INTERNAL ANATOMY. Entomology BIO 3323 COCKROACH DISSECTION PAGE: 1 -

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Ronald Armstrong
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1 INTERNAL ANATOMY Both the Lubber grasshopper and the cockroach are frequently used to introduce internal anatomy of insects and you ll get a chance to look at both during toady's lab. The cockroaches are back, you might remember them from BIO2121? and yes they are alive! That will require some special care in the way we handle them to be sure that we do not have any escapees. That s obviously not a problem with the preserved grasshoppers. We ll use the smaller dissecting dishes which means there should no problem getting you dissected specimen under the microscope to take a closer look. We ll also have some fibre optic illuminators available at the end of some bench so that you can see the fine internal detail a little better. For both the live and preserved specimens observing the internal anatomy is easier if specimens are submerged under either water (preserved specimens) or saline (live specimens). Although at first dissecting under liquid may seem awkward it means that internal tissues remain suspended in the bathing medium rather than collapsing into a single mass. As a consequence it is easier to distinguish the different organs. This is particularly true for the live cockroach and the heart and digestive tract will continue to beat and move if you use saline to cover the animal. COCKROACH DISSECTION The roaches will be knocked out using carbon dioxide and will remain quiet for a few minutes. During that short period of time be sure to completely remove the legs and wings and pin your animal into the dissecting tray with its dorsal side up and pins at the tip of the abdomen and through the sides of the pronotum. As you pin the tip of the abdominal in place gently extend the abdomen to expose intersegmental membranes. As you remove the wings and legs you may have noticed a clear liquid that oozes from the wound. This is haemolymph or blood. Why is it not red? Now that its in place take a look with the dissecting scope and see if you can see pulsating dorsal blood vessel, or heart, through the abdomenal cuticle. In what direction is the haemolymph moving? Starting with the second to last tergum of the abdomen, remove each tergite individually by cutting along the lateral edges from posterior towards the anterior. Be careful when you make the cut you don t want it to be too deep, that may damage underlying organs, especially the heart. Take your time with this step and as you remove the tergite gently tease back any connecting tissues as you remove it. At PAGE: 1 -

2 BIO 3323 Entomology this point the underlying structures appear white or transparent and because you used saline, and were careful, the heart should remain in place and continue to beat. It s the clearer streak running down the centre of the dorsal surface. Two other structures should be visible in your dissection: considerable amount of white fluffy material which is the fat body and numerous glistening tubes that branch and branch throughout the body. These are the tracheal trunks and tracheal branches Figure 1 Circulatory system in a cockroach. BIODIDAC Aorta Segmented vessel Dorsal diaphragm Heart Alary muscle Segmented vesel Circulatory system The circulatory system of an insect is an open system consisting of a heart contained in the dorsal pericardial cavity and the haemocoel which is a series of cavities or sinuses which surround all the internal organs. Trace the heart backwards to the ninth abdominal segment where it ends blindly. At the anterior end the heart extends into the thorax where it becomes a short aorta which opens near the brain. How do you tell the difference between the part that s the heart and the part that s aorta? If you look closely at your the dissection you will see that the heart is composed of a series of repeating diamond shapes (If the dorsal diaphragm remained attached to the tergites as you removed them look there). This is the result of the segmentally arranged alary (or aliform) muscles that are embedded in the wall of the dorsal diaphragm which forms the bottom (ventral) surface of the pericardial cavity. These muscles change the shape of the pericardial cavity, and conjunction with the heart muscles open and close the ostia. Examine the wings of a restrained cockroach that is on display and see if you can see the blood moving through the wing veins. The opening in the wing through which the blood moves are also exten- PAGE: 2 INTERNAL ANATOMY

3 sions of the haemocoel and you shouldn t confuse them with veins as the term is used in vertebrate biology. Respiratory system You will notice large silvery tubes which run along each side of the animal. These are the tracheal trunks which connect to the outside through the spiracular openings and their silvery appearance is because they are filled with air. The trachea are supported internally by a spiral ring of cuticle, the taenidia, which can be observed with the dissecting microscope. Try and trace the tracheal system. You will notice that it extends throughout the whole body and that the tubes become smaller and smaller. What is the difference between a tracheal tube and a trachiole? Carefully remove a piece of the tracheal trunk and place it under a wet mount coverslip so that you can see the detail a little better. We also have prepared slides of the connection between the trachea and a spiracular opening that you can also take a look at. Figure 2 Internal anatomy of the female cockroach. BIODIDAC Crop (foregut) Gizzard Digestive caeca Intestine (midgut) Malpighian tubules Salivary gland Salivary reservoir Rectum Collaterial gland Thoracic ganglia Abdominal ganglia Nerver cord Ovary (composed of 8 ovarioles) Hindgut Fat body The large amounts of white fluffy material that hide just about everything is the fat body. This organ is more than a simple store of fats as its name suggests but is involved in protein synthesis, detoxification and a wide range of metabolic activities essential to an insect. The closest analogy in the vertebrate system is the liver. (Why do we use the term analogy rather than homology?) To observe the other organs you will have to gently remove this tissue. Pick it out a bit at a time and wipe off your forcepts using a piece of paper towel. You might want to do this while you re looking through the dissecting microscope so that you don t pull anything out by mistake - remember PAGE: 3 - INTERNAL ANATOMY

4 BIO 3323 Entomology we re only removing the fat body and trachea that are embedded in it. Digestive system Once you start removing the fat body you should start to see the coiled tube of the digestive system that lies underneath it. Cut forward through the thorax and gently remove the muscle tissue to reveal more of the alimentary tract in this tagma. Did you notice that there wasn t much else other than the muscle in the tagma - Why is that? An insect s gut is made up of three regions: the stomodeum (foregut), mesenteron (midgut) and proctodeum (hindgut). The foregut and hind gut are both lined with cuticle and the midgut contents are contained in a peritrophic membrane. What is the role of the peritrophic membrane? The mouth leads into a tubular pharynx which passes over the tentorium and becomes the oesophagus where it passes out of the head into the thorax. It is at this point that the alimentary tract becomes visible in your dissection. The oesophagus changes into a crop, which is an enlarged sac extending into the abdomen of your specimen (It s also filled with air from when the roaches were knocked out). If you gently move the muscles aside in the mesothoracic area you may be able to see whitish grey salivary glands which lie on either side of the crop. The salivary ducts empty into the buccal cavity through the base of the hypopharynx. Figure 3 Internal anatomy of the male cockroach. BIODIDAC Crop (foregut) Gizzard Digestive caeca Intestine (midgut) Salivary gland Salivary reservoir Thoracic ganglia Nerve cord Abdominal ganglia Malpighian tubules Mushroom bodies Ejaculatory duct Hindgut Gently unwind the coiled portion of the alimentary canal and position it to one side so that all its parts are visible. Where the crop narrows abruptly it becomes the gizzard, or proventriculus which is a short hardened region. The proventriculus marks the junction between the foregut and midgut. The most anterior portion of the PAGE: 4 INTERNAL ANATOMY

5 midgut is also marked by the presence of eight blindly ending tubular outgrowths called the gastric caeca. These outpockets of the midgut provide regions for specialised digestive events. Since the midgut is the only part of the gut that is not lined with cuticle is the principle site of enzyme secretion and nutrient absorption. Why would these processes not occur where there is a cuticular lining? Excretory system The most posterior end of the midgut is marked by the point where the Malpighian tubules join the alimentary canal. These fine tubes that extend throughout the body are involved in the excretory processes and their position suspended in the haemolymph allows them to filter and purify the haemolymph that surrounds them. Remove the tubules and place them on a microscope slide and examine their appearance. We also have some prepared slides for you to look at. The Malpighian tubules mark the beginning of the hind gut which starts with the large intestine or colon. The rectum, which is short and is identifiable by longitudinal striations, is located at the most posterior end of the hindgut. It opens to the exterior through the anus. Reproductive system Be sure that you take a look at the both the male and female reproductive systems - you re responsible for both! How do you identify the difference externally? In females two ovaries lie in the fat body on each side of the abdomen and each is composed of eight ovarioles with each of these producing eggs. The eggs pass down the oviduct and are enclosed within the ootheca, which is a special egg case. The ootheca is then dropped onto the substrate as a white soft structure that immediately hardens and darkens as sclerotization occurs. This forms a strong protective covering for the developing insects inside. If you have an intact ootheca please dispose of it in the 70% ethanol solution that has been provided. This is essential to insure that the building does not become infested with cockroaches. Find the lateral oviducts and although you may not see it you should be aware that they empty into a common oviduct or median oviduct that opens to the exterior via the ovipositor. The reason that you may have trouble seeing the common oviduct is that in this region are large accessory glands which have the appearance of wet spaghetti. They are involved in producing the ootheca which contains the eggs. Located at the junction of the oviducts is a small brown spermatheca which stores sperm received during mating. As the eggs pass this point in the reproductive tract they are fertilised. In the male the testes are difficult to see since they are translucent and dispersed within the mass of the fat body. Each testes is composed of a number of follicles and each of these produces sperm. PAGE: 5 - INTERNAL ANATOMY

6 BIO 3323 Entomology Sperm travel down the vas deferens, to the common medial ejaculatory duct and are subsequently passed to the female during mating. The two large prominent glands with finger like projections are located at the posterior end of the abdomen are the male accessory glands or mushroom bodies. The male accessory glands are involved in producing the seminal fluids that are passed with the sperm to the female and for producing the spermatophore. If you gently push aside the mushroom bodies you will also see a longer finger-like gland, the conglobate gland which also opens into the ejaculatory duct. Nervous system The nervous system is visible once the alimentary system has been pushed to one side. The nerve cord appears as a white line running down the ventral midline of the animal. Remove the saline from your dissecting dish and flood the body cavity of roach with some 70% alcohol. This fixes the tissue and as a consequence the nerve cord will appear whiter, but rather brittle, so be careful as you touch or remove excess tissue that surrounds it. The nerve cord in insects is a paired structure and a single ganglia is located in each segment. The paired nature of the nerve cord may, however be obscured by excessive fatty tissue. You will notice that the last abdominal ganglion appears larger then the other ganglia located within this body tagma. This is the consequence of a fusion of the last few ganglia and this composite ganglia is involved in the reproductive functions. Clear away muscle tissue in the thorax and trace the nerve cord forward and locate the enlarged ganglia in the three thoracic segments. Are they all the same size? Why might they have different sizes? In the thorax apodemes, which are internal inflections of the cuticle, are present and are involved in the muscle insertions. Carefully cut away the wall on the side of the head to expose the large mandibular muscles. Carefully pick away the muscle tissue and you should be able to locate the brain, supraesophagial ganglia which is connected, via circumoesophageal nerve cords to the underlying suboesopgagial ganglia. Prepared slides of sections through the compound eye are also available for you take a look at. GRASSHOPPER DISSECTION. Secure the specimen in the dissection tray, and orient it as you did with roach in the first part of the lab. You will be removing the PAGE: 6 INTERNAL ANATOMY

7 tergites just like you did with the cockroach dissection but the diagram that you have is a sagital section. Brain Aorta Proventriculus Alary muscle Crop Haemocoel Heart Ostia Ovariole Oviduct Rectum Anus Figure 4 Internal anatomy of the female grasshopper. BIODIDAC Pharynx Mouth Subesophagial ganglia Salivary glands Midgut Digestive caeca Vagina Seminal vesicle Malpighian tubules Ventral nerve cord Circulatory system It won t be as easy to see the circulatory system in the grasshopper. Take the microscope and look closely at the dorsal surface before you remove the tergites and you may be able to see the lighter line running down the centre of that surface - that s the heart. If you cut carefully up the sides of the abdomen, starting at the tip of the abdomen you should be able to lift off all of the abdominal tergites in one piece. In the preserved specimen you will have lifted off the dorsal diaphragm at the same time and you should be able to see that, and the alary muscles, involved in pumping blood through the animal Respiratory system The preservative that is used to prepare the grasshoppers has spread throughout the tracheal system and the silvery tubes that we say in the cockroach are now muck darker because they are no longer filled with air. Locate the spiracles along the sides of the abdomen and follow the tracheal trunks as they branch and become smaller. Fat Body Just like in the cockroach the fat body is found all through the abdominal cavity. The preservative has given it a much more crumbly PAGE: 7 - INTERNAL ANATOMY

8 BIO 3323 Entomology texture and its much easier to remove it to expose the underlying organ systems Reproductive systems The main parts of the female and male reproductive system are found on top of the gut. Be sure that you see each! In the female ovarian tubules are arranged linearly down the length of the oviduct and depending on the reproductive state of your animal you should be able to see eggs maturing inside the oviducts. In the cockroach the ovarioles were all joined together at a common point. In the hopper the oviduct extends down the sides of gut and join underneath it to form the unpaired vagina which open to the outside. Later in the dissection when the alimentary tract has been removed you may be able to see the sac-like seminal receptacle where sperm is stored after the female has mated with male. Grasshoppers such as this don t have the elaborate accessory gland system that we saw in the roach. Why? The male system is much simpler and tubular testes lie on top of the digestive tract and joined underneath it to form the sperm duct of vas defrens. It may be hard to see the testes in the grasshopper because the preservative makes them have the same appearance as the fat body. You ll need to look closely using the dissecting scope to see the difference. Digestive system The digestive system of the grasshopper looks very different from that of the roach and one of those differences is that it is not coiled inside the abdomen. Gently remove the reproductive system to expose the underlying gut. The junction of the midgut and hindgut is marked by large digestive caeca that extend both towards the front and back of the animal. Like the cockroach the digestive caeca create a specialised environment for the breakdown if ingested food. To the front of the animal is the oesophagus and ultimately the pharynx that is embedded in the head. The valve at the junction between the foregut and hind gut is a hardened proventriculus that is involved in grinding up the food before it passes into the midgut. As in all insects, the junction between the midgut and hind gut is marked by the location where the Maliphigian tubules empty into the alimentary tract. Behind that is the hindgut and if you dissect far enough back you ll see the larger diameter rectum. Nervous system Remove the remaining tissue and you should be able to see the underlying nerve cord. Be sure to remove the muscles in the thorax as to see the ganglia there. Are they the same size as the abdominal gan- PAGE: 8 INTERNAL ANATOMY

9 glia? What about the size of the very last terminal ganglia compared to the others? PREPARED SLIDES We have a number of prepared slides that better illustrate some of the internal organ systems Tracheal System Prepared slides in insect trachea and their attachment to a thoracic spiracle are available for you to take a look at. This is a complex spiracle and two rather large tracheal trunks and a variety of other small tubes extend from the spiracular opening. Look at the slide under higher power can you see the taenidia? Malpighian tubules Prepared slides of Malpighian tubules are available. The tubule itself is only a cell thick and the hollow lumen is continuous with the digestive system. Compound and simple eyes In the radial section of the compound eye you should be able see both compound eyes and ocelli. In the compound eye distinguish the difference between the dioptric apparatus and the underlying photo receptive part of the eye as they form each of the ommatidia. Behind it you can see the optic lobe of the brain were the nerve ends from the sensory cells collect and integrate the information that they have detected. In the simple ocellus the organization is different and photoreceptive cells are bunched underneath the lens rather then forming ommatidia. How does this effect the way that this photorecptor is believed to function? Flight Muscles - Johnson s organ Microscope slides of the male mosquitoes are availabe. The thorax is transparent enought that you can see the indirect flight musculature. One of the muscle group runs dorsoventrally and the other longitudinally. What is the effect on wing movement when either of these contracts? Compare the male and female antennae. The knob-like base of the male antenna is a it s Johnson s organ which it uses to detect the buzzing of the females wings. PAGE: 9 - INTERNAL ANATOMY

Internal Morphology. 1.Cut the legs and wings (if present) off your specimen. 5.Use forceps to pull skeleton apart, exposing internal systems.

Internal Morphology. 1.Cut the legs and wings (if present) off your specimen. 5.Use forceps to pull skeleton apart, exposing internal systems. Internal Morphology Insect Dissections Often the best approach to understanding internal morphology is by way of a dissection. For this reason, the entire chapter should be treated as a laboratory activity.