Name: preparing and rearing axenic insects with tissue cultured seedlings for host-gut microbiota interaction studies of the leaf beetle
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Description: Watch this Scientific Journal Video about Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle at JoVE.com

This work was funded by the National Natural Science Foundation of China (31971663) and the Young Elite Scientists Sponsorship Program by CAST (2020QNRC001).

1. Insect rearing
<ol>
	<li>Maintain&#160;the P. versicolora population in a growth chamber at the condition of 27 &#176;C and 70 &#177; 5% relative humidity with a photoperiod of 16 h light/8 h dark. Place them in perforated plastic boxes with tiled wet absorbing paper and feed them fresh poplar branches. Spray clean water on absorbing paper to maintain moisture and change the branches every two days.</li>
	<li>Isolate adults for oviposition after pupation. Feed them tender leaves to obtain m...

<ol>
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Preparation of germ-free larvae and obtaining gnotobiotic larvae by reintroducing specific bacterial strains are powerful methods to elucidate the mechanisms underlying host-microbe interactions. Newly hatched larvae obtain gut microbiota in two main ways: vertical transmission from the mother to the offspring or horizontal acquisition from siblings and the environment34. The former can be fulfilled by parental transfer to the offspring through contamination of the egg surface35<...

Similar to mammals, the insect digestive tract is a cavity for food digestion and absorption. Most insects harbor diverse commensal bacteria that thrive in their guts and live on nutrition supplied by hosts1. The gut commensal community has a profound impact on multiple physiological processes in insects, including food digestion and detoxification2,3,4, nutrition and development5,6,7, defense against pathogens and parasites8,9,10,11, chemical communication12,13 and behaviors14,15. Intriguingly, some gut microbiota can be facultatively pathogenic or be manipulated by invading pathogens to aggravate infection, indicating that gut bacteria can be harmful in some cases16,17,18. Gut bacteria can also serve as a microbial resource for biotechnical applications and pest management. For example, lignocellulose-digesting bacteria from phytophagous and xylophagous insects were used to digest plant cells for developing biofuels19. The dispersal of engineered gut symbionts expressing bioactive molecules is a novel and promising tactic to manage agriculture and forestry pests and mosquitoes transmitting infectious diseases19,20,21, which can also be used to improve the fitness of beneficial insects22. Illustrating how a gut bacterium behaves in vivo is thus considered a priority to fully leverage its function and further exploit it for various applications.
Animals can harbor 1 to &#62;1000 symbiotic microbial species in the gut1. As a result, it is difficult to accurately verify how individual bacterial taxa or their assembly perform inside an animal, and whether the host or its microbial partners drive a specific function. Therefore, preparing axenic larvae to obtain gnotobiotic insects by mono- or multi-species colonization is necessary to investigate bacterial function and interaction with insects23. At present, administering antibiotic cocktails and sterilizing the surface of insect eggs are common methods to remove gut bacteria14,24,25,26. However, antibiotic diets cannot eliminate gut bacteria completely and have a negative effect on host insect physiology27,28. Consequently, the use of antibiotic-treated insects may obscure the true abilities of some gut bacteria. Fortunately, surface sterilization of eggs can negate this problem23,29, which has no or negligible effects on experimental insects. Furthermore, artificial diets cannot fully resemble natural insect food, and developing an artificial diet is a costly and labor-consuming process30,31.
The willow leaf beetle, Plagiodera versicolora&#160;(Laicharting) (Coleoptera: Chrysomelidae), is a widespread leaf-eating pest that mainly feeds on salicaceous trees, such as willows (Salix) and poplar (Populus L.)32,33. Here, the willow leaf beetle was used as a representative leaf-eating insect to develop a protocol to prepare and rear a germ-free insect. We exploited plant tissue culture to obtain germ-free poplar leaves to rear P. versicolora axenic larvae from sterilized eggs. The axenic status of P. versicolora larvae was verified via culture-dependent and culture-independent assays. This protocol can maintain axenic insects that better mimic the wild condition than insect rearing with an artificial diet. More importantly, this method is convenient at a very low cost, which increases the feasibility of obtaining axenic insects for future insect-gut microbiota interaction studies, especially for non-model insects without well-developed artificial diets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m syringe filters</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mg/mL NAA stock solution</td>
			<td></td>
			<td></td>
			<td>a. Prepare 0.1 M NaOH solution (dissolve 0.8 g NaOH in 200 mL of distilled water). 
			b. Add 0.2 g NAA in a 250 mL beaker, add little 0.1 M NaOH solution until NAA dissolved, and adjust the final volume to 200 mL with distilled water. 
			c. Filter the solution to remove bacteria with a 0.22 &#181;m syringe filter and a 50 mL sterile syringe, subpackage the solution in 1.5 mL centrifuge tubes and restore at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Sangon Biotech</td>
			<td>F600620</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS stock solution</td>
			<td>Biosharp Life Sciences</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M KOH solution</td>
			<td></td>
			<td></td>
			<td>Dissolve 22.44 g KOH (molecular weight: 56.1) in 200 mL of distilled water and autoclave it for 20 min at 121 &#176;C.</td>
		</tr>
		<tr>
			<td>250 mL and 2,000 mL beakers</td>
			<td>Shubo</td>
			<td>sb16455</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile syringes</td>
			<td>Jinta</td>
			<td>JT0125789</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL measuring cylinder</td>
			<td>Shubo</td>
			<td>sb1601</td>
			<td></td>
		</tr>
		<tr>
			<td>50x TAE stock solution</td>
			<td></td>
			<td></td>
			<td>a.&#160;Dissolve 242 g Tris and 18.612 g EDTA in 700 mL of distilled water. 
			b. Adjust pH to 7.8 with about 57.1 mL of acetic acid. 
			c. Adjust the final volume to 1,000 mL. 
			d. The stock solution was diluted to 1x TAE buffer when used.</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Xingheda trade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-naphthalene acetic acid (NAA)</td>
			<td>Solarbio Life Sciences</td>
			<td>86-87-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbing paper</td>
			<td></td>
			<td></td>
			<td>22.3 cm x 15.3 cm x 9 cm</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sinopharm Chemical Reagent Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Coolaber</td>
			<td>9002-18-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biowest</td>
			<td>111860</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Panasonic</td>
			<td>MLS-3781L-PC</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead-beating homogenizer</td>
			<td>Jing Xin</td>
			<td>XM-GTL64</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA extraction kit</td>
			<td>MP Biomedicals</td>
			<td>116560200</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Saiguo Biotech</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Jiaojie</td>
			<td></td>
			<td>70 mm diameter</td>
		</tr>
		<tr>
			<td>Gel electrophoresis unit</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Signal Green nucleic acid dye</td>
			<td>TsingKe</td>
			<td>TSJ003</td>
			<td></td>
		</tr>
		<tr>
			<td>Germ-free poplar seedlings</td>
			<td></td>
			<td></td>
			<td>Shan Xin poplar from Ludong University in Shandong Province</td>
		</tr>
		<tr>
			<td>Golden Star Super PCR Master Mix (1.1&#215;)</td>
			<td>TsingKe</td>
			<td>TSE101</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Ruihua</td>
			<td>HP400GS-C</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar medium</td>
			<td></td>
			<td></td>
			<td>a. Dissolve 5 g tryptone, 5 g NaCl, 2.5 g yeast extract in 300 mL of distilled water. 
			b. Adjust the final volume to 500 mL, transfer the solution to a 1,000 mL conical flask, and add 7.5 g agar. 
			c. Autoclave the medium for 20 min at 121 &#176;C.</td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>DRAGONLAB</td>
			<td>D1008</td>
			<td></td>
		</tr>
		<tr>
			<td>MS basic medium</td>
			<td>Coolaber</td>
			<td>PM1121-50L</td>
			<td>M0245</td>
		</tr>
		<tr>
			<td>MS solid medium for germ-free poplar seedling culture</td>
			<td></td>
			<td></td>
			<td>a. Dissolve 4.43 g MS basic medium powder and 30 g sucrose in 800 mL of distilled water. 
			b. Adjust the pH to about 5.8 with 2 M KOH by a pH meter. 
			c. Adjust the final volume to 1,000 mL, separate into two parts, transfer into two 1,000 mL conical flasks, and add 2.6 g agar per 500 mL. 
			d. Autoclave for 20 min at 121 &#176;C.</td>
		</tr>
		<tr>
			<td>NanoDrop 1000 spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td></td>
			<td></td>
			<td>1 cm width, used to collect the eggs</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermal Cyclers</td>
			<td>Eppendorf</td>
			<td>6331000076</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Supin</td>
			<td></td>
			<td>90 mm diameter</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>METTLER TOLEDO</td>
			<td>FE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 0.2-2 &#181;L</td>
			<td>Gilson</td>
			<td>ECS000699</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 100-1,000 &#181;L</td>
			<td>Eppendorf</td>
			<td>3120000267</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 20-200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3120000259</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes 2-20 &#181;L</td>
			<td>Eppendorf</td>
			<td>3120000232</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant tissue culture container</td>
			<td>Chembase</td>
			<td>ZP21</td>
			<td>240 mL</td>
		</tr>
		<tr>
			<td>Plastic box</td>
			<td></td>
			<td></td>
			<td>2.35 L</td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sinopharm Chemical Reagent Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primers for amplifying the bacterial 16S rRNA gene</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td>27-F: 5&#8217;-ACGGATACCTTGTTACGAC-3&#8217;, 1492R: 5&#8217;-ACGGATACCTTGTTACGAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sinopharm Chemical Reagent Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sinopharm Chemical Reagent Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steel balls</td>
			<td></td>
			<td>0.25 mm</td>
			<td>used to grind tissues</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sinopharm Chemical Reagent Co. Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans2K plus II DNA marker</td>
			<td>Transgene Biotech</td>
			<td>BM121-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Biosharp Life Sciences</td>
			<td>1115</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>UV transilluminator</td>
			<td>Monad Biotech</td>
			<td>QuickGel 6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Scilogex</td>
			<td>MX-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Willow branches</td>
			<td></td>
			<td></td>
			<td>Sha Lake Park, Wuhan, China</td>
		</tr>
		<tr>
			<td>Willow leaf beetle</td>
			<td></td>
			<td></td>
			<td>Huazhong Agricultural University, Wuhan, China</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Fisher Scientific</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparing and rearing axenic insects with tissue cultured seedlings for host-gut microbiota interaction studies of the leaf beetle

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain into an axenic insect is a powerful method to verify gut microbial function and elucidate the mechanisms underlying gut microbe-host interactions. Administering antibiotics or sterilizing egg surfaces are two commonly used methods to remove gut bacteria from insects. However, in addition to the potential adverse effects of antibiotics on insects, previous studies indicated that feeding antibiotics could not eliminate gut bacteria. Thus, germ-free artificial diets are generally employed to maintain axenic insects, which is a tedious and labor-intensive process that cannot fully resemble nutritional components in natural food. Described here is an efficient and simple protocol to prepare and maintain axenic larvae of a leaf beetle&#160;(Plagiodera versicolora). Specifically, surfaces of the beetle eggs were sterilized, following which germ-free poplar leaves were used to rear axenic larvae. The axenic status of the insects was further confirmed via culture-dependent and culture-independent assays. Collectively, by combining egg disinfection and germ-free cultivation, an efficient and convenient method was developed to obtain axenic P. versicolora, providing a readily transferable tool for other leaf-eating insects.

The life stages of P. versicolora are shown in Figure 1. The adult male is smaller than the adult female (Figure 1A). In the field, the beetle clusters its eggs on a leaf; here, four eggs were detached from a leaf (Figure 1B). The poplar stem segments and seedlings used for axenic insect rearing are shown in Figure 2. The gut of a 3rd instar larva is shown in Fig...

Watch this Scientific Journal Video about Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle at JoVE.com

Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects.

Insect guts are colonized by diverse bacteria that can profoundly impact the host&#39;s physiological traits. Introducing a particular bacterial strain ...

The gut bacteria can affect multiple physiological processes of insect hosts. Preparing and maintaining axenic larvae is a powerful tool to study gut bacteria functions. The main advantages of this protocol are that plant tissue culture is used to obtain germ-free leaves to rear axenic leaf eating insects derived form surface-sterilized eggs, and that this method is not restricted by artificial diets.This method is convenient and increase visibility of maintaining axenic insects for future gut bacteria studies, especially for non-model insects without well-developed artificial diets. Maintain the P.versicolora population in a growth chamber at 27 degrees Celsius and 70%relative humidity, with a 16 hour light and eight hour dark photoperiod. Place the insects in perforated plastic boxes with tilted wet absorbing paper, and feed them fresh poplar branches.Spray clean water on absorbing paper to maintain moisture, and change the branches every two days. Isolate adults for oviposition after pupation, and feed them tender leaves to obtain more eggs. Within 24 hours, collect newly laid eggs and place them on moist absorbing paper for 60 hours to prepare axenic larvae.Prepare MS medium in one milligram per milliliter alpha-naphthalene acetic acid or NAA stock solutions. In a biosafety hood, add 50 microliters of the NAA stock solution to 500 milliliters of MS medium, and shake to mix well. Then pour approximately 50 milliliters per tissue culture container and wait for solidification.Sterilize scalpels and forceps in an alcohol lamp flame in the biosafety hood. Cut three to four centimeter stem segments with apical buds or lateral buds from poplar seedlings at approximately one month of growth, and insert them into the culture medium, one or two stem segments per container. Incubate these stem segments in a growth chamber for approximately 30 days.And use these germ-free leaves to feed the axenic larvae. Autoclave Petri dishes, paint brushes, filter paper, distilled water in a Petri dish containing LB agar medium. Place leaves with adherent eggs in a Petri dish.Then carefully remove the eggs from the leaves using forceps and transfer them to another Petri dish. Wash these eggs with 75%ethanol for eight minutes. Then repeat the wash four times with sterile water.Transfer the eggs onto LB agar medium to preserve moisture for hatching. Place the Petri dish in a growth chamber and wait for the eggs to hatch within 24 hours. In a biosafety hood, tile three pieces of wet filter paper in a Petri dish, and place germ-free poplar leaves on the paper.Collect the larvae and place them on the leaves. Seal the Petri dish with Parafilm, and incubate it in a growth chamber. Change the leaves every two days.For the conventionally reared groups, transfer the eggs from the leaves to a Petri dish containing moist filter paper, and feed these larva with germ-free poplar leaves. Randomly select three first, second and third instar larva from the germ-free and conventionally reared groups. Dissect the third instar larvae with sterile scissors and forceps under a stereo microscope, and collect their guts in microcentrifuge tubes.Collect intact first and second instar larvae in tubes. Add 100 microliters of PBS and three steel balls to the 1.5 milliliter tubes, and grind the tissues into homogenates using a bead beating homogenizer. Add 100 microliters of the homogenate to LB agar medium and plate using a sterile glass spreading rod.Incubate the plates at 37 degrees Celsius for 24 hours. Then observe the bacterial colonies. Extract the total DNA of the previously obtained tissues using a DNA extraction kit, and measure DNA concentration with a spectrophotometer.Amplify the bacterial 16S rRNA gene using universal 16S rRNA primers with PCR, setting up the reaction as described in the text manuscript. Store the PCR products at four degrees Celsius until further analysis. Mix the PCR products with nucleic acid dye and analyze them using electrophoresis on a 1%agarose gel in 1X TAE buffer.Use 10 microliters of a DNA marker as a reference. Observe the gel with a UV transilluminator, and look for the target fragment around 1, 500 base pairs. Although no bacterial colonies were observed in any germ-free group, they were observed in all conventionally reared groups, indicating that larvae from sterilized eggs that were fed tissue cultured poplar leaves contained no bacteria.The 1, 500 base pair PCR bands appeared in all conventionally reared groups. In contrast, no band was observed in the germ-free groups or the negative control, implying that no gut bacteria existed in axenic larvae. For insect species with tiny eggs, the kinds of disinfectants and sterilization duration needed to be optimized.In addition, endophytes in tissue-cultured plants should be notable. This protocol provides a new method to maintain germ-free insects, which is a handy tool to facilitate insect-gut bacteria interaction studies, especially for non-model insects without well-developed artificial diets.

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