Cryoconservation des embryons de l’abeille domestique, Apis mellifera

The honeybee, Apis mellifera, is considered worldwide as one of the most vital species. However,nowadays, a lot of different threats decline their number. The objective of this study is to cryopreservehoneybee embryos in order to preserve its genetic material. To achieve this aim, a lot of steps are to betaken. First, the study of the internal and external structure of the embryos on at 7h-17h maximal agewas performed by confocal microscopy and epi-fluorescence after staining. Secondly, thedechorionation tests and permeabilization of membranes were conducted. A dechorionation on of 17hold embryos was successfully performed with a bleach treatment of 0.3% for 30 seconds and asonication for 15 seconds. Among the 4 permeabilizing agents tested, an enzyme (pronase), twodetergents (saponin and triton) and a solvent (pure heptane), only triton at 0,5% for 3 minutes andheptane for 5 seconds showed positive results on embryos of 17h maximal age. In addition, 0.5%triton for 3 minutes, coupled with bleach or sonication, yielded the best hatching rate for in vitroembryos of 17h maximal age, on average 10%, against only 1% with heptane. The next step is theembryo cryopreservation using vitrification technique.L’abeille domestique, Apis mellifera, est considérée mondialement comme l’une des espèces les plusindispensables à la survie des êtres-vivants. Or de nombreuses menaces font diminuer leur effectif.L’objectif de ce projet est de réussir à cryoconserver des embryons d’abeille domestique afin depréserver son matériel génétique. Pour atteindre cet objectif, de nombreuses étapes sont à franchir.Dans un premier temps, l’étude de la structure interne et externe des embryons de 7h et 17h d’âge aumaximum a été réalisée en microscopie confocale et en épi-fluorescence après coloration. Dans unsecond temps, des tests de déchorionisation et de perméabilisation des membranes de l’embryon ontété réalisés. Un traitement à l’eau de Javel 0,3% pendant 30 secondes ainsi qu’une sonication pendant15 secondes ont été validés pour déchorioniser des embryons de 17h au maximum. Parmi les 4 agentsde perméabilisation testés, une enzyme (la pronase), deux détergents (la saponine et le triton) et unsolvant (l’heptane), le triton 0,5% pendant 3 minutes et l’heptane pur pendant 5 secondes ont étévalidés sur des embryons de 17h. En outre, le triton 0,5% pendant 3 minutes, couplé à l’eau de Javelou à la sonication, a permis d’obtenir le meilleur taux d’éclosion in vitro à partir d’embryons de 17h,en moyenne 10%, contre 1% seulement avec l’heptane. La prochaine étape à réaliser sera lacryoconservation des embryons par vitrification

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis

International audienceHoneybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be avery useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chillsensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes preventpenetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo pre-paration before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization,and passages through cryoprotectant solutions.Apis mellifera ligusticaembryos were collected in artificial cellplugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubatedinvitroovernight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths(B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugsandin vitroincubation device were efficient in producing future embryos hatching. Embryos stained ruby redwith rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilizedembryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitellinemembrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that theseason is an important variability facto