An adapted staining-destaining method to sort soft-bottom macrobenthos mixed with Posidonia oceanica fibers

92   Medit.  Mar.  Sci.,  14/1,  2013,  92-­94

An  adapted  staining-­destaining  method  to  sort  soft-­bottom  macrobenthos  mixed    

with  Posidonia  oceanica¿EHUV

A.  DONNAY1,2_{,  C.  PELAPRAT}2_{,  P.  LEJEUNE}2  _{and  S.  GOBERT}1

1_{MARE Centre, Laboratory of Oceanology, University of Liège, Bat. B6C, B-4000 Liège 1, Belgium} 2_{STARESO SA, Pointe Revellata, BP 33, F-20260 Calvi, France}

Corresponding  author:  [email protected]

Received:  3  February  2012;;  Accepted:  3  September  2012;;  Published  on  line:  26  February  2013

Abstract  

Sorting  of  soft-­bottom  macrobenthos  entangled  with  Posidonia  oceanica¿EHUVLVWLPHFRQVXPLQJDQGWHGLRXVEHFDXVHRI the  weak  color  contrast.  This  paper  describes  a  staining-­destaining  technique  that  produces  good  contrast  between  soft-­bottom   macrobenthos  and  Posidonia  oceanica¿EHUV7KHPHWKRGKDVEHHQWHVWHGRQ&RUVLFDQVDPSOHVLQROLJRWURSKLFDUHDVFKDUDFWHUL]HG E\VPDOOVL]HVRIWERWWRPPDFURIDXQD2XUWHFKQLTXHVDYHVVRUWLQJWLPHDQGLVVLPSOHWRLPSOHPHQW

Keywords:  Staining,  Destaining,  Macrobenthos,  Sorting,  Posidonia  oceanica¿EHUV6RIWERWWRP

Short  Communication Mediterranean  Marine  Science

,QGH[HGLQ:R6:HERI6FLHQFH,6,7KRPVRQDQG6&2386 The  journal  is  available  on  line  at  http://www.medit-­mar-­sc.net

Introduction

Study   of   soft-­bottom   macrobenthos   is   one   of   the   common  methods  used  in  the  assessment  of  coastal  wa-­ WHUTXDOLW\LQPDQ\SDUWVRIWKHZRUOGHJ/DEUXQH  et   al.,  2007;;  Dauvin  et  al.,  2007;;  Mangialajo  et  al.,  2007;;   0DVVp  7KH LPSOHPHQWDWLRQ RI ELRWLF LQGLFHV based  on  this  assemblage  requires  sample  processing.  In   the  Mediterranean  Sea,  the  sorting  is  the  most  time-­con-­ suming   step,   especially   when   Posidonia   oceanica / 'HOLOH¿EHUVFRPLQJIURPDGMDFHQWPHDGRZVDUH present  in  abundance.

Around   Corsica,   the   oligotrophic   seawater   condi-­ WLRQV DUH FKDUDFWHUL]HG E\ VPDOOVL]H VRIWERWWRP PDF-­ UREHQWKRVXQSXEOLVKHGGDWD0RUHRYHUEHFDXVHWKHP.   oceanica  meadow  here  is  well  developed,  dead  leaves  are   RIWHQH[SRUWHGDQGGHJUDGHGRXWVLGHWKHEHGHJ0DWHR 5RPHURDQGLWLVYHU\FRPPRQWR¿QGWKHVH VHDJUDVV¿EHUVPL[HGLQZLWKVDPSOHGVHGLPHQW

*HQHUDOO\ D ÀRWDWLRQ WHFKQLTXH LV XVHG WR VHSDUDWH the  sediment  and  the  lighter  fraction  including  the  dead   macrofauna.   In   our   case,   this   low-­density   fraction   also   includes   the   P.   oceanica ¿EHUV 7R IDFLOLWDWH VRUWLQJ D staining  technique  is  usually  used,  but  all  organic  matter,   LQFOXGLQJSODQW¿EHUVLV  colored.  Therefore,  it  would  be   DGYDQWDJHRXVWR¿QGDQDJHQWZLWKORZVWDLQLQJSRZHURQ P.  oceanica¿EHUV

The   main   objective   of   this   study   was   to   adapt   the   VWDLQLQJ PHWKRG WR IDFLOLWDWH WKH VRUWLQJ RI WKH ÀRDWLQJ fraction   composed   of   soft-­bottom   macrobenthos   and   P.  

oceanica ¿EHUV 0RUHRYHU DV WKH QDWXUDO SLJPHQWDWLRQ RIVRIWERWWRPDQLPDOVLVQHFHVVDU\IRULGHQWL¿FDWLRQWKH possibility  of  destaining  the  fauna  after  the  sorting  step   was  also  investigated.

Material  and  Methods

Sediment  samples  were  collected  along  the  Corsican   coast   at   different   sites   by   scuba   diving   with   an   Ekman   JUDE  Pð DQG WUHDWHG IROORZLQJ WKH ,62 6WDQGDUG ,62

(DFKVDPSOHZDVVHSDUDWHGE\WKHÀRWDWLRQWHFKQLTXH and  only  the  lighter  fraction  was  treated  by  staining.

Four  common  stains  were  tested:  rose  bengal,  meth-­ \OHQHEOXHOXJRODQGHRVLQ>DTXHRXVHRVLQDQG ODERUDWRU\HRVLQ%DVVXJJHVWHGE\:LOOLDPV@

Stains  were  individually  tested  to  achieve  optimum   contrast  between  soft-­bottom  macrobenthos  and  P.  oce-­ anica¿EHUV:KHQWKHEHVWVWDLQZLWKWKHPD[LPXPFRQ-­ WUDVWREVHUYHGZDVGHWHUPLQHGWKHYROXPHRIWKLVVWDLQ POPORUPOSHUJRIPL[WXUHZDVUH¿QHG:KHQ the  best  stain  and  volume  were  found,  different  staining   WLPHVDQGPLQZHUHWHVWHGWRGHWHUPLQH which  time  resulted  in  the  greatest  contrast  between  ani-­ mals  and  P.  oceanica¿EHUV

The  time  saved  using  the  best  stain  was  determined   by   comparing   the   sorting   time   between   two   conditions   XQFRORUHG   macrobenthos   in   a   black   background   basin   FODVVLFDOPHWKRGvs.  colored  macrobenthos  in  a  white  

Medit.  Mar.  Sci.,  14/1,  2013,  92-­94   93

EDFNJURXQG EDVLQ DGDSWHG PHWKRG 7LPLQJV RI WZR sorters,  one  a  beginner  and  one  an  expert,  were  compared.

7ZR GHVWDLQLQJ VROXWLRQV ZHUH WHVWHG  HWKD-­ QRO:LOOLDPV,  DQGDQDONDOLQHVROXWLRQS+  7KLHO7KUHHFRPELQDWLRQVRIGHVWDLQLQJVROXWLRQ were  tested:  ethanol  alone,  alkaline  solution  alone,  and   DQHWKDQRODONDOLQHVROXWLRQPL[HGEDWK7KHRSWL-­ mum  conditions  were  determined  and  then  repeated  sev-­ eral  times  to  verify  performance.

Results

2QO\ HRVLQ VROXWLRQV ZHUH HIIHFWLYH DW LPSURYLQJ the   contrast   between   soft-­bottom   macrobenthos   and   P.   oceanica¿EHUV,QIDFWWKHWZRYDULDQWVRIHRVLQWHVWHG ERWKKDYHDJRRGDI¿QLW\IRUPDFUREHQWKRVDQGZHUHQRW SHUPDQHQWO\¿[HGRQP.  oceanica¿EHUV7DEOH6XE-­ VHTXHQWO\RQO\DTXHRXVHRVLQZKLFKLVFKHDSHUDQG easier  to  obtain,  was  used  for  the  next  steps  of  this  study.

$YROXPHRIPORIHRVLQSHUJRIPL[WXUHJDYH WKHEHVWGLVWLQFWLRQEHWZHHQDQLPDOVDQGSODQW¿EHUV

A  staining  time  of  15  min  resulted  in  good  contrast   DIWHUWKHFOHDQLQJDQGGLVFKDUJLQJVWHSV7DEOH

8VLQJ WKLV SURFHVV ERWK VRUWHUV ZHUH VLJQL¿FDQWO\ faster  when  sorting  a  colored  soft-­bottom  macrobenthos-­ P.  oceanica¿EHUVPL[WKHEHJLQQHUVDYHGRIWLPH DQG WKH H[SHUW VDYHG  ZKHQ WKH\ XVHG WKH DGDSWHG method  vs.WKHFODVVLFDOPHWKRGS”7DEOH

When  the  staining  was  done  immediately  before  sort-­ LQJWKHGHVWDLQLQJFRQVLVWHGRIRQHEDWKZLWKHWKD-­ nol.   When   staining   was   completed   some   hours   before   use,  the  destaining  consisted  of  two  baths  using  alkaline   solution   7DEOH  ,Q WKH ODWWHU FDVH HDFK EDWK ZDV  PLQLQGXUDWLRQWKH¿UVWRQHZLWKRQO\DONDOLQHVROXWLRQ WKHVHFRQGZLWKWKHDONDOLQHVROXWLRQDQGHWKDQROLQ a  1:1  mix.  Finally,  in  all  cases,  organisms  were  stored  in   HWKDQRO

An  outline  of  the  method  is  shown  in  Table  5.

Conclusions

This  paper  describes  an  adapted  technique  to  facili-­ tate  the  sorting  of  macrobenthos  entangled  with  P.  ocean-­ ica¿EHUV7KHRSWLPXPUHVXOWVZHUHREWDLQHGE\VWDLQLQJ WKHÀRWDWLRQSDUWRIDVDPSOHZLWKHRVLQVROXWLRQLQ DSURSRUWLRQRIPORIVROXWLRQSHUJRIPL[WXUHIRU a  coloration  time  of  15  min  followed  by  a  cleaning-­dis-­ charging  step.  This  technique  allows  a  mean  time-­saving   LQWKHVHSDUDWLRQSURFHVVRI7KHGHVWDLQLQJPHWK-­ RGXVLQJHWKDQROVROXWLRQRUDQDONDOLQHVROXWLRQ helps  to  preserve  the  natural  pigmentation  of  organisms   after  a  maximum  time  of  1  h.

Table 1:6WDLQLQJRIVRIWERWWRPPDFUREHQWKRVSODQWÀEHUV Tested  stain Coloration  effect

Rose  bengal $OO¿EHUVDQLPDOVDQGSODQW¿EHUV Methylene  blue $OO¿EHUVDQLPDOVDQGSODQW¿EHUV

/XJRO None  

$TXHRXVHRVLQ Animal  only

(RVLQ% Animal  only

Table 2: Staining time (min) of eosin and observed effects on soft-bottom macrobenthos and on P. oceanicaÀEHUV Time  (min)   Effects  on  soft-­bottom  macrobenthos Effects  on  P.  oceanica¿EHUV

5 Few  organisms,  weakly  colored No  color

10 Weakly  colored Very  weakly  colored

15 Well  colored Weakly  colored,  rapidly  disgorged

30 Well  colored Colored  and  destained  after  2  h

Table 3: Total number (n) of basins sorted, time (min) to sort 25g of sample (uncolored = uncolored macrobenthos sorted on

black background (classical method); colored = colored macrobenthos sorted on white background (adapted method)) and time saving (%).

Total  (n) Uncolored  time  (min) Colored  time  (min) Time  saving  (%) p-­value

Beginner 66 PLQV“VQ  PLQV“VQ  22 p  ”0.05

Expert 71 PLQV“VQ  PLQV“VQ  26 p  ”0.05

Table 4: Method used and observed effect of tested destaining agent.

Destaining  agent Use Effect

(WKDQRO Directly  after  staining *RRGDUWL¿FLDOGHVWDLQ Alkaline  solution A  long  time  after  staining After  two  baths:  good  destain

   Medit.  Mar.  Sci.,  14/1,  2013,  92-­94 Acknowledgements

:HWKDQN/XLVD5RGHIRUKHUKHOSGXULQJWKHH[SHUL-­ PHQW6\OYDLQ3OD]DDQG6WHSKHQ0DXURQIRUWKHLUKHOS during  the  sampling  process,  and  anonymous  referees  for   their  comments.  We  also  thank  Kelly  Stiver  from  Ecolo-­ J\DQG(YROXWLRQDU\%LRORJ\<DOH8QLYHUVLW\DQG,6/9 (GLWLQJ DQG 7UDQVODWLRQ 6HUYLFHV IURP 8QLYHUVLW\ RI /LqJHIRUWKHODQJXDJHUHYLHZLQJ7KLVVWXG\ZDVIXQGHG E\ WKH )UHQFK &RPPXQLW\ RI %HOJLXP 5$&( SURMHFW $5&E\WKH)156)&) DQGWKH6WDUH&DS0HGUHVHDUFKSURMHFW$JHQFHGHO¶HDX 50&DQG&ROOHFWLYLWp7HUULWRULDOHGH&RUVH7KLVSXEOL-­ cation  is  a  MARE  publication  n°229.

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Table 5: Outline of proposed method. Step 1. &OHDQWKHVDPSOHZLWKVHDZDWHUDQGVHSDUDWHWKHKHDY\IURPÀRDWLQJIUDFWLRQVE\WKHÀRWDWLRQWHFKQLTXHUHSHDWLIQHFHVVDU\ 2. :HLJKWKHÀRDWLQJIUDFWLRQWRGHWHUPLQHWKHTXDQWLW\RIHRVLQWREHXVHG 3. &RORUWKHVDPSOHZLWKDQHRVLQVHDZDWHUPL[SURSRUWLRQPORIHRVLQSHUJRIPL[WXUHIRUH[DFWO\PLQ  &OHDQWKHÀRDWLQJIUDFWLRQZLWKVHDZDWHU 5. 3ODFHWKHVLHYHZLWKWKHÀRDWLQJIUDFWLRQLQDER[ZLWKVHDZDWHUIRUGHVWDLQLQJ 6. -XVWEHIRUHVRUWLQJULQVHWKHÀRDWLQJIUDFWLRQDJDLQWKHQSURFHHGWRVRUWLQJ 7. .HHSWKHH[WUDFWHGVRIWERWWRPPDFUREHQWKRVLQHWKDQRO