Sex inducer for efficiently inducing male daphnia magna, induction method and application

Abstract

The invention discloses a sex inducer for efficiently inducing male daphnia magna, an induction method and application, and belongs to the technical field of cladocera male induction. The invention discloses a sex inducer for efficiently inducing male daphnia magna. The sex inducer comprises fenoxycarb. According to the daphnia magna male induction method, the male daphnia magna induction success rate is high and reaches 95% or above, and the obtained male daphnia magna is healthy and good in vitality, can completely develop, has normal male physiological characteristics and can be used for research of cladocera male induction, a reproductive transformation mechanism, a sex determination mechanism and the like; the induced daphnia magna maternal body can still normally grow and grow, and still has normal parthenogenesis capability; the inducer is safe and free of toxic and side effects, sustainable development can be achieved, and cost is saved; the method is simple and easy to operate, and special condition setting and instrument equipment are not needed.

Description

Technical Field

[0001] The invention relates to the technical field of male induction of cladocerans, and in particular to a sex inducing agent for efficiently inducing males of Daphnia cerana, an induction method and an application thereof. Background Art

[0002] Cladocerans, also known as "Daphnia", are a small type of zooplankton commonly found in freshwater. They are short, flattened on the left and right sides, about 1 to 2 mm long, and wrapped in two transparent shells. The second antennae are well developed and are the main organs for swimming. The males and females are dioecious and heteromorphic. In their life history, parthenogenesis and sexual reproduction alternate, which are affected by both the environment and genetics. However, the physiological mechanism of this reproductive transformation is still unclear. Cladocerans reproduce parthenogenetically in the summer, develop directly, lay many eggs, and can develop into females without fertilization. Males will not appear until late autumn and early winter or when the living environment conditions are harsh. Sexually reproduced eggs are called fertilized eggs, or winter eggs, and generally only 1 to 2 eggs are laid each time. It is a cyclical parthenogenesis.

[0003] As a small crustacean with a short generation, many offspring, and easy to breed in the laboratory, Daphnia gracilis is often used as a model organism in the fields of evolution, genomics, and ecotoxicology. Therefore, male individuals that appear as the turning point between the two reproductive transformation modes are extremely important. However, male individuals are difficult or almost non-existent in laboratory water, so it is impossible to obtain sufficient numbers for corresponding research.

[0004] At present, the method of inducing the emergence of male cladocerans is to increase the population density of cladocerans under suitable culture conditions. This method has the following defects: ① Low induction efficiency and high uncertainty: it cannot ensure that males will appear every time they are induced, and it cannot guarantee that enough males will be obtained for the experiment every time they are induced. It can only be repeated and the number of induced males will be increased; ② High cost and large workload: The harsh environment caused by high population density will cause a large number of cladocerans to die, and continuous cultivation is required. Summary of the invention

[0005] The purpose of the present invention is to provide a sex inducing agent, an induction method and an application for inducing males of Daphnia cerana efficiently, so as to solve the problems of extremely low success rate of male induction of cladocerans, great uncertainty of induction effect, high cost, large workload and inability to stably obtain male cladocerans.

[0006] To achieve the above object, the present invention provides a sex inducing agent for effectively inducing maleness of Daphnia cerana, wherein the components of the sex inducing agent include fenoxycarb.

[0007] Preferably, the gender inducing agent is fenoxycarb solid powder dissolved in dimethyl sulfoxide.

[0008] Preferably, the concentration of the sex inducing agent stock solution is 1 mM, and the concentration of the working solution is 0.6 μM.

[0009] A method for inducing male Daphnia cerana using the above-mentioned sex inducing agent for inducing male Daphnia cerana comprises the following steps:

[0010] S1. Selecting the mother Daphnia cerana: Select active and healthy Daphnia cerana in the basic culture water, whose gonads have developed but have not yet been released into the incubation capsule and have not shed before excretion at this stage;

[0011] S2. Induction of male Daphnia cerana: Place the parthenogenetic Daphnia cerana selected in step S1 into the culture water body to which the inducer has been added, and induce the culture for 3 hours;

[0012] S3. After the induction is completed, the parthenogenetic Daphnia cerana is placed in the basic culture water without inducer, and the culture is continued until the parthenogenetic Daphnia cerana discharges the larvae from the incubation sac, and then the parthenogenetic Daphnia cerana that has hatched larvae is picked out, and after the produced larvae are cultured for one week (before the first sexual maturity), the male and female Daphnia cerana cerana larvae are separated into pots.

[0013] Preferably, the basic culture water is tap water that has been irradiated with ultraviolet light and aerated for 2 days.

[0014] Preferably, the induction conditions in step S2 are: population density is 0.5ind. / mL, induction culture temperature is 25°C±2°C, light:darkness=14:10, and light intensity is 3000lx.

[0015] Preferably, in step S3, the cells are cultured in the basic culture water until the parthenogenetic Daphnia sinensis expel the young Daphnia from the incubation capsule at a density of 1×10 5 cells / mL Scenedesmus obliquus was used as food source and conventional feeding was carried out.

[0016] A use of the sex inducing agent as described above that is highly effective in inducing males of Daphnia cerana in male induction of cladocerans.

[0017] An application of the male induction method of Daphnia cerana as described above in male induction of cladocerans.

[0018] Therefore, the sex induction agent, induction method and application of the present invention for efficiently inducing male Daphnia cerana have the following beneficial effects:

[0019] 1. The male induction method of Daphnia cerana provided by the present invention has a high success rate of inducing male Daphnia cerana, reaching more than 95%. The obtained male Daphnia cerana are healthy and vigorous, and can be fully developed and have normal male physiological characteristics. It can be used for the study of male induction, reproductive transformation mechanism and sex determination mechanism of cladocerans;

[0020] 2. The male induction method of Daphnia cerana provided by the present invention can still grow and reproduce normally after induction, and still has the ability to perform parthenogenesis normally, which can achieve sustainable development and save costs;

[0021] 3. The male induction method of Daphnia cerana provided by the present invention is simple and easy to operate, and does not require special conditions and equipment;

[0022] 4. The male sex inducer of Daphnia cerana provided by the present invention is safe, has no toxic side effects, does not affect subsequent scientific research, and does not pose a potential hazard to the health of operators.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0025] Figure 1 This is a photo of the parthenogenetic Daphnia cerana in which the gonads have developed but have not yet been discharged into the incubation capsule in Example 3 of the present invention;

[0026] Figure 2 The egg-carrying parthenogenetic Daphnia cerana in Example 3 of the present invention, whose gonads have been discharged into the incubation capsule;

[0027] Figure 3 is a photograph of a male juvenile Daphnia cerana in test 1 of the present invention;

[0028] Figure 4 This is a photo of the male juvenile Daphnia gracilis developed into an adult male Daphnia gracilis in the first test of the present invention;

[0029] Figure 5 This is a photo of an adult male Daphnia obtained in the basic culture water body in the first test of the present invention;

[0030] Figure 6 This is a photograph showing that a high concentration of inducer causes the death of embryos discharged into the incubation sac in the test of the present invention;

[0031] Figure 7 This is a photo of the death of young Daphnia excreted from the body caused by a high concentration of inducer in the test of the present invention;

[0032] Figure 8 These are photos of the early, middle and late stages of the parthenogenetic Daphnia cerana in the second test of the present invention;

[0033] Fig. 9 It is a statistical diagram of subsequent survival status in the third test of the present invention;

[0034] Fig.10 The present invention tests four kinds of sexual Daphnia cerana in which the gonads have developed but have not yet been discharged into the ovum saddle;

[0035] Fig.11 The winter eggs discharged from the body in the fourth test of the present invention;

[0036] Fig.12 This is a photograph of the mating moment of the adult male Daphnia gracilis induced in the fourth test of the present invention and the sexual Daphnia cerana whose gonads have developed but have not yet been released into the ovum. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0038] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0039] The instruments, equipment, reagents and materials used in the examples were all obtained from commercial sources, wherein the purity of fenoxycarb was 95%, and the purity of dimethyl sulfoxide (DMSO) was analytical grade.

[0040] Embodiment 1

[0041] The Chinese Daphnia gracilis required for the experiment was cultured in the laboratory. The culture method referred to the Chinese invention patent "A semi-automated laboratory cladoceran high-quantity amplification system" with announcement number CN209121007U. The system can simultaneously culture cladocerans and cladoceran bait in the laboratory, and can control the culture temperature to 25°C, the lighting time of the bait culture unit to 24h, and the lighting time of the cladoceran culture unit to light: dark = 14:10.

[0042] The specific steps are as follows:

[0043] 1. Randomly select 10 parthenogenetic Daphnia cerana whose gonads have been discharged into the incubation capsule (already discharged) from the breeding basins of Daphnia cerana cultured in the laboratory, and observe and calculate their average number of eggs, which is 10. It is roughly estimated that the total number of parthenogenetic Daphnia cerana that needs to be expanded is 200.

[0044] 2. The 200 egg-bearing parthenogenetic Daphnia sinensis were selected and placed in an incubation basin containing 4 L of aerated 2-d water. They were fed 10 mL of 1×10 5cells / mL Scenedesmus obliquus algae solution.

[0045] 3. Pick out all the produced Daphnia gracilis and transfer them to a Daphnia gracilis culture basin containing 10L of aerated water. Feed them 20mL of water per day at a density of 1×10 5 cells / mL Scenedesmus obliquus algae solution.

[0046] 4. Before feeding every day, randomly select some larvae and observe their egg-carrying status. When the gonads of most larvae begin to develop, prepare to start the male induction experiment.

[0047] Embodiment 2

[0048] The male sex inducing agent of Daphnia cerana was prepared as follows:

[0049] S2.1. Accurately weigh 3.01 mg of purchased fenoxycarb with a purity of 95%, add it to 1 mL of dimethyl sulfoxide (DMSO) to prepare a mother solution with a fenoxycarb concentration of 10 mM.

[0050] S2.2. Take 1 μL and 100 μL of the stock solution and add 999 μL and 900 μL of DMSO respectively to prepare storage solutions with concentrations of 10 μM and 1 mM.

[0051] Embodiment 3

[0052] The male sex inducer of Daphnia cerana prepared in Example 2 was used to induce male Daphnia cerana, and the concentration of the inducer was initially screened as follows:

[0053] S3.1. Pipette an appropriate amount (more than a dozen) of Daphnia cerana cultured in Example 1 onto a glass slide, carefully remove excess water with a 1 mL syringe, and select active and healthy Daphnia cerana with developed gonads but not yet discharged into the incubation capsule and Daphnia cerana with gonads discharged into the incubation capsule under a dissecting microscope. The dissecting microscope photo of Daphnia cerana with developed gonads but not yet discharged into the incubation capsule is shown in Figure 1 , the dissection microscope photo of the parthenogenetic Daphnia cerana with the gonads expelled into the incubation capsule is shown in Figure 2 .

[0054] S3.2. Take 1 μL, 10 μL of the 10 μM stock solution of the male sex inducer of Daphnia cerana prepared in Example 2 and 1 μL, 10 μL, and 100 μL of the 1 mM stock solution, respectively, and add them to 10 mL of tap water that has been irradiated with UV and aerated for 2 days to make working solutions with concentrations of 0.001 μM, 0.01 μM, 0.1 μM, 1 μM and 10 μM of the male sex inducer of Daphnia cerana, respectively.

[0055] S3.3, Induction of male Daphnia gracilis: Place the parthenogenetic Daphnia gracilis selected in step S3.1 into the working solution of different sex inducer concentrations prepared in step S3.2. The container is a six-well plate, with 10mL of water and 5 parthenogenetic Daphnia gracilis of the corresponding period selected in each well, so that the population density is 0.5ind. / mL. Three parallels are set for each concentration of working solution, with the basic culture water without inducer as the negative control, and the working solution with 100μL DMSO added to the basic culture water as the positive control. Three parallel experiments are also set for the negative control and positive control groups. All were induced and cultured for 3 hours at 25℃±2℃, light:dark=14:10, and light intensity of 3000lx;

[0056] S3.4. After induction, place the parthenogenetic Daphnia cerana in tap water (basic culture water) without inducer, irradiated with ultraviolet light and aerated for 2 days. Continue to culture until all the larvae of Daphnia cerana are expelled from the incubation capsule. During this period, feed each well with 500 μL of 1×10 5 Then the parthenogenetic Daphnia cerana that had hatched larvae were picked out, and after culturing the produced larvae for one week (before the first sexual maturity), the male and female Daphnia cerana cerana larvae were separated into pots.

[0057] Experimental Test 1

[0058] Statistical Example 3 Male Daphnia cerana induction rate

[0059] Count the average number of larvae produced by three parallel groups in each group N T The total number of male larvae produced N M The following formula was used to calculate the average male Daphnia cerana induction rate of three parallel groups: M%, M% = N M / N T , the results are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] As can be seen from Table 1, DMSO as a solvent will not cause male offspring of parthenogenetic Daphnia cerana. The inducer is only effective for parthenogenetic Daphnia cerana when the gonads have developed but have not yet been discharged into the incubation capsule, and the effective concentration is 0.1μM to 10μM. During the experiment, it was also found that the working solution containing 1μM and 10μM inducer will cause the embryos discharged into the incubation capsule to die ( Figure 6 ) or the larvae that have been produced die ( Figure 7 ).

[0064] Photos of male larvae of Daphnia sinensis are available at Figure 3 , the photo of male larvae developing into adult male larvae can be seen in Figure 4 , Figure 5 This is a photo of male Daphnia naturally occurring in routine laboratory culture water. Figure 4 and Figure 5 It can be seen that the male Daphnia cerana obtained by the induction method provided by the present invention has a normal male appearance without any phenotypic defects.

[0065] Embodiment 4

[0066] The male sex inducer of Daphnia cerana prepared in Example 2 was used to induce male Daphnia cerana, and the concentration of the inducer was screened again. The method was exactly the same as that in Example 3, except that:

[0067] The inducer concentrations were 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM, respectively.

[0068] The method of experimental test 1 was used to calculate the induction rate of male Daphnia cerana. The average male Daphnia cerana induction rate M% of three parallel tests in each group is shown in Table 2.

[0069] Table 2

[0070]

[0071]

[0072] As can be seen from Table 2, the effective male induction concentration range of the inducer is between 0.4μM and 1μM, but the working solution containing 1μM inducer still causes the death of embryos discharged into the incubation capsule and the death of produced larvae. The appropriate concentration range of male inducer is 0.4μM to 0.8μM, and the optimal concentration is 0.6μM.

[0073] Experimental Test 2

[0074] Exploring the possible reasons why 2.86% of the young Daphnia produced in the most suitable male-attracting concentration group in Example 4 were female

[0075] Under laboratory culture conditions, Daphnia cerana reproduces every 2 to 3 days. It has been observed that Daphnia cerana molts once before each discharge into the brooding capsule, and will not officially begin to discharge into the brooding capsule until about 12 minutes after molting.

[0076] The male sex inducer of Daphnia cerana prepared in Example 2 was used to induce male Daphnia cerana. The inducer concentration was the optimal concentration (0.6 μM) screened out in Example 4. The method was exactly the same as that in Example 3, except that:

[0077] There are two sexual maturity states of the experimental Daphnia pulex: the gonads have developed but have not yet been released into the brooding sac and no pre-brooding molting has occurred (not molted and not brooded) and the molting has occurred but has not yet been released into the brooding sac (molted but not brooded).

[0078] The method of experimental test 1 was used to calculate the induction rate of male Daphnia cerana. The average male Daphnia cerana induction rate M% of three parallel tests in each group is shown in Table 3.

[0079] Table 3

[0080]

[0081]

[0082] As can be seen from Table 3, the inducer (0.6 μM) is only effective for parthenogenetic Daphnia cerana whose gonads have developed but have not yet been expelled into the brooding capsule and have not undergone pre-expulsion molting (neither expulsion nor expulsion). However, since it is impossible to distinguish between the two states of Daphnia cerana during normal selection, parthenogenetic Daphnia cerana whose gonads have not developed to the late stage can be selected as much as possible to ensure a better male induction effect. The photos of the early, middle and late stages of parthenogenetic Daphnia cerana are shown in Figure 8 .

[0083] Experimental Test Three

[0084] Observation of the physiological state of the parthenogenetic Daphnia cerana that has hatched young Daphnia selected in Examples 3 and 4

[0085] Observe the birth of the female Daphnia sinensis in each well. After all groups have finished giving birth (the juvenile Daphnia are born from the incubation capsule into the water body), transfer the experimental female Daphnia in each group to the water body under the same experimental conditions but without inducers, and count the subsequent survival status and birth status. The subsequent survival status statistics are shown in Fig. 9 The birth and juvenile situation is shown in Table 4.

[0086] Table 4

[0087]

[0088] Experimental Test 4

[0089] Observation of the physiological state of hatched male Daphnia cerana

[0090] When the induced male larvae develop to adulthood, they are mixed with sexual Daphnia cerana (D. sinensis) whose gonads have developed but have not yet ovulated. Fig.10 ) mate, and the winter eggs they lay ( Fig.11 ) were collected and placed in 96-well plates, and the subsequent hatching conditions were observed and counted. The statistical results are shown in Table 5. Fig.12 .

[0091] Table 5

[0092]

[0093]

[0094] Therefore, the male induction method of Daphnia gracilis provided by the present invention has a high success rate of inducing male Daphnia gracilis, reaching more than 95%. The obtained male Daphnia gracilis are healthy and energetic, can be fully developed and have normal male physiological characteristics, and can be used for the research of male induction, reproductive transformation mechanism and sex determination mechanism of cladocerans. After induction, the mother Daphnia gracilis can still grow and reproduce normally and still have the ability to perform parthenogenesis normally. The inducer is safe and has no toxic side effects, can achieve sustainable development, and save costs. The method is simple and easy to operate, and does not require special conditions and instruments.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A sex inducing agent for effectively inducing male sex in Daphnia cerana, characterized in that: Components of sex inducers include fenoxycarb.

2. The highly effective sex inducing agent for inducing maleness of Daphnia cerana according to claim 1, characterized in that: The sex inducing agent is 95% fenoxycarb solid powder dissolved in dimethyl sulfoxide.

3. The sex inducing agent for inducing maleness of Daphnia cerana according to claim 1, characterized in that: The concentration of the sex induction agent stock solution is 1 mM, and the concentration of the working solution is 0.6 μM.

4. A method for inducing male Daphnia cerana using the sex inducing agent for inducing male Daphnia cerana as described in any one of claims 1 to 3, characterized in that: The steps include: S1. Selecting the mother Daphnia cerana: Select active and healthy Daphnia cerana in the basic culture water, whose gonads have developed but have not yet been released into the brooding capsule and have not molted before release; S2. Induction of male Daphnia cerana: Place the parthenogenetic Daphnia cerana selected in step S1 into the culture water body to which the inducer has been added, and induce the culture for 3 hours; S3. After the induction is completed, the parthenogenetic Daphnia cerana is placed in the basic culture water without inducer, and the culture is continued until the parthenogenetic Daphnia cerana discharges the larvae from the incubation capsule, and then the parthenogenetic Daphnia cerana that has hatched larvae is picked out, and after the produced larvae are cultured for one week, the male and female Daphnia cerana larvae are separated into pots.

5. The method for inducing male Daphnia cerana according to claim 4, characterized in that: The basic culture water is tap water that has been irradiated with ultraviolet light and aerated for 2 days.

6. The method for inducing male Daphnia cerana according to claim 4, characterized in that: The induction conditions in step S2 are: population density of 0.5 ind. / mL, induction culture temperature of 25°C±2°C, light:darkness=14:10, and light intensity of 3000 lx.

7. The method for inducing male Daphnia cerana according to claim 4, characterized in that: In step S3, the cells were cultured in the basic culture water until the parthenogenetic Daphnia sinensis expelled the young Daphnia from the incubation capsule at a density of 1×10 5 cells / mL Scenedesmus obliquus was used as food source and conventional feeding was carried out.

8. Use of the sex inducing agent for inducing males of Daphnia cerana as claimed in any one of claims 1 to 3 in inducing males of Cladocerana.

9. Use of the method for inducing males of Daphnia cerana as claimed in any one of claims 4 to 7 in inducing males of Cladocerans.

Images