CN111357692A - Indoor propagation and culture method for daphnia brevicorna - Google Patents

Indoor propagation and culture method for daphnia brevicorna Download PDF

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CN111357692A
CN111357692A CN202010312091.8A CN202010312091A CN111357692A CN 111357692 A CN111357692 A CN 111357692A CN 202010312091 A CN202010312091 A CN 202010312091A CN 111357692 A CN111357692 A CN 111357692A
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daphnia
brevichnia
propagation
culture
culture medium
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曾兆华
游泳
林涛
林岭虹
朱珍珍
方慧玲
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Institute of Plant Protection of FAAS
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/20—Culture of aquatic animals of zooplankton, e.g. water fleas or Rotatoria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/22—Compounds of alkali metals
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/30—Oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs

Abstract

The invention discloses an indoor propagation and cultivation method of daphnia brevichnia, which is characterized in that a propagation culture medium is adopted to propagate and cultivate collected wild daphnia brevichnia females, and then a stable culture medium is adopted to cultivate the young daphnia obtained by propagation so as to ensure the stability of the individual state of the daphnia brevichnia; the stable culture medium comprises the following components: NaHCO 23240 mg/L、CaSO4·2H2O 150mg/L、MgSO4150mg/L, KCl 10mg/L, pH = 6.3-7.8. The operation method is simple, low in cost and easy to master, can realize large-scale propagation and stable culture of the daphnia breuifolia, and provides possibility for using the daphnia breuifolia as a pesticide environment toxicology test material.

Description

Indoor propagation and culture method for daphnia brevicorna
Technical Field
The invention belongs to the technical field of plankton culture, relates to a population culture technology for pesticide environment toxicity tests, and particularly relates to an indoor propagation culture method for daphnia brevichnia.
Background
The cladocera zooplankton (also called daphnia) belongs to cladocera of arthropoda, crustacean, gill poda and dicamba, commonly called as "red worm" or "fish worm", is an important component of zooplankton, mainly takes bacteria, unicellular algae, yeast, protozoa and organic detritus as food, has the characteristics of fast propagation, short growth cycle, easy culture, strong toxicity resistance and the like, is an important link for maintaining the stability of an aquatic ecosystem, is living bait of various larval fishes, and is one of indicator organisms for researching and evaluating the influence of various harmful chemical substances in the environment on organisms. The 1920's daphnia began research for bio-monitoring and developed rapidly after the 40's of the 20 th century. In the experimental methods established by the economic cooperation and development Organization (OECD) leaders of the United nations in 1977-1981, the daphnia magna test is a necessary test item. In 1992, China promulgated a method for testing acute toxicity of water quality and substances to daphnia (daphnia magna) and freshwater fish (zebra fish) (GB/T13266-. Hitherto, daphnia magna is still one of the most important aquatic organisms for toxicity testing in China. The zooplankton species are wide in the breadth of China, the difference of the ecological environment of water areas in the south and north is large, and the species of zooplankton in the cladocera are various, wherein 61 species of fresh water cladocera exist, 5 species of seawater cladocera exist, and 23 species of inland saline water exist. However, the current national standard only adopts a cladocera zooplankton (daphnia magna) which is only naturally distributed in the north of China as an indicator organism for the toxicological evaluation of the pesticide environment is obviously insufficient. Therefore, the search for the cladocera zooplankton distributed in the south of China is indispensable to perfect the pesticide environment toxicological evaluation system.
Daphnia (Daphnia obtusa) With daphnia magnaDaphnia magna) All belong to the genus daphnia, the family daphniaceae, and the major cladocera zooplankton. The daphnia is a wide-temperature world species and is naturally distributed in fresh water areas in the north and south of China. At present, research on daphnia brevichnia at home and abroad mainly focuses on aspects such as morphology, ecology, molecular biology and the like, and no relevant report is provided on whether the daphnia brevichnia can be used as an indicator organism for pesticide environmental toxicological evaluation. The research on the daphnia brevichnia as an indicator organism for pesticide environment toxicological evaluation is mainly to realize the stable indoor culture of a large amount of daphnia brevichnia so as to ensure the experimental consumption.
According to the invention, the growth and development conditions of the daphnia brevichnia cultured in different culture medium formulas are systematically observed to obtain the optimal culture medium formula suitable for the propagation culture of the daphnia brevichnia, so that the propagation speed of the daphnia brevichnia can be accelerated, the individual life of the daphnia brevichnia is prolonged, the individual state of the daphnia brevichnia is kept relatively stable, and the guarantee is provided for the indoor efficient large-scale population growth and propagation of the daphnia brevichnia.
Disclosure of Invention
The invention provides an indoor propagation and culture method for daphnia brevifolia, which aims to solve the problem that the death rate of cultivation of daphnia brevifolia of the same female individual and consistent gene type is more than 10% due to instability of the individual state caused by the shortage of the quantity of the daphnia brevifolia young daphnia in the pesticide environment toxicology test.
In order to achieve the purpose, the invention adopts the following technical scheme:
an indoor propagation and cultivation method for daphnia brevichnia comprises the steps of adopting a propagation medium to propagate and cultivate female daphnia brevichnia, and then adopting a stable medium to cultivate young daphnia obtained by propagation so as to ensure the stability of the individual state of the daphnia brevichnia.
The female daphnia is a second generation of children cultured by taking female daphnia which is collected in the field, separated, domesticated, purified and identified, and has large individual, many pregnant eggs and strong activity.
The propagation medium is M4 culture solution, which is prepared by mixing H with the concentration of 57190 mg/L3BO3Solution, 7210mg/L MnCl2·4H2O solution, 6120mg/L LiCl. H2O solution, 1420mg/L RbCl solution, 3040mg/L SrCl2·6H2O solution, 320mg/L NaBr solution, 1260mg/L Na2MoO4·2H2O solution, 335mg/L CuCl2·2H2O solution, 260mg/L ZnCl2Solution, 200mg/L CoCl2·6H2O solution, 65mg/L KI solution, 43.8mg/L Na2SeO3Solution, 11.5mg/L NH4VO3Mixing 1mL of the solutions, and adding 20mL of 5000mg/L Na2EDTA·2H2O solution and 1991mg/L FeSO4·7H2Mixing and sterilizing the O solution according to the volume ratio of 1:1 to obtain 2LFe-EDTA solution, uniformly mixing and metering the volume to 1L to obtain the product; during propagation and culture, the sheep horn moon-sprout algae is used for feeding.
The daphnia juvenile is non-head-fetus healthy daphnia made by breeding for more than 3 generations in a breeding medium with a birth time of less than 24 h.
The stable culture medium comprises the following components: NaHCO 23240 mg/L、CaSO4·2H2O 150mg/L、MgSO4150mg/L, KCl 10mg/L, pH = 6.3-7.8.
The invention has the beneficial effects that:
1. indoor cultivation of the daphnia brevichnia is carried out according to the method, the reproduction speed of the daphnia brevichnia can be accelerated, the individual life of the daphnia brevichnia is prolonged, the troublesome work of field collection is avoided, and the relative stability of the individual state of the daphnia brevichnia can be ensured;
2. the method is simple in operation method, low in cost and easy to master, and provides possibility for further developing the daphnia brevichnia pesticide environment toxicological test material.
Drawings
FIG. 1 is a microscopic electron micrograph of daphnia brevicorna collected from the field and obtained by screening in the examples.
FIG. 2 is a graph showing the comparison between the first time of the egg-carrying time of daphnia breda and the first time of the output time of daphnia juvenile in different media in example 1.
FIG. 3 is a graph showing the comparison of the average output of daphnia nodosa in different media for the daphnia breda in example 1.
FIG. 4 is a graph showing the comparison of the average maximum body length of the daphnia made in example 1 in different media.
FIG. 5 is a graph showing the comparison of the survival days of daphnia brevifolia young daphnia in example 2 in different media.
FIG. 6 is a graph showing the comparison of the death numbers of daphnia breda in different media in example 2.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Preparing a culture medium: dissolving the components of the culture medium by using distilled water, and measuring the water hardness and the pH of the prepared culture medium by using a multi-parameter water quality analyzer and a pH meter. See tables 1-3 for specific media formulations.
TABLE 1 measurement results of the composition of the culture medium and its pH and water hardness
Figure DEST_PATH_IMAGE002
TABLE 2 preparation of ISO Standard dilution Water
Figure DEST_PATH_IMAGE004
TABLE 3 preparation of stock solutions I and II of Elendt M4 culture solution
Figure DEST_PATH_IMAGE006
Screening young daphnia: in the early morning, a plankton collection net (commonly used No. 3 net, 120 meshes) is adopted to carry out multipoint random sampling on a pond near a grape picking garden of Minhou Nantong town in Fujian province and an upstream river channel, and the depth of a sampled water layer is about 5-15 cm; then filtering most of daphnia by using a 30-mesh screen indoors, selecting the daphnia with better state and larger individual, placing the daphnia in 1L of raw water subjected to filtering and disinfection treatment, standing and culturing, changing 20% of the volume of the raw water every other day, adding equivalent barreled water, feeding the crescent moon algae every day, and domesticating for two weeks to ensure that the daphnia can normally grow; then primarily screening and identifying the daphnia in the collected water sample by means of instruments such as a microscope and the like; under a microscope, the body shape of the patient is long oval, the head is low, no helmet exists, and the ventral side is relatively straight or slightly bent. The shell arc is developed, the rear end is not in an acute angle shape, compound eyes are large, and the back end is close to the vertex of the head; the first antenna is short, and the corner dune is short and high; the posterior abdomen is gradually reduced to the tip of the paw, and 12-15 anal spines are formed; the tail paw is long and slightly bent, two rows of thorns are arranged, the front row is 4-8, the rear row is 10-12, the ventral process is 4, and the identification result is the daphnia (as shown in figure 1); and finally, selecting partial female bodies with large individuals, more eggs and strong activity for culturing the screened and identified daphnia breuifolia, and taking the second-generation daphnia juvenile to carry out formal experiments.
Example 1 screening of daphnia brevicornus propagation Medium
Weighing 50 mL of 11 culture media prepared in advance, adding into 100 mL beakers respectively, adding 5 daphnia larvae with body length of 0.5-0.6 mm into each beaker, repeating 4 times for each culture medium, performing the test at 25 + -1 deg.C in a room with light cycle L =16 h:8 h, and using concentration of 5 × 10 every day during the test55-10 mL of fresh Oenothera capricornutum is fed with daphnia brevifolia.
In the test process, the observation is carried out once every 24 hours, specifically, a disposable rubber dropper is used for randomly absorbing 3 young daphnia in a 100 mL beaker on a glass slide with scales, and the body length (the length from a helmet to a shell thorn part), the brook condition and the reproduction condition of the daphnia are observed and recorded under a dissecting mirror (after each nest of young daphnia is produced and counted, a screen with 40 meshes is used for filtering the young daphnia, and only the original mother daphnia is remained). And after the test is finished, calculating the first egg laying time of the daphnia brevichnia, the first output time of the young daphnia, the average output number of the young daphnia nipponensis and the average maximum body length. The results are shown in Table 4 and FIGS. 2-4.
TABLE 4 growth and reproduction of daphnia brevicornus in different culture media
Figure DEST_PATH_IMAGE008
The result shows that the first time of carrying eggs of daphnia brevichnia in the M4 culture solution is day 4, the first time of producing daphnia juvenile is day 6, and the time required by the daphnia brevichnia is shorter than that in other culture media; the average output numbers of the daphnia breda in the first nest, the second nest and the third nest of the M4 culture solution are respectively 8, 8 and 7, which are all larger than those in other culture media; the average maximum body length of the daphnia is 1.80 mm in the M4 culture solution, which is larger than that in other culture media. Therefore, under the condition of feeding the aenophyta glauca, the first oviposition time of the daphnia brevifolia cultured by the M4 culture solution is shortest, the average output number of the daphnia treiorum nodosum is the largest, the average maximum body length is the longest, and the M4 culture solution is most suitable for being used as a basic culture medium for rapid indoor population propagation of the daphnia brevifolia.
Example 2 Medium screening for Stable culture of daphnia brevicornus
The method comprises the steps of breeding and culturing young daphnia breda for more than 3 generations by using the M4 culture solution which is screened out in the embodiment 1 and is most suitable for breeding the daphnia breda, taking out young daphnia breda with the non-head-fetus healthy period of less than 24 hours as a test material, measuring 50 mL of 11 culture mediums prepared in advance, adding the culture mediums into 100 mL beakers respectively, adding 5 young daphnia breda with the body length of 0.5-0.6 mm into each beaker, and repeating 4 culture mediums. The test was carried out in a climatic chamber at 25 ± 1 ℃ with a light cycle L: D =16 h:8 h, during which no food was added and water was changed 1 time every 2D. And observing every 24h, and recording the survival condition of the daphnia. The results are shown in FIGS. 5-6.
The result shows that the survival life of daphnia breda juvenile is 9 days in ⑤ culture medium, 8 days in ①, ③ and ⑥ culture medium, 7 days in ②, ⑦, barreled water and aerated water culture medium, 6 days in M4 culture medium and 5 days in ④ and ISO standard diluted water, the death rate of daphnia breda juvenile is lower than that of other culture medium in ⑤ culture medium, and the death rate of the daphnia breda juvenile is not more than 10% in the first two days, so that the daphnia breda juvenile cultured in ⑤ culture medium without feeding of Gekko aenophyta has the longest life and high survival rate, namely the daphnia breda juvenile cultured in ⑤ culture medium is most suitable for the growth of daphnia breda juvenile, and the relative stability of the individual state of daphnia breda juvenile is ensured.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (3)

1. An indoor propagation and culture method of daphnia brevichnia is characterized in that: adopting a propagation culture medium to propagate and culture female daphnia brevichnia, and then adopting a stable culture medium to culture the obtained young daphnia brevichnia so as to ensure the stability of the individual state of the daphnia brevichnia;
the stable culture medium comprises the following components: NaHCO 23240 mg/L、CaSO4·2H2O 150mg/L、MgSO4150mg/L, KCl 10mg/L, pH = 6.3-7.8.
2. The indoor propagation and cultivation method of daphnia brevichnia according to claim 1, characterized in that: the propagation medium is M4 culture solution.
3. The indoor propagation and cultivation method of daphnia brevichnia according to claim 1, characterized in that: when the breeding culture is carried out, the moongrass of the cavel is adopted to feed the daphnia brevicornus.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10113095A (en) * 1996-10-14 1998-05-06 Yakult Honsha Co Ltd Method for culturing water flea
CN102138542A (en) * 2011-02-28 2011-08-03 中山大学 Method for reserving, conserving and culturing daphnia magna
CN104273101A (en) * 2014-10-24 2015-01-14 南京大学 Artificial cultivation method of Moina micrura Kurz or Ceriodaphnia cornuta Sars
CN106359206A (en) * 2016-09-30 2017-02-01 广东中科英海科技有限公司 Breed conservation culture method of daphnia magna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10113095A (en) * 1996-10-14 1998-05-06 Yakult Honsha Co Ltd Method for culturing water flea
CN102138542A (en) * 2011-02-28 2011-08-03 中山大学 Method for reserving, conserving and culturing daphnia magna
CN104273101A (en) * 2014-10-24 2015-01-14 南京大学 Artificial cultivation method of Moina micrura Kurz or Ceriodaphnia cornuta Sars
CN106359206A (en) * 2016-09-30 2017-02-01 广东中科英海科技有限公司 Breed conservation culture method of daphnia magna

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
许玉洁 等: "培养基对大型溞（Daphnia magna）生长、繁殖和子代的影响", 《生态毒理学报》 *

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