Overwintering Strategy and Mechanisms of Cold Tolerance in the Codling Moth (Cydia pomonella)

Background

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.

Principal Findings

We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately −15.3°C during summer to −26.3°C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to −15°C, even in partially frozen state.

Conclusion

Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).     

The Crystal Proteins from Bacillus thuringiensis subsp. thompsoni Display a Synergistic Activity Against the Codling Moth, Cydia pomonella

Crystal proteins from Bacillus thuringiensis subsp. thompsoni strain HnC are active against the codling moth, Cydia pomonella, a major pest of orchards. Inclusion bodies purified from strain HnC displayed an LC₅₀ of 3.34 × 10⁻³μg/μl. HnC-purified crystals were tenfold more active than Cry2Aa and Cry1Aa toxins, and 100-fold more toxic than Cry1Ab. The 34-kDa and 40-kDa proteins contained in HnC inclusion bodies were shown to act synergistically. The toxicity of crystal proteins produced by the recombinant B. thuringiensis strain BT-OP expressing the full-length native operon was about tenfold higher than that of the 34-kDa protein. When the gene encoding the non-insecticidal 40-kDa protein, which is not active, was introduced into the recombinant strain producing only the 34-kDa protein, the toxicity was raised tenfold and was similar to that of the strain BT-OP. Received: 25 August 1999 / Accepted: 5 October 1999     

Cydia pomonella granulovirus Genotypes Overcome Virus Resistance in the Codling Moth and Improve Virus Efficiency by Selection against Resistant Hosts

Cydia pomonella granulovirus (CpGV) has been used for 15 years as a bioinsecticide in codling moth (Cydia pomonella) control. In 2004, some insect populations with low susceptibility to the virus were detected for the first time in southeast France. RGV, a laboratory colony of codling moths resistant to the CpGV-M isolate used in the field, was established with collection of resistant insects in the field followed by an introgression of the resistant trait into a susceptible colony (Sv). The resistance level (based on the 50% lethal concentrations [LC₅₀s]) of the RGV colony to the CpGV-M isolate, the active ingredient in all commercial virus formulations in Europe, appeared to be over 60,000-fold compared to the Sv colony. The efficiency of CpGV isolates from various other regions was tested on RGV. Among them, two isolates (I12 and NPP-R1) presented an increased pathogenicity on RGV. I12 had already been identified as effective against a resistant C. pomonella colony in Germany and was observed to partially overcome the resistance in the RGV colony. The recently identified isolate NPP-R1 showed an even higher pathogenicity on RGV than other isolates, with an LC₅₀ of 166 occlusion bodies (OBs)/μl, compared to 1.36 × 10⁶ OBs/μl for CpGV-M. Genetic characterization showed that NPP-R1 is a mixture of at least two genotypes, one of which is similar to CpGV-M. The 2016-r4 isolate obtained from four successive passages of NPP-R1 in RGV larvae had a sharply reduced proportion of the CpGV-M-like genotype and an increased pathogenicity against insects from the RGV colony.     

The Role of Formulation Additives in Increasing the Potency of Cydia pomonella Granulovirus for Codling Moth Larvae,in Laboratory and Field Experiments

Studies were undertaken to improve the biological efficacy of the granulovirus (CpGV) of the codling moth, Cydia pomonella , by evaluating the performance of some formulation additives that might improve virus persistence and/or virus uptake by first instar larvae. Laboratory studies, using a leaf disc bioassay, demonstrated that 15% cane molasses incorporated within a formulation of purified CpGV dramatically reduced the median lethal exposure time (LET 50 ) to CpGV for neonate larvae at a CpGV dosage rate of 10 7 occlusion bodies (OBs) ml -1 . Screening of a range of other compounds showed that sucrose, fructose and sorbitol (at 10% concentrations) and extracts of apple flesh and skin also gave significant reductions in the LET 50 of CpGV formulations containing these ingredients. Pectin, malic acid and &#102 -farnesene did not significantly reduce the LET 50 . In a field trial, molasses included at 15% (v/v) in a CpGV formulation, containing a dosage rate of 10 12 OBs ha -1 , gave as good control of codling moth damage as virus formulations containing the 'sticker' 0.2% skimmed milk at higher dosage rates of 10 13 and 10 14 OBs ha -1 . Studies of CpGV persistence on foliage revealed no significant improvement of virus persistence on apple foliage using 10% or 15% molasses formulations. A second field trial demonstrated that 10% molasses, 10% sorbitol or 0.08% &#102 -farnesene significantly reduced codling moth deep damage to fruit when these ingredients were added to formulations of pure CpGV. Substantial sooty-mould growth ( Cladosporium spp.) was observed on apple foliage treated with formulations containing molasses, indicating that this formulation additive has secondary consequences that would need to be taken into account if molasses was to be used in commercial CpGV formulations. Nonetheless, these studies clearly demonstrate that major biological improvements in CpGV performance can be achieved by the incorporation of formulation additives, including molasses and several other compounds, that probably function as attractants and/or feeding stimulants for codling moth larvae.     

Effect of Delayed Mating on Oviposition Pattern, Fecundity and Fertility in Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Female codling moth ( Cydia pomonella ) were mated either on the day of emergence or 2 or 4 d after emergence. Oviposition was checked daily and fertility (% egg hatch) of eggs laid 1–4 d and 5 + d after mating was determined from a sub-sample of females in each treatment. From these data, net reproduction rate (R₀) was calculated. the effect of delayed mating of males was also determined. Mean fecundity, fertility and net reproduction rate of females mated 4 d after emergence were significantly lower than for females mated either on the day of emergence (day 0) or 2 d after emergence. There were no significant differences between the three treatments in terms of the proportion of females that mated or female longevity, although there was a trend towards increased longevity with increasing delays in mating. Mean longevity of unmated females was significantly greater than that of mated females. Oviposition pattern was similar in females regardless of when they were mated and in all cases maximum oviposition took place 2 d after mating. There was no effect on the fecundity of 2 d old females or on the fertility of their eggs when they were paired with either 2 or 6 d old males. the significance of these findings in relation to control of codling moth by mating disruption is discussed.     

Uptake of Granulovirus from the Surface of Apples and Leaves by First Instar Larvae of the Codling Moth Cydia pomonella L. (Lepidoptera: Olethreutidae)

Cydia pomonella granulovirus (CpGV) has received considerable attention as a potential microbial insecticide for the control of the codling moth (Cydia pomonella L.) , a worldwide pest of apples. Laboratory experiments were established to investigate virus uptake by first instar larvae, using a novel leaf disc bioassay technique. Virus uptake was found to be independent of active feeding and larvae became infected simply by walking or browsing on sprayed leaf disc surfaces in as little time as 3.5 min. Infection increased as a function of time spent on the leaf disc surface and a linear log time/probit mortality relationship could be fitted. The bioassay technique used has potential for the realistic laboratory testing of virus spray formulations. A field experiment showed that virus infection could be contracted by newly hatched codling moth larvae both from the surface of sprayed leaves and sprayed fruit. The potential for exploiting this knowledge for improving spray formulations is discussed.     

Control of Codling Moth,Cydia pomonella (Lepidoptera: Tortricidae), with Steinernema carpocapsae: Effects of Supplemental Wetting and Pupation Site on Infection Rate

Infection of cocooned codling moth (cydia pomonella) larvae by the entomopathogenic nematode Steinernema carpocapsae was studied in three field experiments. Factors that varied within or between experiments included method of application, type of substrate containing cocooned larvae, time when nematodes were applied, seasonal effects, and supplemental wetting before or after nematode application. Conventional air-blast sprayer applications of 0.5–5.0 million infective juveniles (IJs)/tree in fall resulted in ca. 30% mortality of larvae in cardboard trap bands, whereas hand-gun application (2 million IJs/tree) produced mortality of ca. 70%. Application in the evening caused higher larval mortality than application in the morning when no supplemental wetting was used after treatments. Morning and evening applications caused equivalent larval mortality when a postwetting treatment was included. In a trial conducted in midsummer, supplemental wetting, either before or after hand-gun application of 1 million IJs/tree, enhanced nematode-produced mortality. Mortality approached 100% if both pre- and postwetting was used. Larvae in exposed cocoons on apple wood were infected at a higher rate (86%) than those on wood in less exposed positions (73%) or in nonperforated cardboard (72%). Mortality rates for larvae in perforated cardboard were intermediate (77%). Application volumes used to deliver nematodes slightly enhanced infection rate of larvae in some substrates but not others. In one trial, parasitism of codling moth by the wasp Mastrus ridibundus (Ichneumonidae) was negatively correlated with nematode infection of codling moth larvae. Dissections showed that ca. 10% of larvae infected by nematodes had been attacked by the wasp.     

Mating behaviors of Cydia pomonella (Lepidoptera: Tortricidae) as influenced by sex pheromone in electrostatic powder

Entostat is an electrostatically charged wax powder that can adhere strongly to insect cuticle, making it an ideal carrier to deliver pheromone for pheromone-based confusion techniques. We investigated the attractiveness of Cydia pomonella (L.) (Lepidoptera: Tortricidae) males treated with pheromone-laden Entostat powder to naive conspecifics as well as mating behaviors of males after such treatment in a laboratory flight tunnel. Male moths exposed to Entostat containing 1% E,E-8,10-dodecadien-1-ol (codlemone) acquired and retained the largest amount of the powder and became more attractive as point sources to naive moths compared with those treated with powder containing 5 or 10% codlemone. All Entostat-exposed males remained as attractive as a 0.1-mg codlemone lure for up to 24 h in flight tunnel investigations. Male moth orientation to normally attractive sources of codlemone was completely disrupted directly after treatment with Entostat powder. Males' ability to orient to 0.1-mg lures recovered progressively over a 6-d postexposure interval; however, their responses never reached the levels observed with untreated control moths. Entostat-exposed moths retained detectable amounts of codlemone up to 4 d. Our laboratory flight tunnel results suggest that the mode of action of Entostat powder as an autodissemination control tactic may be due to creating both attractive false point sources after exposure to the powder as well as directly inhibiting contaminated males' capability to orient to pheromone.     

Juvenile hormone catabolism and oviposition in the codling moth, Cydia pomonella, as functions of age, mating status, and hormone treatment.

In vitro catabolism of juvenile hormone (JH) in haemolymph of adult female Cydia pomonella was ascribed mainly to juvenile hormone esterase (JHE) activity. No significant differences were noted between virgin and mated females 0-96 h post-emergence. Changes in JHE activity did not appear dependent upon fluctuations in JH titre; conversely, changes in JHE activity could not explain the changes in JH titres. Maximal JHE activity was recorded at 24 h (331.47 +/- 47.25 pmol/h/microl; 355.93 +/- 36.68 pmol/h/microl, virgin; mated insects, respectively) and preceded the peak in JH titres at 48 h. Topical application of JH II (10 ng-10 microg) or fenoxycarb (50 ng) enhanced JHE activity up to 640 and 56%, respectively. Treatment upon emergence with 10 microg JH II induced enzymic activity for less than 24 h, and when 10 microg JH II or 50 ng fenoxycarb were applied, circulating JH titres returned to control levels within 24 h. Oviposition was highly sensitive to exogenous JH and declined significantly with dosages >100 pg. To allow a degree of oocyte maturation before JH treatment, the hormone was administered at 6, 12, 24, or 48 h post-emergence and/or females were mated. Neither measure "protected" the system; oviposition declined immediately after JH application.     

A Theoretical Approach to Study the Evolution of Aggregation Behavior by Larval Codling Moth, <Emphasis Type="BoldItalic">Cydia pomonella</Emphasis> (Lepidoptera: Tortricidae)

Pupation site-seeking larvae of the codling moth, Cydia pomonella, aggregate in response to aggregation pheromone produced by cocoon-spinning conspecific larvae. Larvae that pupate in an aggregation rather than in solitude may experience a lower rate of parasitism by the parasitoid Mastrus ridibundus. Additionally, adults eclosing from a larval aggregation may encounter mates more rapidly at the site of eclosion (on-site) than away from that site (off-site). We employed an evolutionary simulation to determine the effect of several ecological parameters on the evolution of larval aggregation behavior. These parameters included (i) the probability of mate encounter off-site; (ii) the time available for finding a mate; and (iii) the population density of parasitoids and their rate of larval parasitism. The model predicts that larval aggregation behavior is selected for when the probability of off-site mate encounter is low, the time to locate mates is short, and egg-limited parasitoids are at high population levels. We also show that aggregations reduce the risk of parasitism through dilution effects. The parameters found to favour the evolution of larval aggregation behavior are consistent with life history traits exhibited by C. pomonella.     

Early detection of resistance to tebufenozide in field populations of Cydia pomonella L.: methods and mechanisms

Abstract: Four populations of codling moth Cydia pomonella L. were collected as overwintering larvae from apple orchards with different pesticide pressure (S. Michele, Roncafort, Revò and Vervò) in the Trento province (northern Italy). Mortality rate caused by a predetermined discriminating concentration of tebufenozide topically applied on overwintering larvae was evaluated. Neonate F1 progeny of the same populations were assayed for susceptibility to tebufenozide by feeding them on thinning apples treated with an appropriate discriminating dose of the insecticide. The activities of the main enzyme systems involved in the detoxification of insecticides were also evaluated in each population and related to their susceptibility to tebufenozide. The topical test detected a significant loss in susceptibility to tebufenozide in two populations, S. Michele and Roncafort, while all the overwintering larvae collected in the orchards of Revò and Vervò died when treated topically with the discriminating concentration. The apple‐dipping test performed on the neonate larvae showed a highly significant reduction in the susceptibility of the two populations of S. Michele and Roncafort. A less significant reduction in mortality rate was found in the Revò population; however, no statistical difference was found between the Vervò population and the susceptible reference. None of the four field populations significantly differed from the susceptible strain for Glutathione‐S‐transferase and esterase activity. A significantly higher frequency of individuals of the S. Michele and Roncafort populations exhibited a higher mixed function oxidase activity than the susceptible strain. The small resistance ratio values found for the two populations together with the low frequency of individuals exibiting enhanced enzymatic activity, reveals that the selection process was still at the early stage. Because of its efficiency in early detection of resistance to tebufenozide, topical application on diapausing larvae can thus be considered an appropriate, simple and robust tool for implementing resistance monitoring programmes for tebufenozide.     

Enzymatic diagnosis of resistance to deltamethrin in diapausing larvae of the codling moth,Cydia pomonella (L.)

The resistance to deltamethrin was evaluated in diapausing larvae of 14 field populations of the codling moth (Cydia pomonella L.) From the main French orchard areas using biological assays. Resistance to deltamethrin was compared to mixed-function-oxidase (mfo) activity measured at the individual level through ethoxycoumarin-O-deethylase (ECOD) activity using a fluorescence microplate reader. The larvae were collected in corrugated paper band traps in the autumn of 1995. Analysis was previously performed on two laboratory strains, one susceptible and one resistant to deltamethrin, in order to characterize the changes in resistance during diapause development. Resistance to deltamethrin as well as the ECOD activity were stable during the diapause, and ECOD activity was always significantly lower in the susceptible strain than in the resistant one. The ECOD activity was significantly correlated to the frequency of resistant moths in the field populations. This strongly suggested the involvement of the mfo system in the resistance to deltamethrin in these populations. The intra-strain variabilities in ECOD activity of both laboratory and field resistant insects indicated that other resistance mechanisms might also be involved. Further investigations on these mechanisms are needed in order to develop complete diagnostic methods and to define suitable control strategies against each resistant population. Arch. Insect Biochem. Physiol. 39:55–64, 1998. © 1998 Wiley-Liss, Inc.     

Temporal events in the invasion of the codling moth,Cydia pomonella,by a granulosis virus: An electron microscope study

The replication cycle of the granulosis virus of Cydia pomonella, the codling moth, was studied at the cellular and tissue level. Membranelike complexes were observed forming within the remnants of the nucleolus in the cytoplasm of infected cells. Differences in cell polarity relative to the sites of virus entry assembly and budding as well as differences in the temporal aspects of replication were observed between midgut, fat body, and epidermal cells. The progressive spread of virus throughout larval tissues was studied at 24, 32, 48, 56, and 72 hr postinfection. The basal lamina seemed to be an effective barrier for the release of budded progeny virus into the hemocoel and large numbers of budded virus were produced.     

两种苹果蠹蛾性引诱剂诱捕器诱捕效率比较及地面植被的影响

在甘肃省高台县通过对三角胶粘式和水盆式这2种类型苹果蠹蛾Cydia pomonella(L.)诱捕器的田间比较研究。结果显示,二者在诱捕效率上存在的明显差异。在苹果蠹蛾密度较低的条件下,三角胶粘式诱捕器平均日诱捕量为2.50只,比水盆式诱捕器高出近2.84倍,最早监测到成虫的时间也比水盆式诱捕器提前3~4d,因此,三角胶粘式诱捕器具有更高的监测效率。结果还显示地面植被的遮盖极大地降低诱捕器的诱捕效果,严重时会使诱捕器对密度较低的苹果蠹蛾种群的监测功能丧失。     

Populations of Dalapon-decomposing Bacteria in Soil as Influenced by Additions of Dalapon or Other Carbon Sources

The numbers of bacteria capable of decomposing the herbicide dalapon were determined for five soils by the most-probable-number method. Before treatment of the soils with dalapon, the numbers varied from 1,000 to 10,000 cells per g of soil. Incubation of the soils with dalapon resulted in large increases (100-fold) in two of three soils in which dalapon was decomposed rapidly. Lack of increase in numbers in spite of rapid decomposition in the other soil probably indicated breakdown by a chemical process or decomposition by fungi. In the remaining two soils, in which decomposition was slow in one and did not occur in the other, the initial numbers were at the low end of the range and the increase was small on incubation with dalapon. Addition of ground alfalfa or ground corn plant material to a soil did not result in significant increases in the numbers of dalapon-decomposing bacteria, either during or after decomposition of the plant material. Glucose depressed the rate of breakdown of dalapon in the soil and increased dalapon-decomposing Bacillus species rather than Arthrobacter and Agrobacterium species, which were found to increase on incubation with dalapon itself. The most-probable-number method appears to be a valuable tool for pesticide-ecology studies.     

Long flights in Cydia pomonella L. (Lepidoptera: Tortricidae) measured by a flight mill: influence of sex, mated status and age

Abstract. The flight capacity of Cydia pomonella L. (Lepidoptera: Tortricidae) was measured in the laboratory by using computer-linked flight mills. Codling moths showed a large variation in flight capacity between individuals. We defined arbitrarily a longest single flight (LSF) of more than 5 km as an index for long-flyers. About 16.7% of virgin and 10.0% of mated males and 20.0% of virgin and 7.4% of mated females undertook such flights. Based on the LSF and the total distance flown (TDF^we concluded that males and females have little or no difference in flight capacity and that both the within-and between-habitat flights are similar in number and magnitude for both sexes. In the field, females are therefore potentially able to undertake flights of up to 11 km, as reported for males by other authors. This ability was highest at ages of 2–7 days after emergence, i.e. the first third of their lifetime, for virgin and mated male moths and for virgin female moths. Mated females showed peak flight capacity between 1 and 3 days after eclosion, which corresponded with the major egg-laying period. Few long flights were undertaken before oviposition. These findings do not agree with the oogenesis flight syndrome described by other authors, and this theory is believed not to apply to C.pomonella. Our laboratory results are discussed in relation to field experiments in general and hypotheses are developed about the significance of long-flyers for this species.     

Egg adhesion of the codling moth Cydia pomonella L. (Lepidoptera,Tortricidae) to various substrates: II. Fruit surfaces of different apple cultivars

In the late growing season of apples, most eggs of the codling moth, Cydia pomonella L. (Lepidoptera, Tortricidae), of the second and third generations are deposited directly on fruits. The apple fruit surface is densely covered by three-dimensional micro- and nanoprojections, the epicuticular wax crystals, emerging from an underlying wax film. These epicuticular waxes render the apple fruit surface hydrophobic, which could affect the attachment of insect legs and eggs to it. A better survival of the codling moth offspring is expected to be ensured by the selection of suitable oviposition sites by females, as well as by a proper adhesion of deposited eggs to these sites. In this study, we investigated egg adhesion of the codling moth to the fruit surface of different cultivars of the domestic apple, Malus domestica Borkh., by measuring the pull-off force required to detach eggs from fruits. Since surface characteristics may influence insect egg adhesion, the information about morphological and physicochemical properties of the fruit surface is crucial for understanding oviposition site selection by females. In the present study, surface morphology, wettability, and free surface energy of the apple cultivars ‘Boskoop’, ‘Elstar’, ‘Golden Delicious’, ‘Jonica’, and ‘Topaz’ were analyzed. Eggs adhered tightly to the fruit surface of all apple cultivars tested: pull-off forces averaged 63.9 mN. These forces are four- to tenfold stronger than those previously measured on adaxial and abaxial leaf surfaces of the identical apple cultivars. The mechanisms used by the moth to fix its eggs on the waxy surface of apple fruits, and the influence of fruit surface properties on egg glue adhesion are discussed. Furthermore, the results are debated in the context of the oviposition site selection by females, and its role in offspring survival of the second and third generations of the codling moth.