Hepoxilins and trioxilins in barnacles: an analysis of their potential roles in egg hatching and larval settlement
1 School of Biological Sciences, University of Wales Swansea, Singleton
Park, Swansea, SA2 8PP, UK
2 Proteomics Section, Imperial College, Faculty of Medicine, London, W12
ONN, UK
3 School of Marine Science and Technology, University of Newcastle upon
Tyne, Newcastle upon Tyne, NE1 7RU, UK
4 Program in Integrative Biology, Research Institute, The Hospital for Sick
Children, 555-University Avenue, Toronto and the Department of Pharmacology,
University of Toronto, Toronto, Ontario M5G 1X8, Canada
* Author for correspondence (e-mail: a.f.rowley{at}swansea.ac.uk)
Accepted 16 June 2003
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Summary |
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Key words: Elminius modestus, Balanus amphitrite, barnacle egg-hatching activity, larval settlement, hepoxilin, trioxilin, hepoxilin B3, stable analogue, PBT-3
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Introduction |
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The polyunsaturated fatty acid arachidonic acid [AA; 20:4(n-6)] is the
precursor of two major families of bioactive lipids, the prostaglandins (PGs),
arising through the action of cyclooxygenase(s) (COX), and a series of
lipoxygenase (LOX) products that include the leukotrienes and
(poly)hydroxylated eicosanoids (Stanley,
2000). Whereas AA is found mainly in mammalian phospholipids
(Kühn and Borngraber,
1998
), the equivalent predominant C20 polyunsaturated fatty acid
present in aquatic invertebrates is usually eicosapentaenoic acid [EPA;
20:5(n-3); Ackman, 1980
]. The
elongated polyunsaturated fatty acid docosahexaenoic acid [DHA; 22:6(n-3)] is
also present in many aquatic animals and acts as a substrate for LOX
(Holland et al., 1999
) and
aspirin-acetylated COX in terrestrial mammals
(Serhan et al., 2002
).
Within the barnacle life cycle there are two key stages where C20 lipids
(eicosanoids) and C22 fatty acid derivatives are thought to be involved in
signalling pathways that may influence barnacle development (see review by
Clare, 1995). Firstly, the LOX
products 8-hydroxy-5,9,11,14,17-eicosapentaenoic acid
(Hill and Holland, 1992
;
Hill et al., 1988
) and a range
of tri-hydroxy compounds (Hill et al.,
1993
; Song et al.,
1990a
) have been implicated as barnacle egg-hatching factors in
both Elminius modestus and Semibalanus balanoides. In vivo
studies, using extracted seawater in which barnacles (S. balanoides
and Chirona hameri) had previously liberated their larvae, found
trace levels of tri-hydroxy fatty acids, suggesting that these, rather than
the mono-hydroxy fatty acid compounds, were the biologically active compounds
(Song et al., 1990b
).
Secondly, Knight et al. (2000
)
demonstrated that the COX products, prostaglandins E2 and
E3, caused a dose-dependent inhibition of larval settlement in
Balanus amphitrite, while indomethacin, a selective COX inhibitor,
stimulated the settlement process.
Hepoxilins are epoxide metabolites of AA, EPA and DHA derived through the
LOX pathway (Pace-Asciak,
1986; Pace-Asciak et al.,
1983
; Reynaud and Pace-Asciak,
1997
; Fig. 1). In
mammals, hepoxilins act primarily on calcium and potassium channels in
membranes, which ultimately results in a wide range of functions such as
insulin secretion, vascular permeability and vasoconstriction within the aorta
and the activation of neutrophils and platelets (for reviews, see Pace-Asciak,
1993
,
1994
;
Pace-Asciak et al., 1999
). In
invertebrates, their only reported function is in membrane depolarisation,
via a potassium channel-mediated effect in the neurons of the marine
mollusc Aplysia californica
(Piomelli et al., 1989
).
Trioxilins are the biologically inert hydrolysis products of the hepoxilins
(Fig. 1;
Pace-Asciak et al., 1987
;
Pace-Asciak and Lee, 1989
).
Hill et al. (1993
) reported a
number of trioxilins with an 8,11,12-trihydroxy configuration (A-series)
produced by S. balanoides that commonly appeared in fractions with
egg-hatching activity.
|
The current study investigates the generation of trioxilins by E. modestus and B. amphitrite using a combination of high-performance liquid chromatography (HPLC) and gas chromatography (GC), both linked to mass spectrometry (MS). The potential functions of their precursors, hepoxilins, as signalling molecules within the egg-hatching and settlement processes of barnacles are also described.
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Materials and methods |
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Adult Balanus amphitrite Darwin (from Beaufort, NC, USA) were
maintained in aerated, 10 µm-filtered seawater at 22°C, which was
changed every other day, and were fed daily on newly hatched Artemia
(Sanders, Great Salt Lake, UT, USA). Following larval release, nauplii were
cultured in batches (5000; at 1 larva ml1) in
aerated,
0.7 µm-filtered seawater at 28°C on a 16 h:8 h L:D cycle
and were fed daily on Skeletonema costatum (Seasalter Shellfish Ltd,
Whitstable, UK) in 1 litre at
2x105 cells
ml1. Antibiotics were administered to the cultures as
described by Rittschof et al.
(1992
). Under these
conditions, development to the settlement stage cyprid took 45 days.
Cyprids were obtained by filtration (retained on a 250 µm mesh) and stored
in 0.45 µm-filtered seawater at 6°C until use in larval settlement
assays.
Chemicals
Calcium ionophore A23187, arachidonic acid (AA), eicosapentaenoic acid
(EPA), methoxylamine hydrochloride, bis (trimethylsilyl) trifluoroaceamide and
dodecane were purchased from Sigma-Aldrich (Poole, UK). Hepoxilin
A3 was supplied by Biomol Research Laboratories (Plymouth Meeting,
PN, USA). The stable hepoxilin B3 analogue, PBT-3, was prepared as
described in Demin and Pace-Asciak
(1993). All solvents and acids
used in the preparation of material for HPLC-MS and GC-MS were either AnalaR
(BDH, Lutterworth, UK) or HPLC grade (Fisher Scientific UK, Loughborough).
Ethereal diazomethane was prepared from N-methylnitrosotoluene
sulphonamide and alcoholic KOH at 65°C using an Aldrich kit
(Sigma-Aldrich).
Trioxilin A3 used in hatching assays and as HPLC-MS and GC-MS standards was prepared by acidification of hepoxilin A3. Briefly, 100 µl portions of hepoxilin standard (5 µg) were dried under nitrogen, resuspended in 20 µl acetonitrile (20%) and acidified using 50 µl of 0.1% formic acid. Samples were incubated for 20 min, freeze-dried and stored at 80°C in 100 µl methanol until use.
Generation of trioxilins by barnacle tissue
The barnacle extracts used in larval settlement assays and those that were
routinely run on HPLC-MS and GC-MS were prepared from known masses
(0.71.6 g) of soft tissue of either adult barnacles (E.
modestus or B. amphitrite) or known numbers (100010 000)
of larvae (B. amphitrite). Individual species were disrupted on ice
in 5 ml of filtered (0.22 µm) seawater. The crude cell preparations were
stimulated, for 20 min at 16°C, with 5 µmol l1
calcium ionophore A23187 and either 10 µmol l1 AA, 10
µmol l1 EPA or an equal volume of the fatty acid carrier
solvent, ethanol. Debris was removed by centrifugation (2 min, 10 000
g, 4°C) before acidifying the supernatants to pH 3.5 with
10% acetic acid. Samples (4 ml) were loaded onto pre-washed C18
Sep-Pak solid phase extraction cartridges (Waters Chromatography, Watford,
UK), washed with 5 ml ultra-pure water followed by 1 ml hexane and eluted with
methanol (5 ml). Eluants were dried under nitrogen, resuspended in 200 µl
methanol and stored at 80°C until preparation for separation by
HPLC-MS or GC-MS or use in settlement assays.
Preparation of crude barnacle hatching factor
Crude extracts for use as positive controls in hatching assays were
obtained by macerating each adult barnacle species (E. modestus or
B. amphitrite;50 g) in 25 ml 0.22 µm-filtered seawater. After
1 h, the liquid fraction was decanted off and the solid fraction homogenised
twice (1 h each wash) with 25 ml seawater. Seawater fractions were combined
and centrifuged to remove barnacle debris (15 min, 500 g,
4°C). An equal volume of acetone was added to the supernatant, and the
precipitated protein was pelleted by centrifugation (10 min, 500
g, 4°C). The supernatant was centrifuged at 14 000
g for 30 min at 4°C before being filtered (Whatman #1) to
remove any remaining particulates. The supernatant was then rotary evaporated
(40°C) to remove all acetone, and the aqueous fraction adjusted to contain
a final volume of 15% ethanol. The sample was acidified to pH 3.9 with glacial
acetic acid, loaded onto a pre-washed (5 ml methanol followed by 5 ml
ultra-pure water) C18 Sep-Pak, washed with 20 ml ethanol (15%)
followed by 20 ml petroleum ether and finally eluted with ethyl acetate. The
ethyl acetate eluants were stored at 20°C until use in egg-hatching
assays and product identification studies by electron impact gas
chromatographymass spectrometry (GC-EIMS).
High performance liquid chromatographymass spectrometry
(HPLC-MS)
Samples were separated using a Jasco HPLC system on a Spherisorb ODS2 (5
µm, 25 cmx0.46 cm) column (Hichrom) at 1 ml min1
with a 45 min linear gradient of acetonitrile:0.01% formic acid (20:80 to
80:20 v/v). Prior to injection onto the column, samples were dried under
N2 and resuspended in 100 µl of acetonitrile:0.01% formic acid
(20:80 v/v) and 5 µl 10% formic acid. The solvent flow passed through a
Jasco diode-array detector and was then split; 1/13th of the flow was directed
into the electrospray source of a Quattro II triple quadrupole mass
spectrometer (Waters Ltd, Elstree, UK) operated in the negative ion mode, and
the remainder went to a fraction collector. Fractions (1 min) were collected
for 40 min of the HPLC run and, where necessary, were derivatized for
GC-EIMS.
Electron impact gas chromatographymass spectrometry
(GC-EIMS)
Samples were converted to their methoxine O-trimethylsilyl ether
methyl esters for GC-EIMS as described by Knight et al.
(1999). Briefly,
N2-dried samples were methoximated with 50 µl of 2%
methoxylamine hydrochloride for 30 min at room temperature (RT). After two
steps of ethyl acetate extraction, esterification was carried out using
ethereal diazomethane (1 h at RT) and the esters treated with 50 µl
bis(trimethylsilyl) trifluoroaceamide overnight at RT in an N2
atmosphere. Samples were resuspended in 20 µl dodecane for GC-EIMS on a
Trio 100 instrument (Thermoquest, Hemel Hempstead, UK). Chromatography was
carried out on a 30 m DB5 capillary column (Jones Chromatography, Hengoed, UK)
using helium as the carrier gas and a temperature gradient of 10°C
min1 over 150320°C. The GC column was routed into
the mass spectrometer for analysis by EIMS.
Larval settlement assay
The promotion or inhibition of B. amphitrite larval settlement by
ionophore-challenged B. amphitrite tissue, hepoxilin A3
and PBT-3 was investigated using freshly moulted cyprids (day 0) and 3-day-old
cyprids, respectively. All larval settlement assays were performed using
flat-bottomed 24-well culture plates. Each well contained 2 ml of 0.45
µm-filtered seawater, 10 cyprids (day 0 or day 3), and 10 µl of
hepoxilin or PBT-3 dissolved in 20% methanol so as to give a final
concentration of 0.25, 0.5, 5 or 30 µmol l1. Control
plates were run in parallel, each containing either 10 µl methanol (20%) or
10 µl seawater without hepoxilin A3 or PBT-3. All plates were
incubated at 28°C in the dark for up to 48 h and were scored for the
percentage attachment/metamorphosis.
Barnacle egg-hatching activity assay
Immediately prior to their use, E. modestus were carefully removed
from the host mussel shells using a sharp seeker. For each assay, fully
developed paired egg masses were removed intact from a single adult barnacle
and washed for 1 h inrunning seawater. One of the egg masses was transferred
into 250 µl of filtered (0.45 µm) seawater containing a known
concentration of each test compound in an ethanol vehicle. The other egg mass
was placed in 250 µl of a control solution containing an equal amount of
ethanol (<5%) in seawater. All egg masses were incubated for 15 min at
12°C in separate wells of 48-well culture plates and then fixed by the
addition of100 µl of 10% seawater formalin. Numbers of hatched (stage
1 nauplii) and unhatched larvae were counted using a SedgewickRafter
counting chamber. The number of hatched larvae was expressed as a percentage
of the total number of larvae. Any undeveloped larvae found in the centre of
the egg mass were excluded from the count. Any unhatched egg masses were
tested for hatching viability by addition of the crude hatching factor.
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Results |
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Larval settlement
Immediately prior to settlement, the free-swimming cypris larvae of B.
amphitrite were observed searching the plate surface with their
antennules (Fig. 3A). During
the settlement process, larvae initially attached to the substratum by their
antennules, then head-planted down onto the surface and eventually
metamorphosed (moulted) into the new spat
(Fig. 3B).
|
For all experiments, the use of methanol as the solvent carrier (at 0.1%
final incubation concentration) had no significant effect on larval settlement
of B. amphitrite when compared to seawater controls (unpaired
t-test, P>0.05; data not shown). Crude larval (equivalent
to products from 1000 cyprids well1) and adult (equivalent
to products from 0.5 adults well1) extracts from B.
amphitrite both had a stimulatory effect on larval settlement (unpaired
Student's t-tests, P=0.004 and P=0.01 for larvae
and adults, respectively; Fig.
3C). Extracts equivalent to the products from 200 larvae
well1 and
0.1 adults well1 had no
effect on the settlement of B. amphitrite larvae (unpaired Student's
t-tests, P>0.05). Neither hepoxilin A3 nor a
stable analogue of hepoxilin B3, PBT-3, had a stimulatory
(Fig. 3D and 3E, respectively)
or inhibitory (data not shown) effect on the settlement of B.
amphitrite larvae.
Egg-hatching activity
Post-removal from adult E. modestus, the pair of egg masses each
appeared as a tight aggregation of over 1000 torpedo-shaped embryos embedded
in a membrane-bound structure (Fig.
4A,B). Upon addition of the crude egg-hatching factor, the nauplii
larvae rapidly emerged (<5 min) from the egg mass with limbs outstretched
and actively swimming (Fig.
4C,D). Crude hatching extract derived from E. modestus
caused 98.17±0.44% (mean ± 1 S.E.M., N=3) of
larvae in the egg mass to hatch. Similar levels of hatching (>95%) were
observed when E. modestus egg masses were exposed to B.
amphitrite crude hatching extract (data not shown). Authentic trioxilin
A3 had no effect on hatching at any of the concentrations tested
(Fig. 5A), while hepoxilin
A3 caused significant numbers of larvae to hatch at the
106 mol l1 concentration only (paired
t-test, P=0.0037; Fig.
5B). The stable hepoxilin B3 analogue PBT-3 at a
concentration of 107 mol l1 also had a
stimulatory effect on hatching (paired t-test, P=0.0121;
Fig. 5C). A concentration of
106 mol l1 PBT-3 had no significant effect
on larval hatching, although the larvae released were observed to exhibit an
inhibition in their swimming behaviour that was not seen with the other
concentrations of PBT-3.
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Discussion |
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It has previously been suggested that the trioxilins exhibit biological
activity as egg-hatching factors (Hill et
al., 1993; Song et al.,
1990a
). The commercial availability of hepoxilin A3
allowed us to investigate the effects of hepoxilin A3 and trioxilin
A3 on egg hatching. In contrast with earlier reports, trioxilin
A3 exhibited no effects in either assay at concentrations up to
106 mol l1. The epoxide precursor,
hepoxilin A3, was active at 106 mol
l1. Hepoxilin A3 and the stable analogue for
hepoxilin B3, PBT-3, both stimulated the hatching of nauplius
larvae at 106 mol l1 and
107 mol l1, respectively. When
standardised for the levels of hatching in the ethanol controls, the amount of
hatching in the 107 mol l1 PBT-3 treatment
(34.67±3.84%; mean ± 1 S.E.M., N=3) was
approximately four times greater than that observed for the
106 mol l1 hepoxilin A3
treatment (8.83±1.72%, N=6). This difference was presumably a
reflection of the rapid enzymatic breakdown of hepoxilin A3 to the
inert trioxilin A3. Despite the evidence for the involvement of
hepoxilin in the egg-hatching process, the levels of hatching for the
107 mol l1 PBT-3 treatment
(62.33±10.11%, N=3) were much lower than those observed when
the egg mass was exposed to the crude hatching factor (98.17±0.44%,
N=6). The reason for this discrepancy may be due to a number of
possibilities. Firstly, it may be that the process of barnacle egg hatching is
triggered by a number of factors, such as 8-hydroxyeicosapentaenoic acid
(Hill and Holland, 1992
;
Hill et al., 1988
), that may
act synergistically to cause larval release. Secondly, given that the major
precursor fatty acid in barnacles is EPA, it is possible that the EPA product,
hepoxilin A4, or indeed the DHA-derived
10-hydroxy-13,14-epoxydocosapentaenoic acid, might result in higher levels of
larval hatching activity than those observed with the equivalent AA-derived
product, hepoxilin A3. Unfortunately, hepoxilin A4 (or a
stable analogue) is not commercially available, thus the relative effects of
the EPA-derived product versus the equivalent AA-derived product
remain to be determined. Finally, PBT-3 used in the current study is an
analogue for hepoxilin B3, a product that was not observed in
E. modestus. The use of a stable analogue for hepoxilin A3
in larval hatching assays might show greater activity than observed in the
present study.
Mention should also be made of the concentration of hepoxilins required in the current study to potentiate larval hatching. It is questionable whether a concentration as high as 106107 mol l1 hepoxilin would exist naturally in the mantle cavity of barnacles where these unhatched larvae normally reside. However, if synergism exists, where a range of bioactive eicosanoids contribute to the natural barnacle hatching-factor activity, then a cumulative effect could lead to concentrations as high as those used in the current study.
No effects on B. amphitrite settlement were seen with either
hepoxilin A3 or PBT-3, suggesting that these compounds are not
involved in the communication pathways for settlement. However, the
involvement of hepoxilins and trioxilins within the barnacle settlement
process cannot be completely ruled out. If these compounds function as
internal signallers, the concentrations required to permeate the cyprid
exoskeleton (cuticle) may exceed those used in the current study (maximal
concentration used was 30 µmol l1). Indeed, Knight et al.
(2000) required 50 µmol
l1 prostaglandin E2 or prostaglandin
E3 before significant effects on B. amphitrite settlement
were observed.
The mechanism of action of hepoxilins and other inducers of larval egg
hatching is likely to differ greatly from their potential involvement in the
settlement process. Unlike cyprids, which have a thick impervious cuticle,
nauplii have only a thin cuticle that has been shown to be highly porous to
compounds with molecular masses of <800 Da
(Stuart et al., 2002). Thus,
in addition to the possibility of interaction with external receptors,
compounds such as eicosanoids (
350 Da) may also naturally diffuse through
the outer epidermis and make contact with internal receptors. By contrast, the
external cues for settlement are generally thought to be peptidic or
proteinaceous in character and potentially detected by specific receptors on
the cyprid antennules (Clare,
1995
). Rather than acting as exogenous signalling compounds, the
involvement of eicosanoids in larval settlement is more likely to be within
internal signal transduction pathways. An internal role for endogenous
eicosanoids is supported by the finding that indomethacin, a selective COX
inhibitor, stimulates larval settlement
(Knight et al., 2000
).
In summary, trioxilins derived from AA, EPA, 3 AA and DHA were
produced following ionophore challenge in both E. modestus and B.
amphitrite. Although trioxilin A3 did not affect egg hatching,
the parent epoxide, hepoxilin A3, was active at
106 mol l1. The stable analogue PBT-3
showed activity at 10-fold lower concentrations than hepoxilin A3,
presumably due to its stability to hydrolysis. The egg-hatching activity of
barnacle extracts can be ascribed, in part at least, to the hepoxilins. As the
extraction protocol involved acidification, which would hydrolyse hepoxilins,
it is also likely that some bioactivity may reside with some of the numerous
other eicosanoid-like components produced by E. modestus and B.
amphitrite.
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Acknowledgments |
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