Objectives of the labs: External Anatomy of Insects: The Exoskeleton, Head and Mouthparts Exoskeleton exoskeleton structures

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1 Objectives of the labs: 1. Define and correctly use the vocabulary. 2. Examine and identify head, thoracic and abdominal modifications 3. Examine the different anatomical modifications of different insects and thus determine the differences in their lifestyles. External Anatomy of Insects: The Exoskeleton, Head and Mouthparts 1. Exoskeleton Utilize some prepared slides, som and take a thin slice of exoskelton from a grasshopper and other preserved specimens (Tabanus = horsefly, Ishnura = damselfly, Magicicada = cicada, Lethocerus = giant water bug, Peiplaneta americana = cokroach, Apis mellifera = honeybee) to determine the structure of the exoskeleton and associated structures. a) Remember that the exoskeleton covering the insect s body is an extremely thin structure consisting of two basic layers: a cellular epidermis and a non-cellular cuticle, which is further subdivided into epicuticle, exocuticle and endocuticle (together forming the procuticle). When preparing a thin section of exosketon for histological observation, insert the piece of exoskeleton into a styrofoam peanut (in an orientation that allows you to see the skeleton in cross-section when you are done) and make thin sections using a sharp single-edged razor blade (careful, do not cut yourself). You may want to collect the thin section you have created using a drop of water on a slide. Follow this collection by staining the section using methylene blue, which will allow you to stain the nucleated epidermis and highlight the nuclei. Cover the section using a cover slip and observe under the compound miscroscopes provided. b) Use bee in order to observe the setal modifications you may expect in certain insects. Bees are covered in the hair-like setae. Place a drop of glycerine onto a clean slide, pull some setae from the body of a bumble bee (you may need to use the dissecting microscope) using forceps, place it onto the drop of glycerine and cover it with a cover slip. Observe your preparation under the microscope and draw the setae. Also utilize some moths or butterflies you collected and observe the setae in much the same manner. Be sure to take a scrape of the moth or butterfly wings for this part of the exercise, in order to correctly observe the setae. 2. Head and mouthparts Preserved grasshoppers (leaf feeders, with biting and chewing mouthparts to observe head and mouth structures The head is enclosed in a hard, heavily sclerotized, unsegmented, exoskeletal head capsule, or epicranium. The mouthparts are attached ventrally to the capsule, a condition known as hypognathus. The mouth, which you will see later, is located on the ventral surface of the head and is surrounded by the mouthparts. These mouthparts enclose a space, the preoral cavity, from which the mouth opens. The head is more or less ovoid in shape with a nearly vertical anterior surface. Note the composition of the head capsule. Most of the head is enclosed in a single hard piece of exoskeleton known as the epicranium. This is composed of the fused sclerites of the head segments. It covers the dorsal anterior and lateral aspects of the head and is divided into regions. The region of the epicranium anterior to the compound eyes is the frons. The side, or cheek, posterior to the compound eye is the gena. Ventral to the frons is the clypeus. The unpaired clypeus articulates with the epicranium. The short lateral edges of the clypeus are notched. The vertex is the top of the capsule, dorsal to the frons and between the compound eyes. The vertex extends anteriorly above the bases of the antennae as a prominent, pointed, concave 1

2 rostrum. The posterior wall of the head capsule is penetrated by a large aperture, the foramen. Through it pass the organ systems, such as nerve cord, esophagus, salivary ducts, and musculature, connecting the head with the thorax. In an intact specimen the foramen is filled with soft tissue but you can see its circular outline. The occiput (occipital arch) is a narrow band forming the posterior edge of the head capsule arching dorsally over the foramen. pair of large multiarticulate, sensory antennae is attached in deep sockets on the antero-dorsal corners of the head. The antenna consists of two basal articles, the proximal scape and distal pedicel, to which is attached the long multiarticulate filamentous flagellum. The antennae are the first pair of segmental appendages of the insect head and are innervated by the deutocerebrum of the brain. A large, bulging compound eye is located dorsolaterally on each side of the head just posterior to the base of the antenna. Examine the surface of a compound eye and note that it is composed of an uncountable number of small light-receiving units known as ommatidia. The surface of the eye is a specialized, transparent part of the exoskeleton divided into tiny hexagonal cuticular lenses, or corneas, one for each ommatidium. In addition to the two compound eyes there are three tiny, simple eyes, or ocelli. These are not composed of ommatidia. Two of them are lateral ocelli located between the compound eyes and the antennae. The unpaired median ocellus is located on the anterior midline of the head in a pit on the vertical ridge between the two antennae. The labrum, or upper lip, is also located on the vertical anterior face of the head capsule. It is a large movable plate, equipped with muscles, ventral to the clypeus. It bears a small median notch on its ventral border and a median groove on its anterior surface. Some entomologists believe the labrum to be derived from a fused pair of anterior appendages and that the notch and groove represent the line of fusion between the fused right and left appendages. The labrum is the anterior wall of the preoral cavity and it covers the more posterior mouthparts. Its hidden posterior surface bears abundant setae, or bristles. With fine forceps lift the labrum to demonstrate its mobility and to reveal the preoral cavity and the remaining head appendages. The labrum is innervated by the tritocerebrum indicating it is the homolog of the crustacean second antenna and supporting the hypothesis that it is a segmental appendage. Immediately posterior to the labrum is a pair of mandibles. In grasshoppers these massive mouthparts are adapted for biting and chewing. Their heavily sclerotized and strongly toothed median surfaces are apparent when the labrum is moved aside. The mandibles are segmental 2

3 appendages. The toothed, median, cutting surface of the mandible includes a distal (ventral) incisor of sharp shearing teeth and a proximal molar of heavier grinding teeth. You can see these by lifting the labrum. You may later want to remove the mouthparts on one side for closer study but do not do so now. The mandible articulates with the ventral edge of the posterior epicranium (gena). The mandibles lie on either side of the mouth and are the sides of the preoral cavity from which the mouth opens dorsally. The mandibles are operated by powerful muscles with the motion entirely in the transverse plane. Grasshopper mandibles are dicondylic, meaning they are hinge joints, articulating with the epicranium at two points, known as condyles, thus limiting the range of motion to a single plane. A monocondylic articulation, on the other hand, would be a ball and socket joint with a much greater range of motion. Grasp a mandible with fine forceps and move it to demonstrate its motion. The soft, fleshy hypopharynx extends into the preoral cavity as a fold of body wall ventrally from the head capsule just posterior to the mouth. This unpaired, tongue-like structure is not a segmental appendage. It divides the preoral cavity into an anterior cibarium, from which the mouth opens, and a posterior salivarium, into which the duct of the salivary glands open. The appendages immediately posterior to the mandible are the two maxillae. These also articulate with the gena of the epicranium. Each maxilla consists of two basal articles. One, the cardo, articulates with the gena. The other, the stipes, articulates with the cardo. From the stipes arises a spoon-shaped lateral galea and a toothed medial lacinia. The galea and lacinia curve medially, just posterior to the mandibular incisor. The galea hides the lacinia from lateral view. You can roll the maxilla aside on its articulation with the gena to reveal these parts. A large, conspicuous, filamentary, and multiarticulate maxillary palp also arises from the stipes but on its lateral side. The palp is sensory. The last pair of head appendages are fused to form the labium, or lower lip. The labium is easily seen in its entirety by looking at the posterior surface of the ventral head. It forms the posterior boundary of the preoral cavity. The labium articulates with a slender U-shaped arch of exoskeleton (a modified sternite) known as the gula. The labium forms the posterior wall of the preoral cavity. Push the labium posteriorly and look into the preoral cavity. Its anterior wall is the labrum. The mandibles and maxillae are the sides. Inside the cavity you will see the large hypopharynx extending from the ventral wall of the head immediately posterior to the mouth. It is an unpaired fold of the body wall and is not a segmental appendage. The mouth lies between the base of the hypopharynx and the labrum. 3. Different Mouthparts = even though insects do not have teeth like mammals, their 3

4 mouthparts will change and be combined dependent on their feeding style, just like dentition changes in mammals based upon theirs. There are several slides of the different mouthparts of insects, depending on their feeding types and therefore lifestyles. In addition you should study all the preserved insects present in lab (list as mentioned above) in order to see the mothparts on an actual organism. a) Observe the slide of the piercing mouthparts of blood feeding mosquitoes and study the biting moutparts of horseflies, detecting the different mouthparts have been modified. b) Observe the slide of the fly head with the sponging mouthparts of flies that feed on particles that are soluble in saliva. Mandibles are absent, maxillae are non-functional though there are large maxillary palps. The mouth is mainly derived from the labium and forms an elongate proboscis bearing palps, a digital haustellum and sponge-like labella. Look closely at the labella for grooves. c) Examine the slides of the butterfly proboscis. What are the adaptations to these mouthparts? What are the reason for these adaptations. d) Examine mouthparts from all preserved insects and preserved slides of mouthparts present in lab. Determine modifications to these mouthparts, identify the different parts and determine the feeding style of the animals based upon their differences in the mouthparts. In addition you need to determine whether the insect is prognathous, opisthognathous or hypognathous. 4. Antennae a) Slide of different antennal types: i.e. clavate, filiform, plumose, capitate, geniculate. Draw the different antennal types. b) Remove the antenne from some of preserved specimens and observe the antennae, classifying them according to type. Remember than different antennal types will be found in different insect orders and are often considered a useful distinguishing characteristic. 4

5 5. Eyes insects have eyes that are different compared to those of a human, the have compound eyes. Observe the compound eyes on preserved specimens such as the grasshopper (Romalea), the honeybee, the cicada etc. How do compound eyes differe from the tyical mammalian eye? How do these animal see? Do their eyes function in a similar ways as human eyes? How do they differ? 5

6 Thorax 1. External examination of the preserved specimen Start with the examination of the lubber grahopper Romalea because it is one of the easiest specimens to examine. Follow this up by examining the remaining preserved specimen and all of the slides and dried specimens available to you in lab in order to determine anatomical modications and therefore differences in animal color and liestyle. Examine a female and male Romalea under magnification (using either the dissecting microscope or the magnifying glasses provided). The arthropod body is composed of a linear series of segments, some or all of which bear paired jointed segmental appendages. Most arthropods, including insects, are heteronomous, with their segments and their appendages variously modified and grouped in regions, or tagmata, specialized for different functions. The alternative homonomous condition, with all segments and appendages alike over the length of the body, is primitive and among recent tax only apparent in some crustaceans. The three tagmata of the insect body are the anterior head, middle thorax, and posterior abdomen. The head, which shows few external signs of segmentation, bears the eyes, antennae, mouth, and mouthparts. Its major functions are sensory reception and feeding. The thorax, whose primary function is locomotion, is larger and bears three pairs of legs and, typically, two pairs of wings. The abdomen is the largest tagma and is conspicuously segmented. It houses most of the digestive, excretory, and reproductive viscera and its appendages, when present, are specialized for copulation or oviposition. Most abdominal segments lack appendages and those present are highly modified. The external genitalia, at the posterior end of the abdomen, are highly modified segmental appendages. Thorax The heavily sclerotized head and thorax are connected by a short narrow neck, or cervix, whose integument is lightly sclerotized and consequently soft and flexible. The neck and its leathery integument are easiest to see ventrally but are hidden dorsally by the head and thorax. A few 6

7 hard, sclerotized plates, the cervical sclerites, are embedded in its integument. The insect thorax consists of three segments, each of which bears a pair of legs. The legs are segmental appendages. Two of the thoracic segments may bear paired wings, which are complex folds of the body wall not segmental appendages. The three thoracic segments are, in order from anterior to posterior, the prothorax, mesothorax, and metathorax. The meso- and metathoraces are fused rigidly to form an inflexible box housing the flight muscles and bearing the two pairs of wings. Together they are referred to as the pterothorax in reference to the wings they bear (pter = wing). The prothorax is joined to the pterothorax and to the head by flexible articulations. A typical arthropod segment is enclosed in four exoskeletal plates, or sclerites, that form a ring around the segment. These are the dorsal tergite (notum), ventral sternite, and two lateral pleurites. These sclerites are typically joined together by flexible articulating membranes but sometimes they are fused rigidly together. Prothorax: The grasshopper prothorax is covered by an unusually large shieldlike tergal plate called the pronotum. The pronotum covers the prothorax and partly overhangs the mesothorax. The prosternite, located ventrally, is much smaller and bears a conspicuous median prosternal spine, by which may be recognized. The forelegs, or prothoracic legs, articulate with the prothorax. The pleurites of the prothorax, known as propleurites, are small and located between the prosternite and the pronotum. They are fused with both the prosternite and the pronotum. Together the prosternite and propleurites form sockets for the articulation of the forelegs. The propleurite forms the dorsal edge of the socket whereas the prosternite forms the ventral edge. Note the flexible, unsclerotized cuticle forming an articular membrane between the base of the leg and the socket. Rotate the leg around the articulation to demonstrate its mobility. Primitively, each body segment bears a pair of lateral openings, or spiracles, for the intake of air into the tracheal respiratory system. One such spiracle is found on each side of the prothorax. It is located laterally near the border between the prothorax and mesothorax in the soft unsclerotized cuticle under the posterior edge of the pronotum. The prothoracic spiracles are difficult to see because they are hidden by the pronotum. The more posterior spiracles are easier to locate. Pterothorax: The pterothorax comprises the mesothorax and metathorax. Each segment bears a pair of wings and a pair of legs. The wings of the mesothorax are the forewings, or wing covers, and those of the metathorax are the hindwings. The mesothorax bears the middle legs, or mesothoracic legs. The metathorax bears the hindlegs, or metathoracic legs. Dorsally the two segments of the pterothorax are covered by the two sclerotized tergites known as the mesonotum and metanotum. Similarly the ventral surfaces are protected by the mesosternite and metasternite. Note the manner in which sternites join together along their lines of fusion. The metasternite is dovetailed into the mesosternite. The sternite of the first abdominal segment resembles the thoracic sternites and is dovetailed into the posterior edge of the metasternite. Deep invaginations at the corners of the dovetails are apophyseal pits marking the position of important sternal apophyses, or apodemes, extending from the exoskeleton into the interior of the pterothorax to function as an endoskeleton. The sides of the pterothorax are composed of sclerites derived from the pleura of ancient insects. The pleurite on each side of both pterothoracic segments is divided into two smaller plates. The legs articulate between the ventral edge of the pleurites and the sternites. The wings arise between the dorsal edge of the pleurites and the nota (tergites). A spiracle is situated laterally between the pleura of the metathorax and mesothorax. Note that its opening is guarded by two movable lips, or valves. The opening is a straight slit between the two lips. The spiracle opens into the tracheal system. 7

8 Legs Study one of the middle legs. This appendage, which is typical of insect legs, is composed of a linear series of units, or articles. Each article consists of a sclerotized exoskeletal cylinder which contains muscles, tendons, blood, connective tissue, and nerves. Note that while most of the limb exoskeleton is rigid and heavily sclerotized, some parts are flexible and unsclerotized. The unsclerotized regions are the movable articulations between successive articles. Look at some of the articulations under magnification and find these flexible articular membranes. The first (proximal) article is the leg is the coxa (leg base). It is short, wide at the base, narrow distally, and articulates by a broad articulating membrane with the socket formed by the pleurite and sternite of the mesothorax. Articulated with the distal end of the coxa is the even shorter, cylindrical trochanter. The trochanter is fused immovably with the long femur. The femur is one of the two long articles of the leg. The tibia, which is the second of the long articles, articulates with the distal end of the femur. Pay particular attention to the hard parts of the articulation between the femur and tibia. They form a dicondylic hinge joint similar to that in your elbow and knee (but exoskeletal rather than endoskeletal, of course). Such a joint restricts motion to a single plane. You encountered another dicondylic joint between the mandible and epicranium. The tibia is long and slender with teeth on its margins. Next in line along the length of the leg is the tarsus composed of two subdivisions, or tarsomeres. The number of tarsomeres varies among insects. The proximal tarsomere bears three soft, unsclerotized friction pads on its surface to prevent slipping of the tarsus on the substratum. The distal tarsomere is much shorter and has a single friction pad. The final article of the leg is the pretarsus. It extends from the distal end of the tarsus and bears a pair of sclerotized tarsal claws and a single soft friction pad (ariolum). The forelegs are nearly identical to the midlegs but the hindlegs are much large and responsible for the characteristic jumping (saltational) locomotion of grasshoppers. The large coxa of the hind leg is fused with the trochanter and the femur and tibia are much elongated. In addition, the femur is expanded proximally to accommodate the major jumping muscles. This expansion gives it a club or drumstick shape. Wings The two pairs of wings are evaginations or folds of the cuticle and body wall of the two segments of the pterothorax. Each wing is a double layer of body wall consisting largely of cuticle but underlain and secreted by epidermis. The characteristic wing veins of the wings are thickened 8

9 tubes of exoskeleton involved in support. Their primary function is structural but they contain epidermis (which secretes the cuticle), nerves, and blood. Wing veins are divided into longitudinal veins that that extend throughout the length of the wing and cross-veins that connect the longitudinal veins. The longitudinal veins are: Costa (C) Subcosta (Sc) Radius (R) Cubitus (Cu) Media (M) Radial sector (Rs) Media anterior (Ma) Anal veins (A) Media posterior (Mp) The cross veins connecting to the longitudinal veins are: Humeral (h) Radial (r) sectorial (s) Radio-medial (r-m) Medial (m) medio-cubital (m-cu) cubito-anal (cu-a) The forewings of all grasshoppers, including Romalea, are heavy and form a pair of wing covers to protect the more delicate hindwings. Those grasshoppers capable of flight use the hindwings, and not the forewings, for flying. Lift and spread the wing covers (forewings) and examine them with magnification. Note the heavy longitudinal veins and the abundance of small, branching and anastomosing veins arising from them. Make a similar examination of the hindwings. 2. Examination of the different wing types Please utilize the insects you collected during the first lab and some of the common insects on the front table, familiarize yourself with different wing modifications and draw, describe and label the different wing types. 1. Hemelytron the half-sclerotized and half-membranous mesothoraic wing of true bugs (Hemiptera). The metathoraic wing is entirely membranous. 2. Elytron the sclerotized mesothoraic wing of beetles (Coleoptera). 3. Membranous both pairs of wings are membranous for example in bees (Hymenoptera) 4. Haltere the mesothoraic wings are membranous, metathoraic wings are modified into halteres (small knobs on thin stalks) in flies (Diptera) 9

10 5. Tegmen the leathery mesothoraic wing of grasshoppers and cockroaches (Orthoptera) 6. Scaled both wings are covered in scales in butterflies and moths (Lepidoptera). You will need to observe the wings of these animals under the dissecting microscope or using a magnifying glass. 3. Examination of the different leg types Please utilize the insects you collected during the first lab and some of the common insects on the front table, familiarize yourself with different leg modifications and draw, describe and label the different leg types. The leg types are dependent on the animals lifestyle which you should consider during your examinations. 1. Cursorial (running) 2. Natatorial (swimming) 3. Fossorial (digging) 4. Raptorial (grasping) 5. Saltatorial (jumping) 10

11 Abdomen You should again start your exmination using Romalea, the lubber grasshopper and then extend your examination to the remainder of the preserved and dried specimens and slides. The grasshopper abdomen consists of 11 segments and extends posteriorly from the thorax. It is the largest of the tagmata and most of its segments have no appendages. Its segmentation is obvious externally, moreso than that of the other tagmata, and more like that of the ancestral arthropods. With the exception of those at the posterior end, grasshopper abdominal segments are easily recognized and counted. Numbered from anterior to posterior, abdominal segments 1-7 are similar to each other and are typical, unspecialized insect segments. These are the pregenital segments and none bears appendages. Segments 8-9 are reduced and modified and may bear appendages. These are the genital segments and may have appendages modified as external genitalia. The gonopore is on segment 8 but it is hidden by the genitalia. Segments are the postgenital segments. Typically each segment is covered by a large dorsal tergite and a ventral sternite. The two articulate laterally via a flexible, narrow articular membrane. Abdominal sclerites are not as heavily sclerotized as are those of the thorax and head. Eleven tergites are present but some of the posterior segments lack sternites and, as a consequence, there are only eight sternites in females and nine in males. The pleurites are reduced and fused to the ventral edges of the tergites. Most abdominal segments are similar to each other but the anterior and posterior ones show some specializations. The first abdominal segment is fused rigidly with the thorax. You have already seen its sternite dovetailed into the metasternite. Its tergite is mostly hidden by the base of the hindwings. Each side of tergite 1 bears a large aperture covered by a thin cuticular tympanic membrane (= tympanum or eardrum). These are auditory organs for sound transduction. Some species of lubber grasshoppers have stridulating organs with which they produce sound. The spiracle of the first abdominal segment is located on the anterior edge of the tympanum. This spiracle opens into a large air sac attached to the inner surface of the tympanum. Segments 2-7 are similar. The fifth abdominal segment is typical of the pregenital segments. Its tergite forms a large arch over the dorsum and its sternite forms a broad band across the belly. Both sclerites are joined to each other and with adjacent sternites and tergites by flexible articular membranes. A spiracle is located at the extreme anterior ventral corner of the tergite. The posterior abdominal segments are associated with the external genitalia and differ from other segments and between males and females. Some bear modified segmental appendages. The large anus is on segment 11 ventral to tergite 11 (tergite 11 is the epiproct). It is covered dorsally by the single epiproct and flanked by two paraprocts. The ovipositor of females and penis (= aedeagus) of males are ventral to the anus. The posterior abdomens of males and females differ and must be studied independently. Exchange specimens with another student in order to study the opposite sex. Female Externally, female segment 8 is similar to the preceding segments except that its sternite is known as the genital plate. The egg guide is a triangular, median process of the genital plate. Segments posterior to 8 are reduced and lack sternites. The tergites of segments 9 and 10 are reduced and fused laterally although they remain separate dorsally. The 11 th tergite, or epiproct, is rather large and it forms a triangular dorsal shield rather than an arch over the body. Its pointed end projects posteriorly and covers the anus. Associated with it is a lateral paraproct on each side, ventral to the epiproct. The paraprocts are probably the sternites of segment 11. Between 11

12 the 11 th tergite and each of the paraprocts is a small cercus. The cerci are the segmental appendages of segment 11. In some orthopterans, such as crickets, they are long antenniform sensory organs but in Romalea they are very short. The female external genitalia are modified segmental appendages belonging to segments 8 and 9. Together they form the short robust ovipositor extending posteriorly from the abdomen. The ovipositor consists of three pairs of processes, called valvulae, which are used by the female to insert eggs into the ground. Ventralmost is the pair of large, heavily sclerotized first valvulae (= ventral valves). Dorsally is a similar pair of sclerotized third valvulae (= dorsal valves). The first and third valvulae are conspicuous externally. The inconspicuous, membranous second valvulae (= inner valves) are hidden from view by the first and third and will not be seen. The gonopore lies between the first valvulae. The space enclosed by the epiproct, paraprocts, genital plate and ovipositor is the genital chamber. The right and left first and third valvulae together form a hollow shaft, or ovipositor, consisting of four parts, that is used to penetrate the soil. The eggs pass from the gonopore, through the lumen of the shaft, assisted by the second valvulae, and are deposited in masses in the soil. The first valvulae are derived from the appendages of segment 8 whereas the second and third are the appendages of segment 9. Male The male posterior abdomen is simpler than that of the female. Segment 8 resembles segments 2-7 and has an unmodified sternite. Tergites 9-10 are fused ventrally and separate dorsally as in females. Segment 9 possesses a large sternite, the subgenital plate that extends posteriorly ventral to segments It forms a cup enclosing the male external genitalia. Protected and hidden by the plate is the soft (largely unsclerotized) eversible penis (= aedeagus). Although referred to as external genitalia, the penis is not visible externally without moving the paraprocts and subgenital plate aside. Segment 11 is similar to that of females and has a dorsal tergite (= epiproct), lateral paraprocts, and short cerci. The anus is under the epiproct and between the paraprocts. It can be seen by lifting the epiproct. The penis consists of a soft, eversible, bulbous base bearing cuticularized valves, or claspers at its distal end. It contains an extension of the ejaculatory duct through which spermatophores pass during copulation. The gonopore is at the tip of the penis, between the claspers, but will not be seen. The penis lies in the genital chamber. The genital chamber is the posterior space enclosed by the epiproct dorsally, sternite 9 ventrally and the paraprocts laterally. Open it by pinning the posterior tip of sternite 9 (subgenital plate) to the wax. Lift the epiproct and pin it. The space thus revealed is the genital chamber. Its floor is occupied by a large, soft, eversible penis (= aedeagus), which is an intromittent organ. Associated with the penis are dark, hard, sclerites. The male gonopore opens on the midline at the tip of the penis and is flanked by two pairs of slender, dark, sclerotized penis valves that hide it from view. 12

13 What are the differences between male and female cockroaches, cicadas, honey bees? How can you distinguish them? Which of these animals are social, which are eusocial? Examination of abdominal structures Examination of abdominal structures should include observation and drawing of the ovipostor (females, particularly obvious in Orthoptera) and the stingers with poison sacks (Hymenoptera). 13

14 INTERNAL ANATOMY 1. Grasshopper Because of its large size, Romalea is well suited for the study of insect internal anatomy. The study should be conducted with the specimen immersed in fluid (tapwater, Woodring s cricket Ringer s solution, or 40% isopropanol) and with the aid of the dissecting microscope. Procedure: Get a grasshopper and cut off and discard the head, legs and wings. Insert scissors into the opening created when the head was removed and cut posterior through the overlapping portion of the pronotum, above the leg bases and into the abdomen, just above the spiracles (NOTE: Do NOT insert the scissors too deeply or else you will damage the underlying organs and tissues). Proceed cutting to the anus and complete the dissection by making a similar cut on the other side. Carefully lift the dorsal half from the ventral portion. Cut any muscles that may adhere to both halves. The alimental tract should remain in the ventral portion of your dissection. Use fine forceps and fine scissors to separate the middorsal strip of exoskeleton from the underlying soft tissues and remove it. Try to remove the separated pieces of tergite while leaving as much soft tissue as possible behind and intact. If you are careful, the heart will remain with the body. The large body cavity (perivisceral sinus) with the gut should not be visible yet. Instead you will see only the much smaller cavity, the pericardial sinus enclosing the heart.. During the dissection particles and debris will accumulate in the fluid of the dissecting pan and obscure your view of the specimen. When this occurs make sure everything is securely pinned to the wax of the dissecting pan and then gently pour the fluid and particles into the sink. Replace the fluid with clean and continue your study. Hemal System A successful dorsal dissection first reveals the heart in the pericardial sinus. These features must be destroyed to reveal the remainder of the hemocoel and its organs. Accordingly the heart must be studied now, before proceeding to the perivisceral sinus. The arthropod body cavity is a spacious blood space, the hemocoel. In insects it is divided into three longitudinal sinuses by two horizontal diaphragms. The largest of the three is the perivisceral sinus, or perivisceral hemocoel, which contains most of the viscera. The smaller pericardial sinus lies dorsal to the perivisceral and surrounds the heart. The perivisceral and pericardial sinuses are separated by the porous 14

15 dorsal diaphragm. Another small sinus, the perineural, lies ventral to the perivisceral sinus and is separated from it by the ventral diaphragm. The heart is a transparent, slender, longitudinal tube extending the entire length of the perivisceral hemocoel just ventral to the terga and dorsal to the dorsal diaphragm. It is surrounded by the pericardial sinus and blood of the hemocoel. It bears paired segmental ostia and is equipped with alary muscles to expand its lumen in diastole. Its walls contain circular muscles whose function is to constrict its lumen during systole. Once the dorsal strip of exoskeleton is out of the way, you should see a transverse membrane, the dorsal diaphragm, which separates the relatively small, dorsal pericardial sinus from the much larger, more ventral perivisceral sinus. Both sinuses are divisions of the hemocoel and are filled with colorless blood. The dorsal diaphragm is close to the dorsal body wall and is separated from it by a space, the pericardial sinus. The diaphragm will have been destroyed if the initial scissors cuts were too deep. 15

16 The heart is an inconspicuous transparent tube on the dorsal midline of the abdomen and thorax. It is closely attached to the dorsal diaphragm but is just ventral to the dorsal tergites and thus is easily lost when the exoskeletal strip is removed. Look for a narrow longitudinal tube adhering to the dorsal surface of the diaphragm exactly on the midline. Carefully tease away adhering tissue as necessary to improve your view of it. Also present in the pericardial sinus are tubular, branching, silvery tracheae, which should not be confused with the heart. Only the heart lies on the midline and the longitudinal tracheae are lateral to it whereas the occasional transverse tracheae cross it. Paired, segmental alary muscles extend from the heart to the body wall and diaphragm. The muscles fan out to form a thin layer over the upper surface of the dorsal diaphragm. They can be demonstrated by lifting with a minuten nadel. Individual muscles cannot be distinguished by this technique. The heart exhibits inconspicuous segmental swellings equipped with paired, segmental ostia. The swellings are apparent but the ostia will not be seen. Perivisceral Hemocoel and Viscera When you have finished your study of the abdominal hemal system, perivisceral sinus, and external genitalia, remove the legs from both sides by cutting across their trochanters or coxae with medium scissors. Use fine scissors to make a middorsal longitudinal incision through the dorsal diaphragm of the abdomen. This incision should be just deep enough to cut through the thin diaphragm and should not damage the organs in the underlying perivisceral sinus. Pin the abdominal walls aside with stainless steel insect pins as you go. Note the large white air sacs of the tracheal system under the tympana in the anterior abdomen and thorax. Note also the yellow, amorphous fat body lying beneath the diaphragm throughout the hemocoel. Upon reaching the thorax use the medium scissors to remove the heavy dorsal and lateral thoracic sclerites in pieces. Use the medium scissors to cut around a piece, then use fine scissors to cut the muscles holding the piece in place. Use heavier scissors to make the thoracic incision. Thoracic sclerites are usually too heavy to be cut by iridectomy scissors and attempting to do so will ruin them. As you cut through the thoracic sclerites, note and then cut as necessary, the massive flight muscles contained within it. Many of them originate on thoracic nota and must be cut or removed before the sclerites can be removed. Pin the walls of the thorax aside with #4 insect pins. Without further dissection, make a preliminary inspection of the interior of the animal. The cavity occupying most of the interior is the perivisceral sinus of the hemocoel. It is part of the hemal system and is not a coelom. You will see numerous silvery, branching tubes of the tracheal system. Some of them are expanded to form air sacs. The yellow fat bodies may obscure your view of the viscera in the abdominal hemocoel. A slender, transparent, and inconspicuous, longitudinal, middorsal muscle extends the length of the abdomen just dorsal to the chief fat body. In reproductive individuals gonads will occupy much of the space in the 16

17 abdomen. The regionally specialized gut extends lengthwise from the anterior mouth to the posterior anus but is presently covered by fat body and gonad so that little of it is visible. The ventral diaphragm, usually covered by yellow fat body, forms the floor of the perivisceral sinus and separates it from the perineural sinus. We have slides of insect blood available in lab. What does the insect blood look like and how does it compare to human (mammalian) blood? Fat Body The fat bodies extend throughout the hemocoel but are most prevalent in the abdomen. These are sites of intermediary metabolism with a function similar to that of annelid chlorogogen tissue and the vertebrate liver. Usually they are arranged in two sheets or amorphous masses, one near the body wall and the other near the gut, and are supported by connective tissue. In Romalea the fat bodies are thin connective tissue sheets supporting small yellow lobes. The lobes are masses of trophocytes which are bathed in the blood of the hemocoel. The most conspicuous fat body is a sheet covering the surface of the gut in the anterior abdomen. Respiratory System The respiratory system, consisting of 10 pairs of spiracles (two thoracic, eight abdominal), a network of branching tubular tracheae, and several air sacs will be gradually destroyed as you study the organ systems of the perivisceral hemocoel. As you study these systems note the profusion of silvery-white tracheae extending to the organs. Each spiracle on the body surface opens into the system of interconnected tubular trachea. The system includes longitudinal tracheal trunks connecting the segments, branches of the trunk to the three sinuses of the hemocoel and their organs, and transverse commissures connecting right and left. Trachea are ectodermal invaginations of the exoskeleton and are lined by cuticle secreted by their epidermis. They branch repeatedly in a dendritic pattern and their thin walls are reinforced by spirals of chitin called taenidia (taen = band, ribbon). The number of tracheae is related to the metabolic activity of the tissues served. Air sacs are expansions of the tracheae but lack taenidia. The system is divided into cephalic, thoracic, and abdominal subdivisions. A. Make a wetmount of a short piece of trachea and examine it with the compound microscope. Find the taenidia (thickenings of the internal cuticle of the trachea). Alternatively compare the tracheal smear you produced to the prepared slide of the tracheal and spiracles. B. The tracheal system includes several large, silvery, bladder-like expansions called air sacs. Their walls are not reinforced with taenidia and consequently are expansible. They function in ventilation of the tracheal system. In addition to its respiratory role, the tracheal system is important structurally and functions like connective tissue to support the tissues it pervades. The 17

18 gut, as you will soon see, is a good example of an organ system supported by its tracheae. C. Unlike Romalea there are other insects that are not terrestrial but aquatic, hence generally lacking a tracheal system and instead displaying a gill system. Observe the slides of the insect gills and note that these are always from aquatic insects or immatures. Digestive System Remove the fat body from the dorsal hemocoel to reveal the gut under it. Find the large gonad covering the posterior regions of the gut in the posterior hemocoel. Remove the fat body covering the gonad then use fine scissors to free it (the gonad) from its anterior connections with the remaining viscera. Note that most of these connections are tracheae. Locate the gonoducts (oviduct or sperm duct) exiting the gonad laterally. Cut the duct on the left so the gonad can be reflected to the right, thereby revealing the posterior gut but keeping the gonad intact for later study. Like that of other arthropods, the gut consists of anterior ectodermal foregut, middle endodermal midgut, and posterior ectodermal hindgut. The foregut consists of mouth, pharynx, esophagus, crop, and proventriculus and is lined by a cuticle secreted by its epidermal epithelium. The gut begins, of course, with the mouth which opens from the preoral cavity but it cannot be seen from your present vantage point inside the hemocoel. The mouth opens into the short, narrow pharynx, which is entirely contained within the head capsule and consequently is likewise not visible at present. The pharynx leads to the esophagus, which is largely confined to the head but extends a short distance through the foramen magnum to enter the anterior prothorax.. Posterior to the esophagus the gut widens to become the large crop. The separation between esophagus and crop is not distinct on the exterior but will be obvious later when you open the gut. Next, the gut narrows again as it becomes the proventriculus, or gizzard. The proventriculus, which is the last region of the foregut, ends where the proventriculus joins the short midgut. There are slides of the proventriculus to observe in addition to any observation on the preserved specimen. The proventriculus, which is often strongly muscular also contains denticles (constructed of which material?) to aid in grinding the food. Similar structures may already be found all over the stomatodeum (foregut) of some insects, however, in the stomatodeum they are referred to as the intima (slide). Determine and histological detain that is visible, i.e. digestive epithelium etc. An elaborate stomodeal valve, visible only from inside the gut, marks the separation of the foreand midguts. Protruding conspicuously from the periphery of the midgut are six hollow diverticula, the digestive cecae. The remainder of the gut is hindgut, which accounts for about half the length of the gut. The foregut accounts for most of the remaining half with the midgut being short. Internally the proctodeal valve marks the midgut-hindgut junction. The junction of midgut and hindgut is marked externally by clusters of long slender Malpighian tubules. These 18

19 form a tight mass in the vicinity of the junction but also extend anteriorly into the thorax and posteriorly to the posterior end of the abdomen. The hindgut, also known as the intestine, consists of three regions. First is the large ileum followed by the short, narrow colon. The longitudinally ridged rectum is the final region and it opens via the anus to the exterior. Like the foregut, the hindgut is lined by epidermis, which secretes a cuticle. Some gut regions are difficult to distinguish from adjacent regions from the exterior whereas the epithelia lining their lumina are distinct. Use fine scissors to open the entire length of the gut from esophagus to anus with a longitudinal incision. The interior of the esophagus can be seen as it emerges from the head capsule. From the inside, its walls are longitudinally folded whereas those of the crop bear fine oblique ridges best seen at 40X. Each ridge bears a row of tiny, dark, cuticular denticles (= little teeth) which may be seen by focusing carefully with 30-40X. The floor of the crop features a wide, midventral, longitudinal typhlosole composed of a pair of parallel longitudinal ridges flanking a median groove. Observe the typhlosole at 10X. The epithelium lining the proventriculus is longitudinally ridged and the ridges bear tiny conical denticles similar to those of the crop. Find the longitudinal ridges and their denticles by focusing carefully with 40X. Using 10X again, observe the interior of the stomodeal valve at the foregut-midgut junction. The final region of the foregut (proventriculus) is recognized by its numerous, fine, longitudinal ridges whereas the beginning of the short midgut can be identified by its 5-6 wide transverse ridges. Between the two is the conspicuous stomodeal valve marked by six wide, but short, longitudinal ridges. The openings of the digestive cecae are immediately posterior to the ridges of the stomodeal valve and anterior to the transverse ridges. Find at least one and probe it with a small needle to demonstrate that it is hollow and that its lumen is continuous with that of the midgut. The midgut is divided into two regions whose epithelia differ. The anterior midgut bears the wide transverse ridges just mentioned whereas the posterior midgut, which is about the same length, has its walls thrown into inconspicuous longitudinal folds. The longitudinal folds end with the proctodeal valve which marks the end of the midgut and beginning of the hindgut. The openings to the Malpighian tubules are located at the midgut-hindgut junction but will not be seen. Notice that the contents of the hindgut are enclosed in a transparent peritrophic membrane. The walls of the ileum are thin and have inconspicuous longitudinal ridges similar to those of the posterior midgut. These folds become better organized in the colon where they are clearly visible as six distinctive ridges. In the rectum the six ridges remain but become wide and flat-topped. These six flat white bands of cells are known as rectal papillae. 19

20 Excretory System The chief insect excretory organs are the Malpighian tubules, which were seen earlier arising at the midgut-hindgut junction. These are long, slender, hollow diverticula of the gut lumen. In Romalea the tubules are very numerous and very long, extending anteriorly and posteriorly from the midgut-hindgut junction. They extend into the perivisceral hemocoel where they are immersed in blood. Nitrogenous breakdown products of protein metabolism, mostly ammonia, are released into the hemocoel, absorbed by the trophocytes of the fat body, converted to uric acid, and returned to the blood. The uric acid is them absorbed by the tubules, precipitated, and released into the hindgut where it becomes part of the feces. You need to examine the slides of the malpighian tubules as well in order to determine where these sections were taken in the excretory system and how you can tell. Reproductive System Study both male and female reproductive systems by sharing specimens with a classmate. If you have followed instructions, the gonad will be displaced to the right but will be intact and available for study. Male The male system begins with a pair of dorsal testes. The two testes are coalesced over the dorsal midline and appear to be a single testis. 20

21 A sperm duct (=vas deferens) exits each posterior lateral corner of each testis. The ducts are wrapped in fat body and are easy to miss. The left one was cut earlier but should still be present and recognizable. Find it. Each duct extends laterally and posterior to disappear under the gut. Reflect the testis, without cutting the remaining sperm duct, posteriorly to reveal its ventral surface. From the ventral surface each testis can be seen to consist of numerous elongate follicles (= sperm tubes) draining into a central white sperm duct (= vas deferens). Each follicle is a blind ending peritoneal sac lined with epithelium and connecting with the sperm duct by a narrow vas efferens. Each sperm duct curves ventrally around the gut to join the ejaculatory duct which leads to the penis and gonopore. Cut across the gut in the middle of the ileum, free the posterior gut from the body wall, and pin it aside to reveal the ventral regions of the reproductive system. Trace the sperm ducts around the rectum to the ejaculatory duct. Upstream the ejaculatory duct is double (duplex ejaculatory duct) but the two upper arms join each other distally to form a single duct (simplex ejaculatory duct) that enters the penis. Associated with each duplex ejaculatory duct is a bright white seminal vesicle for sperm storage. Numerous blind ending, tubular accessory glands extend from the duplex ejaculatory ducts and form a loose mass around the duct. Some of the accessory glands are bright white and some are transparent. Some are long and some short. Some extend away from the mass and others are confined to it. 21

22 Female The female reproductive system consists of two ovaries, two oviducts and a common vagina opening by the gonopore between the two first valvulae. In addition, an independent seminal receptacle opens to the exterior via a separate receptacular duct and receptacular pore. Sperm stored in the seminal receptacle are used to fertilize the eggs during oviposition. The two ovaries are the most conspicuous feature of the female system because they are large, bright yellow, and dorsal most. They are located in the posterior abdomen, dorsal to the gut and ventral to the dorsal diaphragm. You need remove only the investing layer of fat body and tracheae to reveal them. Each is a cluster of tubular ovarioles, similar to the testicular follicles, emptying into a lateral gonoduct. The two ovaries of reproductively mature females are large and occupy, along with the fat body, most of the space in the dorsal posterior abdomen, between the dorsal diaphragm and the gut. Mature eggs in the ovaries are bright yellow and, since they account for most of the volume of the ovary, impart their color to these organs. Immature eggs are white. The fat body investing the ovaries is also yellow and care must be taken that the two are not confused. Remove as much of the fat body as possible from the ovary. Each ovary is composed of numerous tubes, known as ovarioles, homologous to the follicles, or sperm tubes, of the testis. In each ovary a series of ovarioles drain into a lateral oviduct. Each ovary has an oviduct extending along its outside border. The ovarioles begin upstream as tiny filamentous tubes that gradually increase in diameter moving downstream. At the downstream end the ovariole exhibits its maximum diameter, after which it narrows before joining its oviduct. 22

23 The two oviducts depart from the posterior lateral corner of their respective ovaries and curve far ventrally, around the gut, to coalesce on the midline ventral to the gut and form the single vagina. Observation of the vagina must be postponed, however, in order to find and study the seminal receptacle which is dorsal to it. Cut across the posterior rectum where it joins the anus and pull the intestine forward, between the two ovaries and out of the field of view. This will reveal the last ganglion of the ventral nerve cord and the seminal receptacle. The receptacle is nestled against the curved posterior border of this ganglion. Try to avoid damaging the ganglion as the nervous system has not yet been studied. Remove the third valvulae. The receptacular duct extends to the receptacular pore between the two first valvulae to the spermatheca. The duct is coiled around the vesicle but that is not apparent without careful dissection. The receptacular pore is near the gonopore. The gonopore is at the base of a fingerlike, sclerotized process on sternite 8, the egg guide, located ventrally in the genital chamber. The spermathecal pore is a little dorsal to it. Remove the seminal receptacle, its duct, and the first valvulae. Trace the lateral oviducts from the ovaries posteriorly toward the midline where they join to form the median vagina. The vagina is ventral to all other organs in the posterior hemocoel, including the nervous system. Find the gonopore at the base of the egg guide. Nervous System The nervous system is divisible into the central nervous system (CNS) consisting of the brain and ventral nerve cord, and the peripheral nervous system (PNS) consisting of sensory and motor neurons connecting the CNS with the tissues. The PNS has somatic and visceral 23

24 components but will not be studied in this exercise. The central nervous system consists of a ventral nerve cord in the thorax and abdomen and a dorsal brain in the head. Be sure the specimen is firmly pinned to the dissecting wax and then gently pour the water out of the pan. Replace it with 40% isopropanol. The alcohol will render the nerve cord opaque, white, and easier to see. This effect improves with time. 1. Ventral Nerve Cord: Remove the reproductive system and discard it. Cut across the esophagus and remove the gut so you can see the floor of the perivisceral sinus. The floor is the ventral diaphragm. Through it you can see the perineural sinus and the double ventral nerve cord. The nerve cord is a white band immediately inside the diaphragm. The nerve cord adheres to the ventral surface of the ventral diaphragm. The fat body covers its dorsal surface. Carefully remove the ventral diaphragm and fat body from the nerve cord over the entire length of the abdomen and thorax. In the thorax the massive flight muscles must also be removed. The abdominal region of the cord is easily revealed but considerably more digging is required to find the thoracic region. Remove all tissue necessary to reveal the entire length of the ventral nerve cord. In the thorax removal of the fat body and ventral diaphragm will reveal the salivary glands, of which there are two. Each gland consists of numerous white spherical follicles arranged in clusters, rather like clusters of grapes. The clusters are connected by branches of the two salivary ducts. The ducts extend anteriorly into the head and coalesce to form a single duct which empties into the salivarium of the preoral cavity. Where the salivary glands obscure the ventral nerve cord they must be removed. The ventral nerve cord is conspicuously double and connects a chain of ganglia on the floor of the perineural sinus. The ganglia consist of three large thoracic ganglia, T1-T3 and five smaller abdominal ganglia, A1-A5. Notice the abundant nerves entering and leaving the thoracic ganglia in contrast with the relatively few of the abdominal ganglia. Remember that the thorax is the location of three pairs of legs and two pairs of wings and houses the myriad muscles that operate them. 2. Brain: Dissection of the head capsule to reveal the brain is an optional exercise. The brain is well protected in the heavily sclerotized cranium and exposing can be difficult. Use medium scissors and forceps to open the cranium. Do not attempt to cut or manipulate the hard cranium with your delicate iridectomy scissors or microdissecting forceps. It will ruin them. Cut across the esophagus and remove it. Cut across the posterior cervix to separate the head from the body. Look into the severed cervix and find the foramen magnum. This is the best view of it you have had. Find the severed ventral nerve cord where it enters the foramen magnum. Find the severed esophagus Notice the abundant muscles filling the head capsule. These are responsible for operating the head appendages, especially the mandibles. The brain is dorsal to the esophagus and is connected to the ventral nerve cord by a pair of circumesophageal connectives. With medium scissors make two longitudinal cuts anteriorly through the cranium from the foramen 24

25 magnum, one on each side of the vertex. Make the cuts as shallow as possible to avoid damage to the brain. Extend these incisions anteriorly dorsal to the eyes to the frons. Remove the strip of cuticle thus freed. The space opened if filled with muscles and the esophagus. Insert a #4 insect pin through the labium into the wax to hold the head in place. Cut away the posterior portion of the genae. Use fine forceps to remove the muscles in the posterior head. Do not remove the esophagus, brain, or nerve cord. The muscle has a fibrous texture lacking in nerve tissue. Nerve tissue is yellowish, muscle is dull white. Most of the muscles extend from the cranium to two pairs of thin, flat, transparent mandibular apodemes. The medial apodemes is the larger and extends dorsally almost to the vertex. The later apodeme is smaller. As the muscles are removed the brain will gradually come into view. It consists of the three major paired ganglia of the mandibulate brain which make up the three brain regions. These are the protocerebrum, deutocerebrum, and tritocerebrum, of which the deutocerebrum is dorsalmost. A large optic nerve extends laterally from each protocerebrum to the nearby compound eyes. A pair of circumesophageal connectives extend from the tritocerebrum around the esophagus to unite at the subesophageal ganglion ventral to the esophagus. The paired ventral nerve cord extends posteriorly from the subesophageal ganglion. Remove the soft tissue of the cervix, including its cuticle. Remove all non-nervous tissue surrounding the esophagus, including the medial mandibular apodeme. This will expose a heavy transverse ventral bar of cuticle just ventral to the esophagus. This is the central body of the tentorium, the all-important internal structural brace of the head capsule. The ventral nerve cord enters the head capsule ventral to the tentorium. The subesophageal ganglion is immediately dorsal to the central body of the tentorium. The latter must be removed to reveal the ganglion and the circumesophageal connectives. The subesophageal ganglion represents the combined ganglia of the mandibles, maxillae, and labium. 25