Female and male discrimination method of bagworm

Abstract

To establish a method capable of reliably discriminating a sexuality of a bagworm from one year old.SOLUTION: Whether a dotted small hole is present or absent in a front center part of a web plate of a second part from a terminal end of an abdominal part of a bagworm is checked. An individual having the dotted small hole is determined as a male individual. An individual not having the dotted small hole is determined as a female individual.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for determining the sex of bagworms and a bagworm population selected using the same, as well as a method for synchronizing the growth rate of bagworms, a method for obtaining female bagworms, and a method for harvesting silk. [Background technology]

[0002] Bagworms are the general term for the larvae of moths belonging to the family Psychidae in the order Lepidoptera. The silk thread spun by these insects (bagworm silk thread) has better physical properties than silk thread spun by silkworms and spider silk spun by spiders, and has been attracting attention in recent years as a highly useful new animal-derived natural fiber (Patent Document 1 and Non-Patent Document 1).

[0003] To make bagworm silk practical as a fiber material for industrial use, it is essential to establish techniques for mass-rearing bagworms and breeding them for generations. When mass-rearing bagworms, it is desirable to subject each individual to the same treatment at the same time in terms of production efficiency and workload. To do this, it is necessary to synchronize the growth of each individual, such as the molting and pupation times.

[0004] However, the growth rate of male and female bagworms differs, and the larval period is generally longer for females. Therefore, even if a group of bagworms that hatched on the same day were reared in the same environment, there would inevitably be a difference in growth when males and females were mixed, making it difficult to synchronize their growth.

[0005] In addition, in the case of successive rearing, it is necessary to artificially adjust the mating and egg-laying periods of the bagworm moths. Therefore, it is essential to obtain unmated female moths in the case of successive rearing. However, bagworms build nests immediately after hatching, and spend the entire larval stage thereafter. Although they expose their heads and thorax outside the nest only when moving and feeding, they basically remain hidden in the nest throughout the larval stage. In addition, unlike silkworms (Bombyx mori), bagworms do not spin cocoons during the pupation stage, but instead pupate in the same nest where they spent their larval stage. Since it is not possible to obtain information about the inside of the nest from outside, it is not possible to know the pupation and emergence times, and it is not uncommon for male moths that emerge from the nest to naturally mate with female moths without being noticed. In addition, since female bagworms generally spend their entire lives inside the nest, it is extremely difficult to determine whether the female moths in the nest are unmated or already mated. For these reasons, it has not been possible to obtain unmated female moths easily, reliably, and in large quantities using conventional techniques.

[0006] Furthermore, the stable harvesting of uniform bagworm silk from multiple bagworms is essential for the practical application of bagworm silk. However, the inventors' research has revealed that the width of silk threads spun by middle-aged and older bagworms differs between males and females. Therefore, the uniformity of bagworm silk obtained from a population of mixed male and female bagworms is poor.

[0007] The above problems can be easily solved by rearing bagworms separately by sex, preferably at an early stage.

[0008] However, a method for sexing bagworms has not been established to date. It is generally known that there is a correlation between the age and head width of lepidopteran larvae. In bagworms, the head width generally increases as the larvae (bagworms) age. Non-Patent Documents 2 and 3 disclose that male bagworms, unlike females, have a smaller head width at the final instar than at the subfinal instar. Although sexing can be achieved to some extent by using the head width at the final instar, it is difficult to say that the accuracy is high. In addition, since this method can only be applied at the subfinal to final instar stages, it cannot be used at all to sex young bagworms, especially first instar larvae after hatching and before nest building. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2019 / 003364 [Non-patent literature]

[0010] [Non-Patent Document 1] Osaki, S., 2002, Sen-i-gakkaishi (Textile and Industry) Journal, 58:74-78. [Non-Patent Document 2] Niitsu S., 2010, Butterflies and Moths, 61(3):218-220. [Non-Patent Document 3] Nishida E., 1983, Insects, 51(3):394-411. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to establish a previously unknown method for reliably determining the sex of bagworms from the first instar (first stage).

[0012] Another objective of the present invention is to synchronize the growth rate of individual bagworms in mass-reared populations by separating males and females based on this method, and to harvest bagworm silk of high uniformity. [Means for solving the problem]

[0013] In order to solve the above problems, the present inventors conducted detailed research and found that there are clear differences between males and females in the external morphology of bagworms, even though they are very minute structures. This difference is observed in the first instar larvae immediately after hatching, and it was also revealed that this is a common feature of bagworms regardless of species. The present invention was developed based on this knowledge and provides the following.

[0014] (1) A method for determining the sex of bagworms, comprising a confirmation step of checking for the presence or absence of a small dot-like hole in the anterior center of the abdominal plate of the second segment from the end of the bagworm's abdomen, and a determination step of determining that individuals with the small dot-like hole are male and individuals without the small dot-like hole are female. (2) The method described in (1), further comprising a separation step of separating the bagworms determined to be male or female based on their sex after the determination step. (3) The method described in (1) or (2), further comprising an exposure step of exposing the entire bagworm or the abdominal end from the nest before the confirmation step. (4) A method according to any one of (1) to (3), wherein the bagworm is in the 1st to 6th instar stage. (5) The method described in (4), wherein the bagworms are in the first instar after hatching and before nesting. (6) A method for synchronizing the growth rates of bagworms, comprising a confirmation step of confirming the presence or absence of small dot-like holes in the anterior center of the abdominal plate of the second segment from the end of the abdomen in a number of bagworms of the same age, a determination step of determining whether or not individuals with the small dot-like holes are male and whether or not they are female, a sorting step of separating the bagworms determined to be male or female based on their sex, and a breeding step of breeding the males and females in separate groups. (7) The method described in (6), wherein the bagworms are in the first instar after hatching and before nesting. (8) A method for selectively obtaining female individuals of the bagworm moth, comprising a confirmation step of confirming the presence or absence of small dots in the anterior center of the abdominal plate of the second segment from the end of the abdomen during the larval stage, a female selection step of selecting as female individuals larvae that do not have the small dots, and a rearing step of rearing the selected larvae until the pupal stage or adult stage. (9) A method for harvesting bagworm silk thread, comprising a confirmation step of checking for the presence or absence of small dot-like holes in the anterior center of the abdominal plate of the second segment from the end of the bagworm's abdomen, a determination step of determining whether or not individuals with the small dot-like holes are male and whether or not they are female, a step of separating the bagworms determined to be sexed based on their sex, and a silk harvesting step of harvesting bagworm silk thread from each of the male and female populations. (10) The method described in (9), wherein the bagworm is in the third instar or later. (11) A group consisting of 10 or more bagworms of the same species and up to 6 instars, with a small dot in the anterior center of the abdominal plate of the second abdominal segment from the end of the abdomen. (12) A population consisting of 10 or more bagworms of the same species and up to 6 instars, with no small punctate hole in the anterior center of the abdominal plate of the second abdominal segment from the end of the abdomen. (13) A population described in (11) or (12), in which each bagworm is of the same age. (14) The population described in (13), wherein the bagworms are in the first instar. Effect of the Invention

[0015] According to the method for determining the sex of bagworms of the present invention, the sex of bagworms can be easily and accurately determined from an early stage.

[0016] According to the method for synchronizing the growth rates of bagworms of the present invention, it is possible to synchronize the growth rates of individuals in mass-rearing.

[0017] According to the method for obtaining female Bagworm moths of the present invention, mating and egg collection can be artificially controlled.

[0018] The present invention's method for determining the sex of bagworms, method for synchronizing the growth rate of bagworms, and method for obtaining female bagworms makes it possible to harvest bagworm silk with high uniformity. [Brief description of the drawings]

[0019] [Figure 1] A schematic diagram of the ventral surface of a bagworm to explain the small punctate holes (0106) in a male bagworm. A shows the ventral surface of the entire bagworm extracted from the nest, and B is an enlarged view of the area enclosed by the dashed line in A. [Diagram 2]These are diagrams showing the abdominal end area of ​​males and females of various bagworms. A and B show larvae of the giant bagworm moth (Eumeta japonica), while C and D show larvae of the brown bagworm moth (Eumeta minuscula). A and C are males, while B and D are females. The circular frame shows the area around the anterior center of the abdominal plate of the second segment from the abdominal end of the bagworm. Small dot-like holes can be seen within the frame only in male bagworm individuals (A and C). [Diagram 3] FIG. 1 shows the testes dissected from the bagworms of E. minuscula in Example 1, Sample Nos. 1 to 3. In the figure, A is the testis (arrow) from Sample No. 1, B is from Sample No. 2, and C is from Sample No. 3. [Figure 4] This figure shows the average total larval period (the period from hatching to the onset of pupation) for males and females of E. japonica. Error bars indicate standard deviation. [Diagram 5] This is a diagram showing the average width (thickness) of spin fibers in male and female bagworms of the E. japonica from the third instar onwards. Error bars indicate standard deviation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 1. How to distinguish between male and female bagworms 1-1. Overview The first aspect of the present invention is a method for determining the sex of bagworms. In the method of this aspect, the differences in external morphology between male and female bagworms are confirmed, and the sex is determined based on these differences. According to the method of this aspect, the sex of bagworms can be easily and simply determined, regardless of the type or age of the bagworm.

[0021] 1-2.Definition of Terms As used herein, the following terms are defined. "Baby Moth" is a general term for moths belonging to the Psychidae family of the Lepidoptera order. Moths of the Psychidae family are distributed throughout the world, but the subject of the present specification may be any species. For example, but not limited to, species belonging to Acanthopsyche, Anatolopsyche, Bacotia, Bambalina, Canephora, Chalioides, Dahlica, Diplodoma, Eumeta, Eumasia, Kozhantshikovia, Mahasena, Nipponopsyche, Paranarychia, Proutia, Psyche, Pteroma, Siederia, Striglocyrbasia, Taleporia, Theriodopteryx, and Trigonodoma, etc. For example, species belonging to Eumeta, Mahasena, Metura, Thyridopteryx, etc. are preferred as the subject of the present specification. Specifically, examples include Eumeta japonica, Eumeta minuscula, Mahasena aurea, Metura elongatus, and Thyridopteryx ephemeraeformis. In this specification, when simply referring to bagworms, it refers to adult bagworms regardless of gender, unless otherwise specified. On the other hand, in this specification, bagworm larvae are referred to as "bagworms." In addition, when referring to "female individuals" or "male individuals" in this specification, it indicates the sex of the individual regardless of the developmental stage. However, when referring to "female adult (female adult individual)" and "male adult (male adult individual)," they mean "female moth" and "male moth," respectively.

[0022] As mentioned above, "bagworm" is a general term for the larvae of bagworm moths. Unless otherwise specified, the bagworms covered in this specification can be male or female and of any age. In this specification, bagworms are referred to as young bagworms, middle-aged bagworms, and old bagworms according to their stage of growth. "Young bagworms" refers to bagworms in the early stages of growth. This mainly applies to 1st to 2nd instar individuals immediately after hatching. "Middle-aged bagworms" refers to bagworms in the middle stages of growth. This mainly applies to 3rd to 4th instar individuals. "Old bagworms" refers to bagworms in the later stages of growth. This mainly applies to 5th instar individuals or later.

[0023] As used herein, "subject bagworm" refers to a bagworm that is subjected to the methods described herein.

[0024] As used herein, a "population" refers to a group consisting of multiple individuals. A "male population" refers to a population consisting only of male individuals, and a "female population" refers to a population consisting only of female individuals. A "male / female population" is a general term for a "male population" and a "female population," and is composed of either or both of the populations.

[0025] As used herein, "bagworm silk" refers to silk spun by bagworms. Bagworm silk includes scaffold silk and nest silk.

[0026] In this specification, "nest silk" refers to silk threads that make up the nest, and are spun to bind together leaf and branch pieces, and to create a comfortable environment on the inner walls of the nest, which is the living area.

[0027] As used herein, "scaffold silk" refers to silk that bagworms spin before moving, and functions as a scaffold to prevent them from falling off branches, leaves, etc., while moving. Bagworms usually use the scaffold silk as a foothold to hook the claws of both legs and move in the direction of travel. As a general rule, scaffold silk is thicker and more mechanically strong than nest silk.

[0028] In this specification, "single fiber" refers to the smallest unit filament that constitutes the fiber component of bagworm silk, and is also called monofilament. Single fibers are mainly composed of fibroin-like proteins that constitute bagworm silk. Bagworm silk is spun in the form of difilaments, in which two single fibers are bonded in the longitudinal direction in pairs, and as a rule, single fibers do not exist in the natural state. Therefore, by undergoing an artificial refining process, the adhesive substances between the monofilaments are removed, and the two are separated to obtain single fibers.

[0029] In this specification, the term "spun fiber" refers to the silk thread spun by the bagworm. The spun fiber has a difilament structure in which two single fibers are ejected from the spinnerets located on the left and right sides of the bagworm and bonded together by an adhesive during spinning.

[0030] In this specification, the term "(bagworm) nest" refers to a nest built by a bagworm, and is composed of the silk threads that the bagworm spins and materials such as leaves and branches bound together with the silk threads. The nest is a bag-like structure that can encase the entire body of the bagworm, and can be shaped like a spindle, cylinder, or cone. The bagworm exposes part of its body from the opening of the nest only when feeding or moving, but it basically hides within the nest. Furthermore, a bagworm's nest is made immediately after hatching, and the number of nests increases as the bagworm grows, so in principle, new nests are not built. Furthermore, since the bagworm pupates in the nest, it does not spin a cocoon again before pupation. Therefore, in principle, the bagworm only exposes its entire body from the period after hatching until nest building is completed.

[0031] 1-3. Method The sex-determining method of the present invention includes a confirmation step and a determination step as essential steps, and an exposure step and a separation step as selection steps. Here, each step will be specifically explained using the schematic diagram of the ventral side around the terminal abdominal segment of a bagworm shown in Figure 1.

[0032] (1) Exposure process The "exposure step" is a step of exposing the entire bagworm or the abdominal end from the nest. The "abdominal end" here refers to the area including several abdominal segments from the abdominal end of the bagworm. Bagworms build nests immediately after hatching and spend their entire larval stage in the nest, so in principle the end of their abdomen is not exposed to the outside world. Therefore, the confirmation process described below cannot be carried out directly on bagworms after they have built nests. Therefore, this process is carried out on bagworms after they have built nests prior to the confirmation process described below.

[0033] In this process, there are no limitations on the method of exposure as long as the bagworm can be exposed from the nest in a state where at least the second segment from the end of the abdomen can be visually observed. It is desirable to separate the bagworm in a way that does not cause trauma or excessive stress to the bagworm. For example, there is a method of cutting open the bagworm nest with a blade such as scissors without damaging the bagworm inside and exposing the entire body, or a method of cutting out the area around the nest's outlet (a hole located at the lower end when the nest is suspended and opened to excrete feces) with a blade, enlarging the hole diameter, and then pushing out the bagworm while applying light pressure from outside the nest so that the abdominal end of the bagworm is exposed. This process is not necessary for first-instar bagworms that have hatched but not yet built nests.

[0034] (2) Confirmation process The "confirmation process" is a process in which the presence or absence of a small dot-like hole in the anterior center of the abdominal plate of the second segment from the end of the abdominal cavity of the target bagworm to be sexed is confirmed. In this specification, "the second segment from the end of the bagworm's abdomen" refers to the second segment (0103) forward from the terminal abdominal segment (0102) in the abdominal segment (0101) that constitutes the bagworm's abdomen (0100) shown in Figure 1. This corresponds to the ninth segment counting from the first abdominal segment. The "terminal abdominal segment" here refers to the abdominal segment located at the tail end and having the uropods (0105). "Uropods" refers to a pair of abdominal legs provided at the terminal abdominal segment. Additionally, "abdominal legs" refers to the leg-like organs found in the larval abdomen of lepidopteran insects and the like.

[0035] In this specification, the "anterior central part of the abdominal plate" refers to the anterior central part of the abdominal plate of the exoskeleton that covers the ventral side of the abdominal segment. "Anterior" here refers to the area anterior to the boundary (0107) when the abdominal plate is divided into two equal parts perpendicularly to the anterior-posterior axis. In many cases, this anterior corresponds to a position close to the boundary between the abdominal end and the third segment (0104). Additionally, "central part" here refers to the central part of the left-right axis of the abdominal plate.

[0036] In this specification, the "punctate pores" (0106) refer to small black dots. Strictly speaking, they are not holes like an anus that open from inside the body to the outside, but hole-like depressions. The punctate pores are structures that serve as indicators for sexing in the present invention.

[0037] The size of the punctate pores varies depending on the species and age of the target bagworm, but for example, for old large species of bagworms such as the Japanese bagworm moth (E. japonica), the size will be in the range of approximately 500 μm to 1000 μm in diameter, although this is not limited thereto, and for old medium-sized species of bagworms such as the brown bagworm moth (E. minuscula), the size will be in the range of approximately 100 μm to 500 μm in diameter, although this is not limited thereto.

[0038] In this process, the method for checking the presence or absence of small dot-like holes is not particularly limited. In general, in the case of old bagworms of large or medium-sized bagworm species, the presence or absence of small dot-like holes can be confirmed with the naked eye. However, in the case of young and middle-aged bagworms, especially first-instar bagworms immediately after hatching, the target individuals themselves are small, making it difficult to confirm the small dot-like holes with the naked eye. Therefore, although there is no limitation on the method for checking the presence or absence of small dot-like holes in this process, it is preferable to enlarge the target area using an instrument such as a stereomicroscope to check.

[0039] The age of the bagworms targeted in this process does not matter. They may be from the first instar to the final instar, preferably from the first instar to the sixth instar. However, as mentioned above, bagworms build nests immediately after hatching, and then grow while expanding and remodeling the nest, so in principle, the abdominal end is not exposed to the outside of the nest during the larval stage. Therefore, when carrying out this process on bagworms that have nests, it is necessary to carry out the exposure process. After the determination process described below, a new nest is reconstructed by providing appropriate nesting material to the bagworm that has lost its nest. However, there is a risk of excessive stress on the bagworm and causing it to lose its physical strength. Therefore, although not limited, it is preferable to carry out this process on the first instar larvae after hatching until they build a nest.

[0040] (3) Judgment process The "determination step" is a step of determining whether the individual is male or female based on the punctate pores at the relevant site. As shown in Figures 2A and C, the punctate pores are present only in male individuals. In female individuals, as shown in Figures 2B and D, there is no corresponding structure at the same position, or only an unclear wrinkle-like depression is observed. Therefore, individuals with punctate pores can be determined as male individuals, and other individuals as female individuals. As mentioned above, the presence or absence of small punctate holes can be confirmed from the first to final instars of bagworms. Therefore, in this process, it is possible to determine the sex of bagworms at any stage.

[0041] (4) Sorting process The "separation step" is a step of separating the bagworms determined to be male or female in the determination step based on their sex. This step is a selection step in the sex determination method of the present invention, and may be performed as necessary. In this step, one or more bagworms determined to be male or female may be separated into separate containers for each sex.

[0042] 1-4. Male and female populations of bagworms By using the method for determining the sex of bagworms, a population of bagworms can be separated into a population of male bagworms and a population of female bagworms. A population completely separated by sex can be applied to each method from the second aspect onwards, and is extremely significant in that it can improve the efficiency of breeding management and silk harvesting, thereby reducing breeding costs and increasing the added value of bagworm silk.

[0043] As mentioned above, reliable sex discrimination in bagworms has not been established to date, and therefore it has not been possible to obtain a population consisting of both males and females as in the present invention. However, by using the method for sex discrimination of bagworms, it is possible to obtain a population consisting of only male bagworms of the same species and / or a population consisting of only female bagworms of the same species.

[0044] A population is composed of two or more animals, and preferably 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, or 1,000 or more. On the other hand, there is no upper limit, but it is usually 5 million or less, 1 million or less, 500,000 or less, 100,000 or less, 50,000 or less, 20,000 or less, 10,000 or less, 8,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less.

[0045] Furthermore, the male and female populations of the present invention are, in principle, composed of living bagworms. However, when rearing a male and female population, some of the bagworms may be dead. The term "some" as used herein is not limited to, but may be 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the population.

[0046] The bagworms constituting the male and female population of the present invention are preferably, but not limited to, the same age. More preferably, they are 1st instar bagworms, particularly 1st instar bagworms after hatching and before nesting. Such a male and female population consisting of young bagworms has not been obtainable by conventional techniques.

[0047] Effects According to the method of the present invention for determining the sex of bagworms, it is possible to easily, simply and reliably determine the sex of the bagworm regardless of the type of bagworm or the age of the bagworm. Furthermore, the sex-determining method of the present invention can serve as the basic technology for various inventions from the second aspect onwards in mass-rearing of bagworms.

[0048] 2. How to synchronize the growth rate of bagworms 2-1. Overview A second aspect of the present invention is a method for synchronizing the growth rates of bagworms. Male and female bagworms grow at different rates, with females generally having a longer larval period. Therefore, even if individuals hatched on the same day are reared in the same environment, there will be differences in growth between male and female individuals. According to the method of the present invention, by separating male and female bagworms at an early stage and rearing them in separate groups, it is possible to eliminate the difference in growth between individuals reared in groups.

[0049] 2-2. Method The method of the present invention includes a confirmation step, a determination step, a sorting step, and a rearing step as essential steps, and an exposure step as a selection step. Among these, the explanations of the exposure step, the confirmation step, the determination step, and the sorting step are basically the same as the explanations of the respective steps in the sexing method described in the first embodiment, so duplicate explanations will be omitted. Therefore, here, the characteristic points of this embodiment will be explained below.

[0050] (1) Confirmation process The basic procedure of the confirmation step is similar to that of the first embodiment. However, whereas the bagworms to be sexed in the first embodiment may be one or more than one, the present method is a method for synchronizing the growth rates of multiple individuals, so the number of bagworms to be sexed is multiple.

[0051] Furthermore, the population of bagworms used in the confirmation process of the present invention is, in principle, composed of bagworms of the same age. This is to minimize the growth difference between individuals at the start of the method of the present invention. However, when multiple individuals are kept, some individual differences in growth may occur even among individuals of the same sex, so it is acceptable for individuals that are one age apart from the age that makes up the overwhelming majority in the population to be mixed in.

[0052] The age of the bagworms used in the confirmation process in the present invention does not matter, but for the purposes of the present invention, the earlier the age used, the easier it is to synchronize. Therefore, younger ages are preferred, and 1st instars are more preferred. A group of 1st instars hatched on the same day or within 2 days is even more preferred. A group of 1st instars hatched on the same day is particularly preferred. This is because the greater the difference in time that has passed since the hatching date, the more significant the difference in growth between individuals becomes, making synchronization more difficult. Furthermore, the most preferred age for directly performing this process without going through the exposure process is a group of 1st instars hatched on the same day before nesting.

[0053] (2) Rearing process The "breeding process" is a process in which the population of male individuals (male population) and the population of female individuals (female population) separated in the sorting process are each raised in separate populations.

[0054] The specific rearing method for each group is not limited. It may be normal rearing in which normal feed leaves are fed, or germ-free rearing in which artificial feed is fed under germ-free conditions. Alternatively, the group may be reared in a germ-free environment and then shifted to normal rearing.

[0055] In normal rearing, the bagworms are kept in rearing containers containing groups of males and females at an appropriate temperature and humidity, and food leaves are provided as needed, and feces are regularly removed, etc., so long as the bagworms are constantly fed in a clean environment that is favorable for their growth, and normal rearing methods are used, with no particular limitations.

[0056] On the other hand, in the case of germ-free rearing, although not limited thereto, at least young bagworms immediately after hatching to middle-aged bagworms can be reared under germ-free conditions.

[0057] When rearing bagworms under completely sterile conditions, it is generally difficult to sterilize the bagworms, so it is desirable to rear them from the egg stage. In this case, a sterilization step for sterilizing the surface of the bagworm eggs and a hatching step for hatching the eggs under sterile conditions are carried out before the confirmation step. Also, although not limited thereto, it is preferable to rear the bagworms under sterile conditions even after the hatching step, from the confirmation step to the rearing step.

[0058] The "sterilization step" is a step for sterilizing the surface of the eggs of the Psilocybe in the fish. The purpose of this step is to prevent the introduction of contaminants into the germ-free rearing environment via the eggs.

[0059] The time of the eggs to be subjected to the sterilization treatment does not matter. They may be eggs immediately after laying or immediately before hatching. However, since eggs immediately after laying or immediately before hatching have low resistance to external physical stimuli and have a high mortality rate, eggs that have developed to a certain extent after laying are preferred. For example, eggs several days before hatching are suitable.

[0060] The surfaces of the eggs are sterilized either as egg masses or after being individually separated as necessary. The sterilization method is not limited as long as it can sterilize contaminants such as bacteria attached to the surface of the eggs without affecting the eggs themselves, and any known sterilization method can be used. For example, a method of contacting with ethanol or formalin can be mentioned. More specifically, for example, the obtained eggs can be contacted with 70% ethanol or 2-3% formalin for 20-60 seconds, 30-50 seconds, or 35-40 seconds. The "contact" here refers to immersion, spraying, coating, or a combination thereof. Immersion is preferable.

[0061] After this step, the eggs may be washed once or multiple times with sterilized water. Note that this step is desirably performed under aseptic conditions, such as in a clean bench.

[0062] The "hatching step" is a step in which the eggs obtained after the sterilization step are hatched under sterile conditions. The eggs obtained in the sterilization step are placed under sterile conditions on the nesting material or near the contact boundary between the nesting material and the artificial feed. This is to eliminate or reduce the burden on the bagworms to move around, since the bagworms are the first to begin nesting work after hatching. After that, they are left as they are in an environment suitable for the growth of the bagworms (for example, the optimal growth temperature of 23°C to 27°C).

[0063] On the other hand, if bagworms are reared under aseptic conditions that ensure a certain degree of sterility, the rearing conditions under the completely aseptic conditions can be simplified. For example, first-instar larvae hatched from eggs that have not been sterilized can be placed under aseptic conditions on the nesting material or near the contact boundary between the nesting material and the artificial diet.

[0064] 2-3.Effects By using the method for synchronizing the growth rates of bagworms of the present invention, it is possible to eliminate the difference in growth caused by sex differences within a group by rearing bagworms separately for males and females, and to synchronize their growth rates. This makes it possible to synchronize the timing of food exchange and pupation in a group of bagworms, simplifying rearing management, improving work efficiency in silk thread production, and reducing costs.

[0065] 3. How to obtain female Bagworms 3-1. Overview The third aspect of the present invention is a method for obtaining female individuals of bagworm moths. In order to adjust the timing of mating and egg collection in successive breeding, it is essential to obtain unmated female individuals. However, bagworms build nests immediately after hatching, and after hiding in the nest, they spend the entire larval stage there, and furthermore, females of some species do not leave the nest even after emerging, so it is extremely difficult to confirm and obtain unmated female moths from outside the nest.

[0066] However, according to the method of the present invention, by applying the method of sexing bagworms of the first aspect and separating and rearing the bagworms at an early stage, female bagworms, particularly virgin female bagworms, can be obtained easily and reliably. Furthermore, it is also possible to artificially adjust the timing of mating and egg collection using the virgin female bagworms obtained.

[0067] 3-2. Method The method for acquiring female Psilocybe japonica of the present invention includes a confirmation step, a female selection step, and a rearing step as essential steps, and an exposure step as a selection step. Of these, the exposure step and the confirmation step are basically the same as those described in the first embodiment, and the rearing step is basically the same as those described in the second embodiment, so overlapping explanations will be omitted. Therefore, the following explanation will focus on the female selection step, which is a step unique to this embodiment.

[0068] The bagworms used in the method of the present invention can be either individual or a population, but considering that the main objective is to obtain female individuals under mass breeding, a population is preferred.

[0069] In addition, in the method of the present invention, except for a part of the rearing process, the subject of application is bagworms, which are the larvae of bagworm moths. The age of the bagworms used does not matter, but young bagworms are preferred, and first instar bagworms are more preferred. First instar bagworms after hatching and before nesting are particularly preferred. This is because the omission of the exposure process can reduce the labor, processing time, cost, and burden of bagworms re-nesting.

[0070] (1) Female selection process The "female selection process" is a process in which, after the confirmation process, larvae that do not have punctate holes are selected as females. This process basically follows the same procedures as the determination process and sorting process described above, except that only females are selected from bagworms of unknown sex or from a group of bagworms. After this process, only female bagworms are selected.

[0071] (2) Rearing process The basic procedure of the rearing process of the present invention is similar to that of the third embodiment. In the rearing process of the present invention, the selected female bagworms are reared until they reach the pupal stage or adult stage. As long as the inclusion of male bagworms and / or the invasion of male moths is prevented in this process, the individuals that emerge after this process are virgin female individuals.

[0072] Effects According to the method for obtaining female Bagworm moths of the present invention, it is possible to easily, reliably and in large quantities to obtain unmated adult female Bagworm moths (female moths), which were difficult to obtain using conventional technology due to the difficulty in identifying them and the occasional contamination with mated individuals.

[0073] Furthermore, by using unmated female moths obtained by this method, it is possible to artificially regulate mating and egg collection for successive generations, thereby making it possible to streamline rearing management in mass breeding.

[0074] 4. How to harvest bagworm silk 4-1. Overview The fourth step of the present invention is a method for harvesting bagworm silk. The present inventors' research has revealed that the width of the silk spun by bagworms differs between males and females in old age, even if they are the same species.

[0075] In mass production of bagworm silk, it is important to reproducibly obtain homogeneous silk in order to ensure stable quality and uniform physical properties.

[0076] In the present invention, by applying the method for determining the sex of bagworms described in the first aspect and the method for synchronizing the growth rates of bagworms described in the second aspect, a method is provided for harvesting bagworm silk threads of the same or near-uniform width from a plurality of bagworms, for example.

[0077] 4-2. Method The method for harvesting bagworm silk of the present invention includes a confirmation step, a determination step, a sorting step, and a silk harvesting step as essential steps, and an exposure step and a rearing step as optional steps. Of these, the explanations of the exposure step, confirmation step, determination step, sorting step, and rearing step basically follow the explanations of the corresponding steps described in each of the above-mentioned aspects. Therefore, the matters already explained will be omitted here, and the characteristic points of this method and the silk harvesting step unique to this method will be explained below.

[0078] The subject of the present invention is the larvae of the bagworm moth, i.e., bagworms, which spin silk threads. Furthermore, the method of the present invention is in principle applicable to a population of bagworms of the same species, age and sex.

[0079] The age of the bagworms used does not matter, but as mentioned above, it is preferable that the ages of each individual within a population are the same in principle. However, a difference of one instar before and after molting based on individual differences in growth is acceptable. Bagworms of the third instar or later are preferable. This is because, as shown in the examples described below, the gender difference in silk width becomes significant from the third instar onwards. Therefore, although not limited to this, it is convenient to use the bagworm growth rate synchronization method described in the second aspect of the present invention to synchronize and rear a population of first instar bagworms before nesting, separately for males and females, immediately after hatching, and then apply the method of the present invention to bagworms of each population at an appropriate age, preferably from the third instar or later, more preferably from the fifth instar or later.

[0080] (1) Thread collecting process The "silk harvesting process" is a process in which bagworm silk is harvested from each of the bagworms that have been separated into male and female in the sorting process. In general, the silk threads spun by female bagworms are wider than those spun by male bagworms. This tendency is particularly noticeable in bagworms in the middle or later stages. The specific width of the thread varies depending on the type of bagworm and the age of the bagworm, but for example, the average width of the silk fibers spun by the giant bagworm moth (E. japonica) from the third instar onwards is 6.7 μm for male bagworms and 8.0 μm for female bagworms.

[0081] By separating female and male individuals of the same species and age in the above-mentioned sorting process, it is possible in this process to harvest bagworm silk threads of the same or near-uniform width from multiple bagworms of either sex.

[0082] As used herein, "same width" means that the thread width parallel to the short axis direction per single fiber, or the thread width parallel to the short axis direction per spun fiber, is the same for the single fibers or spun fibers of multiple bagworm silk threads, respectively. On the other hand, as used herein, "quasi-same width" means that the difference in each thread width between multiple bagworm silk threads is so small that it can be said to be substantially the same width. For example, the difference may be 200 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, or 10 nm or less.

[0083] In this process, silk threads derived from male bagworms or female bagworms can be harvested. In addition, silk threads derived from either nest silk threads and / or scaffold silk threads may be harvested in this process, and may be appropriately selected as needed.

[0084] There is no limitation on the method for harvesting bagworm silk from bagworms. For example, the present inventors have developed and filed patent applications for various methods for harvesting bagworm silk. All of these methods are methods for harvesting only bagworm silk that does not contain impurities such as leaf pieces or branch pieces. Although there is no limitation, any of these methods may be used.

[0085] For example, when harvesting continuous long bagworm silk threads, the threads may be harvested based on the method disclosed in Patent No. 6998032, "Method and device for producing long bagworm silk threads." Specifically, the method includes a step of engaging the legs of the bagworm holding the nest with a linear path that has a width less than the maximum left and right leg spread width of the bagworm used and can engage the legs of the bagworm, and causing the bagworm to continuously spin threads along the linear path. To easily carry out this method, the threads may be harvested using the silk harvesting device disclosed in WO2020 / 116503, "Bagus silk harvesting device and method for producing long bagworm silk threads." This silk harvesting device is equipped with a movable linear path that moves in the longitudinal direction and a fixing device that fixes the bagworm, the movable linear path having a width less than the maximum left-right leg spread width of the bagworm fixed to the fixing device and configured to be able to engage the legs of the bagworm, and the fixing device is configured to be positioned so that the fixed bagworm can be engaged with the movable linear path.

[0086] In addition, when harvesting bagworm silk in the form of a lump of silk, the silk can be harvested using the silk harvesting methods disclosed in Patent No. 6960660, Patent No. 7076084, Patent No. 7178735, WO2019 / 203228, and WO2020 / 045381.

[0087] Effects Using this method, homogenized bagworm silk can be harvested reliably. EXAMPLES

[0088] <Example 1: Verification of sex discrimination based on morphological characteristics of bagworms> (the purpose) By confirming that bagworms with punctate slits in the present invention have testes and that individuals without punctate slits do not have testes, the accuracy of the method for determining the sex of bagworms described in the first aspect of the present invention is verified.

[0089] (method) The bagworms used were eight 5th or 6th instar larvae of the giant bagworm moth (E. japonica) and six 3rd to 6th instar larvae of the brown bagworm moth (E. minuscula). Based on the method for determining the sex of bagworms described in the first aspect of the present invention, each type of bagworm was divided into individuals with punctate pores and individuals without punctate pores, and the presence or absence of testes was confirmed by dissection.

[0090] (result) The results are shown in Table 1 (Giant Bagworm Moth) and Table 2 (Brown Bagworm Moth). Figure 3 shows the testes extracted by dissection from brown bagworm moth (Sample No. 1-3).

[0091] [Table 1]

[0092] [Table 2]

[0093] In the table, "Yes" is indicated by "○" and "No" is indicated by "×".

[0094] When comparing the presence or absence of punctate holes in various types of bagworms and the presence or absence of testes, it was found that all individuals with punctate holes in both the giant bagworm moth and the brown bagworm moth had testes, without exception. On the other hand, testes were not found in any bagworms without punctate holes. Therefore, it was confirmed that bagworms with punctate holes were males, and those without punctate holes were females.

[0095] From the above results, it has been proven that the method of the present invention for determining the sex of bagworms is a highly reliable method for determining the sex of bagworms regardless of species.

[0096] <Example 2: Sex discrimination and sex ratio verification of 1st instar bagworms immediately after hatching> (the purpose) It was made clear from Example 1 that it is possible to distinguish between male and female bagworms based on the presence or absence of small punctate holes. Therefore, the sex-distinguishing method of the present invention was applied to a large number of first-instar bagworms immediately after hatching, and it was confirmed that the sex ratio was close to the expected value (1:1) and that the sex-distinguishing method of the present invention is effective from the first instar.

[0097] (method) A total of 502 1st instar bagworms collected from 4 female moths of E. japonica immediately after hatching and before nesting were examined using a stereomicroscope to check for the presence or absence of small punctate holes, and sex determination was performed to confirm the sex ratio. After sex determination, the males and females were reared separately, and after pupation, the pupae were removed and the sex was reconfirmed based on their morphology.

[0098] (result) Table 3 shows the sex ratio results after hatching.

[0099] [Table 3]

[0100] The male / female ratio was 49.6:50.4. A binomial test showed that this difference between males and females was not significant. Therefore, the sex ratio was 1:1, the same as the expected value. Furthermore, reconfirmation after pupation revealed that the method for determining the sex of bagworms of the present invention can also be applied to first-instar bagworms immediately after hatching. These results also suggest that it is possible to artificially control the number of males and females raised at an early stage of bagworm rearing.

[0101] Example 3: Differences in growth rate between males and females of bagworms (the purpose) It is said that the growth rate of male and female bagworms differs, so we investigated how much difference there is between males and females in the entire larval period from hatching to pupation.

[0102] (method) The hatched larvae (Female No. 1, 66 females, 44 males) sexed in Example 2 were placed in a sterile environment containing artificial feed and nesting materials for each sex. All eggs hatched on the same day. After hatching, they were reared in a sterile environment for about 42 days at 24±1°C, 16 hours light: 8 hours dark (16L8D). After that, the bagworms that had grown to the third instar (42 females, 31 males) were transferred to an artificial feed cage and reared at 24±1°C, 16L8D, and the period until pupation was recorded individually. After pupation, the pupae were removed from the cage, and the sex was reconfirmed based on their morphology, and the period from hatching to pupation (total larval period) for both sexes was calculated.

[0103] (result) The results are shown in Figure 4. The average total larval period for males was 138.1 days (n = 20), while the average total larval period for females was 173.2 days (n = 13), a difference of 35.1 days, making it clear that the female larval period was approximately one month longer.

[0104] These results suggest that because there is a difference in growth rate between male and female bagworms, it is not possible to synchronize the rearing of populations containing both sexes, even if they are reared in the same environment. On the other hand, by using the growth rate synchronization method of the present invention, it becomes possible to rear male and female populations separately, and this result suggests that the difference in growth rate due to differences in sex within a population can be eliminated.

[0105] Example 4: Sexual difference in the width of bagworm silk threads (the purpose) Bagworm silk is preferably silk harvested from bagworms of the third instar or higher in terms of thickness, quality, physical properties, etc. Therefore, in this example, we examine whether there is a difference between males and females in the scaffold silk spun by bagworms of the third instar or higher.

[0106] (method) Bagworms of each age from the 3rd instar onwards (males: 3rd to 6th instars; females: 3rd to 8th instars) of E. japonica, which had been sexed using the method for sexing bagworms of the present invention, were placed one by one on a slide glass and allowed to spin scaffold silk threads on the surface. The resulting spun silk threads of bagworms of each age, male and female, were photographed under a microscope and the thread widths were measured.

[0107] (result) The results are shown in Figure 5. The average width of silk threads from male bagworms of the third instar or later was 6.7 μm (n = 1,147), while the average width of silk threads from female bagworms of the third instar or later was 8.0 μm (n = 1,396), making it clear that female bagworms were larger.

[0108] By the way, male bagworms such as the giant bagworm moth and brown bagworm moth completely close the opening of the nest before molting from the 6th to 7th instar and stop feeding, moving, and other activities in preparation for pupation, so silk cannot be harvested from 7th instar male bagworms. On the other hand, female bagworms continue to feed and move even at the 7th and 8th instars, so silk can be harvested. In other words, female bagworms have a longer period in which silk can be harvested than male bagworms, and the obtained bagworm silk is thicker than that of the 6th instar.

[0109] Therefore, it was shown that in order to harvest homogeneous bagworm silk of the same or near-uniform width, or in order to harvest silk over a longer period of time, it is very important to separate male and female bagworms and harvest silk from each population, as this also contributes to the production efficiency of bagworm silk.

Claims

1. A method for determining the sex of a bagworm, comprising the steps of: A confirmation process for confirming the presence or absence of small dots in the center of the front of the abdominal plate of the second segment from the end of the abdomen of the bagworm; and determining whether or not the individual has the punctate pores as a female individual; The method comprising:

2. The method of claim 1, further comprising a sorting step of separating the sex-determined bagworms based on sex after the determination step.

3. The method according to claim 1 or 2, further comprising an exposure step of exposing the entire bagworm or the abdominal end from the nest prior to the confirmation step.

4. The method according to claim 1 or 2, wherein the bagworms are in the 1st to 6th instar stages.

5. The method of claim 4, wherein the bagworms are in their first instar after hatching and before nesting.

6. 1. A method for synchronizing growth rate in a bagworm, comprising: A process of checking the presence or absence of small dot-like holes in the anterior center of the abdominal plate of the second segment from the end of the abdomen in multiple same-age bagworms; determining whether or not the individual has the punctate pores as a female individual; A separation step of separating the sexed bagworms based on their sex; and The breeding process involves raising males and females in separate groups. The method comprising:

7. The method of claim 6, wherein the bagworms are in their first instar after hatching and before nesting.

8. A method for selectively obtaining female individuals of Bagworm Moth, comprising: A confirmation process for confirming the presence or absence of small dot-like holes in the anterior center of the abdominal plate of the second segment from the end of the abdomen in the larval stage; a female selection step of selecting larvae that do not have the punctate holes as females; and A rearing process in which the selected larvae are reared until they reach the pupal or adult stage. The method comprising:

9. A method for harvesting bagworm silk, comprising the steps of: A confirmation process to confirm the presence or absence of small dot-like holes in the front center of the abdominal plate of the second segment from the end of the bagworm's abdomen; determining whether or not the individual has the punctate pores as a female individual; A process of separating the sexed bagworms based on their sex; and Silk harvesting process for harvesting silk from groups of male and female bagworms The method comprising:

10. The method of claim 9, wherein the bagworms are in the third instar or later.

11. A group of 10 or more bagworms of the same species, 6th instar or younger, with a small dot-like hole in the anterior center of the abdominal plate of the second abdominal segment from the end of the abdomen.

12. A group of 10 or more bagworms of the same species, 6th instar or younger, that do not have a small dot in the anterior center of the abdominal plate of the second abdominal segment from the end of the abdomen.

13. 13. A population as described in claim 11 or 12, wherein each bagworm is of the same age.

14. The population of claim 13, wherein the bagworms are in the first instar.

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