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.     

Thermal death kinetics of fifth-instar Plodia interpunctella (Lepidoptera: Pyralidae)

Heat treatments have been suggested as alternatives to chemical fumigants for control of postharvest insects in dried fruits and nuts. Conventional forced hot air treatments heat product too slowly to be practical, but radio frequency treatments are capable of more rapid product heating. While developing radio frequency heat treatments for dried fruits and nuts, the heat tolerance of nondiapausing and diapausing fifth-instar larvae of the Indianmeal moth, Plodia interpunctella (Hübner), was determined using a heating block system developed by Washington State University. Both a 0.5th order kinetic model and a classical empirical model were used to estimate lethal exposure times for temperatures of 44-52 degrees C for nondiapausing fifth-instar larvae. We obtained 95% mortality at exposures suitable for practical radio frequency treatments (< or = 5 min) with temperatures of 50 and 52 degrees C. Diapausing larvae were significantly more tolerant than nondiapausing larvae at the lowest treatment temperature and shortest exposure, but differences were not significant at more extreme temperature-time combinations. Previous studies showed that fifth-instar larvae of the navel orangeworm, Amyelois transitella (Walker), were more heat tolerant than either diapausing or nondiapausing Indianmeal moth larvae. Consequently, efficacious treatments for navel orangeworm would also control Indianmeal moth.     

Conditions for a heat shock response during oogenesis and embryogenesis of the amphibian Pleurodeles waltl

The optimal conditions capable of inducing an increase in HSP70 neosynthesis during development of the urodele amphibian Pleurodeles waltl were determined in this study. These conditions depend on temperature, heat shock duration and recovery duration. In oocytes, a heat shock response was repeatedly obtained at 37°C for 15 min followed by 1 h recovery. These results provided evidence for heat shock response at every stage considered. An increase in HSP70 synthesis was noted throughout oogenesis, but it did not lead to an increase in the amount of soluble HSP70, except for stage VI oocytes. Such results suggest that from stage II to stage IV oocytes, an equilibrium occurs between the HSP70 used and the HSP70 neosynthesized. In contrast, in stage VI oocytes, heat shock led to overproduction of HSP70. During early development, the heat shock response was repeatedly obtained only from the gastrula stage with a 37°C shock and a 15min duration of treatment. Surprisingly, during cleavage stage, the soluble HSP70 total amount increased after heat shock at a time when no HSP70 neosynthesis occurred.     

Control of diapause in codling moth larvae

Diapause in a New Zealand strain of codling moth (Cydia pomonella Linnaeus [Lepidoptera: Olethreutidae]) was induced in larvae by photoperiods of 15 h or less. Once diapause had been initiated, it could not be terminated by any combination of conditions tested for at least 20 days after cocooning. In diapausing larvae a low rate of pupation occurred at 25 °C under a long day (18 h) photoperiod. A high rate of pupation was achieved under a long day regime when larvae were decocooned, and provided with apple as nourishment. Diapause could be terminated predictably in 94–100% of larvae by 1) conditioning at 15 °C and constant darkness for periods of 40–100 days, then 2) chilling at 2±2 °C and constant darkness for 20–50 days followed by 3) any post-chill condition periods at 25 °C, 18 h photoperiod. Complete diapause termination was achieved when 100 days conditioning was followed by 30 days or 50 days post-chill period. Under these conditions, 76% termination occurred in the post-chill period after 10 days, and 93% after 25 days.To terminate diapause in codling moth larvae, we recommend that a 100 days conditioning followed by 30 days chilling and 50 days post chilling periods be used.     

Dosage-mortality and time-mortality studies of a granulosis virus in a laboratory strain of the codling moth,Laspeyresia pomonella

Different doses of a granulosis virus were administered to first- and fifth-instar larvae of the codling moth Laspeyresia pomonella. Virus was very pathogenic for both larval instars. The LD₅₀ values for first- and fifth-instar larvae were 5 and 49 capsules/larva, respectively. However, fifth-instar larvae were much more variable in their response to virus than first-instar larvae. Using probit methods it was calculated that 1 capsule could cause death in about 25% of both larval instars but 1578 capsules were required to cause 70% mortality of fifth-instar larvae as compared to 12 capsules for first-instar larvae. This is the first report of a decided difference in variability of response to virus by two larval instars of the same species. A bimodal response by both larval instars was observed in time-mortality studies. Apparently, about 20% of the larvae were very resistant to virus infections.     

The effect of host stage and temperature on selected developmental parameters of the solitary endoparasitoid Meteorus gyrator (Thun.) (Hym., Braconidae)

Abstract:  The development of the solitary endoparasitoid Meteorus gyrator was compared in the six larval stages of its host, the tomato moth Lacanobia oleracea , and at five constant temperatures. The host instar at the time of parasitism had a marked effect on the larval developmental period of the parasitoid, such that larvae derived from eggs oviposited in first instar hosts took approximately 18 days to egress, whilst those derived from eggs oviposited in sixth instar hosts took <10 days. The weight of cocoons was greatest when oviposition was into final instar hosts, where female cocoons averaged 12.8 mg, and lowest in those derived from eggs oviposited into first instars (9.2 mg). The parasitoid's larval development rate in third instar hosts increased with temperature increments in a linear fashion up to 25°C , after which development times were only marginally increased. At 10°C, the mean larval development time was approximately 90 days and pupal development 35–40 days, whilst at 25°C development times were 10–11 days for larvae and 6–7 days for the pupae. In the majority of cases, overall development times were marginally longer (<1 day) in females than in males.     

Thermal tolerance and heat shock protein synthesis during development in the anuran Lepidobatrachus laevis

Development of the Paraguayan anuran Lepidobatrachus laevis is unusual in that the larvae are obligate carnivores, facultative cannibals and apparently exist at high environmental temperatures in their natural habitat. In the present study, the effect of environmental temperature on the rate of anuran development was investigated. The larvae have a thermotolerance range of 18°C for normal development between 19 and 37°C. The effect of temperature on the rate of development was dramatic; larvae that were incubated at 36.8°C develop to stage 24 (Gosner) in approximately 9 h compared with 24 h for larvae incubated at 19°C. The ability of larvae to survive heat shock was also examined; larvae did not survive a shock of 45°C for 15 min when it was administered at stages 3, 5, 9, 10 or 20. However, using the same heat shock conditions, 50% survival was observed when larvae were shocked at stage 16. To study protein synthesis during heat shock, larvae were pulsed with [³⁵S]-methionine during heat shock and labeled proteins were analyzed by electrophoresis under reducing and denaturing conditions. Larvae synthesized two sets of heat-shock proteins at doublet molecular weights of 83/78 and 62/59 kDa. These proteins were synthesized independently of the stage of development at which the shock was administered or the magnitude of the heat shock.     

Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae

1. Whether Drosophila larvae and pupae naturally experience temperatures that can cause heat damage or death is poorly understood, but bears directly on numerous investigations of the thermal biology and heat-shock response in Drosophila . Accordingly, the temperatures of necrotic fruit, which Drosophila larvae and pupae inhabit, the temperatures of larvae and pupae outside the laboratory, and the levels of the heat-shock protein hsp 70 expressed by larvae in nature were examined.
2. When necrotic fruit was sunlit, internal temperatures rose to levels that can harm indwelling insects. Fruit size and evaporative water loss affected these temperatures. Temperatures of larvae and pupae in the field commonly exceeded 35 °C, with living larvae recorded at >44°C and pupae at >41°C. Natural mortality was evident, presumably because of heat.
3. In the laboratory, these temperatures kill larvae rapidly, with LT₅₀s (time taken for half the sample to be killed) of 30 min at 39 °C, 15 min at 40 °C and 8·5 min at 41 °C. Gradual transfer from 25°C to these temperatures resulted in no lesser mortality than did direct transfer.
4. Hsp 70 levels in lysates of whole larvae were measured by ELISA (enzyme-link immunosorbent assay) with an hsp 70-specific antibody. For larvae within necrotic apples experimentally transferred from shade to sun and within necrotic fruit in situ , hsp 70 levels equalled or exceeded levels detected in parallel laboratory studies of whole larvae or cells in culture.
5. These data provide an ecological context for studies of thermal stress and the heat-shock response in Drosophila that has heretofore been lacking.     

Cocoon-spinning larvae of Oriental fruit moth and Indianmeal moth do not produce aggregation pheromone

1 Mature larvae of the Oriental fruit moth (OFM) Grapholita molesta (Lepidoptera: Tortricidae) and the Indianmeal moth (IMM) Plodia interpunctella (Lepidoptera: Pyralidae) leave their food source in search of suitable pupation sites in which to spin cocoons. These sites are typically well-concealed cracks and crevices within the environment. Such cocooning behaviour is also observed in larvae of the codling moth (CM) Cydia pomonella (Lepidoptera: Tortricidae), which aggregate prior to pupation in response to a pheromone blend produced by cocoon-spinning conspecific larvae.
2 In laboratory experiments, we tested whether cocoon-spinning OFM and IMM larvae produce aggregation pheromones and whether CM larvae are cross-attracted to closely-related OFM larvae.
3 Fifth-instar OFM and IMM larvae were not attracted to, or arrested by, cocoon-spinning conspecifics. Moreover, fifth-instar CM larvae were not cross-attracted to either cocoon-spinning OFM or IMM larvae.
4 Analyses of volatiles released from cocoon-spinning OFM and IMM larvae revealed that both OFM and IMM lack components that are present in the aggregation pheromone of CM larvae. This information may help explain why CM larvae are not cross-attracted to cocooning OFM or IMM larvae.     

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula

Aim:  To determine D - and z -values of Cronobacter species ( Enterobacter sakazakii ) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro-organism.
Methods and Results:  Five Cronobacter species (four C. sakazakii isolates and C. muytjensii ) were heated in reconstituted milk or feeding formula pre-equilibrated at 52–58°C for various times or mixed with powdered milk or feeding formula prior to reconstitution with water at 60–100°C. The D -values of Cronobacter at 52–58°C were significantly higher in whole milk (22·10–0·68 min) than in low fat (15·87–0·62 min) or skim milk (15·30–0·51 min) and significantly higher in lactose-free formula (19·57–0·66 min) than in soy protein formula (17·22–0·63 min). The z -values of Cronobacter in reconstituted milk or feeding formula ranged from 4·01°C to 4·39°C. Water heated to ≥70°C and added to powdered milk and formula resulted in a > 4 log₁₀ reduction of Cronobacter .
Conclusions:  The heat resistance of Cronobacter should not allow the survival of the pathogen during normal pasteurization treatment. The use of hot water (≥70°C) during reconstitution appears to be an effective means to reduce the risk of Cronobacter in these products.
Significance and Impact of the Study:  This study supports existing data available to regulatory agencies and milk producers that recommended heat treatments are sufficient to substantially reduce risk from Cronobacter which may be present in these products.     

Selection of a strain of the granulosis virus of the codling moth with improved resistance against artificial ultraviolet radiation and sunlight

A six-step sequence of selection of the granulosis virus of Laspeyresia pomonella led to a strain with increased resistance to sunlight. Each step consisted of irradiation of dry deposits of the virus on apples with near range ultraviolet light, infection of freshly eclosed codling moth larvae with the irradiated virus, and propagation of the virus thus produced in large fifth-instar larvae. Compared with the original virus, the new strain was 5.6 times more resistant to artificial ultraviolet irradiation in the laboratory and remained twice as long infective in the field.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

A test of fruit varieties on entry rate and development by neonate larvae of the codling moth,Cydia pomonella

The rate of entry by neonate larvae of the frugivorous codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), into fruit material was investigated. We used no‐choice bioassays in climate‐controlled rooms to assay larval entry across four host plant species (apple, pear, quince, walnut) and three varieties within a single fruit species (apple). Larvae successfully entering apples were reared to adulthood, and we collected tissue samples from apples which were successfully colonized in order to determine sucrose concentrations. This information was used to evaluate differences in adult moth size, development time, and pulp sucrose concentration due to apple variety. Four important findings emerged: (1) neonate larvae had the highest frequency of entry (86% of larvae) into apple fruits, compared with pear (78%), quince (56%), and walnut (32%); (2) the frequency of larval entry into immature apples differed across apple varieties, and larval entry rate was highest in variety Golden Delicious (72%), compared with Granny Smith (46%) and Red Delicious (64%); (3) on average, adult moths were larger and development times were shorter on the variety with the highest entry frequency (Golden Delicious); and (4) apple pulp sucrose concentrations were higher for Golden Delicious (17.5 μg mg^?1) than for either Granny Smith (15.9 μg mg^?1) or Red Delicious (15.1 μg mg^?1) varieties, which correlates positively with entry and development data. We conclude that host fruit species and varietals within a species affect the entry rate of neonate codling moth larvae in no‐choice assays. We hypothesize that larval development is influenced by mean sucrose concentrations or other phytochemical differences associated with host fruit varieties.     

Heat-Stress Response of Maize Mitochondria

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

Inhibition of the ubiquitin-proteasome pathway induces differential heat-shock protein response in cardiomyocytes and renders early cardiac protection

The effects of proteasome inhibition (PI) on heat-shock protein (HSP) expression in cardiomyocytes were investigated. Neonatal rat cardiomyocytes were incubated with MG132 (0.1-10 microM) for 1 h. Induction of various HSPs was determined by real-time PCR and Western blotting. PI induced a 2- to 3-fold increase in HSP27, HSP60, and HSP90, and a 18-fold increase in HSP70 mRNA expression, whereas HSP40 levels were unaffected. Western blotting revealed increased protein expression for HSP70 after PI. Similar results were obtained with MG262. HSP induction correlated with enhanced survival of neonatal cardiomyocytes after sublethal heat stress in XTT testing. In papillary muscles, pretreatment with MG132 (10 microM, 90 min) was associated with enhanced recovery of the contractile parameters after a 40-min hypoxia. In these proof-of-principle experiments, we show that PI induces differential heat-shock response in cardiomyocytes, accompanied by enhanced cell survival and functional recovery after various forms of stress.     

Effects of temperature and delayed initial feeding on the survival and growth of Japanese flounder larvae

The effects of the timing of initial feeding (0, 1, 2, 3 and 4 days after yolk exhaustion) and temperature (15, 18 and 21° C) on the point‐of‐no‐return (PNR), survival and growth of laboratory‐reared Japanese flounder Paralichthys olivaceus larvae were studied under controlled conditions. The larvae reached PNR on 7·7, 5·2 and 4·2 days‐post‐hatching (dph) at 15, 18 and 21° C, respectively. At each temperature, larval growth did not differ significantly among the delayed initial feedings 1 day before PNR but decreased significantly in larvae first fed after that. In the treatments where initial feeding was equally delayed, larvae grew significantly faster at 18 and 21° C than at 15° C. The larvae survived apparently better at 15 and 18° C than at 21° C when initial feeding was equally delayed. At each temperature, survival of the larvae first fed before PNR did not differ noticeably, while delayed initial feeding after that apparently reduced their survival. These results indicated that there existed a negatively temperature‐dependent PNR in the Japanese flounder larvae. Survival and growth of the larvae strongly depended on temperature as well as the timing of initial feeding. High temperature accelerated the yolk exhaustion and growth of the larvae and thus reduced their starvation tolerance and survival. To avoid potential starvation mortality and obtain good growth, the Japanese flounder larvae must establish successful initial feeding within 2 days after yolk exhaustion at 15° C and within 1 day at both 18 and 21° C.     

Fruit Bins Washed with Steinernema carpocapsae (Rhabditida: Steinernematidae) to Control Cydia pomonella (Lepidoptera: Tortricidae)

A bin washing apparatus (drencher) was used to treat late-instar codling moth, Cydia pomonella Linnaeus-infested wooden fruit bins with the entomopathogenic nematode Steinernema carpocapsae . Sentinel late-instar codling moth larvae were placed in strips of wood, made from dismantled bins, that were subsequently screwed into the corners of real bins before treatment. Pre-soaking the bins with water prior to treatment and covering the bins with a plastic tarp post-treatment, to maintain a high humidity, significantly increased sentinel codling moth mortality. Treatment for 1 min with 50 S. carpocapsae infective juveniles mL -1 water resulted in more than 80% mortality over 6 h in which the nematodes were continuously pumped through the drencher. The temperature of the water in the bin washer was ≤15°C and the chlorine in the city water was too low to harm the nematodes. The key disadvantage of washing the bins was the labour involved. It may be possible to reduce post-treatment holding time and increase codling moth mortality by holding the bins at a higher temperature post-treatment, however this parameter was not included in the study. The presence of the codling moth granulosis virus within the larvae did not influence the nematode-induced codling moth mortality over a range of concentrations and two temperatures in laboratory trials.     

Effect of hyperthermia on protein synthesis in monolayer and suspension cultures of mouse L cells

Changes in protein synthesis that occurred under the influence of heat shock (HS) in monolayer (L929) and suspension (LS) mouse cell cultures were studied. The rates of protein synthesis determined as 35S-methionine incorporations were seen reduced from the initial level up to 40-60 and 6-13% after HS at 42 and 44 degrees C, respectively. Simultaneously the rate of actin and tubulin syntheses decreased, the decrease being more pronounced in LS cells. According to electrophoresis and autoradiography data, after hyperthermia both the cell cultures were able to synthesize heat shock proteins (HSP), primarily HSP70. After a 40 min HS towards L929 and LS cells at 43 degrees C, the shares of their HSP70 bands in the total label loaded on the gel constituted, resp., 8.8 and 5.4%. The data suggest that L929 cells, with their synthetic activity lower than in LS cells, appear more resistant to HS and are able eventually to synthesize larger amounts of HSP70, compared to the latter.