CHEMORECEPTION IN MYXICOLA INFUNDI BULUm
Jill Anne Ward
June 4, 1976
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Chemoreception in Myxicola
ABSTRACI
The tubiculous polychaete Myxicola infundibulum has
been shoun to utilize chemoreception in connection with its
withdrawal response. The worms were found to be expecially
sensitive to divalent metal cations such as zinc and copper
as they reacted to concentrations as low as 10"'M of these
metal ions.
Chemoreception in Myxicola
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INTRODUCTION
Myxicola infundibulum is a sedentary tubiculous poly-
chaete which responds to stimulation by rapid withdrawal into
its mucus tube (figure 1). The Myxicola escape response has
been studied in detail (MoIntosh, 1921; Nicol, 1948, 1955;
Roberts, 1962a; Dyal 1972; Binstock and Goldman, 1967, 1969)
and has been found to be an extremely rapid all-or-nothing
contraction mediated through a single giant axon without
synapses and effected by giant muscle fibers running longi-
tudinally through the body.
Studies done on sensory behavior in sabellids have shown
withdrawal responses to tactile stimulation of either the
branchial crown or body wall (Roberts 1962a). The reflex has
also been obtained from changes in light intensity and shadow
movement (Dyal 1972).
Chemoreception has been found in several commensal
polychaetes in locating hosts (Mackie and Grant 1974), as
well as in earthworms (Dyal 1972), and a few errant polychaetes
(Mackie and Grant 1974), but it is interesting to study
Myxicola's extremely keen sensitivity to chemicals and its
use as a possible protection device.
Myxicola is particularly good for bioassay due to its
clear-cut response, quick recovery from withdrawal, as well
as being an easy animal to maintain in the lab.
Chemoreception in Myxicola
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MATERIALS AND METHODS
Specimens of Myxicola infundibulum were obtained from
beneath the floats at the Monterey marina. Other invertebrates
Used as homogenates in tests were obtained either at the
marina or from the intertidal region off Hopkins Marine Station.
(see tables 1 and 2). Living animals were kept in running
sea water at 12°C. In order to facilitate chemical application.
the Myxicola were placed in 2" lengths of glass tubing.
which were glued vertically to a Lucite base in a grid for-
mation about 14" apart. These tube arrangements were kept
in plastic tubs with about thirty worms per tub (figure 2).
This separated each worm and allowed all branchial crouns to
be faced upward. Several tubs were used to permit rotation
of test-worms, thus preventing fatigue. Worms were kept
in semi-darkness except when testing, where red light was
Used. This eliminated any responses due to change in light
intensity or shadow movements, since Myxicola do not respond
to red light (Dyal 1972). Disposable syringes with 3" pieces
of plastic tubing attached to the ends were used to apply
the chemicals, allowing for exact measurement, slow controlled
application, little mechanical stimulus, and elimination of
the possibility of contamination (figure 3).
Animal homogenates were made using approximately 10Omg
worm (wet weight) per lml glass-fiber filtered sea water in
a mechanically driven Potter-Elvehjem apparatus. Each
Chemoreception in Myxice
page 5
homogenate was tested at three or more 10-fold dilutions of
the initial concentration. The chemicals tested were mixed
in molar concentrations and diluted 10-fold from 10*9 to at
least 102 molar, except where indicated. Applications were
made to interior base of the plume area. The tub water in-
flow was turned off to allow for maximum sensory reception,
and to minimize dilution of chemicals applied. To eliminate
habituation, the worms were tested only once per hour with
six tests at most in a given 24 hour period, in accordance
with previous studies (Evans 1969, Dyal 1972)
EXPERIMENTS AND RESULTS
Standardization and Controls
The Myxicola were tested with applications of sea
water and a mixture of sea water and graphite particles to
quantify possible reactions to mechanical stimulus. Fifteen
worms were tried with each solution, with only two contractions
total. It was almost always possible to distinguish this
mechanically-stimulated reaction from a chemical one, however,
since the fine-fibered transverse muscles contract uhen
Myxicola first is mechanically stimulated, causing the worm
to slouly close the end of its branchial crown, and possibly
withdraw partially into its tube. Only if the stimulus is
increased in magnitude will the giant fibers contract, giving
a semi-graded response (Roberts 1962, Dyal 1972). For all
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Chemoreception in Myxicola
assays in this paper, only rapid and complete withdrawals
were counted as positive.
Tests were done on Myxicola to determine sensitivity to
osmotic and ph changes. The osmotic assay was made using
filtered instant ocean (plus trace metals) at concentrations
of 50%, 75%, 100%, 150%, and 200% sea water. Worms were also
tested with distilled water. Withdrawal responses occured
only on application of distilled water, 150% and 200% sea
water.
PH sensitivity was tested at phs of 3,4,6,and 7 using
different dilutions of citric acid; pH 5 with KH,PO,; and
pH 7 and 9 using Na,HPO,. 60%-100% worms were sensitive to
acidic phs (especially citric acid), but less than 20% were
receptive to alkaline pH.
Effects of Animal Homogenates
Homogenates of several invertebrates from both the same
habitat as Myxicola (the marina) and also a different one
(intertidal) were assayed to compare Myxicola's sensitivity
to different animals as well as find out whether the chemical
might be species specific or habitat specific, or possibly
due to a predator-prey situation (see tables 1,2.3).
An observation of a live arabellid specimen with a
Myxicola placed together in a fingerbowl for several minutes
revealed that the withdrawal response was not effected by the
presence of a live arabellid.
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Chemoreception in Myxicola
Next, several tests were done using the arabellid
homogenate to characterize the compound active in causing
Myxicola's withdrawal. A homogenate solution made up of .omg
(wet weight) arabellid plus 6ml filtered sea water was centri-
fuged for 15 minutes at 1500 rpm. Activity was found in both
the clear supernatant (80% at .6% solution conc. - .0216 mg
dry arabellid per ml sea water) and in the sediment (not
quantified).
The homogenate was dialysed in a rotating dialyser for
48 hours at 6°C, using a Spectrapore membrane tubing bag (type
2: m.w. cutoff 12,000-14,000) and five outer volumes. The
inside and outside solutions were freeze-dried and reconsti-
tuted to their original water concentration and osmolarity for
the assay, which showed the chemical to be dialysable (at 1%
dilution, 80% Myxicola reacted to outer solution, 10% reacted
to inner solution).
An assay was also done for heat stability. An arabellid
was dried in an oven for 8 hours at 76°C. and then made into
a solution and centrifuged. The supernatant yielded an 80%
reaction at .8% dilution (=.0245mg dry weight per ml sea
water).
The arabellid homogenate was also tested for stability
against oxidation. 30% H,O, was added in excess to a solution
of .5mg worm per ml sea water, then the H202 was evaporated
and the solution reconstituted. The assay resulted in an 80%
reaction at 1% concentration of solution (- .18m9/ml sea water)
Chemoreception in Myxice
page 8
Arabellidae is a family closely related to Lumbriconereis
which has been found to contain nereistoxin' (Deguchi, Nara-
hashi, Haas 1971). The arabellid homogenate was tested on
frog rectus abdominus muscle for any possible inhibition of
the effect of acetyl choline. Several trials yielded no
decrease in the muscle's sensitivity to acetyl choline.
Acetyl choline w as also applied to live Myxicola, as was
carbachol, but both these chemicals failed to elicit the
withdrawal response at concentrations of 1 mg/l0ml sea water!
Effect of Known Chemicals
In the next approach, assays of known chemicals (of
characteristic types) were made in an attempt to find out
what chemical or class of chemicals Myxicola might be sen-
sitive to (tables 4 and 5).
Methylene blue (graph 5) was tested before use as a color-
ing agent in the assay of clear amino acid solutions, but
was found to elicit a reaction in Myxicola at very low con-
centrations. Since zinc chloride is known to be an impurity
in the methylene blue staining solution, it and several
(rauxe)
other transition metals were tried on the Myxicola. Very
clear positive results were obtained at strikingly low con-
centrations of the metals. (Myxicola is much more receptive
nereistoxin is a naturally occuring toxin which blocks
the stimulatory action of acetyl choline on smooth muscle
fiber.
Chemoreception in Myxice
page 9
to these chemicals than any others tried so Far.) Myxicola
reacted to several similar metals, but had different degrees
to sensitivity to different metals.
The tunicate with which Myxicola is closely associated
(Ascidea ceratodes) at the marina concentrates vanadium,
(Goldberg 1970) another divalent metal, so a solution of
Ascidea blood and 10-fold dilutions of it were tried (graph 7).
It is interesting to note however, that the solutions of
V2Ug and Batl, were not especially active in eliciting the
Myxicola response.
DISCUSSION
he experimental results clearly show that Myxicola
conclusively employs chemoreception as another means of
sensing the environment. Preliminary tests show the active
compound to be naturally occuring, at least partially free
in solution, fairly small in molecular weight, heat stable.
and stable against oxidation. All these characteristics
readily support the data showing Myxicola's sensitivity
to metal cations. The positive reactions to mixtures of
organic substances can also be explained in this way since
many of the chemicals asaayed (i.e. casein hydrolysate) are
known to contain trace amounts of metals to which Myxicola
is so sensitive. Two possible reasons for this sensitivity
have to do with Myxicola as prey, and Myxicola's adaptation
to a polluted environment.
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Chemoreception in Myxicnla
Myxicola live at the marina in amongst many other in-
vertebrates, some of which are known to concentrate certain
metals in their bodies (Goldberg 1970). A tunicate, Ascidea
ceratodes, is abundant at the marina and concentrates
vanadium in its blood. The scyphistoma stage of Lyanea capitella
has also been seen in the same environment (Abbott 1976) and
concentrates copper (Goldberg 1970). Myxicola's positive
reactions to several dilutions of Ascidea blood might lead
to the possibility of a sympathetic withdrawal due to the sensing
of an injury in a neighboring animal.
The idea of metal sensitivity as a defense against
pollution is another possibility. Several of the metals meas-
ured are known to be toxic to marine invertebrates in higher
concentrations (Portmann 1972). A response to a slight increase
in metal would enable Myxicola to withdraw into its tube in
case dangerous amounts of toxic metals were diffusing through
the water. These ideas are beyond the scope of this project,
however, but any possible adaptive sensitivity would be advan-
tageous for Myxicola's protection and is worth considering.
It is also interesting that homogenates from animals not
from the same environment as Myxicola generally had the same
effect on Myxicola as animals from the same habitat. This
rules out the possibility of the chemoreception being specific
between a predator and prey, and the widespread acivity clearly
eliminates the possibility of the chemical being a species
specific pheromone.
Chemoreception in Myxicol:
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Since the chemicals had to be applied to the interior
base portion of the branchial croun to be effective in the
assay, it is assumed that the chemoreceptors are located in
this area. This is supported by the fact that nereids have
been found to have chemoreception in their prostomial
cirri (Laverack 1974), which correspond closely to the small
tentacles at the end of Myxicola's prostomium.
Leherally, Myxicola has been shown to be a good, consistant
bioassay for several metals. In addition to responding
immediately to the stimulus, the recovery time is very short.
and accomodation to repeated stimuli is relatively slow.
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Chemoreception in Myxicola
ACKNOULEDGMENTS
I would especially like to thank Dr. F.A. Fuhrman
for all his extremely invaluable assistance in this research,
and Dr. Robin Burnett and Chuck Baxter for their encouragement
and criticism. Thanks also to Gordy Kauhanen for helping with
the set-up design, and to the entire class and faculty for
making my experience at Hopkins so terrific.
Chemoreception in Myxicola
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REFERENCES
Abbott, D.P., Personal Communication
Binstock, L. and L. Goldman (1969) Current and Voltage-Clamped
Studies on Myxicola Giant Axons, J. Gen. Physiol. 54: 730-741
Binstock, L. and L. Goldman (1967) Siant Axon of Myxicola:
Some Membrane Properties as Observed Under Voltage Clamp
Science, 158: 1467-1469
Deguchi, Narahashi, Haas (1971) Mode of Action of Nereistoxin
on the Ne romuscular Transmission in the Frog. Pesticide Biochem.
and Physiol. 1: 196-204
Dyal, J.A. (1972) Behavior of Annelids, Report 441 University
of Waterloo Research Reports in Psychology
Evans, S.M. (1969) Habituation of Withdrawal Response in Nereid
Polychaetes Biological Bulletin 137: 105-117
Goldberg (1970) Review of Trace Element Concentrations In
Marine Organisms Puerto Rico Nuclear Center
Laverack, M.S. (1974) The Structure and Function ofChemoreceptor
Lells p1-48 in"Chemoreception in Marine Organisms"(Grant and
Mackie, eds.) London, Academic Press
Chemoreception in Myxicola
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Mackie, A.M. and P.T. Grant (1974) Interspecies and Intraspecies
Chemoreception by Marine Invertebrates, p 105-141 in
Chemoreception in Marine Organisms"(Grant and Mackie, eds.)
London, Academic Press
MoIntosh, W.C. (1921) A Monograph of British Annelids, vollV,ii
London, Dulau & Co., p307-310
Nicol, J.A.C. (1948) The Giant Nerve-Fibers in the Central
Nervous System of Myxicola (Polychaeta: Sabellidae) Quart.J.
Micro. Sce. 89:1-45
Nicol, J.A.C. and Whitteridge (1955) Conduction in the Giant
Axon of Myxicola, Physiol. Comp Oecol. 4: 101-117
Portmann, J.E., Loxicity Testing with Particular Reference to
Oil-Removino Materials and Heavy Metals, in "Marine Pollution
and Sea Life" (Ruivo, ed.) p 217-221
Roberts, M.B.V. (1962a) The Rapid Response of Myxicola (Grübe)
J. Mar. Bio. Ass. U.K. p 527-539
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