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Brine Shrimp
27-01-04
Copyright 2001 by
Richard Fox
Lander University
Preface
This is one of many exercises available from Invertebrate Anatomy OnLine,
an Internet laboratory manual for courses in Invertebrate Zoology.
Additional exercises can be accessed by clicking on the link. A glossary and
chapters on supplies and laboratory techniques are also available through
this link. Terminology and phylogeny used in these exercises correspond to
usage in the 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.
Systematics
Arthropoda P, Mandibulata, Crustacea sP, Eucrustacea,
Thoracopoda, Phyllopodomorpha, Anostraca C, Artemiidae F (Fig 16-15, 19-90)
Arthropoda P
Arthropoda, by far the largest and most diverse animal taxon, includes
chelicerates, insects, myriapods, and crustaceans as well as many extinct
taxa such as Trilobitomorpha. The segmented body primitively bears a pair of
jointed appendages on each segment. The epidermis secretes a complex
cuticular exoskeleton which must be molted to permit increase in size.
Extant arthropods exhibit regional specialization in the structure and
function of segments and appendages but the ancestor probably had similar
appendages on all segments. The body is typically divided into a head and
trunk, of which the trunk is often further divided into thorax and abdomen.
The gut consists of foregut, midgut, and hindgut and extends the length
of the body from anterior mouth to posterior anus. Foregut and hindgut are
epidermal invaginations, being derived from the embryonic stomodeum and
proctodeum respectively, and are lined by cuticle, as are all epidermal
surfaces of arthropods. The midgut is endodermal and is responsible for most
enzyme secretion, hydrolysis, and absorption.
The coelom is reduced to small spaces associated with the gonads and
kidney. The functional body cavity is a spacious hemocoel divided by a
horizontal diaphragm into a dorsal pericardial sinus and a much larger
perivisceral sinus. Sometimes there is a small ventral perineural sinus
surrounding the ventral nerve cord.
The hemal system includes a dorsal, contractile, tubular, ostiate heart
that pumps blood to the hemocoel. Excretory organs vary with taxon and
include Malpighian tubules, saccate nephridia, and nephrocytes. Respiratory
organs also vary with taxon and include many types of gills, book lungs, and
tracheae.
The nervous system consists of a dorsal, anterior brain of two or three
pairs of ganglia, circumenteric connectives, and a paired ventral nerve cord
with segmental ganglia and segmental peripheral nerves. Various degrees of
condensation and cephalization are found in different taxa.
Development is derived with centrolecithal eggs and superficial cleavage.
There is frequently a larva although development is direct in many.
Juveniles pass through a series of instars separated by molts until reaching
the adult size and reproductive condition. At this time molting and growth
may cease or continue, depending on taxon.
Mandibulata
Mandibulata is the sister taxon of Chelicerata and in contrast has
antennae on the first head segment, mandibles on the third, and maxillae on
the fourth. The brain is a syncerebrum with three pairs of ganglia rather
than the two of chelicerates. The ancestral mandibulate probably had
biramous appendages and a J-shaped gut, posterior-facing mouth, and a
ventral food groove. The two highest level mandibulate taxa are Crustacea
and Tracheata.
Crustacea sP
Crustacea is the sister taxon of Tracheata and is different in having
antennae on the second head segment resulting in a total of 2 pairs, which
is unique. The original crustacean appendages were biramous but uniramous
limbs are common in derived taxa. The original tagmata were head but this
has been replaced by head, thorax, and abdomen or cephalothorax and abdomen
in many taxa. Excretion is via one, sometimes two, pairs of saccate
nephridia and respiration is accomplished by a wide variety of gills,
sometimes by the body surface. The nauplius is the earliest hatching stage
and the naupliar eye consists of three or four median ocelli.
Eucrustacea
Eucrustacea includes all Recent crustaceans except the remipedes. The
taxon is characterized by a primary tagmosis consisting of heat, thorax, and
abdomen although the derived condition of cephalothorax and abdomen is more
common. Eight is the maximum number of thoracic segments.
Thoracopoda
In the ancestral thoracopod the thoracic appendages were turgor
appendages used for suspension feeding in conjunction with a ventral food
groove. Such appendages and feeding persist in several Recent taxa but have
been modified in many others.
Phyllopodomorpha
The compound eyes are stalked primitively although derived sessile eyes
occur in many taxa.
Anostraca C
Anostraca is an ancient line of primitive crustaceans found today in
inland, relictual, waters such as snow-melt pools, wet weather ponds, saline
or alkaline lakes, or similar refugia where there are few or no predators.
Anostracans have no carapace (anostraca = without shell) and are thought
to be similar in many features to the ancestral crustaceans. The body is
elongate and shrimp-like and with little regional specialization of segments
or appendages. The heart is a long dorsal tube extending most of the length
of the body and equipped with segmental ostia. Anostracans are gonochoric
and the gonads are paired, tubular coelomic derivatives. The excretory
organs are saccate nephridia derived from coelomic remnants.
The gut is J-shaped with paired digestive ceca. Most anostracans are
suspension feeders and/or bottom scrapers. The thoracic appendages are broad,
flat phyllopods resembling those of the ancestral crustacean. The
exoskeleton is thin, flexible, only weakly sclerotized and is not calcified.
The ladder-like nervous system is typical of primitive arthropods (and
annelids) and exhibits no cephalization of ventral ganglia. Development
includes nauplius and zoea larvae.
Laboratory Specimens
Anostracans are the best examples of primitive crustacean morphology
readily available for study in introductory invertebrate zoology courses. Of
the crustaceans usually available for study in teaching laboratories,
anostracans are the most primitive and provide the closest approximation to
the presumed condition of the ancestral crustaceans.
Brine shrimp, in the genus Artemia, inhabit inland saline lakes worldwide.
They are tolerant of a wide range of salinities ranging from almost fresh
water to saturated. Artemia is easy to rear from inexpensive, commercially
available eggs and is thus easily studied alive at any time of the year.
Artemia franciscana is the most common North American species. It occurs in
Great Salt Lake and is the species whose eggs are sold commercially in North
America. This species produces desiccation-tolerant resting eggs that remain
viable in dry condition for several years. These eggs can be purchased
inexpensively from Carolina Biological, Ward's Natural Science, or at local
pet stores. With little effort the eggs can be hatched to produce living
nauplius, advanced larvae, and adults for the laboratory. Rearing
instructions are provided at the end of this exercise. Artemia is restricted
to saline lakes and consequently cannot be collected locally in most places.
Anostraca is a homogeneous taxon and its members are similar to each
other so that any species can be used for this study. If possible, living
specimens should be used but preserved specimens will also serve. Preserved
specimens are suitable for the study of external anatomy but are much
inferior for observation of internal structures. Fairy shrimps, such as
Eubranchipus, occur in temporary ponds and ditches, are morphologically
similar to Artemia, and can be used for this study. Fairy shrimps are
widespread but their occurrence is spotty and unpredictable and they are not
available alive from most supply houses. If they are available from local
wet weather ponds they are recommended for this exercise. Commercially
prepared wholemount slides of Eubranchipus are less satisfactory than
unmounted preserved specimens.
External Anatomy
Use the dissecting microscope to study an adult brine shrimp (Artemia) or
fairy shrimp (e.g. Eubranchipus). If the animal is alive, place it in a
small square watch glass of chloroform-saturated water (See Supplies and
Recipes Chapter). If the specimen is preserved, place it in a dish of
tapwater. Use your minuten nadeln to manipulate the specimen and study it
with the dissecting microscope.
Tagmata
The anostracan body is divided into three tagma; head, thorax, and
abdomen (Fig 1). The head and its appendages are specialized, as they were
in the ancestral crustacean. The thoracic appendages are similar to each
other and are unspecialized phyllopods. The abdomen lacks appendages.
Head
The head at the anterior end is composed of five coalesced segments as it
is in all crustaceans (Fig 1). It bears a pair of stalked, lateral, compound
eyes and a single, median, unstalked naupliar eye at the anterior end (Fig
2, 3). The eyes, although stalked, are not considered to be segmental
appendages.
The small chemosensory first antennae are the appendages of the first
head segment (Fig 1, 2, 3, 19-11A). They are uniramous, and unjointed.
The second antennae are larger and are sexually dimorphic. Those of adult
males are very large and modified to form a clasping organ to hold the
female during copulation (Fig 19-11A). They are composed of two articles.
Female second antennae are smaller, about the length of the first antennae
but much thicker, and are composed of a single article (Fig 2). Determine
the sex of your specimen.
The labrum, or upper lip, is a large, median, ventral fold of body wall
arising just posterior to the bases of the second antennae (Figs 1, 2). It
is not paired and is not a segmental appendage. It extends posteriorly and
covers the ventral surface of the head, including the mouth.
The two, oval, bulging mandibles lie on either side of the head and are
the appendages of the third head segment (Figs 1, 2, 3). The mandibles curve
medially and touch each other on the midline where the ventral borders bear
teeth.
The mouth is on the ventral midline between the two mandibles (Fig 2). It
may be necessary to move the labrum aside to see the ventral ends of the
mandibles and the mouth.
The first and second maxillae are small and difficult to see. The first
maxilla is larger than the second and bears a bundle of anteriorly directed
setae on its medial edge (Fig 2, 3). The first maxillae are immediately
posterior to the mandibles on the ventral surface of the head and are used
to transfer food from the thoracic appendages to the mouth. The tiny,
conical second maxillae are vestigial and bear a few setae and the
nephridiopores (Fig 2, 3).
The adult excretory organs are the two maxillary glands, or coxal glands,
in the segment of the second maxillae where they form conspicuous bulges on
its dorsolateral surfaces (Fig 3). These are typical crustacean saccate
nephridia. Their coiled ducts may be visible within the bulges. The
maxillary glands open via the nephridiopores on the second maxillae.
Thorax
The remainder of the body is the segmented trunk consisting of an
anterior, limb-bearing thorax and limbless, posterior abdomen. The thorax
consists of 11 independent segments. No carapace is present and, since none
of the thoracic segments is fused with either the head or with each other,
there is no cephalothorax.
Each thoracic segment bears a ventral pair of leaflike thoracopods (=
thoracic appendages) known as phyllopods (Figs 1, 3). The 11 pairs of
phyllopods are similar to each other and exhibit no regional specialization,
differing only in size. The phyllopods are turgor appendages in which the
exoskeleton is thin and flexible and blood pressure is required to keep the
appendages stiff. The phyllopods are used for swimming, feeding, and
respiration.
The gross features of the phyllopod are visible with the dissecting
microscope but observation of detail requires preparation of a wet mount and
examination with the compound microscope. At present you should use the
dissecting microscope to study intact phyllopods while they are attached to
the animal (Fig 3). Later you will remove a phyllopod and make a wetmount.
Do not remove a phyllopod until you have completed the study of the internal
anatomy and then return to the following description for a detailed study.
For now, find only the larger features visible with the dissecting
microscope.
There are similarities between the parts of the phyllopods and the
ancestral biramous appendage but proposed homologies between these parts are
not universally accepted. The appendages are functionally uniramous although
they have parts that are thought to be homologous to the two rami of a
biramous appendage. Each appendage is flat and leaflike (phyll = leaf) and
thus resembles the phyllopodous portion of the ancestral biramous mixopod.
There is no stenopodous (cylindrical) portion.
Find the large basal protopod attached along its dorsal edge to the body
(Fig 4, 19-11B). Several processes extend from the lateral and medial
borders of the protopod. Any process from the lateral border of a crustacean
limb is an exite and any process from the medial border is an endite. You
can see some of these with the dissecting microscope but they will be
clearer later under the compound microscope.
Five or six endites, some of which are very small, extend from the medial
margin of the protopod (Fig 4, 19-11B). The proximal and distal endites are
the largest and easiest to see. Three much smaller, and harder to see,
middle endites lie between the proximal and distal endites. Note the array
of medially directed setae on the endites.
The densely setose proximal endite is often referred to as the gnathobase.
Its setae form a setal comb, or filter, of finely spaced setae used to
filter food particles from the water. The large distal endite may be
homologous to the endopod of the ancestral biramous appendage.
The three large exites on the lateral margin of the protopod are easily
seen with the dissecting microscope. The proximal and middle exites do not
bear setae. The middle exite is the epipod, which was once thought to be a
gill although it now appears to be involved in osmoregulation.
The distal exite may be homologous to the exopod of the biramous
appendage. It is the only process attached to the protopod by an
articulation. It bears long plumose natatory setae used for swimming.
Plumose setae are feather-like to increase their effectiveness as oars.
Later you can inspect them with high magnification to see the side branches.
Feeding
Most anostracans are suspension feeders although a few are carnivores
that consume other species of anostracans. A longitudinal, midventral food
groove lies between the gnathobases of the phyllopods (Fig 3, 19-12)). The
mouth faces the anterior end of the groove. Swimming movements of the
phyllopods draw a water current into the groove. The water is then forced
laterally through the setal comb on the gnathobases and food particles are
prevented from leaving the groove. The food is moved anteriorly in the
groove by the setae of the gnathobases. At the anterior end it is entangled
in mucus from the labrum and transferred to the mouth by the setae of the
first maxillae.
>1a. Place a living active brine shrimp in an 8-cm culture dish or square
watch glass with a few milliliters of brine. Add a drop or two of yeast/Congo
red suspension and observe the animal with the dissecting microscope. Red
food particles quickly accumulate on the setal comb of the phyllopods and in
the food groove. This colors the food groove red making it easy to visualize.
In a few minutes the red material appears in the anterior gut, rendering it
highly visible as well. Set the dish aside and return to it later in
conjunction with your study of the digestive system. <
Genital Region
The two segments posterior to the thorax are the genital segments (Fig 1)
and bear the external genitalia. Females have a conical pouch called the
brood sac (= ovisac) which may contain eggs (Fig 1, 5). Males bear a pair of
tubular, retractile penes which can be extended to four times their resting
length. The retracted penes are visible posterior to the last pair of
phyllopods in male specimens.
Abdomen
The abdomen consists of the six segments posterior to the genital region
and is nearly cylindrical (Fig 1). The posterior end of the body is the
telson. There is a caudal furca with two short rami on the end of the telson.
None of the abdominal segments bears appendages. The anus is located on the
telson at the base of the caudal furca (Fig 5).
Internal Anatomy
Living specimens should be used for the study of internal anatomy if
possible. Many internal features are not visible in preserved material due
to its opacity. Living specimens should be studied with the dissecting
microscope in small dishes of chloroformed saltwater (for Artemia) or
pondwater (fairy shrimp). This should be supplemented by examination of
wholemounts of small specimens with the compound microscope. Instructions
for preparing the wholemount are provided in the next section.
Digestive System
The gut is a simple tube extending the length of the animal (Fig 1,
19-11A). Most of it is easy to see in living specimens, especially if the
animal has fed recently on a yeast/Congo red suspension. The mouth is
located on the ventral midline of the head between the opposing surfaces of
the mandibles (Fig 3). The short vertical esophagus extends dorsally from
the mouth to open into the stomach above the mouth (Fig 3). The mouth and
esophagus are easiest to see in wholemounts of living specimens viewed from
the side with the compound microscope. The mouth and esophagus make up the
foregut and arise during ontogeny from the stomodeum, an invagination of
surface ectoderm.
The stomach is an expanded region of the gut in the middle of the head (Figs
3, 6). Two spherical digestive ceca bulge from the anterolateral walls of
the stomach (Fig 6).
The intestine is a long tube extending posteriorly from the stomach
through the thorax and most of the abdomen (Fig 1, 5). The stomach, ceca,
and intestine make up the midgut and are endodermal derivatives. The midgut
is the site of enzyme secretion, digestion (hydrolysis), and absorption. It
is surrounded by the hemocoel and bathed in blood so that uptake of
materials occurs across its thin walls.
The intestine joins the short rectum, or hindgut, in segment 4 of the
abdomen (Fig 1, 6). The hindgut, like the foregut, is ectodermal and is
lined by a chitinous exoskeleton. It is responsible for formation of fecal
pellets and opens to the exterior via the anus between the caudal furcae.
The anus is equipped with a sphincter.
The rectum develops from an ectodermal invagination, the proctodeum.
Early in development the rectum is not yet continuous with the midgut being
separated from it by a partition. Focus with high power on the junction of
the mid- and hindguts and see if there seems to be a flow of particles from
one to the other.
1b. Return to the animal left in the yeast/Congo red suspension earlier (or
make such a preparation now). Use the dissecting microscope to observe the
shrimp as it swims in its dish and look at the gut. By now the entire length
of the digestive tract should be filled with red particles, making it easy
to see. It takes only about 15 minutes for yeast to move the length of the
gut. Remove the shrimp from the dish and make a wetmount with it for
examination with the compound microscope. The regions of the gut should be
clearly visible with the compound microscope. Save this slide and refer to
it as you study the remaining internal organ systems. Add tapwater to the
slide as necessary so that it does not dry out. <
Hemal System
The anostracan hemal system is a good example of the primitive arthropod
condition and it is easy to see how it could develop from the dorsal blood
vessel of an ancestor.
The heart is a long, median, dorsal tube extending the length of the
trunk (Fig 1). It may be visible in living, and sometimes preserved,
material with the dissecting microscope but it is best studied with the
compound microscope using wholemounts of small living shrimp.
>1c. Prepare a wetmount of a small (about 5 mm), living, unanesthetized
brine shrimp. Arrange the shrimp on a slide so you will have a side view of
the abdomen and then place a coverslip over the animal. The coverslip will
immobilize the specimen but will not immediately stop the heart. Study the
animal with the compound microscope and find a place where you can see the
abdomen or thorax in side view.
Find the large, tubular intestine, which will be opaque if the animal has
been feeding. The transparent, also tubular, heart lies dorsal to the
intestine and is easily seen in good preparations (Fig 1). It is surrounded
by the pericardial sinus, which is a region of the hemocoel and is not a
coelomic space. Anteriorly the heart becomes the short aorta which empties
into the anterior hemocoel.
Look for the paired openings, the ostia, in the lateral walls of the
heart. Most trunk segments have a pair. The easiest one to see, however, is
the large, unpaired terminal ostium at the posterior end of the heart (Fig
1). Ostia are valved pores in the wall of the heart that admit blood from
the pericardial sinus into the heart lumen.
Note the beating of the heart. There is no obvious peristalsis and the
entire heart appears to contract simultaneously. Anteriorly it opens into
the hemocoel, whose spaces extend throughout the tissues of the animal.
Elevated pressure in the heart lumen closes the ostia so that blood must
exit through the aorta at the anterior end.
Look for small spherical, or ovoid corpuscles and use them as markers to
visualize the flow of the blood. The corpuscles tend to move posteriorly in
the pericardial sinus outside the heart. Watch closely and you may see some
of them pass through ostia in the walls of the heart and then reverse
direction and move anteriorly once inside the lumen of the heart. They move
in surges associated with contractions of the heart. Entry of corpuscles
into the heart is easiest to see in the terminal ostium in the sixth
abdominal segment. Sometimes the blood contains hemoglobin in solution in
the plasma. Hemoglobin is most likely to be present in animals from water
with low dissolved oxygen concentrations. There may be sufficient pigment to
impart a pinkish color.<
The heart is surrounded by a special compartment of the hemocoel, the
pericardial sinus (Fig 16-7). In living and preserved specimens this area is
relatively free of solid tissues and is transparent. It is separated from
the rest of the hemocoel, which lies ventral to it, by a perforated,
horizontal septum through which blood flows on its way back to the heart.
Contractions of the heart force blood out its open anterior end of the aorta
into the body hemocoel. Blood flows through the hemocoel and over the
tissues while making its way posteriorly. It is aided in its flow by
movements of the appendages and their muscles. Blood flows from the body
hemocoel into the pericardial sinus through the perforations in the
horizontal partition and then passes posteriorly in the sinus and enters the
ostia of the heart.
Respiratory and Excretory /
Osmoregulatory Systems
Most gas exchange is accomplished across the permeable surfaces of the
phyllopods.
The two maxillary glands in the segment of the second maxilla (Figs 1, 3)
are usually referred to as excretory organs but, in fact, their role is
largely osmoregulatory and they have little to do with the excretion of
metabolic wastes. Nitrogen is lost as ammonia across the phyllopod surfaces.
Each maxillary gland consists of an enclosed end sac, derived from a
coelomic space, from which a long excretory duct leads to the nephridiopore
located on the tiny second maxilla. The duct wraps around the end sac and
its coils can be seen through the integument on the side of the head (Fig
1). The gland is surrounded by hemocoel and bathed with blood. The
epithelium of the end sac is equipped with podocytes and forms an
ultrafiltrate of the blood into the lumen of the end sac. The ultrafiltrate
is modified as it passes down the duct to the exterior. Artemia is an
efficient osmoregulator and is strongly euryhaline, being tolerant of an
impressively wide range of salinities.
Artemia can keep its blood hyposmotic to environments more saline than
about 10 parts per thousand. This is something most marine invertebrates
cannot do, at least not to the same extent. Artemia drinks brine and
actively secretes salts from the maxillary glands, epipods, and gut. The
maxillary glands can produce urine four times as salty as the blood.
Maintenance of a hyposmotic blood is facilitated by the impermeability of
most of the integument. The exoskeleton, with the exception of the epipods,
is impermeable to salts. The epipods are major sites of active salt
secretion. Artemia belongs to a predominantly freshwater taxon and
presumably evolved from freshwater, not marine, ancestors.
>1d. The permeability of the epipods in comparison with the
impermeability of the rest of the body surface can be demonstrated with
silver nitrate. Remove some small brine shrimp (about 5 mm is a convenient
size) from their dish of saltwater. Wash the salt from the outside of the
body by placing them in a dish of distilled water for a minute or two. With
a pipet, transfer one shrimp to a square watch glass of 0.002M silver
nitrate. When the shrimp stops swimming in a few minutes remove it to a
microscope slide. Place it ventral side down on the slide so the phyllopods
are splayed to the sides and add a coverslip. Examine the specimen with the
compound microscope. Silver nitrate reacts with chloride ions to form an
insoluble, opaque silver chloride precipitate that turns brown in light.
Inspect the shrimp for large dark opaque areas of concentrated silver
chloride. Such areas can form only where a permeable integument allows
chlorides from the blood to come in contact with silver nitrate. Does the
silver chloride seem to be localized or widespread? Pay particular attention
to the phyllopods. Does any particular area of the phyllopod appear to be
especially permeable? If so, which? Is this observation consistent with what
you already know about the functions of the parts of the phyllopod? <
Nervous System
The nervous system is difficult to study in whole specimens, either
living or preserved. It consists of a dorsal brain, paired circumenteric
connectives, and double, ventral nerve cord with segmental ganglia. The
brain is a mass of translucent tissue surrounding the naupliar eye in the
dorsal, anterior part of the head. You may be able to see it in living
specimens.
The sensory system includes the median naupliar eye which appears in the
earliest larval instar and persists throughout life. It consists of three
black pigment cups. Two cups face laterally and one points ventrally. Two
stalked, lateral compound eyes, each composed of numerous black ommatidia
are also present.
Reproductive System
The gonads of both sexes are paired tubes located dorsolaterally in the
posterior thorax and anterior abdomen (Fig 1). They may be faintly visible
as transparent, elongate sacs beside the intestine. They are easiest to see
in wetmounts of small, living specimens.
Reproduction
During mating the male approaches the dorsal side of the female and holds
her with his enlarged second antennae. The male twists his body around the
female, inserts the penes into the brood pouch, and deposits sperm. The
partners remain coupled for several hours during which copulation may occur
every few minutes. Eggs are released unto the brood pouch where they are
fertilized and covered with a shell.
Two kinds of eggs are produced. One has a thin shell and hatches in the
brood pouch. The other, known as the resting egg, has a heavy shell and can
remain viable for several years out of water and then hatch when immersed in
saltwater. This second type of egg is collected and sold by supply houses
and aquarium shops. Both egg types hatch into nauplius larvae.
Appendages
now to the postponed study of the thoracic appendages. Use
your nadeln and fine forceps to remove a phyllopod from your anesthetized
specimen and make a wholemount with it. Select an appendage from near the
middle of the thorax for this purpose. Examine the slide with the compound
microscope. Return to the description of thoracopods which appeared earlier
in this exercise and find the structures you were unable to see with the
dissecting microscope. Be sure to examine the plumose swimming setae of the
endopod and consider how their structure is adapted for swimming.
1e. Make a wholemount of a phyllopod if you have not already done so. Use
100 and 400X of the compound microscope to compare the many types of setae
characteristic of the different areas of the phyllopod.
The setae of the exopod (distal exite) are large, strong and plumose, or
pinnately branched. Plumose setae have lateral branches from the central
shaft and resemble a feather. These are swimming setae, or natatory setae,
that function as paddles to increase the surface area of the appendage and
enhance its effectiveness in swimming. The lateral branches of the seta
increase the effective surface area of the seta and make a little oar of it.
The setae of the endopod (distal endite) are short, fairly stout, and
finely serrate. These are scraping setae used to scrape algae from hard
substrata.
Setae on the three middle endites are similar but are heavier, generally
longer and more coarsely serrate. They are also scraping setae and these
three endites are sometimes called "claws".
The setae on the proximal endite are fine and delicate. They are very
finely plumose although that is not apparent unless the light is perfectly
adjusted. These are the filter setae of the setal comb used to remove food
from the water as it exits the sides of the food groove. Does the structure
of these different kinds of setae seem to be adapted to their functions? <
Behavior
Adult anostracans swim constantly using the phyllopodous thoracic
appendages. Waves of motion pass along the series of phyllopods to draw
water and food particles into the food groove and to create an effective
stroke with the swimming setae so that locomotion and feeding are
accomplished simultaneously. Artemia also feeds by scraping algae from hard
surfaces. Anostracans can make sudden quick moves by flexing the abdomen.
The caudal furca is also equipped with plumose swimming setae.
1f. With a dissecting microscope and incident illumination observe a
living, active adult brine shrimp swimming in a dish of water. Note the
orientation of the animal in the water. The preferred orientation is with
the dorsum down but Artemia can swim right side up also. The filter-feeding
mechanism works best when upside down. Which appendages are used for
locomotion? Do the antennae seem to be involved in swimming? Does the animal
ever stop swimming? Do you see any evidence of feeding by scraping the
bottom? Watch for movement resulting from flicking the abdomen. How does it
differ from motion produced by phyllopods? <
Artemia, with its three eyes, is sensitive to light intensity and
exhibits highly variable responses to light. In general, Artemia is
positively phototactic at low light intensities and photonegative at medium
and high intensities. The response varies, however, depending on the
physiological condition of the animal, wavelength, age, salinity, pH, and
metabolic condition.
>1g. Place a dish containing larvae in a part of the room where it will
receive uneven illumination. Leave the dish for 15 minutes or so and then
observe the distribution of shrimp. Do they seem to be clustered in any
particular region of the dish? How does this relate to the light source? You
may want to design a series of more carefully controlled experiments to
determine the effect of light intensity, life history stage, salinity,
wavelength, or temperature. <
Most animals exhibit what is called the "dorsal light reaction" in
response to the sun's rays by maintaining their dorsal surface up. A few
animals, such as Artemia, backswimmers (hemipterous insects), and the fish
louse, Argulus (a crustacean), reverse this response and exhibit a "ventral
light reaction" and keep the ventral surface pointed toward light.
Consequently, brine shrimp (and fairy shrimp) normally swim upside down
(ventral side up), because in nature the light is overhead. A brine shrimp
in a dish on the stage of a dissecting microscope with substage
illumination, however, may reverse its orientation and swims with the
ventral side down.
>1h. Place a brine shrimp or fairy shrimp in a small culture dish and put
it on the stage of the dissecting microscope with the incident lamp on and
the substage lamp off. Observe the swimming orientation of the animal. Turn
the incident lamp off and the substage (transmitted) lamp on. Do this
several times noting the response of the animal. <
Larvae
A laboratory culture of Artemia will provide representatives of the major
larval stages of a typical crustacean life cycle. Artemia requires about 14
molts to reach its terminal size and achieves sexual maturity in about 12
molts. The stages between successive molts are instars. Artemia larval
stages are the nauplius, which includes a metanauplius, and the zoea. No
megalops is present. A nauplius is a crustacean larva that swims with head
appendages, whereas a zoea is an older larva that swims with thoracic
appendages. The megalops, which is older still, swims with abdominal
appendages.
In Artemia the larvae hatch with few segments and gradually increase the
number to 19 with successive molts. The mitotically active teloblast areas
in the telson add new segment buds with each molt until the characteristic
number for the species is achieved. New limb buds appear on existing
segments with each molt. The 19 trunk segments of Artemia are added in
groups, rather than one with each molt.
Nauplius
Use a fine pipet to select several larvae of each of as many different
sizes as possible from the laboratory culture. Transfer them to a small
(8-cm) culture dish of chloroform-saturated saltwater or add a few drops of
chloroform to the culture dish and wait for the larvae to become
anesthetized. When the larvae stop moving, transfer some of the smallest to
a glass slide and make a wetmount with them. Try to arrange the larvae so
some have the venter facing up and others the dorsum. Support the coverslip
with wax feet and press it gently against the larvae but do not crush or
distort them. Refer to the Techniques chapter for instructions on making wax
feet.
With the compound microscope find one of the smallest larvae, which
should be a nauplius (Fig 7, 19-8). Artemia hatches as a nauplius with a
short body consisting of the first three head segments and a short trunk but
with no external segmentation.
Stored yolk gives the body an orangish color and renders it nearly opaque.
Due to this opacity it is difficult to discern internal structures. (The
interior is much easier to see in an older metanauplius in which the yolk
reserves have been exhausted and the tissues are transparent.) The nauplius
is lecithotrophic, living off its yolk reserves without feeding.
Find the dark, red or black, median naupliar eye at the anterior end of
the head. It consists of three pigment cup ocelli. The brain may be visible
surrounding the eye in older, more transparent embryos. No carapace is
present in adult or larval anostracans.
The anteriormost appendages are the small uniramous first antennae. The
second antennae are the largest appendages of the nauplius and are the
principal swimming organs (Fig 7). They are equipped with long natatory
swimming setae and are biramous. The chief swimming setae are on the exopod.
The third pair of appendages is the uniramous mandibles. The labrum is a
large, thin, ventral fold of body wall arising between the second antennae
and the mandibles, immediately anterior to the mouth (Fig 7B). It is not a
segmental appendage.
The posterior end of the body is the telson (Fig 7). The telson contains
the teloblast areas that produce the mesoderm from which new segments are
fashioned. Buds produced at the anterior edge of the telson differentiate
into new segments. The youngest segments are closest to the telson and the
oldest are anterior.
>1i. With the dissecting microscope observe a few active Artemia larvae
in a small culture dish of salt solution or seawater. Watch one swim and try
to see which appendages it uses. Swimming with a single pair of appendages
is erratic and jerky. Compare this motion with the smooth swimming of adults.
Adult swimming utilizes 11 pairs of appendages and is much smoother. <
Metanauplius
Try to find a small transparent larva about the size of the opaque,
orange nauplii but with a longer trunk. Such a larva will be a metanauplius.
The metanauplius is a slightly older larva but, since it swims with its head
appendages, is still considered to be a nauplius.
The metanauplius stage includes several molts and instars and lasts for
several days. It ends when the anterior thoracic limbs become functional in
the tenth instar. The metanauplius begins feeding when its yolk reserves are
exhausted.
The metanauplius has an unsegmented head to which is attached an elongate
thorax. The anterior end of the thorax is visibly segmented. The internal
organs of these slightly older larvae are easier to see than are those of
the nauplius.
The gut extends from the head posteriorly to the telson. The mouth is on
the ventral surface of the head, between the bases of the mandibles, under
the labrum but you probably cannot see it. The stomach is wider than the
rest of the gut and is situated dorsal to the mouth to which it is connected
by a vertical esophagus. Two diverticula, the digestive ceca, extend
anterolaterally from the stomach.
The intestine extends posteriorly from the stomach. In the nauplius it is
relatively short but it is long, narrow, and getting longer in the
metanauplius. The posterior end of the gut is hindgut, or rectum, and it
differs from the midgut in appearance. Its walls are nearly colorless
whereas those of the midgut are reddish brown with the remaining yolk. The
rectum opens to the exterior via the anus at the end of the telson.
Sessile lateral eyes appear in the metanauplius or zoea. They require
several molts to develop their stalks.
Zoea
The zoea stage, consisting of many instars, follows the metanauplius. The
zoeal sequence begins with a long transition period (known as the protozoea)
in which both the head and thoracic appendages are involved in swimming.
Loss of its swimming setae and cessation of swimming movements by the
second antennae signals the beginning of the true zoeal stage. (In Artemia
this period is sometimes referred to as the postlarva. The larva resembles
the adult but is smaller.) During this period the limb primordia continue
developing into functional limbs and the animal grows to its adult size. The
animal becomes an adult when all appendages are present, complete, and
functional but molting continues every few days through an adult life of
several months.
>1j. If protozoea or zoea are available, place a dish with a few larvae
on the stage of a dissecting microscope and watch them swim. Compare the
swimming with that of the nauplius. The swimming motion becomes
progressively smoother as more and more pairs of phyllopods replace the
single pair of antennae. <
References
Abbott DP. 1987. Observing Marine Invertebrates. Stanford Univ. Press,
Stanford. 380p.
Anderson DT. 1967. Larval development and segment formation in the
branchiopod crustaceans Limnadia stanleyana King (Conchostraca) and Artemia
salina (L.) (Anostraca). Aust. J. Zool. 15:47-91.
Bond RM. 1937. A method for rearing Artemia salina, in Needham, J. G. et
al., Culture Methods for Invertebrate Animals. Comstock, Ithaca. 205-206.
Brown FA. (ed) 1950. Selected Invertebrate Types. Wiley, New York. 597p.
Browne RA. 1993. Sex and the single brine shrimp. Natural History,
102(5):34-39.
Bullough WS. 1958. Practical Invertebrate Anatomy (2nd ed). MacMillan,
London. 483p.
Green J. 1981. Crustaceans, in Dales, R. P. (ed) Practical Invertebrate
Zoology. MacMillan, London. pp. 261-302.
Heath H. 1924. The external development of certain phyllopods. Jour.
Morphol. 38:453-483.
Linder F. 1941 (1946?). Contributions to the morphology and the taxonomy
of the Branchiopoda Anostraca. Zool. Bidrag. Uppsala 20:101-302.
Martin JW. 1992. Branchiopoda, pp 25-224 in Harrison, F. W. & A. G. Humes
(eds.). 1992. Microscopic Anatomy of Invertebrates vol. 9 Crustacea.
Wiley-Liss, New York.
Moore WG. 1957. Studies on the laboratory culture of Anostraca. Trans.
Am. Micros. Soc. 76:159-173.
Pardi L, Papi F. 1961. Kinetic and tactic responses, in T. H. Waterman,
The Physiology of Crustacea II. Academic Press, New York. p 365-399.
Pennak RW. 1989. Fresh-water Invertebrates of the United States, 3 ed.
Wiley, New York. 628p.
Personne G. et al. (eds.). 1980. The Brine Shrimp, Artemia, vols 1-3.
Universa Press, Wetteren, Belgium.
Ruppert EE, Fox RS, Barnes RB. 2004. Invertebrate Zoology, A functional
evolutionary approach, 7th ed. Brooks Cole Thomson, Belmont CA. 963 pp.
Walley LJ. 1969. Studies on the larval structure and metamorphosis of
Balanus balanoides (L.). Philos. Trans. Roy. Soc. London, Ser B 256:237-280.
Williamson, D. I. 1982. Larval morphology and diversity, pp. 43-110 in D.
E. Bliss (ed), The Biology of Crustacea vol 2. Academic Press, New York.
Supplies
Dissecting microscope
Compound microscope
8-cm culture dish or square watch glass
Yeast / Congo red suspension (See Supplies and Recipes chapter)
Slides and coverslips
Beeswax
plastic Pasteur pipets
chloroform-saturated seawater
iodine-free salt (e.g. ice cream salt)
distilled water
5-10 ml 0.002M silver nitrate
Brine shrimp (Artemia) eggs or colony
It is convenient to have a self-perpetuating colony in which nauplii,
zoea, postlarvae, and adults are always available. Alternatively, the
necessary life history stages can be produced by adding eggs to saltwater 48
hours, 96 hours, 1 week and three weeks prior to the day of the laboratory
in which they are needed.
Brine Shrimp Cultures
Brine shrimp are easily reared in a salt solution (brine) of almost any
salinity. A suitable concentration can be achieved using 40 g of non-iodized
salt (e.g. ice cream salt or non-iodized table salt) per liter of
chlorine-free fresh water. Use pond, spring, distilled, or aged tapwater.
The brine should be in a shallow, non-metallic (i.e. glass) pan or dish with
a large exposed surface area and shallow depth (about 2-3 cm). Do not
aerate.
With a lab marker, mark the original level of the brine on the side of
the container and add distilled water as needed replace evaporative losses
and maintain this level. Do not replace evaporative losses with more brine.
Do not cover the container as this restricts the oxygen supply.
To have adults and all larvae stages on the day of the laboratory
exercise, begin about 3-4 weeks prior to the day for which the animals are
needed. Add a tiny pinch of Artemia eggs to the brine in the shallow dish.
Add an additional tiny pinch of eggs every 4 days until about a week before
the animals are needed and then add a tiny pinch of eggs daily. Eggs will
begin hatching about 24-48 hours after being placed in the brine.
The second day after the first eggs hatch, a feeding regimen should be
instituted and maintained thereafter for the life of the culture. Prepare a
suspension of one package baker’s yeast in 100 ml of fresh water and store
it covered, in a refrigerator (See Supplies and Recipes chapter). Each day,
use a pipet to add enough of the suspension to render the water in the
culture slightly cloudy. Do not overfeed, do not add too many eggs, and do
not aerate. Keep the yeast suspension refrigerated. Do not add more yeast
than the shrimp can remove before the next feeding. The water should never
become opaque rather should be transparent or nearly so.
The population density must be low enough that sufficient oxygen can
enter the water by diffusion from the surface. Artemia larvae are sensitive
to increased carbon dioxide concentrations but under the conditions
described can be reared without artificial aeration. Aeration damages the
older, more fragile larvae and adults but does not harm nauplii and
metanauplii.
Larvae can be reared to maturity by this method and, in fact, continuous
cultures can be maintained with the adults of the first generation producing
eggs for subsequent generations. Nauplii will be available 24-48 hours after
the eggs are added to the water. Other larval stages will follow in turn.
Adults will appear in about three weeks and will be seen mating (swimming in
tandem) shortly after. Once adults appear, it is no longer necessary to add
eggs. Mature females produce thin-walled eggs which hatch in the brood
chamber and are released as larvae to perpetuate the colony.
If only nauplii or metanauplii are needed, feeding is not necessary and
the culture can be in a deeper container, such as a gallon jar, and aerated
with an airstone. Under these conditions the population density can be
vastly increased. Start such a high density culture (aerated) with 5 ml dry
eggs per 4 liters of saltwater 36-48 hours prior to the time the larvae are
needed. A culture with this density must be aerated vigorously. Aeration
does not damage the nauplii but will kill the more delicate older instars
and adults.
Sources
Artemia resting eggs are available from biological supply houses, pet
shops, and aquarium stores.
Living Eubranchipus are available Apr 1 to May 15 from Nebraska
Scientific, 3832 Leavenworth St., Omaha, Nebraska 68105.
Preserved Eubranchipus are available from Carolina Biological and Ward's
Natural Science.
Prepared wholemount slides of small fairy shrimps are available from
Carolina Biological and Triarch.
Prepared slides of Artemia nauplii are available from Triarch.
Ward's markets, under the name "Living Fossil" culture, vials of soil
from the bottom of temporary ponds that contain eggs of fairy shrimp,
tadpole shrimp, and cladocerans. Adults can be reared from this soil.
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