CN105340792A - Method for screening beach shellfish larvae microalgae baits - Google Patents
Method for screening beach shellfish larvae microalgae baits Download PDFInfo
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- CN105340792A CN105340792A CN201510644349.3A CN201510644349A CN105340792A CN 105340792 A CN105340792 A CN 105340792A CN 201510644349 A CN201510644349 A CN 201510644349A CN 105340792 A CN105340792 A CN 105340792A
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- shellfish
- algae
- micro
- fatty acid
- larvae
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention provides a method for screening beach shellfish larvae microalgae baits. The method is used for accurately screening beach shellfish larvae high-quality microalgae baits by quantitatively detecting composition of fatty acid and sterol in shellfish bodies after beach shellfish larvae intake mixed microalgae. The method provided by the invention is short in culture period, avoids probable influences of water-quality environmental conditions on the culture effect, and can be used for screening microalgae on the basis of the composition of fatty acid and sterol in the shellfish bodies. Therefore, the effect of the baits more fits in with selective ingestion and assimilation of the shellfish bodies during actual growth, so that the baits can be taken as high-quality baits for shellfish larvae.
Description
Technical field
The invention belongs to aquaculture bait culture technique field, be specifically related to a kind of method of screening intertidal shellfish seedling Mirco algal food.
Background technology
The intertidal shellfish output such as current China " clam, blood clam, razor clam " account for marine shellfish cultivation gross yield nearly 40%.And in intertidal shellfish nursery, the kind of Mirco algal food directly decides the success or failure of nursery.Long-term practice finds, during different microalgae of throwing something and feeding in seedling raising process, intertidal shellfish larval growth speed difference is very large, and the grown direct impact of micro-algae kind on intertidal shellfish seedling is described.In actual intertidal shellfish seedling reproductive process, we need to select the Mirco algal food having an obvious bait effect to the intertidal shellfish young to carry out, and expansion is numerous throws something and feeds.
To intertidal shellfish young bait effect how certain bait micro-algae, general all by different types of micro-algae of throwing something and feeding to intertidal shellfish, bait effect [the Zhu Yurui etc. of corresponding micro-algae are judged by the growth rate and survival rate observing corresponding shellfish seedling, the impact that 2010.5 kinds of micro-algaes grow 4 kinds of beach juvenile mollusks, oceanographic research, 28 (3): 60-66].But these means need long cultivation cycle, in addition, condition of water quality, environmental condition all can have an impact to the growth rate of seedling and survival rate.So often need finally to determine the bait effect of different microalgae to shellfish by repetition test repeatedly.Therefore, the method providing the micro-algae of a kind of effective detection as shellfish bait is just needed.And, generally adopt the mixing of two or more micro-algaes of throwing something and feeding to throw something and feed mode at present, and this mode need to cultivate different micro-algaes, causes the cost increase of bait.
Summary of the invention
The invention provides a kind of method of screening intertidal shellfish seedling Mirco algal food, namely to ingest the composition of the acid of shellfish body fat and sterol after the micro-algae of mixing by quantitatively detecting the intertidal shellfish young, accurately screen the method for intertidal shellfish seedling high-quality Mirco algal food, thus make up the deficiencies in the prior art.
Method of the present invention, comprises following step:
1) acquisition of standard group PCA shot chart
Throw something and feed shellfish larvae as standard group with the micro-algae of mixing, measures fatty acid and the sterol composition of shellfish larvae after some cycles of throwing something and feeding, then principal component analysis (PCA) is carried out to fatty acid and sterol composition, obtain PCA shot chart;
2) under same cultivating condition, cultivate as experimental group to the shellfish larvae micro-algae of different types of single of throwing something and feeding respectively, fatty acid and the sterol composition of different experiments group shellfish larvae is measured after same period of throwing something and feeding, then principal component analysis is carried out to fatty acid and sterol composition, select the bait of micro-algae as this shellfish of the experimental group close with standard group according to the distribution screen of each experimental group on PCA shot chart.
Described shellfish is the shellfish of mudflat aquaculture, such as mud blood clam, blood clam, clam, razor clam of hanging, moerella irideseens, clam etc.
The present invention micro-algae used is all in being positioned at exponential phase of growth, and concentration reaches 80 ~ 120cell μ l
-1shi Jinhang throws something and feeds;
Shellfish of the present invention is thrown something and fed before Mirco algal food, needs at the hungry 8-10h of not bait throwing in husky filter is not substantially containing the clean natural sea-water of any micro-algae with residual bait in emptying stomach as far as possible.
The present invention uses gas chromatography-mass spectrography to carry out the quantitative analysis of fatty acid and sterol.
Adopt method of the present invention, determine that the bait micro-algae of mud blood clam is Chaetoceros or chrysophyceae.
Compared with prior art, the invention has the advantages that: cultivation cycle is short, avoid water quality environment condition to the issuable impact of culture effect, the micro-algae filtered out is formed based on shellfish larvae fatty acid sterols, its bait effect more gears to actual circumstances the design choices of shellfish larvae in process of growth and assimilation, thus can be used as the selection of shellfish larvae high-quality bait.
Accompanying drawing explanation
Fig. 1: based on first and the 3rd principal component scores figure (A) and second and the 3rd principal component scores figure (B) of the principal component analysis of mud blood clam seedling fatty acid sterols
On figure, sample is respectively the throw something and feed mud blood clam seedling (IG-PV) of P.viridis, single of single and throws something and feeds the mud blood clam seedling of C.calcitrans (TG-CC), I.galbana (TG-IG) and N.oculata (NO); Mix throw something and feed I.galbana and N.oculata (TG-IG-NO), I.galbana and P.viridis (TG-IG-NO), C.calcitrans and I.galbana (TG-CC-IG), C.calcitrans and P.viridis (TG-CC-PV), C.calcitrans and N.oculata (TG-CC-NO) and all 4 kinds of micro-algaes between two and entirely mix the mud blood clam seedling (TG-MIX) of throwing something and feeding.
Embodiment
The present invention is by measuring fatty acid sterols composition to the shellfish larvae of throw something and feed mixing Mirco algal food and single Mirco algal food, and carry out PCA analysis, on PCA shot chart, shellfish larvae sample if thrown something and fed after certain micro-algae is with mixing the shellfish larvae sample of Mirco algal food of throwing something and feeding relatively, just can illustrate that this micro-algae is preferentially ingested by this shellfish and assimilated, this algae in actual shellfish larvae cultivating process, just can be selected as the high-quality bait of this shellfish larvae.The present invention applies this technology, substantially reduces incubation very long in conventional art, and avoids water quality environment condition to the issuable impact of culture effect, and the Mirco algal food effect the filtered out assimilation of ingesting of shellfish larvae in process that more gears to actual circumstances is selected.
Principal component analytical method (principalcomponentsanalysis used in the present invention, PCA) be a kind of multivariate data statistical method, two-dimensional space shows the PCA shot chart of material clustering distribution, can intuitively sample room material composition similar or non-similarity [VlietEVetal., 2008.Anovelinvitrometabolomicsapproachforneurotoxicityte sting, proofofprincipleformethylmercurychlorideandcaffeine.Neur oToxicology, 29 (1): 1 – 12].
The micro-algae of described mixing completes after the various micro-algae mixing by this shellfish reported.
By the following examples the present invention is described in further detail.
Embodiment 1
In September, 2014 is in Bao Zhi Aqua Sciences Inc. of Fujian Province, select the conventional 4 kinds of marine microalgaes of intertidal shellfish, respectively: the Nannochloropsis oculata (Nannochloropsisoculata) of the Chaetoceros (Chaetoceroscalcitrans) of Bacillariophyta, the Isochrysis galbana (Isochrysisgalbana) of Chrysophyta and pavlova viridis (Pavlovaviridis), Eustigmatophyceae algae door is cultivated, the cultivation seawater (salinity is 20) of micro-algae, after 0.45 μm of acetate fiber membrane filtration, boils cooling.Add NMB
3nutrient solution (100mgL
-1kNO
3, 10mgL
-1kH
2pO
4, 0.25mgL
-1mnSO
4h
2o, 2.50mgL
-1feSO
47H
2o, 10mgL
-1eDTA-Na
2, 6 μ gL
-1vB
1, 0.05 μ gL
-1vB
12), add sodium silicate (20mg.L simultaneously
-1) as the silicon source of Chaetoceros.Micro-algae algae kind [natural daylight in the conical flask of 2500mL, cultivation temperature is (20 ± 2) DEG C] cultivate 1 week after, they are proceeded in the white bucket of plastics of 50L, about 1 week is cultivated again, when micro-algae density reaches 80 ~ 120cell μ l under natural daylight, natural temperature (25 ~ 29 DEG C)
-1in time, throws something and feeds to shellfish seedling.
Growing, wide, height is respectively 25, 15 and 6cm white plastic basin (experiment basin) in add the sand filter seawater that salinity is 20 respectively, and add the thick 200 DEG C of dried disinfectings of 1-2mm and through the filtered fresh ooze of the nylon bolting silk of mesh 75 μm, be that the mud blood clam children shellfish sample of 1.5g average length 0.68 ± 0.05mm is evenly spilled in 10 groups of experiment basins (often organizing parallel 3) respectively by weight in wet base, to throw in enough corresponding tables 1 the micro-algae of single respectively at about 08:00 and the 20:00 moment in each experiment basin or mix micro-algae (equal proportion mixing), cultivate at natural temperature 25.2-31.7 DEG C after 7 days, collect sample, residual bait in the emptying stomach of the hungry 8-10h of not bait throwing in the clean natural sea-water through husky filter, fatty acid sterols analysis is carried out after freeze drying, the growth (table 2) of 30 young shellfishes is measured in random sampling simultaneously.
The food species that table 1 is selected in throwing something and feeding and testing
It is as follows that mud blood clam children shellfish fatty acid sterols analyzes concrete steps: get freeze drying and the children of the 0.2g mud blood clam after pulverizing shellfish sample of milling, with reference to the Bligh-Dyer method [BlighEG after improvement, DyerWJ.1959.ARapidMethodLipidExtractionandPurification, CanJBiochemPhysiol, 37:911-917] extract total fat; Total fat adds 2mL5-6%KOH methanol-water (V/V=4:1) solution after extracting, inflated with nitrogen 1min, sealing, 60 DEG C of water-bath saponification 2h, cooling, saponification liquor adds 15mL saturated nacl aqueous solution 1mL, HCl (1:1) is used to adjust pH to be less than 1 again, chloroform: n-hexane (V/V=1:4) 6mL divides three extractions, after rotavapor under vacuum drying, adds 0.5mL14%BF
3-CH
3oH solution, inflated with nitrogen 1min, sealing, 60 DEG C of water-bath esterification 1h, cooling, divides 3 extractions with n-hexane 6mL, and merge extract, wash once with 2mL redistilled water, upper liquid shifts out, and adds 1g anhydrous sodium sulfate, and water suction 12h, pipettes upper liquid, N
2add the excessive BSTFA of 100 μ L after drying up, sealing, 60 DEG C of water-bath 2h, N2 dries up, n-hexane constant volume, carries out GC-MS analysis.GC condition: injector temperature 250 DEG C, carrier gas is high-pure helium, column flow rate 0.81mL/min, pressure 73.0Kpa before post, post initial temperature 150 DEG C, keeps 3.5min, rises to 200 DEG C with 20 DEG C/min, keep 5min, then rise to 280 DEG C/min with 5 DEG C/min, keep 30min.1 μ L is flow to, split ratio 50:1 with the EI source analysis time-division.MS condition: bombard ionization source (EI) analysis with electronics, electron energy is 70eV, ion source temperature 200 DEG C, and interface temperature 250 DEG C, chooses SCAN pattern, and mass scan range is 40-600, solvent delay 3.5min.Finally according to the fragment ion quality collection of illustrative plates of component each in GC-EIMS-TIC, by identifying with reference to fatty acid and sterol standard spectrogram and bibliography NIST storehouse and WILEY library searching.The percentage composition (table 2 represents with the percentage and percentage contents that account for fatty acid and sterol total amount) of fatty acid and sterol constituent is calculated with area normalization method.
Table 2: fatty acid sterols composition (%) of mud blood clam children shellfish after different bait of throwing something and feeding
The measurement result of fatty acid sterols input SIMCA-P-12.0 analysis software (UmetricsAB company of Sweden) is carried out principal component analysis.On the shot chart that software exports, (Fig. 1) can be observed, mixing chrysophyceae of throwing something and feeding is close with bar husband algae or sample (on figure TG-IG-PV and the TG-IG-NO) merchandiser of Nannochloropsis oculata chrysophyceae (on figure TG-IG) of solely throwing something and feeding, and solely the throw something and feed sample of Ba Fuzao (on figure TG-PV) or Nannochloropsis oculata (on figure TG-NO) of merchandiser is far apart; Equally, mixing Chaetoceros of throwing something and feeding is close with bar husband algae or sample (on figure TG-CC-PV and the TG-CC-NO) merchandiser of Nannochloropsis oculata Chaetoceros (on figure TG-CC) of solely throwing something and feeding, and solely the throw something and feed sample of Ba Fuzao (on figure TG-PV) or Nannochloropsis oculata (on figure TG-NO) of merchandiser is far apart; Mixing is thrown something and fed, and then entirely to mix the sample (TG-MIX) of throwing something and feeding with four kinds of algaes close for the sample (TG-CC-IG) of chrysophyceae and Chaetoceros.Visible, mixing throw something and feed after the fatty acid sterols composition merchandiser kind of mud blood clam children shellfish throw something and feed chrysophyceae or single throw something and feed Chaetoceros mud blood clam children shellfish fatty acid sterols close, illustrate that mud blood clam children shellfish preference is ingested and assimilated chrysophyceae or Chaetoceros, these two kinds of algaes are the high-quality Mirco algal food of this intertidal shellfish seedling that the present invention filters out.As proof, from 4 groups of single throw something and feed micro-algae after 7 days mud blood clam children shellfish growth also can find out (table 3), the throw something and feed sample grown speed of Chaetoceros and chrysophyceae of single is obviously fast than throw something and feed Ba Fuzao and Nannochloropsis oculata of single, illustrates that the result that this screening technique obtains is correct effective.
Table 3: single throw something and feed Mirco algal food after 7 days mud blood clam children shellfish size length (mm)
Claims (7)
1. screen a method for intertidal shellfish seedling Mirco algal food, it is characterized in that, described method comprises following step:
1) acquisition of standard group PCA shot chart
Throw something and feed shellfish larvae as standard group with the micro-algae of mixing, measures fatty acid and the sterol composition of shellfish larvae after some cycles of throwing something and feeding, then principal component analysis (PCA) is carried out to fatty acid and sterol composition, obtain PCA shot chart;
2) under same cultivating condition, cultivate as experimental group to the shellfish larvae micro-algae of different types of single of throwing something and feeding respectively, fatty acid and the sterol composition of different experiments group shellfish larvae is measured after same period of throwing something and feeding, then principal component analysis is carried out to fatty acid and sterol composition, select the bait of micro-algae as this shellfish of the experimental group close with standard group according to the distribution screen of each experimental group on PCA shot chart.
2. the method for claim 1, is characterized in that, described shellfish is the shellfish of mudflat aquaculture.
3. method as claimed in claim 1 or 2, it is characterized in that, described shellfish is mud blood clam, blood clam, clam, razor clam of hanging, moerella irideseens or clam.
4. the method for claim 1, is characterized in that, described micro-algae is positioned at exponential phase of growth, and concentration reaches 80 ~ 120cell μ l
-1.
5. the method for claim 1, is characterized in that, described shellfish is before Mirco algal food of throwing something and feeding, and in the first natural sea-water filtering through sand, hungry 8-10h is with bait residual in emptying stomach.
6. the method for claim 1, is characterized in that, described method uses gas chromatography-mass spectrography method to carry out the quantitative analysis of fatty acid and sterol.
7. the application of method according to claim 1 in the bait micro-algae of screening mud blood clam, is characterized in that, the bait micro-algae of the mud blood clam of method screening according to claim 1 is Chaetoceros or chrysophyceae.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931179A (en) * | 2021-04-12 | 2021-06-11 | 浙江省海洋水产研究所 | Method for large-scale directional culture of juvenile mytilus coruscus baits |
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2015
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931179A (en) * | 2021-04-12 | 2021-06-11 | 浙江省海洋水产研究所 | Method for large-scale directional culture of juvenile mytilus coruscus baits |
| CN112931179B (en) * | 2021-04-12 | 2022-11-22 | 浙江省海洋水产研究所 | Method for large-scale directional culture of juvenile mytilus coruscus baits |
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