CN101099452B - Laboratory artificial cultivating method for intertidal zone marine sponge - Google Patents
Laboratory artificial cultivating method for intertidal zone marine sponge Download PDFInfo
- Publication number
- CN101099452B CN101099452B CN2006100471255A CN200610047125A CN101099452B CN 101099452 B CN101099452 B CN 101099452B CN 2006100471255 A CN2006100471255 A CN 2006100471255A CN 200610047125 A CN200610047125 A CN 200610047125A CN 101099452 B CN101099452 B CN 101099452B
- Authority
- CN
- China
- Prior art keywords
- sponge
- laboratory
- intertidal zone
- culture
- growth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- 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
Landscapes
- Cultivation Of Seaweed (AREA)
- Cultivation Of Plants (AREA)
Abstract
The present invention relates to a method for making intertidal sponge undergo the process of artificial cultivation under the laboratory condition to obtain biomass. Said method includes the following steps: collecting sponge in intertidal zone, cleaning impurity and dirts from sponge surface in laboratory, cutting sponge into small blocks, making temporary culture in culture vessel, then fixing the small block sponge on the carrier, placing it into a culture device to make temperature-controlled photophobic culture, adding bait algae as main body food, making culture for 10-20 days, so that the biomass maxmum value of primary sponge growth can be obtained.
Description
Technical field
The present invention relates to the marine biotechnology field, is to adopt sponge explant cultural method to produce the technology of spongy biological amount in the laboratory, specifically set up a kind of in the laboratory method of artificial culture intertidal zone marine sponge.
Background technology
In the at present known marine organisms, sponge is the largest source of bioactive natural product, bioactive natural product as antitumor, antimycotic even anti-HIV, and other animals and plants in the high Yu Haiyang of probability of the reactive compound that discovery obtains from sponge, so the sponge drug development becomes marine drug hot of research and development in recent years.But activity substance content is few in the cavernous body, generally has only 10 of weight in wet base
-8" medicine source undersupply problem " promptly do not have enough sponges and measures the active substance that extracts some and can supply the deep research of medicine, making some medicines only rest on the clinical trial stage can't continue, thereby " medicine source undersupply problem " restrictive factor of just having become the sponge drug development obviously to lag behind.Multiple mode is attempted going to address this problem but scabrous key technology is all arranged, and sponge cell culture technology for example also not have at present the cell-line that foundation can continuous passage; Chemosynthesis costs an arm and a leg, step complexity, practical feasibility are too low; Generally believe that at present the breed of sponge industrial artificial is considered to one of mode more likely that can solve " medicine source undersupply problem ".The report of external relevant sponge breed aspect mostly relates to the sponge at deep-sea, they culture for the land for growing field crops that the breed of sponge explant concentrates in the seawater mostly, build metal truss, cement dish or set up rope at the water body of depth of water 5-10m, fix on it after sponge gathered stripping and slicing, but owing to be subjected to the influence of weather such as uncertain storm, sponge death, taken away often by storm and to take place, it is lower, uncertain relatively large to cause the land for growing field crops to culture success rate.Stone1970 once reported under the open-air self-sow state of film sponge in great numbers with its growth area and to increase about 2 times as index between 5 months, increased average every month approximately 40%, these 5 months also was the month of youngster's spongy biological amount rising unique in 1 year.Industrial aquaculture is because the controlled shortcoming that can avoid physioclimate to change the influence that sponge is grown of condition.Document also once had the data that is reported in laboratory aquarium breed sponge, but its objective is the growth rhythm of research variety classes and region sponge, investigated external environmental condition to the sponge growth effect, for example the temperature of Pei Yanging, salinity, ammonia nitrogen etc.The sponge of different growing environments and kind, to the requirement difference of growing environment, the kind difference of the active substance that is contained in the body is so the sponge artificial culture under the laboratory condition can be regulated and control according to natural environment.The present invention be directed to the cultural method of intertidal zone marine sponge, under experiment indoors artificial controlled condition, cultivate intertidal zone marine sponge and obtain biomass for the researcher use of doing its related fields research.
Summary of the invention
It is effective and simple to operate to the purpose of this invention is to provide a cover, thereby can carry out the method that the spongy biological amount is produced in indoor controlled cultivation to intertidal zone marine sponge.
The method principle: intertidal zone marine sponge is a lower animal of seeking the non-selectivity filter food of growing admittedly, and regeneration capacity is stronger, under suitable external environment, is attached at continued growth on other carriers but cut an explant from sponge.Its influence that is subjected to seasonal temperature of growing of film sponge in great numbers that is grown in the temperate zone is bigger, presents that spring and summer seasonal growth is vigorous, the growth rhythm of autumn and winter season decline, cultivates for the indoor control of sponge and transfers part that theoretical foundation is provided.
For achieving the above object, the technical solution used in the present invention is:
A kind of intertidal zone marine sponge laboratory artificial cultivating method that is used for, laboratory are manually made lower animal-sponge that the suitable growth environment is cultivated intertidal zone set growth; Sponge self-sow environment with manual simulation intertidal zone, laboratory set growth is produced the spongy biological amount; Concrete steps, after the sponge collection that is grown on the reef of intertidal zone, put into immediately in the keg of containing seawater, take back the laboratory and remove sponge surface impurity and foul, then sponge is cut into the fritter of suitable size, putting into glass jar supports temporarily, be fixed on the carrier then, place in the incubator, temperature control, lucifuge are cultivated, throw something and feed feed algae as main body food, attach situation, metamorphosis and biomass situation of change as evaluation index, cultivate the maximum biomass that can reach former generation sponge growth in 10-20 days with sponge.The present invention has overcome the sponge laboratory and has cultured growth phase to problem slow, that the cycle is long.
Specific operation process is:
When 1) sponging on the reef of intertidal zone, seashore, select bright-colored, surperficial relatively flat, projection is short and sturdy, surface clean (the impurity foul is less) sponge places sponge after the collection in the keg of containing seawater immediately;
2) after sponge is taken back the laboratory, when carefully clearing up sponge body surface impurity foul, have in the spongy tissue than high concentration of sediment stone grain, then abandon selecting other sponges,, then carefully take out with tweezers if the stone grain is less and relatively large in the tissue if run into tweezers and injection needle;
3) aseptic operation cutter cutting sponge, suitable size is the about 3-9cm of area
2, to put into glass jar and supported 2-7 days temporarily, the wound that forms during to its cutting and collection tentatively heals, and finishes preliminary callus process, and temperature adapted when gathered on foster temporarily temperature and seashore, and then was adjusted to cultivation temperature gradually;
4) sponge is fixed on to place in the reactor on the carrier with fine rule and cultivates, and the carrier size is about 4-8 times of sponge size;
5) control sponge culture density 2-15 ‰ (m/v), water temperature 15-25 ℃, oxyty 5-9mg/L;
6) sunlight direct projection sponge is avoided in the place that hides one deck brown paper or directly reactor is placed on no sunlight direct projection, glass jar outside;
7) in the culture period, keep cultivating the NH in the water body
4 +Concentration is lower than 1mg/L;
8) concentration of throwing something and feeding is 2 * 10
4-2 * 10
5The unit cell algae as food;
Every day is changed water in quiet cultivation, and sponge is a main body food with the feed algae of throwing something and feeding etc.;
It is the algae concentration of throwing something and feeding according to streamflow calculating every day incubator planted agent that stream adds training method;
8) in the incubation, sponge attaches and sprawls, metamorphosis is taken pictures and record with digital camera, and biomass is weighed with weight in wet base (wet sponge quality), is converted into the weight in wet base increased percentage after the weighing, and formula is as follows: S=(M
n-M
0)/M
0* 100%, M
0, M
tBe respectively sponge and cultivating the weight in wet base of the 0th day and n days;
9) look the different cultivations of sponge collection season state 10-20 days, can reach the maximum biomass of former generation sponge growth, winter-spring season was cultivated 15-20 days, and summer and autumn cultivated 10-15 days.
The present invention compared with prior art has following advantage:
1. technology is complete relatively, first film sponge in great numbers is provided a cover complete method and is used for laboratory artificial cultivating.
2. simple to operation, sponge carrier making, fixing means, temperature control, illumination control, dissolved oxygen concentration control etc. are all very simple, reach requirement easily.
3, be widely used, the method can be used for producing in a short time film sponge biomass of former generation in great numbers; Can provide sponge to study the excellent material of international difficult point one cell culture by the good sponge of training quality; Can carry out the go down to posterity comparison of culture studies and targeted activity material regulation and control of spongy tissue piece.
In a word, the present invention can provide film sponge in great numbers effectively to cultivate under the controlled condition of laboratory and produce sponge biomass of former generation, technology is more complete relatively, easy and simple to handle, produce the spongy biological amount in indoor controlled cultivation, for the sponge industrial aquaculture provides technical support, thereby, has wide application prospect for " the medicine source undersupply problem " that solves the spongy biological medicine proposes a kind of new way.
Description of drawings
Fig. 1 is 5L water purification cultivating system figure; Wherein: 1 sponge is cultivated glass jar, and 2 sponges attach carrier and orange sponge, 3 breather pipes, 4 gas stones, 5 air pumps; 6 waters cylinders, 7 temperature controllers, 8 desks, 9 waters water storage tanks, 10 shallow water pumps, 11 water-bath pipes;
Fig. 2 is 5L flowing water cultivating system figure; Wherein: 12 air, 13 ozone generators, 14 stirring arms, 15 bait algae liquid, 16 mixing tanks, 17 air filter membranes, 18 sponge incubators, 19 waste collection grooves, 20 heat exchangers, 21 seawater, 22 Seawater Treatment grooves, 23 remove the albumen device;
Fig. 3 is outward appearance growth conditions figure in the film sponge incubation in great numbers; Wherein: a is for cultivating initial sponge state; Sponge state when b finishes for cultivating;
Fig. 4 is film sponge in great numbers biomass change curve in the hydrostatic incubation;
Fig. 5 adds biomass change curve in the incubation for film sponge in great numbers at stream;
Fig. 6 is the GC-MS spectrogram of 5 α in the sponge extract-Cholestan-3 beta-ol;
Fig. 7 is 5 α-Cholestan-3 beta-ol calibration curve.
Embodiment
Below by specific embodiment method of the present invention and result are described, the present invention uses and buys deep-sea water as breeding seawater, is used for the cultivation of sponge breed and feed algae; Weight in wet base adopts analytical balance to take by weighing quality, gets the sponge growth quality by the loss of weight method, and mercurial thermometer is measured the interior actual water temperature of cultivation water body and is convenient to regulate; Portable dissolved oxygen meter is measured the air mass flow that dissolved oxygen concentration is convenient to regulate the air pump supply in the water body, and the cultivation of bait algae adopts filtration sterilization seawater conical flask to cultivate, and nutritive salt adopts the general Kang Wei mill nutritive salt of algae culture.
5L hydrostatic system is cultivated.
Intertidal zone film sponge in great numbers (Hymeniacidon perleve) is collected in DaLian, China Ling Shui bridge intertidal zone in July, 2005, and sponge surface impurity and foul are less, choose the about 2 * 2cm of area during collection
2Fritter, take back the laboratory and be placed in the 12L glass jar of containing seawater and supported 2 days temporarily, support temperature temporarily and be controlled at about 20 ± 1 ℃.Cultivating system as shown in Figure 1.Cultivate initial, be fixed on sponge on the frosted glass carrier of 4 * 4cm with line, put into the 5L glass jar that fills seawater for totally 15, then with the glass jar arm in temperature is about 20 ± 2 ℃ waters, air pump and gas distributor bubbling air, dissolved oxygen meter is surveyed dissolved oxygen in the water body, the size of regulating the bubbling air flow makes dissolved oxygen concentration in the water in 7 ± 0.5mg/L scope, the glass jar outside covers with brown paper and prevents that sunlight from shining directly on the cavernous body, change water l00% every day, change and will store up the corvee seawater before the water earlier and be adjusted to sponge cultivate identical temperature in the waters, change throw something and feed behind the water Nitzschia closterium minutissima and the equilateral chrysophyceae of ball as food, concentration respectively is 2 * 10
5/ ml.In the culture period,, cultivate sponge weight in wet base canopy and stop experiment being in plateau every the weight in wet base of 2 days weighing sponges and carrier.With 5 α-Cholestan-3 beta-ol as the targeted activity material in the film cavernous body in great numbers, after cultivating end, the content of targeted activity material when testing initial and end with GC-MS logotype method detection cavernous body is inherent, the GC-MS spectrogram of 5 α in the sponge extract-Cholestan-3 beta-ol as shown in Figure 6, according to 5 α-Cholestan-3 beta-ol content in peak area and calibration curve (Fig. 7) calculation sample.The result, in the incubation, it is more bright-coloured when initial that the sponge color becomes, sponge has bigger attaching and sprawls on carrier, change has taken place in form, has become from original smooth surface to have a lot of full long projection (sprout) shapes (as shown in Figure 3), and this sprout endogenous cells content is abundant than other positions of sponge, be the good material of sponge cell culture, the NH in the incubation in the water body
4 +Concentration is lower than 1mg/ (as shown in table 1) all the time, tests end in the 11st day, and survival rate reaches 100%, and the spongy biological amount rises to 140% (as shown in Figure 4) of original weight in wet base, increases average every day to surpass 4%.5 α-Cholestan-3 beta-ol content in the cavernous body also 6.244mg/g dry weight sponge before the experiment of mountain has risen to 10.478mg/g dry weight sponge (as table 3), is about experiment 1.5 times when initial.
Table 1 is cultivated the interior NH of different incubation time water bodys down for hydrostatic
4 +The concentration table;
| NH4(mg/L) | 0 | 1 | 3 | 5 | 7 | 9 |
| |
0.045 | 0.606 | 1.159 | 0.855 | 0.872 | 0.648 |
| |
0.045 | 0.655 | 1.174 | 0.815 | 0.741 | 0.621 |
Table 2 is cultivated the interior NH of different incubation time water bodys down for flowing water
4 +The concentration table;
| Incubation time (my god) | 4 | 6 | 8 | 10 | 12 | 14 | 16 |
| Ammonia nitrogen concentration (mg/L) in the flowing water water body | 0.264 | 0.587 | 0.511 | 0.313 | 0.293 | 0.398 | 0.382 |
Table 3 is cultivated interior 5 α of cavernous body-Cholestan-3 beta-ol content table down for hydrostatic;
| Experimental point | Test initial (0day) | Experiment finishes (11day) |
| Peak area | 5410 | 11033.5 |
| 5 α-Cholestan-3 beta-ol content (mg/g dry weight sponge) | 6.244 | 10.278 |
5L stream adds system and cultivates.Film sponge in great numbers (Hymeniacidon perleve) is collected in DaLian, China Ling Shui bridge intertidal zone in July, 2005, takes back the laboratory and is placed on temporary supporting 2 days in the 12L glass jar of containing seawater, and temperature is controlled at 18-20 ℃.Cultivating system as shown in Figure 2.Cultivate initial, be fixed on sponge on the circular frosted glass carrier of the about 5cm of diameter with line, put into the 5L liquid that fills seawater and rise incubator for totally 15, discharge is 20L/day, the control of incubator coated outside cooling tube is cultivated water temperature at 20 ± 1 ℃, and air pump and gas distributor provide sponge required oxygen, and whole system places indoor no sunlit place, throw something and feed Nitzschia closterium minutissima, the equilateral chrysophyceae of ball as food, and concentration respectively is 2 * 10
5/ ml, the NH in the water body is cultivated in control in the incubation
4 +Concentration is no more than 1mg/L.In the culture period, measured the spongy biological amount every 2 days, the sponge apparent condition stops experiment when decay occurring.With 5 α-Cholestan-3 beta-ol as the targeted activity material in the film cavernous body in great numbers, the content of targeted activity material when initial and end are tested with the method detection cavernous body inherence of GC-MS logotype in cultivation end back, the result, in the incubation, it is more bright-coloured when initial that the sponge color becomes, and sponge has bigger attaching on carrier sprawls, and change has taken place form, become from original smooth surface and to have had a lot of full long overshooting shapes, the interior NH of water body in the incubation
4 +Concentration is lower than 0.6mg/L (as shown in table 2) all the time, tests end in the 16th day, and the sponge survival rate is 87%.The spongy biological amount rises to 140% (as shown in Figure 5) of original weight in wet base, the increment of average every day has all surpassed 4%, 5 α-Cholestan-3 beta-ol content in the cavernous body has also risen to 10.733mg/g dry weight sponge (as table 4) by the 6.721mg/g dry weight sponge before the experiment, is about experiment 1.5 times when initial.
Table 4 is cultivated interior 5 α of cavernous body-Cholestan-3 beta-ol content volumeization table down for flowing water;
| Experimental point | Test initial (0day) | Experiment finishes (11day) |
| Peak area | 7499.5 | 16412.3 |
| 5 α-Cholestan-3 beta-ol content (mg/g dry weight sponge) | 6.721 | 10.733 |
Claims (2)
1. one kind is used for the intertidal zone marine sponge laboratory artificial cultivating method, and lower animal-sponge that the suitable growth environment is cultivated intertidal zone set growth is manually made in the laboratory; It is characterized in that: after sponge is gathered in the intertidal zone, at first remove sponge surface impurity and foul after taking back the laboratory, cut into fritter then, putting into incubator supports temporarily, be fixed on then on the carrier, put into incubator temperature control, lucifuge cultivation, the feed algae of throwing something and feeding is as main body food, attach situation, metamorphosis and biomass situation of change as evaluation index with sponge, cultivate the maximum biomass that can reach former generation sponge growth in 10-20 days;
Cutting process is: adopt aseptic operation cutter cutting sponge, suitable area size is 3-9cm
2The carrier size is 4-8 a times of sponge area; Sponge carrier is selected glass or plastic material, and sponge selects fine rule to fix.
2. in accordance with the method for claim 1, it is characterized in that: sponge will be supported 2-7 days after taking back the laboratory temporarily, and the wound that forms during to its cutting and collection tentatively heals, and temperature adapts when supporting the collection of temperature and seashore temporarily, and then be adjusted to cultivation temperature, 15-25 ℃ of sponge cultivation temperature gradually.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100471255A CN101099452B (en) | 2006-07-05 | 2006-07-05 | Laboratory artificial cultivating method for intertidal zone marine sponge |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100471255A CN101099452B (en) | 2006-07-05 | 2006-07-05 | Laboratory artificial cultivating method for intertidal zone marine sponge |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101099452A CN101099452A (en) | 2008-01-09 |
| CN101099452B true CN101099452B (en) | 2010-07-21 |
Family
ID=39034057
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006100471255A Expired - Fee Related CN101099452B (en) | 2006-07-05 | 2006-07-05 | Laboratory artificial cultivating method for intertidal zone marine sponge |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101099452B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071166B (en) * | 2009-11-20 | 2012-12-26 | 中国科学院大连化学物理研究所 | Method for tissue artificial culture of hymeniacidon perleve by using program for temperature regulation and control |
| CN102721434B (en) * | 2012-06-19 | 2014-12-10 | 哈尔滨理工大学 | Intelligent control testing system for researching intertidal benthos based on wireless network |
| CN102901800B (en) * | 2012-10-24 | 2014-07-09 | 厦门大学 | Automatic simulation device for intertidal zone environmental change |
| CN105010186B (en) * | 2015-07-15 | 2018-04-10 | 厦门大学 | The method of sponge young directional induction settlement and metamorphosis |
| CN105123561B (en) * | 2015-07-15 | 2018-12-04 | 厦门大学 | The method of spongia transplanting |
| CN111670846B (en) * | 2020-04-20 | 2021-10-15 | 厦门大学 | In-situ cultivation method for sponge larvae |
-
2006
- 2006-07-05 CN CN2006100471255A patent/CN101099452B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 张卫,薛凌云,付晚涛,张骁英,刘元令,刘淑芬,金美芳.繁茂膜海绵的实验室养殖.中国水产科学12 4.2005,12(4),430-436. |
| 张卫,薛凌云,付晚涛,张骁英,刘元令,刘淑芬,金美芳.繁茂膜海绵的实验室养殖.中国水产科学12 4.2005,12(4),430-436. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101099452A (en) | 2008-01-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104170618B (en) | Zero-emission no pollution high-efficiency agriculture ecological recycle system | |
| CN104430079B (en) | Vegetable, Oryza sativa L., flower planting support, with net cage set, the apparatus and method combined | |
| CN101099452B (en) | Laboratory artificial cultivating method for intertidal zone marine sponge | |
| CN211241202U (en) | Three-dimensional co-culture device for seawater-resistant vegetables and aquatic animals | |
| CN102356746A (en) | Technology for cultivating organic fruit and vegetable matrix | |
| CN101811776B (en) | Method for processing aquaculture wastewater containing antibiotics by utilizing plant floating bed | |
| CN102668831A (en) | Submerged plant breeding blanket and application thereof | |
| CN104126494A (en) | Zostera marina indoor long-term cultivation method and device | |
| CN108911153A (en) | A kind of ecological restoring method of eutrophication water | |
| CN107691077A (en) | A kind of potted plant method of keeping a full stand of seedings of mangrove | |
| CN102657071B (en) | Method for culturing phytoplankton with predominant chlorophyta | |
| CN101773072A (en) | Method for culturing isolated microspore of common head cabbage to obtain regeneration plant | |
| EP3340772B1 (en) | A system for cultivating aquatic plants and method thereof | |
| CN104823898A (en) | Sillago sihama fry mouth opening bait culturing, selecting and feeding method | |
| CN101715743A (en) | Method for culturing low-salt resistant juvenile stichopus | |
| CN107751050B (en) | Batched rhinogobio ventralis fry breeding method | |
| CN105961230A (en) | Cultivation and planting method for madrepore in natural sea area | |
| CN101613673A (en) | A kind of collection of enteromorpha linza spore and cultural method | |
| CN101322477B (en) | Method for artificially collecting and cultivating larva of intertidal belt sponge with embryo | |
| CN109169177A (en) | A kind of environment protection flower pot and its medium production method with fermented cow dung matrix | |
| CN201160430Y (en) | Intermediate cultivation apparatus for juvenile blue crab | |
| CN109430120A (en) | Mangrove area cultivates mudskipper method using marine culture wastewater | |
| CN103663699A (en) | Method for controlling eutrophic water body by selecting grass type lake native aquatic plant | |
| CN104872027B (en) | Facility and method for indoor large-scale cultivation of larvae and juveniles of Sillago sihama using prawn cultivation wastewater | |
| CN107176689A (en) | A kind of pond waters ecological purification system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100721 Termination date: 20130705 |