CN103651265B - A kind of microelectrode detects the method for coral larva calcification rate - Google Patents
A kind of microelectrode detects the method for coral larva calcification rate Download PDFInfo
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Abstract
The invention discloses a kind of method that microelectrode detects coral larva calcification rate.It first gathers adult coral, put into incubator, treat that coral discharges Metamorphore, collect Metamorphore, allow Metamorphore be attached on attachment, then Metamorphore is put into micro-bottle respirator that seawater is housed, carry out T1/T2h illumination/dark culturing, constantly measure the oxygen change of seawater with oxygen microelectrode simultaneously and measure the pH value change of seawater with pH microelectrode, and collecting data, not have the seawater of Metamorphore according to same process in contrast; Measure the initial basicity before illumination cultivation or before dark culturing in addition, described T1h and T2h is within the scope of 0 ~ 12h; According to the photosynthetic and respiration of the oxygen change calculations young, utilize carbonate eqrilibrium system model CO2SYS to calculate the photosynthetic pH change caused with respiration, calculate the illumination calcification rate of coral larva and dark calcification rate.
Description
Technical field:
The invention belongs to coral calcification research field, be specifically related to a kind of method that microelectrode detects coral larva calcification rate.
Background technology:
Coral reef and the relevant ecosystem have important ecological functions, and it is that many marine organisms provide the place of laying eggs, breed, perch and hiding harmful animal, have the function that protection coastline and Ecological sightseeing tourism etc. are important.Due to the pressure of climatic variation, socioeconomic development and mankind's activity, coral cover of living reduces rapidly, and coral reef bio-diversity declines.If can successfully obtain its coral larva and effectively monitor every physical signs of coral larva, identifying contributing to the principal element affecting coral larva growth, promoting the recovery of coral reef ecologic system.
Due to the collection of coral larva, the difficulty of cultivating and detecting, the research of coral larva is also relatively less.The basic physiological parameter of coral larva, as calcification rate detects mainly through drying the method for weighing, the young can be killed, and makes experiment not have sustainability.At present for coral larva, also there is no the undamaged detection method of live body.Although the researcher of China has researched and analysed the developmental state (Li Yuan superfine 2007) of beauty Acropora egg mother cell, and the process (Huang Jieying etc. 2011) of process and the larvae development of laying eggs to expansion rose coral and sturdy Acropora with trickle sem observation, the people such as Zhang Chenglong (2010) application buoyant force weighing method calculates the calcification rate method of adult coral, but the physical signs of the young is as the but nobody's research of photosynthesis and calcification.
Summary of the invention:
The object of this invention is to provide a kind of can fast detecting, the loss that obtains data in real time, decrease sample size in operating process, the microelectrode that improve detection efficiency detects the method for coral larva calcification rate, and this method has simple, cheap, accurate and efficient feature.
Microelectrode of the present invention detects the method for coral larva calcification rate, it is characterized in that, comprises the following steps:
A, collection adult coral, put into incubator, treat that coral discharges Metamorphore, collect Metamorphore, allow Metamorphore be attached on attachment, then Metamorphore is put into micro-bottle respirator that seawater is housed, carry out T1/T2h illumination/dark culturing, constantly measure the oxygen change of seawater with oxygen microelectrode simultaneously and measure the pH value change of seawater with pH microelectrode, and collecting data, not have the seawater of Metamorphore according to same process in contrast; Measure the initial basicity before illumination cultivation or before dark culturing in addition, described T1h and T2h is within the scope of 0 ~ 12h, can select according to specific needs;
B, photosynthetic and respiration according to the oxygen change calculations young, carbonate eqrilibrium system model CO2SYS is utilized to calculate the photosynthetic pH change caused with respiration, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, according to the hydrogen ion of actual measurement and the difference of oxygen concentration change, can calcification rate be calculated:
Specific formula for calculation:
Gross photosynthesis speed=(DO
lT0-DO
lT1h– DO
l contrasts change)/T
Dark respiratory rate=(DO
dT0-DO
dT2h– DO
d contrasts change)/T
DO
lT0the dissolved oxygen concentration before illumination cultivation, DO
lT1hthe dissolved oxygen concentration after illumination cultivation T1 hour, DO
l contrasts changebe (there is no the seawater of the young) in control sample illumination cultivation T1 hour dissolved oxygen concentration change, DO
dT0the dissolved oxygen concentration before dark culturing, DO
dT2hthe dissolved oxygen concentration after dark culturing T2 hour, DO
d contrasts changebe (there is no the seawater of the young) in control sample dark culturing T2 hour dissolved oxygen concentration change, T is incubation time;
According to Barnes(1983) calculate the formula of calcification rate:
Illumination calcification rate=(gross photosynthesis speed × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
Dark calcification rate=(dark respiratory rate × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
In formula, Q is carbon ratio example (being approximately 1:1), TA ' is that the initial basicity before illumination cultivation or before dark culturing (detects with drop method, TA ' before illumination cultivation is corresponding with calculating illumination calcification rate, TA ' before dark culturing is corresponding with the dark calcification rate of calculating), BA and HA is before illumination cultivation respectively or boric acid before dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software, belong to common practise, software for calculation can be:
http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA with HA before illumination cultivation is corresponding with calculating illumination calcification rate, BA with HA before dark culturing is corresponding with the dark calcification rate of calculating), BA ' and HA ' is that boric acid after illumination cultivation or after dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software respectively, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing is corresponding with the dark calcification rate of calculating with HA '), K and K ' is by formula:
AH and aH ' be respectively illumination cultivation initial time and illumination cultivation after hydrogen ion concentration (=10
-pH, detect with pH meter), k
1and k
2be the dissociation constant of carbonate respectively, T is incubation time;
Calculate the illumination calcification rate of coral larva and dark calcification rate thus.
The normal healthy coral of growth that described adult coral preferably gathers from seabed, gathers before the release young.
Compared with prior art, the present invention has the following advantages:
1, compare the detection method of traditional calcification rate, the present invention have detected the photosynthetic and respiration of coral larva alive under having illumination and not having illumination condition first, by calculating O in water body
2with the slight change of pH, calculate the photosynthesis of coral larva and calcification rate, it has simple, cheap, accurate and efficient feature, there is no people at home and carries out research in this respect in Coral Reef Region.
2, the present invention is by the method for microelectrode, significantly improves the photosynthetic and respiratory measuring accuracy of coral, has filled up the blank of this respect at home and abroad;
3, the present invention can fast detecting, obtain data in real time.Decrease the loss of sample size in operating process, improve detection efficiency.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1
The microelectrode of the present embodiment detects the method for coral larva calcification rate, and its concrete steps are as follows:
(1) in July, 2013, at 2 to 5m depth of water place, Coral Reefs of Luhuitou, Sanya, the cup-shaped coral of deer horn (Pocillopora damicornis) that is normal, healthy, that also do not discharge the young is grown from gathering, take back laboratory, then put into 15L culture vessel, treat that coral discharges the Metamorphore of ellipse or circle, collect Metamorphore, after Metamorphore sinks to the bottom, the young is allowed to be attached to plastic culture dish about 15 days;
(2) treat that the young is attached to plastic culture dish, take out culture dish, with knife blade, around the young, plastic culture dish is cut into 1cm
2fritter, note do not encounter the young.
(3) micro-bottle respirator 10%(v/v of 5ml) watery hydrochloric acid cleaning after, distilled water cleans 3 times;
(4) 1cm of the young will be attached with
2fritter put into micro-bottle respirator, pour into net filter (200 order) seawater.Test and be made up of three Duplicate Samples and a control sample not loading the young.In vitro illumination level is ~ 200 μm of ol/cm2/s.Sample light application time is 12 hours, and interlunation is 12 hours.
(5) the initial basicity of seawater before detecting illumination cultivation by titration or before dark culturing.Be placed on by micro-bottle respirator on the support of microelectrode Study system of UNISENSE company, oxygen microelectrode and pH microelectrode are inserted in the seawater of micro-bottle respirator and detect DO and pH, data are sent to computer in real time.
(6) according to the photosynthetic and respiration of the oxygen change calculations young, carbonate eqrilibrium system model CO2SYS is utilized to calculate the photosynthetic pH change caused with respiration, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, according to the hydrogen ion of actual measurement and the difference of oxygen concentration change, can calcification rate be calculated:
Specific formula for calculation:
Gross photosynthesis speed=(DO
lT0-DO
l12h– DO
l contrasts change)/T
Dark respiratory rate=(DO
dT0-DO
d12h– DO
d contrasts change)/T
DO
lT0the dissolved oxygen concentration before illumination cultivation, DO
l12hthe dissolved oxygen concentration of illumination cultivation after 12 hours, DO
l contrasts changethe dissolved oxygen concentration change that the illumination cultivation that (do not have the seawater of the young) in control sample is cultivated 12 hours, DO
dT0the dissolved oxygen concentration before dark culturing, DO
d12hthe dissolved oxygen concentration after dark culturing cultivates 12 hours, DO
d contrasts changebe the dark culturing dissolved oxygen concentration of 12 hours that (do not have the seawater of the young) in control sample change, T is incubation time;
According to Barnes(1983) calculate the formula of calcification rate:
Illumination calcification rate=(gross photosynthesis speed × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
Dark calcification rate=(dark respiratory rate × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
In formula, Q is carbon ratio example (being approximately 1:1), TA ' is that the initial basicity before illumination cultivation or before dark culturing (detects by titration, TA ' before illumination cultivation is corresponding with calculating illumination calcification rate, TA ' before dark culturing is corresponding with the dark calcification rate of calculating), BA and HA is before illumination cultivation respectively or boric acid before dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software, belong to common practise, software for calculation can be:
http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA with HA before illumination cultivation is corresponding with calculating illumination calcification rate, BA with HA before dark culturing is corresponding with the dark calcification rate of calculating), BA ' and HA ' is that boric acid after illumination cultivation or after dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software respectively, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing is corresponding with the dark calcification rate of calculating with HA '), K and K ' is by formula:
AH and aH ' be respectively illumination cultivation initial time and the hydrogen ion concentration (=10 of illumination cultivation after 12 hours
-pH, detect with pH meter), k
1and k
2be the dissociation constant of carbonate respectively, T is incubation time;
Calculate the illumination calcification rate of coral larva and dark calcification rate thus.
Therefore, to apply the photosynthesis of coral larva that micro-breathing electrode system detects be illumination calcification rate is 0.98 ± 0.2% μm of ol d
-1(mean value ± standard deviation), dark calcification rate is 0.52 ± 0.12% μm of ol d
-1(mean value ± standard deviation).
The conventional method of young calcification is: cultivate the young more than 2 days, detects young weight differential, calculates the recruitment of every day.Conventionally detect, the calcification rate of short-term cannot be detected, more cannot differentiate coral larva illumination and dark calcification rate.Conventionally detect, our the coral larva calcification rate of research is 0.85 ± 0.16% μm of ol d
-1(this includes illumination and dark calcification rate).Between the illumination and the calcification rate of dark of the present embodiment, illustrate that according to the method for microelectrode detection coral larva calcification rate of the present invention be accurately thus.
Embodiment 2
The microelectrode of the present embodiment detects the method for coral larva calcification rate, and its concrete steps are as follows:
(1) in April, 2013, at 2 to 5m depth of water place, Coral Reefs of Luhuitou, Sanya, Acropora (Acropora sp.) that is normal, healthy, that also do not discharge the young is grown from gathering, take back laboratory, then put into 15L culture vessel, treat that coral discharges the Metamorphore of ellipse or circle, collect Metamorphore, after Metamorphore sinks to the bottom, the young is allowed to be attached to plastic culture plate about 2 months;
(2) take out culture plate, with knife blade, the young is separated from bottom.
(3) micro-bottle respirator 10%(v/v of 5ml) watery hydrochloric acid cleaning after, distilled water cleans 3 times;
(4) young is put into micro-bottle respirator, pour net filter (200 order) seawater into.Test and be made up of three Duplicate Samples and a control sample not loading the young.In vitro illumination level is ~ 200 μm of ol/cm2/s.Sample light application time is 12 hours, and interlunation is 12 hours.
(5) micro-bottle respirator is placed on the support of microelectrode Study system of UNISENSE company, oxygen microelectrode and pH microelectrode are inserted in the seawater of micro-bottle respirator and detects DO and pH, data are sent to computer in real time, and the initial basicity of seawater before detecting illumination cultivation by titration or before dark culturing.
(6) according to the photosynthetic and respiration of the oxygen change calculations young, carbonate eqrilibrium system model CO2SYS is utilized to calculate the photosynthetic pH change caused with respiration, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, according to the hydrogen ion of actual measurement and the difference of oxygen concentration change, can calcification rate be calculated:
Specific formula for calculation:
Gross photosynthesis speed=(DO
lT0-DO
l12h– DO
l contrasts change)/T
Dark respiratory rate=(DO
dT0-DO
d12h– DO
d contrasts change)/T
DO
lT0the dissolved oxygen concentration before illumination cultivation, DO
l12hthe dissolved oxygen concentration of illumination cultivation after 12 hours, DO
l contrasts changethe dissolved oxygen concentration change that the illumination cultivation that (do not have the seawater of the young) in control sample is cultivated 12 hours, DO
dT0the dissolved oxygen concentration before dark culturing, DO
d12hthe dissolved oxygen concentration after dark culturing cultivates 12 hours, DO
d contrasts changebe the dark culturing dissolved oxygen concentration of 12 hours that (do not have the seawater of the young) in control sample change, T is incubation time;
According to Barnes(1983) calculate the formula of calcification rate:
Illumination calcification rate=(gross photosynthesis speed × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
Dark calcification rate=(dark respiratory rate × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T)
In formula, Q is carbon ratio example (being approximately 1:1), TA ' is that the initial basicity before illumination cultivation or before dark culturing (detects with drop method, TA ' before illumination cultivation is corresponding with calculating illumination calcification rate, TA ' before dark culturing is corresponding with the dark calcification rate of calculating), BA and HA is before illumination cultivation respectively or boric acid before dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software, belong to common practise, software for calculation can be:
http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA with HA before illumination cultivation is corresponding with calculating illumination calcification rate, BA with HA before dark culturing is corresponding with the dark calcification rate of calculating), BA ' and HA ' is that boric acid after illumination cultivation or after dark culturing and hydroxide ion basicity (calculate gained by salinity, temperature and pH CO2SYS software respectively, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing is corresponding with the dark calcification rate of calculating with HA '), K and K ' is by formula:
AH and aH ' be respectively illumination cultivation initial time and the hydrogen ion concentration (=10 of illumination cultivation after 12 hours
-pH, detect with pH meter), k
1and k
2be the dissociation constant of carbonate respectively, T is incubation time;
Calculate the illumination calcification rate of coral larva and dark calcification rate thus.
Therefore, to apply the photosynthesis of coral larva that micro-breathing electrode system detects be illumination calcification rate is 0.79 ± 0.1% μm of ol d
-1(mean value ± standard deviation), dark calcification rate is 0.68 ± 0.14% μm of ol d
-1(mean value ± standard deviation).
Conventionally detect, our the coral larva calcification rate of research is 0.70 ± 0.1% μm of ol d
-1(this includes illumination and dark calcification rate).Between the illumination and the calcification rate of dark of the present embodiment, illustrate that according to the method for microelectrode detection coral larva calcification rate of the present invention be accurately thus.
Claims (2)
1. microelectrode detects a method for coral larva calcification rate, it is characterized in that, comprises the following steps:
A, collection adult coral, put into incubator, treat that coral discharges Metamorphore, collect Metamorphore, allow Metamorphore be attached on plastic culture dish, treat that Metamorphore is attached to plastic culture dish, take out culture dish, with knife blade, around Metamorphore, plastic culture dish is cut into 1cm
2fritter, then will the 1cm of Metamorphore be attached with
2fritter put into micro-bottle respirator that seawater is housed, or gather adult coral, put into incubator, treat that coral discharges Metamorphore, collect Metamorphore, Metamorphore is allowed to be attached on attachment, again Metamorphore is separated the micro-bottle respirator put into and seawater is housed, carry out T1/T2 h light/dark culturing, constantly measure the oxygen change of seawater with oxygen microelectrode simultaneously and measure the pH value change of seawater with pH microelectrode, and collect data, not have the seawater of Metamorphore according to same process in contrast; Measure the initial basicity before illumination cultivation or before dark culturing in addition, described T1 hour and within T2 hour, be in 0 ~ 12 hours window;
B, according to the gross photosynthesis speed of the oxygen change calculations young and dark respiratory rate, the pH change utilizing carbonate eqrilibrium system model CO2SYS to calculate photosynthetic and respiration to cause:
Specific formula for calculation:
Gross photosynthesis speed=(DO
lT0-DO
lT1h– DO
l contrasts change)/T
Dark respiratory rate=(DO
dT0-DO
dT2h– DO
d contrasts change)/T
DO
lT0the dissolved oxygen concentration before illumination cultivation, DO
lT1hthe dissolved oxygen concentration after illumination cultivation T1 hour, DO
l contrasts changethe dissolved oxygen concentration change of in control sample illumination cultivation T1 hour, DO
dT0the dissolved oxygen concentration before dark culturing, DO
dT2hthe dissolved oxygen concentration after dark culturing T2 hour, DO
d contrasts changebe the dissolved oxygen concentration change of in control sample dark culturing T2 hour, T is incubation time;
Calculate the formula of calcification rate:
Illumination calcification rate=(gross photosynthesis speed × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T), in formula, Q is carbon ratio example, TA ' is the initial basicity before illumination cultivation, BA and HA is boric acid and hydroxide ion basicity before illumination cultivation respectively, and BA ' and HA ' is boric acid and hydroxide ion basicity after illumination cultivation respectively;
Dark calcification rate=(dark respiratory rate × Q+TA ' × (K-K ')-K × (BA+HA)+K ' × (BA '+HA '))/(2 × (K-0.5) × T), in formula, Q is carbon ratio example, TA ' is the initial basicity before dark culturing, BA and HA is boric acid before dark culturing and hydroxide ion basicity respectively, and BA ' and HA ' is boric acid after dark culturing and hydroxide ion basicity respectively;
K and K ' is by formula:
AH and aH ' be respectively illumination cultivation initial time and illumination cultivation after hydrogen ion concentration, k
1and k
2be the dissociation constant of carbonate respectively, T is incubation time;
Calculate the illumination calcification rate of coral larva and dark calcification rate thus.
2. microelectrode according to claim 1 detects the method for coral larva calcification rate, it is characterized in that, described adult coral is the normal healthy coral of growth gathered from seabed, gathers before release Metamorphore.
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| CN112931304B (en) * | 2021-01-11 | 2022-08-19 | 中国科学院南海海洋研究所 | Method for improving attachment of coral larvae |
Citations (5)
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|---|---|---|---|---|
| US4587021A (en) * | 1983-12-19 | 1986-05-06 | University Of South Alabama | Inhibition of the formation of inorganic or biological CaCO3 -containing deposits by a proteinaceous fraction obtained from CaCO3 -forming organisms |
| KR100571997B1 (en) * | 2005-03-03 | 2006-04-17 | 이화여자대학교 산학협력단 | Method of collecting and culturing larvae of korean soft corals |
| KR100574762B1 (en) * | 2005-03-03 | 2006-04-27 | 이화여자대학교 산학협력단 | Method of collecting and culturing larvae of korean soft corals |
| CN101769842A (en) * | 2009-12-31 | 2010-07-07 | 中国科学院南海海洋研究所 | Weighing device and weighing method for coral buoyance suitable for field application |
| CN101806693A (en) * | 2010-03-16 | 2010-08-18 | 中国科学院南海海洋研究所 | Method for monitoring growth and calcification rate of hermatypic coral in natural sea area |
-
2013
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4587021A (en) * | 1983-12-19 | 1986-05-06 | University Of South Alabama | Inhibition of the formation of inorganic or biological CaCO3 -containing deposits by a proteinaceous fraction obtained from CaCO3 -forming organisms |
| KR100571997B1 (en) * | 2005-03-03 | 2006-04-17 | 이화여자대학교 산학협력단 | Method of collecting and culturing larvae of korean soft corals |
| KR100574762B1 (en) * | 2005-03-03 | 2006-04-27 | 이화여자대학교 산학협력단 | Method of collecting and culturing larvae of korean soft corals |
| CN101769842A (en) * | 2009-12-31 | 2010-07-07 | 中国科学院南海海洋研究所 | Weighing device and weighing method for coral buoyance suitable for field application |
| CN101806693A (en) * | 2010-03-16 | 2010-08-18 | 中国科学院南海海洋研究所 | Method for monitoring growth and calcification rate of hermatypic coral in natural sea area |
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