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

Summary

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.

Abstract

Insect guts are colonized by diverse bacteria that can profoundly impact the host'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 (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.

Introduction

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 detoxification^2,3,4, nutrition and development^5,6,7, defense against pathogens and parasites^8,9,10,11, chemical communication^12,13 and behaviors^14,15. 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 cases^16,17,18. 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 diseases^19,20,21, 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 >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 bacteria^14,24,25,26. However, antibiotic diets cannot eliminate gut bacteria completely and have a negative effect on host insect physiology^27,28. Consequently, the use of antibiotic-treated insects may obscure the true abilities of some gut bacteria. Fortunately, surface sterilization of eggs can negate this problem^23,29, 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 process^30,31.

The willow leaf beetle, Plagiodera versicolora (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.

Protocol

1. Insect rearing

1. Maintain the P. versicolora population in a growth chamber at the condition of 27 °C and 70 ± 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.
2. Isolate adults for oviposition after pupation. Feed them tender leaves to obtain more eggs.
3. Collect newly laid eggs (within 24 h). Place the eggs on moist absorbing paper for 60 h to prepare axenic larvae.
   ​NOTE: Newly laid eggs will hatch after ~72 h. The best time to sterilize egg surfaces is the day before hatching; otherwise, the number of successfully hatched eggs will decrease.

2. Germ-free poplar culturing

1. Prepare Murashige and Skoog (MS) medium and 1 mg/mL α-naphthalene acetic acid (NAA) stock solutions (see the Table of Materials).
2. In a biosafety hood, add 50 µL of 1 mg/mL NAA stock solution to 500 mL of MS medium, shake to mix well, and pour ~50 mL per tissue culture container and wait for solidification.
3. Prepare scalpels, alcohol lamp, and forceps; sterilize the scalpels and forceps in the alcohol lamp flame in the biosafety hood.
4. Cut 3-4 cm stem segments with apical buds or lateral buds from poplar seedlings at ~1 month of growth (germ-free tissue-cultured seedlings), and insert them into the culture medium, one or two stem segments per container.
5. Incubate these stem segments in a growth chamber at 25 °C and 50 ± 10 cd light intensity with a photoperiod of 16 h light/8 h dark for approximately 30 days. Use the germ-free leaves to feed the axenic larvae.

3. Egg surface sterilization and rearing axenic larvae

1. Autoclave Petri dishes, paintbrushes, filter paper, distilled water, and a Petri dish containing LB (Luria-Bertani) agar medium.
2. Place leaves with adherent eggs in a Petri dish, carefully remove the eggs from the leaves using forceps, and transfer them to another Petri dish.
   NOTE: This step should be performed very carefully because the eggs are adherent to the leaves.
3. Wash these eggs with 75% ethanol for 8 min, and repeat the wash four times with sterile water.
4. Transfer the eggs onto LB agar medium to preserve moisture for hatching.
   NOTE: Using LB agar medium can help to verify whether the disinfected egg is germ-free.
5. Place the Petri dish in a growth chamber and wait for the eggs to hatch within 24 h.
6. In a biosafety hood, tile three pieces of wet filter paper in a Petri dish, 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 them in a growth chamber at 27 °C and 70 ± 5% relative humidity with a photoperiod of 16 h light/8 h dark.
   NOTE: The tissue-cultured leaves are delicate and can lose water quickly. Thus, using moist filter paper is necessary when transferring the leaves to the Petri dish.
7. Change the leaves every two days.
8. For the conventionally reared groups, transfer the eggs from the leaves to a Petri dish containing moist filter paper and feed these larvae with germ-free poplar leaves.

4. Verification of axenic larvae with culture-dependent assays

1. Randomly select three 1^st, 2^nd, and 3^rd instar larvae from the germ-free and conventionally reared groups.
2. Dissect the 3^rd instar larvae with sterile scissors and forceps under a stereomicroscope and collect their guts in microcentrifuge tubes. Collect intact 1^st and 2^nd instar larvae in tubes.
   NOTE: Collect whole 1^st and 2^nd instar larvae as they are too small to dissect, and keep their guts intact.
3. Add 100 µL of phosphate-buffered saline and three steel balls to 1.5 mL tubes and grind the tissues into homogenates using a bead-beating homogenizer.
4. Add 100 µL of the homogenate to LB agar medium and plate using a sterile glass spreading rod.
5. Place the plates at 37 °C for 24 h and observe the bacterial colonies.

5. Verification of axenic individuals with culture-independent assays

1. Extract the total DNA of tissues (obtained in section 4) using a DNA extraction kit.
2. Measure DNA concentration using a spectrophotometer at 260 nm.
3. Amplify the bacterial 16S rRNA gene using universal 16S rRNA primers with PCR. Set up the reaction system with 18 µL of 1.1x PCR master mix (see the Table of Materials), 0.5 µL of 27F primer (5'-ACGGATACCTTGTTACGAC-3'), 0.5 µL of 1495R primer (5'-ACGGATACCTTGTTACGAC-3') and 100 ng template DNA. Set the PCR conditions at 95 °C for 3 min; 28 cycles of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min; followed by 72 °C for 10 min. Store the PCR products at 4 °C until further analysis.
4. Mix the PCR products with nucleic acid dye and analyze them using electrophoresis on a 1% agarose gel in 1x TAE buffer. Use 10 µL of a DNA marker as a reference.
5. Observe the gel with a UV transilluminator and look for the target fragment around 1,500 bp.

Results

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 3^rd instar larva is shown in Fig...

Discussion

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 surface^35<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm syringe filters	Millipore	SLGP033RB
1 mg/mL NAA stock solution			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 µm syringe filter and a 50 mL sterile syringe, subpackage the solution in 1.5 mL centrifuge tubes and restore at -20 °C.
1.5 mL microcentrifuge tubes	Sangon Biotech	F600620
10x PBS stock solution	Biosharp Life Sciences	BL302A
2 M KOH solution			Dissolve 22.44 g KOH (molecular weight: 56.1) in 200 mL of distilled water and autoclave it for 20 min at 121 °C.
250 mL and 2,000 mL beakers	Shubo	sb16455
50 mL sterile syringes	Jinta	JT0125789
500 mL measuring cylinder	Shubo	sb1601
50x TAE stock solution			a. 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.
75% ethanol	Xingheda trade
α-naphthalene acetic acid (NAA)	Solarbio Life Sciences	86-87-3
Absorbing paper			22.3 cm x 15.3 cm x 9 cm
Acetic acid	Sinopharm Chemical Reagent Co. Ltd
Agar	Coolaber	9002-18-0
Agarose	Biowest	111860
Autoclave	Panasonic	MLS-3781L-PC
Bead-beating homogenizer	Jing Xin	XM-GTL64
DNA extraction kit	MP Biomedicals	116560200
EDTA	Saiguo Biotech	1340
Filter paper	Jiaojie		70 mm diameter
Gel electrophoresis unit	Bio-rad	164-5052
Gel Signal Green nucleic acid dye	TsingKe	TSJ003
Germ-free poplar seedlings			Shan Xin poplar from Ludong University in Shandong Province
Golden Star Super PCR Master Mix (1.1×)	TsingKe	TSE101
Growth chamber	Ruihua	HP400GS-C
LB agar medium			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 °C.
Mini centrifuge	DRAGONLAB	D1008
MS basic medium	Coolaber	PM1121-50L	M0245
MS solid medium for germ-free poplar seedling culture			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 °C.
NanoDrop 1000 spectrophotometer	Thermo Fisher Scientific
Paintbrush			1 cm width, used to collect the eggs
Parafilm	Bemis	PM-996
PCR Thermal Cyclers	Eppendorf	6331000076
Petri dishes	Supin		90 mm diameter
pH meter	METTLER TOLEDO	FE20
Pipettes 0.2-2 µL	Gilson	ECS000699
Pipettes 100-1,000 µL	Eppendorf	3120000267
Pipettes 20-200 µL	Eppendorf	3120000259
Pipettes 2-20 µL	Eppendorf	3120000232
Plant tissue culture container	Chembase	ZP21	240 mL
Plastic box			2.35 L
Potassium hydroxide (KOH)	Sinopharm Chemical Reagent Co. Ltd
Primers for amplifying the bacterial 16S rRNA gene	Sangon Biotech		27-F: 5’-ACGGATACCTTGTTACGAC-3’, 1492R: 5’-ACGGATACCTTGTTACGAC-3’
Sodium chloride (NaCl)	Sinopharm Chemical Reagent Co. Ltd
Sodium hydroxide (NaOH)	Sinopharm Chemical Reagent Co. Ltd
Steel balls		0.25 mm	used to grind tissues
Stereomicroscope	OLYMPUS	SZ61
Sucrose	Sinopharm Chemical Reagent Co. Ltd
Trans2K plus II DNA marker	Transgene Biotech	BM121-01
Tris base	Biosharp Life Sciences	1115
Tryptone	Thermo Fisher Scientific	LP0037
UV transilluminator	Monad Biotech	QuickGel 6100
Vortexer	Scilogex	MX-S
Willow branches			Sha Lake Park, Wuhan, China
Willow leaf beetle			Huazhong Agricultural University, Wuhan, China
Yeast extract	Thermo Fisher Scientific	LP0021