies of sub-
lethal effects of insecticides , is the emphasis on the need for a new dialogue on the methods
that are currently used for selecting chemicals for control o f insect populations.In an ex-
treme case , a chemical could be rejected for economic or environmental reasons when it was
assessed solely on the basis o f mortali ty that could have been effective in IPM at a fraction of
the cost and with a minimal environmental impact.We documented sublethal effects of eser-
ine on the female calling , pheromone production of female O .furnacalis , and these effects
could be expected to interfere w ith female s reproductive capacity and caused a decreasing of
the mating rate.Finally , it w ill results in a decreasing of the population density.There-
fore , our research works in this area may help to provide basic knowledge to develop a new
technique of controlling insect pests in the future.
Additional studies of the modes of action of neurotoxic chemicals are even more crit ical
to the search for new behavioral disputants than to the search for new true insecticides.For
example , in the future it may be possible to identify chemical structures of new neurotrans-
mitters from the CNS.These chemical structure of the neurotransmitters could provide the
useful information that would help to guide synthesis of effective chemical analogues.
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Cusson , M.and J.N.McNeil　1989　I nnervation of juvenile hormone in the regulation of pheromone release
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Hunt , R.E.and K.F.Haynes 　1990　Periodicity in the quantity and blend ratios of pheromone components
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Raina , A.K.and J.A.Klun 　1984　 Brain factor control of sex pheromone production in the female corn
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Tang , J.D., R.T.Charlton , R.T.Carde et al.　 1987　 Effect of allatectomy and ventral nerve cord tran-
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亚致死剂量的毒扁豆碱对亚洲玉米螟 Ostrinia furnacalis化学通讯系统的干扰作用
杜智化　杜家纬　周弘春　许少甫
中国科学院昆虫研究所 ,上海 200025
胸部点滴亚致死剂量的毒扁豆碱对亚洲玉米螟化学通讯产生明显的干扰作用 , 同时抑制了雌蛾求偶
活动和性信息素释放量 , 且剂量越高 , 抑制作用越强 , 但不影响求偶高峰期出现的时间;顺反异构体比例
也受影响 , 且其变异系数不能控制在 10%以内 , 表明亚致死剂量的毒扁豆碱处理使亚洲玉米螟化学通讯
不能控制在较为狭窄的范围之内。受毒扁豆碱影响后的通讯系统的恢复实验表明:随时间的推移 , 雌蛾
求偶百分数和性信息素释放量呈回升趋势 , 但不能完全恢复。 27ng/ ♀剂量处理的当天 、第二天 、第四天
的求偶百分数分别为正常雌蛾的 50%、78%、83%, 释放量为正常雌蛾的 40%、40%、80%。顺反异构体比
例的变异系数从第二天起能控制在 10%以内。
关键词　　亚洲玉米螟　性信息素　求偶行为　毒扁豆碱　亚致死剂量　回复
256 　 ENTOMOLOGIA SINICA　Vol.7 , No.3 , September 2000