CN104651235A - Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait - Google Patents
Thalassiosira pseudonana and application of thalassiosira pseudonana as mercenaria mercenaria larva breeding bait Download PDFInfo
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- CN104651235A CN104651235A CN201510092775.0A CN201510092775A CN104651235A CN 104651235 A CN104651235 A CN 104651235A CN 201510092775 A CN201510092775 A CN 201510092775A CN 104651235 A CN104651235 A CN 104651235A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention provides a thalassiosira pseudonana Guh005 with the preservation number of CCTCC NO.M2015044. A specific nutrient solution formula is adopted for carrying out three-stage amplification culture on the thalassiosira pseudonana and is used for feeding after Mercenaria mercenaria D-shaped planula larva metamorphosis is finished for 36 hours, and feeding effect is the same as the conventional shell-fish bait. Under the culture by virtue of the nutrient solution formula provided by the invention, outdoor quick three-stage expanding propagation can be carried out on the marine diatom under the condition that illumination intensity is 2000-8000Lux, and especially the marine diatom can be a priority selection of a bait for breeding Mercenaria mercenaria larvae in rainy days.
Description
Technical field
The invention belongs to aquaculture bait to cultivate and feeding technology field, be specifically related to a strain Thalassiosira pseudonana and the application as mercenaria mercenaria linnaeus nursery bait thereof.
Background technology
Mercenaria mercenaria linnaeus (Mercenaria mercenaria), also known as quahog, cherrystone, northern curtain clam, be under the jurisdiction of lamellibranchiata (Lamillibranchia), curtain clam order (Venus clam), Veneridae (Veneridae), curtain clam genus (Venus), it is a kind of relatively large economic mussels, maximum individual shell is long is that U.S.'s thing is littoral more than 100mm. country of origin, be adapted to the cultivation of different substrate, the speed of growth is very fast, disease resistance is strong, is the most popular economic shellfish in United States consumes market.Last century end is incorporated into China, day by day increases at present in coastal area of china cultivation scale.Current seed breeding concentrates on southeastern coast mostly, the main season of its nursery is in the annual 3-5 month, now nursery area rainy weather is more mostly, bait micro-algae reproduction speed under this weather that China's shellfish seed breeding is conventional is very slow, can not meet the bait of these kind seed ramp needs far away.This just needs to find a kind of micro-algae kind that biomass reproduction speed is very fast under production rainy weather in season.
Summary of the invention
The object of this invention is to provide a strain Thalassiosira pseudonana and the application as mercenaria mercenaria linnaeus nursery bait thereof, the one strain Thalassiosira pseudonana Guh005 (Thalassiosira pseudonana Guh005) that namely can breed rapidly under separation screening to Low light intensity from ocean basin, and as the application of mercenaria mercenaria linnaeus nursery bait, thus make up the deficiencies in the prior art.
One aspect of the invention relates to a strain Thalassiosira pseudonana Guh005 (Thalassiosira pseudonanaGuh005), be preserved in the China typical culture collection center being positioned at Wuhan University of Wuhan City, deposit number on January 18th, 2015: CCTCC NO:M2015044.
The present invention provides this strain Thalassiosira pseudonana as the application of seed bait in mercenaria mercenaria linnaeus seed breeding on the other hand;
The present invention also provides the cultural method of above-mentioned Thalassiosira pseudonana, is to carry out third stage culture preparation; Wherein adopt nutrient solution formula as follows at one-level cultivation stage:
100mg KNO is added with in often liter of seawater
3, 20mg KH
2pO
4, 10mg Na
2siO
3, 0.20mgMnSO
4h
2o, 5.0mg FeSO
47H
2o, 20mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12;
At secondary cultivation stage, adopt nutrient solution formula for being added with 50mg KNO in often liter of seawater
3, 15mgKH
2pO
4, 10mg Na
2siO
3, 2.50mg FeSO
47H
2o, 10mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12;
In the third stage culture stage, adopt nutrient solution formula for being added with 20mg urea, 20mgKH in often liter of seawater
2pO
4, 10mg Na
2siO
3;
Thalassiosira pseudonana of the present invention, it cultivates intensity of illumination is 2000-8000Lux.
The present invention filters out a strain Thalassiosira pseudonana Guh005 that can breed rapidly Low light intensity from natural waters; under culture condition provided by the invention; with other Mirco algal food effectiveness comparison; will considerably beyond the bait micro-algae of other routines in overcast and rainy reproduction speed; can throw something and feed after mercenaria mercenaria linnaeus hatches two days; bait effect is compared with other conventional bait micro-algaes does not have notable difference; the bait demand of wet weather mercenaria mercenaria linnaeus seed propagation in scale in season can be met, improve the stability that this shellfish produces.
Embodiment
For the various components that substratum of the present invention is used, be medicine conventional in microdisk electrode, the invention reside in the interworking effect used between medicine.And during Thalassiosira pseudonana is cultivated, except the selection of substratum, other cultural method all can adopt the cultural method of published marine microalgae.For the cultivation of mercenaria mercenaria linnaeus seedling, also select disclosed method.
Below in conjunction with case study on implementation, the present invention is described in detail.
Embodiment 1: the separation and purification of Thalassiosira pseudonana Guh005
Is taken from the seawater sample in prawn culturing pond, Shan Yu village, Weng Yang town, From Yueqing County, Zhejiang city in May, 2010, choose after single algae cell with kapillary under inverted microscope, be put in 96 orifice plates and cultivate, substratum used by cultivation is f/2 substratum (Guillard and Ryther 1962, Guillard 1975), culture temperature 25 ± 1 DEG C, Light To Dark Ratio 12h:12h, timing shake twice every day.Under different illumination intensity (100-10000Lux), screen the marine microalgae all can normally bred within the scope of 1 strain intensity of illumination 2000-8000Lux---the strain of Thalassiosira pseudonana Guh005 algae, and be preserved in the China typical culture collection center being positioned at Wuhan University of Wuhan City, deposit number on January 18th, 2015: CCTCC NO.M2015044.The algae strain of the present invention's screening can grow within the scope of salinity 5-30, temperature 10-30 DEG C, is a wide salt, eurythermic kind, under Low light intensity, especially possesses the reproduction speed being obviously better than other micro-algaes.
Embodiment 2: the enlarged culturing of Thalassiosira pseudonana Guh005
Be inoculated in the glass spinner flask of 2500mL by Thalassiosira pseudonana Guh005 algae kind, be equipped with and carry out one-level and cultivate nutrient solution used in Erlenmeyer flask, nutrient solution formula is added with 100mg KNO in often liter of seawater
3, 20mg KH
2pO
4, 10mg Na
2siO
3, 0.20mg MnSO
4h
2o, 5.0mg FeSO
47H
2o, 20mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12, sea water salinity is 20.5, but the salinity of seawater can change within the scope of 5-30.During cultivation every day shaking flask 3-6 time, at (20 ± 2) DEG C, natural light is cultivated 1 week density and is reached 5000cell μ L
-1fill air continuously in the white plastic bucket of the rear access in left and right 50L and carry out secondary cultivation (inoculum density 40-50cell μ L
-1), the nutrient solution formula that secondary is cultivated is for being added with 50mgKNO in often liter of seawater
3, 15mg KH
2pO
4, 10mg Na
2siO
3, 2.50mg FeSO
47H
2o, 10mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12, at natural light, temperature (20 ~ 25 DEG C), cultivate about 5d again, density reaches 2000 ~ 3000cell μ L
-1rear access 15-30m
2cement pit (inoculum density 20-30cell μ L
-1), the water level 1m of sand filter seawater, fills air jet flow continuously, and third stage culture liquid formula is added with 20mg urea, 20mg KH in often liter of seawater
2pO
4, 10mg Na
2siO
3, cultivate 3-5d at natural light, temperature (20 ~ 25 DEG C) after, density reaches 1000-1200cell μ L
-1after throw something and feed.
Under table 1 different illumination conditions, the numerous multiple of expansion of three strain algaes compares
In order to compare the reproduction speed of micro-algae under outdoor different weather condition in actual production, be added with the above-mentioned 20m that salinity is 20.5 water temperatures, 18 ~ 24 DEG C of sand filter seawaters
3cement pit in, use third stage culture liquid formula of the present invention, parallel three cultivation pools of each algae under same weather condition, inoculate Platymonas helgolandica var 5cell μ L respectively
-1, Thalassiosira pseudonana 20cell μ L
-1, Chaetoceros 20cell μ L
-1, every day 10:00 ~ 16:00 every half an hour with photometer measurement nutrient solution surface light intensity, the 3rd afternoon 16 counts culture density with blood counting chamber, and result gets average.
Cultivation results shows (table 1), under the weather condition of the relatively high intensity of illumination of continuous sunny, (in Measuring Time section, light intensity is more than 8000Lux, most times light intensity exceedes photometer measurement scope), in three days, to expand numerous multiple the highest for Chaetoceros, reaches 61.5 times; It is relatively high that flat algae and Thalassiosira pseudonana under medium tenacity illumination weather condition (weather is cloudy is main, light intensity 4000 ~ 9000Lux in Measuring Time section) expand numerous speed, reaches 64 times and 53 times respectively; And (weather wet weather is main under relatively low illumination weather condition, light intensity 1200 ~ 3300Lux in Measuring Time section), Thalassiosira pseudonana demonstrates the numerous speed of the strongest expansion, overcast and rainyly still reach 43.5 times, slightly there is no statistically difference lower than the proliferative speed under cloudy weather, other two kinds of micro-algaes are expanded numerous speed and then greatly reduce, and expand numerous multiple far below 20 times.Visible, the numerous ability of expansion that Thalassiosira pseudonana is higher under having other Low light intensity not available for micro-algae, because mercenaria mercenaria linnaeus seed breeding is in the annual 3-5 month mostly in period, now many places are in overcast and rainy season, so this algae is particularly suitable for the Mirco algal food kind of this season as outdoor cultivation.
Embodiment 3: Thalassiosira pseudonana is thrown something and fed mercenaria mercenaria linnaeus seedling
Mercenaria mercenaria linnaeus floating larvae is cultivated in the cement pit filling 8.5 × 3.5 × 1.2m of air (long × wide × dark) continuously.During cultivation, the nature seawater of salinity 25 enters pond after sand filter, and mercenaria mercenaria linnaeus spawning and fertilization hatching at culture temperature 22-25 DEG C, 18h-22h to D shape larva, chrysophyceae of now throwing something and feeding maintains chrysophyceae density 5-8cell μ L in water body
-1left and right, starts Thalassiosira pseudonana of throwing something and feeding after 36h, and every day throws something and feeds at twice, and after throwing something and feeding first, Thalassiosira pseudonana density domination is at 30cell μ L
-1left and right, later along with the growth of mercenaria mercenaria linnaeus increases feeding volume every day gradually, remains on 2-3cell μ L with Measures of Algae in Water Body density before throwing something and feeding at every turn
-1for good, under 22-25 DEG C of temperature condition, 7-11 days settlement and metamorphosis are juvenile mollusk.
Juvenile mollusk is also cultivated in same cement pit, the nature seawater of salinity 25 enters pond after sand filter, add through 200 DEG C of hyperthermia dryings and ooze after 200 orders (diameter 75m) nylon silk cover filtering, the ooze at the bottom of pond is made to reach 1 ~ 2mm thick, mercenaria mercenaria linnaeus juvenile mollusk about the 0.5kg of just attachment is thrown in every mouthful of pond, throws something and feeds at twice every day through the 1000-1200cell μ L of third stage culture
-1thalassiosira pseudonana, each feeding volume 1m of first day
3left and right, after progressively increase feeding volume along with the growth of juvenile mollusk, clear for reference with the razor clam juvenile mollusk filter food of substantially being hung of Measures of Algae in Water Body before throwing something and feeding at every turn, cultivate 5 days at temperature (20 ~ 25 DEG C), juvenile mollusk weight can reach more than 2-3kg.
In order to determine that just this marine diatom needs mercenaria mercenaria linnaeus Metamorphore to grow into which in stage and can throw something and feed, in the glass cylinder of 1L, add the sand filter seawater that salinity is 25, and add the 10/mL just abnormal mercenaria mercenaria linnaeus larva for 12h, 24h, 36h, 48h after D shape, D shape (each specification larva all throw something and feed on pretreatment chrysophyceae) respectively, air-charging incubation at temperature (21 ~ 24 DEG C), throw something and feed respectively chrysophyceae and Thalassiosira pseudonana (density controls respectively 5,8,10,12,15cell μ L
-1), basis of microscopic observation after 3h, the mercenaria mercenaria linnaeus larva being less than 36h throw something and feed Thalassiosira pseudonana larva stomach in color be obviously shallower than the larva of chrysophyceae of throwing something and feeding, and measure size under selecting 20 seedling microscopes after the 2d that continues to throw something and feed and observe its growth velocity, the mercenaria mercenaria linnaeus larva being less than 36h to throw something and feed after Thalassiosira pseudonana growth obviously slower than chrysophyceae (table 2); And be greater than the mercenaria mercenaria linnaeus larva of 36h, the larva stomach of Thalassiosira pseudonana of throwing something and feeding is full equally with the stomach stomach of chrysophyceae of throwing something and feeding, and growth does not have significant difference (table 2, table 3), so the present invention is that 36h just throws something and feeds Thalassiosira pseudonana after mercenaria mercenaria linnaeus D shape larval metamorphosis.
Table 2, to throw something and feed the growth of mercenaria mercenaria linnaeus Metamorphore after chrysophyceae and Thalassiosira pseudonana 2d respectively
In order to compare this marine diatom with other bait micro-algaes to the bait effect of mercenaria mercenaria linnaeus Metamorphore, have selected present the sector generally acknowledge and a large amount of use Chaetoceros, chrysophyceae, flat algae, carry out single to mercenaria mercenaria linnaeus Metamorphore to throw something and feed experiment together with Thalassiosira pseudonana, the D shape Metamorphore that salinity is the sand filter seawater of 20.5 and the abnormal 36h of 10/mL is added, respectively enough micro-algaes of single kind of throwing something and feeding (chrysophyceae, Chaetoceros, Thalassiosira pseudonana control cell density 20-30cell μ L in the glass beaker of 1L
-1, flat algae cell density controls at 3-5cell μ L
-1, get 20 seedlings after the 2d that throws something and feeds and measure length, result gets average (table 3).Visible, to throw something and feed flat algae in this stage, mercenaria mercenaria linnaeus larval growth wants obviously slow than the larva of other algae of throwing something and feeding, and the bait effect of Thalassiosira pseudonana does not have notable difference with chrysophyceae and Chaetoceros.
Table 3 is thrown something and fed the size (length, μm) of (2d) mercenaria mercenaria linnaeus Metamorphore before and after 4 kinds of micro-algaes respectively
In order to compare this marine diatom with other bait micro-algaes to the bait effect of mercenaria mercenaria linnaeus juvenile mollusk, in the white plastic basin of 28cm × 15cm × 10cm (long × wide × high), add salinity is the sand filter seawater of 20.5 and the ooze same with cement pit, is evenly spilled into the juvenile mollusk of 1g weight in wet base, length 0.57 ± 0.12mm.Enough micro-algaes of single kind of throwing something and feeding (chrysophyceae, Chaetoceros, Thalassiosira pseudonana control cell density 100 ~ 120cell μ L in each experiment basin
-1, flat algae cell density controls at 15 ~ 20cell μ L
-1), every 4 ~ 5d changes fresh water and ooze and random sampling is calculated to be motility rate, and get 20 seedlings and measure length, under 18 ~ 25 DEG C of natural temperatures, cultivate 12d is an experimental period. and each sample parallel tests 3 times, and result gets average (table 4).Visible, under this specification, the juvenile mollusk of throwing something and feeding with hang the razor clam juvenile mollusk speed of growth and other 3 kinds of micro-algaes that Thalassiosira pseudonana is thrown something and fed is without significant difference.
Table 4 is thrown something and fed the size (length, mm) of (12d) mercenaria mercenaria linnaeus juvenile mollusk before and after 4 kinds of micro-algaes respectively
Claims (6)
1. miniature hailian seaweed is intended in a strain, it is characterized in that, the deposit number of the miniature hailian seaweed of described plan is CCTCC NO.M2015044.
2. the miniature hailian seaweed of plan according to claim 1 is as the application of shellfish bait.
3. apply as claimed in claim 2, it is characterized in that, described shellfish is mercenaria mercenaria linnaeus.
4. cultivate a method for the miniature hailian seaweed of plan according to claim 1, it is characterized in that, described method is prepared by third stage culture; Wherein adopt nutrient solution formula as follows at one-level cultivation stage:
100mg KNO is added with in often liter of seawater
3, 20mg KH
2pO
4, 10mg Na
2siO
3, 0.20mgMnSO
4h
2o, 5.0mg FeSO
47H
2o, 20mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12;
At secondary cultivation stage, adopt nutrient solution formula for being added with 50mg KNO in often liter of seawater
3, 15mgKH
2pO
4, 10mg Na
2siO
3, 2.50mg FeSO
47H
2o, 10mg EDTA-Na
2, 6 μ g VB
1with 0.05 μ g VB
12;
In the third stage culture stage, adopt nutrient solution formula for being added with 20mg urea, 20mgKH in often liter of seawater
2pO
4, 10mg Na
2siO
3.
5. method as claimed in claim 4, it is characterized in that, described cultivation stage, its intensity of illumination is Low light intensity.
6. method as claimed in claim 5, it is characterized in that, described Low light intensity is 2000Lux.
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|---|---|---|---|
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Cited By (4)
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| CN105199959A (en) * | 2015-10-22 | 2015-12-30 | 湛江恒兴南方海洋科技有限公司 | Macro-element nutrient salt formula for large-scale culture of Thalassiossira sp. |
| CN105713838A (en) * | 2016-05-05 | 2016-06-29 | 宁波大学 | High-vitality cell preservation method for thalassiosira pseudonana |
| CN109287534A (en) * | 2018-11-22 | 2019-02-01 | 宁波大学 | A kind of marine shellfish cultivating pool microalgae group manual intervention system and application method |
| CN113016676A (en) * | 2019-12-25 | 2021-06-25 | 宁波大学 | Method for cultivating litopenaeus vannamei through directional culture of diatoms |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105199959A (en) * | 2015-10-22 | 2015-12-30 | 湛江恒兴南方海洋科技有限公司 | Macro-element nutrient salt formula for large-scale culture of Thalassiossira sp. |
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| CN105713838B (en) * | 2016-05-05 | 2019-10-11 | 宁波大学 | A kind of high vigor cell method for preserving of Thalassiosira pseudonana |
| CN109287534A (en) * | 2018-11-22 | 2019-02-01 | 宁波大学 | A kind of marine shellfish cultivating pool microalgae group manual intervention system and application method |
| CN113016676A (en) * | 2019-12-25 | 2021-06-25 | 宁波大学 | Method for cultivating litopenaeus vannamei through directional culture of diatoms |
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