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.
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 >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 (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.
1. Insect rearing
- 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.
- Isolate adults for oviposition after pupation. Feed them tender leaves to obtain more eggs.
- 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
- Prepare Murashige and Skoog (MS) medium and 1 mg/mL α-naphthalene acetic acid (NAA) stock solutions (see the Table of Materials).
- 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.
- Prepare scalpels, alcohol lamp, and forceps; sterilize the scalpels and forceps in the alcohol lamp flame in the biosafety hood.
- 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.
- 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
- Autoclave Petri dishes, paintbrushes, filter paper, distilled water, and a Petri dish containing LB (Luria-Bertani) agar medium.
- 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.
- Wash these eggs with 75% ethanol for 8 min, and repeat the wash four times with sterile water.
- 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.
- Place the Petri dish in a growth chamber and wait for the eggs to hatch within 24 h.
- 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.
- 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 larvae with germ-free poplar leaves.
4. Verification of axenic larvae with culture-dependent assays
- Randomly select three 1st, 2nd, and 3rd instar larvae from the germ-free and conventionally reared groups.
- Dissect the 3rd instar larvae with sterile scissors and forceps under a stereomicroscope and collect their guts in microcentrifuge tubes. Collect intact 1st and 2nd instar larvae in tubes.
NOTE: Collect whole 1st and 2nd instar larvae as they are too small to dissect, and keep their guts intact.
- 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.
- Add 100 µL of the homogenate to LB agar medium and plate using a sterile glass spreading rod.
- Place the plates at 37 °C for 24 h and observe the bacterial colonies.
5. Verification of axenic individuals with culture-independent assays
- Extract the total DNA of tissues (obtained in section 4) using a DNA extraction kit.
- Measure DNA concentration using a spectrophotometer at 260 nm.
- 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.
- 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.
- Observe the gel with a UV transilluminator and look for the target fragment around 1,500 bp.
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 Figure 3, and gut segments are marked with white brackets.
Although no bacterial colonies were observed in any germ-free group, they were observed in all conventionally reared groups (Figure 4), indicating that larvae from sterilized eggs that were fed tissue-cultured poplar leaves contain no bacteria. The ~1,500 bp PCR bands appeared in all conventionally reared groups. In contrast, no band was observed in the germ-free groups or the negative control (Figure 5), implying no gut bacteria existed in axenic larvae. There were no differences in larval development time, survival rate, or appearance between germ-free and conventionally reared P. versicolora larvae. However, the body mass of germ-free larvae was slightly higher than that of conventionally reared larvae on the 5th day, although the masses will become similar before pupation16. These results confirmed the feasibility of this protocol to prepare and rear axenic larvae.
Figure 1: Life stages of the willow leaf beetle, Plagiodera versicolora. (A) Female and male adults; (B) eggs; (C) 1st instar larva; (D) 2nd instar larva; (E) 3rd instar larva; and (F) pupa. Scale bars = 1 mm. Please click here to view a larger version of this figure.
Figure 2: Poplar stem segments and seedlings. (A) Stem segments with apical buds; (B) stem segments grew roots after 10 days; (C) a one-month-old seedling. Scale bars = 1 cm. Please click here to view a larger version of this figure.
Figure 3: The gut of a third-instar larva. Foregut, midgut, and hindgut are labeled with brackets. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 4: Confirmation of efficacy of gut bacteria elimination by culturing gut or whole insect homogenates on LB agar plates. No bacteria were observed in larvae fed germ-free poplar leaves, whereas bacteria were observed in conventionally reared groups. 1st, 2nd, and 3rd instar larvae were used for the assay. Three larvae were randomly selected in each group. Abbreviations: GF = germ-free larvae; CR = conventionally reared larvae. Please click here to view a larger version of this figure.
Figure 5: Confirmation of the efficacy of gut bacteria elimination by PCR assay using universal 16S rRNA gene primers. The target band of the 16S rRNA gene is ~1,500 bp. 1st, 2nd, and 3rd instar larvae were used for the assay. No target band was observed in the GF groups. Abbreviations: NC = negative control; GF = germ-free; CR = conventionally reared. Please click here to view a larger version of this figure.
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. Thus, it is highly feasible to obtain axenic larvae by sterilizing insect egg surfaces27,28,29. Administration of a cocktail of several antibiotics is another way to develop axenic insects but has several disadvantages28. In contrast, egg surface sterilization followed by an axenic diet is better for developing axenic insects23,28,29.
The eggs of most leaf-consuming beetles are visible, easily acquired, and simple to disinfect. This is the main reason why a simple reagent (75% ethanol) and shorter time (8 min) were used for disinfection compared to similar studies (e.g., 40 min of egg disinfection for stinkbug Plautia stali with 75% ethanol and formaldehyde to obtain axenic insects; 6 min of egg surface sterilization of Drosophila with 1% active chlorine and 75% ethanol; and 10 min of egg surface sterilization of red palm weevil Rhynchophorus ferrugineus with 10% sodium hypochlorite solution)23,29,36. For insect species with tiny eggs, the use of disinfectors and sterilization duration needs to be optimized as treatment can significantly impact the results.
In some cases, germ-free artificial diets are employed for axenic insect rearing after egg surface sterilization29,37. However, developing a suitable artificial diet for insects is a tedious and labor-consuming process. Nutrients in the diet extensively influence insect physiology (e.g., development time, immunity) and gut microbiota6,35. Thus, a qualified artificial diet for an insect should contain a similar nutritional composition to the natural food, which is difficult to achieve, especially for phytophagous insects. In this protocol, we fed insects with axenic host plants, which overcomes the shortcomings of an artificial diet. Importantly, as with the poplar plant used here, it is also not difficult to obtain tissue-cultured seedlings from many economically important crops such as tobacco, potato, tomato, wheat, and rice by seed surface-sterilization or stem section disinfection38. Of note, endophytes in plants may exist in tissue-cultured seedlings39 and can be eliminated through shoot tip meristem culture technology40. In conclusion, this protocol provides a new method to maintain germ-free insects, which is a handy tool to facilitate insect-gut bacteria interaction studies.
The authors have no conflicts of interest to disclose.
This work was funded by the National Natural Science Foundation of China (31971663) and the Young Elite Scientists Sponsorship Program by CAST (2020QNRC001).
|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|
|Bead-beating homogenizer||Jing Xin||XM-GTL64|
|DNA extraction kit||MP Biomedicals||116560200|
|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|
|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.
|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|
|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|
|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|
|Willow branches||Sha Lake Park, Wuhan, China|
|Willow leaf beetle||Huazhong Agricultural University, Wuhan, China|
|Yeast extract||Thermo Fisher Scientific||LP0021|
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