Effects of developmental state on low-temperature physiology of the alfalfa leafcutting bee, Megachile rotundata.

The success of agriculture relies on healthy bees to pollinate crops. Commercially managed pollinators are often kept under temperature-controlled conditions to better control development and optimize field performance. One such pollinator, the alfalfa leafcutting bee, Megachile rotundata, is the most widely used solitary bee in agriculture. Problematically, very little is known about the thermal physiology of M. rotundata or the consequences of artificial thermal regimes used in commercial management practices. Therefore, we took a broad look at the thermal performance of M. rotundata across development and the effects of commonly used commercial thermal regimes on adult bee physiology. After the termination of diapause, we hypothesized thermal sensitivity would vary across pupal metamorphosis. Our data show that bees in the post-diapause quiescent stage were more tolerant of low temperatures compared to bees in active development. We found that commercial practices applied during development decrease the likelihood of a bee recovering from another bout of thermal stress in adulthood, thereby decreasing their resilience. Lastly, commercial regimes applied during development affected the number of days to adult emergence, but the time of day that adults emerged was unaffected. Our data demonstrate the complex interactions between bee development and thermal regimes used in management. This knowledge can help improve the commercial management of these bees by optimizing the thermal regimes used and the timing of their application to alleviate negative downstream effects on adult performance.

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.

Thermal history of alfalfa leafcutting bees affects nesting and diapause incidence.

Variable spring temperatures may expose developing insects to sublethal conditions, resulting in long-term consequences. The alfalfa leafcutting bee, Megachile rotundata, overwinters as a prepupa inside a brood cell, resuming development in spring. During these immobile stages of development, bees must tolerate unfavorable temperatures. In this study, we tested how exposure to low temperature stress during development affects subsequent reproduction and characteristics of the F1 generation. Developing male and female M. rotundata were exposed to either constant (6°C) or fluctuating (1 h day-1 at 20°C) low temperature stress for 1 week, during the pupal stage, to mimic a spring cold snap. Treated adults were marked and released into field cages, and reproductive output was compared with that of untreated control bees. Exposure to low temperatures during the pupal stage had mixed effects on reproduction and offspring characteristics. Females treated with fluctuating low temperatures were more likely to nest compared with control bees or those exposed to constant low temperature stress. Sublethal effects may have contributed to low nesting rates of bees exposed to constant low temperatures. Females from that group that were able to nest had fewer, larger offspring with high viability, suggesting a trade-off. Interestingly, offspring of bees exposed to fluctuating low temperatures were more likely to enter diapause, indicating that thermal history of parents, even during development, is an important factor in diapause determination.

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.

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.

Body and Wing Allometries Reveal Flight-Fecundity Tradeoff in Response to Larval Provisioning in Osmia lignaria (Hymenoptera: Megachilidae).

Variation in body size has important implications for physical performance and fitness. For insects, adult size and morphology are determined by larval growth and metamorphosis. Female blue orchard bees, Osmia lignaria, (Say) provision a finite quantity of food to their offspring. In this study, we asked how provision-dependent variation in size changes adult morphology. We performed a diet manipulation in which some larvae were starved in the final instar and some were given unlimited food. We examined the consequences on adult morphology in two ways. First, allometric relationships between major body regions (head, thorax, abdomen) and total body mass were measured to determine relative growth of these structures. Second, morphometrics that are critical for flight (wing area, wing loading, and extra flight power index) were quantified. Head and thorax mass had hyperallometric relationships with body size, indicating these parts become disproportionately large in adults when larvae are given copious provisions. However, abdominal mass and wing area increased hypoallometrically with body size. Thus, large adults had disproportionately lighter abdomens and smaller wing areas than smaller adults. Though both males and females followed these general patterns, allometric patterns were affected by sex. For flight metrics, small adults had reduced wing loading and an increased extra flight power index. These results suggest that diet quantity alters development in ways that affect the morphometric trait relationships in adult O. lignaria and may lead to functional differences in performance.

Fluctuating temperatures extend longevity in pupae and adult stages of the sepsid Themira biloba.

Fluctuating Thermal Regimes (FTR), where organisms are held at low temperatures with a brief, daily warm pulse, have been shown to increase longevity in adult insects and improve pupa survival while reducing sublethal effects. We used FTR to extend the longevity and thus generation time of the fly species Themira biloba (Diptera: Sepsidae). T. biloba can be maintained in continuous culture and requires an insecticide-free dung substrate for larval growth and development. Our objective was to decrease labor and consumable materials required to maintain insect species in critical scientific collections using FTR. We extended pupation time from 4 days up to 8 weeks with no increase in mortality, and mean adult longevity was increased from 12 days to 50 days. FTR is a valuable tool for reducing the investment required to maintain rare and exotic insects.

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.

Metamorphosis is induced by food absence rather than a critical weight in the solitary bee, Osmia lignaria.

Body size is an important phenotypic trait that correlates with performance and fitness. For determinate growing insects, body size variation is determined by growth rate and the mechanisms that stop growth at the end of juvenile growth. Endocrine mechanisms regulate growth cessation, and their relative timing along development shapes phenotypic variation in body size and development time. Larval insects are generally hypothesized to initiate metamorphosis once they attain a critical weight. However, the mechanisms underlying the critical weight have not been resolved even for well-studied insect species. More importantly, critical weights may or may not be generalizable across species. In this study, we characterized the developmental aspects of size regulation in the solitary bee, Osmia lignaria We demonstrate that starvation cues metamorphosis in O. lignaria and that a critical weight does not exist in this species. Larvae initiated pupation <24 h after food was absent. However, even larvae fed ad libitum eventually underwent metamorphosis, suggesting that some secondary mechanism regulates metamorphosis when provisions are not completely consumed. We show that metamorphosis could be induced by precocene treatment in the presence of food, which suggests that this decision is regulated through juvenile hormone signaling. Removing food at different larval masses produced a 10-fold difference in mass between smallest and largest adults. We discuss the implications of body size variation for insect species that are provided with a fixed quantity of provisions, including many bees which have economic value as pollinators.

Mechanisms underpinning the beneficial effects of fluctuating thermal regimes in insect cold tolerance.

Insects exposed to low temperature often have high mortality or exhibit sublethal effects. A growing number of recent studies have shown beneficial effects of exposing insects to recurrent brief warm pulses during low-temperature stress (fluctuating thermal regime, FTR). The physiological underpinnings of the beneficial effects of FTR on cold survival have been extensively studied over the past few years. Profiling with various '-omics' techniques has provided supporting evidence for different physiological responses between insects exposed to FTR and constant low temperature. Evidence from transcriptomic, metabolomic and lipidomic studies points to a system-wide loss of homeostasis at low temperature that can be counterbalanced by repair mechanisms under FTR. Although there has been considerable progress in understanding the physiological mechanisms underlying the beneficial effects of FTR, here we discuss how many areas still lack clarity, such as the precise role(s) of heat shock proteins, compatible solutes or the identification of regulators and key players involved in the observed homeostatic responses. FTR can be particularly beneficial in applied settings, such as for model insects used in research, integrated pest management and pollination services. We also explain how the application of FTR techniques in large-scale facilities may require overcoming some logistical and technical constraints. FTR definitively enhances survival at low temperature in insects, but before it can be widely used, we suggest that the possible fitness and energy costs of FTR must be explored more thoroughly. Although FTR is not ecologically relevant, similar processes may operate in settings where temperatures fluctuate naturally.

Cues for cavity nesters: investigating relevant zeitgebers for emerging leafcutting bees, Megachile rotundata.

Photoperiod is considered the universal zeitgeber, regulating physiological processes in numerous animals. However, for animals in light-restricted habitats (e.g. burrows or cavities), thermoperiod may be a more important cue. Our study tested this hypothesis in the alfalfa leafcutting bee, Megachile rotundata, which nests in cavities and undergoes development within a brood cell. We assessed the role of environmental cues (thermoperiod and photoperiod) on the process of adult emergence by examining: (1) whether those cues direct circadian rhythms, (2) which cue is more dominant and (3) how sensitive developing bees and emergence-ready adults are to cues. Although we found that 20% of light penetrates the brood cell, and bees respond to photoperiod by synchronizing emergence, thermoperiod is the dominant cue. When presented with a conflicting zeitgeber, bees entrained to the thermophase instead of the photophase. When temperature cues were removed, we observed free-running of emergence, indicating that underlying circadian mechanisms can be synchronized by daily fluctuations in temperature. We also found that emerging bees were highly sensitive to even small increases in temperature, entraining to a ramp speed of 0.33°C h-1 The response and sensitivity to temperature cues suggest that M. rotundata evolved a temperature-mediated clock to time emergence from light-restricted cavities.

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.

Physiological responses to fluctuating temperatures are characterized by distinct transcriptional profiles in a solitary bee.

Exposure to stressful low temperatures during development can result in the accumulation of deleterious physiological effects called chill injury. Metabolic imbalances, disruptions in ion homeostasis and oxidative stress contribute to the increased mortality of chill-injured insects. Interestingly, survival can be significantly increased when chill-susceptible insects are exposed to a daily warm-temperature pulse during chilling. We hypothesize that warm pulses allow for the repair of damage associated with chill injury. Here, we describe transcriptional responses during exposure to a fluctuating thermal regime, relative to constant chilled temperatures, during pupal development in the alfalfa leafcutting bee, Megachile rotundata, using a combination of RNA-seq and qPCR. Pupae were exposed to either a constant, chilled temperature of 6°C, or 6°C with a daily pulse of 20°C for 7 days. RNA-seq after experimental treatment revealed differential expression of transcripts involved in construction of cell membranes, oxidation-reduction and various metabolic processes. These mechanisms provide support for shared physiological responses to chill injury across taxa. The large number of differentially expressed transcripts observed after 7 days of treatment suggests that the initial divergence in expression profiles between the two treatments occurred upstream of the time point sampled. Additionally, the differential expression profiles observed in this study show little overlap with those differentially expressed during temperature stress in the diapause state of M. rotundata While the mechanisms governing the physiological response to low-temperature stress are shared, the specific transcripts associated with the response differ between life stages.

Transcriptional responses to fluctuating thermal regimes underpinning differences in survival in the solitary bee Megachile rotundata.

The transcriptional responses of insects to long-term, ecologically relevant temperature stress are poorly understood. Long-term exposure to low temperatures, commonly referred to as chilling, can lead to physiological effects collectively known as chill injury. Periodically increasing temperatures during long-term chilling has been shown to increase survival in many insects. However, the transcripts responsible for this increase in survival have never been characterized. Here, we present the first transcriptome-level analysis of increased longevity under fluctuating temperatures during chilling. Overwintering post-diapause quiescent alfalfa leafcutting bees (Megachile rotundata) were exposed to a constant temperature of 6°C, or 6°C with a daily fluctuation to 20°C. RNA was collected at two different time points, before and after mortality rates began to diverge between temperature treatments. Expression analysis identified differentially regulated transcripts between pairwise comparisons of both treatments and time points. Transcripts functioning in ion homeostasis, metabolic pathways and oxidative stress response were up-regulated in individuals exposed to periodic temperature fluctuations during chilling. The differential expression of these transcripts provides support for the hypotheses that fluctuating temperatures protect against chill injury by reducing oxidative stress and returning ion concentrations and metabolic function to more favorable levels. Additionally, exposure to fluctuating temperatures leads to increased expression of transcripts functioning in the immune response and neurogenesis, providing evidence for additional mechanisms associated with increased survival during chilling in M. rotundata.

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.

Nonconstant Thermal Regimes Enhance Overwintering Success and Accelerate Diapause Development for Smicronyx fulvus (Coleoptera: Curculionidae).

Recent populations of the red sunflower seed weevil, Smicronyx fulvus LeConte (Coleoptera: Curculionidae), have been inconsistent or declining, particularly in North Dakota. Consequently, research on weevil biology, including development of resistant germplasm, has been limited. To determine whether cold storage of diapausing larvae could be improved, nonconstant temperature treatments (fluctuating thermal regime [FTR] and thermoperiod [TP]) were tested versus a constant 6°C for storage up to 365 d. Both alternate temperature treatments produced more adult weevils than constant 6°C for short (42, 91 d) storage, while all temperature treatments were good (≥60% adult emergence) at moderate term (182 d) cold storage, and FTR was best for long (365 d) periods. Excluding the 14-d storage period, which produced too few weevils for comparison, each doubling of cold storage time (e.g., from 42 to 91 d, 91 to 182 d), usually decreased the number of days to 50% relative emergence by ∼10 d. After 365 d of larval storage, emerged S. fulvus adults successfully infested sunflowers in a plant growth chamber, with damage per female similar to that observed in field trials. Compared with previous efforts to store weevil larvae, the method of collection and FTR storage is either more effective (greater adult emergence and reduced parasitism) or more time-efficient, and should permit year-round research using S. fulvus adults. Because successful emergence under FTR was >75% after 365 d, additional research would be required to determine the maximum effective duration of cold storage for S. fulvus.

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.

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.

Nesting cavity diameter has implications for management of the alfalfa leafcutting bee (Hymenoptera: Megachilidae).

Body size influences performance in many bee species and may be influenced by nesting cavity diameter in cavity-nesting bees. Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae) is a commercially-managed, solitary cavity-nesting bee. In M. rotundata body size has low heritability and is strongly influenced by the size of the larval provision and the diameter of the nesting cavity. Commercial nesting boxes have cavities that are 7 mm in diameter. Our goal was to examine the effects that nesting cavity diameter has on M. rotundata body size and performance by manipulating the size of cavities that are available for nesting. We provided bees with nesting cavities that ranged in size from 4 to 9 millimeters in 1 mm increments. To assess body size we measured mass and intertegular span. To assess performance we measured wing area, wing loading, sex, overwintering survival, pollen ball occurrence, and diapause status in the offspring. We also examined the reproductive output from the different nest cavity diameters. We found that the 8 mm cavities reared bees with the largest mass, and 4 mm cavities reared bees with the smallest mass. We determined that the 7 mm nesting cavity is optimal for offspring yield, the 8 mm nesting cavity is optimal for performance, and the 5 mm nesting cavity may be optimal for conservation efforts of other cavity-nesting bees. Based on the desired outcome of the bee managers, nest sizes differing from the standard may provide an advantage.