CN103651265A - Method for detecting coral larva calcification rate by means of microelectrodes - Google Patents

Method for detecting coral larva calcification rate by means of microelectrodes Download PDF

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CN103651265A
CN103651265A CN201310590037.XA CN201310590037A CN103651265A CN 103651265 A CN103651265 A CN 103651265A CN 201310590037 A CN201310590037 A CN 201310590037A CN 103651265 A CN103651265 A CN 103651265A
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coral
illumination
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young
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CN103651265B (en
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袁翔城
李秀保
黄晖
袁涛
张浴阳
江雷
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South China Sea Institute of Oceanology of CAS
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a method for detecting the coral larva calcification rate by means of microelectrodes. The method comprises the steps of collecting adult corals, placing the collected adult corals in an incubator, collecting planulae released by the corals to enable the planulae to be attached to an attachment, placing the planulae into a micro respirator bottle filled with sea water, conducting illumination/darkness cultivation for T1/T2h and measuring the change of oxygen in the sea water continuously with an oxygen microelectrode and the change of the PH value of the sea water with a PH microelectrode at the same time, collecting data, comparing the data with data acquired from the same treatment on sea water not containing planulae, measuring the initial alkalinity before illumination/darkness cultivation, calculating photosynthesis and respiration of larvae according to the change of oxygen, calculating the change of the PH value caused by photosynthesis and respiration according to a carbonate balance system model CO2SYS, and obtaining the illumination calcification rate and darkness calcification rate of the coral larvae through calculation, wherein the T1h and T2h are between 0h and 12h.

Description

A kind of microelectrode detects the method for coral young calcification rate
Technical field:
The invention belongs to coral calcification research field, be specifically related to a kind of method that microelectrode detects coral young calcification rate.
Background technology:
Coral reef and the relevant ecosystem have important ecological functions, and it provides the place of laying eggs, breed, perching and hide harmful animal for many marine organisms, have the important functions such as protection coastline and Ecological sightseeing tourism.Due to the pressure of climatic variation, socioeconomic development and mankind's activity, the coral cover of living reduces rapidly, and coral reef bio-diversity declines.If can successfully obtain its coral young and every physical signs of the coral young is effectively monitored, by contributing to identification to affect the principal element of coral larval growth, promote the recovery of coral reef ecologic system.
Due to the collection of the coral young, the difficulty of cultivating and detecting, the research of the coral young is also relatively less.The basic physiological parameter of the coral young, the method for mainly weighing by oven dry as calcification rate detects, and the young can be killed, and makes experiment there is no sustainability.For the coral young, also there is no the undamaged detection method of live body at present.Although the researcher of China has researched and analysed the developmental state (Li Yuan superfine 2007) of beauty Acropora egg mother cell, and with trickle sem observation to the lay eggs process (Huang Jieying etc. 2011) of process and larvae development of expansion rose coral and sturdy Acropora, the people such as Zhang Chenglong (2010) application buoyant force weighing method has been calculated the calcification rate method of adult coral, but the physical signs of the young is as photosynthesis and but nobody's research of calcification.
Summary of the invention:
The object of this invention is to provide a kind of can fast detecting, obtain data in real time, reduced the loss of sample size in operating process, the microelectrode that has improved detection efficiency detects the method for coral young calcification rate, simple, cheap, accurate and efficient feature that this method has.
Microelectrode of the present invention detects the method for coral young 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, simultaneously constantly with the oxygen that oxygen microelectrode is measured seawater, change and measure with pH microelectrode the pH value variation of seawater, and collect data, with the seawater that there is no a Metamorphore according to same processing in contrast; Initial basicity before measuring in addition illumination cultivation or before dark culturing, described T1h and T2h are within the scope of 0～12h, can select according to specific needs;
B, according to the photosynthetic and respiration of the oxygen change calculations young, utilizing carbonate eqrilibrium system model CO2SYS to calculate pH photosynthetic and that respiration causes changes, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, difference according to the hydrogen ion of actual measurement and oxygen concentration variation, can calculate calcification rate:
Specific formula for calculation:
Gross photosynthesis speed=(DO lT0-DO lT1h– DO l contrasts variation)/T
Dark respiratory rate=(DO dT0-DO dT2h– DO d contrasts variation)/T
DO lT0the dissolved oxygen concentration before illumination cultivation, DO lT1hthe dissolved oxygen concentration after illumination cultivation T1 hour, DO l contrast changethat in control sample, (seawater that there is no the young) dissolved oxygen concentration of illumination cultivation T1 hour changes, DO dT0the dissolved oxygen concentration before dark culturing, DO dT2hthe dissolved oxygen concentration after dark culturing T2 hour, DO d contrasts variationbe that in control sample, (seawater that there is no the young) dissolved oxygen concentration of dark culturing T2 hour changes, 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 are respectively that boric acid and the hydroxide ion basicity before illumination cultivation or before dark culturing (is calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, software for calculation can be: http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA before illumination cultivation is corresponding with calculating illumination calcification rate with HA, BA before dark culturing is corresponding with the dark calcification rate of calculating with HA), BA ' and HA ' are respectively after illumination cultivation or the boric acid after dark culturing and hydroxide ion basicity (are calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing and HA ' with to calculate dark calcification rate corresponding), K and K ' are by formula:
K = aH · k 1 + · k 1 · k . 2 + a H 2 aH · k 1 + 2 · k 1 · k . 2
K ′ = a H ′ · k 1 + · k 1 · k . 2 + a H ′ 2 a H ′ · k 1 + 2 · k 1 · k . 2
AH and aH ' are respectively the hydrogen ion concentration (=10 of illumination cultivation when initial and after illumination cultivation -pH, with pH meter, detect), k 1and k 2be respectively the dissociation constant of carbonate, T is incubation time;
Calculate thus illumination calcification rate and the dark calcification rate of the coral young.
The normal healthy coral of growth that described adult coral preferably gathers from seabed gathers before discharging the young.
Compared with prior art, the present invention has the following advantages:
1, compare the detection method of traditional calcification rate, the present invention has detected the coral young alive first to be had illumination and there is no the photosynthetic and respiration under illumination condition, by calculating O in water body 2with the slight change of pH, calculate photosynthesis and the calcification rate of the coral young, it has simple, cheap, accurate and efficient feature, there is no at home people and in Coral Reef Region, carries out research in this respect.
2, the present invention, by the method for microelectrode, has significantly improved 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.Reduce the loss of sample size in operating process, improved detection efficiency.
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment 1
The microelectrode of the present embodiment detects the method for coral young calcification rate, and its concrete steps are as follows:
(1) in July, 2013,2 to the 5m depth of water places in Coral Reefs of Luhuitou, Sanya, from gathering the growth cup-shaped coral of deer horn normal, healthy, that also do not discharge the young (Pocillopora damicornis), take back laboratory, then put into 15L culture vessel, treat that coral discharges ellipse or circular Metamorphore, collects Metamorphore, after Metamorphore sinks to the bottom, allow the young 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 not encountering the young.
(3), after watery hydrochloric acid micro-bottle respirator 10%(v/v of 5ml) cleans, distilled water cleans 3 times;
(4) 1cm of the young will be attached with 2fritter put into micro-bottle respirator, pour net filter (200 order) seawater into.Experiment is comprised of three Duplicate Samples and a control sample that does not pack the young into.In vitro illumination level is～200 μ mol/cm2/s.Sample light application time is 12 hours, and be 12 hours interlunation.
(5) by the initial basicity of seawater before titration detection illumination cultivation or before dark culturing.Micro-bottle respirator is placed on the support of microelectrode Study system of UNISENSE company, oxygen microelectrode and pH microelectrode is 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, utilizing carbonate eqrilibrium system model CO2SYS to calculate pH photosynthetic and that respiration causes changes, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, difference according to the hydrogen ion of actual measurement and oxygen concentration variation, can calculate calcification rate:
Specific formula for calculation:
Gross photosynthesis speed=(DO lT0-DO l12h– DO l contrasts variation)/T
Dark respiratory rate=(DO dT0-DO d12h– DO d contrasts variation)/T
DO lT0the dissolved oxygen concentration before illumination cultivation, DO l12hthe dissolved oxygen concentration of illumination cultivation after 12 hours, DO l contrast changethat the dissolved oxygen concentration that in control sample, (seawater that there is no the young) illumination cultivation is cultivated 12 hours changes, DO dT0the dissolved oxygen concentration before dark culturing, DO d12hthat dark culturing is cultivated the dissolved oxygen concentration after 12 hours, DO d contrasts variationbe that in control sample, (seawater that there is no the young) the dark culturing dissolved oxygen concentration of 12 hours changes, 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 are respectively that boric acid and the hydroxide ion basicity before illumination cultivation or before dark culturing (is calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, software for calculation can be: http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA before illumination cultivation is corresponding with calculating illumination calcification rate with HA, BA before dark culturing is corresponding with the dark calcification rate of calculating with HA), BA ' and HA ' are respectively after illumination cultivation or the boric acid after dark culturing and hydroxide ion basicity (are calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing and HA ' with to calculate dark calcification rate corresponding), K and K ' are by formula:
K = aH · k 1 + · k 1 · k . 2 + a H 2 aH · k 1 + 2 · k 1 · k . 2
K ′ = a H ′ · k 1 + · k 1 · k . 2 + a H ′ 2 a H ′ · k 1 + 2 · k 1 · k . 2
AH and aH ' are respectively that illumination cultivation is when initial and the hydrogen ion concentration (=10 of illumination cultivation after 12 hours -pH, with pH meter, detect), k 1and k 2be respectively the dissociation constant of carbonate, T is incubation time;
Calculate thus illumination calcification rate and the dark calcification rate of the coral young.
Therefore the photosynthesis of, applying the coral young that micro-breathing electrode system detects is that illumination calcification rate is 0.98 ± 0.2% μ mol d -1(mean value ± standard deviation), dark calcification rate is 0.52 ± 0.12% μ mol d -1(mean value ± standard deviation).
The conventional method of young calcification is: cultivate the young more than 2 days, detect young weight differential, calculate the recruitment of every day.According to conventional method, detect, cannot detect the calcification rate of short-term, more cannot differentiate the illumination of the coral young and dark calcification rate.According to conventional method, detect, our the coral young calcification rate of research is 0.85 ± 0.16% μ mol d -1(this has comprised illumination and dark calcification rate).Between the illumination of the present embodiment and the calcification rate of dark, illustrate that thus according to the method for microelectrode detection coral young calcification rate of the present invention be accurately.
Embodiment 2
The microelectrode of the present embodiment detects the method for coral young calcification rate, and its concrete steps are as follows:
(1) in April, 2013,2 to the 5m depth of water places in Coral Reefs of Luhuitou, Sanya, from gathering growth Acropora normal, healthy, that also do not discharge the young (Acropora sp.), take back laboratory, then put into 15L culture vessel, treat that coral discharges ellipse or circular Metamorphore, collects Metamorphore, after Metamorphore sinks to the bottom, allow the young be attached to plastic culture plate about 2 months;
(2) take out culture plate, with knife blade, the young is separated from bottom.
(3), after watery hydrochloric acid micro-bottle respirator 10%(v/v of 5ml) cleans, distilled water cleans 3 times;
(4) young is put into micro-bottle respirator, pour net filter (200 order) seawater into.Experiment is comprised of three Duplicate Samples and a control sample that does not pack the young into.In vitro illumination level is～200 μ mol/cm2/s.Sample light application time is 12 hours, and be 12 hours interlunation.
(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 detect DO and pH, data are sent to computer in real time, and before detecting illumination cultivation by titration or the initial basicity of seawater before dark culturing.
(6) according to the photosynthetic and respiration of the oxygen change calculations young, utilizing carbonate eqrilibrium system model CO2SYS to calculate pH photosynthetic and that respiration causes changes, because the calcium ion of coral precipitation 1mol can discharge 2mol basicity, difference according to the hydrogen ion of actual measurement and oxygen concentration variation, can calculate calcification rate:
Specific formula for calculation:
Gross photosynthesis speed=(DO lT0-DO l12h– DO l contrasts variation)/T
Dark respiratory rate=(DO dT0-DO d12h– DO d contrasts variation)/T
DO lT0the dissolved oxygen concentration before illumination cultivation, DO l12hthe dissolved oxygen concentration of illumination cultivation after 12 hours, DO l contrast changethat the dissolved oxygen concentration that in control sample, (seawater that there is no the young) illumination cultivation is cultivated 12 hours changes, DO dT0the dissolved oxygen concentration before dark culturing, DO d12hthat dark culturing is cultivated the dissolved oxygen concentration after 12 hours, DO d contrasts variationbe that in control sample, (seawater that there is no the young) the dark culturing dissolved oxygen concentration of 12 hours changes, 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 are respectively that boric acid and the hydroxide ion basicity before illumination cultivation or before dark culturing (is calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, software for calculation can be: http:// cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS_v2.1/download, BA before illumination cultivation is corresponding with calculating illumination calcification rate with HA, BA before dark culturing is corresponding with the dark calcification rate of calculating with HA), BA ' and HA ' are respectively after illumination cultivation or the boric acid after dark culturing and hydroxide ion basicity (are calculated gained by salinity, temperature and pH with CO2SYS software, belong to common practise, BA ' after illumination cultivation is corresponding with calculating illumination calcification rate with HA ', BA ' after dark culturing and HA ' with to calculate dark calcification rate corresponding), K and K ' are by formula:
K ′ = a H ′ · k 1 + · k 1 · k . 2 + a H ′ 2 a H ′ · k 1 + 2 · k 1 · k . 2
K = aH · k 1 + · k 1 · k . 2 + a H 2 aH · k 1 + 2 · k 1 · k . 2
AH and aH ' are respectively that illumination cultivation is when initial and the hydrogen ion concentration (=10 of illumination cultivation after 12 hours -pH, with pH meter, detect), k 1and k 2be respectively the dissociation constant of carbonate, T is incubation time;
Calculate thus illumination calcification rate and the dark calcification rate of the coral young.
Therefore the photosynthesis of, applying the coral young that micro-breathing electrode system detects is that illumination calcification rate is 0.79 ± 0.1% μ mol d -1(mean value ± standard deviation), dark calcification rate is 0.68 ± 0.14% μ mol d -1(mean value ± standard deviation).
According to conventional method, detect, our the coral young calcification rate of research is 0.70 ± 0.1% μ mol d -1(this has comprised illumination and dark calcification rate).Between the illumination of the present embodiment and the calcification rate of dark, illustrate that thus according to the method for microelectrode detection coral young calcification rate of the present invention be accurately.

Claims (2)

1. microelectrode detects a method for coral young 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, simultaneously constantly with the oxygen that oxygen microelectrode is measured seawater, change and measure with pH microelectrode the pH value variation of seawater, and collect data, with the seawater that there is no a Metamorphore according to same processing in contrast; Initial basicity before measuring in addition illumination cultivation or before dark culturing, described T1h and T2h are within the scope of 0～12h;
B, according to the photosynthetic and respiration of the oxygen change calculations young, utilize carbonate eqrilibrium system model CO2SYS to calculate pH photosynthetic and that respiration causes and change:
Specific formula for calculation:
Gross photosynthesis speed=(DO lT0-DO lT1h– DO l contrasts variation)/T
Dark respiratory rate=(DO dT0-DO dT2h– DO d contrasts variation)/T
DO lT0the dissolved oxygen concentration before illumination cultivation, DO lT1hthe dissolved oxygen concentration after illumination cultivation T1 hour, DO l contrast changethat in control sample, the dissolved oxygen concentration of illumination cultivation T1 hour changes, DO dT0the dissolved oxygen concentration before dark culturing, DO dT2hthe dissolved oxygen concentration after dark culturing T2 hour, DO d contrasts variationbe that in control sample, the dissolved oxygen concentration of dark culturing T2 hour changes, 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)
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 illumination cultivation or before dark culturing, BA and HA are respectively boric acid and the hydroxide ion basicity before illumination cultivation or before dark culturing, BA ' and HA ' are respectively after illumination cultivation or the boric acid after dark culturing and hydroxide ion basicity, and K and K ' are by formula:
K = aH · k 1 + · k 1 · k . 2 + a H 2 aH · k 1 + 2 · k 1 · k . 2
K ′ = a H ′ · k 1 + · k 1 · k . 2 + a H ′ 2 a H ′ · k 1 + 2 · k 1 · k . 2
AH and aH ' are respectively the hydrogen ion concentration of illumination cultivation when initial and after illumination cultivation, k 1and k 2be respectively the dissociation constant of carbonate, T is incubation time;
Calculate thus illumination calcification rate and the dark calcification rate of the coral young.
2. microelectrode according to claim 1 detects the method for coral young calcification rate, it is characterized in that, described adult coral is the normal healthy coral of growth gathering from seabed, before discharging the young, gathers.
CN201310590037.XA 2013-11-20 2013-11-20 A kind of microelectrode detects the method for coral larva calcification rate Expired - Fee Related CN103651265B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105352999A (en) * 2015-10-12 2016-02-24 临沂大学 Ex-situ determination method for pH value of plant sap
CN111543360A (en) * 2020-05-20 2020-08-18 中国水产科学研究院黄海水产研究所 Method for measuring deposition rate of calcium carbonate for culturing shellfish and application
CN112931304A (en) * 2021-01-11 2021-06-11 中国科学院南海海洋研究所 Method for improving attachment of coral larvae

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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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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
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Publication number Priority date Publication date Assignee Title
CN105352999A (en) * 2015-10-12 2016-02-24 临沂大学 Ex-situ determination method for pH value of plant sap
CN111543360A (en) * 2020-05-20 2020-08-18 中国水产科学研究院黄海水产研究所 Method for measuring deposition rate of calcium carbonate for culturing shellfish and application
CN112931304A (en) * 2021-01-11 2021-06-11 中国科学院南海海洋研究所 Method for improving attachment of coral larvae
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

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