A fruit fly probiotic for repairing irradiation damage and its application

Abstract

This invention relates to the field of microbial technology, and proposes a fruit fly probiotic for repairing irradiation damage and its application. The fruit fly probiotic is *Enterobacter leucobacter* (…). Enterobacter ludwigii EL1, with accession number CCTCC NO:M 2025376. This invention discovers that the probiotic Enterobacter leuciscus EL1 in fruit flies can effectively repair the damage to the ecological adaptability of fruit flies caused by irradiation sterility doses. After irradiation, feeding with the probiotic significantly restored the flight ability, mating competitiveness, survival rate, and lifespan of Orientia citrinum and Drosophila melanogaster, with no significant difference in various physiological indicators compared with the control group.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a fruit fly probiotic for repairing irradiation damage and its application. Background Technology

[0002] Oriental fruit fly ( Bactrocera dorsalis ), South Asian fruit fly ( Zeugodacus tau The fruit fly (Aegilops spp.) is a significant pest of the fruit fly family. It has a wide host range, a short life cycle, and a high reproductive capacity, with rapid generational alternation, making it extremely prone to outbreaks and severe outbreaks. Control is extremely difficult, seriously harming the economic development and trade of the fruit and vegetable industry. It has been listed as an import quarantine pest by many countries, including my country. In recent years, it has shown a continuous northward spread, and the damage is becoming increasingly severe. Currently, the main control method for this type of fruit fly is using adult traps, but sex pheromones can only attract and kill males, resulting in relatively high costs and limited control effectiveness.

[0003] In recent decades, the sterile insect technique (SIT) has emerged as a green pest control strategy as an alternative to pesticides. Due to its target specificity and minimal environmental impact, it has been widely adopted by many countries for the control of the Mediterranean fruit fly. Radiation-based SIT is an effective means of integrated pest management (AW-IPM) at the regional level and is widely used to control and eliminate crop pests and public health pests. Currently, radiation-based SIT is widely used in the Mediterranean fruit fly (Mediterranean fruit fly). Ceratitis capitata Queensland fruit fly ( Bactrocera tryoni ), guava fruit fly ( Bactrocera correct Control of various fruit fly pests, including the oriental fruit fly and the citrus fruit fly.

[0004] However, radiation treatment can cause numerous irreversible damages to insects. Multiple reports indicate that radiation-treated pupae have low emergence rates and exhibit poor ecological adaptability in sterile males. For example, it reduces the mating competitiveness of the Mediterranean fruit fly and affects the mating ability of the West Indian fruit fly (Anopheles cerana). Anastrepha obliqua Flight capability and lifespan. These shortcomings increase the cost of implementing SIT technology, hindering its widespread adoption.

[0005] The insect gut contains a wide variety of microorganisms, primarily bacteria, fungi, and viruses, which interact in complex ways with the host. Gut probiotics play a crucial role in host growth, development, metabolism, and reproduction, helping the host break down ingested cellulose, providing nutrition, resisting exogenous pathogens, and improving insect rearing efficiency. Currently, research on the role of insect gut microorganisms in repairing radiation damage is limited, and suitable microorganisms are scarce. Summary of the Invention

[0006] In view of this, the present invention proposes a fruit fly probiotic that can repair irradiation damage and its application.

[0007] The technical solution of this invention is implemented as follows: Firstly, this invention provides a fruit fly probiotic for repairing irradiation damage, wherein the fruit fly probiotic is Enterobacter leucobacter (…). Enterobacter ludwigii EL1, accession number CCTCC NO:M 2025376.

[0008] Secondly, the present invention also provides the application of fruit fly probiotics in the preparation of repair agents for damage caused by X-ray radiation.

[0009] Thirdly, the present invention also provides the application of fruit fly probiotics in feed additives.

[0010] Based on the above technical solutions, preferably, the feed additive contains Enterobacter leuciscus (Lycium barometz). Enterobacter ludwigii EL1 viable count ≥10 7 CFU / g.

[0011] Fourthly, the present invention also provides the application of fruit fly probiotics in promoting the growth and reproduction of fruit flies.

[0012] Based on the above technical solutions, the preferred fruit flies are the Oriental fruit fly and the South Asian fruit fly.

[0013] The fruit fly probiotic of the present invention, which can repair radiation damage and its application, has the following advantages over the prior art:

[0014] The present invention contains Enterobacter leukogene ( Enterobacter ludwigii EL1 can effectively repair the damage to the ecological adaptability of fruit flies caused by irradiation sterility dose. After irradiation, feeding with probiotics significantly restored the flight ability, mating competitiveness, survival rate and lifespan of the Oriental fruit fly and the South Asian fruit fly. There was no significant difference in various physiological indicators compared with the control group. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] The fruit fly probiotic of this invention is Enterobacter leucis ( Enterobacter ludwigiiEL1 was deposited on March 4, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2025376.

[0017] The Enterobacter leucis EL1 of this invention can repair radiation-damaged fruit flies (Bactrocera dorsalis). Bactrocera dorsali s) and South Asian fruit fly ( Zeugodacus tau It can also be used as a feed additive to promote the growth and reproduction of fruit flies and improve the wild ecological adaptability of male citrus fruit flies and South Asian fruit flies.

[0018] The fruit fly probiotic of the present invention will be further described in detail below with reference to the embodiments.

[0019] Example 1: Obtaining Probiotic Strains

[0020] Isolation of gut microbiota from fruit flies

[0021] All forceps and culture dishes used for dissection were autoclaved beforehand, and the lab bench and stereomicroscope stage were sprayed with 75% alcohol for disinfection before the dissection began. The oriental fruit flies to be dissected were first frozen on ice, then surface-sterilized in 75% alcohol for 3 minutes, and rinsed three times in PBS buffer.

[0022] Subsequently, an appropriate amount of PBS buffer was added to the culture dish containing the oriental fruit fly, and the dish was dissected under a microscope. After dissection, the oriental fruit fly intestinal sample was placed in a 1.5 mL EP tube containing an appropriate amount of LB and grinding beads. After grinding evenly, the sample was diluted to form an intestinal bacterial suspension for intestinal bacterial extraction.

[0023] Identification of gut bacteria in fruit flies

[0024] Intestinal bacterial suspensions were spread onto NA plates and incubated at 37°C for 12-24 hours, observing single colony growth. Single colonies were then picked and inoculated into LB broth and incubated at 37°C for 12 hours. The species of the strains were identified using RT-PCR and 16S rRNA sequencing.

[0025] Several enteric bacterial strains, including *Enterobacter leucovorin*, *Enterobacter hominis*, *Providensia rubella*, *Klebsiella pneumoniae*, *Providensia rubella* reticularis, and non-decarboxylated *Levitraceta*, were obtained from *Bacteroides citrinum* and *Bacteroides aurea*, respectively. *Enterobacter leucovorin* promoted the growth, development, and reproduction of *Bacteroides citrinum*. This strain was deposited on March 4, 2025, at the China Center for Type Culture Collection (CCTCC) located at Wuhan University in Wuhan, Hubei Province. The naming and accession number of the strain are as follows:

[0026] Enterobacter leukemia ( Enterobacter ludwigii) EL1, CCTCC NO:M 2025376.

[0027] Enterobacter leukemia ( Enterobacter ludwigii EL1 belongs to the class Gammaproteobacteria ( Gammaproteobacteria ) Enterobacteriaceae ( Enterobacteriales Enterobacteriaceae ( Enterobacteriaceae Enterobacteriaceae ( Enterobacter ).

[0028] Enterobacter leukemia EL1 is a Gram-negative bacterium that is motile, does not produce pigment, and is rod-shaped. When grown on eosin methylene blue agar, the colonies are pink, round, with neat edges and a smooth, moist surface.

[0029] Example 2 Preparation of Probiotic Feed Additive for Fruit Flies

[0030] The preserved Enterobacter leucis EL1 was inoculated at a rate of 5% (v / v) into 100 mL of LB liquid medium (1 g tryptone, 0.5 g yeast extract, 1 g NaCl, 90 mL deionized water, pH adjusted to 7.0 with 5 mol / L NaOH, volume brought to 100 mL, and sterilized at 120 °C for 20 min). The culture was incubated at 30 °C and 150 rpm on a shaker. The OD value of the bacterial culture at 600 nm was measured to be 1 using a spectrophotometer.

[0031] Then, dissolve 2.5g of honey, 5g of yeast extract, and 15g of sucrose in the above bacterial solution, and then add 18g of wheat bran (bacterial agent carrier) and mix thoroughly to prepare a probiotic feed additive. Add deionized water to a final volume of 150g. The effective live bacteria count of the probiotics in this feed additive is 10. 7 CFU / g.

[0032] Example 3: Application of Probiotic Feed Additives in Fruit Flies

[0033] I. Effects of probiotic feed additives for fruit flies on the growth, development, and reproduction of normal fruit flies

[0034] The fruit fly larvae used in the examples were fed with artificial feed consisting of 2.5g honey, 5g yeast extract, 15g sucrose and 18g wheat bran, which were thoroughly mixed and then deionized water was added to 100g. The treatment group was supplemented with 66g of the insect probiotic feed additive prepared in Example 2, while the control group was supplemented with probiotic carrier (50ml sterile water + 16g wheat bran). The mixture was stirred evenly and then used.

[0035] The collected fruit fly eggs were added to the feed, approximately 400 eggs per group. Rearing conditions: 27±2℃, humidity 70±5%, light cycle (L:D) 12h:12h. After 10 days, the larvae were transferred to sand treated at 120℃ to pupate. The pupal weight, emergence rate, and total egg production within 15 days after mating were measured in both the control and treatment groups. The results are shown in Table 1.

[0036] Table 1. Effects of probiotics on various indicators of growth, development, and reproduction in the oriental fruit fly.

[0037]

[0038] The results in Table 1 show that, compared with the control group, the pupal weight, emergence rate, and egg production of the insect probiotic feed additive treatment group were significantly increased (Table 1, p<0.05, t-test), indicating that the addition of Enterobacter leucis EL1 to the feed can promote the growth and reproduction of fruit flies.

[0039] II. The Repairing Ability of Probiotic Feed Additives for Irradiation-Damaged Fruit Flies

[0040] Fruit fly larvae were pupated in sand. 3000 final-instar pupae in the treatment group (48 hours before emergence) were irradiated with 100 Gy of X-rays, while 2000 final-instar pupae in the control group were not irradiated with X-rays. After adult emergence, male and female flies were reared separately.

[0041] The control group (unirradiated male insects) and treatment group A (irradiated male insects) were fed 20g of sucrose: yeast (3:1) with sterile water to a final volume of 150g. Treatment group B (irradiated male insects) was fed 20g of sucrose: yeast (3:1) and the probiotic feed additive from Example 2 (equal volume to the sterile water in the control group) to a final volume of 150g.

[0042] Five days after feeding, various biological indicators of the control group, treatment group A, and treatment group B were tested: adult flight ability (7 days old), mating competitiveness, and lifespan. The results are shown in Tables 2 and 3.

[0043] Table 2. Effects of probiotics on various indicators of adult oriental fruit flies after irradiation.

[0044]

[0045] Table 2 shows that, compared with treatment group A, feeding irradiated male oriental fruit flies with Enterobacter leucobacter EL1 significantly improved their flight distance, mating competitiveness, and lifespan, bringing them close to the control group.

[0046] Table 3. Effects of probiotics on various indicators of adult South Asian fruit flies after irradiation.

[0047]

[0048] Table 3 shows that, compared with treatment group A, feeding irradiated male *Drosophila melanogaster* with *Enterobacter leucovorin* EL1 significantly improved the males' flight distance, mating competitiveness, and lifespan, nearly restoring them to the levels of the control group. This is consistent with the results in Table 1. This indicates that *Enterobacter leucovorin* EL1 and *Providencebrinus PV1* of this invention have the effect of repairing irradiation damage.

[0049] The above results indicate that the fruit fly probiotics and their feed additives provided by this invention significantly improve the wild ecological adaptability of male fruit flies (Bactrocera dorsalis) and male fruit flies (Flycocytos spp.) and significantly repair the radiation damage of male fruit flies, providing a scientific and effective method for improving the efficiency of insect sterility technology (SIT).

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Dacnusia irradiation damage repairing probiotic, characterized in that: The probiotics in the fruit fly are Enterobacter leucobacter (Lewseriformis) Enterobacter ludwigii EL1, accession number CCTCC NO:M 2025376.

2. Use of the Bactrocera probiotic bacteria according to claim 1 for the preparation of a restorer for restoring the damage caused by X-ray radiation in Bactrocera flies, characterized in that: The fruit flies are Bactrocera dorsalis (Hendel) Bactrocera dorsali and Bactrocera correcta (Stainton) Zeugodacus tau .

3. Use of Bactrocera probiotics according to claim 1 for the preparation of a Bactrocera feed additive, characterized in that: The fruit flies are Bactrocera dorsalis (Hendel) Bactrocera dorsali and Bactrocera correcta (Stainton) Zeugodacus tau .

4. Use according to claim 3, wherein: In the feed additive, the viable cell number of Enterococcus faecium (Eli) Enterobacter ludwigii ) is ≥ 10 7 CFU / g.

5. The use of Bactocera (Dacus) ciliatus probiotics according to claim 1 for promoting the growth and reproduction of Bactocera (Dacus) ciliatus, characterized by: The fruit flies are Bactrocera dorsalis (Hendel) Bactrocera dorsali and Bactrocera correcta (Stainton) Zeugodacus tau .

Images