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May 2019

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Preparation of Red Palm Weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) Germ-free Larvae for Host-gut Microbes Interaction Studies
用于宿主肠道微生物相互作用研究的红棕象甲(稻铁甲虫)(鞘翅目:椰象鼻虫科)无菌幼虫的制备    

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Abstract

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a devastating pest of palm trees worldwide. RPW gut is colonized by diverse bacterial species which profoundly influence host development and nutritional metabolism. However, the molecular mechanisms behind the interactions between RPW and its gut microbiota remain mostly unknown. Antibiotics are usually employed to remove gut bacteria to investigate the impact of gut bacteria on insect fitness. However, administration of antibiotics cannot thoroughly remove gut bacteria for most insect species. Therefore, establishing germfree (GF) organisms is a powerful way to reveal the mutual interactions between gut bacteria and their insect hosts. Here, we describe a protocol to generate and maintain RPW GF larvae, being completely devoid of gut bacteria in laboratory. RPW GF larvae were established from the dechorionated fresh eggs which were reared on the sterilized artificial food under axenic conditions. The establishment of GF larvae set a solid foundation to deeply elucidate the molecular mechanisms behind the interactions between RPW and its gut microbiota.

Keywords: Rhynchophorus ferrugineus (红棕象甲), Germfree larvae (无菌幼虫), Gut microbiota (肠道菌群), Insect symbiosis (昆虫共生), Host physiology (寄主生理)

Background

Red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthorodae), is one of the most noxious pest of palm trees in the world (Li et al., 2009; Ju et al., 2011; Al-Dosary et al., 2016; Peng et al., 2016). It is native to south Asia and Melanesia, but recently it has dramatically spread to European, African, some American and other Asian countries (OEPP/EPPO, 2008; Shi et al., 2014). The larva of RPW is the main destructive agent which feed on tender tissues and sap in the trunk from the apical growing point of palms. RPW larvae complete its entire developmental time inside the palm trunk until newborn adults fly out for locating new oviposition sites (Kaakeh, 2005; OEPP/EPPO, 2008; El-Mergawy and Al-Ajlan, 2011). The concealed infestation behavior of the pest makes its control more challenging. Currently, the control measures against this insect pest include the use of synthetic insecticides, phyto-sanitation, pheromone-based mass trapping and releasing some biological control agents, such as entomopathogenic fungi, bacteria and nematodes (Murphy and Briscoe, 1999; Faleiro, 2006; Mazza et al., 2014; Pu and Hou, 2016). However, due to the occurrence of pesticide resistance and limitations of other control measures, it is urgent to develop some sustainable, eco-friendly and economical manage strategies that can effectively protect palm trees from this insect pest.

Like mammals, insects live in the symbiotic associations with gut microbiota which play thecritical roles in many host physiological processes, containing development, digestion and detoxification, immunity and chemical communication (Douglas, 2009, 2010 and 2015; Engel and Moran, 2013). Gut bacteria are the excellent agents for paratransgenesis by using an engineered symbiont to express some specific bioactive molecules that impair insect fitness to achieve pest control (Engel and Moran, 2013). Therefore, elucidating the interactions between insect pests and their symbiotic microbes could provide some important implications for the development of novel pest management tactics (Butera et al., 2012; Crotti et al., 2012).

Owing to its enormous economic and environmental losses, RPW has attracted great attention in recent years. Recently, some preliminary investigations have deciphered gut bacterial components of RPW larvae and adults. It has been determined that RPW gut is colonized by bacteria mainly from Enterobacteriaceae, Lactobacillaceae, Acetobacteriaceae, Entomoplasmataceae, Enterococcaceae and Streptococcaceae (Jia et al., 2013; Tagliavia et al., 2014; Montagna et al., 2015; Muhammad et al., 2017). Moreover, we have discovered that a secretory protein, RfPGRP-LB, and the NF-ĸB like transcription factor, RfRelish, regulate gut immunity to modulate the homeostasis of RPW gut microbiota (Dawadi et al., 2018; Xiao et al., 2019). Furthermore, it has also been found that gut microbiota of RPW can affect its host growth and development by modulating its nutrition metabolism (Muhammad et al., 2017; Habineza et al., 2019). Unfortunately, we found gut bacteria of RPW larvae cannot be removed thoroughly by the administration of antibiotic cocktails (Muhammad et al., 2017). Increasing evidence strongly suggest that GF animal models are one of pivotal tool for dissecting the crosstalks between animal and their gut microbiota (Grover and Kashyap, 2014; Koyle et al., 2016; Kietz et al., 2018). Therefore, to investigate the role of residential gut microbiota on RPW physiology, RPW GF larvae were generated to decipher the host-gut bacteria interplays. Compared with the administration of antibiotics, our protocol in the report can successful and easily to establish and maintain RPW GF larvae. It is also easy to generate the gnotobiotic RPW larvae with specified gut bacterial species.

Materials and Reagents

  1. Perforated plastic bottles (330 ml, 70mm Ø, 107mm height; Jiafeng Horticultural Products Co. Ltd., Chongqing, China)
  2. Conical flask
  3. Petri dishes (90 mm Ø, Yancheng Huida Medical Instruments Co. Ltd., Jiangsu, China)
  4. Paintbrush (1 cm width), used to collect the fresh eggs
  5. 0.22 µm syringe filters (Pall Corporation, USA)
  6. Spatula (20 cm long and 4 cm wide at the tip)
  7. PCR tubes (Biosharp Life Sciences, Hefei, China)
  8. 1.5 ml microcentrifuge tubes (Biosharp Life Sciences, Hefei, China)
  9. Metal sieve with pore size 60 µm (Sigma-Aldrich Co. Ltd., Switzerland, catalog number: Z289744)
  10. Aluminum foil
  11. Parafilm (Bemis PM-996, USA)
  12. 10 ml sterile syringes (Hamilton® Syringes, Sigma-Aldrich Co. Ltd., Switzerland)
  13. Filter paper (90 mm Ø, Fuyang Special Paper Industry Co. Ltd., Hangzhou, China)
  14. Sugarcane, Saccharum sinensis Roxb. (Zhangzhou City, Fujian Province, China)
  15. Primers for amplifying the bacterial 16S rRNA gene (27F: 5ʹ-AGAGTTTGATCATGGCTCAG-3ʹ, 1492R: 5ʹ-TACGGYTACCTTGTTACGACTT-3ʹ) (Sangon BioTech. Co. Ltd., Shanghai, China)
  16. Trans2K® DNA marker (Transgene BioTech. Co. Ltd., Beijing, China)
  17. Distilled water
  18. Ethanol (Sigma-Aldrich, CAS number: 64-17-5)
  19. Sodium hypochlorite (NaClO) (Sigma-Aldrich, CAS number: 7681-52-9)
  20. Antibiotics
    1. Kanamycin (Sigma-Aldrich, CAS number: 70560-51-9)
    2. Tetracycline (Sigma-Aldrich, CAS number: 64-75-5)
    3. Gentamycin (Sigma-Aldrich, CAS number: 1405-41-0)
    4. Erythromycin (Sigma-Aldrich, CAS number: 114-07-8)
  21. Tryptone (OXOID, catalog number: 2406419)
  22. Sodium chloride (NaCl) (Sigma-Aldrich, CAS number: 746398)
  23. Potassium chloride (KCl) (CAS number: 7447-40-7)
  24. Anhydrous sodium phosphate dibasic (Na2HPO4) (CAS number: 7558-79-4)
  25. Hydrochloric acid (HCl) (CAS number: 7647-01-0)
  26. Monopotassium phosphate (KH2PO4) (CAS number: 7778-77-0)
  27. Yeast extract (CAS number: 8013-01-2)
  28. Agar (CAS number: 9002-18-0)
  29. Sodium hydroxide (NaOH) (CAS number: 221465)
  30. DNA extraction kit (DNeasy blood and tissue kit, Qiagen, Germany)
  31. 6x DNA Loading buffer (TRANS®, Transgene BioTech. Co. Ltd., Beijing, China)
  32. 0.5x TAE buffer (Tris-Acetate EDTA)
  33. 2x Taq PCR Mastermix (Tiangen Biotechnology, Beijing, China)
  34. Tris-HCl (Solarbio Life Sciences Co. Ltd., Beijing, China)
  35. Glacial acetic acid (CAS number: 64-19-7)
  36. Ascorbic acid
  37. Sucrose
  38. Casein
  39. Corn flour
  40. Avicel
  41. Cholestrol
  42. Choline chloride
  43. Inositol
  44. Potassium sorbate
  45. Sodium p-hydroxybenzoate
  46. Disodium EDTA (CAS number: 6381-92-6)
  47. Agarose (Transgen. Biotech. Co. Ltd., Beijing, China)
  48. TRANS® Ethidium bromide Gel Stain (Transgen. Biotech. Co. Ltd., Beijing, China)
  49. 10x PBS stock solution (see Recipes)
  50. Antibiotic stock solution (see Recipes)
  51. LB agar medium (see Recipes)
  52. 10x TAE stock solution (see Recipes)
  53. The artificial food for RPW larvae (see Recipes)

Equipment

  1. Heal Force safe-1200LC biosafety cabinet (Heal Force Bio-meditech Holdings Co. Ltd., Shanghai, China)
  2. Autoclave (Shennan LDZF-75KB-II, China)
  3. SCIENTZ-48 tissue lyser (Ningbo Scientz Botecthnology Co., Ltd, China)
  4. Haier refrigerator (Haier Co. Ltd., Qingdao, China)
  5. Scissors (World Precision Instruments, Sarasota, USA)
  6. Pointed tip tweezers (3.9 x 3.0 x 1.0 inches, World Precision Instruments, Sarasota, USA)
  7. Forceps with curved flat rounded tips (1.65 mm x 0.05 mm x 120 mm, width x thickness x length, World Precision Instruments, Sarasota, USA)
  8. Yiheng® Incubator (500 x 460 x 800 mm, Shanghai Yiheng Instruments Co. Ltd., China) was employed to rear the axenic larvae (The conditions were set at 37 °C and dark for 24 h)
  9. BIO-RAD T100TM PCR thermocycler (Bio-Rad Co. Ltd., USA) 
  10. Eppendorf 5804 R Centrifuge (Eppendorf Co. Ltd., Germany)
  11. Gel electrophoresis unit (Liuyi Instruments Co. Ltd., Beijing, China)
  12. Nikon® Stereomicroscope (Nikon Co. Ltd., Japan)
  13. Saifu ZRX-260 incubator for maintaining the RPW lab population (260 L, Ningbo Experimental Instrument Co. Ltd, China)
  14. Nanodrop 1000 (Thermo SCIENTIFIC, USA)
  15. Microwave (Midea Co. Ltd., Guangdong, China)
  16. Magnetic stirrer (Ronghua Experimental Instrument Co. Ltd., Jintan, Jiangsu, China)
  17. BSA124S Electric balance (Sartorius Co. Ltd., Germany)
  18. UV transilluminator (Peiqing Science and Technology Co. Ltd., China)
  19. Laminar hood (Heal Force Safe-1200LC)

Software

  1. IBM SPSS Statistics (22.0)

Procedure

  1. Insect rearing and maintaining
    1. RPW lab population was established and maintained by the adults which were trapped in Pingtan District (119°32′ E, 25°31′ N) of Fuzhou City, Fujian Province and Jinshan campus of Fujian Agriculture and Forestry University (119°30′ E, 26°08′ N). RPW individuals were fed with sugarcane stems in the incubator (Saifu ZRX-260, Ninbo Experimental Instrument Co. Ltd., China) at the condition of 27 ± 1 °C, 75 ± 5% relative humidity, and a photoperiod of 24 h dark for larvae and 12 h light/12 h dark for adults (35 mm x 10 mm, length x width, Figure 1).


      Figure 1. The four different life stages of red palm weevil, Rhynchophorus ferrugineus

    2. A female and male RPW adult were kept in the perforated plastic bottle. Provide them with sugarcane stem slices (30 mm x 15 mm, length x width) for feeding and oviposition. The food was changed every three days.
    3. The fresh eggs within 12 h after being laid were collected by cutting the sugarcane stem slices into much thinner ones, for dechorionation to generate RPW GF larvae.

  2. Preparation of artificial food and antibiotic stock solutions
    1. Make RPW artificial food (see Recipe 1) and prepare antibiotics stock solutions (see Recipe 2).
    2. Syringe filter (0.22 µm) the antibiotics stock solution and store in aliquots.
    3. Prepare the artificial food in an autoclavable 1 L conical flask and sterilize it using an autoclave for 20 min at 121 °C.
    4. Add the antibiotic cocktail (600 mg/kg, 150 mg/kg each) and 1 g ascorbic acid to the autoclaved artificial food when the temperature drops to about 55 °C.
    5. After solidification, prepare a parallel batch of the artificial diet without antibiotics, replaced by the same volume of sterile distilled water.
    6. After solidification, prepare aliquots of the artificial food in Petri dishes and seal it with parafilm to avoid the potential microbial contamination during changing food for RPW individuals.
      Note: Carry out this step in the biosafety cabinet.
    7. Diet can be stored at 4 °C for later use.

  3. Generating RPW GF individuals via egg dechorionation
    1. Sterilize all the tools, containing paintbrush, metal sieve, spatula (20 cm x 4 cm, length x width), tweezers and forceps, with 75% ethanol.
    2. Turn on the UV light for 30 min to sterilize the surface of laminar hood and other materials inside.
    3. Divide the eggs into two groups: a) non-dechorionated (control group) and b) dechorionated eggs. Use 30 eggs in each group.
    4. For eggs dechorionation, process the dechorionated eggs with the following steps as Koyle et al. (2016):
      1. Wash the freshly laid eggs with 10% sodium hypochlorite solution (NaClO) for 3-5 min and then filter them through the metal sieve.
      2. Rinse these eggs two times with 75% ethanol and filter through the metal sieve.
      3. Wash these eggs twice with sterilized distilled water to wash off the bleach (NaClO) and ethanol and filter the water carrying dechorionated eggs through the metal sieve. For the eggs in control group, they are only washed with the autoclaved distilled water and then filter the water carrying eggs through a metal sieve.
    5. Observe the dechorionated eggs under a stereomicroscope and we found that the dechorionated eggs became white as compared to the controls (Figure 2).


      Figure 2. Non-dechorionated and dechorionated eggs of red palm weevil, Rhynchophorus ferrugineus. (A) Non-dechorionated eggs are only washed with autoclaved distilled water only and (B) dechorionated eggs were washed with 10% sodium hypochlorite solution (NaClO), 75% ethanol and sterilized distilled water.

    6. Transfer the dechorionated eggs to the sterilized Petri dishes (90 mm Ø) with some UV sterilized moist absorbent cotton inside the laminar hood (Heal Force Safe-1200LC). The eggs are allowed to hatch inside the laminar hood at the condition of 27 ± 1 °C and 75 ± 5% relative humidity in the dark.
    7. Observe the eggs for hatching every 24 h. Two to three days later, they will hatch.
    8. Transfer the neonatal larvae to the new sterilized Petri dishes with 3 g solid artificial diet (Step C6), with or without antibiotics. Refresh the food every two days.
    9. Maintain the RPW larvae at 27 ± 1 °C, 75 ± 5% relative humidity, and 24 h dark photoperiod.
    10. Change the artificial food every three days.
    11. To verify the efficiency of our protocol, four groups are designated here: 1) dechorionated eggs + food with antibiotics, 2) dechorionated eggs + food without antibiotics (DNA), 3) non-dechorionated eggs + food with antibiotics (NDA), and 4) non-dechorionated eggs + food without antibiotics (CR conventionally reared). The larvae in Groups 1 and 3 are maintained in the laminar hood, while two other groups, be served as the positive and negative control, are kept in the incubator (Ninbo Experimental Instrument).

  4. Verification of germfree RPW individuals with culture-dependent and -independent assays
    Gut dissection and homogenization
    1. To validate the GF status of RPW, randomly select three larvae (the 5th instar larvae, Figure 3) from each treatment as a replicate for dissection, each group compromised at least three replicates.
    2. To remove the potential effect of associated microbes on the body surface of RPW larvae, submerge the specimens in 10% NaClO for 30 s, 75% ethanol for 90 s and two rinses with sterile distilled water. This step is completed inside the laminar hood.
    3. Dissect the guts under a stereomicroscope using sterilized scissors and forceps in a clean Petri dish with sterile PBS.
    4. Homogenize the collected guts in 1.5 ml microcentrifuge tube containing 1 ml sterile PBS, using a tissue lyser (48 tissue lyser Ningbo Scientz BioTech. Co. Ltd, China).


      Figure 3. The gut from the fifth RPW larvae

    Verification of germfree RPW individuals with culture-dependent assays
    1. Serially dilute (10-1-10-4 fold) the gut homogenate with sterile PBS.
    2. Plate 100 µl diluted homogenate on the LB agar plate in triplicate.
    3. Incubate the plates aerobically at 37 °C for 24 h and then check for the bacterial growth.
    4. As expected, no bacterial colonies appear on the plates containing gut homogenates from the dechorionated eggs provided with food supplemented with antibiotics (Dechorionated eggs + food with antibiotics, Figure 4). However, bacterial colonies grow up on the plates containing gut homogenates from other three groups (Figure 4). Consequently, the evidence suggests that RPW GF larvae were generated successfully.


      Figure 4. Culture-dependent verification of bacterial growth in the guts of RPW larvae from different treatment cohorts. No bacterial colony is observed in the larvae guts from germ-free (GF) group: dechorionated eggs reared on antibiotic treated food (Dechorionated + food with antibiotic). However, bacterial colonies were found in other three treatment cohorts: DNA (dechorionated eggs + food without antibiotics), NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).

    Confirmation of germfree RPW individuals with culture-independent assays
    1. The lysed guts in Step D4 (gut dissection and homogenization) were employed to extract the total gut bacteria DNA with DNeasy blood and tissue Kit (Qiagen) following the manufacturer guidelines.
    2. Repeat the final elution twice in 80 µl buffer AE for improving DNA yield.
    3. Quantify the purity and concentration of the extracted DNA by running on 1% gel stained with ethidium bromide and using Nanodrop 1000 (Thermo Scientific, USA).
    4. Prepare the 25 µl PCR reaction system:
      50 ng template DNA
      1 µl of each forward (27F: 5ʹ-AGAGTTTGATCATGGCTCAG-3ʹ) and reverse primer (1492R: 5ʹ-TACGGYTACCTTGTTACGACTT-3ʹ)
      12.5 µl of 2x Taq PCR Mastermix (Tiangen Biotechnology Beijing, China)
      Up to 25 µl with PCR grade ddH2O
    5. Set up a negative control for PCR by adding 1 µl PCR grade ddH2O as a template while other reagents remain the same.
    6. Run PCR reactions with the thermal conditions as in Table 1.

      Table 1. The thermal conditions of Bacterial 16R rRNA-based PCR assays


    7. Detect the PCR products with electrophoresis on 1% agarose gel. Prepare a 1% agarose gel by dissolving 1.0 g agarose in 100 ml 0.5x TAE buffer and microwave the solution until it has completely dissolved.
    8. Carefully pour the gel solution in an agarose gel cast tray to avoid bubbles formation and let it polymerize for at least 30 min.
    9. Place the gel in the electrophoresis unit filled with 0.5x TAE buffer.
    10. Mix 4 µl of PCR products with 1 µl loading buffer and add to the wells of the gel.
    11. Load 5 µl 2,000 bp DNA marker and run the gel for 30 min at 90 V.
    12. Visualize the gel under the UV light to detect the target band with the expected size of 1,500 bp.
    13. No target PCR band is observed in RPW GF larvae, it presents in other three groups (Figure 5). This result is line with that of culture-dependent method.


      Figure 5. Verification of gut bacteria-derived PCR products with bacterial 16S rRNA-based PCR assays. GF (Dechorionated eggs + food with antibiotics), DNA (dechorionated eggs + food without antibiotics) NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).

  5. Conclusion
    Here, we developed a protocol to generate RPW germ-free larvae for further investigation on the interplays between RPW larvae and their associated gut bacteria. This method requires strict sterile conditions, in other words, each step in our protocol should be completed inside the laminar hood. Taken together, following our reported protocol here, RPW GF larvae can be generated from dechorionated eggs which are reared on sterilized artificial food under axenic conditions.

Data analysis

Run a One-way Analysis of Variance (ANOVA) to determine the significance in bacterial CFUs across the different treatment groups. Significant differences were determined in the number of bacterial colonies across different cohorts. The guts of RPW individuals from DNA and NDA group were colonized by significantly less bacteria colonies as compared to CR insects (Figure 6).


Figure 6. Quantification of bacterial colony forming units (CFUs) in the guts of RPW larvae from different treatment groups. The number of CFUs of each plate was counted 24 h after incubation at 37 °C. A One-way ANOVA (Tukey’s HSD post hoc multiple comparisons) was run to detect the statistical differences of bacterial CFU across the treatments. The letters indicate significance at P < 0.05. GF (Dechorionated eggs + food with antibiotics), DNA (dechorionated eggs + food without antibiotics), NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).

Recipes

  1. The artificial food for RPW larvae (Table 2)

    Table 2. Composition of artificial diet used for RPW rearing


  2. Antibiotic stock solution (Table 3)

    Table 3. Preparation of antibiotics stock and working concentrations


  3. 10x PBS stock solution
    1. Prepare PBS stock solution (10x) by dissolving 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4 into 800 ml distilled water in a conical flask
    2. Mix it well on a magnetic stirrer and adjust pH to 7.4 with HCl in a final volume of 1 L
    3. Autoclave the solution for 20 min at 121 °C and store in aliquots at 4 °C
  4. LB agar medium
    1. Take 5 g tryptone, 5 g NaCl, 2.5 g yeast extract, 7.5 g agar and add it to a 400 ml distilled water. Mix it well in a 1 L conical flask.
    2. Adjust the pH to 7.0 with NaOH in a final volume of 500 ml
    3. Autoclave the media for 20 min at 121 °C
    4. Let the agar cool down to 55 °C and then pour (20 ml) it into sterilized Petri dishes
    5. Let it polymerize for about 40 min and then seal it with parafilm
    6. Store the LB agar plates in the fridge at 4 °C
  5. 10x TAE stock solution
    1. Prepare TAE stock solution (10x) by dissolving 48.4 g Tris, 11.4 ml of glacial acetic acid and 3.7 g disodium EDTA in 800 ml distilled water
    2. Stir the solution until dissolved completely and make the final volume 1 L with distilled water. There is no need to sterilize the solution and store it at room temperature

Acknowledgments

We are very indebted to the financial support from National Natural Science Foundation of China (31470656), the National Key Research and Development Project of China (2017YFC1200605) and Natural Science Foundation of Fujian Province (2018J01705).This protocol was modified from our previous work (doi:10.3389/fmicb.2019.01212) which has been published in Frontiers in Microbiology.

Competing interests

The authors declare that they do not have any competing interest.

Ethics

Rhynchophorus ferrugineus individuals are treated according to the standard ethical protocol.

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  23. OEPP/EPPO (2008). Data sheets on quarantine pests: Rhynchophorus ferrugineus. Bulletin OEPP 38: 55-59. 
  24. Peng, L., Miao, Y. and Hou, Y. (2016). Demographic comparison and population projection of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) reared on sugarcane at different temperatures. Sci rep-UK 6: e31659. 
  25. Pu, Y. and Hou, Y. (2016). Isolation and identification of bacterial strains with insecticidal activities from Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae). J Appl Entomol 140: 617-626. 
  26. Shi, Z. H., Lin, Y. T. and Hou, Y. M. (2014). Mother-derived trans-generational immune priming in the red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera, Dryophthoridae). Bull Entomol Res 104(6): 742-750.
  27. Tagliavia, M., Messina, E., Manachini, B., Cappello, S. and Quatrini, P. (2014). The gut microbiota of larvae of Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae). BMC microbiol 14(1): 136. 
  28. Xiao, R., Wang, X., Xie, E., Ji, T., Li, X., Muhammad, A., Yin, X., Hou, Y. and Shi, Z. (2019). An IMD-like pathway mediates the intestinal immunity to modulate the homeostasis of gut microbiota in Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae). Dev Comp Immunol 97: 20.

简介

红掌象鼻虫(RPW), Rhynchophorus ferrugineus Olivier,是全世界毁灭性的棕榈树害虫。RPW肠道被多种细菌殖民,这些细菌深刻影响宿主的发育和营养代谢。但是,RPW及其肠道菌群之间相互作用的分子机制仍然未知。通常使用抗生素去除肠道细菌,以研究肠道细菌对昆虫适应性的影响。但是,抗生素的使用不能彻底清除大多数昆虫的肠道细菌。因此,建立无菌(GF)生物是揭示肠道细菌与其昆虫宿主之间相互作用的有力方法。在这里,我们描述了一种生成和维护RPW GF幼虫的协议,该协议在实验室中完全没有肠道细菌。RPW GF幼虫由去皮的新鲜鸡蛋制成,并在无菌条件下饲养在无菌人工食品上。GF幼虫的建立为深入阐明RPW及其肠道菌群之间相互作用的分子机制奠定了坚实的基础。

【背景】红棕榈象鼻虫(Olivier)(Coleoptera:Dryophthorodae)是世界上最有害的棕榈树害虫之一(Li et al。 ,2009年; Ju 等人,2011年; Al-Dosary 等人,2016年; Peng 等人,2016年)。它原产于南亚和美拉尼西亚,但最近已广泛传播到欧洲,非洲,一些美洲和其他亚洲国家(OEPP / EPPO 2008; Shi et al。,2014)。RPW的幼虫是主要的破坏剂,从棕榈的顶端生长点取食嫩组织和树干中的汁液。RPW幼虫在掌干内完成整个发育时间,直到成年成年人飞出来寻找新的产卵位点(Kaakeh,2005; OEPP / EPPO,2008; El-Mergawy和Al-Ajlan,2011)。害虫的隐蔽侵扰行为使其控制更具挑战性。目前,针对这种害虫的控制措施包括使用合成杀虫剂,植物卫生,基于信息素的物质诱捕和释放一些生物控制剂,例如昆虫病原真菌,细菌和线虫(Murphy和Briscoe,1999; Faleiro,2006)。 ; Mazza et al。,2014; Pu and Hou,2016)。但是,由于出现了抗药性和其他控制措施的局限性,迫切需要制定一些可持续,环保和经济的管理策略,以有效保护棕榈树免受这种害虫的侵害。



像哺乳动物一样,昆虫与肠道菌群共生,它们在许多宿主生理过程中起着至关重要的作用,包括发育,消化和排毒,免疫力和化学交流(Douglas,2009、2010和2015; Engel和Moran,2013)。肠道细菌是通过工程化的共生体表达某些特定的生物活性分子(会损害昆虫适应性以实现害虫控制)的极佳的转基因媒介(Engel和Moran,2013)。因此,阐明害虫及其共生微生物之间的相互作用可能为新型害虫治理策略的发展提供重要意义(Butera et al。,2012; Crotti et。,2012年)。



RPW由于其巨大的经济和环境损失,近年来引起了极大的关注。最近,一些初步研究已经破译了RPW幼虫和成虫的肠道细菌成分。已确定RPW肠道被细菌克隆,这些细菌主要来自肠杆菌科,乳杆菌科,醋杆菌科,内质体科,肠球菌科和链球菌科(贾等人。 ,2013年; Tagliavia等人,2014年;蒙大拿州等人,2015年;穆罕默德等人,2017年)。此外,我们发现分泌蛋白 Rf PGRP-LB和NF-ĸB样转录因子 Rf Relish调节肠道免疫以调节RPW的稳态肠道菌群(Dawadi等,2018; Xiao等,2019)。此外,还发现RPW的肠道菌群可以通过调节其营养代谢来影响其宿主的生长和发育(Muhammad et al。,2017; Habineza et al。 ,2019)。不幸的是,我们发现使用抗生素鸡尾酒无法彻底清除RPW幼虫的肠道细菌(Muhammad et al。,2017)。越来越多的证据强烈表明,GF动物模型是剖析动物与其肠道菌群之间串扰的关键工具之一(Grover和Kashyap,2014; Koyle等,2016; Kietz等) 。,2018年)。因此,为了研究住宅肠道菌群对RPW 生理的作用,生成了RPW GF幼虫来解释宿主-肠道细菌之间的相互作用。与抗生素管理相比,报告中的方案可以成功,轻松地建立和维持RPW GF幼虫。也容易产生具有特定肠道细菌种类的gnotobiotic RPW幼虫。

关键字:红棕象甲, 无菌幼虫, 肠道菌群, 昆虫共生, 寄主生理

材料和试剂

  1. 穿孔塑料瓶(330毫升,直径70毫米,高度107毫米;嘉丰园艺产品有限公司,重庆,中国)
  2. 锥形烧瓶
  3. 培养皿(直径90毫米,江苏盐城汇达医疗器械有限公司)
  4. 画笔(1厘米宽),用于收集新鲜的鸡蛋
  5. 0.22 µm注射器过滤器(美国颇尔公司)
  6. 刮铲(长20厘米,尖端宽4厘米)
  7. PCR试管(Biosharp Life Sciences,合肥,中国)
  8. 1.5 ml微量离心管(Biosharp Life Sciences,合肥,中国)
  9. 孔径为60 µm的金属筛(瑞士Sigma-Aldrich Co. Ltd.,目录号:Z289744)
  10. 铝箔
  11. 封口膜(美国Bemis PM-996)
  12. 10 ml无菌注射器(Hamilton ®注射器,瑞士Sigma-Aldrich Co. Ltd.)
  13. 滤纸(90毫米直径,阜阳特种纸业有限公司,中国杭州)
  14. 甘蔗(Semcharum sinensis) Roxb。(中国福建省漳州市)
  15. 用于扩增细菌16S rRNA基因(27F:5 ' -AGAGTTTGATCATGGCTCAG-3 ' ,1492R:5 ' -TACGGYTACCTTGTTACGACTT-3 ' )(上海生物工程有限公司,上海,中国)
  16. Trans2K ® DNA标记(Transgene BioTech。Co. Ltd.,中国北京)
  17. 蒸馏水
  18. 乙醇(Sigma-Aldrich,CAS号:64-17-5)
  19. 次氯酸钠(NaClO)(Sigma-Aldrich,CAS号:7681-52-9)
  20. 抗生素
    1. 卡那霉素(Sigma-Aldrich,CAS号:70560-51-9)
    2. 四环素(Sigma-Aldrich,CAS号:64-75-5)
    3. 庆大霉素(Sigma-Aldrich,CAS号:1405-41-0)
    4. 红霉素(Sigma-Aldrich,CAS号:114-07-8)
  21. 胰蛋白((OXOID,目录号:2406419)
  22. 氯化钠(NaCl)(Sigma-Aldrich,CAS号:746398)
  23. 氯化钾(KCl)(CAS号:7447-40-7)
  24. 无水磷酸氢二钠(Na 2 HPO 4 )(CAS编号:7558-79-4)
  25. 盐酸(CAS号:7647-01-0)
  26. 磷酸一氢钾(KH 2 PO 4)(CAS号:7778-77-0)
  27. 酵母提取物(CAS号:8013-01-2)
  28. 琼脂(CAS号:9002-18-0)
  29. 氢氧化钠(CAS号:221465)
  30. DNA提取试剂盒(DNeasy血液和组织试剂盒,德国恰根)
  31. 6x DNA上样缓冲液(TRANS ®,转基因生物技术有限公司,中国北京)
  32. 0.5x TAE缓冲液(Tris-Acetate EDTA)
  33. 2x Taq PCR Mastermix(天健生物技术有限公司,中国北京)
  34. Tris-HCl(Solarbio生命科学有限公司,中国北京)
  35. 冰醋酸(CAS号:64-19-7)
  36. 抗坏血酸
  37. 蔗糖
  38. 酪蛋白
  39. 玉米粉
  40. 阿维切尔
  41. 胆固醇
  42. 氯化胆碱
  43. 肌醇
  44. 山梨酸钾
  45. 对羟基苯甲酸钠
  46. EDTA二钠(CAS号:6381-92-6)
  47. 琼脂糖(转基因生物技术有限公司,中国北京)
  48. TRANS ®溴化乙锭凝胶染料(Transgen。Biotech。Co. Ltd.,中国北京)
  49. 10x PBS储备溶液(请参阅食谱)
  50. 抗生素原液(请参阅食谱)
  51. LB琼脂培养基(请参见食谱)
  52. 10倍的TAE储备溶液(请参阅食谱)
  53. RPW幼虫的人工食品(请参见食谱)

设备

  1. Heal Force safe-1200LC生物安全柜(Heal Force Bio-meditech Holdings Co. Ltd.,中国上海)
  2. 高压釜(中国深南LDZF-75KB-II)
  3. SCIENTZ-48组织裂解仪(中国宁波宁波生物技术有限公司)
  4. 海尔冰箱(中国青岛海尔有限公司)
  5. 剪刀(世界精密仪器公司,美国萨拉索塔)
  6. 尖头镊子(3.9 x 3.0 x 1.0英寸,世界精密仪器公司,美国萨拉索塔)
  7. 带弯曲的圆形尖端的镊子(1.65毫米x 0.05毫米x 120毫米,宽度x厚度x长度,世界精密仪器公司,美国萨拉索塔)
  8. 使用Yiheng ®孵化器(500 x 460 x 800 mm,中国上海宜亨仪器有限公司)来饲养轴突幼虫(条件设置为37°C,黑暗24小时)
  9. BIO-RAD T100 TM PCR热循环仪(美国Bio-Rad Co. Ltd.)
  10. Eppendorf 5804 R离心机(德国Eppendorf有限公司)
  11. 凝胶电泳仪(六一仪器有限公司,中国北京)
  12. Nikon ®体视显微镜(日本尼康有限公司)
  13. 赛富ZRX-260培养箱,用于维持RPW实验室人数(260升,中国宁波实验仪器有限公司)
  14. Nanodrop 1000(Thermo SCIENTIFIC,美国)
  15. 微波(中国美的电器有限公司)
  16. 电磁搅拌器(江苏金坛市荣华实验仪器有限公司)
  17. BSA124S电子天平(德国Sartorius Co. Ltd.)
  18. 紫外线透射仪(中国北青科技有限公司)
  19. 层流罩(Heal Force Safe-1200LC)

软件

  1. IBM SPSS Statistics(22.0)

程序

  1. 昆虫的养护
    1. 被困在福建省福州市平潭区(北纬119°32′,北纬25°31′)和福建农林大学金山校区(北纬119°30′)的成年人建立并维持了RPW实验室种群。 ,26°08′N)。在27±1°C,75±5%相对湿度和24 h暗光条件下,在孵化器(Saifu ZRX-260,Ninbo实验仪器有限公司,中国)中给RPW个体喂甘蔗茎。适用于成虫的幼虫和12小时亮/ 12小时黑暗(35毫米x 10毫米,长x宽,图1)。


      图1。红色棕榈象鼻虫(Rhynchophorus ferrugineus)的四个不同生命阶段

    2. 将雌性和雄性RPW成虫放在带孔的塑料瓶中。为它们提供甘蔗茎切片(30毫米x 15毫米,长x宽)以供喂养和产卵。每三天更换一次食物。
    3. 产下12小时后将新鲜的鸡蛋切成薄片,将甘蔗茎切片切成薄片,以进行去绒毛,产生RPW GF幼虫。

  2. 制备人造食品和抗生素原液
    1. 制作RPW人造食品(请参见配方1)并准备抗生素储备溶液(请参见配方2)。
    2. 针筒过滤器(0.22 µm)将抗生素储备液分装。
    3. 在可高压灭菌的1 L锥形瓶中准备人造食品,并使用高压灭菌器在121°C下灭菌20分钟。
    4. 当温度降至约55°C时,将抗生素混合物(600 mg / kg,每个150 mg / kg)和1 g抗坏血酸添加到高压灭菌的人造食品中。
    5. 固化后,准备一批平行的不含抗生素的人工饮食,用等体积的无菌蒸馏水代替。
    6. 固化后,在培养皿中准备好等分的人造食品,并用封口膜密封,以免RPW个人更换食物时潜在的微生物污染。
      注意:在生物安全柜中执行此步骤。
    7. 饮食可以保存在4°C以便以后使用。

  3. 通过卵去绒毛作用产生RPW GF个体
    1. 用75%的乙醇消毒所有工具,包括画笔,金属筛,刮铲(20 cm x 4 cm,长x宽),镊子和镊子。
    2. 打开紫外线灯30分钟,以消毒层流罩的表面和内部的其他材料。
    3. 将鸡蛋分为两组:a)未去皮蛋(对照组)和b)去皮蛋。每组使用30个鸡蛋。
    4. 对于蛋的去绒毛,请按照Koyle et al。(2016)的以下步骤处理去蛋的蛋:
      1. 用10%的次氯酸钠溶液(NaClO)清洗刚产下的鸡蛋3-5分钟,然后通过金属筛过滤。
      2. 将这些鸡蛋用75%的乙醇冲洗两次,然后通过金属筛过滤。
      3. 将这些鸡蛋用无菌蒸馏水洗涤两次,以洗去漂白剂(NaClO)和乙醇,并通过金属筛过滤载有去壳蛋的水。对于对照组的鸡蛋,只能用高压灭菌的蒸馏水洗净,然后通过金属筛过滤载有鸡蛋的水。
    5. 在立体显微镜下观察去壳蛋,我们发现去壳蛋与对照相比变成白色(图2)。


      图2.红色棕榈象鼻虫, Rhynchophorus ferrugineus 的未去壳和去壳蛋。(A)仅用高压灭菌蒸馏水洗涤未去壳的鸡蛋,(B)去皮蛋用10%次氯酸钠溶液(NaClO),75%乙醇和无菌蒸馏水洗涤。

    6. 将经过去绒毛的鸡蛋转移到无菌的培养皿中(直径90毫米),并在层流罩(Heal Force Safe-1200LC)内用一些经过紫外线消毒的湿吸收棉。在黑暗中,蛋可以在27±1°C和75±5%相对湿度的条件下在层流罩内孵化。
    7. 每隔24小时观察卵以进行孵化。两到三天后,它们会孵化。
    8. 将新生幼虫转移到带有3 g固体人工饮食(步骤C6)(有或没有抗生素)的新无菌培养皿中。每两天刷新一次食物。
    9. 将RPW幼虫维持在27±1°C,75±5%相对湿度和24 h黑暗光周期中。
    10. 每三天更换一次人造食品。
    11. 为了验证我们协议的效率,此处指定了四组:1)去壳蛋+含抗生素的食物,2)去壳蛋+不含抗生素(DNA)的食物,3)非去壳蛋+含有抗生素(NDA)的食物,以及4)非去皮蛋+不含抗生素的食物(常规饲养CR)。第1和第3组中的幼虫保留在层流罩中,而其他两个组(分别作为阳性和阴性对照)保留在培养箱中(Ninbo实验仪器)。

  4. 通过培养依赖性和非依赖性测定验证无菌RPW个体
    肠解剖和均质化
    1. 为了验证RPW的GF状态,从每个处理中随机选择三个幼虫(第5龄幼虫,图3)作为解剖的重复样本,每组至少折衷了三个重复样本。
    2. 为了消除相关微生物对RPW幼虫体表的潜在影响,将标本浸入10%NaClO中30秒钟,浸入75%乙醇中90秒钟,并用无菌蒸馏水冲洗两次。此步骤在层流罩内部完成。
    3. 在无菌显微镜下,在干净的培养皿中,用消毒的剪刀和镊子在立体显微镜下解剖肠壁。
    4. 使用组织溶解器(48个组织溶解器,宁波信实生物技术有限公司,中国)在装有1 ml无菌PBS的1.5 ml微量离心管中匀浆收集的肠。


      图3.第五个RPW幼虫的肠道

    通过培养依赖性分析验证无菌RPW个体
    1. 用无菌PBS连续稀释(10 -1 -10 -4 倍)肠匀浆。
    2. 一式三份,将100 µl稀释的匀浆物在LB琼脂平板上。
    3. 在37°C下需氧培养板24小时,然后检查细菌生长。
    4. 如预期的那样,在含有添加了抗生素的食物的去蛋壳蛋的肠匀浆中的板上没有细菌菌落出现(去蛋壳蛋+含有抗生素的食物,图4)。但是,细菌菌落在含有其他三组肠道匀浆的平板上长大(图4)。因此,有证据表明成功产生了RPW GF幼虫。


      图4.不同治疗组的RPW幼虫肠道中细菌生长的培养依赖性验证。在无菌(GF)组的幼虫肠道中未观察到细菌菌落:抗生素处理过的食物(去皮+含抗生素的食物)。但是,在其他三个治疗队列中发现了细菌菌落:DNA(去皮蛋+未添加抗生素的食物),NDA(未去皮蛋+已添加抗生素的食物)和常规饲养的CR(未去皮蛋+未添加抗生素的食物)。

    通过与培养无关的测定法确认无菌RPW个体
    1. 按照制造商的指导,使用DNeasy血液和组织试剂盒(Qiagen),将步骤D4中裂解的肠道(肠道解剖和均质化)用于提取总肠道细菌DNA。
    2. 在80 µl缓冲液AE中重复两次最终洗脱,以提高DNA产量。
    3. 通过在1%溴化乙锭染色的凝胶上运行,并使用Nanodrop 1000(Thermo Scientific,美国),对提取的DNA的纯度和浓度进行定量。
    4. 准备25 µl PCR反应系统:
      50 ng模板DNA
      1μl的每个前向(27F:5 ' -AGAGTTTGATCATGGCTCAG-3 ' )和反向引物(1492R:5 ' -TACGGYTACCTTGTTACGACTT-3 ' )
      12.5 µl 2x Taq PCR Mastermix(天健生物技术有限公司,中国北京)
      最高25 µl,PCR级ddH 2 O
    5. 通过添加1 µl PCR级ddH 2 O作为模板,设置PCR阴性对照,而其他试剂保持不变。
    6. 在表1所示的热条件下运行PCR反应。

      表1.基于细菌16R rRNA的PCR测定的热条件


    7. 在1%琼脂糖凝胶上电泳检测PCR产物。通过将1.0 g琼脂糖溶解在100 ml 0.5x TAE缓冲液中,并用微波将溶液完全溶解,来制备1%琼脂糖凝胶。
    8. 小心地将凝胶溶液倒入琼脂糖凝胶铸盘中,以避免形成气泡,并使其聚合至少30分钟。
    9. 将凝胶放入装有0.5x TAE缓冲液的电泳装置中。
    10. 将4 µl PCR产物与1 µl上样缓冲液混合,并添加到凝胶孔中。
    11. 加载5 µl 2,000 bp DNA标记,并在90 V下运行凝胶30分钟。
    12. 在紫外光下可视化凝胶以检测预期大小为1,500 bp的目标条带。
    13. 在RPW GF幼虫中未观察到目标PCR带,它出现在其他三个组中(图5)。此结果与文化相关方法的结果相符。


      图5.使用基于细菌16S rRNA的PCR测定法验证肠道细菌衍生的PCR产品。 GF(去壳蛋+含抗生素的食物),DNA(去壳蛋+不含抗生素的食物)NDA(非去皮蛋+含抗生素的食物)和常规饲养的CR(非去皮蛋+不含抗生素的食物)。

  5. 结论
    在这里,我们开发了一个协议来生成RPW无菌幼虫,以进一步研究RPW幼虫与其相关的肠道细菌之间的相互作用。此方法需要严格的无菌条件,换句话说,本协议中的每个步骤都应在层流罩内完成。综上所述,按照我们在此报告的操作规程,RPW GF幼虫可以由去皮蛋产生,将其在无菌条件下饲养在无菌人工食品上。

数据分析

运行方差单向分析(ANOVA),以确定不同治疗组中细菌CFU的重要性。确定了不同队列中细菌菌落数量的显着差异。与CR昆虫相比,来自DNA和NDA组的RPW个体的肠道被细菌菌落的数量明显减少(图6)。


图6.不同处理组的RPW幼虫肠道中细菌菌落形成单位(CFU)的定量。在37°C孵育24小时后,计算每块板的CFU数量。运行单向方差分析(Tukey的HSD事后多重比较)以检测各处理过程中细菌CFU的统计差异。字母表示 P &lt; 0.05。GF(去皮蛋+含抗生素的食物),DNA(去皮蛋+不含抗生素的食物),NDA(非去皮蛋+含抗生素的食物)和常规饲养的CR(未去皮蛋+不含抗生素的食物)。

菜谱

  1. RPW幼虫的人造食品(表2)

    表2。用于RPW饲养的人工饮食组成


  2. 抗生素原液(表3)

    表3.抗生素储备液和工作浓度的制备


  3. 10x PBS储液
    1. 通过溶解80 g NaCl,2 g KCl,14.4 g Na 2 HPO 4 和2.4 g KH 2 制备PBS储备溶液(10x)将PO 4 放入锥形瓶中的800毫升蒸馏水中
    2. 在磁力搅拌器上充分混合,并用HCl将pH调节至7.4,最终体积为1 L
    3. 在121°C下高压灭菌溶液20分钟,并在4°C下等分储存
  4. LB琼脂培养基
    1. 取5克胰蛋白,、 5克氯化钠,2.5克酵母提取物,7.5克琼脂,将其加入400毫升蒸馏水中。将其在1 L锥形瓶中充分混合。
    2. 用NaOH将pH调节至7.0,最终体积为500 ml
    3. 在121°C下高压灭菌介质20分钟
    4. 让琼脂冷却至55°C,然后将(20 ml)琼脂倒入灭菌的培养皿中
    5. 让其聚合约40分钟,然后用石蜡膜密封
    6. 将LB琼脂板保存在4°C的冰箱中
  5. 10倍TAE储备液
    1. 通过在800 ml蒸馏水中溶解48.4 g Tris,11.4 ml冰醋酸和3.7 g EDTA二钠来制备TAE储备溶液(10x)
    2. 搅拌溶液直至完全溶解,并用蒸馏水制成最终体积1L。无需对溶液进行灭菌并将其保存在室温下

致谢

我们非常感谢中国国家自然科学基金(31470656),中国国家重点研究开发项目(2017YFC1200605)和福建省自然科学基金(2018J01705)的财政支持,该协议是根据我们以前的工作修改而来的(doi:10.3389 / fmicb.2019.01212)已发布在微生物学前沿中。

利益争夺

作者宣称他们没有任何竞争利益。

伦理

根据标准的伦理规范对 Rhynchophorus ferrugineus 个体进行治疗。

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引用:Muhammad, A., Habineza, P., Hou, Y. and Shi, Z. (2019). Preparation of Red Palm Weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) Germ-free Larvae for Host-gut Microbes Interaction Studies. Bio-protocol 9(24): e3456. DOI: 10.21769/BioProtoc.3456.
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