Monarch butterfly conservation through male germplasm cryopreservation.

Monarch butterfly (Danaus plexippus L.) populations have declined in North America. The International Union for Conservation of Nature (IUCN) recently classified the species as endangered, sparking public concern and conservation efforts. Our approach to conservation is through cryopreservation of germinal cells and tissue. The goal of this study was to develop a cryopreservation protocol for monarch spermatozoa to ensure successful long-term storage. Cryopreserved sperm cells would provide a reserve of monarch germplasm, which could be utilized in the event of population loss. In this study, sperm cell bundles collected from male monarch butterflies were cryopreserved in a cryoprotective medium and stored in liquid nitrogen. To determine the post-cryopreservation sperm cell viability, a subsample of preserved sperm bundles were thawed rapidly, and their viability was qualified using a sperm live/dead stain. We are presenting a protocol to preserve and store genetic material and viable sperm bundles of the monarch butterfly. To date, this is the first report of successful cryopreservation of monarch germplasm which sets the foundation for cryostorage and could be extensible to other vulnerable lepidopterans.

Thermal biology and overwintering behavior of the red sunflower seed weevil (Coleoptera: Curculionidae).

Most natural mortality of the red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), occurs while larvae overwinter in the soil. To test the hypothesis that S. fulvus mortality is related to low temperatures, experiments were used to (i) evaluate the temperature at which larvae freeze (= supercooling point [SCP]), (ii) assess possible vertical movement between entry into the soil in fall and adult emergence in summer, and (iii) determine if realistic soil temperatures could explain patterns of overwintering mortality. Mean SCP for groups of S. fulvus larvae differed between years and months, but only ranged from -20.93 to -22.68 °C. Most overwintering larvae were found within 6 cm of the soil surface, but larvae appeared to move 1-2 cm deeper between pairs of successive sample dates (September to January, January to April). Significant larval mortality that occurred between January and April 2021 was tentatively attributed to a period in February where daily minimum soil temperatures ranged from -8 to -12 °C. When overwintering under control conditions (constant 4 °C) was interrupted with week-long exposure to -4, -8, or -12 °C in a cold bath, significant S. fulvus mortality was seen for temperatures at or below -8 °C. Combined results suggest that mortality of overwintering S. fulvus is likely caused by continuous exposure to low temperatures that may not be cold enough to freeze larvae. Additionally, the shallow overwintering by S. fulvus supports the idea that routine farm management, including tillage and herbicide incorporation, may help limit populations of this sunflower pest.

Sperm can't take the heat: Short-term temperature exposures compromise fertility of male bumble bees (Bombus impatiens).

Bumble bee (genus Bombus) populations are increasingly under threat from habitat fragmentation, pesticides, pathogens, and climate change. Climate change is likely a prime driver of bumble bee declines but the mechanisms by which changing climates alter local abundance, leading to shifts in geographic range are unclear. Heat tolerance is quite high in worker bumble bees (CTmax âˆ¼ 48-55 °C), making it unlikely for them to experience these high temperatures, even with climate warming. However, the thermal tolerance of whole organisms often exceeds that of their gametes; many insects can be sterilized by exposure to temperatures well below their upper thermal tolerance. Male bumble bees are independent from the colony and may encounter more frequent temperature extremes, but whether these exposures compromise spermatozoa is still unclear. Using commercially-reared Bombus impatiens colonies, males were reared in the lab and spermatozoa were exposed (in vivo and isolated in vitro) to sublethal temperatures near lower and upper thermal tolerance (CTmin and CTmax, respectively). Heat exposure (45 °C for up to 85 min) reduced spermatozoa viability both for whole males (in vivo; control = 79.5 %, heat exposed = 58 %, heat stupor = 57.7 %) and isolated seminal vesicles (in vitro; control = 85.5 %, heat exposed = 62.9 %). Whole males exposed to 4 °C for 85 min (in vivo; control = 79.2 %, cold = 72.4 %), isolated seminal vesicles exposed to 4 °C for 85 min (in vitro; control = 85.5 %, cold = 85.1 %), and whole males exposed to for 4 °C for 48 h (in vivo; control = 88.7 %, cold = 84.3 %) did not differ significantly in spermatozoa viability. After<85 min at 45 °C, males had significantly reduced spermatozoa viability, suggesting that short-term heat waves below CTmax could strongly reduce the fertility of male bumble bees with potential population-level impacts.

Vitrification of Lepidopteran Embryos-A Simple Protocol to Cryopreserve the Embryos of the Sunflower Moth, Homoeosoma electellum.

Embryos of the sunflower moth, Homoeosoma electellum (Hulst), were cryopreserved after modification to the method that was previously described for Pectinophora gossipiella. The workflow to develop the protocol consisted of methods to weaken the embryonic chorion followed by the application of various methods to disrupt the sub-chorionic wax layer. These steps were necessary to render the embryos permeable to water and cryoprotectants. Initially, the embryos were incubated at 21° and 24 °C, and the development of the double pigment spots/eyespot and eclosion were tracked every two hours. The embryos at 24 °C showed eyespots as early as 30 h, while in the case of the embryos that were incubated at 21 °C, there was a developmental delay of approximately 20 h. The embryos at 24 °C showed peak eclosion between 55 and 70 h, and the embryos at 21 °C eclosed between 80 and 100 h of development. Estimating this range is crucial for the purposes of stage selection and treatment initiation for cryopreservation protocol development for the embryos. The control hatch percentage at either developmental temperature was >90%, and the sodium hypochloride, 2-propanol and alkane-based treatments reduced the embryo hatchability to <10%. Hence, a modified surfactant-hypochlorite mixture-was used to destabilize the chorion and solubilize the hydrophobic lipid layers. Water permeability assessments using the dye-uptake method show that polysorbate 80 in combination with sodium hypochlorite alone is capable of permeabilizing the embryo as efficiently as sequential hypochlorite-alkane treatments, but with significantly higher hatch rates. A vitrification medium consisting of ethane diol and trehalose was used to dehydrate and load the embryos with the cryoprotective agent. The median hatch rates after vitrification were 10%, and maximum was 23%.

Vitrification of manually stage-selected embryos of Drosophila have significantly higher survival and emergence - Consequences for insect germplasm storage.

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 and >72% 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.

Cryopreservation of seminal vesicle derived spermatozoa from Bombus impatiens and Apis mellifera - Implications for artificial insemination of bumble bees.

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.

Comparison of Fluctuating Thermal Regimes and Commercially Achievable Constant-Temperature Regimes for Short-Term Storage of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae).

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.

Physiological responses to cryoprotectant treatment in an early larval stage of the malaria mosquito, Anopheles gambiae.

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.

A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization.

A non-activating semen diluent does not cause motility or acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of honey bee spermatozoa stored in ambient conditions were assessed 60 days pre-cryopreservation and 24 h post-cryopreservation. Seven variations of a Tris-based non-activating diluents (FEM1 - FEM7) were compared to samples treated with conventional activating diluent and untreated semen. Semen viability (membrane integrity) was assessed after short- and long-term storage at 14.0 ± 0.2 °C. The non-activating medium FEM7 contained more viable spermatozoa than the activating medium, 24 h after cryopreservation (67.6 ± 10.9% and ~4%, respectively). After 60 days, 22.0 ± 7.8% of spermatozoa was viable in non-activating medium versus 0.0 and 60.8 ± 12.3%, in conventional media and untreated controls, respectively. Hence FEM7 was used to cryopreserve bee semen and subsequently inseminate honey bee queens. The quality of brood produced by the queens was assessed 30-60 days after insemination. The percentage of worker-bee offspring (produced from successfully fertilized eggs) was ~75% for both the non-activating medium and the conventional extender medium. Our results indicate that a non-activating medium possesses significant advantage over the conventional activating medium if the semen requires storage after treatments such as cryopreservation. The percentage of female offspring (from fertilized eggs) produced by queens inseminated with semen diluted in either the activating or non-activating medium did not differ from one another.

Thermoprofile Parameters Affect Survival of Megachile rotundata During Exposure to Low-Temperatures.

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).

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata.

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.

Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae).

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.

The geometric framework for nutrition reveals interactions between protein and carbohydrate during larval growth in honey bees.

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.

Cryopreservation of Embryos of the Mediterranean Fruit Fly Ceratitis capitata Vienna 8 Genetic Sexing Strain.

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.

Stage selection and restricted oviposition period improves cryopreservation of dipteran embryos.

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 <2% for 15 and 60 min samples, respectively. Significantly higher hatching rates were obtained for cryopreservation after manual selection of specific embryonic developmental stages from the dechorionated samples. Post-cryopreservation hatching rate for stage-selected M. domestica embryos was 86.5 ± 5.5% compared to 33.3 ± 4.5% for embryos staged only by an overall visual confirmation. In the case of L. sericata, the hatching percentage was 79.0 ± 11.1 for stage-selected embryos compared to 17.0 ± 3.9% without individual selection.

Cryopreservation of embryos of Lucilia sericata (Diptera: Calliphoridae).

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 for > 10 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 for < 12 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.

Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae).

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%.

Synchrotron x-ray visualisation of ice formation in insects during lethal and non-lethal freezing.

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.

Short-term hardening effects on survival of acute and chronic cold exposure by Drosophila melanogaster larvae.

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.

Slow and stepped re-warming after acute low temperature exposure do not improve survival of Drosophila melanogaster larvae.

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