Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth,Cydia pomonella

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (T_m) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.     

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).     

Temporal dynamics of parasitoid assemblages parasitizing the codling moth

Population dynamics of parasitoid–host interaction is primary important knowledge to develop an efficient biological control strategy of insect pests. We analyzed the seasonal dynamic of the parasitoid community of the codling moth in two sites in South-Eastern France, which differed by the number of codling moth annual generations. Parasitism was estimated by sampling both young larvae collected within apple fruits and mature larvae in band traps wrapped around the trunk of the apple trees. Parasitism rates differed between sites and between young and mature larvae. Parasitism rate were higher in young larvae (29% in average) than in the mature ones (21% in average) and globally increased along the season among cohorts of mature codling moth larvae (from 4% to 34%). The three most abundant species in the parasitoid community – Ascogaster quadridentata, Pristomerus vulnerator and Perilampus tristis – were observed at both sites, in each codling moth cohort and in both young and mature larvae. Among all the parasitoids, the proportion of Perilampus – an hyper-parasitoid attacking both Ascogaster and Pristomerus primary parasitoids – increased among the codling moth cohorts (from 9% to 53%) whereas the proportion of Ascogaster decreased (from 82% to 35%). This shed light on the importance to characterize the dynamic of the whole trophic network (including hyperparasitism) to design biological control strategies.     

Cold hardiness adaptations of codling moth, cydia pomonella

The cold hardiness adaptations of natural and laboratory reared populations of the codling moth, Cydia pomonella, were examined. Hemolymph, gut, and whole body supercooling points (SCPs), 24-h LT50s, polyhydroxy alcohol concentrations, hemolymph freezing points, and hemolymph melting points were determined. Nondiapausing codling moth larvae do not have appreciable levels of ice nucleators in the hemolymph or gut. Whole body supercooling points were higher than hemolymph supercooling points. For nondiapausing larvae, LT50s were significantly higher than both the whole body and the hemolymph supercooling points, indicating the presence of chill sensitivity. As the larvae left the food source and spun a cocoon, both hemolymph and whole body SCPs decreased. Diapause destined larvae had significantly lower hemolymph SCPs than nondiapausing larvae, but whole body SCPs were not significantly different from nondiapausing larvae of the same age. The LT50s of diapause destined and diapausing larvae were significantly lower than that of nondiapausing larvae. Codling moths are freezing intolerant, with LT50s close to the average whole body supercooling point in diapause destined and diapausing larvae. The overwintering, diapausing larvae effectively supercool to avoid lethal freezing by removal of ice nucleators from the gut and body without appreciable increase of antifreeze agents such as polyols or antifreeze proteins.     

Seasonal changes in supercooling capacity and major cryoprotectants of overwintering Asian longhorned beetle (Anoplophora glabripennis) larvae

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The insecticidal activity of foliage from New Zealand conifers

When incorporated into artificial diets, the milled foliages of many New Zealand conifers are toxic to larvae of housefly (Musca domestica), codling moth (Laspeyresia pomonella) and light-brown apple moth (Epiphyas postvittana). The most toxic foliages are those of Podocarpus nivalis which contains diterpene lactones, and Dacrydium laxifolium, which has a high concentration (1%) of phytoecdysones. The insecticidal activities of the plants are discussed in relation to their insect associations, chemistry and taxonomy.     

Control of diapausing codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in wooden fruit bins,using entomopathogenic nematodes (Heterorhabditidae and Steinernematidae)

Stacked wooden fruit bins are frequent overwintering sites for overwintering diapausing codling moth larvae. Control strategies against the codling moth (Cydia pomonella) (Lepidoptera: Tortricidae) in South Africa have been hampered by the reinfestation of orchards from nearby stacked infested fruit bins and by the movement of infested bins between orchards. Worldwide, wooden fruit bins are systematically being replaced with plastic bins, however in South Africa this will not be accomplished in the near future. The objective of this study was to evaluate the potential of two recycled commercially available entomopathogenic nematode (EPN) species, Heterorhabditis bacteriophora and Steinernema feltiae, as well as of a local species, Steinernema yirgalemense, to disinfest miniature wooden fruit bins under controlled conditions in the laboratory. After dipping miniature bins loaded with codling moth larvae in a suspension of 25?IJs/mL of each of the three EPN species, under optimum conditions of temperature and humidity, the highest percentage of control was obtained using S. feltiae (75%). The addition of adjuvants significantly increased S. feltiae infectivity to >95%, whereas it did not result in a significant increase in H. bacteriophora or S. yirgalemense infectivity.     

Integrated control of apple pests in New Zealand 10. Population dynamics of codling moth in Nelson

Abstract

Sampling methods are described for estimating the population density, mortality, and natality of a univoltine population of codling moth attacking mature apple trees (cv. ‘Delicious’) at Nelson, New Zealand. These methods were used to construct life tables for the species over eight generations (1967–68 to 1974–75) on trees variously sprayed and not sprayed with ryania in an integrated control programme. Bait traps provided a sensitive measure of seasonal adult population density. Analysis of the life tables shows that migration of adults was the main key factor and that overwintering larval mortality (particularly that due to bird predation), fecundity, and ryania also made a major contribution to variation in generation mortality. In the absence of ryania the resident population usually increased between generations, whereas it usually decreased when ryania sprays were applied. The density dependence of overwintering larval mortality was due to bird predation, and the inverse density dependence of larval mortality from ryania was due to changes in the site of fruit entry with larval population density. Fecundity was density independent, and inconclusive evidence was obtained on the density dependence of migration. The wide variation in fecundity is attributed primarily to weather conditions. The impact on control strategy of the above key factors, density dependence, and total natural mortality is discussed. Ryania is found to be uneconomic, whereas the granulosis virus of codling moth and male removal with pheromone traps show promise as future control methods. The need to eliminate reservoirs of codling moth close to orchards under integrated pest control is emphasised. Regulation of codling moth populations at Nelson on neglected, unsprayed trees appears to result from intraspecific competition for fruits and cocooning sites, and weakly density-dependent mortality of mature larvae when seeking cocooning sites and while overwintering in their cocoons. Variation in fecundity also cohtributes to fluctuations in abundance of the species. In contrast, at low density in an integrated control programme no intraspecific competition was evident; migration, winter mortality, and fecundity were the main determinants of abundance. This illustrates the need to study pest populations at densities similar to those tolerable commercially.     

Ascorbic acid influence on larval blood cell number and susceptibility to bacterial and fungal infection in the codling moth, Laspeyresia pomonella (Lepidoptera: Tortricidae)

The optimum concentration of ascorbic acid in a semisynthetic diet for codling moth larvae was 0.6–0.8%. The number of circulating hemocytes and the percentage of prohemocytes and phagocytes decreased in larvae reared on the diet with suboptimal and supraoptimal ascorbic acid levels. These larvae were significantly more susceptible to Bacilus thuringiensis and Beauveria bassiana.     

Resistance to the pink potato aphid, Macrosiphum euphorbiae, in two accessions of Lycopersicon hirsutum f. glabratum

An integrated programme of pheromone-mediated mating disruption using Isomate-C®, post-harvest removal of fruit, and trapping overwintering larvae with cardboard tree bands, was used to control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in four commercial organic apple orchards in Cawston, British Columbia during 1989–1992. One application of 1000 dispensers ^{– 1} on May 1 delivered estimated seasonal totals of 16.6, 16.5 and 19.9 g of E,E-8,10-dodecadien-1-ol [=codlemone] ^{– 1}in 1990, 1991 and 1992, respectively, at median rates of 8.4, 8.3, and 13.3 mg · < ha^–1 · ha^–1 during dusk flight periods of first brood and 5.3, 4.7 and 4.6 mg · ^{– 1}· ha^–1 in second brood, respectively. Over this 3-year period damage from codling moth at harvest ranged from 0.08 to 2.4%, and averaged 60.7% in these four organic orchards, while damage in five conventional orchards receiving sprays of azinphosmethyl ranged from 0.02 to 1.85%, and averaged 0.5%. Damage in an experimental orchard that was banded only, ranged from 43.5 to 56.7%, and averaged 48.9%. Between 1990 and 1992 cumulative male catches in Pherocon 1-CP wing traps baited with 10 mg of codlemone declined by 52% and densities of overwintering codling moth larvae declined an average of 49.5% in all organic orchards. Overwintering populations in the banded experimental orchard showed an increase of 57.7% during this study period. We conclude that an integrated programme of pheromone-mediated mating disruption, post-harvest fruit removal and tree banding, controls codling moth effectively enough to make organic apple production viable in British Columbia.     

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.     

Field trials of CpGV virus isolates overcoming resistance to CpGV-M

The Cydia pomonella granulovirus (CpGV) has been used for many years as biological agent for codling moth control in apple orchards. Resistance to the Mexican strain of CpGV was detected in orchards in Germany, France and Italy. A laboratory insect colony was started from insects collected in a French resistant orchard. It was named RGV. Various virus isolates were identified as active against this resistant insect colony. Field tests were carried out in 2007 to test if the two virus isolates CpGV-I12 and NPP-R1 were effective in the field. Although these virus isolates were not able to reduce insect caused fruit damages, they significantly reduced the overwintering insect populations. NPP-R1 was subjected to eight passages on RGV larvae (NPP-R1.8) that improved its biological activity on RGV larvae. 2008 field trials were set up to test this improved virus strain, compared to CpGV-I12 and Madex plus active on RGV. These tests confirmed the ability to control both in susceptible and resistant insect populations.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Mortality factors affecting immature stages of codling moth,Cydia pomonella (Lepidoptera: Tortricidae), and the impact of parasitoid complex

Codling moth, Cydia pomonella Linnaeus (Lepidoptera: Tortricidae), is a serious pest of apples worldwide. This study aimed to evaluate the mortality rate of codling moth eggs, larvae and pupae in the field in commercial and neglected apple and walnut orchards over two years, and to investigate the biodiversity and intensity of parasitoids associated with codling moth in the orchards. Five patches of wax paper containing 1-day-old codling moth eggs were placed in a neglected orchard in order to evaluate parasitism rates. Corrugated cardboard bands were placed around the trunk of 15 trees during late spring and the beginning of summer through to fruiting season to capture and measure parasitism of codling moth larvae. 5285 larvae in total were collected during this study. Mortality rate (egg?+?larvae?+?pupae) varied between the commercial and neglected orchards, reaching a maximum of (42.89% and 66.67%) in neglected apple orchards and (61.03% and 74.76%) in the neglected walnut orchard in 2003 and 2004, respectively. Trichogramma cacoeciae (Hymenoptera: Tichogrammatidae) was the only egg parasitoid recorded. Eight hymenopteran larval and pupal parasitoids belonging to several subfamilies were recorded: Cheloninae, Agathidinae, Cremastinae, Haltichellinae, Chalcidinae, Anomalinae, and Pteromalinae and one dipteran belonging to Tachininae. In conclusion, mortality factors, mainly by parasitoids, are contributing to a general reduction in codling moth larvae populations particularly in neglected orchards. The hymenopteran Ascogaster quadridentata and the dipteran Neoplectops pomonellae can contribute to biological control programmes against codling moth in the coastal region and other regions.     

Hybridisation and selective breeding for improvement of low temperature activity of the entomopathogenic nematode Steinernema feltiae

Steinernema feltiae is used to control overwintering larvae of the codling moth Cydia pomonella L. Application is in autumn when efficacy can be limited by low temperature. The objective of this study was to screen for low temperature activity among wild type populations of S. feltiae, hybridise most active strains and further improve low temperature activity by subjection of a hybrid strain to selective breeding. Significant variation was recorded among 22 S. feltiae strains. The temperature at which 50 % (AT₅₀) and 10 % (AT₁₀) of the dauer juveniles (DJs) were active ranged between 2.9 to 5.8 °C and 0.95 to 3.5 °C, respectively. The mean AT₅₀ of 22 S. feltiae strains was 3.83 °C. The five most active strains were crossed. The hybrid strain HYB01 was more active at low temperature than parental and other hybrid strains with an AT₅₀ of 0.52 °C and an AT₁₀ of 0.09 °C. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella, but was recovered after seven selection cycles with exposure to lowering temperatures. The heritability for the low temperature activity was calculated at h ² = 0.45. Negative trade-off effects on virulence against C. pomonella and reproduction on the same insect were not reported. The most virulent strain was a commercial strain with an LD₅₀ of 30.2 at 8 °C and 37.2 DJs per cocooned instar at 15 °C, followed by the selected hybrid with 48.1 and 47.4 DJs, respectively. Offspring production reached 15.000 DJs per instar at 8 °C and was only half at 15 °C. The results well document the potential of a breeding programme for enhancement of the activity of S. feltiae at lower temperature with the objective to improve the control potential of overwintering codling moth C. pomonella.     

Entomopathogenic nematodes for control of diapausing codling moth (Lepidoptera: Tortricidae) in fruit bins

Fruit bins infested with diapausing larvae of codling moth larvae, Cydia pomonella (L.), are a source of reinfestation of orchards and may jeopardize the success of mating disruption programs and other control strategies. Bins are not routinely treated for control of overwintering codling moth before placing them in orchards. Entomopathogenic nematodes provide a noninsecticidal alternative to methyl bromide that could be applied at the time bins are submerged in dump tanks at the packing house for flotation of fruit. Diapausing codling moth larvae in miniature fruit bins were highly susceptible to infective juveniles of Steinernema carpocapsae (Weiser). Immersion of bins in suspensions of S. carpocapsae ranging from 5 to 100 infective juveniles per milliliter of water resulted in 68-100% mortality. Immersion times of 1 or 5 min in suspensions with 5 infective juveniles of S. carpocapsae per milliliter of water, with and without Tween 80 (0.01%), yielded essentially the same mortality of codling moth larvae. Highest mortalities in codling moth larvae (88%) after treatment of bins in suspensions of 5 infective juveniles of S. carpocapsae per milliliter of water were observed after incubation for 24 h at 25 degrees C and 70% RH. Lowest mortalities (37%) were observed after incubation at 15 degrees C and 35% RH. Comparative tests conducted with Heterorhabditis marelatus Liu & Berry, Steinernema kraussei (Steiner), and S. carpocapsae with 5 infective juveniles per milliliter of water resulted in 21.7, 53.9, and 68.7% mortality, respectively. The use of miniature fruit bins as described in this article provides an effective means of assessing nematode efficacy without the cumbersome size of commercial bins.     

The susceptibility of the pea moth, Cydia nigricana, to infection by the granulosis virus of the codling moth, Cydia pomonella

Laboratory studies demonstrated that neonate larvae of the pea moth, Cydia nigricana, are susceptible to infection with a granulosis virus (CpGV) isolated from the codling moth, Cydia pomonella. Comparative LC₅₀ values for C. nigricana and C. pomonella are 1.90 × 10⁵ and 1.54 × 10⁴ capsules/ml of diet, respectively. The virus extracted from CpGV-infected pea moth larvae is serologically related, and probably identical, to CpGV.     

Key elements in the successful control of diapausing codling moth,Cydia pomonella (Lepidoptera: Tortricidae) in wooden fruit bins with a South African isolate of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae)

The non-insecticidal control strategies currently being implemented in South African orchards for the control of codling moth, Cydia pomonella (L.) may be hampered by wooden fruit bins being infested with diapausing codling moth larvae, acting as a potential source of re-infestation. Key factors contributing to the success or failure of an entomopathogenic nematode application were investigated using the SF 41 isolate of Heterorhabditis zealandica in laboratory bioassays with wooden minibins. Under operational conditions, an application rate of 100 IJs/mL (LD₉₀₌102 IJs/mL) effectively controlled codling moth larvae in these bins, and for further laboratory bioassays, the LD₅₀ value of 18 IJs/mL (?25 IJs/mL) was identified as the discriminating dosage. Maximum mortality was attained when bins were pre-wet for at least 1 min (>90% RH) and maintained at maximum humidity (>95% RH) post-treatment for at least 3 days (LT₉₀=73 h), to ensure nematode survival and subsequent satisfactory infection of diapausing codling moth larvae. Tarping bins achieved the desired high level of humidity required. Furthermore, adjuvants (specifically Reverseal 10?) also improved an application. The study conclusively illustrated that if all the above-mentioned conditions are met, H. zealandica has the potential to successfully disinfest wooden fruit bins of codling moth.     

The ontogeny of cold tolerance in the gall fly, Eurosta solidagensis

The lower lethal temperature of many insects indicates an overwintering flexibility as a result of either extensive supercooling or production of cryoprotectants. Ontogenetically, the gall fly (Eurosta solidagensis) utilizes both means of seasonal cryoprotection. All stages except third instar larvae demonstrate supercooling points well below the lowest temperature normally experienced by that particular stage. The third instar larvae exhibit a high supercooling point but are well protected by a cryoprotectant system consisting of glycerol, sorbitol, and trehalose. Glycerol is accumulated, possibly from triglyceride sources, during early autumn and reaches plateau levels (0·6 M) by early winter. Sorbitol synthesis is delayed until freezing exposures and reaches a plateau with glycerol at 0·3 M. It is not until mid-winter that peak trehalose levels are reached (300 mg %). All cryoprotectant levels are a reflection of haemolymph concentrations.Laboratory acclimation experiments further quantify these results. Trehalose synthesis is time and temperature dependent and appears to be affected by developmental processes.