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.
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.
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-symbiont interactions in model symbiotic systems. Axenic insects can be produced by sterilizing the egg stage and providing sterile food to the larvae and adults6,7. Axenic insects are of great significance and are widely used in biological research. For instance, a study conducted by Somerville et al.8 demonstrated that diamondback moths inoculated with Enterobacter cloacae improved the adaptability of transgenic males.
Delia antiqua Meigen is an economically important pest of onions and other Liliaceae crops worldwide, with its larvae damaging the bulbs of onions and other Liliaceae crops9. D. antiqua is mainly found in temperate climates and is widespread in onion-cultivation areas of the Americas, Europe, and Asia. If not properly controlled, it can cause crop losses in onions (Allium cepa L.), garlic (Allium sativum L.), shallots (Allium fistulosum L.), and leeks (Alliumchoenoprasum L.) ranging from 50% to 100%10,11. The larvae feed on the below-ground parts of plants, and this feeding causes the seedlings to wilt and eventually die. In addition, damaged plants can allow pathogens to enter, leading to bulb rotting12. Even if the plants are not completely consumed by the larvae, the damage they cause renders the onion plants unmarketable and results in economic losses.
Insects are closely associated with gut microbiota, and most insect guts contain a variety of symbiotic bacteria that thrive on the nutrients provided by the host13,14. Jing et al.15 showed that the primary function of the intestinal symbiotic community is to provide essential nutrients, followed by functions related to digestion and detoxification. In certain cases, gut bacteria can serve as a microbial resource for pest management purposes. Consequently, studying the individual gut bacteria's performances and specific functions within the body of D. antiqua is desirable. Therefore, preparing axenic larvae is particularly important for studying the interactions between specific bacterial strains and insects16. Currently, a commonly used method to eliminate insect gut bacteria is the use of an antibiotic combination to eradicate associated microbes17,18,19. Unlike using antibiotics alone, which can only reduce microbial numbers, axenic rearing of insects allows for control over the composition and quantity of microorganisms, enabling more accurate validation of gut microbiota functionality.
Thus, this article introduces a protocol for preparing and rearing axenic D. antiqua. Axenic larval food is obtained by utilizing high-temperature sterilization of natural diets combined with half-fermented foods. The eggs are sterilized following an experimental protocol to obtain axenic eggs, and finally, axenic larvae are cultured from the axenic eggs. The axenic rearing system was carried out for only one generation for the experiment. This will provide convenience for studying the interaction between insects and gut microbiota.
D. antiqua are obtained from the field of Fanzhen, Taian.
1. Preparation of sterile diets
2. Acquisition of axenic eggs
3. Rearing of axenic larvae
4. Validation of axenic larvae with culture-dependent assays
5. Validation of axenic larvae with culture-independent assays
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 Figure 6. The egg stage of non-axenically reared D. antiqua is 2.83 ± 0.29 days, and that of axenically reared D. antiqua is 2.83 ± 0.29 days, with no significant difference between the two (Independent t-test, t = 0.00, p = 1.00). The normal larval stage lasts for 13.83 ± 0.76 days, while the axenic larval stage lasts for 14.50 ± 0.50 days, with no significant difference between the two (Independent t-test, t = 1.27, p = 0.27). The pupal stage of normally reared D. antiqua is 17.33 ± 0.76 days, and that of axenically reared D. antiqua is 18.00 ± 0.50 days, with no significant difference between the two (Independent t-test, t = 1.27, p = 0.27). The survival time of adults is 81.50 ± 1.00 days for normal rearing and 80.33 ± 0.76 days for axenic rearing, with no significant difference between the two (Independent t-test, t = 1.61, p = 0.18). It can be observed that there is no significant difference in the developmental time between axenic D. antiqua and those raised under normal conditions.
Culture-dependent assays showed that no colonies were detected in the larval intestines (Figure 7). Furthermore, PCR analysis based on bacterial 16S rRNA revealed the presence of a band at around 1500 bp (Figure 8A), identified as Wolbachia (The GenBank accession number for this nucleotide sequence is: OR564190), an endosymbiotic bacterium. However, no bands were observed in the PCR analysis targeting fungal ITS regions (Figure 8B). This indicates that the larvae have achieved axenic state. Axenic larvae are of great significance for studying the mechanisms of interaction between D. antiqua and microorganisms, as well as for preventing and controlling the damage caused by D. antiqua.
Figure 1: The rearing devices and food for D. antiqua. (A) The white part of the scallion for larvae. (B) Water dispenser for adults. (C) Food for adults. (D) Collection device for the eggs. Please click here to view a larger version of this figure.
Figure 2: Components of diets preparation. (A) Ground scallions. (B) Fermented scallions. (C) Fermented scallion residue and filtrate. (D) Prepared diets. Please click here to view a larger version of this figure.
Figure 3: The process of egg transfer. (A) Eggs in the cell sieve. (B) 1 mL pipette without the end tip. (C) 1 mL pipette with eggs. (D) Eggs transferred to diets. Please click here to view a larger version of this figure.
Figure 4: Life stages of axenic D. antiqua. (A) Eggs. (B) Larvae. (C) Pupa. (D) Female and male adults. Please click here to view a larger version of this figure.
Figure 5: The process of dissecting larval intestines. (A) The head and tail of larva. (B) The larva with head and tail removed. (C) Separated intestinal and body tissues. (D) An intact intestine after dissection. Please click here to view a larger version of this figure.
Figure 6: Developmental time of each growth stage of normally reared and axenically reared D. antiqua. No significant difference in developmental time between axenically reared and normally reared D. antiqua. Independent t-test, p < 0.05. Please click here to view a larger version of this figure.
Figure 7: Confirmation of the sterility of D. antiqua by randomly selecting three larvae and incubating gut homogenates on TSA and PDA agar plates. No bacteria were observed in larvae fed sterile diets. (A) Axenic larvae on TSA. (B) Normal larvae on TSA. (C) Axenic larvae on PDA. (D) Normal larvae on PDA. Please click here to view a larger version of this figure.
Figure 8: Confirmation of the sterility of D. antiqua by PCR analysis using universal 16S rRNA primers and fungal ITS primers. The target band of the 16S rRNA gene is ~1,500 bp. A band of approximately 1500 bp was observed and identified as the endosymbiotic bacterium Wolbachia. The target band of the ITS gene is 500-750 bp. No target band was observed in the AF groups. (A) 16S rRNA electropherogram. (B) ITS electropherogram. Abbreviations: M = marker; NC = negative control; NF = normal feeding; AF = axenic feeding. Please click here to view a larger version of this figure.
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 fed P. xylostella with an artificial diet containing rifampicin to clear the gut bacteria. However, antibiotic treatment cannot completely eliminate gut bacteria and has many side effects on host insects. These include changes in metabolic enzyme activity, impaired gut function, inhibition of growth, development, and reproduction, as well as increased mortality rates23,24,25. Consequently, the functionality of the insect gut microbiota may be masked after antibiotic treatment. Fortunately, this issue can be overcome by chemically disinfecting the surface of the eggs, allowing for the production of axenic insects with relative ease16. Comparatively, the combination of surface disinfection of the eggs followed by feeding with sterile diets is more effective and feasible than antibiotic treatment26,27.
Obtaining axenic D. antiqua primarily relies on utilizing axenic eggs and sterile diets. Compared to other insect species, the eggs of D. antiqua are relatively small. Previously employed disinfection methods, such as briefly soaking the eggs in 0.1% sodium dodecylbenzene sulfonate followed by a 5 min treatment with a 2% hydrogen peroxide solution to obtain axenic cockroach eggs28, or using a 10% sodium hypochlorite solution for 10 min to surface disinfect eggs of the red palm weevil29, are not suitable for disinfecting D. antiqua eggs. Previous studies on the disinfection of eggs of Diptera species, such as utilizing hydrogen peroxide solutions used as a hand disinfectant and hydrogen peroxide as a surface disinfectant to sterilize Lucilia sericata (Diptera: Calliphoridae) eggs30, exist. Therefore, optimizing the choice of disinfectant and disinfection time becomes necessary. After experimentation, it has been found that using 0.26% NaClO and 75% EtOH, disinfecting the eggs for 0.5 min and repeating the process three times can achieve sterilization of D. antiqua eggs.
After obtaining axenic eggs, another crucial aspect of rearing axenic D. antiqua is providing them with sterile diets for the larvae in vitro. Although artificial diets cannot fully replicate natural food, studies31,32,33 have demonstrated the feasibility of rearing insects using artificial diets. The nutritional content of artificial diets can affect the immune system and adaptability of insects34. Therefore, it is important for artificially prepared diets to contain similar nutritional components to natural food. This will help ensure that the reared insects receive the necessary nutrients for their development, immune response, and overall health. In this study, scallions serve as the primary component of sterile diets for D. antiqua. Half-fermented diets provide better nutrients for D. antiqua, contributing to their growth and development. Additionally, mixing the larval homogenate with the diets aims to facilitate pre-digestion of the diets by the gut microbiota outside the body. This is because intestinal bacteria have an effect on insect feeding and digestion35. Using this method to prepare sterile diets for D. antiqua simplifies the process and significantly improves the efficiency of diet preparation.
Bacterial symbionts transmitted maternally within cells are widely present in insects36. Wolbachia, an intracellular bacterium transmitted maternally, is present in numerous insects37. Wolbachia is most commonly known for its role in reproductive manipulation, as it can bias the sex ratio of the host's offspring towards producing more infected females38. After verifying the axenic state of D. antiqua larvae, it was found that the larvae were essentially axenic after disinfection, except for the endosymbiotic bacteria Wolbachia, present in the cytoplasm of ovarian tissue cells in D. antiqua and transmitted vertically across generations through the cytoplasm of egg cells. As for the method of eliminating Wolbachia, antibiotic treatment is a common approach. In summary, this article presents a method for rearing axenic D. antiqua. According to the protocol, axenic D. antiqua can be successfully bred. Furthermore, this method is characterized by its simplicity and low cost, providing a new approach for rearing other axenic insects and increasing the feasibility of studying the interactions between insects and gut microbiota.
The authors have nothing to disclose.
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).
0.22 μM filter bottle | Thermo Scientific | 450-0045 | |
0.22 μM Syringe Filter | Biosharp | BS-QT-011 | |
100-mesh sieve | Zhejiang Shangyu Jinding Standard Sieve Factory | No Catalog numbers | |
1x PBS solution | Solarbio | P1020 | |
2x Taq PCR Master Mix | GENVIEW | GR1113-1ML | |
5.2% NaClO solution | Sinopharm Chemical Reagent Co., Ltd. | 80010428 | |
500 mL Conical flask | Thermo Scientific | 4103-0500 | |
50 mL vented centrifuge tube | JET BIOFIL | BRT-011-050 | |
50x TAE buffer | GENVIEW | GT1307 | |
Agar powder | Ding Guo | DH010-1.1 | |
Biochemical incubator | STIK | 21040121500010 | |
Cell sieve | SAINING | 5022200 | |
Choline chloride | Sangon Biotech | A600299-0100 | |
ddH2O | Ding Guo | PER018-2 | |
Disposable grinding pestle | JET BIOFIL | CSP-003-002 | |
DNA extraction kit | Sangon Biotech | B518221-0050 | |
DNA Marker | Sangon Biotech | B600335-0250 | |
Ethanol absolute | Sinopharm Chemical Reagent Co., Ltd. | 10009218 | |
Filter paper | NEWSTAR | 1087309025 | |
Food processor | Guangdong Midea Life Electric Appliance Manufacturing Co., Ltd. | WBL25B26 | |
Illuminated incubator | Shanghai ESTABLISH Instrumentation Co., Ltd. | A16110768 | |
L-Ascorbic acid | Sangon Biotech | A610021-0100 | |
L-shaped spreader | SAINING | 6040000 | |
Nutrient agar medium | Hope Bio | HB0109 | |
Scissors | Bing Yu | BY-103 | Purchase on Jingdong |
Shock incubator | Shanghai Zhichu Instrument Co., Ltd. | 2020000014 | |
Sucrose | GENVIEW | CS326-500G | |
Super Green nucleic acid dye | Biosharp | BS355A | |
Super-clean table | Heal Force | AC130052 | |
TSB | Hope Bio | HB4114 | |
Vacuum pump | Zhejiang Taizhou Seeking Precision Vacuum Pump Co., Ltd. | 22051031 | |
Yeast extract | Thermo Scientific | LP0021B |