Laboratory 7

Hormonal Control Systems

The control of blood glucose in crustacea

Prelaboratory Preparation Required

Objectives

• To familiarize students with ablation and replacement as a means of investigating endocrine control systems.
• To give students an opportunity to design and conduct an experiment to investigate the nature of endocrine regulation in crustaceans.
• To collect the present data in support of the hypothesis that blood sugar regulation, like many other endocrine factors in crustaceans, is mediated by a neuralheamal organ located in the sinus gland.

Prelab preparation

Ablation

The Ablation of the sinus glands must be compleated at least 24 hours prior to your experiments. We will do this during Mondays lecture time so be certain that at least one lab partner brings a dissection kit. You will be provided with 36 crabs.

Hypothesis and experimental design

You need to think about your model and formulate your hypothesis prior to coming to lab.  As you read on you will se that you will require several hypothesis in order to fully cover the question of hormonal control in crabs.  You will be provided with 36 crabs so work this into your design.

We will have a discussion of all of your input in lab and develop a concensus hypothesis and protocol.  So that every one is working on the same experiment and we can combine data for statistical purposes

You also need to formulate your experimental design in detail don't forget controls.  If your hypothesis are in the form of an if then statment then your experimental design becomes very clear.  Each lab team should submit a brief statement of your model, hypothesis (if then staetment), and an outline of your lab protocol.   This is not formal writing you don't need references.  Just outline exactly what you are going to do including statistical methods.

Introduction

The Nature of the Problem

Both vertebrates and invertebrates have specialized neurons in the central nervous system that produce neurohormones.  These neurosecretory cells combine the best features of the nervous and endocrine systems.  They function like endocrine glands by secreting hormones into the circulatory system, and like neurons, they conduct impulses.  The neurohypophysis (posterior pituitary) of the vertebrates is a familiar example of a neuroendocrine organ.  The cell bodies of neurons in the hypothalamus produced the hormones, ADH and oxytocin.  They are transported to the posterior pituitary along axons of the hypothalamus-hypophyseal tract.  The hormones are stored until specific stimuli trigger their release. Thus the posterior pituitary is a neuralhemal organ, a site at which neurohormones (stored in synaptic vessicles) are released into the blood.

The eyestalks of invertebrates such as crustacean contain both neurosecretory cells, the X organ, and a neuroendocrine (neuroheamal) organ, the sinus glan.  Together these structures make up ths sinus gland complex  (SGC) The X-organ and sinus gland are the production and release site for the various eyestalk hormones (Carlisle & Knowles, 1959). Cell bodies of neurons in the the X organ receives input from central nervous system ganglia and the swollen axon ends of these neurons terminate in the sinus gland.  Thus the SGC intergrates the functions of the nervous system and the endocrine system that is quite analogous to the vertebrate hypothalmous - pituatary (anterior and posterior) complex.

The literature suggests that X organ sinus gland complex is responsible for: Molt inhibition, Sugar metabolism, Metabolic rat,e Depression of gonad development, Pigment dispersion, Body protein metabolism, Salt and water metabolism, and Heart rate.  The X-organ Sinus gland complex is the control station from the brain to almost allo all bodily functioning. It relays its messages via the SG release site into the hemolymph. Some of the hormones synthesized in and released from this system are molt inhibiting hormone (MIH), ovary inhibiting hormone (aka vitellogenin inhibiting hormone (VIH)), gonad-inhibiting hormone (GIH), and two mandibular organ inhibiting hormones (MOIH1 & MOIH2). The X-organ sinus gland complex controls molting by having inhibitory control over the Y-organ via molting inhibitory hormone (MIH). It also controls the synthesis of MF from the MO by the release of inhibitory hormones MOIH1 and MOIH2. Ovary-inhibiting hormone or VIH originates in the medulla terminalis of the X-organ and prevents vitellogenesis from occurring, which might be the case within a molt phase (Lockwood, 1968). GIH also prevents gonad development from occurring at inappropriate times, by inhibition via the Y-organ. GIH has been associated with the prevention of yolk deposition rather than cellular multiplication (Subramoniam, 2000).  

Some Crustaceans have been demonstrated to produce a hyperglycemic hormone (diabetogenic hormone) that acts to elevate blood sugars. This circulating hormone acts on the hepatopancreas to promote the release of glucose into the hemolymph.  This action is very similar to glucagon, a hormone released by the pancreas of the vertebrates.  In vertebrates adrenalin, a hormone associated with the ‘flight or fight’ response also facilitates the release of blood glucose that would be required by an animal to act on the stress that resulted in adrenalin release.

As a starting point, therefore, it would be appropriate to assume that the control of blood glucose is mediated by the release of hyperglycemic hormone by the sinus gland complex. Using vertebrates as your model it would also be appropriate to assume that, adrenalin may also play an independent role in the release of glucose as it does in vertebrates.  You are to conduct experiments based on the working hypothesis that the control of blood glucose is mediated through the sinus gland that adrenalin may be involved. With the materials on hand, you should be able to gather sufficient data to support or refute these points.

Procedures

If your experiments involve ablation of the sinus gland, this operation will have to be completed at least 48 hours prior to your experiments. The surgery is traumatic and you might expect to lose a few animals. Each lab group will require a minimum of two ablated animals and should not prepare more than four for logistic reasons.

These surgical procedure must be performed at least 48 hours in advance of the experiment.

Ablation of the sinus gland

• Lift the entire eye stalk out of the orbit with a forceps.
• With a small sharp scissors snip through the base of the eye stalk proximal to the sinus gland.
• Immediately pack the orbit with cotton or tissue to prevent exsanguination.  It is important to pack the orbit tightly; the blunt end of a probe or pen work nicely.
• Maintain the crab out of water for about ten to fifteen minutes while watching for signs of blood loss. This will allow time for clotting to occur while providing an opportunity to repack the wound if bleeding persists.
• Return the crabs to the labeled holding tank) and continue to watch for blood loss.
• It is prudent to check up on your patients after a few hours as well as on a daily schedule.
• I would advise labeling the animals so that they can be recognized after they move from tank to tank.

Sinus gland extract

Your experiment involves the replacement of the sinus gland hormones. It is very important to retain the eye stalks that you have removed. Place them in any suitable container and store in the freezer. Alternatively you can extract them now.

• Place two to four eyestalks in a 1.500 ml micro centrifuge tube and crush them with a plastic micro pestle.
• Add 0.5 ml of crab ringers and homogenize vigorously
• Spin for 10 min at full speed in a Sorval microfuge
• Decant into a fresh tub
• Re extract with 0.5ml of crab ringers
• Combine the extracts and heat in boiling water fro 10 min.
• Spin at high speed for 10 min and decant into a fresh centrifuge tube and bring up to 1ml total volume.

Note:  You should avoid disarming any animal used in this experiment. It is more appropriate to tape the claws shut with plastic electrical tape. Crabs that are disarmed and can not defend themsevesf will respond differently to stress

General procedure for obtaining blood - The pretreatment control sample

The easiest way to collect data in this type experiment is to to use auto controls.  That is, you will take a resting blood sample from one organism, then treat the same crab andand then take a second post treatment sample.  Your data then meets the assumptions for a paired T test, or anova, and you can calculate values like % change

The pretreatment control should be from unstressed crabs, so It is very important that you work quickly and discreetly, being certain not to disturb the other crabs.

• Capture a crab and place it on a flat surface dorsal side up in such a way that you can bend the swim fin downward.
• Puncture the articulating membrane and withdraw about 200 ul of blood With a large (16-18g) needle.
• It is important to keep the needle directed upward along the inner surface of the shell and to avoid inserting it deep enough to end up in the viscera.
• If you experience difficulty in withdrawing blood, rotate the syringe slowly to aid in unclogging the needle. Transfer the 200 microliters of the blood to a centrifuge tube (1500 microliters) and shake aggressively to promote clotting.
• Spin for 5 min at full speed in a Sorval microfuge
• If clotting is not complete repeat shaking or use a micro pestle to homogenize the blood and repeat the centrifugation.
• This sample constitutes your pretreatment control sample. Make certain that it and the crab is properly labeled and that the label matches or you will loose the ability to use the very powerfull paired sample T test.

• Note: You can not cut the swim fin in this experiment as you can not stop the bleeding after the required sample has been obtained
• Note: Work very rapidly, 30-60 seconds, no more - for the entire procedure. Remember this is to be a sample from an unstressed crab.
• Hint: A two-person team is the most effective approach.  Net the crab and place it on the lab bench.  One person cover the anterior end of the crab (the pain producing part) with the net and then firmly pin the chelapeds to the bench with one hand.  The second person can then quickly take a sample with out risking personal blood loss.

Adrenalin treatment

• Be certain that you have an initial unstressed hemolymph sample and that it is labeled with the same code as the crab.
• Then inject 0.1 ml of a 1:1000 adrenaline hydrochloride solution in each animal. 
• Put them back into a shallow tray with 4 or 5 cm of water.
• Resample 10 minutes later. Again be certain that the sample labels corisponds to the unstressed sample from the same crab

Stress treatment

The most repeatable way to stress the crabs is to expose them to a toxicant, chloroform.

• Be certain that you have an initial unstressed hemolymph sample and that it is labeled with the same code as the crab. 
• Crabs are then placed in a chloroform bath (sea water sateurated with chloroform) for about 20 seconds and returned to “clean” water in a shallow tray.  Do not place these animals in the same container as the other crabs. Use a separate tray for recovery as the crabs will leach out enough chloroform to affect others.
• After 10 minutes resample the crabs blood.

Sinus gland replacement

• Be certain that you have an initial unstressed hemolymph sample and that it is labeled with the same code as the crab.
• Inject 0.1 ml of your previously prepared eyestalk preparation.
• After 10 min take a blood sample as described above.

Glucose determination

Caution: The color developed in this assay is quite dependant on assay conditions. All of the samples in a group (stressed, adrenalin etc) must be run at the same time with their own control to insure that the assay provides precise values.

The glucose LiquiColor Enzymatic-colorimetric test in the appendices will allow you to accurately determine the glucose concentration of the blood samples obtained above.  This assay is a commercial colorimetric assay for determination of glucose in clinical samples. The manual in the appendix is a copy of the manufactures sinstructions and these instructions are intended for humans. The standards and dillutions may need to be adjusted to compensate for this. The results of this assay can be read from a an Ocean Optics specrtorphotometer.

Important note: 

The glucose assay was designed for Homo sapiens not Callenecties sapdus which has a considerably lower blood glucose. You can compensate for this by adding 40ul of blood and standard or DI instead of 10ul suggested in the human protocol.  Of course if you do this you need to drop the concentration of the standard by the same ratio from 100mg.dl to 25 mg/dl to prevent the assay from exceeding 1.00 AU.  The calculation allso needs to be modified to AU sample/AU standard *25mg.

Report

Include both graphic and tabular presentation of your data in your report (All values should be reported in mM/L). Using your data, come to some conclusion as to the role of both the sinus gland and adrenalin in the regulation of blood glucose in the decapod crustacean. The data tables were arranged with paired sample T tests in mind but of course you wont be able to make compairisons between treatments. You can choose and alternative statistic to accomplish this if you wish. Can you say anything about the chenical nature of the hormone?  ie can you exclude any of the major chemical classes of hormones? Graphs and comments are required - a formal report is optional and data dependant but this experiment has yeiled useful data for 20 years in a row.

Walter I. Hatch
wihatch@smcm.edu

November 3, 2014