Invertebrate Anatomy OnLine
Molgula manhattensis ©
Copyright 2001 by
is one of many exercises available from Invertebrate
Anatomy OnLine , an
Internet laboratory manual for courses in Invertebrate Zoology. Additional
exercises, a glossary, and chapters on supplies and laboratory techniques are
also available at this site. Terminology
and phylogeny used in these exercises correspond to usage in the Invertebrate
Zoology textbook by Ruppert, Fox, and Barnes (2004). Hyphenated
figure callouts refer to figures in the textbook. Callouts
that are not hyphenated refer to figures embedded in the exercise. The glossary
includes terms from this textbook as well as the laboratory exercises.
Tunicata sP, Ascidiacea C,
Molgulidae F (Fig
is characterized by a suite of apomorphies including a dorsal hollow nerve cord,
notochord, pharyngeal gill slits, and a post anal tail (Fig 29-1). The
ancestor was a fishlike deuterostome that swam using alternating contractions of
right and left longitudinal axial muscles to create undulations of the body. The
flexible, incompressible notochord prevented these contractions from compressing
the body while allowing lateral deflection. The
chordate central nervous system is a hollow, median, longitudinal nerve cord
formed in the embryo by an invagination of surface ectoderm whose original
function was probably sensory reception. Paired pharyngeal gill slits connect
the lumen of the pharynx with the exterior and originally functioned in
suspension feeding with respiration being added later. A
muscular tail posterior to the anus is, although commonplace in chordates, an
unusual feature not found in other taxa. It
is an extension of the axial musculature and is the chief locomotory organ. An
additional apomorphy is the endostyle, a region of pharyngeal endoderm, that
secretes iodated compounds, either mucus or hormones.
Tunicata (= Urochordata) sP
are highly derived and less like the ancestral chordates than are
cephalochordates or vertebrates. At
some time in the life cycle all possess a notochord, dorsal hollow nerve cord,
pharyngeal gill slits, postanal tail, and endostyle but only the gill slits and
endostyle are present in adults. Tunicates use the pharyngeal gill slits for
suspension feeding. The larva is much more chordate-like than the adult and
resembles a tadpole or fish, has all the chordate apomorphies, and is known as
the tadpole larva. Metanephridia are absent and coelom is reduced to a
pericardial cavity and gonads. As in cephalochordates the gut is dominated by an
enormous pharynx surrounded by a water-filled atrium but unlike
cephalochordates, it is U-shaped with the mouth and anus anterior. Tunicates may
be benthic or planktonic and solitary or colonial. All are marine.
is traditionally divided into Ascidiacea (the benthic sea squirts in three taxa;
Aplousobranchia, Phlebobranchia, and Stolidobranchia), Thaliacea (the pelagic
salps), and Appendicularia (the pelagic larvaceans). Recent
molecular evidence and reevaluation of morphological evidence, however, suggests
that Ascidiacea is paraphyletic and Tunicata should be reorganized into three
different higher taxa (Fig 29-32). In
this reorganization Stolidobranchia would be one higher taxon. Phlebobranchia
plus Thaliacea would be the second taxon. Aplousobranchia
plus Appendicularia is the final tunicate taxon. For now, however, the
traditional classification will be followed.
is usually taken as representative of Tunicata, at least for the purposes of
introductory laboratory exercises. Ascidians, or sea squirts, are sessile filter
feeders that, as adults, bear little resemblance to their chordate relatives. Ascidians
have a living, external, cellular exoskeleton, or tunic, underlain by epidermis. The
tunic resembles connective tissue, except it isoutside the
epidermis, and consists of cells, a secreted extracellular matrix, and ground
of it is a cellulose-like polysaccharide. In
many ascidians blood vessels cross the epidermis to enter the tunic, a feature
found in no other animal.
gut is U-shaped and both openings are anterior, with the anus dorsal to the
gut is dominated by an enormous pharynx whose wall is perforated by numerous
tiny gill slits. The pharynx is
surrounded by a water-filled atrium into which the gill slits open and which
itself opens to the sea. It
is both respiratory organ and filter-feeding device. Water
and food particles enter the pharynx and the water passes through the gill slits
to the atrium and then out the siphon. Food,
entangled in mucus secreted by the endostyle, remains in the gut and passes
posteriorly to be digested.
hemal system includes a heart, vessels, and blood spaces in the connective
tissue. The heart is enclosed in a pericardial cavity derived from the ancestral
pattern of blood flow resembles that of the cephalochordates and early
vertebrates except that the heart reverses direction periodically and the blood
thus flows in both directions through the system. Ascidians
have no structure recognizable as a kidney.
are simultaneous hermaphrodites and the gonoducts open into the atrium. Some
ascidians are solitary and may be relatively large. Others
are colonial with tiny individual zooids in a common tunic.
gonad*s) are on the inner surface of the body wall. The
epicardium is absent or represented by a renal sac.
Aplousobranchia, and Stolidobranchia are the three higher ascidian taxa. In
stolidobranchs the gonads are on the inside surface of the body wall beside the
pharynx and not in the gut loop. No epicardium is present but Molgulidae has
renal sacs, which are derived from the epicardium. The neural gland is dorsal to
the cerebral ganglion. The stolidobranch pharyngeal lining is strongly pleated
and has transverse and longitudinal blood vessels. Molgulidae has spiral gill
can be solitary or colonial. The
plane of the tadpole tail is vertical.
manhattensis, the sea grape, is a solitary sea squirt of shallow water on
the coast of most of Europe and Britain (Norway to Portugal) and on the Atlantic
and Gulf coasts of the United States from Maine to Texas. This
exercise is written specifically for Molgula but
could also be used with several other genera.
wishing to use locally collected sea squirts have several choices depending on
their geographic location. Of
the two common solitary ascidians on the southeastern coast of the Unites
States, Styela plicata (Stolidobranchia:
Styelidae) and Molgula
manhattensis, the latter is the best subject for dissection even though it
is much smaller than Styela. The
thick tunic of Styela is
very difficult to remove and is a formidable deterrent to dissection. Ascidia
Ascidiidae) occurs in the northeastern United States and is present in some
parts of the Southeast (North Carolina and Florida). It is a good choice when
available. Molgula, Ascidia
interrupta or Ciona
intestinalis can be used in the
northeastern United States. Ciona
Cionidae) is one of the most widely distributed ascidians. It
is common on the northern European and North American Atlantic and Pacific
coasts. Ciona is
often used in teaching laboratories and another exercise in this series
describes its anatomy ( Invertebrate
Anatomy OnLine ).
the west coast of North America Ascidia
ceratodes is almost identical to Ascidia
interrupta but is smaller, 50-70
occurs from British Columbia to the Gulf of California in shallow water and is
usually common, sometimes abundant. Pyura
Pyuridae) is about the size of Molgula
manhattensis and occurs from
Alaska to southern California where it can be common.
it is smooth, globular, translucent, and often grows in clusters, Molgula
manhattensis goes by the common
name of "sea grape" (Fig 29-12G). Large
specimens are about 2 cm in diameter but much of this mass is tunic and seawater
so the animal inside is closer to 1 cm. The
dissection is best performed on living specimens but preserved material can be
used if necessary. A dissecting microscope should be used throughout and living
specimens should be relaxed in isotonic magnesium chloride.
a large Molgula in
a culture dish of seawater (or tapwater if preserved) on the stage of the
dissecting microscope and examine with the lowest power. If
it is alive and you are careful to avoid disturbing it, the animal will probably
relax, extend its siphons, and begin pumping water through its pharynx.
body is usually ovoid and slightly flattened from side to side (right to left). The
two siphons that
protrude conspicuously from one end of the body are easily found but not so
easily told apart. Try
to distinguish them at this time but do not despair if you cannot be sure which
is which. You
will be able to tell them apart later when you get inside.
siphon is situated at the
end of the oval outline of the animal whereas theatrial siphon is
close to it but on one side of the oval. If
you have a living specimen and allow it to extend the siphons they can more
easily be distinguished. The
opening of the buccal siphon is surrounded by six small conical lobes whereas
that of the atrial siphon has four larger lobes. These
lobes can be seen when the siphons are not extended but are not as obvious then. The
atrial siphon is usually the longer and narrower of the two.
you have identified the two siphons, you can use them as landmarks to orient the
buccal siphon is anterior and
the atrial siphon is dorsal. The
end of the sphere opposite the buccal siphon is posterior and
the side opposite the atrial siphon is ventral. The
plane of symmetry passes through both siphons and divides the animal into right and left halves.
sure you understand the feeding process before dissecting your specimen. The
buccal siphon opens into the anterior end of the gut, most of which is an
enormous pharynx with minutely perforated walls. The
perforations are the gill slits, or stigmata. The
pharynx is surrounded by an invaginated seawater space known as the atrium. The
atrium connects with the exterior through the atrial siphon.
walls of the pharynx are covered by a moving sheet of mucus that forms a
fine-meshed sieve (Fig 29-15D). The
mucus is secreted by the endostyle, located mid-ventrally in the pharynx. The
mucus is moved dorsally from the endostyle by frontal cilia on the walls of the
pharynx so it covers the walls and gill slits.
water current is generated by lateral cilia in the gill slits. Water
and food particles enter the buccal siphon and move into the pharynx lumen. The
water passes through the mucous net, through the gill slits, and into the
then returns to the sea through the atrial siphon.
particles too large to pass through the mucous net are retained in the pharynx.
The mucus is gathered by the dorsal lamina on the dorsal midline of the pharynx.
Cilia of the dorsal lamina move the mucus and food posteriorly into the stomach
where they are digested. The gut empties via an anus into the atrium.
gonads also empty into the atrium. Digestive
wastes and gametes are carried out of the atrium by the feeding current as it
exits the atrial siphon.
some seawater/carmine suspension to the water in the dish and gently swirl the
carmine about so it is dispersed throughout the water in the dish. Under
magnification and against a lighted background (transmitted light), observe the
openings of the siphons and look for the movement of carmine, or other
particles, into or out of the siphons. You
should be able to discern a steady flow of water into the buccal siphon and
another stream out of the atrial siphon.
incurrent flow will be easier to see because of the carmine particles in the
water but the outflow should have no particles and will be best detected by
watching for the displacement of the water surrounding the opening of the atrial
are there no carmine particles in the outflow? <
a Pasteur pipet to deliver a little 0.1% methyl green/seawater or
carmine/seawater directly to the opening of the extended buccal siphon. The
dye will probably enter the siphon, be detected by sense organs, and then be
forcibly ejected as the animal contracts the body wall muscles and empties the
pharynx and atrium through the siphons. <
the tip of a pipet or hypodermic needle into the buccal siphon and inject a
little 1% methyl green/seawater into the closed pharynx. Remove
the needle and allow the animal to recover from the experience, reextend its
siphons, and begin pumping water again. When
the pharyngeal cilia begin beating, the dye will be seen exiting the atrial
sure to distinguish between the slow gentle ciliary current and the abrupt,
forceful muscular current. How
is it that dye will cross the pharynx and leave the atrial siphon when carmine
particles will not?
up the squirt and hold it over a towel well away from your microscope and books,
and squeeze it gently. Make
sure the siphons are not pointed at the microscope when you do this. If
it is healthy, it should contract its body wall muscles and eject streams of
water from one or both siphons as it empties the pharynx and atrium. Much
of the volume of a functioning, feeding sea squirt is seawater and the animal
will become much smaller when that water is expelled. <
covered by a thick, translucent tunic,
to which the name “tunicate” alludes. The
tunic is the outermost layer of the body
wall which consists of tunic
and mantle. The mantle, in turn, consists of the epidermis, connective tissue,
circular and longitudinal muscles. The tunic is a secreted exoskeleton but is
far from being dead and inert as exoskeletons typically are. It
is composed of a matrix of cellulose (tunicin), protein fibers, cells, and
proteoglycan ground substance. In
many tunicates it also contains blood vessels. The
tunic is unique in being a living exoskeleton that grows as the animal grows and
does not require molting.
cells are derived from the connective tissue mesenchyme of the body wall, not
from the epidermis which underlies it. It
is thought that the cellulose, however, is secreted by the epidermis. The
tunic is sometimes thought of as an external connective tissue lying outside the
epidermis, muscles and internal connective tissue of the mantle lie beneath the
tunic and cannot be seen yet.
closely at the surface of the tunic. It is probably fouled with silt, sand, and
may be other animals, including small Molgula,
growing on it. Some
areas of the surface bear small, hairlike extensions of the tunic, called papillae that
are used to hold sand and other particles to the surface. A
roughened, irregular area or projection, usually at the posterior end, marks the
former point of attachment of the tunic to the substratum. Sometimes
the substratum is another tunicate, often of the same species.
the sea squirt from the dish and scrub it gently with a toothbrush to remove
most of the adhering debris. Return
it to the dish. Most
of the remaining debris will probably be in the vicinity of the siphons, which
is where the papillae are best developed. The
papillae should be easier to see now and some of the viscera should be visible
through the body wall. Easiest
to see are the intestine and orange or brown pyloric gland (= liver) associated
with the stomach wall at the posterior end of the dorsal surface.
next task is to remove the tunic without damaging the animal within. This
is not difficult but it must be done with care and in accordance with the
soft body of a sea squirt is connected to the tunic only at
the siphons so it is fairly easy to remove the animal once the tunic has been
sure you know the locations of the sagittal plane and midline. Remove
the animal from the dish and hold it gently between your thumb and forefinger. With
a sharp scalpel,
make a median longitudinal incision through the tunic (only) beginning at one
siphon and extending around the perimeter of the animal to the other siphon. Keep
the incision on the midline all the way around the dorsal, posterior, and
ventral perimeter. Use
short, repetitive, slicing strokes of the scalpel and do not put unnecessary
pressure on the animal with either your fingers or the scalpel.
soon as you make the first penetration completely through the tunic, the soft
body will ooze through the break in the tunic. Avoid
cutting in the region of the break as it is important that you do not cut the
soft body. Continue
cutting the tunic on either side of the breakthrough and work your way
completely around the specimen.
the pressure from your fingers will cause the entire animal to squeeze out
through the buccal siphons. This
is not the same as oozing out through a cut in the tunic and, while not
disastrous, it is not desirable. If
it happens, the animal will be intact and usable but inside out. It
must be separated from the tunic and turned right side out before you can use
an inside out animal without realizing it can be very confusing. Such
an animal will be enclosed in its own pharynx.)
you have extended the incision completely around the animal, from one siphon to
the other, the two halves of the tunic will peel away revealing the soft animal,
enclosed in the mantle, within. The
process may remind you of peeling a grape.
the two sides will not separate easily from each other, use the dissecting
microscope to look for small areas where the two halves of the tunic remain
attached to each other and carefully cut them without cutting the adjacent body. Whereas
the tunic is now free of the body, the rest of the body wall (mantle) is still
intact and should remain so for the time being.
you separate the two sides of the tunic, you will notice that the animal is
firmly attached to the tunic only at the two siphons. If
you are observant, you may notice a delicate, transparent blood vessel running
from the body to the tunic on each side of the tunic. Working
under magnification, use your fine forceps to pull gently the siphonal tissues
and free them from their attachment to the tunic.
the texture and consistency of the tunic and then set it aside. Place
the animal in a small dissecting pan of seawater deep enough to cover it. An
empty sardine can with a wax bottom makes a good dissecting pan for Molgula. The
animal should not be placed in magnesium chloride until after the heart has been
you are dissecting a preserved animal, cover it with tapwater.
the buccal and atrial siphons and orient the animal with the right side facing
upward, toward you. The
animal is still enclosed in the remaining body wall, or mantle,
and only the exoskeleton has been removed so far. The
mantle is primarily connective tissue and muscle with the thin epidermis over
its surface. The
muscles are poorly developed in Molgula. They
are best developed around the siphons where they form sphincters to close the
siphons (Fig 29-19). The muscle layer
consists of widely separated transverse, longitudinal and oblique strands. With
carefully adjusted light and a dark background, you can see these strands in the
viscera are partly obscured by the translucent mantle but the outlines of most
of the organs can be seen through it. Do
not remove the mantle at this time, but be aware that because of it you are not
getting a clear view of the organs.
at the animal's right surface with the buccal siphon on your right
and away from you as in Figure 1. Most
of the tissue inside the mantle is the pharynx but it is difficult to observe
with the mantle intact.
Figure 1. The
right side of Molgula
manhattensis with the tunic
removed (after van Name, 1945). Uro79L.gif
most conspicuous feature on this side is the renal
sac (= renal organ, kidney). The
renal sac is a unique and characteristic feature of Molgulidae. It
is thought to be homologous to the epicardium of other ascidians and the
epicardium may be a remnant of the coelom. The renal sac is slightly curved and
has a longer greater
curvature and shorter lesser
renal sac is a turgid, transparent, sausage- or bean-shaped sac located in the
posterior half of the squirt (Fig 1, 29-22C). It
has no duct connecting it to the exterior. It
is a storage kidney that accumulates and stores uric
acid crystals, which are easily seen as a whitish precipitate in the
cannot break down the uric acid from nucleic acid metabolism and it is stored
for life by various mechanisms in different species. Only
molgulids have a single, large renal sac for this purpose. The
nitrogen from protein metabolism is lost more conventionally as ammonia across
the permeable surfaces of the body. No
excretory organ has been found in ascidians.
squirts are hermaphroditic and Molgula has
gonads consisting of an ovary and testis on each side. The
gonads of stolidobranchs are embedded in the part of the mantle that forms the
wall of the atrium. The right
hermaphroditic gonad is
located on the anterior, lesser curvature of the renal sac, between the sac and
the siphons, and almost touching the sac (Fig 1). It
is composed of both ovary and testis. Its
long axis is roughly parallel to the animal's dorso-ventral axis and is parallel
to that of the renal sac and it is about the same length and width as the sac.
gonads appear through the translucent tissues of the mantle as an indistinct
whitish lobed organ. The long ovary is
surrounded by the testis. A
white oviduct extends
along its long axis. The testis is
composed of many small spherical lobules that may remind you of eggs, which they
are not. The
testis is located on the periphery of the ovary, along both its margins on the
left but only on the posterior margin on the right. You
will see the gonads more clearly after you remove the mantle.
The heart is
located on the concave lesser curvature (anterior margin) of the renal sac, on
the right side of the animal (Fig 29-22C). It
is best to locate it now, before removal of the mantle. It
is between the renal sac and the right gonad and extends along most of the
length of the lesser curvature of the renal sac. It
is enclosed in a coelomic space, the pericardium, but that is not apparent (Fig
diaphanous, transparent walls of the heart are the inner walls of the
pericardium and are difficult to recognize as such unless the heart is beating
(Fig 29-21B). Waves
of contraction run from one end of the heart to the other every few seconds. The
billowing walls of the pulsating heart look like a diaphanous curtain waving in
a gentle breeze. If the animal is in seawater you may see the heart beat but not
if it is in magnesium chloride.
and record the beats, or peristaltic waves, per minute. The
heart of urochordates is unusual in that it periodically pauses and then
reverses direction. Note
the direction in which the peristaltic waves propagate and watch for a few
minutes to see the beat reverse direction. What
is the approximate length of time between reversals? <
body cavity of ascidians is a hemocoel consisting of open, unlined sinuses and
vessel-like channels. The
body is supplied by the heart via two blood channels, or vessels (Fig 29-21A). Each
end of the heart opens into one of these channels. The
ventral end of the heart connects via some of the ventral channels to the
ventral side of the pharynx, endostyle, and test. The
dorsal end of the heart connects, via the dorsal channel, to the dorsal side of
the pharynx, the viscera, and the test. Both
parts of the hemal system end in the same fine capillary-like channels in the
half of the system alternately serves as veins, then arteries, and then veins
again as the heart periodically reverses its beat.
you have finished your study of the heart, you may replace the seawater in your
dissecting pan with isotonic magnesium chloride if you wish. The
heart will stop beating in magnesium chloride. It
will continue beating for hours in seawater.
the animal over and look at the left side (Fig 2). The
most conspicuous feature on this side is the postpharyngeal gut which
appears as a hairpin loop along the periphery of the animal. It
begins on the dorsal side near the atrial siphon with a short esophagus which
you cannot see now.
Figure 2. The
left side of Molgula manhattensis with
the tunic removed (after van Name, 1945). Uro80L.gif
esophagus leaves the pharynx and empties into the anterior end of the stomach which
is almost completely hidden by the large, conspicuous, brown or orange (in life) pyloric
stomach exits the pyloric gland and begins the outer leg of the hairpin loop of
the intestine (Fig 2, 29-15A). The
stomach is swollen proximally but tapers gradually to become theintestine. The
intestine extends ventrally and then doubles back on itself, passes by the
stomach to empty via the anus into the atrium, neither of which are visible at
this time. The left
gonadis located in the interior of the gut loop. Its
long axis roughly coincides with the animal's antero-posterior axis.
your specimen back over so the right side faces you. Insert
the finest point of your finest scissors in the atrial siphon and cut through
its mantle wall to open the atrium. Continue
cutting through the mantle in a ventral direction across the body anterior to
the right gonad. Do
not cut the gonad.
the ventral end of the gonad turn the incision posteriorly around the gonad and
renal sac and then dorsally along the posterior margin (greater curvature) of
the renal sac. Stop
cutting when you reach the dorsal end of the sac.
gonad and renal sac are now freed on three sides but remain intact and attached
dorsally, at the base of the atrial siphon. The
oviduct has not been harmed. The atrium has
been opened and is most of the space you see surrounding the organs. Deflect
the renal sac and right gonad dorsally, cutting the delicate, transparent blood
vessels that connect them to
the body as necessary. With
most of the right body wall deflected, look again at the lesser curvature of the
renal sac and see if the heart is still beating. If
it is, it will be easier to see from this, the right, side.
gonads are now visible. With fine scissors snip the ventral end off the tip of
the right gonad (including part of the ovary and testis). Set
the tip aside for the moment and inspect the cut surface of the ovary and note
that it is hollow. Carefully
insert the tip of a minuten nadel in
at the opposite, dorsal end of the ovary and find the wide, short oviduct where
it exits the ovary and is free of it. Follow
the oviduct to its terminus at the female
gonopore where it empties
into the atrium. Insert
a nadel into
the gonopore and see that it slips easily into the lumen of the hollow ovary. The
ovarian lumen is a remnant of the coelom. Eggs
are shed into it, move through the oviduct, enter the atrium, and are then
released through the atrial siphon.
the tip of the gonad in a drop of seawater (tapwater if using preserved
material) on a slide, tease it apart, and make a wet mount with it. Examine the
slide with the compound microscope and locate the large irregular eggs. Find
an isolated egg and note that it is surrounded by numerous small follicle cells. Use
high power to look at the remains of the testis that you teased apart. If
you have a living specimen, you should see numerous tiny swimming sperm. Observe
them and make a sketch of one of them. <
the right gonad is deflected, look at its inner (left) surface and find the
sperm duct, or vas deferens. It
runs longitudinally along the margin of the testis and on the left surface of
the ovary. It
receives numerous dendritic branches from the testis and empties via several seminal
papillae into the atrium,
but not at the atrial siphon.
to most accounts, Molgula
manhattensis is an oviparous
species in which gametes are released to the sea where fertilization and
development occur. Some
species ofMolgula are
viviparous and retain eggs in the atrium where they are fertilized and develop. These
species brood their embryos and release tadpole larvae from the atrium. Some
viviparous species are easily confused with M.
manhattensis and collections
from the American northeast may contain some specimens of a viviparous species.
your specimen has large eggs and/or embryos in the atrium, it is one of the
viviparous species and you should take advantage of the opportunity to study
development in species that brood their embryos. First,
inform the instructor of your good fortune and be prepared to share the embryos
with the rest of the class.
a wetmount with a supported coverslip, in seawater, of several of the embryos. Look
at the embryos with the compound microscope. You
can probably find all developmental stages from unfertilized eggs to tadpole
will be about the same size.
eggs will not have an
elevated egg membrane but will have a large, conspicuous, clear, spherical
400X, yolk platelets can
easily be seen in the cytoplasm of the unfertilized egg, and of all other
stages, for that matter. Zygotes will
resemble the unfertilized egg in being a single cell but they will have an
elevated egg membrane. Look
for two-cell, four-cell,
and more advanced stages up to tadpole larvae.
numerous small cells around the periphery of the embryo, but inside the egg
membrane, are "test" cells. Their
function and origin is not well understood.
follicle cells can be seen adhering to the outside of the egg membrane. These
cells are spherical when they surround developing ova but flatten as the egg
of the embryos will probably be recognizable tadpole
larvae curled within the egg
membrane (Fig 29-24). The
tail of the larva is wrapped around the body. The
large vacuolated cells of the notochord can
be seen easily. The
black statolith in the larval cerebral vesicle can also be seen but no ocellus
is present in Molgula tadpoles. Using
400X, focus on the surface of the embryo and you will see the numerous small
individual cells of its epidermis. Their
nuclei can also be seen.
very large, thin, delicate pharynx underlies
most of the mantle and extends dorsally and posteriorly from the buccal siphon. The
open space surrounding the pharynx is the atriumand
it is continuous with the sea outside via the atrial siphon, as you have
atrium is an ectodermal invagination that surrounds the pharynx on the dorsal
and lateral, but not the ventral, surfaces.
tear or cut the numerous connections between the mantle and the pharynx on the
right side but avoid damaging the pharynx. These
points of attachment of the connective tissue of the mantle to the wall of the
pharynx are the sites of entry of blood vessels into the pharyngeal wall. Remove
the mantle from the right side of the specimen.
a wetmount of a small square of the mantle from the vicinity of the siphons and
examine it with the compound microscope. Note
the widely spaced, relatively weak transverse, longitudinal, and oblique muscle
the mantle removed you will have a better look at the renal
if the heart is still visible. If
it is still beating take advantage of the clearer view you now have and make any
observations you were unable to make earlier with the mantle still in place.
you have removed the mantle, the wall of the pharynx (variously
known as the branchial basket, branchial sac, pharyngeal basket, or pharyngeal
sac) will be exposed. Most
of the tissue you see inside the mantle is the pharynx.
fine scissors to open the pharynx by cutting through the right pharyngeal wall
from the anterior buccal siphon to the posterior esophagus (Fig 29-15A). To
do this insert one blade of a fine scissors in the buccal siphon and cut
posteriorly on the right side until you reach the posterior end of the pharynx
at the pyloric gland. Deflect
and pin the pharyngeal wall and examine the interior. Relocate
the opened buccal siphon at the anterior end and the opening to the esophagus at
the opposite end.
pharyngeal wall is perforated by minute gill
slits (= stigmata). Find
an area of the pharynx wall that is over a dark background and look at it with
the highest power of your dissecting microscope. Against
such a background you can barely make out the maze of tiny, elongate, curved
gill slits in the wall. The
gill slits of molgulids are spirals rather then the more usual ovals (Fig
longitudinal ridges or folds, the branchial
pleats, are present on each side (right and left) of the inner wall of
the pharynx. These
pleats are an apomorphy of Stolidobranchia (Fig 29-26D). Several
longitudinal blood vessels are contained within each pleat but the number
vessels of different sizes
exit the longitudinal vessels and run between successive branchial pleats and
their vessels. The
largest ones are easily seen and are about the same size as the longitudinal
vessels in the pleats. The
pharynx is well supplied with blood vessels and functions as the respiratory
surface as well as a filter for feeding.
the dorsal and ventral midlines of the pharynx and locate the endostyle on
the ventral midline (Fig 29-15A,B,C). It
extends from the base of the buccal siphon to the posterior end of the pharynx. It
is a narrow, wavy, ciliated groove lying between two wide ridges (Fig 29-15C). It
is wide and conspicuous and easily recognized. It
superficially resembles one of the many branchial folds in the pharynx wall but
on closer inspection will be seen to be entirely different.
endostyle is composed of ciliated and glandular cells. The
glandular cells secrete an iodine-containing mucous net (Fig 29-15D) and the
endostyle is homologous to the vertebrate thyroid gland (which is also an
endodermal derivative of the floor of the pharynx). Frontal
cilia on the inner surface of the pharynx move the mucous net dorsally to cover
the inner wall and the gill slits. Water
passes through the gill slits to the atrium but food particles are retained by
the mucous net and remain in the lumen of the pharynx. The mucous net greatly
reduces the pore size of the pharyngeal filter making it possible to utilize
smaller food particles.
the dorsal midline of the pharynx, opposite the endostyle, is the less
conspicuous dorsal lamina ( Fig
is a narrow, longitudinal, ciliated ridge and gutter. It
begins at the base of the buccal siphon and runs to the esophagus.
the buccal siphon the dorsal lamina is joined by two circumferential ciliated
ridges, the peripharyngeal
bands that begin at the
anterior end of the endostyle and run around both sides of the top of the
pharynx at the base of the buccal siphon (Fig 3).
mucus and trapped food moves dorsally from the endostyle and is gathered by the
dorsal lamina and rolled into a string. The
ciliary current of the lamina moves the mucus/food string posteriorly to enter
the esophagus. In
many tunicates (eg Ciona)
the dorsal lamina is replaced by a longitudinal row of papillae, the dorsal
languets, that perform the same function.
fine scissors, remove a small piece of pharyngeal wall being sure to include a
branchial pleat and some transverse vessels. Make
a wet mount, being sure the tissue is not folded over itself, and examine it
with the compound microscope. Note
the spectacular array of short, curved gill
at them with 100X, then 400X. Find
the conspicuous fringe oflateral cilia on
the margins of the gill slits. These
are the cilia that generate the flow of water in the buccal siphon, through the
gill slits, and into the atrium. Regardless
of length, the width of the gill slits is uniform and is about double the length
of the lateral cilia. Another
set of cilia, the frontal cilia, move the mucus net dorsally from the endostyle
to the dorsal lamina. Frontal
cilia are more difficult to see in this view as they extend vertically out of
the plane of the slide and parallel to your line of vision so you are looking at
them in end view.
your examination of the wetmount, look for longitudinal
blood vessels in the
branchial folds (Fig 29-26D). Look
for transverse blood vessels leaving
the longitudinal vessels and running perpendicular to them. Many
sizes of transverse vessels are present and they branch repeatedly to supply the
walls of the pharynx with blood. The
spaces between the gill slits are the blood spaces and they contain abundant
corpuscles. If the blood is moving,
they are easily recognized for what they are and the extent of the blood vessels
into all the connective tissue spaces between the gill slits is readily apparent
to the dissected animal and use a Pasteur pipet to place some carmine particles
suspended in seawater on the surface of the endostyle, pharyngeal wall, and
dorsal lamina. Watch closely for
ciliary currents. <
a drop of 1% methyl green/seawater on the inside surface (the surface facing
you) of the left wall of the pharynx. Some
of the dye may be moved dorsally by the frontal cilia but most of it should be
moved rapidly through the walls of the pharynx by the lateral cilia.<
Neural Gland and Cerebral
the anterior end of the dorsal lamina, on the dorsal midline between the atrial
and buccal siphons, is a large, spherical dorsal
tubercle (Fig 3, 29-23). The
two peripharyngeal bands that encircle the base of the buccal siphon pass around
the dorsal tubercle to join the dorsal lamina.
surface of the dorsal tubercle bears a C- or horseshoe-shaped groove that is the
opening of a ciliated duct that leads to the neural gland (Fig 29-23). Through
this opening seawater enters the neural gland from whence it is admitted to the
hemal system to replace fluid lost from the blood vessels of the gills.
opaque, oval neural gland is
easily seen in the tissue dorsal to and close to the dorsal tubercle (Fig 3). It
is about the same size as the tubercle. A
homology between the neural gland and the vertebrate anterior pituitary gland,
as well as the echinoderm madreporite and stone canal, has been proposed. Despite
its name, the neural gland contains no neurons and has no nervous role.
nervous system of adult ascidians is very simple and the dorsal hollow nerve
cord and sense organs of the larva are absent. The cerebral
ganglion, or brain, is a long, narrow (fusiform), opaque, white (in
life) structure lying between the dorsal tubercle and the neural gland (Fig
long axis parallels the dorsal lamina. The
neural gland is dorsal to the cerebral ganglion in the stolidobranchs, whereas
in most ascidians the positions are reversed and the ganglion is dorsal to the
gland. A nerve
cord exits each end of the
ganglion and both branch soon after leaving the ganglion.
small, branched buccal tentacles surround
the opening of the buccal siphon into the pharynx (Fig 3, 29-15A). These
tentacles are sensory and, when stimulated with chemicals or touch, elicit
contraction of the mantle muscles and expulsion of the water in the pharynx,
along with the offending material. You
may have seen the effect of these tentacles when you tried to introduce dyes or
carmine into the buccal siphon. The
tentacles also prevent the entry of large particles. The
ring of tentacles marks the end of the buccal cavity and beginning of the
Figure 3. View
of the dorsal midline of the anterior end of the pharynx of Ciona
intestinalis, viewed from inside the pharynx. The
pharynx has been opened and its walls deflected. Uro77La.gif
Posterior Digestive System
the dorsal lamina to the postero-dorsal corner of the pharynx and relocate the
opening of theesophagus. In
life the esophagus and its opening are white but are surrounded by the orange or
brown mass of the pyloric
gland (= liver, = hepatic
the nadel into
the opening of the esophagus and trace it posteriorly to the stomach.
the left wall of the pharynx so the entire double loop of the gut is exposed. You
are looking at the right side of the gut. Find
the opening of the esophagus again and insert the fine point of your fine
scissors into it. Cut
along the length of the esophagus, stomach, and intestine.
inside the lumen of the gut and observe that the pyloric gland is the thickened,
folded, glandular epithelium of the stomach.
stomach gradually narrows to become the intestine but
the separation is indistinct. A
large typhlosole, or
longitudinal fold of the gut wall begins at the distal end of the esophagus and
bulges into the intestine for almost its entire length.
intestine may contain mucous
fabricated by the dorsal lamina from mucus secreted by the endostyle. Trace
the intestine for its entire length, opening it as you go. It
eventually ends at the anus,
which opens into the atrium at the base of the atrial siphon. Note
that the typhlosole ends just short of the anus.
inner, or right, surface of the left gonad is apparent in the lesser curvature
of the gut loop. It
is composed of a central, hollow ovary with a lobulated, peripheral testis on
a commercially prepared wholemount of a tadpole larva of unknown species. Very
little internal structure is discernable in most commercial slides but you can
see the general shape of the larva and a few structures. It
consists of an ovoid anterior body with
a long, slender, muscular, posterior tail. In
some preparations a thick, transparent, almost invisible tunicsurrounds
the body. The notochord is
usually visible in the center of the tail. Within
the body the cerebral vesicle (=
sensory vesicle) should be apparent. It
contains at least one (as inMolgula), but more often two, opaque
is a statocyst for
gravity detection and the other is an eyespot for
cerebral vesicle will become the cerebral ganglion of the adult. Two adhesive
papillae are present at the
extreme anterior end. These
will be used to attach to the substratum when the larva leaves the plankton and
begins its sessile adult life. The
gut, with nonfunctional pharynx, endostyle, and intestine are usually an
amorphous mass whose details cannot be recognized in these slides. Buccal and
atrial siphons, although present, are neither open nor functional.
J, Hancock A. 1907. The
British Tunicata, vol II. Ray
Society, London. 164p,
E.J.W. 1965. The
Biology of Hemichordata and Protochordata . Oliver
and Boyd, Edinburgh. 176p.
NK. 1950. The
Tunicata, with an account of the British species. Ray
Society, London. 354p.
P, Cloney RA. 1997.
Urochordata: Ascidiacea, pp 221-347 in Harrison FW, Ruppert
EE (eds.) Microscopic anatomy
of invertebrates. Wiley-Liss, New York. 537pp.
Kleinholz LH. 1950. Molgula
manhattensis. in F.
A. Brown, Selected Invertebrate Types. Wiley,
New York. 597p.
TJ, Haswell WA. 1921. A
Text-book of Zoology, vol II. MacMillan. London.
Ruppert EE, Fox RS,
Barnes RB. 2004.
Invertebrate Zoology, A functional evolutionary approach, 7 th ed.
Brooks Cole Thomson, Belmont CA. 963 pp.
Name WG. 1945. The
North and South American ascidians. Bull.
American Mus. Nat. Hist. 84:1-476, 31 pls.
8-cm culture dish
Small dissecting pan (sardine tin with wax bottom)
# 1 stainless steel insect pins
Dissecting set with microdissecting tools
Isotonic magnesium chloride
Molgula manhattensis ,
living (preferred) or preserved.
0.1% methyl green/seawater
1% methyl green seawater
manhattensis are available from
Woods Hole Marine Biological Lab. Preserved specimens from Wards Natural