Method and device for inducing antimicrobial peptide of fly

Abstract

[Problem] To provide a method for inducing an antimicrobial peptide in the body of an insect. [Solution] The present invention provides: a method for inducing an antimicrobial peptide in an insect by damaging the insect, said method being characterized by irradiating the outer covering of the insect with a laser and damaging the insect by opening a hole passing through the outer covering of the insect with a laser beam in a state of not causing, to the insect, damage that prevents recovery; a CO2 laser irradiation device; a device used in the method for inducing an antimicrobial peptide in the body of an insect, said device comprising a positional information acquisition means for acquiring positional information that pertains to the insect and a means for transmitting, to the positional information that pertains to the insect, the positional information that pertains to the insect and that has been acquired by the positional information acquisition means; and use of a CO2 laser in the method for inducing an antimicrobial peptide in the body of an insect by opening a hole passing through the outer covering of the insect.

Description

Method and device for inducing antimicrobial peptides in flies

[0001] The present invention relates to a method and apparatus for inducing (producing) antimicrobial peptides in insects, particularly insects, such as houseflies.

[0002] Antimicrobial peptides are a general term for peptides formed by chains of amino acids that exhibit antibacterial properties against bacteria, viruses, fungi, etc., and are expected to be used in the development of new antibacterial drugs and disease prevention measures. In particular, feed additives containing antibiotics have led to the emergence of super bacteria that are resistant to antibiotics used in animals and humans, and regulations on their use have been strengthened. Therefore, there is a demand for the development of antibacterial peptides as feed additives that contain safe and environmentally friendly alternatives to antibiotics.

[0003] Patent Document 1 (Japanese Patent No. 3661549) describes a partial peptide of a 26-kDa protease derived from the flesh fly, which exhibits antibacterial activity, and an antibacterial agent containing the same. This polypeptide is a 26-kDa protease that appears only in the intestine of mid-pupal stage larvae of the flesh fly, and is a linear polypeptide consisting of 239 amino acids that exhibits selective antibacterial activity against Gram-positive bacteria.

[0004] Wounding organisms, such as insects, to induce antimicrobial peptides has long been used. For example, Patent Document 2 (Japanese Patent No. 2671912) describes the induction of antimicrobial proteins in the hemolymph of Taiwanese rhinoceros beetle larvae upon wounding. Furthermore, Non-Patent Document 1 (Kawasaki et al. Drug Discoveries & Therapeutics 2017, pages 1-5, Advance Publication) confirms that hemolymph collected from needle-wounded housefly larvae exhibits significant antimicrobial activity against both gram-positive and gram-negative bacteria, such as Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, since the antimicrobial activity is predominantly maintained in the hemolymph even after heat treatment, wounded larvae are considered a potential therapeutic resource for antimicrobial agents.

[0005] The above-mentioned wounding process generally involves pricking each individual insect with a needle, which is an inefficient and labor-intensive process. Housefly larvae reach their maximum size just before pupation, but even at that stage, each insect weighs only 25 mg. Inflicting appropriate wounds on each individual is labor-intensive and requires skill, and the method of wounding can result in differences in the induction effect. For example, the induction effect varies depending on which part of the housefly larvae is pierced with the needle. While some reports suggest that stimulating the fat body is sufficient, the exact mechanism by which antimicrobial peptides are induced has yet to be elucidated.

[0006] Patent Document 3 (JP 2009-268448 A) discloses a device that isolates flesh fly larvae from food and keeps them hydrated, then cools and anesthetizes them, stings them, and induces the production of antimicrobial peptides. This patent involves image processing of a large number of third-instar larvae on a tray, determining the positional information of each third-instar larva, and then stinging each third-instar larva from directly above with a needle that moves up and down. In this case, the larvae are stabbed to the point of almost penetrating the larvae, but the wound closes within a few minutes due to their self-healing ability, preventing leakage of body fluids.

[0007] However, the proteins that make up the larvae's cuticle are hard, and if even 10 larvae are injured with a needle, the needle tip will become rounded, and further attempts to injure the larvae will kill them. This necessitates frequent needle replacement, which requires time and expense and prevents mass production. Therefore, instead of injuring fly larvae with a needle, a method has been proposed in which fly larvae are genetically modified and then used to induce (produce) antimicrobial peptides to produce biological feed additives (see Patent Document 4 (Chinese Patent No. CN1144597)).

[0008] The inventors have tried various methods to find a less costly method than injuring the larvae with a needle, such as "dropping hydrochloric acid or sulfuric acid onto the larvae," "stimulating the larvae with the mycelium and spores of mushrooms that parasitize the larvae, such as Pupal Pupal," and "stressing the larvae by sudden temperature changes," but have not found a method that effectively induces antimicrobial peptides in housefly larvae.

[0009] The inventors wondered whether it would be possible to effectively induce antimicrobial peptides in housefly larvae by injuring the larvae with laser irradiation, but simply irradiating the housefly larvae with a laser resulted in the complete annihilation of the larvae. The inventors believed this was due to the laser output being too high, and repeated experiments with reduced laser irradiation output succeeded in increasing the survival rate of the larvae after laser irradiation, but were unable to induce antimicrobial peptides. In other words, although the survival rate increased when "irradiating housefly larvae with a laser," the antimicrobial peptides could not be induced.

[0010] Japanese Patent No. 3661549 Japanese Patent No. 2671912 JP 2009-268448 A Chinese Patent No. CN1144597

[0011] Kawasaki et al. Drug Discoveries & Therapeutics 2017, page 1-5, Advance Publication

[0012] An object of the present invention is to provide a method for efficiently inducing (producing) antimicrobial peptides in housefly larvae, which can increase the survival rate of housefly larvae when irradiated with a laser and also increase the efficiency of inducing antimicrobial peptides. Another object of the present invention is to provide an antimicrobial peptide production method and apparatus that can increase the efficiency of antimicrobial peptide production. A still further object of the present invention is to use a laser to efficiently induce (produce) antimicrobial peptides in housefly larvae.

[0013] The first object of the present invention is a method for inducing antimicrobial peptides in insects by damaging the insects, which method comprises irradiating the insect's cuticle with a laser, and damaging the insect by creating a hole through the insect's cuticle with a laser beam, without causing irreparable damage to the insect.

[0014] In a preferred embodiment of the present invention, the insects are house fly larvae, the laser is a CO2 laser, and the holes penetrated through the cuticle of the house fly larvae have a diameter of 1-300 μm.

[0015] In a preferred embodiment of the present invention, the insect larvae are immobilized at temperatures just below freezing and then irradiated with a laser.

[0016] In a preferred embodiment of the present invention, after laser irradiation, the laser-irradiated housefly larvae are transferred to an environment with a wet-bulb temperature of 20°C to 35°C and prevented from pupating for 3 to 48 hours, allowing the larvae to induce antimicrobial peptides themselves.

[0017] In a preferred embodiment of the present invention, housefly larvae are placed on a wet surface containing a near-osmotic liquid maintained at 20°C to 35°C and exposed to the liquid to inhibit pupation.

[0018] In a preferred embodiment of the present invention, the device used in the method of the present invention includes a CO2 laser irradiation device, a position information acquisition means for obtaining position information of insects, and a means for transmitting the position information of insects obtained by the position information acquisition means to the device.

[0019] In a preferred embodiment of the invention, the apparatus further comprises means for cooling the insect larvae to a temperature just below freezing to immobilize the insect larvae.

[0020] A third object of the present invention is the use of a CO2 laser in a method for introducing antimicrobial peptides into the body of an insect by drilling holes through the insect's cuticle.

[0021] 1. A conceptual diagram for explaining the method of the present invention. 2. A conceptual diagram when a laser beam is scanned and irradiated onto a group of housefly larvae. 3. A conceptual diagram when the antimicrobial peptide induction method of the present invention is executed using an image processing device. 4. A conceptual diagram showing the position information of a group of housefly larvae obtained by binarizing an image captured by a camera of the group of housefly larvae. 5. A flow chart of image processing used in one embodiment of an image processing system. 6. A screen display of the housefly abdominal position obtained from precise position information. 7. A conceptual diagram of one embodiment of image processing when identifying the abdomen of each housefly. 8. A diagram summarizing the antibacterial activity measurement results of the examples of the present invention and comparative examples, with the vertical axis of the figure representing the colony forming units (CFU) of Staphylococcus aureus. 9. A photograph showing that a hole of approximately 250 μm was created in the epidermis of the housefly larvae when laser irradiation was performed in Example 1. 10. A photograph showing that no hole was created in the epidermis of the housefly larvae when the laser beam was irradiated onto the housefly larvae when UV laser irradiation was performed in Comparative Example 5.

[0022] The term "insects" generally refers to arthropod insects, but in this specification, the term is used to include a variety of small animals including earthworms, spiders, centipedes, snails, etc., without being bound by strict academic classification. The present invention is preferably applicable to flies, black soldier flies, mealworms, crickets, silkworms, etc., and is particularly preferably applicable to flies, black soldier flies, and mealworms.

[0023] Although the present invention will be described herein using the house fly (Musca domestica) as an example, the present invention is not limited to house flies and can be applied to any "insects," including insects, as long as the method of the present invention is applicable. Furthermore, although the present invention will be described with respect to insect larvae, the method of the present invention can be applied not only to insect larvae but also to pupae and adults.

[0024] Houseflies inhibit fungal growth by competing for nutrients with symbiotic Gram-negative bacilli on their surface, and it is thought that houseflies induce antimicrobial peptides when their bodies are injured. Therefore, injury to the body can promote the production of antimicrobial peptides.

[0025] As used herein, "a state in which irreparable damage to the insects is not caused" means a state in which the insects do not die before inducing antimicrobial peptides. In the method of the present invention, an antimicrobial peptide induction period of 3 to 48 hours is required after laser irradiation, and it is desirable that the insects do not die during this antimicrobial peptide induction period. Generally, the insects are preserved by drying, freezing, or refrigeration, and then provided as feed. Since the antimicrobial peptides may be lost when the insects are allowed to pupate, they are generally not allowed to pupate.

[0026] Preparation of insects Housefly larvae can be supplied by the method described in the applicant's patents (Patent Document 5, Patent No. 5579122).

[0027] Methods for growing and rearing other insects to which the method of the present invention can be applied, such as earthworms, mealworms, crickets, silkworms, etc., are described in numerous patent documents.

[0028] In the method of the present invention, eggs laid by adult houseflies are grown, the larvae are reared, and the third-instar larvae before pupation are used. While Patent Document 5 above causes the larvae to produce antimicrobial peptides by scratching their skin, in the present invention, holes are made in the larvae's skin by laser irradiation to cause them to produce antimicrobial peptides.

[0029] Laser irradiation "Drilling a hole through the insect's cuticle with a laser beam" means that the laser beam does not simply stress the insect, but actually drills a hole through the insect's cuticle. The type of laser capable of drilling a hole through the insect's cuticle, as well as the intensity and irradiation time of the laser beam, depend on the type of insect and the type of laser irradiation device used, and can be easily determined by experiment.

[0030] In the case of adult houseflies, the size of the hole penetrating the insect's cuticle is generally in the range of 1 to 300 μm, preferably in the range of 10 to 250 μm. If the size is less than 1 μm, the effect of the present invention cannot be expected, and if it exceeds 300 μm, the damage to the adult houseflies will be significant.

[0031] Any type of "laser" can be used as long as it is capable of drilling a hole through the insect's cuticle, but a CO2 laser is generally used. Various laser irradiation devices are commercially available, but in the present invention, commercially available devices can be preferably used as laser markers used to engrave (print) marks on various workpieces. In particular, in the present invention, a laser marker that can automatically control the laser beam in three axes according to the three-dimensional shape of the insect workpiece can be advantageously used. Such laser markers are commercially available, for example, from Keyence Corporation.

[0032] FIG. 1 is an explanatory diagram of the implementation of the method of the present invention using a commercially available laser marker. Generally, the method of the present invention can be implemented by scanning a laser beam (2) from a head (1) left and right and up and down within the X-Y plane to sequentially form through-holes (i.e., print desired symbols) in a number of insects (4) placed in a container, such as a tray (3). In actual use, all that is required is to input the necessary parameters (laser output, scan speed, pulse frequency, etc.) into a computer (6) using a monitor (60) and keyboard (61). It is preferable to follow the settings recommended by the laser marker device manufacturer for the various parameters.

[0033] Because a group of housefly larvae (4) placed in a container (3) actively move around, the position of each insect changes over time, making it difficult to determine the exact location of each insect. However, in the present invention, it is sufficient that the laser beam hits the housefly larvae (4). Therefore, by scanning the laser beam with the device shown in FIG. 1 and irradiating it at predetermined intervals, the laser beam will hit at least some of the group of housefly larvae (4). In this case, some larvae (4) will be hit by the laser beam multiple times, and some larvae (4) will not be hit by the laser beam. However, by finding the optimal conditions through experiments that increase the probability of hitting the housefly larvae (4), optimal scanning and irradiation conditions can be selected.

[0034] [Figure 2] conceptually shows two typical marking patterns (pattern A and pattern B) for carrying out the method of the present invention. Pattern A in [Figure 2] is a conceptual diagram of a method in which the laser beam is irradiated (scanned) in a pattern of line segments of a fixed length, while pattern B is a conceptual diagram of a method in which the laser beam is irradiated in a dot pattern. Either method can be used to drill holes that penetrate the insect's cuticle. Commercially available laser markers are capable of marking (printing) desired marks (symbols) on a workpiece by irradiating the workpiece with a laser beam in a continuous or pulsed manner, so the method of the present invention can be easily carried out with a commercially available laser marker.

[0035] As mentioned above, in the case of housefly larvae, the third instar larvae move around actively, so it is not possible to mark the exact position of each larva. In other words, the position of the insect (workpiece) changes over time. Therefore, it is preferable to stop the movement of the larvae when irradiating them with the laser.

[0036] There are various methods for stopping the movement of larvae, but generally, housefly larvae can be stopped by cooling them to just below freezing. In addition to using this temperature change, larvae can also be stopped by using chemicals, such as anesthetics like CO2 gas, or by physical restraint.

[0037] When housefly larvae are cooled to just before freezing to stop their movement, the larvae will die if they freeze, but their movement becomes significantly slower at temperatures below 10°C, so by doing so, it is possible to irradiate and injure multiple housefly larvae as evenly as possible with a laser beam. In practice, the movement of the larvae can be stopped, or almost stopped, by arranging a group of third-instar larvae on a flat surface in a tray cooled to about 3°C ​​in a refrigerator. By scanning and irradiating a group of housefly larvae (4) that have stopped moving with a laser beam, it is possible to drill holes as evenly as possible in multiple housefly larvae. It is preferable to first perform a test print to adjust the position and spacing of the holes.

[0038] The intensity and irradiation time of the laser beam depend on the type of laser irradiation device and the type of insect, and can be determined through experiments. For example, when using a CO2 laser marker with a wavelength of 10.6 μm and an output of 30 W, a hole penetrating the cuticle of a single housefly larva can be created by applying a heat quantity equivalent to 0.0096 J to the larva. The intensity and irradiation time of this laser beam can be determined through experiments depending on the laser irradiation device used. The CO2 laser creates a hole in the cuticle of the housefly larvae using heat. While the effects of this hole creation on the inside of the housefly larvae are currently unknown, the laser irradiation method of the present invention does not kill the housefly larvae and effectively induces antimicrobial peptides.

[0039] When the laser skin irradiation method is performed without precisely detecting the position of the housefly larvae, some of the housefly larvae will die depending on the location where the laser beam hits. For example, if the laser beam hits a vital part such as the heart, the housefly larvae will die. The actual survival rate may be as low as 80%. In this case, the dead housefly larvae may be removed as needed, but they can also be provided as feed without being removed.

[0040] Method using image processing When using a commercially available laser marker as is, it is not possible to precisely measure the position information of the insect (workpiece), so some insects (workpieces) are not hit by the laser beam, and some insects (workpieces) are hit by multiple laser beams. Also, the laser beam may be irradiated onto a location where no insect (workpiece) is present. Therefore, the efficiency of the method of the present invention can be improved by image processing the image of the insect (workpiece).

[0041] [Figure 3] is a conceptual diagram of the case where image processing is used in the method of the present invention, with a CCD camera (8) added to the laser marker of [Figure 1]. While the built-in camera of the laser marker can be used, it is preferable to install a small camera (CMOS camera) or CCD camera (CCD sensor) independently of a commercially available laser marker, or to attach it externally to the laser marker. The method of the present invention can be easily implemented by adding a CCD camera (8) to the laser marker of [Figure 1].

[0042] As shown in Figure 3, the image captured by the CCD camera (8) is sent to a PC (6), where it is processed and the position information of the insect (workpiece) is calculated, and this position information is used by a controller (7) (Figure 1) to control the movement of the laser beam from the laser marker head (1). Some commercially available laser markers can directly import WORD data or bitmap data for laser marking, so simply by adding image processing to the laser marker, the movement of the laser beam can be easily controlled based on accurate position information.

[0043] After an image of a group of housefly larvae (4) placed in a container, such as a tray (3), is taken with a CCD camera (8), the tray (3) is moved under the laser marker head (1). This movement can be automatic or manual. Simply put, the support (9) for the tray (3) can be a conveyor belt, which moves the tray in the direction of the arrow. A dragonfly (31) (see Figures 4 and 7) can be used as a reference position for comparing the image taken by the CCD camera (8) with the image taken by the position detection camera built into the laser marker head (1).

[0044] Other means and devices may be incorporated into the device of Fig. 3. For example, a means for cooling the housefly larvae to stop their movement, such as a freezing means, a means for uniformly distributing the swarm of housefly larvae, such as a vibrating device, or an automatic tray (3) loading and unloading device may be incorporated.

[0045] Image processing can generally be performed using simple software built into a PC (or added later). Figure 4 is a conceptual diagram of an image obtained by simple image processing using binarization. In this case, camera images of a group of housefly larvae captured by a CCD camera (8) are sent to a PC (6), where image processing is performed to extract only the housefly larvae, easily obtaining a binarized image of the housefly larvae (41) as shown in Figure 4. The binarized position information of the group of housefly larvae obtained in this way can sometimes be used directly in the laser marker. Preferably, the binarized position information is further processed, if necessary, to remove noise, etc., to calculate the position information of the insects (workpieces), and this position information is sent to the laser marker's controller (7), which then controls the movement of the laser beam from the laser marker's head (1) based on commands from the controller (7).

[0046] Obtaining more precise position information If you want to irradiate a specific location on an insect (workpiece), for example, only the abdominal part of a housefly, with a laser beam, you need to send more precise position information to the controller (7). In this case, you can use AI image processing technology, etc. Commercially available AI image processing software can be used for this processing.

[0047] Figure 5 is a flowchart illustrating an example of basic operations for obtaining more precise location information when implementing the method of the present invention on houseflies. (1) "Image Acquisition" involves capturing images of a swarm of houseflies using a camera or other device. (2) "Image Preprocessing" involves normalizing the images, reducing noise, and performing other image processing. (3) "Feature Extraction" involves extracting housefly features using a pre-trained convolutional neural network (CNN), such as ResNet, MobileNet, or YOLO. (4) "Region of Interest Detection" involves identifying areas in the image where housefly abdomens may be present. (5) "Abdomen Detection" involves further refining the detected regions using additional neural network or image processing techniques to specifically identify housefly abdomens. (6) "Postprocessing" involves refining the results by filtering out false positives and adjusting bounding boxes. (7) "Output" displays and saves the final result showing the XY coordinate position of the housefly abdomen in the image.

[0048] Figure 6 shows a screen display of, for example, the position of the abdomen of a housefly (42) obtained from precise position information obtained, for example, according to the flowchart shown in Figure 5. The laser marker controller (7) controls the movement of the laser beam from the head (1) based on this position information.

[0049] [Figure 7] is a conceptual diagram of image processing for detecting the "individual regions" (10) of each housefly (4) and identifying its abdomen (43, black circle). In this case, it is also possible to predetermine how to handle overlapping housefly (11).

[0050] After laser irradiation, the houseflies are left for 3 to 48 hours to induce antimicrobial peptides in their bodies. For example, to prevent the larvae from pupating, the houseflies are transferred to an environment with a wet-bulb temperature of 20 to 35°C and maintained there for 3 to 48 hours to induce antimicrobial peptides in the larvae themselves.

[0051] After antimicrobial peptide induction, the antimicrobial peptides are recovered from the houseflies. The tip of the housefly is cut, hemolymph is allowed to leak from the cut end, and the hemolymph is collected in a tube on ice. To remove hemocytes, the hemolymph is centrifuged at 100 g for 10 minutes, and the supernatant is collected and stored at -30°C.

[0052] Antibacterial activity measurement: Bacteria are plated on agar medium and cultured according to standard methods to measure antibacterial activity and evaluate the ability to induce antibacterial peptides. Bacteria that can be selected include Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

[0053] The drawings used in the above description are conceptual diagrams or partially enlarged views, and the dimensions are not exact. Furthermore, the image processing and location information acquisition methods are not limited to those described above. Examples of the present invention are described below, but the present invention is not limited to the following examples.

[0054] Example 1 The following equipment and methods were used: Laser equipment: A Keyence ML-Z9620 CO2 laser marker was used. This is a CO2 laser marker with a simultaneous X, Y, and Z three-axis scanning system, and is capable of irradiating a working space of 100 x 100 mm or 300 x 300 mm with a laser beam of 10.6 μm wavelength and 30 W output power using a scanning system.

[0055] Laser irradiation method: A glass tray (100 x 100 mm) was cooled to a temperature just below the freezing point of housefly larvae (approximately 3°C), and approximately 1,000 housefly larvae were placed inside. The larvae were then arranged flat, with no overlapping. The movement of housefly larvae was significantly reduced on the cooled tray. Using the CO2 laser marker described above, a 1 mm x 1 mm grid marking pattern was created. The laser beam was irradiated onto the group of housefly larvae using the "scan printing" method, with the printing line length set to 0.1 mm and the scan speed set to 250 mm / s. The laser power was set to 80% of the output. The amount of heat applied was equivalent to 0.0096 J.

[0056] Induction of antimicrobial peptides Wet gauze soaked in physiological saline at a concentration close to the osmotic pressure of the larvae was kept at 20°C to 35°C, and the laser-irradiated housefly larvae were left on it for 24 hours to induce antimicrobial peptides in the bodies of the housefly larvae.

[0057] After 24 hours, the tip of the larvae was cut with sharp scissors, and the hemolymph was allowed to leak from the cut end and collected in a tube on ice. Approximately 500 μl of hemolymph was collected from approximately 500 larvae. After centrifugation at 100 g for 10 minutes to remove hemocytes, the supernatant was collected and stored at -30°C.

[0058] Antibacterial Activity Measurement Staphylococcus aureus was cultured in LB medium and bacteria in the logarithmic growth phase (OD600 = 0.15-0.3) were used for measurement. Staphylococcus aureus in the medium (2 μl) was mixed with 18 μl of a body fluid sample. The bacteria / body fluid measurement mixture was incubated at 30°C for 1 hour and then diluted with Mueller-Hinton II medium. The diluted measurement mixture (100 μl) was plated on an agar medium and cultured at 30°C for 1 day. After culture, the colony forming units (CFU) of Staphylococcus aureus were calculated.

[0059] The colony-forming units (CFU) of Staphylococcus aureus in the diluted test mixture (blank) was 167 x 10 5 CFU / ml.

[0060] In contrast, when laser irradiation was performed using the method of the present invention, the colony forming units (CFU) of Staphylococcus aureus was 0 (zero) CFU / ml, indicating that the proliferation of Staphylococcus aureus could be effectively inhibited.

[0061] FIG. 9 is a photograph showing that holes of about 250 μm were formed in the cuticle of a housefly larva when laser irradiation was performed in Example 1.

[0062] Example 2 The same procedure as in Example 1 was repeated, but the marking pattern was changed to a 1 mm x 4 mm grid. In this case, the colony forming units (CFU) of Staphylococcus aureus were also 0.5 x 10 5 Similar to the results of Example 1, it was shown that the proliferation of Staphylococcus aureus could be effectively inhibited at CFU / ml or less.

[0063] Example 3 The same operations as in Example 2 were repeated, but the laser beam irradiation method was changed from the "scan printing" method to the "fixed-point printing" method. The fixed-point printing time was 1 ms, and the amount of heat applied in this case was equivalent to 0.0240 J, which is about 2.5 times that of Example 2 in terms of heat amount. In this case, the colony forming units (CFU) of Staphylococcus aureus were also 2.3 x 10 5 Similar to the results of Example 2, it was shown that the proliferation of Staphylococcus aureus could be effectively inhibited at CFU / ml or less.

[0064] Example 4 The same procedure as in Example 3 was repeated, except that the intervals between the fixed-point prints were changed to a 1 mm x 1 mm grid. In this case, the colony-forming units (CFU) of Staphylococcus aureus were also 0.5 x 10 5 Similar to the results of Example 3, it was shown that the proliferation of Staphylococcus aureus could be effectively inhibited at CFU / ml or less.

[0065] Example 5 After laser irradiation, soil was mixed with physiological saline, and the larvae were left in the soil for 24 hours. Thereafter, the same procedure as in Example 4 was repeated. In this case, the colony forming units (CFU) of Staphylococcus aureus were 22 x 10 5 It was shown that the inhibitory effect on the growth of Staphylococcus aureus decreased at CFU / ml or less.

[0066] Comparative Examples 1 to 3 The same operations as in Example 3 were repeated, except that a UV laser marker was used as the laser irradiation device instead of a CO2 laser marker. The UV laser marker used was a Keyence MD-U1020C, which is capable of emitting a laser beam with a wavelength of 355 nm and a printing output of 2.5 W. The printing method in Comparative Examples 1 to 3 was the same "fixed-point printing" method as in Example 3. The irradiation conditions and the results obtained (colony forming units (CFU) of Staphylococcus aureus) are summarized in Table 1 below.

[0067]

[0068] [Figure 10] is a photograph of the cuticle of a housefly larva when the laser beam hit it during UV laser irradiation in Comparative Example 3, and shows that no holes could be made in the cuticle of the housefly larva. In the case of a UV laser, a narrow laser beam of about 40 μm can be irradiated, but the effect of inhibiting the growth of Staphylococcus aureus is low. This is thought to be because no holes could be made in the cuticle of the housefly larva.

[0069] Comparative Examples 4 to 7 Comparative Example 4 is a "control" (see below). In Comparative Example 5, antimicrobial peptides were induced in the bodies of housefly larvae by manually pricking them with a needle using a conventional method, without using a laser. The needle used was a stainless steel needle with a diameter of 0.3 mm, which was inserted into the abdomen of the housefly larvae. In Comparative Example 6, for comparison with Example 5, soil was mixed with saline solution after pricking a housefly larvae with a needle in the same manner as in Example 5, and the larvae were left in the soil for 24 hours. Comparative Example 7 is a "blank" (see below).

[0070] Except for not irradiating with a laser, antimicrobial peptides were induced in the same manner as in Example 1. The irradiation conditions and the results (colony forming units (CFU) of Staphylococcus aureus) of Comparative Examples 4 to 7 are summarized in Table 2 below.

[0071]

[0072] The "control" (Comparative Example 4) in "Table 2" is a case of housefly larvae that were not irradiated with laser and not pierced with a needle. When antimicrobial peptides were induced in the same manner as in Example 1, the colony-forming units (CFU) of Staphylococcus aureus were 88 x 10 5 The "blank" (Comparative Example 7) was prepared by measuring the antibacterial activity in the same manner as in Example 1, except that the hemolymph of housefly larvae was not included. The colony-forming units (CFU) of Staphylococcus aureus in this "blank" were 167 x 10 5 CFU / ml.

[0073] The following points can be observed from the results in "Table 2": 1) The growth inhibitory effect of the present invention (Examples 1 to 5) on Staphylococcus aureus is significantly higher than that of UV laser (Comparative Examples 1 to 3). 2) The growth inhibitory effect of the present invention (Examples 1 to 5) on Staphylococcus aureus is also higher than that of conventional methods (Comparative Examples 5 and 6). This indicates that "opening holes through the insect's cuticle with a laser beam" is necessary for the growth inhibitory effect on Staphylococcus aureus. 3) Even untreated housefly larvae (control of Comparative Example 4) have a certain growth inhibitory effect on Staphylococcus aureus compared to the blank (Comparative Example 7).

[0074] REFERENCE SIGNS LIST 1 Head of laser irradiation device 2 Laser beam 3 Tray 4 Housefly larvae 6 PC (position detection device) 7 Controller 8 Camera 9 Support stand

Claims

1. A method for inducing antimicrobial peptides in insects by damaging them, comprising irradiating the insect's cuticle with a laser, and damaging the insect by creating holes through the insect's cuticle with the laser beam, without causing irreparable damage to the insect.

2. The method of claim 1, wherein the insects are housefly larvae and the laser is a CO2 laser.

3. The method according to claim 1, wherein the diameter of the holes penetrating the cuticle of the housefly larvae is in the range of 1 to 300 μm.

4. The method according to claim 1, wherein the insect larvae are immobilized at a temperature just below freezing and then irradiated with a laser.

5. The method according to claim 2, wherein after laser irradiation, the laser-irradiated housefly larvae are transferred to an environment with a wet-bulb temperature of 20°C to 35°C and allowed to not pupate for 3 to 48 hours, thereby inducing antimicrobial peptides in the larvae themselves.

6. The method of claim 4, wherein housefly larvae are placed on a wet surface containing a near-osmotic liquid maintained at 20°C to 35°C and exposed to the liquid to inhibit pupation.

7. An apparatus for use in the method of inducing antimicrobial peptides in the bodies of insects described in claim 1, comprising a CO2 laser irradiation device, a position information acquisition means for obtaining position information of the insects, and a means for transmitting the position information of the insects obtained by the position information acquisition means to the insects.

8. The apparatus of claim 7 further comprising means for cooling the insect larvae to a temperature just below freezing to immobilize the insect larvae.

9. Use of a CO2 laser in a method to induce antimicrobial peptides into the insect's body by drilling holes through the insect's cuticle.

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