Application of a strain of psychrophilic bacteria and its fermentation broth in inducing gravid female mosquitoes to lay eggs

Abstract

This invention relates to the field of microbial technology, and more particularly to the application of a marine psychrophilic bacterium and its fermentation broth in inducing housefly oviposition. The marine psychrophilic bacterium of this invention is *PSMA1*, and its fermentation broth has a significant effect on inducing housefly oviposition. Based on this, this invention also provides a method for housefly control and targeted reproduction using its fermentation broth, which can environmentally and efficiently regulate the oviposition and reproductive behavior of houseflies, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of a near-shore psychrophilic bacillus and its fermentation broth in inducing houseflies to lay eggs. Background Technology

[0002] Houseflies ( Musca domestica The housefly, belonging to the family Muscidae in the order Diptera, is a widespread sanitary pest that not only threatens human health but also spreads various pathogens, severely impacting environmental hygiene. For a long time, housefly control has primarily relied on chemical pesticides. While these are effective in the short term, long-term use leads to pesticide resistance in houseflies, pollutes the environment, and even disrupts the balance of ecosystems. Therefore, there is an urgent need to explore a green, environmentally friendly, and sustainable housefly control strategy to achieve effective control while protecting the ecological environment.

[0003] Meanwhile, houseflies are also important resource insects, possessing significant economic and ecological value in areas such as biotransformation, waste utilization, and protein production. Improving their oviposition efficiency and reproductive capacity is crucial for large-scale application in promoting the resource utilization of houseflies. Therefore, developing an environmentally friendly and efficient technology for regulating housefly oviposition and promoting reproduction will help enhance their resource utilization value, provide important reference for large-scale housefly breeding and application, and offer new ideas for achieving green housefly control goals. In summary, housefly oviposition regulation strategies are expected to promote the transformation of houseflies from "pests" to "beneficial insects" while balancing resource development and ecological protection, providing important support for sustainable agricultural development and environmental governance.

[0004] Microorganisms are widely distributed in nature and have developed complex and diverse relationships with insects over a long evolutionary process. These microorganisms directly or indirectly affect the physiology and behavior of insects. For example, when insects choose hosts, volatile substances produced by some microorganisms on the host's surface have a significant attraction effect; some bacteria that establish symbiotic relationships with insects can participate in the synthesis of insect pheromones, thereby affecting insect aggregation, social behavior, and interactions between multiple species; certain microorganisms in the environment can also alter the composition of insect gut microbiota, affecting insect foraging behavior, etc.

[0005] Microbial metabolites often serve as important chemical signaling substances during insect oviposition, significantly influencing their oviposition behavior. For example, studies have shown that in the fruit fly (Drosophila melanogaster),... Drosophila melanogaster It can accurately identify the yeast (Saccharomyces cerevisiae) Saccharomyces cerevisiaeGrapes infected with *Saccharomyces cerevisiae* showed a higher preference for feeding and oviposition. This preference was closely related to volatile substances produced by the metabolism of *Saccharomyces cerevisiae*, such as ethanol, acetic acid, acetone, 2-phenylethanol, and 3-methyl-1-butanol. Furthermore, geosmin, produced by the metabolism of bacteria, fungi, and cyanobacteria, has also been shown to attract insects, including *Aedes aegypti*. Aedes aegypti (This sentence appears to be incomplete and requires further context.) It mentions that *Geostrobin* can select environments rich in cyanobacteria as oviposition sites for larvae. The text also mentions *Geostrobin*, which is likely a typo and should be removed. Cochliomyia hominivorax Bacteria isolated from animal wounds and inoculated into fresh blood can significantly attract blowflies to lay eggs by releasing volatile substances. Similarly, *Serratia marcescens* (a type of bacteria) Serratia entomophila Stimulation can effectively increase the growth of tobacco bud moth ( Heliothis virescens While increasing the number of eggs laid, it also causes them to lose the ability to identify suitable spawning grounds.

[0006] Utilizing microorganisms and their metabolites to regulate insect physiology and behavior can provide significant applications in agricultural pest control, vector-borne insect management, resource insect development, and ecological protection. Currently, there are no reports on the influence of microorganisms and their metabolites on the oviposition behavior of houseflies. Summary of the Invention

[0007] This invention aims to screen target microorganisms that regulate housefly oviposition behavior and develop an environmentally friendly and efficient technology for regulating housefly oviposition and promoting reproduction. This technology will provide technical support for the concentrated trapping and killing of housefly eggs in housefly control and will also provide important basis and reference for the large-scale and efficient breeding and application of houseflies.

[0008] To solve the above-mentioned technical problems, the present invention provides a near-shore psychrophilic bacillus ( Psychrobacter maritimus The strain is a near-shore psychrophilic bacillus, PSMA1, with accession number CGMCC.No.33679.

[0009] Through extensive screening of microorganisms, this invention has found that the fermentation broth of the aforementioned near-shore psychrophilic bacillus PSMA1 has a significant effect on inducing houseflies to lay eggs.

[0010] The marine psychrophilic bacterium PSMA1 of this invention was obtained by screening from livestock and poultry manure (sheep manure). This strain was deposited on February 28, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Its classification and nomenclature are: marine psychrophilic bacterium. Psychrobacter maritimus The accession number is CGMCC No. 33679.

[0011] Furthermore, the present invention provides a microbial inoculant containing the aforementioned near-shore psychrophilic bacillus or its fermentation broth.

[0012] Furthermore, the present invention provides a fermentation broth, which is obtained by fermentation of the aforementioned near-shore psychrophilic bacillus.

[0013] Preferably, the fermentation medium used is LB medium.

[0014] Preferably, the fermentation temperature is 28℃~32℃.

[0015] Most preferably, the fermentation temperature is 30°C.

[0016] Preferably, the volatiles in the fermentation broth include methoxybenzoxime, dimethyl trisulfide, 2-ethylhexanol, phenethyl alcohol, benzothiazole, dimethyl phthalate, 3,5-di-tert-butylphenol, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisobutyl phthalate, and dibutyl phthalate.

[0017] Furthermore, the present invention provides the application of the fermentation broth in at least one of the following aspects:

[0018] (1) To lure houseflies to lay eggs;

[0019] (2) Preparation of the product; the product is used to induce houseflies to lay eggs.

[0020] Preferably, the product is an oviduct.

[0021] Furthermore, the present invention provides a method for controlling houseflies, comprising: using the fermentation liquid to induce houseflies to lay eggs, and then centrally treating the housefly eggs.

[0022] Furthermore, the present invention provides a method for targeted breeding of houseflies, comprising: adding the fermentation liquid to the housefly breeding environment to promote targeted egg laying by houseflies and improve the breeding efficiency of houseflies.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention provides a near-shore psychrophilic bacillus strain whose fermentation broth has a significant effect on inducing housefly oviposition. Based on this, this invention also provides a method for housefly control and targeted breeding using its fermentation broth, which can environmentally and efficiently regulate housefly oviposition and reproduction, and has broad application prospects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the experimental setup for oviposition preference behavior.

[0026] Figure 2This is the result of a daily oviposition selection test of houseflies on the fermentation broth of *PSMA1*, a bacterium found in nearshore psychrophilic organisms; where A represents the daily oviposition amount of houseflies in the fermentation broth-containing oviposition substrate (experimental group) and the LB medium-containing oviposition substrate (control group); B represents the daily oviposition selection rate of houseflies in the fermentation broth-containing oviposition substrate (experimental group) and the LB medium-containing oviposition substrate (control group). and They represent in P <0.05 and P The difference was significant at the <0.01 level. Representative at P The difference was significant at the <0.0001 level.

[0027] Figure 3 This is a statistical chart showing the total number of eggs laid by houseflies over 7 days in an oviposition substrate containing fermentation broth (experimental group) and an oviposition substrate containing LB medium (control group). Indicates in P The difference was significant at the <0.0001 level.

[0028] Figure 4 This is a graph showing the results of volatile composition analysis in the fermentation broth of the near-shore psychrophilic bacterium PSMA1. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Example 1: Culture of marine psychrophilic bacillus PSMA1

[0031] Prepare LB liquid medium and LB agar. The LB liquid medium is prepared as follows: accurately weigh 10 g peptone, 5 g yeast extract, and 10 g NaCl, dissolve in 800 mL distilled water, adjust the pH to 7.2, and bring the volume to 1 L. Autoclave at 121℃ for 20 min, then cool to room temperature. The LB agar agar formulation is as follows: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH adjusted to 7.2. Remove the near-shore psychrophilic bacillus PSMA1 stored in 25% glycerol (final concentration) from a -80℃ freezer and thaw slowly on ice. After complete thawing, dilute the bacterial culture with LB liquid medium (without agar) to different gradients (10...). -1 ~10 -9 100 μL of the diluted solution was spread onto LB agar plates. The plates were incubated at 30°C for 48 h, and colony growth was observed. Target strains with good growth and uniform colony morphology were selected, and individual colonies were picked and placed in fresh LB liquid medium with shaking at 180 rpm and incubated overnight at 30°C for subsequent fermentation broth preparation.

[0032] Example 2: Preparation of fermentation broth of marine psychrophilic bacillus PSMA1

[0033] 1. Inoculation with bacterial suspension: Under aseptic conditions, the *PSMA1* bacterial suspension prepared in Example 1 was diluted to 1.0 × 10⁻⁶. 8 CFU / mL (concentration determined by plate count), 10 μL of diluted bacterial culture was inoculated into 100 mL of LB liquid medium.

[0034] 2. Fermentation Culture: Place the inoculated culture medium in a shaker and ferment at 180 rpm and 30℃ for 72 hours. During fermentation, observe the color change and turbidity of the culture medium regularly to ensure normal bacterial growth and the production of metabolites. After fermentation, the fermentation broth is pale yellow with an OD value in the range of 1.8-2.0.

[0035] 3. Centrifugation: Transfer the fermentation broth to centrifuge tubes and place them in a high-speed centrifuge pre-cooled to 4°C. Centrifuge at 13000 g for 10 min to separate the supernatant and precipitate. The precipitate consists of bacterial cells and other particulate matter, while the supernatant is a solution of metabolic products produced by the fermentation strain.

[0036] 4. Collect the supernatant: Carefully aspirate the supernatant after centrifugation, avoiding disturbing the precipitate. Transfer the supernatant to a glass bottle to obtain the fermentation broth of *PSMA1*, a psychrophilic bacterium found in nearshore environments. This fermentation broth can be used directly for subsequent experiments on housefly oviposition behavior. The remaining fermentation broth should be stored at -20°C for later use. Before freezing, it can be aliquoted into multiple clean, sterile glass bottles to avoid the loss of volatile components due to repeated freeze-thaw cycles.

[0037] Example 3: Housefly oviposition behavior experiment

[0038] To verify the inducing effect of fermentation broth of the nearshore psychrophilic bacterium PSMA1 on oviposition in houseflies, the following behavioral experiment was designed:

[0039] 1. Experimental subjects and grouping

[0040] Houseflies used in the experiment: Healthy houseflies 5-6 days after emergence were selected from the laboratory housefly breeding system to ensure they were sexually mature and in the egg-laying period. Each experimental group contained 10 pairs (20 houseflies) with a male-to-female ratio of 1:1.

[0041] Experimental Groups: The experiment was divided into an experimental group and a control group. The spawning substrate in the experimental group was a mixture of 15 g wheat bran and 35 mL of fermentation broth of *PSMA1*, a near-shore psychrophilic bacterium prepared in Example 2. The spawning substrate in the control group was a mixture of 15 g wheat bran and 35 mL of LB liquid medium. Both groups were configured with 4 replicates.

[0042] 2. Experimental Apparatus and Design

[0043] Insect cage design: The experiment used 30 cm × 30 cm × 30 cm insect cages. Each cage contained clean water and a food dish (a 1:1 mixture of milk powder and brown sugar). Figure 1 As shown in the diagram. Oviposition cups for the experimental and control groups were placed at opposite ends of the cage diagonally to ensure the houseflies could freely choose their oviposition location.

[0044] Preparation of oviposition cups: Take 50 mL disposable plastic cups as oviposition cups, add oviposition substrate to the experimental group and control group respectively, mix well, and then place them in the rearing cage. Ensure that the oviposition cups of the two groups are symmetrically positioned in the cage to avoid the influence of positional preference on the experimental results.

[0045] 3. Experimental Procedure

[0046] Adaptation period: Place the houseflies in the breeding cage and allow them to adapt to the environment for 24 hours to eliminate the interference of environmental changes on their behavior.

[0047] Experiment begins: Oviposition cups are updated daily at 19:00. New experimental and control group oviposition cups are placed diagonally opposite each other in the rearing cage, while the previous day's oviposition cups are collected. The experiment continues for 7 days, with the above steps repeated daily to ensure data continuity and accuracy.

[0048] 4. Egg count

[0049] Each day, the oviposition cups were clearly labeled to distinguish between the experimental and control groups, and the specific date and rearing cage number were recorded. The substrate in the collected oviposition cups was removed, and the number of eggs was counted under a microscope.

[0050] 5. Recording and Analysis of Experimental Results

[0051] The number of eggs in the oviposition cups of the experimental and control groups was recorded daily. Statistical software (SPSS and GraphPad) was used for data analysis and graphing. Independent samples t-tests were used to compare the differences in egg count and oviposition selection rate between the experimental and control groups.

[0052] 6. Other precautions

[0053] To avoid interference from other environmental factors on housefly behavior, the experiment was conducted under constant temperature and humidity conditions (28℃, relative humidity 60%-70%). Water and food were changed daily to ensure the health and reproductive capacity of the houseflies. The oviposition substrate for both the experimental and control groups was freshly prepared and used immediately to avoid changes in substrate composition or loss of volatile substances due to prolonged storage.

[0054] 7. Experimental Results and Analysis

[0055] Data was collected over seven consecutive days. An independent samples t-test was used to analyze the differences in egg production and selection rate between the experimental and control groups. The results are as follows: Figure 2 and Figure 3 As shown, the daily number of eggs laid and the egg selection rate in the experimental group were significantly higher than those in the control group throughout the entire experimental period. P <0.05), and the total number of eggs laid in the experimental group over 7 days was also significantly higher than that in the control group ( P The value of <0.05 indicates that the fermentation broth of the nearshore psychrophilic bacterium PSMA1 has a significant oviposition-attracting effect on houseflies.

[0056] Example 4: Determination of volatile composition in fermentation broth of marine psychrophilic bacillus PSMA1

[0057] To further investigate the material basis of the oviposition-inducing effect of the fermentation broth of *PSMA1*, a near-shore psychrophilic bacterium, on housefly oviposition, gas chromatography-mass spectrometry (GC-MS) was used to qualitatively analyze the volatile compounds in the fermentation broth. The specific operation steps are as follows:

[0058] 1. Volatile Extraction

[0059] Solid-phase microextraction (SPME) was used to extract volatile compounds from the fermentation broth. The specific steps are as follows:

[0060] Sample preparation: Add 5 mL of fermentation broth of marine psychrophilic bacillus PSMA1 to a brown sample vial, set up 3 replicates, and incubate the sample vial in a 37℃ water bath for 30 min to equilibrate.

[0061] Extraction procedure: Place a magnetic stir bar in the sample vial and stir the liquid at 600 rpm to promote the release of volatile compounds. Insert the SPME extraction head (coated with 50 / 30 μm DVB / CAR / PDMS) into the sample vial and extract at 37°C for 30 min.

[0062] 2. GC-MS detection conditions

[0063] After extraction, the SPME extraction head was immediately inserted into the injection port of the gas chromatography-mass spectrometry (GC-MS) instrument for detection, with the specific parameters as follows:

[0064] Gas chromatography conditions:

[0065] Column: DB-5MS (30 m × 0.25 mm × 0.25 μm).

[0066] Carrier gas: high-purity helium, flow rate 1.0 mL / min.

[0067] Temperature program: Initial temperature 40℃, hold for 5 min; then increase to 150℃ at a rate of 5℃ per minute; then increase to 225℃ at a rate of 10℃ per minute, hold for 10 min.

[0068] Injection method: splitless injection, injection port temperature is 270℃.

[0069] Mass spectrometry conditions:

[0070] Ion source: Electron impact (EI) mode.

[0071] Scan range: m / z 35 - 400.

[0072] Ion source temperature: 250℃, quadrupole temperature: 250℃.

[0073] 3. Data Collection and Analysis

[0074] Total ion current (TIC) chromatograms of the test samples were obtained using GC-MS, and the retention times and peak areas of volatile compounds were recorded. Combined with mass spectrometry data, the mass spectrometric fragmentation characteristics of the corresponding compounds were analyzed to preliminarily determine the chemical composition. The detected mass spectrometry data were compared with the NIST20 mass spectrometry library to identify the types of volatile compounds in the fermentation broth.

[0075] 4. Experimental Results and Analysis

[0076] Analysis results of volatile compounds in the fermentation broth of *PSMA1*, a marine psychrophilic bacterium, are as follows: Figure 4 As shown, its volatile composition mainly includes: methoxybenzoxime, dimethyl trisulfide, 2-ethylhexanol, phenethyl alcohol, benzothiazole, dimethyl phthalate, 3,5-di-tert-butylphenol, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisobutyl phthalate, and dibutyl phthalate, etc., indicating that these substances are metabolites produced by the near-shore psychrophilic bacterium PSMA1 during fermentation, and it is speculated that they may be key chemical signaling molecules that have the effect of inducing housefly oviposition in the fermentation broth.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of near-shore psychrophilic bacillus ( Psychrobacter maritimus ), characterized in that, It is a near-shore psychrophilic bacillus PSMA1, with accession number CGMCC.No.33679.

2. A microbial inoculant, characterized in that, It includes the near-shore psychrophilic bacillus or its fermentation broth as described in claim 1.

3. A fermentation broth, characterized in that, It is prepared by fermentation of the near-shore psychrophilic bacillus described in claim 1.

4. The fermentation broth according to claim 3, characterized in that, The fermentation process uses LB medium.

5. The fermentation broth according to claim 3, characterized in that, The fermentation temperature is 28℃~32℃.

6. The fermentation broth according to claim 3, characterized in that, The volatiles in the fermentation broth include methoxybenzoxime, dimethyl trisulfide, 2-ethylhexanol, phenethyl alcohol, benzothiazole, dimethyl phthalate, 3,5-di-tert-butylphenol, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, diisobutyl phthalate, and dibutyl phthalate.

7. The use of the fermentation broth according to any one of claims 3 to 6 in at least one of the following aspects: (1) To lure houseflies to lay eggs; (2) Preparation of the product; the product is used to induce houseflies to lay eggs.

8. An oviductor, characterized in that, It contains the fermentation broth according to any one of claims 3 to 6.

9. A method for controlling houseflies, characterized in that, include: The fermentation broth according to any one of claims 3 to 6 is used to induce houseflies to lay eggs, and then the housefly eggs are centrally processed.

10. A method for the targeted reproduction of houseflies, characterized in that, include: Adding the fermentation liquid according to any one of claims 3 to 6 to the housefly breeding environment promotes housefly directional egg laying.

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