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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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-symbio....

Protocol

D. antiqua are obtained from the field of Fanzhen, Taian.

1. Preparation of sterile diets

  1. 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.......

Representative Results

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.......

Discussion

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

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
0.22 μM filter bottleThermo Scientific450-0045
0.22 μM Syringe FilterBiosharpBS-QT-011
100-mesh sieveZhejiang Shangyu Jinding Standard Sieve FactoryNo Catalog numbers
1x PBS solutionSolarbioP1020
2x Taq PCR Master MixGENVIEWGR1113-1ML
5.2% NaClO solutionSinopharm Chemical Reagent Co., Ltd.80010428
500 mL Conical flaskThermo Scientific4103-0500
50 mL vented centrifuge tubeJET BIOFILBRT-011-050
50x TAE bufferGENVIEWGT1307
Agar powderDing GuoDH010-1.1
Biochemical incubatorSTIK21040121500010
Cell sieveSAINING5022200
Choline chlorideSangon BiotechA600299-0100
ddH2ODing GuoPER018-2
Disposable grinding pestleJET BIOFILCSP-003-002
DNA extraction kitSangon BiotechB518221-0050
DNA MarkerSangon BiotechB600335-0250
Ethanol absoluteSinopharm Chemical Reagent Co., Ltd.10009218
Filter paperNEWSTAR1087309025
Food processorGuangdong Midea Life Electric Appliance Manufacturing Co., Ltd.WBL25B26
Illuminated  incubatorShanghai ESTABLISH Instrumentation Co., Ltd.A16110768
L-Ascorbic acidSangon BiotechA610021-0100
L-shaped spreaderSAINING6040000
Nutrient agar mediumHope BioHB0109
ScissorsBing Yu BY-103Purchase on Jingdong
Shock incubatorShanghai Zhichu Instrument Co., Ltd.2020000014
SucroseGENVIEWCS326-500G
Super Green nucleic acid dyeBiosharpBS355A
Super-clean tableHeal ForceAC130052
TSBHope BioHB4114
Vacuum pumpZhejiang Taizhou Seeking Precision Vacuum Pump Co., Ltd.22051031
Yeast extractThermo ScientificLP0021B

References

  1. Al-Asmakh, M., Zadjali, F. Use of germ-free animal models in microbiota-related research. Journal of Microbiology and Biotechnology. 25 (10), 1583-1588 (2015).
  2. Bhattarai, Y., Kashyap, P. C. Germ-free mice model for studying host-....

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Axenic Delia AntiquaHalf fermented Sterile DietsMicrobial EcologyGut MicrobiotaWolbachiaAntibiotic TreatmentInsect microflora InteractionsCulture dependent And Culture independent AssaysOnion Pest

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