A method for sex determination using rhinoceros beetle larvae feces

Abstract

To provide a method for determining sex without contacting a beetle larva.SOLUTION: A sex determination method using droppings of a beetle larva includes a determination step which has unevenness of the surface of the droppings as a first index, a proportion of an area of the droppings and length of the droppings as a second index, and determines sex by combining the first index and the second index.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The disclosure of the present application relates to a method for determining the sex of rhinoceros beetle larvae using feces.

[0002] Rhinoceros beetles are known as the "king of insects" and are known as a popular large beetle, along with stag beetles. Male rhinoceros beetles, in particular, have large horns on their heads, making them popular with both children and adults. For this reason, it is known that sexing can be done at the larval stage. One method for sexing rhinoceros beetles is to identify them as males by identifying a V-shaped mark near the lower abdomen and hindgut of third-instar larvae (see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Inter-University Research Institute Corporation, National Institute for Basic Biology, Department of Evolutionary and Developmental Biology [Retrieved February 4, 2022], Internet<URL:https: / / www.nibb.ac.jp / niimilab / column / 16_001.html> Summary of the Invention [Problem to be solved by the invention]

[0004] However, the method of checking for the V-shaped mark near the lower abdomen and hindgut of third-instar larvae requires (1) direct hand contact with the larvae, and (2) if the larvae are curled up, they must be stretched out in order to visually inspect the lower abdomen and hindgut. This puts a great deal of strain on the larvae when determining whether they are male or female, which can weaken them.

[0005] The present invention has been disclosed to solve the above-mentioned problems. As a result of intensive research, the present inventors have newly discovered that it is possible to discriminate between male and female rhinoceros beetle larvae without contact by using the roughness of the surface of the feces of rhinoceros beetle larvae as a first index, the ratio of the feces area to the length as a second index, and combining the first index and the second index.

[0006] That is, an object of the disclosure in this application is to provide a method for distinguishing between males and females of rhinoceros beetle larvae based on their feces. [Means for solving the problem]

[0007] The disclosure of the present application relates to the following method for determining the sex of rhinoceros beetle larvae using feces.

[0008] (1) A method for determining the sex of rhinoceros beetle larvae using their feces, the method comprising: The method includes a discrimination step of determining sex by using the degree of unevenness of the feces surface as a first index, the ratio of the feces area to the feces length as a second index, and combining the first index and the second index. Discrimination method. (2) The first index is selected from convexity, contour length divided by the approximate perimeter of the approximate ellipse, and circularity divided by the minor axis of the approximate ellipse. The determination method described in (1) above. (3) The second index is selected from the area divided by the Feret diameter, the area divided by the length of the major axis of an ellipse approximation, and the area divided by the length of the long side of a rectangle approximation. The determination method according to (1) or (2) above. (4) The discrimination process is Using the first and second indices, the outlier rate of the squared Mahalanobis distance of the larval feces was calculated using the Mahalanobis-Taguchi method. The calculated abnormal value rate of the larvae's feces is compared with a threshold value calculated from the abnormal value rate of the squared Mahalanobis distance of the female. If the rate is above the threshold, the larvae are identified as males, and if it is below the threshold, the larvae are identified as females. The determination method according to any one of (1) to (3) above. [Effects of the Invention]

[0009] The method for sexing using feces of rhinoceros beetle larvae disclosed in the present application makes it possible to sex the rhinoceros beetle larvae without contacting them. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram for explaining an outline of an embodiment of the discrimination method. [Figure 2] FIG. 2 is a diagram for explaining an outline of an embodiment of the discrimination method. [Figure 3] FIG. 3 is a photograph substituted for a drawing, and is an example of a photograph of feces taken in Example 1. [Figure 4] FIG. 4 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 1. [Figure 5] FIG. 5 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 1. [Figure 6] FIG. 6 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 1. [Figure 7] FIG. 7 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 1. [Figure 8] FIG. 8 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 1. [Figure 9] FIG. 9 is a graph showing the rate of abnormal values ​​(horizontal axis) for each larva (vertical axis) analyzed in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0011] The sex determination method using rhinoceros beetle larvae feces disclosed in the present application is described in detail below. Note that the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc., in order to facilitate understanding. Therefore, the disclosure of the present application is not necessarily limited to the position, size, range, etc., disclosed in the drawings.

[0012] In addition, in this specification, (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field. (3) The description of the shape includes not only the exact shape of the description, but also the shape that can be understood as roughly ____ shape. This is interpreted as:

[0013] (Embodiment of a method for determining sex using feces of rhinoceros beetle larvae) An embodiment of a method for determining the sex of rhinoceros beetle larvae using feces (hereinafter, sometimes simply referred to as "the method") will be described with reference to Figures 1 and 2. Figures 1 and 2 are diagrams for explaining an outline of an embodiment of the method.

[0014] The discrimination method includes a discrimination step in which the roughness of the surface of the rhinoceros beetle larvae's feces is used as a first index, the ratio of the feces' area to its length is used as a second index, and the first index and the second index are combined to discriminate between males and females.

[0015] In this specification, "rhinoceros beetle" refers to an insect belonging to the order Coleoptera, family Scarabaeidae, subfamily Dynastinae, and tribe Dynastinae. Many of the popular rhinoceros beetles with large horns on their heads belong to tribe Dynastinae, and the following 13 genera are known. The names of well-known beetles within these genera are also listed. Hercules beetle: Hercules beetle, Neptune beetle, Saturn beetle, and Stag beetle Genus Stag Beetle: Elephas beetle, Geass beetle, Anubis beetle, Acteon beetle, Rex beetle, Mars beetle, Janus beetle, and Dwarf beetle Genus Stag Beetle: Sawtooth Stag Beetle, Pisaro Stag Beetle, Stinging Stag Beetle, Atlas beetle genus: Atlas beetle, Caucasus beetle, Mohrenkamp beetle Genus Centaurus: Centaurus beetle, Gabon beetle Genus Stag Beetle: Stag Beetle, Thai Stag Beetle, Himalayan Stag Beetle Genus: Stag beetle, black stag beetle Genus: Three-horned beetle, Three-horned beetle Genus: Stag beetle, woolly beetle Genus: Japanese rhinoceros beetle Genus: Rust beetle Genus: David's beetle Genus: Stag beetle, giant stag beetle

[0016] As mentioned above, many of the popular beetles with large horns on their heads belong to the tribe Scarabaeidae. Therefore, the discrimination method disclosed in the present application can be suitably used to discriminate between the sexes of larvae of the tribe Scarabaeidae, but is not technically limited to the tribe Scarabaeidae. Similar to the tribe Scarabaeidae, the method can also be applied to the tribe Scarabaeidae, Scarabaeidae, Scarabaeidae, Scarabaeidae, and Scarabaeidae, which belong to the subfamily Scarabaeidae.

[0017] There are no particular restrictions on the growth stage of rhinoceros beetle larvae (hereinafter sometimes simply referred to as "larvae"), as long as feces can be collected, and they may be in the first, second, or third instar.

[0018] Larvae are generally raised in insect enclosures with leaf mold placed inside. Rhinoceros beetles crush the leaf mold with their strong jaws, and then use intestinal bacteria to break it down and absorb the residue, forming it into bale-shaped feces. Therefore, larval feces can be collected, for example, by sieving it from the leaf mold. The number of feces required per larva for sex determination is at least two, but the more feces, the more evenly distributed they can be. Examples of feces include, but are not limited to, 5 or more, 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, 110 or more, 120 or more, 130 or more, 140 or more, and 150 or more.

[0019] Next, the indexes used in the discrimination method will be described with reference to FIG. 1. The first index, the degree of unevenness of the surface of the feces, is not particularly limited as long as it can represent the unevenness (convexoconcave) of the surface of the feces. For example, (1) Convexity (Solidity) (2) [Perimeter] divided by [approximate perimeter of ellipse (= 4 × minor axis of ellipse approximation)] (Perim / 4Mi), (3) [Circularity] divided by [Minor axis of ellipse approximation] (Ciru / Mi), etc.

[0020] The definitions of the terms used in the first index are as follows: Note that convexity and circularity are not shown in Figure 1. (a) Convexity (Solidity) This is a number that indicates the lack of indentation in the shape; the larger the indentation, the smaller the value, and the closer to 1.0 the less indentation there is. It can be calculated by dividing the area of ​​the feces by the area of ​​the convex hull. The convex hull is the smallest convex set that contains a given set. For example, it can be said to be the shape that the rubber band creates when it surrounds feces. (b) Perimeter (Perim) The length of the faeces outline. (c) Circularity (Circ) [4π×Area]÷[(contour length) 2 ]. 1.0 is a perfect circle, and the closer it is to 0, the more elongated it becomes. (d) Minor axis of ellipse approximation The length of the minor axis when approximating the feces as an ellipse.

[0021] The second index, the ratio of fecal area to fecal length, is not particularly limited as long as it is an index that can be expressed as a ratio including the fecal area and the fecal length. For example, (4) Area divided by Feret's diameter (A / F), (5) [Area] divided by [Major axis length of ellipse approximation] (A / Ma), (6) Area divided by the length of the long side of the approximate rectangle (height), etc.

[0022] The definitions of the terms used in the second index above are as follows: (e) Area (A) Projected area of ​​feces. (f) Feret's diameter The longest distance between two parallel lines when the feces is sandwiched between two lines. (g) Major axis length of ellipse approximation (Major) The length of the major axis when the feces is approximated as an ellipse. (h) Length of the long side of the approximate rectangle (Height) The length of the long side of the feces when approximating it as a rectangle.

[0023] The indices described in (1) to (6) above (in other words, the values ​​described in (a) to (h)) can be determined by analyzing photographs of larval feces using the image analysis software ImageJ.

[0024] The present inventors conducted various studies focusing on larval feces and discovered that there are differences in fecal shape features between males and females. More specifically, they found that the proportion of feces showing outliers (samples indicated by the arrows in Figure 2) (outlier rate: MD) was higher in male feces compared to female feces, as shown in Figure 2. Note that outliers refer to data that have a different correlation pattern from the data in the unit space, as shown by the arrows in Figure 2. Furthermore, the outlier rate refers to the number of samples showing outliers among the feces of larva X divided by the total number of fecal samples of larva X. The discrimination process is not particularly limited as long as it can combine the first and second indices and discriminate between males and females based on differences in fecal shape features. Discrimination methods that can be used in the discrimination process include, but are not limited to, the Mahalanobis-Taguchi algorithm, decision tree analysis, random forests, support vector machines (SVMs), and deep learning (DNNs).

[0025] An example of using the Mahalanobis-Taguchi method in the discrimination process will be explained in more detail. The pattern diagnosis using the Mahalanobis-Taguchi method was used to determine the abnormal value rate (MD in Figure 2) of the feces collected from larva X. 2 The abnormal value rate of feces of larva X is calculated by comparing it with a threshold value calculated from the abnormal value rate of the squared Mahalanobis distance of females, and if it is above the threshold value, it is determined to be male, and if it is below the threshold value, it is determined to be female.

[0026] The Haranobis-Taguchi (MT) method is a well-known pattern recognition technique that is effective in identifying the characteristics of objects. In this embodiment of the discrimination method, the female feces is used as the reference (unit space), and the distance between each variable in the feces of larva X, whose sex is to be discriminated, and the variable data group in the unit space is calculated and evaluated using the following formula:

number

[0027] The threshold value may be determined appropriately taking the following points into consideration. (A) The threshold value varies depending on what is used as the first index (e.g., at least one selected from the above (1) to (3)) and the second index (e.g., at least one selected from the above (4) to (6)). Therefore, the threshold value may be determined appropriately depending on the index used. (a) The threshold value may be determined based on the feces of any one female, or may be determined using the feces of multiple female groups. When determining the threshold value using the feces of multiple female groups, the maximum abnormal value rate among the abnormal value rates of the female groups should be used as the threshold value. (c) Even if the same indicator is used as the first and second indicators, the breeding environment, such as the type of rhinoceros beetle and the type of food, may affect the shape of the feces. When sexing larvae from the same breeder, the threshold determined from the feces of rhinoceros beetles that were previously waited to reach adulthood and identified as female may be used as the threshold for that breeder. Furthermore, when sexing only larvae from a first-time breeder, the threshold may be determined, if necessary, based on the feces of any female identified by conventional methods or genetic analysis from multiple larvae.

[0028] Regarding decision tree analysis, random forest, SVM (support vector machine), and DNN (deep learning), they may be carried out according to known procedures, except that the first index and the second index are used in combination.

[0029] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. [Example]

[0030] Example 1 [Rearing larvae and collecting feces] First-generation adult rhinoceros beetles (genus: Rhinoceros beetles) collected in Nagasaki Prefecture in 2019 were reared for generations. Twenty third-generation larvae (GU1-20) hatched from eggs of second-generation adults were reared at Gifu University, and feces from third-instar larvae were collected by sieving from leaf mold. To ensure a uniform rearing environment, leaf mold (Marukan Bio Larval Rearing Mat) and individual larvae were placed in an 1804 mL polypropylene container. The collected feces were dried on a hot plate at 140°C to prevent mold growth. Adult beetles emerged in July 2021 and were identified as males or females by the presence or absence of horns. Two beetles (GU7 and GU12) failed to pupate. Of the 18 adults, eight were males and 10 were females.

[0031] [Analysis by Mahalanobis-Taguchi method] Feces from 18 adult third-instar larvae (collected between January and May 2021) were lined up and photographed with a digital camera. Figure 3 shows an example of a photograph. In this example, approximately 100 to 300 feces were photographed, although this number varied depending on the larvae. The photographs were binarized using ImageJ (ver. 1.53a, National Institutes of Health, USA), and the following were calculated: convexity (Solidity), contour length (Perim), circularity (Circ), minor axis of ellipse approximation (Minor), area (A), Feret's diameter (F), major axis length of ellipse approximation (Ma), and long side length of rectangular approximation (H). Using the feces of GU14 (female) as the reference (unit space), and the combinations shown in Table 1 as the first and second indices, the Mahalanobis distance (MD) was used to evaluate the distance between each variable in the feces of the remaining 17 individuals and the variable data group in the unit space. The abnormal value rate (horizontal axis) for each larva (vertical axis) is shown in Figures 4 to 8. In addition, the maximum abnormal value rate of the female group is set as the threshold, and the percentage (%, correct answer rate) of male abnormal value rates greater than the threshold is shown in Table 1 and Figures 4 to 8.

[0032] [Table 1]

[0033] As shown in Figures 4 to 8, it was confirmed that by using the first and second indices shown in Table 1, it was possible to distinguish the sex of larvae from feces with a high probability. In particular, when convexity (solidity) was used as the first index, the accuracy rate was 100% regardless of the type of second index. Furthermore, when area / Feret diameter was used as the second index, the accuracy rate was 100% regardless of the type of first index. Therefore, it was confirmed that it is particularly preferable to use convexity (solidity) as the first index and area / Feret diameter as the second index.

[0034] <Example 2> Instead of the Japanese rhinoceros beetle (genus Dorcus) used in Example 1, seven sexed third-instar larvae of the Hercules beetle (genus Dorcus) [two females (GUF1 and GUF2) and five males (GUM1 to GUM5)] were used, and the Mahalanobis distance (MD) outlier rate for each larva was calculated using the same procedure as in Example 1, except that the feces of GUF1 (female) was used as the reference (unit space). The results are shown in Figure 9. Note that Figure 9 shows an example in which convexity (solidity) was used as the first index and area / Feret diameter was used as the second index. As is clear from Figure 9, it was confirmed that sexing of Hercules beetles was also possible.

[0035] From the above results, it was confirmed that the following effects can be achieved by using the determination method disclosed in the present application. (1) It is possible to distinguish between male and female larvae without touching them, which prevents the larvae from weakening. (2) Sex can be determined by analyzing the feces images. Therefore, unlike the conventional method of determining sex using V marks, even non-experts can distinguish between males and females. (3) Sex determination does not require the larvae themselves, as long as the images are available. Therefore, if images are provided via communication services such as the Internet, the sex of larvae raised by breeders in any location can be determined without the need to move the larvae. [Industrial Applicability]

[0036] The discrimination method disclosed in the present application can be used to distinguish between male and female rhinoceros beetle larvae, and is therefore useful for the insect industry, such as rhinoceros beetle breeders.

Claims

1. A method for determining the sex of rhinoceros beetle larvae using feces, the method comprising: a discrimination step of using the degree of unevenness of the surface of the feces as a first index, the ratio of the area of ​​the feces to the length of the feces as a second index, and combining the first index and the second index to discriminate between males and females; The first index is selected from convexity, contour length divided by approximate perimeter of an approximate ellipse, and circularity divided by minor axis of an approximate ellipse. Discrimination method.

2. The second index is selected from the area divided by the Feret diameter, the area divided by the length of the major axis of an ellipse approximation, and the area divided by the length of the long side of a rectangle approximation. The method of claim 1 .

3. The determination process is Using the first index and the second index, the outlier rate of the squared Mahalanobis distance of the larvae feces is calculated by the Mahalanobis-Taguchi method; The calculated abnormal value rate of the larvae's feces is compared with the threshold value calculated from the abnormal value rate of the square of the female Mahalanobis distance. If it is above the threshold, it is determined to be male, and if it is below the threshold, it is determined to be female. The method according to claim 1 or 2.

Images