Activation of gonads and disruption of imaginal diapause in the apple blossom weevil , Anthonomuspomorum ( Coleóptera : Curculionidae ) , with juvenoids in laboratory and field trials

A possibility of using synthetic analogues of juvenile hormone (juvenoids) to disrupt imaginal diapause of the apple blos­ som weevil, Anthonomus pomorum females was demonstrated. Out of three preparations tested (methoprene, fenoxycarb and W-328) methoprene and fenoxycarb appeared to be effective. Sensitivity to juvenile hormone analogues develops early after imagi­ nal emergence (even before the female starts to feed) and lasts throughout the whole acstivo-hibernation dormancy. Although the ju­ venoids could stimulate the onset of oogenesis at any time during diapause, the propensity of the ovaries to form normal eggs developed only during hibernation part of the dormancy; in earlier stages of diapause accumulation of yolk was observed but ma­ tured eggs were not produced. Methoprene treatment caused marked increase of locomotory activity accompanied with decrease of dry weight, increase of water content, depletion of trehalose resources, decrease of cold hardiness and, finally, 100% mortality within four weeks in the weevils treated during their feeding or aestivation stages. Although similar changes were observed in the treated pre-feeding weevils, they later recovered and survived until next spring without apparent loss of cold hardiness. A possibility of designing a control method based on this principle is discussed and the results of small-scale field trials that support its plausibil­ ity are reported.


INTRODUCTION
The apple blossom weevil, Anthonomus pomorum (L.) is an important pest of apple trees throughout the Holarctic region (Kletnm, 1937;Massee, 1954;Reijbroek, 1983;Wildbolz, 1992).It is a univoltine species with aestivohibernation dormancy (Masaki, 1980).In the spring (March, April) when day temperatures reach certain threshold (5° for crawling, fooding and mating, and 10° for flying) the overwintering beetles leave their dormancy sites and search for the host trees where they feed and mate, and the females lay eggs in the blossom buds (Duan et al., 1996).Infested buds become sterile and the blos soms never open.Larval and pupal development proceeds inside the buds and the next generation of beetles emerges in May or June.Young adult weevils feed on the paren chyma of apple leaves for about a month and then leave the trees and search for shelters having a suitable micro climate (bark crevices, soil, litter, etc.), some of them mi grating short distances to a nearby forest or hedge.Dormancy includes an obligatory reproductive diapause, during which epigamic behaviour and development of the gonads are suppressed (Čtvrtečka & Žďárck, 1992).The dormant beetles show special physiological adaptations (cold hardiness and accumulation of polyols) that are also a part of the diapause syndrome (Košťál & Šimek, 1996).A period of chilling is necessary for completion of dia pause development; thenceforth the beetles are kept inac tive by cold quiescence until the onset of favourable conditions in the spring.
Availability of a suitable food (sprouting apple tree buds) is a necessary prerequisite for initiation of gonadal development of the females but not for sexual receptivity.The males acquire sexual maturity earlier than females, and the initiation of their postdiapause development does not require food (Ctvrtecka & Zd'arek, 1992).Little is known about how this reproductive arrest, which may last nearly 10 months, is regulated in the apple blossom wee vil.
Diapause supports survival during adverse conditions by halting development at a specifically adapted stage.The fitness of an insect to survive through winter depends on physiological adaptations of the organism to cold (Lee & Denlinger, 1991 for review).These adaptations are hormonally potentiated and in the case of reproductive diapause, juvenile hormone (JH) and its synthetic ana logues and mimics (juvenoids) can precociously terminate the diapause of many insects (Denlinger, 1985).A long time ago it was suggested that juvenoids might be used to control insect pests that undergo an imaginal diapause by interrupting their reproductive arrest during a period un suitable for reproduction (Bowers & Blickenstaff, 1966).The idea was attractive because many pest species enter diapause (Tauber et al., 1986), and diapause of many spe cies was thought to be susceptible to disruption (Chippen dale, 1982;Staal, 1982).As far as we know, no pest control system based on this concept has been field tested for any insect except for a scutellerid bug, Eurygaster inlegriceps Putzeys (Kontev et al., 1974), in spite of the fact that laboratory experiments demonstrated the ability of JH and its analogues to precociously terminate diapause in many insects (see Denlinger, 1985 for review).The main reason for the reluctance of researchers to test the practicability of this approach in large scale field experi ments could be the fact that relatively high doses per ani mal are needed to affect diapause development in laboratory tests and the effect is often reversible.The idea was revived by Krysan (1990a,b) who showed in labora tory and field experiments that reproductive diapause in the pear psylla, Cacopsylla pyricola (Foerster) can be pre cociously terminated by minute amounts of a juvenoidbased insecticide, fenoxycarb.
The present paper reports the results of our study aimed at (i) investigating the possibility of using juvenoids to disrupt imaginal diapause of the apple blossom weevil fe males in the laboratory and (ii) evaluating possibilities of designing a method based on this principle that could eventually become a viable IPM strategy to control the pest.The efficacy of three selected juvenoids to disrupt diapause was first compared in laboratory tests and the most promising one was chosen for small-scale field trials.In order to better understand the mechanism of arti ficial interruption of diapause, the effects of a hormonal analogue on changes in some physiological characteris tics of dormancy (cold hardiness, water content, dry weight, presence of some cryoprotectants) were also in vestigated.

Insects
Adult weevils that had emerged from pupae collected in apple tree orchards near Prague, Liberec and České Budějovice, Czech Republic (49-50.5°N)were used for all experiments.The beetles were allowed to emerge in rearing cages (50 x 50 x 50 cm) and, except for the winter season, the cages were kept out doors in a shaded shelter so that the insects were exposed to natural temperature, humidity and photoperiod.For summer feeding the weevils were offered fresh apple twigs with leaves three times a week for six weeks until all beetles spontaneously terminated their feeding activity and began to seek shelter in the fallen dry leaves to begin their aestivo-hibernation.The weevils were then transferred into 500 ml wax-paper cups filled with dry leaves and the cups were closed with nylon netting and placed in an inverted position over a wet Styrofoam layer to create a humidity gradient inside the cup.At the end of October cups with the beetles were transferred to a dark cabinet with con trolled temperature of 3-5°C.
Adult beetles used for all experiments were grouped in four eco-physiological stages: Stage 1 -beetles less than 24 h after eclosion from pupae, without access to food.
Stage 2 -beetles feeding for 7 to 20 days after eclosion.Stage 3 -aestivating beetles in summer after having been fed ad libitum.
The exact dates and duration of feeding are specified in the Results.

Laboratory tests
For topical application the juvenoids were dissolved in ace tone and applied in the volume of 0.5 pi per specimen.Controls were treated with acetone alone.For investigation of ovarial de velopment treated weevils were kept in large petri dishes pro vided with a water vial and fresh apple tree leaves at 23-25°C.A week later the weevils were dissected under a stereoscopic microscope and the degree of ovarian development evaluated according to a classification scale, the stages of which are de scribed in the Results.For evaluation of the effects ovarial indi ces were calculated for each experimental group based on the mean degree of ovarial activation reached on day 7 after the treatment.
For investigation of the survival rate, polyol content, dry weight, water content and cold hardiness, treated weevils were taken at three different eco-physiological stages (1,2 and 3 as described above), treated with either methoprene (2.5 pg) or acetone, returned to natural conditions and subjected to proce dures that are fully described by Košťál & Simek (1996).

Field trials
For application to the leaves, methoprene was dissolved in acetone, and emulsified in water in three concentrations (25, 5 and 1 g .f).The preparations were sprayed as 0.01% Tweenwater emulsion to both sides of the leaves on a twig ca.50 cm long bearing approximately 50 leaves.Tween 20 (Fluka AG) was used as an emulsifier.From the amount of emulsion sprayed it was calculated that each square centimetre of the leaf surface received an average of 320, 64 and 13 pg of the active compound, respectively.The weevils were released to the twigs after the water had evaporated.For the outdoor experiments the treated twigs on apple trees were enclosed in nylon netting be fore the weevils were released.In the laboratory the treated twigs were kept in ajar with water in 50 x 50 x 50 cm breeding cages.

Statistics
Dependence of probability of activation of gonads on two ex perimental factors (dose and life stages) was modelled using a generalised linear model (GLM) with binomial distribution of dependent variable and logit link function (McCullagh & Nelder, 1989).The test of significance of the factors was based on the analysis of deviance table.A two-tailed t-test was used for the remaining evaluations.

Morphological effects of juvenoids on the female gonads
Precocious development of the ovaries induced by ju venile hormone analogues applied before and during the feeding period (in May and June, stages 1 and 2), and during the aestivation period (from July to October, stage 3) was atypical in comparison with normal development of the ovaries occurring after aestivo-hibernation in spring.Three distinct stages of growth and development of the ovaries could be distinguished.The ovarioles of stage I were short, undifferentiated and entirely transpar ent.The ovaries of other stages had no parallel with nor mal developmental stages as classified by Ctvrtecka & Zd'árek (1992).The ovarioles of stage II were elongated and wider than those of stage I, their vitellarium was not differentiated and the calyx slightly enlarged to the size of a ripe oocyte.The ovarioles of stage III were still larger than those of stage II, the vitellarium showed no differen tiation of oocytes but appeared to be packed with a mass of yolk.The yolk may have partially descended to the ca lyx where the oocytes were enveloped with thin and in distinct membranes (Fig. 1).The oocytes never developed into ripe eggs, even if the females were offered apple tree leaves for feeding.On the contrary, if development of the gonads was activated by a juvenoid during the overwin tering period (from November to March, stage 4) the in duced changes were comparable to ovarian development occurring during natural reactivation in the spring, i.e. vi tellogenesis began and if the females were offered apple tree buds for feeding, the eggs developed.Unfed females did not develop eggs.

Comparison of the effects of three juvenoids on activation of the ovaries
The objective was to select the most suitable JH ana logue for field experiments out of three preparations that were either available commercially (fenoxycarb, methoprene) or locally synthesised in large quantities as a po tential insecticide (W-328).The juvenoids were topically applied in two doses (0.5 and 2.5 pg) to freshly emerged stage 1 females and stage 2 females that had been allowed to feed on apple tree leaves for 10 days before application (Table 1).All three juvenoids caused activation of the go nads both in pre-feeding and feeding females, but the lat ter were significantly more sensitive to the juvenoids than the freshly eclosed females.Among the three juvenoids, methoprene and fenoxycarb appeared to be more effective than W-328.Because the ovarial indices of females treated with the lower dose of methoprene were higher than in the females treated with the same dose of the other two juvenoids, methoprene was selected for all further experiments.

Changes in sensitivity to methoprene during adult stages of the female
Methoprene was applied in two concentrations to the female weevils at four adult stages of their life cycle, namely to freshly emerged (pre-feeding) females, females after 10 days of feeding, inactive females during their aestivation period in September, and inactive females dur ing their hibernation period in November (Table 2).Ex cept for the lower dose (0.5 pg) applied to freshly eclosed females (stage 1), application of either dose caused ovar ial activation in practically all females treated at later de velopmental stages (2, 3 and 4).Thus the effect of dose was not significant at the level 0.05 (F = 7.12, P = 0.76), while the effect of stage was (F = 11.58,P = 0.037).In other words, the probability of activation of gonads of stage 1 females differs from that of females of later life stages.Some degree of ovarial activation was also ob served in the acetone-treated weevils and also in nontreated overwintering females (not shown in the table) indicating a low degree of spontaneous activation before and during diapause.Mortality of weevils treated with 1.06 1 Mean degree of ovarial activation according to the scale explained in the methods. 2 After 10 days of feeding on fresh apple tree leaves.2.5 pig methoprene at stage 1 differed markedly from that of weevils treated at stages 2 and 3, but did not differ from mortality of controls treated with acetone at the same stage (Fig. 2).While weevils of stages 2 and 3 did not survive after methoprene treatment for more than a month, ca.25% of beetles treated at stage 1 survived till the next spring, and so did the controls.

The effects of methoprene applied to apple tree leaves
Freshly eclosed weevils were allowed to feed on apple tree leaves treated with three different doses (320, 64 and 13 pg.cm'2of the leaf surface) of methoprene, and the de gree of activation of their gonads was evaluated and com pared with that of females treated topically with 2.5 pg of methoprene and allowed to feed on untreated leaves.Acetone-treated females feeding on untreated leaves served as controls (Table 3).All doses of methoprene ap plied to the leaves on which the weevils fed caused acti vation of the gonads in the majority of the females, no matter if the cage with the apple tree twigs was kept in doors or if the treatment was done in the orchard using leaves of a growing tree.Consistent with the previous ex periments, all feeding females treated topically with methoprene showed activated gonads while none from the acetone treated group did so.

The effect of methoprene on some physiological attributes of dormancy
Substantial differences of the juvenoid effect on cold hardiness (measured as a capacity to survive exposure to low temperature for 5 days) were observed between freshly emerged, pre-feeding weevils (stage 1) and postfeeding aestivating ones (stage 3) (Table 4).Application of methoprene to the beetles of stage 1 had no effect on their cold hardiness assessed during overwintering (i.e.eight months after treatment).In the beetles of stage 2, our original intention was to assess cold hardiness also during the most relevant, hibernation part of the life cycle.But no beetle of stage 2 survived methoprene treat ment till winter (Fig. 2).Thus, in the beetles of stage 3 we assessed cold hardiness only one week after treatment when the beetles were still in aestivation.Substantially impaired survival was observed in the methoprene-treated group in comparison with the acetone-treated controls af ter exposure to -1 1°C (Table 4).Water content was significantly elevated in weevils of all three life stages one week after treatment with methoprene.Dry weight decreased significantly only in beetles of stages 1 and 2; a similar trend was apparent but insignificant (p = 0.13, t-test) also in the beetles of stage 3 (Table 5).
The content o f glucose was insensitive to the treatment with methoprene in the beetles of all three life stages (Ta ble 6).Depletion of trehalose after treatment with methoprene was observed in the aestivating beetles of stage 3 (Table 6).
Behavioural observations.In the control (acetonetreated) or untreated weevils, locomotory activity regu larly decreased after termination of feeding; the weevils searched for shelters and ultimately became lethargic.Ap plication of methoprene, both during feeding (stage 2) and during aestivation (stage 3), dramatically elevated ac tivity of the beetles.They perpetually walked or flew but did not seek shelters.Beetles treated with methoprene at stage 1 behaved normally.

DISCUSSION
It is generally understood that reproductive diapause in female insects is caused by a lack of hormones that regu late gonadal functions and epigamic behaviour (Denlinger, 1985), and addition of this hormone or its analogues after a certain refractory phase following emer gence can induce reproduction.Reproductive diapause may occur only in females, while males of some species are able, under favourable conditions, to mate at any time after their gonads become mature, if receptive females are available (Zd'arek & Kontev, 1975).If reproductive dia pause does occur in the male, it is usually controlled by the same endocrine mechanism as in the females, i.e. by a shut-down of the corpora allata (Pener, 1992;Watanabe & Tanaka, 1998).
In the present study the potency of exogenous juvenoids to disrupt obligatory diapause of apple blossom weevil females was investigated.Our earlier results indi cated that female weevils were indeed sensitive to the ju venoid methoprene (Ctvrtecka & Zd'arek, 1992).The analogue accelerated ovarian development and sexual Table 4.The effects of methoprene (2.5 pg) on cold hardiness, measured as a capacity to survive the exposure to low temperature for 5 days, in A. pomorum adults of three different life stages.maturation in winter and spring (i.e. during and after hi bernation), and it did so in the absence of food.Here we have confirmed the previous findings and complemented them with a study of the action of the juvenoid in earlier adult stages of the female's life cycle, namely shortly af ter the imaginal emergence, during feeding and later in the period of summer inactivity (aestivation).The gonads of a few untreated females showed slightly advanced de velopment in the autumn and winter, indicating that some spontaneous activation of reproductive processes may be gin before hibernation.This observation is paralleled by the observation that spermathecae of a small percentage of females (ca.20%) dissected in the autumn contained sperm (Ctvrtečka, unpubl.).
Our present results thus indicate that female sensitivity to JH analogues develops very early in the imaginal stage, even before the female starts to feed, and the period of sensitivity lasts throughout adult life.It indicates that the postemergence refractory phase in the female is very short, if it exists at all.The water content and dry weight of methoprene-treated pre-feeding weevils were also dif ferent from those of controls, indicating their physiologi cal responsiveness to the hormonal treatment.However, sensitivity of freshly emerged weevils to juvenoids was lower than in older ones, because the amount of exoge nous hormone required to activate the gonads in prefeeding females was higher than in those that had already consumed some food.The low sensitivity of the youngest beetles to the juvenoid was also apparent from mortality data.While feeding and post-feeding (aestivating) wee vils invariably died within one month after the treatment, the mortality curve of pre-feeding beetles followed practi cally the same course as that of acetone-treated controls resulting in a similar proportion of beetles of both groups surviving till next spring.
Although the exogenous hormonal signal can stimulate the onset of oogenesis at any time, the propensity of the ovaries to produce matured eggs in response to the hor monal stimulus develops much later, namely during the hibernation part of the dormancy.In earlier stages only accumulation of yolk in the ovarioles can be observed.
Besides the effects of the JH analogue on the gonads we also investigated its effects on some other physiologi cal characteristics of dormancy.Cold hardiness is one of the important aspects of the diapause syndrome in A. poinoruni.It develops during the aestivation part of dor mancy and further increases during hibernation, after the onset of low autumnal temperatures (Košťál & Simek, 1996).Only the weevils treated with methoprene at stage 1 survived till hibernation and their winter cold har diness was similar to controls.The weevils treated with methoprene during aestivation (stage 3) decreased their cold hardiness within one week, which suggests that their diapause was disrupted.A similar loss of cold hardiness accompanying artificial termination of adult reproductive diapause by juvenoid treatment was observed in a chrysomelid beetle, Aulacophora nigripennis (Watanabe & Tanaka, 1998).
The increase of water content and concomitant decrease of dry weight were observed in A. pomorum after treat ment with juvenoid.Such a pattern of changes again evokes an interpretation that, after the juvenoid treatment, the weevils switched from inactive, energy-saving status typical for diapause to the higher rate of metabolism typi cal for an active state.This switch was further docu mented by two additional facts: (1) a markedly elevated locomotory activity in the juvenoid-treated beetles, and (2) depletion of trehalose (a main resource of easily ac cessible metabolic fuel) in the weevils of stage 3; a simi lar depletion was not observed in the weevils of stage 2, 'Ten individuals were pooled for each treatment and analysed as one sample using GC-MS technique (Košťál & Šimek, 1996).
probably because these weevils could continuously re plenish their resources by feeding.Thus, although activation of gonads and other physio logical changes were observed in response to methoprene treatment in the pre-feeding beetles (stage 1), they proba bly recovered fully later and survived in similar numbers as controls until the next spring having their capacity to overwinter unimpaired.However, from the practical point of view it is important that no beetles of either sex treated at stages 2 and 3 survived the hormonal treatment for more than four weeks.Our data suggest that the treated insects died because metabolism was elevated and no or limited food was supplied.
The outcome of our small-scale field trials implied that the encouraging results obtained in the laboratory could be expected under natural field conditions.However, more extensive field tests are needed to substantiate this argument.Nevertheless, the present results do imply a po tential use of pesticides based on juvenoid action for the control of the apple blossom weevils.Juvenoid basedpesticides, such as fenoxycarb (Charmillot & Pasquier, 1992) or W-328 and related preparations (Kuldovâ & Matolin, pers.commun.)have been tested as ovicides for use as an alternative management strategy for a major ap ple orchard pest, the codling moth (Cydia pomonella).Possibly some such insecticides could be used for com bined control of the codling moth and the apple blossom weevil in an 1PM programme.Because the flight of first generation moths roughly coincides with the feeding pe riod of newly emerged apple blossom weevils, both pests could possibly be treated with a single application.

Fig. 1 .
Fig. 1.Stages of the ovarioles after activation by a juvenoid during the aestivation period.

Table 1 .
The effects of three juvenoids on activation of gonads of A. pomorum females before and after maturation feeding.(The degree of activation was evaluated 7 days after the treatment.)

Table 2 .
The effects of methoprene on activation of gonads of A. pomorum females in different adult stages.(The degree of acti vation was evaluated 7 days after treatment.)

Table 3 .
The effects of methoprene on activation of gonads of A. pomorum females that were allowed to feed on apple tree leaves treated with different doses of the juvenoid.

Table 5 .
The effects of methoprene (2.5 pg) on water content and dry weight when applied at different life stages o f A. pomorum adults.(Evaluation was done 7 days after treatment.)

Table 6 .
The effects of methoprene (2.5 pg) on the trehalose and glucose contents in adult stages of A. pomorum.(Contents of the sugars were evaluated 14 days after treatment1.)