Name: Author Spotlight: Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development
Uploaded: 2023-12-22T14:55:00
Description: Watch this Scientific Journal Video about Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development at JoVE.com

This work was supported by National Natural Science Foundation of China (32272530), the New Twenty Policies for University in Jinan Project (2021GXRC040), Major Scientific and Technological Innovation Projects in Shandong Province (2021TZXD002), and the Science and Education Integration Project of Qilu University of Technology (2022PYI009, 2022PY016, 2022PT105).

D. antiqua are obtained from the field of Fanzhen, Taian.
1. Preparation of sterile diets
<ol>
	<li>Peel off the outer layers of the scallions and discard the green leaves. Keep the white part of the scallions (Figure 1A) and wash them with sterile water, repeating the rinsing process three times. Cut the white part of the scallions into 1-2 cm diced pieces using scissors (see Table of Materials) sterilized with 75 % Et...

Studying Development of Axenic &lt;em&gt;Delia antiqua&lt;/em&gt; and Ensuring Bacteria-Free Growth

<ol>
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Z., Qi, F. H., Wang, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+dominant+roles+of+insect+gut+bacteria:+digestion,+detoxification,+or+essential+nutrient+provision.">Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision.</a> Microbiome. 8 (1), 38 (2020).</li><li>Kietz, C., Pollari, V., Meinander, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+germ-free+drosophila+to+study+gut-microbe+interactions:+protocol+to+rear+Drosophila+under+axenic+conditions.">Generating germ-free drosophila to study gut-microbe interactions: protocol to rear Drosophila under axenic conditions.</a> Current Protocols in Toxicology. 77 (1), e52 (2018).</li><li>Schretter, C. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+red+palm+weevil+Rhynchophorus+Ferrugineus+(Olivier)+(Coleoptera:+Dryophthoridae)+germ-free+larvae+for+host-gut+microbes+interaction+studies.">Preparation of red palm weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) germ-free larvae for host-gut microbes interaction studies.</a> Bio-Protocol. 9 (24), e3456 (2019).</li><li>Bavani, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterilization+of+Lucilia+sericata+(Diptera:+Calliphoridae)+Eggs+for+maggot+debridement+therapy.">Sterilization of Lucilia sericata (Diptera: Calliphoridae) Eggs for maggot debridement therapy.</a> Journal of Medical Entomology. 59 (3), 1076-1080 (2022).</li><li>Han, L. Z., Li, S. B., Liu, P. L., Peng, Y. F., Hou, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+artificial+diet+for+continuous+rearing+of+Chilo+suppressalis+(Lepidoptera:+Crambidae).">New artificial diet for continuous rearing of Chilo suppressalis (Lepidoptera: Crambidae).</a> Annals of the Entomological Society of America. 105 (2), 253-258 (2012).</li><li>Bezerra, C. E. S., Amaral, B. B., Souza, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+Chrysoperla+externa+larvae+on+artificial+diets.">Rearing Chrysoperla externa larvae on artificial diets.</a> Neotropical Entomology. 46 (1), 93-99 (2017).</li><li>Feng, H. Q., Jin, Y. L., Li, G. P., Feng, H. 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Insects possess a highly complex gut microbiota20,21, necessitating the use of axenic insects inoculated with specific gut microbial strains for studying insect-microorganism interactions. The preparation of axenic insects is crucial for such research endeavors. Antibiotic treatment is a method used to eliminate gut microbiota. For example, Jung and Kim22 fed Spodoptera exigua with penicillin, while Raymond23</su...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;M filter bottle</td>
			<td>Thermo Scientific</td>
			<td>450-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;M Syringe Filter</td>
			<td>Biosharp</td>
			<td>BS-QT-011</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mesh sieve</td>
			<td>Zhejiang Shangyu Jinding Standard Sieve Factory</td>
			<td>No Catalog numbers</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS solution</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>2x Taq PCR Master Mix</td>
			<td>GENVIEW</td>
			<td>GR1113-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5.2% NaClO solution</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>80010428</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Conical flask</td>
			<td>Thermo Scientific</td>
			<td>4103-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL vented centrifuge tube</td>
			<td>JET BIOFIL</td>
			<td>BRT-011-050</td>
			<td></td>
		</tr>
		<tr>
			<td>50x TAE buffer</td>
			<td>GENVIEW</td>
			<td>GT1307</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Ding Guo</td>
			<td>DH010-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>STIK</td>
			<td>21040121500010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sieve</td>
			<td>SAINING</td>
			<td>5022200</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline chloride</td>
			<td>Sangon Biotech</td>
			<td>A600299-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Ding Guo</td>
			<td>PER018-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable grinding pestle</td>
			<td>JET BIOFIL</td>
			<td>CSP-003-002</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA extraction kit</td>
			<td>Sangon Biotech</td>
			<td>B518221-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Marker</td>
			<td>Sangon Biotech</td>
			<td>B600335-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>NEWSTAR</td>
			<td>1087309025</td>
			<td></td>
		</tr>
		<tr>
			<td>Food processor</td>
			<td>Guangdong Midea Life Electric Appliance Manufacturing Co., Ltd.</td>
			<td>WBL25B26</td>
			<td></td>
		</tr>
		<tr>
			<td>Illuminated&#160; incubator</td>
			<td>Shanghai ESTABLISH Instrumentation Co., Ltd.</td>
			<td>A16110768</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sangon Biotech</td>
			<td>A610021-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-shaped spreader</td>
			<td>SAINING</td>
			<td>6040000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient agar medium</td>
			<td>Hope Bio</td>
			<td>HB0109</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Bing Yu</td>
			<td>&#160;BY-103</td>
			<td>Purchase on Jingdong</td>
		</tr>
		<tr>
			<td>Shock incubator</td>
			<td>Shanghai Zhichu Instrument Co., Ltd.</td>
			<td>2020000014</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>GENVIEW</td>
			<td>CS326-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Green nucleic acid dye</td>
			<td>Biosharp</td>
			<td>BS355A</td>
			<td></td>
		</tr>
		<tr>
			<td>Super-clean table</td>
			<td>Heal Force</td>
			<td>AC130052</td>
			<td></td>
		</tr>
		<tr>
			<td>TSB</td>
			<td>Hope Bio</td>
			<td>HB4114</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Zhejiang Taizhou Seeking Precision Vacuum Pump Co., Ltd.</td>
			<td>22051031</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Scientific</td>
			<td>LP0021B</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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.

The life stages of D. antiqua are depicted in Figure 4. The complete life cycle comprises eggs, larvae, pupa (Figure 4C), and adults (Figure 4D). They are cultivated in sterile centrifuge tubes, and their appearance and survival rate are indistinguishable from D. antiqua raised under non-axenic conditions. The growth and development times for each stage of D. antiqua can be found in Fi...

Watch this Scientific Journal Video about Axenic Rearing of Delia antiqua to Detect the Impact of Intestinal Microbes on Its Growth and Development at JoVE.com

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 ...

rearing-axenic-delia-antiqua-with-half-fermented-sterile-diets

Research

JoVE Journal

Biology

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.

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.