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Abstract 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.

IntroductionWarming summer temperatures have the potential to harm managed pollinators, impacting both summer performance and overwintering success. The alfalfa leafcutting bee, Megachile rotundata, is a solitary bee used for commercial pollination of alfalfa. M. rotundata undergoes facultative diapause in the prepupal stage. Prepupae that diapause early in the season are exposed to warm temperatures for a longer period of time than the individuals that start diapause closer to fall, which may reduce lipid reserves required for overwintering survival. Warm temperatures may also contribute to pollen ball incidence, which is when a provision is present but there is no sign of a larva in the brood cell. Our goal was to identify factors that regulate diapause and pollen ball incidence and examine effects of pre-wintering field conditions on post-overwintering energy reserves in M. rotundata.MethodsNest boxes were installed near Fargo, ND, Laramie, WY, and Las Cruces, NM, which exposed bees to different photoperiods and thermal regimes. Three nest boxes were placed at each site. We monitored nesting conditions and diapause and pollen ball incidence throughout the season. Lipids, sugars, and glycogen reserves were measured in adults after overwintering.Results and DiscussionOur models indicate that most of the variation in diapause incidence was explained by nest, with individuals within a nest tending to have the same diapause outcome. This suggests that the environmental conditions experienced by the mother, or genetic predisposition, influences offspring diapause. We also found evidence that high cavity temperatures can cause diapause aversion. In addition, our study is the first to link high nesting cavity temperatures to increased pollen ball incidence. Exposure to stressful temperatures during development and early diapause resulted in an increase in adult lipid reserves after overwintering. Adult sugar and glycogen reserves were not affected by exposure to warm temperatures during development and early diapause. In conclusion, maternal effects and temperature were important factors for diapause and pollen ball incidence in M. rotundata with macronutrient reserves similar for early and late season bees.

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

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.

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.

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.