Name: Collecting Axenic Eggs and Rearing of Axenic Delia antiqua Larvae on Half-Fermented Sterile Diets
Uploaded: 2023-12-22T14:55:00
Description: Watch this Scientific Journal Video about Collecting Axenic Eggs and Rearing of Axenic Delia antiqua Larvae on Half-Fermented Sterile Diets at JoVE.com

collecting axenic eggs and rearing of axenic delia antiqua larvae on half-fermented sterile diets

Studying Development of Axenic Delia antiqua and Ensuring Bacteria-Free Growth

Axenic animals, defined as animals in which no viable microorganisms or parasites can be detected, are valuable experimental models for studying host-microorganism interactions1,2. Insects, the largest group of invertebrates, can form symbiotic relationships with microorganisms3. Axenic insects can be used to study host-symbiont interactions in symbiotic systems4. For example, Nishide et al.5 established a practical sterile rearing procedure for the malodor worm Plautia stali, enabling reliable and rigorous analysis of host-symbiont interactions in model symbiotic systems. Axenic insects can be produced by sterilizing the egg stage and providing sterile food to the larvae and adults6,7. Axenic insects are of great significance and are widely used in biological research. For instance, a study conducted by Somerville et al.8 demonstrated that diamondback moths inoculated with Enterobacter cloacae improved the adaptability of transgenic males.
Delia antiqua Meigen is an economically important pest of onions and other Liliaceae crops worldwide, with its larvae damaging the bulbs of onions and other Liliaceae crops9. D. antiqua is mainly found in temperate climates and is widespread in onion-cultivation areas of the Americas, Europe, and Asia. If not properly controlled, it can cause crop losses in onions (Allium cepa L.), garlic (Allium sativum L.), shallots (Allium fistulosum L.), and leeks (Alliumchoenoprasum L.) ranging from 50% to 100%10,11. The larvae feed on the below-ground parts of plants, and this feeding causes the seedlings to wilt and eventually die. In addition, damaged plants can allow pathogens to enter, leading to bulb rotting12. Even if the plants are not completely consumed by the larvae, the damage they cause renders the onion plants unmarketable and results in economic losses.
Insects are closely associated with gut microbiota, and most insect guts contain a variety of symbiotic bacteria that thrive on the nutrients provided by the host13,14. Jing et al.15 showed that the primary function of the intestinal symbiotic community is to provide essential nutrients, followed by functions related to digestion and detoxification. In certain cases, gut bacteria can serve as a microbial resource for pest management purposes. Consequently, studying the individual gut bacteria&#39;s performances and specific functions within the body of D. antiqua is desirable. Therefore, preparing axenic larvae is particularly important for studying the interactions between specific bacterial strains and insects16. Currently, a commonly used method to eliminate insect gut bacteria is the use of an antibiotic combination to eradicate associated microbes17,18,19. Unlike using antibiotics alone, which can only reduce microbial numbers, axenic rearing of insects allows for control over the composition and quantity of microorganisms, enabling more accurate validation of gut microbiota functionality.
Thus, this article introduces a protocol for preparing and rearing axenic D. antiqua. Axenic larval food is obtained by utilizing high-temperature sterilization of natural diets combined with half-fermented foods. The eggs are sterilized following an experimental protocol to obtain axenic eggs, and finally, axenic larvae are cultured from the axenic eggs. The axenic rearing system was carried out for only one generation for the experiment. This will provide convenience for studying the interaction between insects and gut microbiota.

Author Spotlight: Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development 

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets

Our field of research is microbial ecology, and we want to explore inside microbial interactions by rearing axenic Delia antiqua. Questions will be answered on the diversity of intestinal fungi of Delia antiqua and their influence on the growth and development of Delia antiqua. A simple method for rearing of axenic Delia antiqua with half-fermented sterile diets has been established and only one identified Wolbachia strain was detected in each instar of Delia antiqua with PCR.In contrast to antibiotic treatments, this protocol completely eliminates gut bacteria and doesn't cause many side effects to the host insects. The function of the insect gut microbiota is also not marked. It is more effective and affordable than antibiotic treatment.

Watch this Scientific Journal Video about Collecting Axenic Eggs and Rearing of Axenic Delia antiqua Larvae on Half-Fermented Sterile Diets at JoVE.com

Collecting Axenic Eggs and Rearing of Axenic Delia antiqua Larvae on Half-Fermented Sterile Diets  

Collecting Axenic Eggs and Rearing of Axenic Delia antiqua Larvae on Half-Fermented Sterile Diets

After rearing male and female Delia antiqua adults, remove the other position device from the cage. Pour the sand and garlic clove into a 500 milliliter beaker. Using tap water, flush the remaining eggs from the garlic into the beaker.Moisten a 100 meshed sieve with tap water, and pour the water containing floating eggs from the beaker into the sieve. Once the eggs are dried, use a brush to gently sweep them into a Petri dish. Place the collected eggs onto a sterile cell sieve.Prepare two Petri dishes, one with 30 milliliters of 0.26%sodium hypochlorite, and another with 30 milliliters of 75%ethanol. Soak the cell sieve with eggs in the sodium hypochlorite solution for 0.5 minutes. Then transfer it to the ethanol for another 0.5 minutes.Using a pair of sterile scissors, cut the tip of a one milliliter pipette. Then using the cut end tip pipette, transfer the eggs from the ethanol to a 50 milliliter tube containing dyads. After removing the excess ethanol from the tube, close the lid and place the tube in a self-sealing bag.Place a cotton piece at the opening of the bag for air circulation, and place the bag in a 25 degree Celsius incubator. The complete life stages of axenic D.antiqua comprise eggs, larvae, pupae, and adults. No significant difference in developmental time between axenically reared and normally reared Delia antiqua was observed.

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Biology

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth ...