The circulatory system is vital to the survival of vertebrates and invertebrates because it helps to transport oxygen and nutrients to various parts of the body. The main features of a circulatory system include a liquid that transports substances, machinery to pump the liquid, which in most cases is referred to as a heart (Tieck, 2011).
The muscles making up the hearts of most organisms are called cardiac muscles and are made of myocardial cells. These muscles contain proteins such as actin and myosin, which confer the cardiac muscles the ability to contract, which leads to the pumping of the heart and the propulsion of the circulatory fluid to different body parts. Two main types of circulatory systems exist: open and closed. In an open circulatory system, the circulatory fluid (hemolymph) is contained within a cavity referred to as a hemocoel and combines with the interstitial fluid (Noordergraaf, 2012).
Conversely, in a closed circulatory system, blood is contained in a network of blood vessels and moves unidirectionally to and from the heart. In an open circulatory system, the movement of the animal together with the heartbeat causes the hemolymph to flow around the organs inside the body cavity. The heart has orifices known as ostia through which the hemolymph enters the heart. In so doing, there is an interchange of gas and nutrients. Little energy is used in the operation of an open circulatory system. However, its efficiency is less than that of the closed circulatory system because limited amounts of blood can be conveyed to metabolically active organs and tissues with high oxygen requirements.
Most crustaceans including crayfish have neurogenic hearts. Neurogenic hearts cannot beat in the absence of neural involvement from the cardiac ganglion (Kuklina, Kouba, & Kozák, 2013). The baseline or resting heart rate are established by the neural feedback. Crayfish (Procambarus clarkii) are decapod crustaceans that dwell in fresh to partly salty waters. Their food includes fish, small invertebrates, flesh, aquatic plants, and debris. Since they dwell in water, crayfish use gills for gaseous exchange. However, they can survive outside of water for short periods provided that their gills are moistened to prevent them from drying out.
The objective of this experiment was to determine the impact of low temperature and stress on the heartbeat of crayfish as well as the effective concentrations of the neurotransmitters acetylcholine, serotonin and epinephrine on crayfish heart rate.
Procedure
The output of the impedance converter was connected to the recording device followed by the attachment of the electrode cables to the green input binding posts. Two lengths of wire of approximately 12 inches in length were cut. About 2-3 mm of the insulation covering on one end of the wires was burnt off. The crayfish specimen was obtained and secured against a foam pad in readiness for the experiment. An 18g needle was then used to bore two tiny holes through the covering of the cephalothorax.
Care was taken to avoid further drilling once a drop of hemolymph appeared just after boring through the shell. The wire with the exposed end was placed into one of the holes and held in place with a small amount of cyanoacrylate adhesive over the hole and a small amount of Zip kicker. The procedure was repeated for the second wire on the other side of the crayfish. The animal was then put in a Tupperware container followed by the addition of 100 ml of Freshwater Crustacean Saline solution whose temperature was measured. The 1 cm stripped ends of the wires were hooked onto the input posts at the back of the impedance converter. The LabTutor module called ‘Crayfish Heart Rate’ was started to record the heart rate of the specimen.
The baseline heart rate was determined by covering the container with a piece of aluminum foil and allowing the animal to rest for about 10 minutes before collecting the readings. The readings were obtained for 5 to 10 minutes ensuring that the heart rate was steady. The start and end of the baseline heart rate trace were annotated upon clicking the stop button. The stressed heart rate was obtained by uncovering the container and initiating the heart trace rate.
The impact of serotonin on the heart rate was obtained by adding 1ml of 1mM stock solution of serotonin to the water using a blue tip of the micropipette. The animal was allowed to sit with the foil cover on for the neurotransmitter to equilibrate between the holding water and the hemolymph of the animal. The heart rate was measured using the same procedure as the basal heart rate.
The solution was then replaced with Freshwater Crustacean Saline. A similar procedure was repeated for acetylcholine and epinephrine. The covered container was placed in a bucket with two-thirds of ice to determine the impact of temperature on crayfish heart rate. The animal was left to equilibrate for 5 minutes after which the temperature of the water was measured and recorded. The heart rate was measured as before and recorded. The heart rate for each of the annotated sections was determined and compared to the baseline rate.
Data and Results
It was observed that the average heartbeat was highest under the influence of serotonin followed by stress. Acetylcholine and serotonin increased the heart rate from the baseline while cold temperatures decreased the heart rate from the baseline.
Table 1: The Average Crayfish Heart Rates at the Baseline, Stress, Cold and Various Neurotransmitters
The concentration of the neurotransmitters was calculated as follows:
If 1mM is present in 1000 ml of the stock solution, the concentration of the neurotransmitter in 1 ml is equal to (1 ml×1 mM) ÷ 1000 ml= 0.001 mM.
Discussion
Opening the container signified water stress to the crayfish, which responded by trying to look for a source of water. The increased movement amplified the metabolic activities that consequently increased the heart rate. Reducing the temperature of the water forced the crayfish to adapt accordingly by lowering its body temperature.
The resulting decrease in temperature reduced the activity of enzymes that were responsible for contraction of the contractile proteins found in the cardiac muscles thus reducing the heart rate. Animals with the capacity to adjust their body temperature to match that of their environs are referred to as poikilotherms (Culos & Tyson, 2014). It is hypothesized that living organisms will respond to stress by making adjustments to avoid the stressful conditions hence the observed occurrences.
Neurotransmitters control the heart rate of vertebrates and invertebrates (Holsinger & Cooper, 2012). Serotonin is a neurotransmitter that controls the heart rate in invertebrates. Serotonin hastens contractions and elevates the heart rate of these organisms. Therefore, it is hypothesized that introducing serotonin to the crayfish’s environment will increase the heart rate because the origin of contraction of the crayfish heart is neuronal stimulation. In crustaceans, the concentration of serotonin in the hemolymph is proportional to the level of aggression.
The heart tissues were suffused in hemolymph containing serotonin, which stimulated the muscles of the heart to contract thus increasing the heart rate. The findings of this study corroborated those reported by Listerman, Deskins, Bradacs, and Cooper (2000). In their study, injections of serotonin to yield systemic concentrations of about 100 nM to 10 mM were reported to elevate the heart rate significantly for hours. In this study, the concentration of serotonin was 0.001mM, which was equivalent to 1000000 nM. However, the systemic concentration of the neurotransmitter was unknown.
The innate ganglion cell pool of the crustacean heart has an unstructured rhythm. A series of impulses transmitted to the nerve fibers linking the ganglion cells with the heart muscle fibers is responsible for the heartbeat. The frequency of these impulses is determined by substances that affect the regulatory nerves. Since neurotransmitters accelerates nerve impulses, it is hypothesized that acetylcholine will increase the heart rate. Acetylcholine increased the heart rate from the baseline to 94.7. These findings were in agreement with those reported by Listerman et al. (2000) that perfusion of crayfish’s environment leads to an increase in the frequency and amplitude of the heartbeat.
Epinephrine increased the heart rate of the crayfish from the baseline average of 76.7 to an average of 94.0. Epinephrine is responsible for the fight or flight reactions that help humans respond to danger. However, in crustaceans, high concentrations of adrenaline have been shown to decrease the heart rate initially as the animal adapts followed by an increase in heart rate as the metabolic activity is increased. Epinephrine is known to elevate the heart rate in invertebrates and vertebrates. Therefore, it is hypothesized that introducing it to the crayfish’s environment will raise the heart rate.
Available information regarding heart rate in crustaceans indicates that factors such as body size, respiration, stress, activity, temperature, and neurotransmitters influence the heart rate of crustaceans (Loganathan, Devi, & Kalaiarasi, 2016). The findings of this experiment corroborated these observations when very small quantities of these neurotransmitters were used (0.001mM). Therefore, it can be concluded that small amounts of neurotransmitters can alter the heart rate of crayfish.
References
Culos, G. J., & Tyson, R. C. (2014). Response of poikilotherms to thermal aspects of climate change. Ecological Complexity, 20 (2014), 293-306.
Holsinger, R. C., & Cooper, R. L. (2012). Effect of environment and modulators on hindgut and heart function in invertebrates: Crustaceans and Drosophila. Proceedings of the Association for Biology Laboratory Education, 33, 68-85.
Kuklina, I., Kouba, A., & Kozák, P. (2013). Real-time monitoring of water quality using fish and crayfish as bio-indicators: A review. Environmental monitoring and assessment, 185(6), 5043-5053.
Listerman, L. R., Deskins, J., Bradacs, H., & Cooper, R. L. (2000). Heart rate within male crayfish: Social interactions and effects of 5-HT. Comparative Biochemistry and Physiology, Part A 125 (2000), 251–263.
Loganathan, P., Devi, M. R., & Kalaiarasi, M. V. (2016). Study on the impact of drugs on heart and cardio-vascular system in crabs inhabiting freshwater, marine and brackish waters. International Journal of Current Research and Review, 8(1), 36.
Noordergraaf, A. (2012). Circulatory system dynamics (Vol. 1). New York: Elsevier.
Tieck, S. (2011). Circulatory system. Edina, Minnesota: ABDO Publishing.