Germ Cell Isolation and Cryopreservation from Reproductive Organs of Brown Mealworm

SIMPLE SUMMARY: Long-term breeding of mealworms leads to in inbreeding weakness, such as decreased egg production, decreased survival rate, and increased growth period. In this study, we isolated and freezed germ cells from the superior brown mealworm. Male and female adult brown mealworms were distinguished according to the presence or absence of protrusion at the tip of the five segments from the abdomen. We observed sperm and ovarioles in anatomically isolated testes and ovaries. The mechanical and enzymatic (collagenase I) methods were used to isolate ovarioles from adult female brown mealworms. In the enzymatic method, most were torn and burst as the membrane of the ovarioles was damaged by collagenase I, unlike the mechanical method. We also cryopreserved the ovaries of female brown mealworms using slow freezing and vitrification. We found that the yolk sac was completely damaged in the ovaries after slow freezing. However, only partial damage was achieved in the vitrification group compared to the control group (no freezing). The newly developed vitrification method with alginate-encapsulated ovarioles maintained the yolk sac in the ovarioles but was evenly distributed. This study provides basic data for reproductive studies of other useful insects. ABSTRACT: This study aimed to isolate and freeze germ cells from the superior brown mealworm. Styrofoam diet changes were observed for 20 days to determine whether mealworms were useful insects for decomposing Styrofoam. The average weight of mealworms before the Styrofoam diet was 500 mg, which decreased to 336 mg at D20 after their diet. To preserve mealworms with excellent Styrofoam-degrading ability, we first isolated the reproductive organs of mealworms, testes, ovaries, sperms, and ovarioles. Morphologically, male and female adult brown mealworms were distinguished according to the presence or absence of a protrusion at the tip of the fifth segment of the abdomen. Sperms and ovarioles were observed in anatomically isolated testes and ovaries. We compared mechanical and enzymatic (collagenase I) methods to effectively isolate ovarioles from adult female brown mealworms. For the enzymatic method, most were torn and burst as the membrane of the ovarioles was damaged by collagenase I, unlike the mechanical method. To preserve the superior genetic resources of mealworms, we cryopreserved the ovaries of female brown mealworms using slow-freezing and vitrification. Histological analysis showed that the yolk sac was completely damaged in the ovaries after slow-freezing. However, only partial damage was achieved in the vitrification group compared to the control group (no freezing). The newly developed vitrification method with alginate-encapsulated ovarioles maintained the yolk sac in the ovarioles but was evenly distributed. These results provide basic data for reproductive studies of other useful insects and contribute to the biobanking and fertility preservation of superior mealworm germ cells and endangered insects.