This method can help answer key questions in the molecular biology and genetic spheres of Hymenoptera, such as sex determination systems. The main advantage of this technique is that we can easily induce inbreeding lines over the ant Vollenhovia Emery, which are essential for genetic mapping and the molecular status. So, this mission can provide insight into the sex determination system of a specific ant and it may also be applied to other ants and other Hymenoptera species, such as wasps.
To begin the protocol, collect V.Emeryi colonies during early summer. Transfer the ant specimens from the collected branches to an artificial plaster nest with a glass cover, using an aspirator. Maintain the colonies in the artificial nest at 25 degrees C, under a 16 to eight hour light-dark cycle.
Provide tap water with a wash bottle to keep the plaster moist every other day at the same time. Next, add about 100 micrograms of dry cricket powder wrapped in aluminum foil and a brown sugar water-filled tip to the colony, using a 20 microliter tip every other day until new reproductive ants emerge. It is important to collect micro colonies from the fields and to provide them enough food to gain new queens and males.
First, stop individuals from moving by placing colonies in a room with a controlled environment set at four degrees Celsius for 15 minutes. Remove the mid-legs of 30 worker ants using forceps under a stereoscope to distinguish the F-zero generation from the workers produced in subsequent inbreeding crosses. Then transfer the ants into a new smaller plaster nest for inbreeding crosses.
Next, add three to four larvae or pube into a plaster nest containing workers. Transfer a long winged queen and a male into a previously prepared plaster nest for inbreeding crosses. For this step, it is important to maintain experimental crossing colony at the same state as that of a normal colony.
It is difficult to induce inbreeding just by placing males and females together. Keep the colonies at 25 degrees Celsius under a 16 to eight hour light, dark cycle with food and water provided until the queen loses her wings and lays eggs. Check the experimental colony everyday under a stereo microscope.
After setting up the inbreeding crosses between the F-one offspring, eggs can be observed under a stereoscope. After the F-one queen begins to lay eggs, remove the F-one males and larvae or pube from the nest to prevent the F-one generation and the F-two generation from mixing. Keep the colonies under the same conditions until the F-two offspring emerge.
Remove one leg of an F-zero queen using forceps. Transfer the leg to a 1.5 milliliter micro tube containing 100 microliters of a chill agent. Under a stereoscope dissect a female abdomen in a glass dish filled with 300 microliters of ultra pure water using forceps.
Dissimilate the spermatica containing the sperm from mated males. Peel away the tissue of the spermatica and isolate the sperm from the tissue of the female using insect pins. Using a micro pie pad, transfer the sperm into a 1.5 milliliter micro tube containing 100 microliters of a chill agent.
Incubate the samples from the F-zero queen and sperm at 95 degrees Celsius for 20 minutes. Flash centro fuse the micro tube and store the samples at four degrees Celsius. After confirming egg production by the siv-mated F-one queen, extract the DNA of the queen using the shed wings or one mid leg and genotype.
Next, extract the DNA of an F-one male by genotyping one leg. To evaluate the fertility of the male ants from the inbreeding crosses, dissect internal reproductive organs in a glass dish with 400 microliters of PBS solution using forceps. Then remove the PBS and replace it with four percent paraformaldihyde using a micro pie pad.
Fix the tissue by incubating the samples in PFA for forty minutes in room temperature. After fixation, wash the tissues five times with 400 microliters of PBS using a micro pie pad. Dilute DAPI solution to one milligram per milliliter in PBS.
Remove the PBS and add approximately 300 microliters of the diluted DAPI staining solution to the tissue. Incubate the tissue for 15 minutes under dark conditions a room temperature. Following the incubation, wash the tissue five times with 400 microliters of PBS and transfer the tissue to the center of a glass slide using forceps.
Then mount the tissue on mounting medium containing TRITC tetramethylrhodamine. Finally observe the samples with a confocal laser scanning microscope using the 20 or 60 3x objective lenses. Microscopic images of testes of Haploid male v Emeryi show healthy fibrous tissue or sperm which are absent in Diploid male v Emeryi, which did not show healthy fibrous tissue.
The male reproductive organs of Diploid male v Emeryi did not produce sperm and their testes did not develop, suggesting that males produced in inbreeding crosses are sterile. After its development, this technique paved the way for us to explore sex myatony in a constant in Vollenhovia Emeryi for the fast timing ant species.
