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Embryonic selection for vitrification and cryostorage in Drosophila and other dipterans is generally carried out by gross observation of the embryonic development at a constant temperature. In this study, the effect of embryo developmental temperature (19, 20 and 21 °C) on the stage specific convergence of the embryonic development to the developmental stages 15-17, which are relevant for cryopreservation, was studied in a flightless mutant strain of Drosophila melanogaster and compared with the Ore-R strain. The temperature that allowed for the best convergence to stage 16 was chosen for further selection and treatment of the embryos. The converged embryos (SS) were directly treated or further manually sorted (MS) for the requisite developmental stage to reduce the number of non-converged embryos. These selected embryos were then permeabilized and cryopreserved. While at all the three incubation temperatures the embryos exhibited convergence peaks, it was only at 20 °C and at hour 22 that a maximum number of stage 16 embryos converged and remained at a much higher proportion than the other developmental stages in both the strains. When permeabilized, MS embryos showed higher mean viability and hatching proportion compared to SS embryos (wingless: ∼0.70 vs. ∼0.58; Ore-R: ∼0.77 versus 0.54). Upon vitrification, the manually selected embryos hatched and survived at significantly higher mean rates than the converged embryos at stage 16 (wingless: 0.32 vs. ∼0.08; Ore-R: 0.47 vs. 0.15) after adjusting for permeabilization mortality. The maximum proportion hatch after vitrified storage that could be obtained by this method was 0.74 for both the wingless and Ore-R strains. More than 55% of the larvae pupated and72% of the pupae eclosed in MS and vitrified wingless stage 16. In Ore-R, well over 85% of the larvae pupariated and eclosed as flight capable flies.

The development of cryopreservation protocols for Anopheles gambiae could significantly improve research and control efforts. Cryopreservation of any An. gambiae life stage has yet to be successful. The unique properties of embryos have proven to be resistant to any practical cryoprotectant loading. Therefore, we have chosen to investigate early non-feeding first instar larvae as a potential life stage for cryopreservation. In order to determine an appropriate cryoprotective compound, larvae were treated with progressively better glass-forming cryoprotective mixtures. Toxicity evaluation in combination with calorimetry-based water content and supercooling point depression assessments were used to determine the cryoprotectants that could be used for cryostorage of viable larvae. Approximately 35–75% of the larvae were viable after reasonably high osmotic and biochemical challenge. This study provides ample evidence for an active osmoregulatory response in the Anopheles larvae to counter the permeation of cryoprotectants from the surrounding medium. The data show a strong correlation between the larval mortality and water content, indicating an osmoregulatory crisis in the larva due to certain cryoprotectants such as the higher concentrations of ethane diol (ED). The observations also indicate that the ability of the larvae to regulate permeation and water balance ceases at or within 20 min of cryoprotectant exposure, but this is strongly influenced by the treatment temperature. Among the compound cryoprotectants tested, 25% ED + 10% dimethyl sulfoxide (DMSO) and 40% ED + 0.5 M trehalose seem to present a compromise between viability, larval water content, supercooling point depression, and glass forming abilities.

Interrupting the spring incubation of Megachile rotundata (F.) with a period of low-temperature storage for synchronizing the bees’ emergence with crop bloom is an essential part of M. rotundata management. Previously, we demonstrated that bees exposed to thermoperiods (TPs) during low-temperature storage have higher survival rates than bees exposed to constant temperatures. But changing the temperature in the large mass of bees commonly found in most commercial settings would place considerable stress on the chambers’ refrigeration system. Reducing the difference between a TP’s cryophase and thermophase would decrease the stress on the refrigeration system. Therefore, we investigated a range of TPs with cryophases (12 h) of 6, 12, or 15°C and thermophases (12 h) of 15 or 18°C and compared the survival rates of these bees against bees exposed to constant temperatures of 12, 15, or 18°C. For eye-pigmented pupae, the TP 6–18°C and the control fluctuating thermal regime (FTR; 6°C with a daily 1-h pulse at 20°C) had the highest survival rates for the 2 yr tested. For the constant-temperature storage protocols, constant 15 and 18°C were either equivalent or lower survival than the control FTR. For emergence-ready adults, the 6–18°C TP had the highest survival rates. The constant 15°C and the control FTR had equivalent survival rates. Under the current constraints imposed by a commercial chamber’s refrigeration system, interrupting M. rotundata spring incubation by exposing the developing bees to constant temperatures of 15–18°C is currently the best option for commercial operations.

Insects exposed to low temperature stress can experience chill injury, but incorporating fluctuating thermoprofiles increases survival and blocks the development of sub-lethal effects. The specific parameters required for a protective thermoprofile are poorly understood, because most studies test a limited range of thermoprofiles. For example, thermoprofiles with a wave profile may perform better than a square profile, but these two profiles are rarely compared. In this study, two developmental stages of the alfalfa leafcutting bee, Megachile rotundata, eye-pigmented pupae, and emergence-ready adults, were exposed to one of eight thermoprofiles for up to 8 weeks. All the thermoprofiles had a base of 6°C and a peak temperature of either 12°C or 18°C. The duration at peak temperature varied depending on the shape of the thermoprofile, either square or wave form. Two other treatments acted as controls, a constant 6°C and a fluctuating thermal regime (FTR) with a base temperature of 6°C that was interrupted daily by a single, 1-h pulse at 20°C. Compared with constant 6°C, all the test thermoprofiles significantly improved survival. Compared with the FTR control, the thermoprofiles with a peak temperature of 18°C outperformed the 12°C profiles. Bees in the eye-pigmented stage exposed to the 18°C profiles separated into two groups based on the shape of the profile, with higher survival in the square profiles compared with the wave profiles. Bees in the emergence-ready stage exposed to 18°C profiles all had significantly higher survival than bees in the FTR controls. Counter to expectations, the least ecologically relevant thermoprofiles (square) had the highest survival rates and blocked the development of sub-lethal effects (delayed emergence).

This study evaluates the efficacy of a cryopreservation protocol for spermatozoa derived from the accessory testis of male Bombus impatiens. It is also the first report of successful cryopreservation of bumble bee spermatozoa. The spermatozoa viability was compared with the similarly treated honey bee spermatozoa derived from its accessory testis. The semen was frozen using a yolk-free non-activating buffer containing dimethyl sulphoxide and stored in liquid nitrogen for 24 h to ~14 days. Thereafter, the frozen samples were thawed rapidly and assessed by staining with live/dead differentiating fluorescent dyes. Semen viability in cryopreserved samples (55.8 ± 14.0%) was significantly different than controls (96.2 ± 10.5%). Similar assessment with A. mellifera resulted in 82.2 ± 7.0% viable cryopreserved spermatozoa versus 99.4 ± 0.1% in controls. A similar proportion of the sperm cells were also capable of motility upon dilution of the extender medium with phosphate buffered saline. The proportion of viable accessory testis derived sperm cells obtained post-cryopreservation was estimated to be sufficient to initiate long term storage and artificial insemination programs.

In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects.

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA sequencing (RNA-seq). However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory-reared and -maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata, entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory- and field-maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect.

Embryos of two dipteran species (Musca domestica and Lucilia sericata) were assessed for an effective sampling time that would result in the highest post-cryopreservation hatch rate, with a primary goal to define species-specific egg collection periods and the effects of manual stage selection on post cryopreservation yield. The effects of the time taken to collect eggs on, (a) the proportion of embryos reaching a specific developmental stage between 17 and 20 h of development, and (b) the post-cryopreservation hatch rate were assessed. Permeabilization treatment applied at any stage of embryonic development did not significantly reduce embryo viability. Eggs collected over longer durations significantly reduced the number of embryos available in a specific developmental stage amenable to cryopreservation. Hatch percentage after cryopreservation of the embryos of M. domestica collected over a 60 min period was 10.7 ± 8.7% compared to 31 ± 5% for the eggs collected for just 15 min. Similarly, percent hatch in L. sericata resulted in 17.0 ± 3.9 and

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA sequencing (RNA-seq). However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory-reared and -maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee

In holometabolous insects, larval nutrition affects adult body size, a life history trait with a profound influence on performance and fitness. Individual nutritional components of larval diets are often complex and may interact with one another, necessitating the use of a geometric framework for elucidating nutritional effects. In the honey bee, Apis mellifera, nurse bees provision food to developing larvae, directly moderating growth rates and caste development. However, the eusocial nature of honey bees makes nutritional studies challenging, because diet components cannot be systematically manipulated in the hive. Using in vitro rearing, we investigated the roles and interactions between carbohydrate and protein content on larval survival, growth, and development in A. mellifera. We applied a geometric framework to determine how these two nutritional components interact across nine artificial diets. Honey bees successfully completed larval development under a wide range of protein and carbohydrate contents, with the medium protein (∼5%) diet having the highest survival. Protein and carbohydrate both had significant and non-linear effects on growth rate, with the highest growth rates observed on a medium-protein, low-carbohydrate diet. Diet composition did not have a statistically significant effect on development time. These results confirm previous findings that protein and carbohydrate content affect the growth of A. mellifera larvae. However, this study identified an interaction between carbohydrate and protein content that indicates a low-protein, high-carb diet has a negative effect on larval growth and survival. These results imply that worker recruitment in the hive would decline under low protein conditions, even when nectar abundance or honey stores are sufficient., Summary: Protein to carbohydrate ratio affects the survival and growth rate of larval worker honey bees in vitro. Protein and carbohydrate content of food interact in complex ways to alter growth rate, development time and survival in worker bound honey bee larvae.

Embryos of Lucilia (Phaenicia) sericata (Meigen) (Diptera Calliphoridae), the green blowfly, were successfully cryopreserved by vitrification in liquid nitrogen and stored for 8 yr. Embryos incubated at 19 degrees C for 17 h after oviposition were found to be the most appropriate stage to cryopreserve. Removal of the embryonic surface water was done using 2-propanol before the alkane treatment to permeabilize the embryo. Exposure to 2-propanol for10 s caused necrotic tissue damage in the embryos. Among the alkanes used, hexane was found to be a superior permeabilizing solvent compared with heptane or octane, with embryo hatching rates on par with the controls. Treatment with the vitrification solution for12 min was insufficient to vitrify the embryos. Treatment time in the solution beyond 15 min reduced embryo viability. However, the percentage of embryos vitrifying upon exposure to liquid nitrogen vapor remained constant after 12 min of treatment. Long-term storage was initiated in 2004, and the mean hatch percentage recorded then for the short-term cryopreserved embryos was 9.51%. When the long-term stored samples were retrieved in 2012, 8.47% of the embryos hatched, 66.36% larvae pupariated, and 36.96% of the pupae eclosed. Recent optimization of the technique has resulted in a hatch rate of 34.08 +/- 15.5%, of which 67.5% of the larvae pupariated and 72% of the pupae eclosed to normal flies.

The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer, and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. After the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant with water as evidenced by a significant lowering of the supercooling point of the cryoprotectant-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was approximately 7%.

The Mediterranean fruit fly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. During the last decades, area-wide pest management (AW-IPM) approaches with a sterile insect technique (SIT) component have been used to control populations of this pest in an effective and environment-friendly manner. The development of genetic sexing strains (GSS), such as the Vienna 8 strain, has been played a major role in increasing the efficacy and reducing the cost of SIT programs. However, mass rearing, extensive inbreeding, possible bottleneck phenomena and hitch-hiking effects might pose major risks for deterioration and loss of important genetic characteristics of domesticated insect. In the present study, we present a modified procedure to cryopreserve the embryos of the medfly Vienna 8 GSS based on vitrification and used this strain as insect model to assess the impact of the cryopreservation process on the genetic structure of the cryopreserved insects. Forty-eight hours old embryos, incubated at 24°C, were found to be the most suitable developmental stage for cryopreservation treatment for high production of acceptable hatch rate (38%). Our data suggest the absence of any negative impact of the cryopreservation process on egg hatch rate, pupation rates, adult emergence rates and stability of the temperature sensitive lethal (tsl) character on two established cryopreserved lines (flies emerged from cryopreserved embryos), named V8-118 and V8-228. Taken together, our study provides an optimized procedure to cryopreserve the medfly Vienna 8 GSS and documents the absence of any negative impact on the genetic structure and quality of the strain. Benefits and sceneries for utilization of this technology to support operational SIT projects are discussed in this paper.

The emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae) (EAB), an invasive wood-boring beetle, has recently caused significant losses of native ash (Fraxinus spp.) trees in North America. Movement of wood products has facilitated EAB spread, and heat sanitation of wooden materials according to International Standards for Phytosanitary Measures No. 15 (ISPM 15) is used to prevent this. 2 In the present study, we assessed the thermal conditions experienced during a typical heat-treatment at a facility using protocols for pallet wood treatment under policy PI-07, as implemented in Canada. The basal high temperature tolerance of EAB larvae and pupae was determined, and the observed heating rates were used to investigate whether the heat shock response and expression of heat shock proteins occurred in fourth-instar larvae. 3 The temperature regime during heat treatment greatly exceeded the ISPM 15 requirements of 56 ◦ C for 30 min. Emerald ash borer larvae were highly tolerant of elevated temperatures, with some instars surviving exposure to 53 ◦ C without any heat pre-treatments. High temperature survival was increased by either slow warming or pre-exposure to elevated temperatures and a recovery regime that was accompanied by up-regulated hsp70 expression under some of these conditions. 4 Because EAB is highly heat tolerant and exhibits a fully functional heat shock response, we conclude that greater survival than measured in vitro is possible under industry treatment conditions (with the larvae still embedded in the wood). We propose that the phenotypic plasticity of EAB may lead to high temperature tolerance very close to conditions experienced in an ISPM 15 standard treatment.

This study evaluates characteristics commonly used to define insect quality or fitness by using a complement of three species of tephritid fruit flies (Diptera: Tephritidae) obtained from cryopreserved embryos. Anastrepha ludens (Loew), Anastrepha suspensa (Loew), and Ceratitis capitata (Wiedemann) were used to assess embryo to adult emergence and adult longevity, flight ability, mating ability, fecundity, and genetic variability after cryopreservation. With the three species tested, embryo survival was reduced by 50–70% whereas adult eclosion seemed unaffected by cryogenic treatment. Laboratory cage survival of cryopreserved A. ludens paralleled that of controls when tested with or without food and water posttreatment. With C. capitata, field cage survival was also similar when the adult progeny of cryopreserved parents was compared with that of untreated flies of the same age. Assessment of flight ability of cryopreserved A. ludens over a 19-d period by using a flight mill showed no statistical difference when compared with the untreated groups over the same time period. Flight ability within field cages for newly emerged progeny of cryopreserved C. capitata also mirrored that of the controls. Observed matings occurring within laboratory cages containing equal numbers of A. ludens males and females did not differ from comparable control groups. Furthermore, male progeny obtained from cryopreserved C. capitata parents competed equally with untreated males for mates while housed in field cages. A laboratory analysis of fertility and fecundity of A. suspensa revealed that males mated with control females were unaffected by cryopreservation as embryos, whereas cryopreserved females exhibited a significantly reduced fecundity when mated with control males. The fecundity of C. capitata progeny of cryopreserved parents also did not differ from control levels while caged under laboratory conditions. A random amplified polymorphic DNA assay of the genetic diversity of A. ludens comparing cryopreserved males with control males showed that the coefficient of similarity was ≥85%. This study indicates that embryo cryopreservation had little or no effect on the reproduction, longevity and flight of the species tested and can be used to support maintenance of insect stocks and control programs supported by the mass-rearing process.

Chill-susceptible insects are able to improve their survival of acute cold exposure over both the short term (i.e. hardening at a relatively severe temperature) and longer term (i.e. acclimation responses at milder temperatures over a longer time frame). However, the mechanistic overlap of these responses is not clear. Four larval stages of four different strains of Drosophila melanogaster are used to test whether low temperature acclimation (10 °C for 48 h) improves the acute cold tolerance (LT90, ∼2 h) of larvae, and whether acclimated larvae still show hardening responses after brief exposures to nonlethal cold or heat, or a combination of the two. Acclimation results in increased cold tolerance in three of four strains, with variation among instars. However, if acclimation is followed by hardening pre-treatments, there is no improvement in acute cold survival. It is concluded that short-term thermal responses (e.g. hardening) may be of more ecological relevance to short-lived life stages such as larvae, and that the mechanisms of low temperature hardening and acclimation in D. melanogaster may be antagonistic, rather than complementary.

This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae.

The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer, and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. After the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant with water as evidenced by a significant lowering of the supercooling point of the cryoprotectant-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was approximately 7%.

Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3(rd) instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above -14 degrees C, and non-diapausing 3(rd) instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.

We quantified the variation and plasticity in cold tolerance among four larval stages of four laboratory strains of Drosophila melanogaster in response to both acute (2h of cold exposure) and chronic ( approximately 7h of cold exposure) cold exposure. We observed significant differences in basal cold tolerance between the strains and among larval stages. Early larval instars were generally more tolerant of acute cold exposures than third-instar larvae. However, wandering larvae were more tolerant of chronic cold exposures than the other stages. Early stages also displayed a more pronounced rapid cold-hardening response than the later stages. Heat pre-treatment did not confer a significant increase in cold tolerance to any of the strains at any stage, pointing to different mechanisms being involved in resolving heat- and cold-elicited damage. However, when heat pre-treatment was combined with rapid cold-hardening as sequential pre-treatments, both positive (heat first) and negative (heat second) effects on cold tolerance were observed. We discuss possible mechanisms underlying cold-hardening and the effects of acute and chronic cold exposures.

We tested the hypothesis that slow rewarming would improve the ability ofDrosophila melanogasterMeigen (Diptera: Drosophilidae) larvae to survive acute low-temperature exposure. Four larval stages (1st, 2nd, and 3rd instars, including wandering-stage 3rd instars) of four wild-type strains were exposed to –7 °C for periods of time expected to result in 90% mortality. Larvae were then directly transferred to their rearing temperature (21 °C) or returned to this temperature either in a stepwise fashion (pausing at 0 and 15 °C) or by slow warming at 1 or 0.1 °C/min. We observed a reduced rapid cold-hardening effect and no general increase in survival of acute chilling in larvae rewarmed in a stepwise or slow fashion, and we hypothesize that slow rewarming may result in accumulation of chill injuries.