A method for assessing the reproductive fitness of a feed by determining an indicator of ovarian development

Abstract

This invention provides a method for assessing the reproductive adaptability of natural enemies to feed by using ovarian development indicators, including: (1) establishing an experimental population: natural enemies are fed different prey or feed and are raised and bred under environmental conditions of 27±1℃, 70±5% relative humidity, and 16L:8D photoperiod; (2) dissection of the female adult reproductive system: the reproductive system is completely removed by anesthesia, wing removal, and separation of the dorsal plate; (3) determination of ovarian development indicators at key ages: the ovaries of female adults at key ages and under different feeds are dissected, and parameters such as the number of eggs, ovarian tube length, and mature egg volume are counted. The ovarian development progress of *Triplophysa fasciata* under different feeds is compared to assess reproductive adaptability.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for evaluating the reproductive adaptability of natural enemies to feed by ovary development indicators (taking the example of Orius sauteri), and belongs to the technical field of biological control natural enemy reproduction. BACKGROUND

[0002] In the large-scale breeding of natural enemy insects, the primary task is to obtain a large number of high-quality natural enemy insects in the shortest possible time, and one of the important factors restricting the efficiency of large-scale breeding is the nutrition of feed. A high-quality feed can greatly shorten the production cycle of natural enemy insects, improve production efficiency, and reduce cost input by promoting growth and reproduction.

[0003] The eggs of oviparous insects often contain almost all the nutrients required for the development of offspring. Therefore, food intake plays an important role in female reproduction, such as providing different host plants, which can significantly affect the ovary development of Lygus lucorum and thus affect the level of fecundity. Studies have shown that nutrition is closely related to insect ovary development, especially the process of oogenesis. During oogenesis, the yolk proteins synthesized by the fat body will bind to the yolk receptors on the plasma membrane surface of the oocyte through the follicular space, and then be absorbed by the oocyte through endocytosis. During yolk formation, the eggs laid by female insects may approach or exceed their body weight, at which time, if the female insect is to synthesize enough yolk proteins to ensure a high level of fecundity, it needs to intake sufficient nutrients.

[0004] The insect of the genus Orius belongs to the order of Insecta, the family of Pentatomidae, and the subfamily of Lyssae. It is a kind of commercialized predatory insect, which is applied to tobacco, corn, fruit trees and vegetables to control the larvae of various pests of Lepidoptera, Hemiptera and Coleoptera. In recent years, the domestic large-scale rearing has been gradually realized, but the selection of feed is still a key problem. Domestic scholars have studied the artificial and insect-derived feed of Orius: from 1985 to 1986, the Guangdong Institute of Entomology introduced the original population of Orius from Thailand, and reared it indoors with rice moths and galleria mellonella. Then, the blood lymph extracted from the pupae of Antheraea pernyi was tried to be used for rearing, and it was found that the Orius could develop normally. After that, Tetsuya and Sadao reared the Orius with frozen larvae of Noctuidae, although the life cycle was prolonged and the body weight of the adult was reduced, but the reproductive ability was not significantly affected, and the interference of live prey on the molting of the Orius could be avoided to some extent. Gupta compared the development parameters of the Orius feeding on frozen silkworm pupae, rice moths and galleria mellonella, and the results showed that the frozen silkworm pupae could be used as the feed for large-scale rearing of the Orius. Jingyao Gong et al. reported the preparation method of the artificial feed of the Orius, and the main components were maggot powder, beef liver, egg yolk and blood lymph of Antheraea pernyi pupae. Compared with the Tenebrio molitor, the biological parameters of the Orius reared with the artificial feed had no significant difference. The above studies have laid a certain foundation for the selection of the feed of the Orius.

[0005] In summary, the ovarian development index is an important index for reflecting the influence of feed nutrition on the natural enemy insects. The study on the influence of the feed on the reproductive ability of the Orius by measuring the ovarian development index also proves that the method is feasible, and the method of selecting the feed of the natural enemy by further studying the ovarian development has not been reported yet. SUMMARY

[0006] The purpose of the present application is to provide a method for evaluating the reproductive adaptability of natural enemies to feed by using the ovarian development index on the basis of the previous research. The method analyzes the ovarian development dynamics of the natural enemy insects reared with different feeds by means of systematic dissection, ovarian grading and determination of key age development indexes, and determines the internal influence of different feeds on the reproductive ability of the natural enemy insects, which has important significance for the selection of the feed of the natural enemy insects.

[0007] A method for evaluating the adaptability of natural enemies to feed by using the ovarian development index of the Orius, comprising the following steps:

[0008] (1) Establishment of experimental population

[0009] Different natural enemy insects should be reared in a unified way according to their growth, development and reproductive characteristics, so as to provide sufficient insect sources for the follow-up experiments.

[0010] Orius sauteri: 5-10 newly laid egg masses were selected from each diet feeding group and reared in an artificial climate chamber or incubator at 27±1°C, 70±5% RH, and a photoperiod of 16L:8D. Egg to 2nd instar nymphs were reared in 9 cm diameter plastic Petri dishes (1 egg mass per dish); 3rd to 5th instar nymphs were transferred to 10 cm diameter, 8 cm high square rearing boxes (about 10 individuals per box); adults were transferred to 10 cm diameter, 10 cm high circular oviposition boxes (3-4 pairs per box) after eclosion. Folded A4 paper strips were provided in the rearing boxes for resting and oviposition, which also increased the rearing space and avoided excessive interference between individuals. Since eggs and 1st instar nymphs (hereafter referred to as N1) do not feed, only provide cotton soaked with 5% honey water, and after developing to N2, provide sufficient and appropriate prey or artificial diet.

[0011] (2) Dissection of female reproductive system

[0012] The dissection methods of different natural enemy insects may vary, and the following main steps should be followed: anesthesia, cutting off the wings and other tissues and organs that block the abdomen, separating the dorsal plate or ventral plate to expose the abdominal contents, picking out the digestive system and fat body and other non-target organs, completely removing the reproductive system, taking photos and recording, and measuring related indicators.

[0013] Orius sauteri: Take female adults that have been eclosed for more than 24 hours, and use a stereoscopic microscope to dissect the reproductive system in PBS buffer (pH = 7.2-7.4). Place the dissected ovaries and other organs into single concave glass slides, and use a micro imaging recorder to take photos. The specific method is as follows: First, starve the collected female adults for 12 hours to empty the residual contents in the gut, anesthetize them with CO2 in a canning jar, and then remove them after they lose consciousness. Use dissection scissors to cut off one pair of forewings, one pair of hindwings, six legs, and a small shield from the base. Cut the intersegmental membrane between the dorsal plate and the ventral plate. Then, insert a No. 1 insect needle from the posterior position of the prothoracic dorsal plate, fix the dorsal plate upward in the center of the wax disc using two No. 5 insect needles, and add a certain amount of PBS buffer. Carefully lift the dorsal plate with a dissection forceps and dissection needle, and remove it. Use a dissection forceps and a dissection knife to remove the fat body and digestive organs above the reproductive system. Carefully cut the suspensory ligament at the front end of the bilateral ovaries with a dissection knife, and then remove the entire abdominal segment connected to the posterior genital cavity. Carefully pull the entire reproductive system onto a single concave glass slide, and add PBS buffer for photography. If the ovary is too large, a transparent plastic cover can be used instead of a single concave glass slide. During the dissection process, use toilet paper to absorb and add PBS buffer at any time to improve dissection efficiency.

[0014] (3) Determination of key time nodes of ovary development

[0015] To reduce the workload and difficulty, only select some key age nodes to determine the relevant indicators. According to the different life spans of different natural enemy insects, the post-oviposition period is determined, such as the rapid development period, the stable period, the beginning of oviposition, the peak oviposition period, and the decline period. According to the needs, some periods are selected for dissection analysis.

[0016] Orus oviposition: Select female adults that have taken different feeds after emergence, and dissect the ovaries at key ages: rapid development period (1 day), stable period (3 days), beginning of oviposition (7 days), peak oviposition period (14 days), and decline period (21 days).

[0017] (4) Determination of ovary development-related indicators

[0018] Collect female adults of different feeds at key ages after emergence, and dissect the complete ovaries in PBS buffer according to the method described in (2). Carefully place the dissected ovaries into a single concave slide or transparent plastic cover, drop in an appropriate amount of PBS buffer, and use a micro-image recorder to record the number of eggs at different development stages, measure the length and width of mature eggs, and calculate the volume of mature eggs. Among them, the length and width of mature eggs refer to the average length and width of the first mature egg at the end of the ovary tube. After collecting ovaries at each age, the ovaries of Orus are classified according to the length of the ovary tube, the development level of oocytes, and the deposition of yolk proteins.

[0019] (5) Judgment of egg development stage

[0020] The development stage of the egg is divided into pre-yolk occurrence, yolk occurrence, and mature period according to the development progress of the oocyte and the deposition of yolk proteins. The main judgment criteria are as follows:

[0021] Pre-yolk occurrence: The egg is milky white and transparent, with no obvious yolk protein deposition;

[0022] Yolk occurrence: Obvious yolk protein deposition can be seen, and there is obvious deposition under low light irradiation;

[0023] Mature period: The yolk protein deposition process is basically completed, and a hard eggshell has been formed.

[0024] (6) Calculation of egg volume

[0025] Different natural enemy insects should choose different formulas according to the characteristics of the eggs.

[0026] Orus: We consider the mature egg of Orus as an ellipsoid, and according to the formula for the volume of an ellipsoid: V = 4πabc / 3, we obtain the volume of the Orus egg V e Approximate calculation formula:

[0027]

[0028] wherein, l is the long axis length of the egg, and b is the short axis length of the egg.

[0029] Compared with the prior art method, the present application has the following advantages:

[0030] 1. The difference in the ovary development index is used to analyze the influence of different feeds on the fecundity of natural enemy insects, which is more comprehensive and accurate than screening the feeds only by the length of the development period and the high and low of the egg production;

[0031] 2. Only a few key adult day-old ovary development indexes need to be determined, which is less laborious and more convenient than the determination of the whole adult stage;

[0032] 3. It is not necessary to scale up the rearing, only a small amount of natural enemy insects need to be carefully reared, and 30-50 adult insects are obtained for each feed to complete the experiment, which reduces the workload and difficulty. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a schematic diagram of the egg rearing device of the rod-shaped earwigs;

[0034] Figure 2 It is a schematic diagram of the nymph rearing device of the rod-shaped earwigs;

[0035] Figure 3 It is a schematic diagram of the adult rearing device of the rod-shaped earwigs;

[0036] Figure 4 It is a schematic diagram of the eggs at different development stages;

[0037] Figure 5 It is a basic structure diagram of the female reproductive system of the rod-shaped earwigs.

[0038] In the figure, 1-1 is the first instar nymph of the rod-shaped earwigs, 1-2 is a plastic culture dish, 1-3 is a cotton ball, 2-1 is a cover with breathable holes, 2-2 is the second to fifth instar nymphs of the rod-shaped earwigs, 2-3 is a folded A4 paper strip, 2-4 is prey or feed, 3-1 is the adult of the rod-shaped earwigs, 4-1 is the egg at the pre-yolk formation stage, 4-2 is the egg at the yolk formation stage, 4-3 is the mature egg, 5-1 is the suspensory ligament, 5-2 is the terminal filament, 5-3 is the follicular sac, 5-4 is the ovary tube, 5-5 is the ovary tube plug, 5-6 is the ovary tube handle, 5-7 is the ovary tube calyx, 5-8 is the lateral oviduct, 5-9 is the median oviduct, 5-10 is the genital atrium, 5-11 is the accessory gland, and 5-12 is the spermatheca. DETAILED DESCRIPTION

[0039] The present application will be further described below in combination with the embodiments:

[0040] Embodiment 1

[0041] Establishment of experimental populations of H. spinicollis fed with different diets

[0042] Spodoptera exigua, Spodoptera exigua, Tenebrio molitor and artificial diet (reference to the preparation method proposed by Gong Jingyao in 2019) were selected for the experiment, respectively. Each diet treatment group selected 5-10 new egg blocks and was reared in an artificial climate chamber with a temperature of 27±1℃, humidity of 70±5%, and a photoperiod of 16L:8D. Egg-2nd instar nymph stage groups were reared in 9 cm diameter plastic culture dishes (1 egg block / dish); 3-5th instar nymphs were transferred to 10 cm diameter, 8 cm high square feeding boxes (about 10 heads / box); adult moths were transferred to 10 cm diameter, 10 cm high circular oviposition boxes (3-4 pairs / box) after emergence. 1st instar nymphs were provided with cotton soaked with 5% honey water, and 2nd instar nymphs were provided with sufficient age-appropriate prey or artificial diet.

[0043] The reproductive system of H. spinicollis female adults was dissected, including the following steps:

[0044] (1) The collected female adults were starved for 12 h, and the intestinal residues were emptied. They were anesthetized with CO2 in a canning bottle, and then removed after losing consciousness. A pair of forewings, a pair of hindwings, six feet and a small shield were cut off from the base using dissection scissors. The intersegmental membrane between the dorsal plate and the ventral plate was cut open, then a No. 1 insect needle was inserted from the posterior position of the prothoracic dorsal plate, the dorsal plate was fixed upward in the center of the wax plate, two No. 5 insect needles were used to fix the two sides of the dorsal plate, and a certain amount of PBS buffer was added.

[0045] (2) The dorsal plate was carefully removed using dissection forceps and dissection needles, and the fat body and digestive organs above the reproductive system were removed using dissection forceps and dissection knives.

[0046] (3) The suspensory ligament at the front end of the bilateral ovary was carefully cut off using a dissection knife, and the part of the abdominal segment connected to the posterior genital cavity was removed in one piece. The entire reproductive system was carefully pulled into a single concave glass slide, and PBS buffer was added for photography.

[0047] The key age ovary development indicators of H. spinicollis fed with different diets were determined, including the following steps:

[0048] (1) Female adults fed with different diets were collected at the rapid development stage (1 day), stable stage (3 days), beginning of oviposition (7 days), peak oviposition period (14 days) and decline period (21 days) after adult emergence, respectively. The complete ovary was dissected in PBS buffer.

[0049] (2) Carefully place the dissected ovary into a single concave slide or transparent plastic cover, drop in an appropriate amount of PBS buffer, and use a micro image recorder to record the number of oocytes at different developmental stages, measure the length of the ovarian tube (the main part of the ovarian tube), and the length and breadth of mature oocytes to calculate the volume of mature oocytes.

[0050] Conduct a comprehensive analysis, including the following steps:

[0051] (1) Use Microsoft Excel for data statistics.

[0052] (2) The mean and standard error of the number of oocytes at different stages, the length of the ovarian tube, and the volume of mature oocytes at different ages are subjected to single-sample t-test using IBM SPSS Statistics. The comparison of the number of oocytes, the length of the ovarian tube, and the volume of oocytes between different feed-treated populations is performed using one-way ANOVA, and multiple comparisons are made using Tukey's HSD method. Finally, the data are plotted using GraphPad prism 8.

[0053] (3) Compare the ovarian development progress of Orius sauteri under different feed treatments and evaluate the reproductive adaptability.

Claims

1. A method for evaluating the reproductive fitness of a feedstuff by determining an indicator of ovarian development, characterized in that, The method comprises the following steps: Anatomy of female reproductive system of H. quadripunctata: The collected female adults were starved for 12 h, and the intestinal residues were emptied. The insects were anesthetized with CO2 in a canning bottle. The pair of forewings, the pair of hindwings, six legs and the scutellum were cut off from the base using dissecting scissors. The intersegmental membrane between the dorsal plate and the ventral plate on both sides was cut open. Then, a No. 1 insect needle was inserted from the posterior position of the prothoracic dorsal plate, and the dorsal plate was fixed on the central wax plate. Two No. 5 insect needles were used to fix the dorsal plate on both sides. A certain amount of PBS buffer was added. The dorsal plate was lifted and removed using a dissecting forceps and a dissecting needle. The fat body and the digestive organs above the reproductive system were removed. The suspensory ligament at the front end of the ovary was cut off. The abdominal segments connected to the rear end of the genital cavity were taken out in one piece. The entire reproductive system was dragged into a single concave glass slide, and PBS buffer was added for subsequent experiments. Measurement of ovary development indicators of key age natural enemies: female adults fed with different feeds were collected at the rapid development period, the stable period, the beginning of oviposition, the peak oviposition period and the decline period after the adults emerged, respectively. The complete ovary was dissected in the PBS buffer. The dissected ovary was carefully placed in a single concave glass slide or a transparent plastic cover, and an appropriate amount of PBS buffer was added. The number of eggs at different development stages was recorded using a micro image recorder. The length of the ovary tube, the length and width of mature eggs, and the volume of mature eggs were measured. The reproductive adaptability of the natural enemies to different feeds was determined by comparing the ovary development indicators of female adults fed with different feeds.

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