HELGOL,~NDER MEERESUNTERSUCHUNGEN Helgol~nder Meeresunters. 49, 797-804 (1995)
P h e r o m o n e s in marine algae: a technical approach
G. G a s s m a n n 1, D. G. M i i l l e r 2 & P. Fritz 3
1 Biologische Anstalt Helgoland, Zentrale Hamburg; Notkestrai3e 31, D-22607 Hamburg, Germany
2 Universit~t Konstanz, Fakult~t ffir Biologie; Postfach 5550, D-78434 Konstanz, Germany
3 Normag, Labor- und Verfahrenstechnik GmbH; Peldstrafle 1, Postfach I259, D-65719 Hofheim am Taunus, Germany
ABSTRACT: It is now well known that many marine organisms use low-molecular volatile substan- ces as signals, in order to coordinate activities between different individuals. The study of such pheromones requires the isolation and enrichment of the secretions from undisturbed living cells or organisms over extended periods of time. The Grob-Hersch extraction device, which we describe here, avoids adverse factors for the biological materials such as strong water currents, rising gas bubbles or chemical solvents. Furthermore, the formation of sea-water spray is greatly reduced. The application of this technique for the isolation of pheromones of marine algae and animals is described.
I N T R O D U C T I O N
T h e first t h r e e sexual p h e r o m o n e s of m a r i n e a l g a e (ectocarpene: Mfiller, 1967; Mfiller et al., 1971; f u c o s e r r a t e n e : Miiller & J a e n i c k e , 1973, J a e n i c k e & Seferiadis, 1975; multifidene: Miiller, 1974; J a e n i c k e et al., 1974) w e r e isolated from s e a w a t e r c o n t a i n i n g f e m a l e g a m e t e s by a p p l y i n g a simple C O 2 - c o o l e d trap. This kind of p u r g e - a n d - t r a p p r o c e d u r e is p r o n e to a lot of d r a w b a c k s : l a r g e a m o u n t s of biological m a t e r i a l s are n e e d e d , w h i c h is v e r y t i m e - c o n s u m i n g ; the e n r i c h m e n t factor is v e r y low; the risk of losing the collected, volatile s u b s t a n c e s is high; the s e p a r a t i o n of the e x c r e t i o n p r o d u c t from the w a t e r p h a s e is i n c o m p l e t e , a n d the c r o s s - c o n t a m i n a t i o n from outside is d e s t r u c - tive. B e t w e e n 1973-1975 the Grob family, a n d Grob & Zfircher (1976), i n t r o d u c e d an e l e g a n t p u r g e - a n d - t r a p p r o c e d u r e d e s i g n e d as a closed-loop system. This extraction t e c h n i q u e avoids all the d r a w b a c k s of a cold trap. N e v e r t h e l e s s , one single d i s a d v a n t a g e c o m m o n to all p u r g e - a n d - t r a p p r o c e d u r e s r e m a i n e d : the spray formed b y rising and b u r s t i n g gas b u b b l e s in solute-rich a q u e o u s m e d i a like seawater. E a c h rising and bursting gas b u b b l e creates s e v e r a l film a n d jet droplets, w h i c h quickly form s e a - s a l t crystals w h e n d r y i n g (Blanchard & Syzdek, 1975; Wu. 19801. An i n c o m p l e t e s e p a r a t i o n of the p h a s e s a n d a fast c l o g g i n g of the c h a r c o a l filter trap are the c o n s e q u e n c e s of this p h e n o m e n o n .
798 G. G a s s m a n n , D. G. Miiller & P. Fritz
oxygen, b u t also completely avoids the jet-droplet formation by b u r s t i n g b u b b l e s . A l t h o u g h it constituted significant progress, this work r e m a i n e d largely u n n o t i c e d by the scientific community. T h e c o m b i n a t i o n of both G r o b ' s a n d Hersch's p r i n c i p l e s leads to a n extraction device which is especially suitable for i n v e s t i g a t i o n s in s e a w a t e r .
MATERIALS AND M E T H O D S
The p r o c e d u r e for the a c c u m u l a t i o n of female g a m e t e s from field material or cultured algae are described in various reports by Miiller et al. b e t w e e n 1971-1988. T h e t e c h n i q u e for bio-assays of n a t u r a l a n d synthetic p h e r o m o n e s have b e e n d e s c r i b e d b y Mfiller (1976).
A 2-din 3 Hersch vessel e q u i p p e d with a cooling jacket a n d a n all-glass h e a t a b l e filter holder (cf. Boland et al., 1984) was m a n u f a c t u r e d by N o r m a g (Hofheim, G e r m a n y ) . T h e adsorptive filter trap, loaded with 1.5 m g of activated charcoal a n d p a c k e d as a n a r r o w slice b e t w e e n stainless steel sieves, was p u r c h a s e d from BrechbLihler (Schlieren, Switzer- land). A n inert (stainless steel a n d graphite) m a g n e t i c a l l y - c l u t c h e d g a s - c i r c u l a t i n g micro- p u m p (G 24/02; 160 g weight) was o b t a i n e d from H. Brey ( M e m m i n g e n , G e r m a n y ) .
A g e n e r a l description of the closed-loop stripping process a n d a d e t a i l e d report of the extraction p r o c e d u r e of the charcoal filter trap with organic solvents were g i v e n b y Grob (1973), Grob & Grob (1974), Grob et al. (1975), Grob & Ztircher (1976), a n d C o o n e y et al. (1983), In our work, the charcoal filters w e r e e l u t e d with 30-~1 d i - c h l o r o - m e t h a n e .
G a s - c h r o m a t o g r a p h i c separations a n d detections of algal extracts w e r e carried out on Carlo Erba gas chromatographs (Type GC 2900) e q u i p p e d with d u a l o n - c o l u m n injectors a n d flame-ionisation detectors (FID). Each extract was s e p a r a t e d on fused silica capillary c o l u m n s coated with stationary phases of different polarities ( C a r b o w a x 20M or OV101). H y d r o g e n (30-50kPa) was u s e d as carrier gas. We used the following t e m p e r a - ture p r o g r a m m e : 25 ~ for 5 rain isothermal. 5 ~ final t e m p e r a t u r e 220 ~ Signals were recorded by electronic integrators (Spectra-Physics [SP-4100 a n d SP-4200]).
Purified c o m p o u n d s from algal extracts were o b t a i n e d by the following t e c h n i q u e : after each a n a l y t i c a l g a s - c h r o m a t o g r a p h i c r u n of a n algal extract, the e l e c t r o d e - h e a d of the FID was removed, a n d the flame on the FID-tip e x t i n g u i s h e d . T h e e n d of the analytical c o l u m n was p r e p a r e d for c o n n e c t i o n to a n N - s h a p e d micro-trap m a d e of a 100- r a m - l o n g glass capillary tube filled with silica-gel particles. T h e trap w a s cooled with sohd CO2 a n d c o n n e c t e d to the c o l u m n w h e n e v e r a p e a k of interest p a s s e d a selected r e t e n t i o n time w i n d o w 10 to 20 s e p a r a t e p e a k s could be selectively t r a p p e d b y this method. Each trap l o a d e d with a single p e a k w a s i n s t a n t l y s e a l e d at its e n d s with a micro- b u r n e r (propane/oxygen), a n d stored in a fridge until some of the silica-gel (Spherosil) particles w e r e r e m o v e d a n d u s e d as artificial p h e r o m o n e sources i n a bio-assay.
P h e r o m o n e s in m a r i n e algae 799
RESULTS
T h e closed-loop stripping (CLS) device according to Grob is shown in Figure 1A. The construction principle is simple. A bottle, partially filled with the a q u e o u s sample, is e q u i p p e d with stripping utilities consisting of a gas p u m p (P), a n adsorptive filter (F), a n d a gas dispersion t u b e inserted deep d o w n into the a q u e o u s phase a n d e n d i n g with a
L
/
P
A
m
8
Fig. I. Operational scheme of a closed-loop purge-and-trap device according to Grob (A) and to Grob-Hersch (B)
800 G. G a s s m a n n , D. G. Mfiller & P. Fritz
C o m p a c t - K a i t e t h e r m o s t a t
L t [::,
[- ] q
. . . ; , , . _ , . j
GROB'SChes A k t i v k o h l e - F ~ ( t e f
Fig. 2. Diagram of a Grob-Hersch extraction device with a 2-din 3 Hersch vessel {GH-CLS AusblasgefaB) with cooling jacket, a heatable all-glass filter holder qPr~zisionsfilterhalter), a gas- circulating pump (Saug-Druckpumpe), a filter with a 1.5 mg active charcoal packing (Grob'sche- Aktivkohle-Filter), power supplies {Labor-Netzger~t), and a cryostate {Compact-K~iltethermostat)
Plowrn~
r n e s S U n g
P h e r o m o n e s in m a r i n e a l g a e 801
t h e s e c o n d i t i o n s t h e s t r i p p i n g e f f i c i e n c y falls b a c k to t h e n o r m a I l e v e l . This e f f e c t c a n b e e a s i l y q u a n t i f i e d b y m e a s u r i n g t h e d e - o x y g e n a t i o n of w a t e r s t r i p p e d w i t h u l t r a - p u r e n i t r o g e n in a n o p e n H e r s c h v e s s e l . W h i l e t h e p r o p e r H e r s c h p r o c e s s ( o p e n u p p e r orifice) d i m i n i s h e s t h e o x y g e n l e v e l b e l o w t h e d e t e c t i o n limit of 0.1 ~tg k g -1 0 2 w i t h i n a f e w h o u r s , t h e s i m p l e G r o b - t y p e s t r i p p i n g c o n d i t i o n ( u p p e r orifice i m m e r s e d in t h e l i q u i d p h a s e ) r e a c h e s its l i m i t at a n 0 2 l e v e l of a b o u t 100 #g k g -1, e v e n a f t e r p r o l o n g e d s t r i p p i n g t i m e s ( G a s s m a n n , 1973).
In m a r i n e b i o l o g y , t h e G r o b - H e r s c h e x t r a c t i o n d e v i c e is i d e a l l y s u i t e d for t h e d e t e c t i o n of v o l a t i l e n a t u r a l p r o d u c t s l i k e a l g a I p h e r o m o n e s . F e m a l e g a m e t o p h y t e s , m a t u r e o o g o n i a or fertile e g g s c a n b e i n c u b a t e d i n s i d e a H e r s c h v e s s e l u n d e r o p t i m a l c o n d i t i o n s . Salinity, t e m p e r a t u r e , n u t r i e n t s a n d i l l u m i n a t i o n c a n b e a d m i n i s t e r e d a n d
i
i Laminaria
digitata ?
~,1 n- C15
88
i y
Fig. 3. Gas chromatogram of volatile excretions from the e g g s of Laminaria du'gitata (Phaeophyceae, Laminariales). 0.2 ~1 (of 30-~1 di-chloro-methane filter extract) were on-column injected onto a Supelcowax 10, fused silica capillary column (60 m • 0.32 mm x 0.25 ~m). Hydrogen as carrier gas (50 kPa). Temperature programme: 25 ~ (5 min) - 5 ~ - 220 ~ Detector: FID. Carlo-Erba gas
802
G. G a s s m a n n , D. G. Mfiller & P. Fritzcontrolled, while c o n t i n u o u s or i n t e r m i t t e n t extraction of volatile e x c r e t i o n p r o d u c t s t a k e s place.
F i g u r e 3 shows a gas c h r o m a t o g r a m of volatile e x c r e t i o n p r o d u c t s from m a t u r e
Lammaria digitata
e g g s i n c l u d i n g the s e x u a l p h e r o m o n e l a m o x i r e n e . M o s t of the p r o m i - n e n t p e a k s are h y d r o c a r b o n s w i t h a C l l c a r b o n s k e l e t o n a n d their e p o x i d e s . N - p e n - t a d e c a n e (n-C15) is p a r t of the u n s p e c i f i c b a c k g r o u n d , w h i c h is p r o d u c e d b y m a n y m a r i n e algae.1.
~
Ectocarpene1971
2.
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Fucoserratene
1973
3.
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Muttifidene1974
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1982
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1984
9.
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1985
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,~-,,,.~,,~__
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1985
11.
Caudoxirene
1988
0
P h e r o m o n e s in m a r i n e a l g a e 803 T h e G r o b - H e r s c h C L S d e v i c e w a s u s e d to e s t a b l i s h t h e o c c u r r e n c e of f u c o s e r r a t e n e as t h e s p e r m a t t r a c t a n t in s e v e r a l s p e c i e s of t h e g e n u s F u c u s (Mfiller & G a s s m a n n , 1978). S u b s e q u e n t l y , s e v e r a l n e w s e x u a l p h e r o m o n e s (Fig. 4) c o u l d b e d e t e c t e d a n d t h e i r s t r u c t u r e e l u c i d a t e d (Mfiller et al., 1979, 1981a, 1981b, 1982, 1984, 1985; M a r n e r et al., 1984; B o l a n d et al., 1985, 1987). By a p p l i c a t i o n of t h e s a m e t e c h n i q u e s , t h e s e x u a l p h e r o m o n e of t h e m a r i n e p o l y c h a e t e P l a t y n e r e i s d u m e r i l l i i c o u l d b e e n r i c h e d a n d i d e n t i f i e d as a k e t o n e w i t h a Cs c a r b o n - s k e l e t o n ( 5 - m e t h y l - 3 - h e p t a n o n e ) ( Z e e c k et al., 1988).
C O N C L U S I O N
T h e s u c c e s s f u l a p p l i c a t i o n of t h e G r o b - H e r s c h C L S to t h e s t u d y of a l g a l p h e r o m o n e s i l l u s t r a t e s t h a t this t e c h n i q u e is e x c e l l e n t l y s u i t e d to s t u d y v o l a t i l e c o m p o u n d s in s a l i n e m e d i a . Its a p p l i c a t i o n c a n b e e x p a n d e d to t h e e x t r a c t i o n of m o d e r a t e l y p o l a r s u b s t a n c e s b y t h e a d d i t i o n of salt (such as Na2SO4) to a s o l u t e - p o o r s o l u t i o n (Friant & Suffet, 1979). S u c h t r e a t m e n t s c a n shift t h e v a p o u r - p h a s e p a r t i t i o n i n g into t h e d e s i r e d d i r e c t i o n . T h i s t e c h n i q u e c a n e x t e n d t h e r a n g e of t h e e x t r a c t i o n p a t t e r n , w h i c h n o r m a l l y e n d s at t h e b o i l i n g p o i n t of o c t a d e c a n e (C18H38; b.p. 316.1 ~ to i n c l u d e t h e b o i l i n g - p o i n t r a n g e f r o m d o c o s a n e
(C22H46;
b.p. 368.6 ~ to t r i c o s a n e (C23H48; b.p. 380.2 ~ G a s s m a n n , 1993, u n p u b l . ) .Lately, w e h a v e b e e n u s i n g t h e H e r s c h v e s s e l for s t u d i e s of t h e p h o s p h i n e b i o s y n - t h e s i s in t h e h y d r o - a n d b i o s p h e r e . In this c o n t e x t , t h e H e r s c h v e s s e i p r o d u c e s o x y g e n - f r e e s o d i u m - h y d r o x i d e solution, w h i c h is e s s e n t i a l for t h e d e t e c t i o n of p h o s p h i n e in PH3- b e a r i n g m a t r i c e s ( G a s s m a n n & S c h o r n , 1993; G a s s m a n n & G l i n d e m a n n , 1993).
A c k n o w l e d g e m e n t . The financial support provided for the second author, Dieter G. Mi~ller, by the Deutsche Forschungsgemeinschaft (DFG), is gratefully acknowledged.
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