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

Estudando o desenvolvimento de Delia antiqua axênica e garantindo o crescimento livre de bactérias

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.

Autor em destaque: criação axênica de Delia antiqua para detectar o impacto dos micróbios intestinais em seu crescimento e desenvolvimento 

Criação de Delia antiqua axênica com dietas estéreis semi-fermentadas

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  

Coleta de Ovos Axênicos e Criação de Larvas de Delia antiqua Axênicas em Dietas Estéril Semifermentadas

Depois de criar machos e fêmeas adultos Delia antiqua, remova o outro dispositivo de posição da gaiola. Despeje a areia e o dente de alho em um copo de 500 mililitros. Usando água da torneira, lave os ovos restantes do alho no copo.Umedeça uma peneira de 100 malhas com água da torneira e despeje a água contendo ovos flutuantes do copo na peneira. Depois que os ovos estiverem secos, use um pincel para varre-los suavemente em uma placa de Petri. Coloque os ovos coletados em uma peneira de células estéreis.Prepare duas placas de Petri, uma com 30 mililitros de hipoclorito de sódio a 0,26% e outra com 30 mililitros de etanol 75%. Mergulhe a peneira celular com ovos na solução de hipoclorito de sódio por 0,5 minutos. Em seguida, transfira para o etanol por mais 0,5 minutos.Usando uma tesoura estéril, corte a ponta de uma pipeta de um mililitro. Em seguida, usando a pipeta da ponta da extremidade cortada, transfira os ovos do etanol para um tubo de 50 mililitros contendo díades. Depois de remover o excesso de etanol do tubo, feche a tampa e coloque o tubo em um saco auto-selante.Coloque um pedaço de algodão na abertura do saco para circulação de ar e coloque o saco em uma incubadora de 25 graus Celsius. Os estágios completos de vida de D.antiqua axênico compreendem ovos, larvas, pupas e adultos. Não houve diferença significativa no tempo de desenvolvimento entre Delia antiqua criada axenicamente e normalmente criada.

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Research

JoVE Journal

Biology

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

57 visualizações.  Qilu University of Technology (Shandong Academy of Sciences). Depois de criar machos e fêmeas adultos Delia antiqua, remova o outro dispositivo de posição da gaiola. Despeje a areia e o dente de alho em um copo de 500 mililitros. Usando água da torneira, lave os ovos restantes do alho no copo.Umedeça uma peneira de 100 malhas com água da torneira e despeje a água contendo ovos flutuantes do copo na peneira. Depois que os ovos estiverem secos, use um pincel para varre-los suavemente em uma placa de Petri. Coloque os ovos coletados em uma pe...

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

Rearing Axenic Delia antiqua with Half-Fermented Sterile Diets

Axenic insects are obtained from sterile artificial rearing systems using sterile media. These insects, characterized by their small size, short growth cycle, and low feed requirements, are ideal for studying the relationship between microorganisms and hosts. The gut microbiota significantly influences the physiological characteristics of insect hosts, and introducing specific strains into axenic insects provides a method for verifying gut microbial functions. Delia antiqua, a threatening pest in the order Diptera, family Anthomyiidae, and genus Delia, primarily feeds on onions, garlic, leeks, and other vegetables of the family Liliaceae. Its larvae feed on the bulbs, causing rotting, wilting, and even death of entire plants. By rearing axenic larvae, follow-up studies can be conducted to observe the effects of intestinal microflora on the growth and development of D. antiqua. Unlike the method involving antibiotic elimination of associated microbes, this article presents a low-cost and high-efficiency approach to raising axenic D. antiqua. After surface sterilization of D. antiqua eggs, half-fermented sterile diets were used to raise larvae, and the axenic state of D. antiqua was verified through culture-dependent and culture-independent assays. In conclusion, the combination of insect egg sterilization and the preparation of sterile diets for larval culture has enabled the development of an efficient and simple method for obtaining axenic D. antiqua. This method provides a powerful approach to studying insect-microflora interactions.

A simple procedure for rearing axenic Delia antiqua with half-fermented sterile diets is described. Only one Wolbachia strain was detected in each instar of axenic D. antiqua using PCR.

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

Biologia

Preparation of Sterile Diets Incorporating Scallions and Insect Larvae for the Rearing of Delia antiqua

Preparation of Sterile Diets Incorporating Scallions and Insect Larvae for the Rearing of Delia antiqua  

To begin, peel off the outer layers of the scallions and discard the green leaves. Sterilize scissors with 75%ethanol, and cut the white part of the scallions into one to two centimeter diced pieces. Weigh 50 grams of diced scallions and place them into a food processor.Add 50 milliliters of sterilized water into a food processor and grind the scallions five times for two minutes each. Using tweezers, transfer ten thirds instar larvae of Delia Antigua into a 10 milliliter centrifuge tube containing 10 milliliters of PBS. Grind the larvae with a disposable grinding pestle.Pour the pasty scallion liquid and larval grinding liquid into a 500 milliliter conical flask. Wrap the flask mouth with two layers of transparent ceiling film and place the flask in a shaking incubator for 12 hours. Cut out seven layers of square gauze with 10 centimeter sides and stack them together to form a simple filter device.After incubation, slowly pour the fermented scallion liquid onto the gauze, collecting the filtrate into a 500 milliliter flask. Place the wet filter paper on the Buckner funnel. Then pour the filtrate and perform suction filtration three times with different filter paper using a vacuum pump.Perform another filtration using a 0.22 micron filter bottle and a vacuum pump. After filtration, screw on the cover of the suction filter bottle. Add 0.24 grams of choline chloride and 0.56 grams of L-ascorbic acid in a separate 10 milliliter centrifuge tube.Then add three milliliters of deionized water and shake the tubes vigorously. Sterilize the solutions using a five milliliter syringe and three 0.22 micron syringe filters in 10 milliliter tubes. Add two grams of agri powder and six grams of TSB powder in a 500 milliliter conical flask.Then add 100 milliliters of deionized water and stir the mixture using a glass rod until well mixed. Seal the flask using two layers of transparent sealing film. Wrap the fermented scallions and unfermented scallions in aluminum foil.Sterilize the scallions and culture medium in an autoclave at 121 degrees Celsius for 20 minutes. While gently swirling the flask, pour the sterilized choline chloride, L-ascorbic acid solution, and filtrate into the culture medium. Add fermented and unfermented scallions into the culture medium and shake vigorously.Finally, pour the diets into the 50 milliliter centrifuge tubes.

Preparação de dietas estéreis incorporando cebolinha e larvas de insetos para a criação de Delia antiqua

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.