Telomere length is longer following diapause in two solitary bee species.

The mechanisms that underlie senescence are not well understood in insects. Telomeres are conserved repetitive sequences at chromosome ends that protect DNA during replication. In many vertebrates, telomeres shorten during cell division and in response to stress and are often used as a cellular marker of senescence. However, little is known about telomere dynamics across the lifespan in invertebrates. We measured telomere length in larvae, prepupae, pupae, and adults of two species of solitary bees, Osmia lignaria and Megachile rotundata. Contrary to our predictions, telomere length was longer in later developmental stages in both O. lignaria and M. rotundata. Longer telomeres occurred after emergence from diapause, which is a physiological state with increased tolerance to stress. In O. lignaria, telomeres were longer in adults when they emerged following diapause. In M. rotundata, telomeres were longer in the pupal stage and subsequent adult stage, which occurs after prepupal diapause. In both species, telomere length did not change during the 8 months of diapause. Telomere length did not differ by mass similarly across species or sex. We also did not see a difference in telomere length after adult O. lignaria were exposed to a nutritional stress, nor did length change during their adult lifespan. Taken together, these results suggest that telomere dynamics in solitary bees differ from what is commonly reported in vertebrates and suggest that insect diapause may influence telomere dynamics.

Metabolic and transcriptomic characterization of summer and winter dormancy in the solitary bee, Osmia lignaria.

The solitary bee Osmia lignaria is a native pollinator in North America with growing economic importance. The life cycle of O. lignaria provides a unique opportunity to compare the physiological and molecular mechanisms underlying two ecologically contrasting dormancies within the same species. O. lignaria prepupae become dormant during the summer to avoid high temperatures. Shortly after adult eclosion, they enter a second dormancy and overwinter as diapausing adults. To compare these two dormancies, we measured metabolic rates and gene expression across development as bees initiate, maintain, and terminate both prepupal (summer) and adult (overwintering) dormancies. We observed a moderate temperature-independent decrease in gas exchange during both the prepupal dormancy after cocoon spinning (45 %) and during adult diapause after eclosion (60 %). We sequenced and assembled a high-quality reference genome from a single haploid male bee with a contiguous n50 of 5.5 Mbp to facilitate our transcriptomic analysis. The transcriptomes of dormant prepupae and diapausing adults clustered into distinct groups more closely associated with life stage than dormancy status. Membrane transport, membrane-bound cellular components, oxidoreductase activity, glutathione metabolism, and transcription factor activity increased during adult diapause, relative to prepupal dormancy. Further, the transcriptomes of adults in diapause clustered into two groups, supporting multiple phases of diapause during winter. Late adult diapause was associated with gene expression profiles supporting increased insulin/IGF, juvenile hormone, and ecdysone signaling.

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.

Effects of temperature on metabolic rate during metamorphosis in the alfalfa leafcutting bee.

Spring conditions, especially in temperate regions, may fluctuate abruptly and drastically. Environmental variability can expose organisms to temperatures outside of their optimal thermal ranges. For ectotherms, sudden changes in temperature may cause short- and long-term physiological effects, including changes in respiration, morphology, and reproduction. Exposure to variable temperatures during active development, which is likely to occur for insects developing in spring, can cause detrimental effects. Using the alfalfa leafcutting bee, Megachile rotundata, we aimed to determine if oxygen consumption could be measured using a new system and to test the hypothesis that female and male M. rotundata have a thermal performance curve with a wide optimal range. Oxygen consumption of M. rotundata pupae was measured across a large range of temperatures (6-48°C) using an optical oxygen sensor in a closed respirometry system. Absolute and mass-specific metabolic rates were calculated and compared between bees that were extracted from their brood cells and those remaining in the brood cell to determine whether pupae could be accurately measured inside their brood cells. The metabolic response to temperature was non-linear, which is an assumption of a thermal performance curve; however, the predicted negative slope at higher temperatures was not observed. Despite sexual dimorphism in body mass, sex differences only occurred in mass-specific metabolic rates. Higher metabolic rates in males may be attributed to faster development times, which could explain why there were no differences in absolute metabolic rate measurements. Understanding the physiological and ecological effects of thermal environmental variability on M. rotundata will help to better predict their response to climate change.

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.

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 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%.

Effects of Temperature and Wildflower Strips on Survival and Macronutrient Stores of the Alfalfa Leafcutting Bee (Hymenoptera: Megachilidae) Under Extended Cold Storage.

Megachile rotundata (F.) is an important pollinator of alfalfa in the United States. Enhancing landscapes with wildflowers is a primary strategy for conserving pollinators and may improve the sustainability of M. rotundata. Changing cold storage temperatures from a traditionally static thermal regime (STR) to a fluctuating thermal regime (FTR) improves overwintering success and extends M. rotundata's shelf life and pollination window. Whether floral resources enhance overwintering survival and/or interact with a thermal regime are unknown. We tested the combined effects of enhancing alfalfa fields with wildflowers and thermal regime on survival and macronutrient stores under extended cold storage (i.e., beyond one season). Megachile rotundata adults were released in alfalfa plots with and without wildflower strips. Completed nests were harvested in September and stored in STR. After a year, cells were randomly assigned to remain in STR for 6 months or in FTR for a year of extended cold storage; emergence rates were observed monthly. Macronutrient levels of emerged females were assessed. FTR improved M. rotundata survival but there was no measurable effect of wildflower strips on overwintering success or nutrient stores. Timing of nest establishment emerged as a key factor: offspring produced late in the season had lower winter survival and dry body mass. Sugars and glycogen stores increased under FTR but not STR. Trehalose levels were similar across treatments. Total lipid stores depleted faster under FTR. While wildflowers did not improve M. rotundata survival, our findings provide mechanistic insight into benefits and potential costs of FTR for this important pollinator.

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.

Environmental impacts on diapause and survival of the alfalfa leafcutting bee, Megachile rotundata.

Megachile rotundata exhibits a facultative prepupal diapause but the cues regulating diapause initiation are not well understood. Possible cues include daylength and temperature. Megachile rotundata females experience changing daylengths over the nesting season that may influence diapause incidence in their offspring through a maternal effect. Juvenile M. rotundata spend their developmental period confined in a nesting cavity, potentially subjected to stressful temperatures that may affect diapause incidence and survival. To estimate the impact of daylength and nest cavity temperature on offspring diapause, we designed a 3D printed box with iButtons that measured nest cavity temperature. We observed nest building throughout the season, monitored nest cavity temperature, and followed offspring through development to measure diapause incidence and mortality. We found that daylength was a cue for diapause, and nest cavity temperature did not influence diapause incidence. Eggs laid during long days had a lower probability of diapause. Siblings tended to have the same diapause status, explaining a lot of the remaining variance in diapause incidence. Some females established nests that contained both diapausing and nondiapausing individuals, which were distributed throughout the nest. Nest cavities reached stressful temperatures, which decreased survival. Mortality was significantly higher in nondiapausing bees and the individuals that were laid first in the nest. In conclusion, we demonstrate a maternal effect for diapause that is mediated by daylength and is independent of nest box temperature.

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.

Size constrains oxygen delivery capacity within but not between bumble bee castes.

Bumble bees are eusocial, with distinct worker and queen castes that vary strikingly in size and life-history. The smaller workers rely on energetically-demanding foraging flights to collect resources for rearing brood. Queens can be 3 to 4 times larger than workers, flying only for short periods in fall and again in spring after overwintering underground. These differences between castes in size and life history may be reflected in hypoxia tolerance. When oxygen demand exceeds supply, oxygen delivery to the tissues can be compromised. Previous work revealed hypermetric scaling of tracheal system volume of worker bumble bees (Bombus impatiens); larger workers had much larger tracheal volumes, likely to facilitate oxygen delivery over longer distances. Despite their much larger size, queens had relatively small tracheal volumes, potentially limiting their ability to deliver oxygen and reducing their ability to respond to hypoxia. However, these morphological measurements only indirectly point to differences in respiratory capacity. To directly assess size- and caste-related differences in tolerance to low oxygen, we measured critical PO2 (Pcrit; the ambient oxygen level below which metabolism cannot be maintained) during both rest and flight of worker and queen bumble bees. Queens and workers had similar Pcrit values during both rest and flight. However, during flight in oxygen levels near the Pcrit, mass-specific metabolic rates declined precipitously with mass both across and within castes, suggesting strong size limitations on oxygen delivery, but only during extreme conditions, when demand is high and supply is low. Together, these data suggest that the comparatively small tracheal systems of queen bumble bees do not limit their ability to deliver oxygen except in extreme conditions; they pay little cost for filling body space with eggs rather than tracheal structures.

Body size allometry impacts flight-related morphology and metabolic rates in the solitary bee Megachile rotundata.

Body size is related to many aspects of life history, including foraging distance and pollination efficiency. In solitary bees, manipulating the amount of larval diet produces intraspecific differences in adult body size. The goal of this study was to determine how body size impacts metabolic rates, allometry, and flight-related morphometrics in the alfalfa leafcutting bee, Megachile rotundata. By restricting or providing excess food, we produced a range of body sizes, which allowed us to test the effect of body size on allometry, the power required for flight, and amount of energy produced, as measured indirectly through CO2 emission. The power required during flight was predicted using the flight biomechanical formulas for wing loading and excess power index. We found larger bees had higher absolute metabolic rates at rest and during flight, but smaller bees had higher mass-specific metabolic rates at rest. During flight, bees did not have size-related differences in mass-specific metabolic rate. As bees increase in size, their thorax and abdomens become disproportionately larger, while their wings (area, and length) become disproportionately smaller. Smaller bees had more power available during flight as demonstrated by flight biomechanical formulas. Smaller body size was advantageous because of a reduced power requirement for flight with no metabolic cost.

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.

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.

Microclimate Temperatures Impact Nesting Preference in Megachile rotundata (Hymenoptera: Megachilidae).

The temperature of the nest influences fitness in cavity-nesting bees. Females may choose nest cavities that mitigate their offspring's exposure to stressful temperatures. This study aims to understand how cavity temperature impacts the nesting preference of the solitary bee Megachile rotundata (Fabricius) under field conditions. We designed and 3D printed nest boxes that measured the temperatures of 432 cavities. Nest boxes were four-sided with cavity entrances facing northeast, northwest, southeast, and southwest. Nest boxes were placed along an alfalfa field in Fargo, ND and were observed daily for completed nests. Our study found that cavity temperature varied by direction the cavity faced and by the position of the cavity within the nest box. The southwest sides recorded the highest maximum temperatures while the northeast sides recorded the lowest maximum temperatures. Nesting females filled cavities on the north-facing sides faster than cavities on the south-facing sides. The bees preferred to nest in cavities with lower average temperatures during foraging hours, and cavities that faced to the north. The direction the cavity faced was associated with the number of offspring per nest. The southwest-facing cavities had fewer offspring than nests on the northeast side. Our study indicates that the nesting box acts as a microclimate, with temperature varying by position and direction of the cavity. Variation in cavity temperature affected where females chose to nest, but not their reproductive investment.

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.