Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Summary

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

Abstract

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

Introduction

The microbiota (i.e., Archaea, Bacteria, Eukarya, and viruses) play crucial roles in maintaining host health and contributing to the development of various diseases by influencing physiological and pathological processes through symbiotic interactions within the intestinal barrier, epithelial surface, and mucin functions in individuals^1,2,3. The composition of the microbiota across different life stages, from infancy to juvenility, adulthood, and aging, as well as its presence in various locations such as nares, oral, skin, and gut sites, is dynamic....

Protocol

The fish experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Chongqing and the Institutional Animal Care and Use Committee of Chongqing Medical University, China, as well as the standards for experimental animals issued by the State Bureau of Quality and Technical Supervision (Approval ID: GB14922-2001 to GBT14927-2001). Zebrafish (Danio rerio, wild type, AB strain) were sourced from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in the laboratory following previously reported procedures³⁶.

1. Adult zebrafish maintenance and e....

Results

The GF zebrafish models can be efficiently produced by utilizing the abundant eggs spawned by pairs of zebrafish, with the protocol optimized based on previous GF fish models³⁵. A single 6-well plate can culture approximately 30-48 embryos/larvae, allowing for ample data collection and statistical analysis. After sterile treatment, the GF embryos are cultured in a clean incubator till hatching to larvae at 48-72 h, and changed GZM daily with the detection of collected samples, which is crucial to .......

Discussion

Critical steps within the protocols of GF fish and GF food preparation
During the generation of GF fish models, several critical steps were involved, including the preparation of sterile materials, sterilization of embryos, daily renewal of GZM, collection of various samples, and the sterile examination of each sample using multiple methods. Among these steps, the initial treatment of embryos is fundamental and decisive for the success of GF models. Controlling agents, their concentrations, and tre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AB-GZM	Amphotericin:Solarbio;  kanamycin:Solarbio; Ampicillin:Solarbio.	Amphotericin:CAS:1397-89-3; kanamycin:CAS: 25380-94-0; Ampicillin:CAS: 69-52-313.	49.6 mL GZM, 50 µL amphotericin stock solution (250 µg/mL), 25 µL kanamycin stock solution (10 mg/mL), and 250 µL ampicillin stock solution (20 mg/mL).
1.5 mL, 15 mL, 50 mL EP tubes	biosharp	BS-15-M	To collect samples, and hold agents
2.4 g/L NaClO	XILONG SCIENTIFIC Co., Ltd.	CAS: 7681-52-9	Diluted with 8% sodium hypochlorite aqueous solution.
6-well plates, 24-, 48- well plates	LABSELECT	11112	To culture fish
Aeronomas	NCBI database	No.MK178499	2019-JPP-ESN
Anaerobic TSA plates	tryptone:Oxoid ; soy peptone:Solarbio ;NaCl:Biosharp; agar powder:BioFroxx.	tryptone:LP0042B; soy peptone:Cat#S9500; NaCl:BS112; agar powder:9002-18-0.	The TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, and 6 g agar powder under the anaerobic system.
Anaerobic work station	GENE SCIENCE	E200G	Bacterial isolation, sterile testing
Analysis	GraphPad Prism 5	v6.07	To analysis the data
API 20 E kits	BioMerieux SA, France	No.1005915090	Ref 20100 Kits to detect bacterial metabolism
Artemia (Brine shrimp)	Shangjia Aquarium Co., Ltd.	Aquamaster brand	Artemia cysts, and brine shrimp eggs
Auto cycle system for fish culture	Ningbo Hairui Technology Co., Ltd	No Cat	Maintain the fish
Autoclave	Zeal Way	G154DWS	Prepare the materials
BHI Aerobic	Coolaber	Cat#PM0640	BHI medium was prepared, wherein 100 mL medium included 3.7 g BHI powder.
BHI Anaerobic	Coolaber	Cat#PM0640	BHI medium was prepared and divided into anaerobic tubes under the anaerobic system.
Biochemical incubator	LongYue Co., Ltd	SPX	For fish and plates
Biosafety cabinet	Haier	HR40-IIA2	Sterile treatment and testing
Bleaching agent of 0.02 g/L NaClO	XILONG SCIENTIFIC Co., Ltd.	CAS: 7681-52-9	Working solution with sodium hypochlorite (NaClO) concentration: Diluted with 8% sodium hypochlorite aqueous solution or 166.6 uL 6% sodium hypochlorite with 500 mL distilled water.
Blood plates	sheep blood:Solarbio	Cat. NO. TX0030	Sterile-defibrinated sheep blood was added into TSA to prepare 5% blood plates.
Cell culture flask	Corning	430639	To culture fish
CM-Dil dyes	Molecular Probes	Cat#C7000	To label the bacteria
Constant temperature shaking incubator	Peiving Co., Ltd	HZQ-X100	Bacterial culture
Database	NCBI	Bacteria and Archaea database	Link: Archaea FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Archaea/ Bacteria FTP: ftp://ftp.ncbi.nlm.nih.gov/refseq/TargetedLoci/Bacteria/
Disposable Pasteur pipette	biosharp	bs-xh-03l	Used to change water, and transfer eggs
Disposable petri dish	biosharp	BS-90-D	To culture fish
DNA kits	Solaribio	Cat#D1600	Bacterial genomic DNA extraction kits
Electric pipette	SCILOGEX	Levo me	Change water
Exiguobacterium	NCBI database	No.MK178504	2019-JPP-ESN
GZM	Sea salt:LANDEBAO Co., Ltd.	No Cat	Composed of 1 L of water and 1.5 mL of sea salt solution (40 g/L), autoclaved. The content of sea salt in the GZM solution was 60 mg/L.
Laboratory pure water system	Hitech Co., Ltd	Prima-S15	Prepare the agents
Microscope	Nikon	SMZ18	With fluorescent light to observe fish larvae
PCR kits	TIANGEN	Cat#ET101	Taq DNA Polymerase kit
Pipette	LABSELECT	sp-013-10	Change water
Povidone iodine (PVP-I)	Aladdin	Lot#H1217005	Aqueous solution povidone iodine 0.4 g/L pure water.
Timing converter	PinYi Co., Ltd	AL-06	To regulate the light
TSA plates	tryptone:Oxoid ; soy peptone:Solarbio ;NaCl:Biosharp; agar powder:BioFroxx.	tryptone:LP0042B; soy peptone:Cat#S9500; NaCl:BS112; agar powder:9002-18-0.	TSA plates were prepared with 400 mL medium containing 6 g tryptone, 2 g soy peptone, 2 g NaCl, 6 g agar powder.
TSB Aerobic	tryptone:Oxoid ; soy peptone:Solarbio ;NaCl:Biosharp;	tryptone:LP0042B; soy peptone:Cat#S9500; NaCl:BS112;	TSB medium was prepared, wherein 400 mL medium included 6 g tryptone, 2 g soy peptone, and 2 g NaCl.
TSB Anaerobic	tryptone:Oxoid ; soy peptone:Solarbio ;NaCl:Biosharp;	tryptone:LP0042B; soy peptone:Cat#S9500; NaCl:BS112;	TSB medium was prepared and divided into the anaerobic tubes under the anaerobic system.
Ultra-clean workbench	Airtech	SW-CJ-2FD	Sterile treatment and testing
Ultra-pure flow system for fish culture	Marine Biological Equipment company	No Cat	Produce water for fish
Vibrio	NCBI database	No.MK178501	2019-JPP-ESN