Invertebrate Anatomy OnLine
Papilio polyxenes ©
Carrot Caterpillar
30jun2006
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 links to the left. A glossary and chapters on supplies and laboratory techniques are also available. 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.
Systematics
Arthropoda P, Mandibulata sP, Tracheata, Hexapoda SC, Insecta C, Dicondylia, Pterygota, Metapterygota, Neoptera, Eumetabola, Holometabola, Lepidoptera O, Papilionoidea SF, Papilionidae F, Papilioninae sF, Papilio polyxenes (Fig 16-15, 20-14, 20-15, 21-23)
Arthropoda P
Arthropoda, by far the largest and most diverse animal taxon, includes chelicerates, insects, myriapods, and crustaceans as well as many extinct taxa. The body is segmented and 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. The body is typically divided into a head and trunk, of which the trunk is often itself 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. 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 and from 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 sP
Mandibulata includes arthropods in which the third head segment bears a pair of mandibles. As currently conceived this taxon includes myriapods, hexapods, and crustaceans. Appendages may be uni- or biramous and habitats include marine, freshwater, terrestrial, and aerial.
Tracheata
Myriapods and hexapods share tracheae and a single pair of antennae and are sister taxa in Tracheata. Crustaceans, which have gills and lack tracheae, are excluded and form the sister group.
Hexapoda SC
The body is divided into three tagmata; head, thorax, and abdomen. Appendages are uniramous and a single pair of antennae is present. Three pairs of legs and two pairs of wings are found on the thorax of most adults. Hexapod legs are uniramous although there is increasing evidence that they evolved from multiramous appendages of their ancestors. Gas exchange is accomplished by trachea. Excretory organs are Malpighian tubules and the end product of nitrogen metabolism is uric acid. There is relatively little cephalization of the nervous system. Insects are gonochoric with copulation and internal fertilization.
Insecta C
Most hexapods are insects. A few hexapod taxa (orders) lack wings and have primitive mouthparts recessed into the head and belong to Entognatha, the sister taxon of Insecta. Insects have ectognath mouthparts and the adults (imagoes) of most taxa have wings.
Pterygota
The winged insects. These insects are derived from a winged common ancestor. Adults of most taxa have wings although they have been lost in some.
Eumetabola
Juveniles have no ocelli and there are six or fewer Malpighian tubules.
Holometabola
The final larval instar pupates and undergoes a radical metamorphosis in which it is converted to an imago, or adult. The imago is sexually mature and in most taxa has wings whereas larvae are immature and wingless. During metamorphosis many or most larval tissues are dismantled and adult structures built anew. Wings, for example, are manufactured from clusters of undifferentiated cells known as imaginal discs but not from preformed wingpads as in pauro- and hemimetabolous insects.
Lepidoptera O
Butterflies and moths. Order consisting of the polyphyletic “moths”, skippers (Hesperoidea), and scudders (Papilionoidea). Skippers and scudders are together known as “ butterflies”. Wings, body, appendages covered with pigmented, dust-like epidermal scales or hair-like setae. Adults with highly derived sucking mouthparts and liquid diet. Adult mouthparts consist of large labial palps and a coiled tubular proboscis derived from the maxillary galeae. Mandibles absent. Larvae herbivorous with typical chewing mouthparts. Larval labial glands modified as silk glands. Holometabolous complete metamorphosis in a pupal stage. In many moths pupation occurs in a silk cocoon but most butterflies have no cocoon and the pupa is known as a chrysalis, sometimes associated with a few silk fibers. Lepidopteran pupae are obtect, with the appendages attached to the body over their entire length.
Laboratory Specimens
Adult swallowtails are large, familiar butterflies of many species in the lepidopteran family Papilionidae. This relatively small, mostly tropical family contains about 500-600 extant species of large, brightly colored, often sexually dimorphic, butterflies known as swallowtails. The world’s largest butterflies are swallowtails.
One species, Papilio polyxenes (= P. joanae ) (Papilio is Latin for "butterfly"), is known as the Black Swallowtail, or American Swallowtail. The imago has black wings with white or orange spots, depending on the subspecies. The larva, known as the Carrot Caterpillar or carrot “worm”, feeds on members of the carrot family, Apiaceae and are regular inhabitants of herb gardens in which parsley, dill, or fennel are grown.
Like that of most butterflies, the life cycle consists of egg, five larval instars, pupa, and imago. Butterflies are holometabolous insects in which the body undergoes a radical metamorphosis during the pupal instar. During metamorphosis wings develop de novo from imaginal discs (clusters of undifferentiated embryonic cells) in the larval thorax and muscles are reorganized for flight. The mouthparts are converted from the primitive chewing design suited for feeding on plant tissue to highly derived sucking mouthparts adapted for ingesting liquids. The gut is reorganized to accommodate the change in diet. The antennae form anew from imaginal discs in the head and the reproductive system develops. The prolegs are completely lost and the adult compound eye develops near the old larval ommatidia. The nervous system is reorganized, in part through condensation and cephalization of ganglia.
Larvae are green with black circumferential stripes over the dorsum of each segment. Some of these stripes lie in the center of the segments and are partially interrupted by yellow spots. Others are located on the anterior borders of the segments and not interrupted by yellow spots. The venter is white with black spots. The caterpillar is smooth and naked with tiny inconspicuous setae on the body. The setae of the prolegs, while tiny, are much more abundant than those of the body, although all are visible only with magnification. Color photographs and discussions are available at Oehlke (undated) and USGS (undated).
This exercise is written for living or freshly sacrificed larvae but preserved specimens can be used if necessary, although color and texture descriptions may not be applicable to preserved material. Fresh material is far preferable to preserved. Ideally the dissection should be conducted with a recently collected specimen whose gut is full. A dissecting microscope should be used. The exercise does not include adults.
External Anatomy
Examine a living, active (i.e. unanesthetized) late instar larva of the Black Swallowtail, Papilio polyxenes. Active caterpillars are difficult to manipulate but, with a little patience, most aspects of the external anatomy can be studied without anesthetization and it is best to do so to take advantage of the opportunity to observe locomotion and behavior. (If desired, however, the caterpillar may be anesthetized immediately to avoid the inconvenience of dealing with an uncooperative specimen.) Sometimes the caterpillar will sit quietly on the tip of your index finger, which can then be manipulated under the lens of a dissecting microscope to provide the desired view of the specimen. With Papilio larvae, you will have to put up with the stinky odor of the repugnatorial substance released from an, eversible, orange tentacle on the thorax.
The larva is caterpillar-like, i.e. eruciform, in having a more or less cylindrical body and well-developed sclerotized head capsule, thoracic legs and abdominal prolegs (Fig. 1). The body is smooth and naked, with inconspicuous, scattered, short setae, visible only with magnification. The body consists of a small anterior head, middle thorax, and large posterior abdomen. The thorax and abdomen together make up the trunk. The head is connected to the thorax by a short, soft neck, or cervix. Paired, jointed, sclerotized, segmental, appendages are present on the head and thorax but are lacking on the caterpillar abdomen. Paired, unjointed, segmental, fleshy prolegs are found on the abdomen. The body wall is heavily pigmented and opaque so that no preview of the viscera is possible prior to dissection.
Head
The head (Fig 21-1A,B*) is enclosed in a white, black, and yellow sclerotized head capsule, or epicranium, at the anterior end of the body (Fig. 1). It consists of several fused segments and bears the appendages of those segments and the mouth. The component segments of the head are not recognizable and the head appears to be unsegmented.
The posterior dorsum of the epicranium is the vertex, its sides are the genae, and the front is the frons (Fig. 2). A conspicuous Y-shaped epicranial suture divides the capsule into right and left halves. The fork of the “Y” is formed by two frontal sutures whereas the stem is the coronal suture.
The frons and clypeus lie between the two frontal sutures. The triangular frons is dorsal and the clypeus is situated on its ventral border. The bilobed labrum articulates with the ventral edge of the clypeus. The labrum is the anterior border of the preoral cavity (Fig 21-7). The preoral cavity is a space surrounding the mouth and formed by the ring of mouthparts around the mouth; labrum anteriorly, mandibles and maxillae laterally, and labium posteriorly.
Figure 1. External anatomy of a generalized caterpillar. Redrawn and modified from Snodgrass (1935). Lepid63L.gif
Insects are usually considered to have three pairs of mouthpart appendages; mandibles, maxillae, and labium. Most entomologists do not consider the labrum to be a segmental appendage. Posterior and ventral to the labrum are the large, dark, heavily sclerotized mandibles, each with a row of fine teeth on its free median margin. The cutting edges of the two mandibles oppose each other on the midline and are used to cut tissue from the plant. If your specimen is alive and active you may see the mandibles in operation. The mandibles form the sides of the preoral cavity and lie beside the mouth. Use a pair of fine forceps to move the mandibles apart and reveal the mouth. The mouth is plugged by the soft hypopharynx, or tongue, immediately posterior to it (Fig 21-7). With the mandibles held aside, push the hypopharynx posteriorly to open the mouth. The hypopharynx is a fold of the body wall and is not a segmental appendage and is not paired. The mandibles, mouth, and hypopharynx will be easier to study after you have anesthetized your specimen.
Orient the caterpillar on its back with its venter facing up, toward you, and study the two remaining mouthparts. The maxillae and labium are small, inconspicuous, and best studied after the specimen is anesthetized. Immediately posterior to each mandible is a maxilla consisting of a large, sclerotized, basal stipes from which protrudes a tiny, lateral, biarticulate maxillary palp and a medial galea bearing strong apical spines (Fig. 2). During metamorphosis the galea is transformed into the large sucking proboscis characteristic of adult lepidopterans (Fig 21-3). The labium, which is derived from a fused pair of posterior head appendages, lies on the midline between the two maxillae. Its large bulbous anterior portion bears a pair of vestigial labial palps at its base. The labial palps become greatly enlarged in adults and, along with the maxillary proboscis, are the only functional adult mouthparts. The median spinneret lies between the two palps. You may notice strands of silk associated with the caterpillar. The silk is extruded from the spinneret.
Figure 2. Dorsal view of the head capsule of the larvae of Papilio polyxenes. Anterior is at the top. Lepid61L.gif
Posteriorly the head capsule is penetrated by a large opening, the foramen magnum, through which the gut, nerve cord, silk ducts, hemocoel and tracheae pass between head and thorax. The foramen is filled with soft tissue and is obscured when the head is attached to the thorax but you can make out its outline by pushing the soft tissue of the cervix aside. With fine forceps, pull the head forward to expose the soft, unsclerotized cervix connecting it with the trunk.
Antero-laterally each side of the capsule bears an ocularium consisting of a C-shaped ring of the colorless, transparent cornea, or lenses, of six large isolated ommatidia, sometimes referred to as ocelli but lepidopterans have no true ocelli (Fig. 2). The ocularium is a derived compound eye with six discontiguous facets. The ommatidia are obscured by a patch of black pigment and are best found by using higher magnification to look at the anterior corner of the sides of the head capsule. The corneas look like tiny clear beads on the surface of the epicranium.
A short, inconspicuous, cylindrical antenna can be seen lateral to each mandible. The antennae are between the mandibles and the ommatidia. The antennae and ommatidia should be revisited after you have anesthetized the specimen and it ceases to resist your efforts to examine it.
Thorax
The insect thorax consists of three segments, prothorax, mesothorax, and metathorax, each of which bears a pair of sclerotized, jointed thoracic legs known as the forelegs,midlegs, and hindlegs, respectively (Fig. 1). In caterpillars each thoracic leg consists of a series of cuticular cylinders decreasing in diameter distally. The largest cylinder is the proximalcoxa, which articulates with the body (Fig. 3, 21-1E). It is followed in turn by the femur, which is fused with a vestigial trochanter, then the tibia, and finally a biarticulate tarsus. The second tarsal article is a darkened, sclerotized, hooked claw.
The prothorax bears a sclerite, the pronotum, or prothoracic shield, covering its dorsal surface. An oval black prothoracic spiracle can be seen on each side of the prothorax near the edge of the pronotum (Fig. 1). These are actually the spiracles of the mesothoracic segment that have migrated anteriorly. The mesothorax and metathorax have neither sclerotized nota nor spiracles.
Figure 3. The forelegs of the catalpa caterpillar, Ceratomia catalpae. Lepid59L.gif
The prothorax has a transverse dorsal slit from which a forked orange tentacle, the osmeterium, can be protruded (Fig. 1). As you handle a living caterpillar, it will undoubtedly evert this tentacle and release a malodorous repugnatorial substance consisting of 2-methyl-buteric acid and isobuteric acid. You are invited to touch the tip of finger to the osmeterium and then sniff the finger to fully experience the aroma. You will be reminded of the experience for the next hour or so until the compounds dissipate.
The osmeterium is a forked, or Y-shaped, invagination of the body surface and is normally retracted out of sight into the hemocoel of the dorsal thorax (Fig. 1). On superficial examination it appears to be two tentacles but these are, in fact, united at their bases. Elevation of blood pressure in the hemocoel everts the osmeterium and retractor muscles withdraw it. The repugnatorial substances are secreted by the epithelium of the invagination and not by a discrete gland. Osmeteria are characteristic of papilionid larvae.
Abdomen
The abdomen consists of a succession of ten segments and is hemispherical, or loaf-shaped in cross section, i.e. rounded on top and flattened ventrally (Fig. 1). Abdominal segments 3-6 and 10 each bear a pair of soft abdominal prolegs. Those of the 10 th segment are the anal prolegs. The prolegs make good landmarks for recognizing the segments by number. Each proleg terminates in a central sucker bordered by a row of sclerotized hooks, or crochets. The suckers can be used to grasp smooth surfaces whereas the crochets function primarily in attaching to silk or other rough surfaces.
Abdominal segments 1-8 each bear a pair of oval black spiracles which open into the extensive tracheal system (Fig. 1).
Segment nine lacks either spiracles or prolegs. Segment 10 bears the anal prolegs and is covered dorsally by a lightly sclerotized suranal sclerite (Fig. 1). The anus can be found hidden under this sclerite.
If you are fortunate, your caterpillar may present you with one or more fecal pellets. If so, dissect one at about 20X and demonstrate to yourself that it is enclosed in a delicateperitrophic membrane (Fig 21-9). Inside the membrane you will find the uniformly sized and shaped bites of plant tissue cut from the host by the mandibles.
Internal Anatomy
The remainder of the exercise should be performed on an anesthetized or recently sacrificed specimen. It makes little difference which. Anesthetize (or kill) a caterpillar by immersing it in 7% ethanol (non-denatured) in a small, wax-bottom dissecting pan. Dissecting pans made from anchovy fillet tins are ideal for most caterpillars and fit conveniently on the stage of a dissecting microscope. The animal will succumb in 5-10 minutes.
Spend a few minutes observing features that were difficult to see (such as the ommatidia, head appendages, mouth, spiracles, thoracic legs, prolegs, crochets, and anus) when the animal was active.
Your specimen may periodically extend and retract the crochets of the prolegs. Watch the crochets under high magnification of a dissecting microscope during one of these cycles. At rest the curved crochets are deployed and would engage silk strands or another rough substratum. When withdrawn they release their hold on the substratum and permit movement of the proleg.
Examine a spiracle at about 40X. Each spiracle consists of a black, sclerotized, cuticular ring surrounding a recessed atrium from which the spiracular trachea arises. The atrium is covered and protected by a filter apparatus consisting of a ring of pale, closely spaced setae. The atrium and tracheae lie under the filter and cannot be seen from the surface. A slit-like atrial orifice penetrates the center of the filter and opens into the atrium below it. Insert a minuten nadel into the orifice to demonstrate its presence and continuity with the atrium. Use the nadel to demonstrate that the filter is composed of bristles extending toward the orifice from the surrounding ring. In most insects, including Papilio, the spiracle is equipped with a valve, under muscular control, that allows the animal to control evaporation from the tracheal system and thus is important in water conservation.
Find the articles (coxa, femur, tibia, tarsus with terminal claw) of a thoracic leg using 40X.
The insect body cavity is the hemocoel (Fig 16-7), which is filled with circulating blood that bathes the tissues and transports food, wastes, and hormones (but not oxygen). Organs and tissues, including the gut, nerve cord, fat bodies, salivary glands, and excretory organs, are suspended in the hemocoel and surrounded by blood. In insects, the hemocoel is usually divided by two horizontal septa (dorsal and ventral diaphragms) into three longitudinal sinuses. The dorsal pericardial sinus encloses the heart, the middle perivisceral sinus is by far the largest and contains the gut and other viscera. The small, ventral perineural sinus surrounds the ventral nerve cord. The following dissection opens the hemocoel for a study of its organs.
" Insert one blade of a fine scissors under the posterior edge of the suranal sclerite and cut anteriorly through the body wall well to the right of the middorsal line so that the median heart and pericardial sinus are not damaged. Use #1 stainless steel insect pins to secure the edges of the body wall against the floor of the dissecting pan. Begin at the posterior end and work your way anteriorly, inserting each pin at a 45 ° angle and stretching the body wall laterally and anteriorly as you pin it.
The heart will end up on the left side of the incision. Remember this so you can find it later after it is pinned to the dissecting pan. The perivisceral sinus and dorsal blood vessel will ideally remain attached to the dorsal body wall but they may adhere to the mass of fat body and connective tissue surrounding the gut and be torn away from the body wall. Watch for these structures as you make your longitudinal incision and try to keep them with the body wall. Avoid cutting deeper than the body wall. Much of the space in the hemocoel is filled with the amorphous white or yellow, leafy fat body in addition to tracheae and muscles but the dominant structure is the gut. It may be necessary to detach some of the fat body from the body wall in order to pin the latter. Extend the incision anteriorly to the posterior border of the head capsule but do not cut into the capsule. Larvae of parasitoid wasps and/or flies may occur in large numbers in the hemocoel and should be removed and counted if present.
The body cavity opened by this incision is the perivisceral sinus, a division of the hemocoel (Fig. 4, 5, 16-7). It is filled with blood (hemolymph). The much smaller pericardial sinus, which is also filled with blood, surrounds the heart and has been pinned aside with the body wall.
Hemal System
The insect hemal system consists of a tubular dorsal blood vessel extending the length of the body immediately under the dorsal midline and enclosed in the pericardial sinus (Fig. 4, 5, 16-7). Posteriorly, for most of the length of the abdomen, the vessel is the contractile heart. The vessel continues anteriorly through the thorax to the head as the non-contractile aorta.
Look for the heart in the pericardial sinus attached to the body wall and pinned to the wax of the dissecting pan on the left side of your specimen. It may still be beating weakly. The heart is a transparent, colorless tube on the dorsal midline. It is difficult to see. Anteriorly the aorta and pericardial sinus are closely invested by two discrete, narrow bands of fat body and the aortacan be seen between the two bands. The heart is not bordered by discrete bands of fat body and is harder to see. Find the aorta and trace it posteriorly to find the heart. The heart has muscular walls penetrated by segmental pairs of ostia. Large, translucent, strap-like, obviously fibrous dorsal longitudinal body wall muscles lie on either side of the heart and are reminiscent of the continuous muscle layers of an annelid rather than the individual muscles of an arthropod. Individual muscles are also present.
Fat Body
In Papilio the fat bodies are conspicuous in the perivisceral sinus as seemingly amorphous white or yellow leafy sheets attached to the body wall and extending throughout the hemocoel. The system typically consists of a peripheral fat body forming a layer attached to the body wall and a perivisceral fat body associated with the gut wall as well as smaller concentrations with specific organs. Note the tracheae extending to the fat bodies.
Figure 4. Internal anatomy of a generalized caterpillar viewed from the left. Two of the three left Malpighian tubules have been truncated for clarity. Redrawn and modified from Snodgrass (1935). Lepid64L.gif
The fat body is a large, multipurpose, mesodermal organ consisting of cells involved in intermediary metabolism, protein synthesis, and storage of lipids, carbohydrates, proteins, uric acid, and energy. In most insects the chief period of feeding, growth, and energy storage is as juveniles for adults feed relatively little or not at all. The energy accumulated by juveniles is stored in the fat body and later used to support the activities, including dispersal and reproduction, of the imago. The thin morphology, only one or two cells thick, exposes a large surface area to the hemolymph and places all cells in contact with it for the exchange of materials.
Digestive System
The digestive system fills most of the space in the perivisceral hemocoel. It lies on the ventral midline where it is associated with the extensive white or yellow fat body and receives abundant tracheal tubes. The salivary (silk) glands and Malpighian tubules are close to the gut and should be protected from damage during the dissection.
The gut consists of anterior ectodermal foregut, middle endodermal midgut, and posterior ectodermal hindgut, which may themselves be regionally specialized. Note the numerous branches of the visceral tracheal system extending to the gut. Move the lobes of the fat body aside, or remove them entirely, as necessary to improve your view of the gut. Do not damage the inconspicuous salivary glands or Malpighian tubules. The two salivary glands (silk glands) are long, clear, unbranched tubes whereas the Malpighian tubules, are small twisted (zigzag) tubes. Both are long and looped.
The foregut of Papilio, which is lined by cuticle, consists of mouth, pharynx, esophagus, and crop. Its function is chiefly storage and to connect the mouth with the midgut. The pharynx and esophagus are of small diameter and are largely enclosed within the head capsule. You have already seen the mouth. The esophagus can be seen emerging from the capsule as it widens to become the crop in the prothorax (Fig. 4). Both have thin transparent walls. The crop is a storage chamber occupying most of the space in the thoracic perivisceral sinus. It is probably full of recently ingested food particles cut from the host plant by the mandibles.
The midgut is a little larger in diameter than the crop and it occupies most of the anterior abdomen from the metathorax posteriorly to about segment 8. Foregut and midgut are separated by the stomodeal (cardiac) valve. The walls of the midgut are thicker than those of the foregut and are opaque or translucent. Abundant tracheae can be seen radiating from each spiracle to the nearby region of the midgut. These are part of the visceral tracheal system which will be explained later. Avoid damaging the tracheae. The midgut epithelium is responsible for synthesizing and secreting hydrolytic enzymes and for absorption of the products of hydrolysis. Most hydrolysis takes place in the midgut lumen.
The midgut narrows abruptly in abdominal segment 8 to form the proctodeal valve separating midgut from hindgut. The hindgut, which is cuticularized like the foregut, consists of a short intestine (anterior intestine) in segments 8-9, a short rectum (posterior intestine) in segments 9-10, and the anus, which you have already seen. Water and salts are reclaimed from the feces and fecal pellets are formed and stored in the hindgut. The intestine and rectum are separated from each other by a constriction. Later you will open the gut and see the valves more clearly.
A pair of glands opening on the labium and accordingly known as the labial glands function in most insects as salivary glands and secrete saliva with hydrolytic enzymes adapted for the diet of the species. Such labial glands are sometimes referred to as salivary glands. In some taxa, most notably Lepidoptera (butterflies and moths), Trichoptera (caddisflies), and Hymenoptera (bees, ants, and wasps), the larval labial glands secrete, not saliva, but a proteinaceous silk used for a variety of purposes and are known as silk glands. In addition, caterpillars have a pair of mandibular glands, opening on the mandibular segment, that secrete saliva whose enzymes assist in the digestive process. The mandibular glands probably will not be seen.
In Papilio each silk gland is a long, unbranched, colorless, translucent tube lying to the right or left of the gut in the perivisceral hemocoel (Fig. 4). A common duct from the spinneret arises within the head capsule, where you cannot see it, and bifurcates to form the right and left silk gland ducts. These narrow, transparent ducts can be seen emerging laterally from the foramen magnum to enter the thorax where they are embedded in fat body. The silk ducts are small in diameter but increase in diameter to become the silk glands. Each gland extends the length of the trunk to the posterior abdomen and is free of the fat body over part of its length.
In the caterpillar of the silk moth, Bombyx mori, secretory cells in the epithelium of the posterior region of the gland secrete fibroin, the major silk protein, composed of about 40% glycine. The central region of the gland secretes other proteins whose function is to hold fibroin strands together. Note the tracheae extending to the glands.
Although the true salivary glands do not secrete saliva, caterpillars also have a pair of mandibular glands and these do secrete saliva whose enzymes assist in the digestive process. The mandibular glands probably will not be seen.
Respiratory System
A typical insect respiratory system of interconnected tracheae is present in Papilio larvae and its major features can be demonstrated by observing a spiracle from inside the body. The left first abdominal spiracle is probably the best to use for this purpose but any clearly visible, undamaged spiracle with intact tracheae can be used. Because of the position of our initial incision, the tracheae are less likely to have been cut on the left side.
Tracheae arise from each spiracle and branch dendritically (tree-like) to supply nearby tissues and organs with oxygen and remove carbon dioxide from them (Fig. 5). Each spiracle is associated with clusters of tracheae radiating outward from the spiracle and with unbranched longitudinal tracheal trunks connecting adjacent spiracles. The branched tracheae supply nearby tissues with oxygen whereas by connecting spiracles, the longitudinal trunks form a single continuous system extending throughout the caterpillar. The tracheal system is complex and ramifies throughout the body to provide oxygen to all tissues. The hemal system plays no role in oxygen transport in insects. In the course of your dissection you have probably noticed the pervasiveness of the tracheal system. Some of the tracheae of your immersed specimen may still contain air. This renders them white or silver and makes them easier to see.
In general, each spiracle opens into a short chamber, the atrium, which connects with the lateral longitudinal trunk. These trunks extend along the row of spiracles and connect them. From the junction arise three major clusters of segmental tracheae, each with many branches. Together these supply the nearby tissues with oxygen. The dorsal segmental trachea and its many branches extend to the dorsal body wall and heart musculature. The visceral segmental trachea sends branches to the gut, fat body, silk glands, and other organs. The visceral segmental tracheae are the easiest to see of the three clusters. The ventral segmental trachea supplies the ventral body wall musculature and nerve cord.
Fig. 5. Diagrammatic cross section of a generalized insect abdominal segment. Redrawn from Snodgrass (1935). Lepid65L.gif
In some segments the right and left dorsal segmental tracheae join each other across the dorsal midline to form a dorsal transverse tracheal commissure. Similarly some ventral segmental tracheae form ventral transverse commissures. Additional longitudinal trunks may be associated with the gut and dorsal body wall. These are smaller than the lateral longitudinal tracheal trunks. Note that, because of the longitudinal and transverse connections, the tracheae form a single interconnected system so that all parts of the body, whether or not they have spiracles, are supplied with tracheae and oxygen. The head and some thoracic segments, for example, lack spiracles but nevertheless are provided with oxygen by the system.
" Use fine scissors to remove a piece of a lateral longitudinal trunk and make a wet mount with it. Examine the preparation with 100X, then 400X of the compound microscope. Note the chitinous rings, known as taenidia, that reinforce the walls of the trachea and hold it open, much like the cartilaginous rings that hold your trachea open.
Excretory System
The excretory system consists of nephrocytes in the hemocoel and Malpighian tubules, also in the hemocoel. Lepidopteran larvae (and adults) have six Malpighian tubules, three arising on each side, right and left, of the midgut-hindgut junction in the vicinity of the proctodeal valve, at the constriction between midgut and intestine (Fig. 4, 16-9). On each side the cluster of three arises from a common ampulla evaginated from the gut. The tubules are supplied with tracheae. Proximally, the tubules emerge from the gut at about the level of abdominal segment 8-9 and loop back and forth in the perivisceral hemocoel, making them difficult to count. To further complicate the pattern in Lepidoptera, the distal (posterior) ends of the six tubules enter the connective tissue of the rectum wall and end there, out of sight, and there are thus no free ends of the tubules. The connections of both ends of the tubules with the gut are difficult to demonstrate in gross dissection. The tubules are small in diameter and in this species are uniformly zigzagged in the manner of the rick-rack used by seamstresses to decorate clothing (Fig. 6). They may be yellow or whitish.
Fig 6. A short section of a Malpighian tubule of Papilio polyxenes. Lepid62L.gif
Nephrocytes are scattered throughout the hemocoel but most are concentrated in the dorsal diaphragm surrounding the heart. They are not conspicuous in Papilio as they are in some caterpillars. Nephrocytes are thought to be storage kidneys that absorb and sequester a variety of particles and dyes but they do not phagocytose bacteria.
Digestive System Interior
" Make a longitudinal, middorsal incision along the entire length of the gut to expose its lumen, which is probably filled with small pieces of dill or parsley or whatever the caterpillar was eating. In the midgut the food mass of leaf or stem particles is enclosed in a thin transparent peritrophic membrane (Fig 21-9). Use a plastic Pasteur pipet to remove the leaf fragments. Note that the peritrophic membrane interferes with your attempts to suck the gut contents into the pipet and the contents tend to remain packaged in the membranous tube. Note also that this is not true of contents in the crop, where there is no peritrophic membrane.
The foregut (crop) is separated from the midgut by an inconspicuous stomodeal valve, which in Papilio, is manifest as an orange circle of thickened gut wall (Fig. 4). In many insects the valve is more elaborate.
The peritrophic membrane is secreted by cells in the vicinity of the stomodeal valve. Note that there is no peritrophic membrane anterior to the foregut-midgut junction. The peritrophic membrane is secreted by the midgut epithelium to enclose the food mass. It is a porous mesh with the microvilli of the midgut epithelium protruding through its pores.
The midgut walls are opaque due to the thick, orange mucosal epithelium which lines the lumen. The mucosal epithelium is secretory and absorptive. The walls are folded transversely. Observe the lining of the midgut with about 30X of the dissecting microscope. The brush border of microvilli of the absorptive mucosal epithelium is visible as a greenish-blue iridescent outline caused by the microvilli acting as a diffraction grating. This iridescence is less evident in Papilio than it is in some caterpillars such as Ceratomia and Calpodes. Posteriorly the midgut epithelium is much thicker and more intensely colored. A short length of midgut with lightly pigmented walls connects the midgut with the hindgut.
The midgut and hindgut are separated by a thick, muscular proctodeal valve (pyloric valve) visible as a ring of tissue encircling the lumen. The crest of the valve bears a ring of small sclerites. The anterior intestine also has a ring of small sclerites but these are in patches of fine sclerites whereas those of the proctodeal valve are larger solitary sclerites. The walls of the intestine and rectum are marked by thick longitudinal folds, or rugae. Find the anus from inside the rectum.
Osmeteria
Locate the orange osmeterium in the prothorax if you have not already done so. Each fork resembles the finger of a glove and is an invagination of the body surface. The osmeterium turns inside out as it is everted and that of your specimen may be either everted pr retracted. Grasp one end and pull it to retract or evert it as appropriate. Look for the retractor muscles whose function is to withdraw the invagination into the hemocoel. Rapid eversion is accomplished by elevated hemocoelic pressure.
Nervous System
Papilio has a primitive, largely uncephalized nervous system. In its primitive condition the insect central nervous system includes the tripartite brain, or supraesophageal ganglion, consisting of proto-, deuto- and tritocerebrum and the ventral nerve cord consisting of segmental ganglia joined by paired longitudinal connectives (Fig 16-11). The brain innervates the eyes (protocerebrum), antennae (deutocerebrum), and labrum (tritocerebrum). The tritocerebrum is connected with the double longitudinal ventral nerve cord by a pair of circumesophageal connectives. Immediately posterior to the connectives is the subesophageal ganglion consisting of the fused ganglia of the mandibles, maxillae, and labia. In caterpillars the segmental ganglia of the thorax and abdomen remain independent of each other and are spaced along the nerve cord.
" Cut the tracheae on the right side of the gut, move the gut to the left, and pin it so the ventral midline is revealed. Remove fat bodies as necessary to reveal the ventral midline of the thorax and abdomen being careful that you do not damage the nerve cord on that midline. Do not attempt to remove the small ventral fat bodies associated with the nerve cord. The white segmental ganglia and nerve cord are easy to see on the midventral body wall after the gut is moved aside. For most of its length the longitudinal connectives are coalesced to form the nerve cord.
The brain and subesophageal ganglia are far anterior and can best be seen by tilting the head capsule and looking into the foramen magnum. Using 20X of the dissecting microscope, move the gut aside as necessary to reveal the brain dorsal to the gut and the subesophageal ganglion ventral and posterior to it (Fig. 4). The two are joined by the pair ofcircumesophageal connectives. The subesophageal ganglion, which serves the mandibles, maxillae, and labium, is in the posterior head capsule. It is connected by a pair of short connectives with the first thoracic ganglion. The second thoracic ganglion is well separated from the first thoracic ganglion and connected to it by a pair of long, well-separated connectives. The third thoracic ganglion is far posterior to the second and is connected with it by a pair of long connectives, also widely separated.
Abdominal ganglia 1-6 are widely spaced along the length of the abdomen and are connected by coalesced connectives, which form an apparently single ventral nerve cord. With a forceps or tiny needle you can demonstrate that the cord actually consists of two contiguous longitudinal connectives. Abdominal ganglia 7-8 are coalesced and appear to be a single ganglion. Several nerves radiate posteriorly from the eighth ganglion but the nerve cord itself ends with the eighth ganglion. The combined longitudinal connectives, whether they are coalesced or separate, form the ventral nerve cord (Fig. 4, 5, 16-11).
Examine a ganglion, such as the first abdominal, with 40X and find the pair of inconspicuous segmental nerves exiting the ganglion laterally and connecting it with the periphery. Note that the nerve cord, ganglia, and nerves are supplied with tracheae. Do not confuse segmental nerves with tracheae
Note that ventral longitudinal body wall muscles, similar to the dorsal muscles seen beside the heart, lie beside the nerve cord.
Reproductive System
The reproductive system of caterpillars is rudimentary and will develop in the pupa during metamorphosis. Small testes, however, are present in late instar larvae and spermatogenesis occurs in these larvae and in the pupa. Eggs develop in the pupa. Development of both internal and external genitalia occurs during metamorphosis in the pupa.
*Hyphenated figure call-outs, such as this one, refer to figures in Ruppert, Fox, and Barnes (2004). Those without hyphenation refer to figures embedded in this exercise.
References
Borror DJ, Triplehorn CA, Johnson NF . An introduction to the study of insects, 6 th ed. Saunders College Publishing, Philadelphia. 875pp.
Chapman RF. 1998. The insects, Structure and function, 4 th ed. Cambridge Univ. Press, Cambridge. 769 pp.
Comstock JH. 1930. An introduction to entomology. Comstock Pub., Ithaca. 1044 pp.
Gillott C. 1995. Entomology, 2 nd ed. Plenum Press, New York. 798 pp.
Oehlke W. undated. Papilio polyxenes asterius: the Black Swallowtail. http://www3.pei.sympatico.ca/~david.fraser/rasteriu.htm
Ross HH. 1965. A textbook of entomology, 3 rd ed. John Wiley & Sons, New York. 539pp.
Ruppert EE, Fox RS, Barnes RB. 2004. Invertebrate Zoology, A functional evolutionary approach, 7 th ed. Brooks Cole Thomson, Belmont CA. 963 pp.
Snodgrass RE . 1935. Principles of insect morphology. McGraw-Hill, New York. 667 pp.
USGS . Undated. Butterflies of North America, Black Swallowtail (Papilio polyxenes). US Geological Survey, Northern Prairie Wildlife Research Center. www.npwrc.usgs.gov/resource/distr/lepid/bflyusa/usa/895.htm
Supplies
1 living, freshly collected, late instar Papilio polyxenes caterpillar
1 small (anchovy tin) wax-bottom dissecting pan
1 dissecting microscope
1 compound microscope (can be shared by several students or the entire class)
1 microdissecting forceps
1 microdissecting (iridectomy) scissors
2 minuten nadeln with applicator stick handles
20 # 1 stainless steel insect pins
7% non-denatured ethyl alcohol
1 plastic Pasteur pipet