testing plant defensive compounds effect on Artemia salina heart rate

Testing Plant Defensive compounds effect on Artemia salina heart rate. Gianna Calabrese Nova Southeastern University Biology 1510 (Section DA4) March 11, 2019 Abstract The goal of the experiment is to be able to understand the effect of the Plant defensive compounds on small organisms. The lab includes using two plant defensive compounds, coffee and lemongrass and watch how it alter the preys heart rate. The null hypothesis is the heart rate of the A. salina will not increase when toxins are added and the alternate hypothesis is the heart rate of the A. salina will increase when toxins are added. This was tested by adding one drop of the control seawater and counting the number of flaps for 5 seconds and multiplying it by 12 to find the fbm of the small organism, A. salina. The average control fbm was 129 fbm. When the plant defensive compounds were added and followed the same process. The A. salina heart rate increased for both variables. The fbm for coffee was 130 and the fbm for lemongrass was 134. The t statistics for the control and coffee was 0.1. The t-statistics for the control and lemon grass was 0.9. Lastly the t-statistics for coffee and lemon grass was 0.8. The critical value for the control and the plant defensive value was 2.069.Overall, the major conclusions of the experiment are the plants use these defense compounds in order to protect themselves from predators. They produce toxins which alter the heart rate and homeostasis of the predator. Over time, the predator becomes custom to the side effect of the eating the toxin and will avoid it. Introduction The main purpose of this lab is to investigate in the different effects of the plants defensive compounds on small organism’s heart rate. The null hypothesis for this experiment is the heart rate of the A. salina will not increase when toxins are added. Plants chemical defenses take form of allelochemicals, which are toxins that cause harm to the attacker. These toxins are secondary metabolites produced by the host plant and serve to disrupt behavior , growth/development or overall survival of the herbivore. ( Bio II Lab Manual , 2019) When predators try to eat these plants, the plants will emit their defensive compounds which will cause them to have an alter in behavior and life style. In this experiment, the small organism used as a test subject to the defensive compounds is a A. salina. Artemia salina L. (Artemiidae), the brine shrimp larva, is an invertebrate used in the alternative test to determine toxicity of chemical and natural products. (Parra et al., 2004). Using A. salina as a test subject is more ethical according to scientists. To use an Artemia salina assay in toxicological tests that screen a large number of extracts for drug discovery in medicinal plants. This is because in this case, aseptic techniques are not required, and thus A. salina assays could replace the more ethically challenging MTT assay that requires animal serum.( Rajabi etal.,2015) These organisms are mainly used due to their circulatory system being similar to or the same as small predators who fed on plants. Small organisms have an open circulatory system like A. salina . Like many molluscs, arthropods have an open circulatory system( Reece et.al , 2014) Very similar to small organisms such as insects have well developed sensory organs. In the experiment two different plant defensive compounds were used coffee and lemon grass. Coffee provides one of the biggest drugs in the world, Caffeine. Coffee is from the Coffea sp., Which is the genus plant made famous for its effect as a stimulant. The active ingredient in coffee is caffeine.( Bio II Lab Manual , 2019) Caffeine is a stimulant, which will connect to your adenosine receptor and keep you more awake and alert Caffeine is a methylxanthine whose primary biological effect is the competitive antagonism of the adenosine receptor. ( Chou, T. & Benowitz, N, 2003) The coffee plants uses the caffeine to ward off small organisms that would prey on their leaves. Caffeine affects the small species heart rate make it difficult for them to survive. It has an effect on the cardiovascular system with a slight increase in blood pressure and heart output.(Ramalakshmi, & Raghaven, 2010,) Caffeine is an alkaloid that stimulates the central nervous system causing in balance in homeostasis and an increase in heart rate. Lemon grass (Cymbopogan Citratus) which is a plant commonly found and used in Southeast Asia. Lemon grass extracts an oil which can be harmful to small organisms such as insects. Lemon grass oil contains a high concentration of a chemical component called Citral ( Lemon oil facts, 2018) Citral is known to a toxin for small organisms which is lethal to their survival. Both plant defensive compounds protect the plants against their predators and alter the heart rate of their predators. The null hypothesis is the heart rate of the A. salina will not increase when toxins are added and the alternate hypothesis is the heart rate of the A. salina will increase when toxins are added. Methods For this experiment , a transfer pipette was used to extract a single Artemia salina onto a slide. The A. salina ( specimen) had enough water where the gill was still able to function, but not be able to actively swim. All excess water was removed with a kimwipe. The specimen was located under a dissecting microscope. The control that was used for this experiment was seawater. The two plant defensive compound variables used were coffee and lemon grass. First, the control was tested. A drop of seawater was placed onto the slide. The microscope light was off as it waited for approximately a minute for incubation. Then, using a timer the number of leg flaps were counted for 5 seconds. The number of counted flaps in that time period was then multiplied by 12 to get the total of flaps per minute. The experiment is repeated with two additional A. salina. Each specimen went through 3 trials before the next specimen. The average flaps per minute for the control was 129 fpm. Now the same experiment is repeated but instead of the control, the first plant defensive compound variable, coffee is used. The A. salina was transferred from the transfer pipette onto the slide. A drop of coffee was placed onto the slide while the microscope light was off. The specimen incubated in the coffee for approximately a minute. Once the minute was up, the microscope light was turned on and the amount of leg flaps were counted for 5 seconds. Once the total of legs flaps for 5 seconds was found, it was multiplied by 12 to find the new flaps per minute. As stated before, the experiment is repeated with two additional A. salina. Each specimen went through 3 trials before the next specimen. The average flaps per minute for the coffee was 130 fpm. Finally, the last plant defensive compound variable, lemon grass, is tested. Again, The A. salina was transferred from the pipette onto the slide. A drop of lemon grass was placed onto the slide while the microscope light was off. The specimen incubated in the lemon grass for approximately a minute. After a minute, the microscope light was turned on and the number of leg flaps were counted for 5 seconds. Once the value of legs flaps was counted for the 5 seconds, the value id then multiplies by 12 to find the flaps per minute. As repeated in the previous statements, , The experiment is repeated with two additional A. salina. Each specimen went through 3 trials before the next specimen. The average flaps per minute for the lemon grass was 134. Results Table 1: Average Flaps per minute of the A. salina when incubated by the plant defensive compounds Control Coffee Lemongrass Average 129 130 134 StDev 16.9 18.5 16.8 Variance 287 340 281 n 24 24 24 Table 2: Statistical analysis of the average flaps per minute of the A.salina by the plant defensive compounds | x?1- x?2 | 0-52990s12n1+s22n2 00s12n1+s22n2 t stat Control v. Coffee 0.48611 5.1 0.1 Control v. Lemongrass 4.4861 4.9 0.9 Coffee v. Lemongrass 4.0000 5.1 0.8 Critical t-value = 2.06865761 Figure 1: Average Flaps per minute of the A. salina when incubated by the plant defensive compounds the graph shows a visual representation of the average heart rate by the flaps per minute. The lowest average heart rate the A.salina had was the control group of 129 fpm and with a minimum value was 58 fbm and a maximum value of 196 fbm. Coffee had the second highest average heart rate of 130fpm with a minimum value of 60 fbm and the maximum value of 224 fbm. The lemon grass had the largest average heart rate of 134 fpm and a minimum value of 48 fbm and the maximum value of 226 fbm. Discussion In this experiment, the experimenters tested the effect of the plant defensive compounds on the small organism’s heart rate. In table 1, The results of the average flaps per minute increased when coffee and lemon grass was added. The largest average of flaps per minute was the Lemongrass. Lemon grass had 139 fpm and coffee had 130 fbm. These toxins did put an effect on the small organism and caused their heart rate to increase. Shown in Figure 1, is clearly indicated on the chart that lemon grass has a higher average than the coffee and the control. We see the effect because the averages shown on the graph are higher than the control. Which means the coffee and lemon grass did affect the small organisms heart rate. Also, according to table 1 the standard deviation of the coffee was the highest. The coffee had the largest amount of differences in the numbers. The standard deviation for coffee was 18.5. Also found in the experiment that the control and lemon grass had high standard deviations. According to table 1, The control had a standard deviation of 16.9 and the lemon grass had a standard deviation of 16.8. From this data I can conclude there may have been sources of error. Through the statistical analysis, the null hypothesis fail to reject. Due to the t- statistics was not equal or greater than the critical value. According to Table 2, the t statistics for the control and coffee was 0.1. The t-statistics for the control and lemon grass was 0.9. Lastly the t-statistics for coffee and lemon grass was 0.8. The critical value for the control and the plant defensive value was 2.069. Due to the critical value being larger than the t- statistics, the null hypothesis: the heart rate of the A. salina will not increase when toxins are added, was failed to be rejected. This also leads to the conclusion that there were sources of error. Compared to other studies completed in the past, the null hypothesis was rejected. In the experiment Caffeine as a repellent for slugs and snails, the snails died to the coffee being toxic and causing their heart rate to increase significantly and lethal to them. As stated, while field-testing caffeine as a toxicant against an introduced frog pest that infests potted plants in Hawaii, we discovered that large slugs were killed by spray applications containing 1–2% caffeine. To test whether caffeine solutions could be used to remove or kill large slugs that attack potted plants, we allowed Veronicella cubensis (Pfeiffer) to bury themselves in the soil in the pots, and then thoroughly wetted the soil with a 2% caffeine solution. After 3.5 h, only 25% of the slugs remained in the soil; after 48 h, all slugs had left the soil and 92% were dead.”(Hollingsworth et.al, 2002) The other studies showed it should have impacted the heart rate significantly and lead to death. Also, lemon grass should have also done the same effect to the heart rate. The study showed the insects, Stapf, are repellent to lemon grass and will die if exposed to the lemongrass oi. It would cause an increase in heart rate then lead to death. This attracted twelve insects as the highest number at a time after a period of 180 min (?82% repellency). The third sample was treated and used immediately after treatment following the same procedure. After a period of 180 mins, the highest number of insects found around the sample at a time was three (?95% repellency). Within 48 h, a great number of the insects were found dead and the remaining insects were weak. Their death may be due partly to the insecticidal properties of the lemongrass oil and partly to starvation.” (Adeniran, O. I. & Fabiyia, E. , 2012) Based on the similar studies conducted, it leads to the conclusion that there was error during this experiment. Possible sources of error can be not waiting a minute to let the toxic incubate before reading the heart rate. This could of caused the results to skewed. Another source of error can be waiting too long to record the number of leg flaps and the specimen had already been poisoned and died. One last possible source of error is putting too much water allowing the A. Salina to swim which would cause the brine shrimp to swim and not fully incubate the toxicants given causing an inaccurate number of leg flaps. References Adeniran, O. I. & Fabiyia, E. (2012) A cream formulation of an effective mosquito repellent: a topical product from lemongrass oil (Cymbopogon citratus) Stapf Bio II Lab Manual (2019) Testing Plant Defensive Compounds. Nova Southeastern University Biology Department. Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. (2004, November 10). Retrieved from https://www.sciencedirect.com/science/article/pii/S0944711304700574 Hollingsworth, R. G., Armstrong, J. W., & Campbell, E. (2002, June 27). Pest Control: Caffeine as a repellent for slugs and snails. Retrieved from https://www.nature.com/articles/417915a Lemongrass Oil Facts (Definitive Safety Guide). (2018, July 09). Retrieved from https://www.peststrategies.com/pest-guides/chemicals/lemongrass-oil/ Chou, T. & Benowitz, N.(2003, August 11). Caffeine and coffee: Effects on health and cardiovascular disease. Retrieved from https://www.sciencedirect.com/science/article/pii/074284139400048F Rajabi, S., Ramazani, A., Hamidi, M., & Naji, T. (2015, February 24). Artemia salina as a model organism in toxicity assessment of nanoparticles. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344789/ Ramalakshmi, K., & Raghaven, B. (2010, June 03). Caffeine in Coffee: Its Removal. Why and How? Retrieved from https://www.tandfonline.com/doi/abs/10.1080/10408699991279231 Reece, J. B., Urry, L. A., Cain, M. L. 1., Wasserman, S. A., Minorsky, P. V., Jackson, R., & Campbell, N. A. (2014). Campbell biology (Eleventh edition.). Boston: . Appendix Table 1: Control Coffee Lemongrass Average =AVERAGE(B6:B29) =AVERAGE(C6:C29) =AVERAGE(D6:D29) StDev =STDEV(B6:B29) =STDEV(C6:C29) =STDEV(D6:D29) Variance =VAR.S(B6:B29) =VAR.S(C6:C29) =VAR.S(D6:D29) n =COUNT(B6:B29) =COUNT(C6:C50) =COUNT(D6:D50) Table 2: 142240088900s12n1+s22n2 00s12n1+s22n2 | x?1- x?2 | t stat Control v. Coffee =ABS(B2-C2) =SQRT((B4/B5)+(C4/C5)) =G46/H46 Control v. Lemongrass =ABS(B2-D2) =SQRT((B4/B5)+(D4/D5)) =G47/H47 Coffee v. Lemongrass =ABS(C2-D2) =SQRT((C4/C5)+(D4/D5)) =G48/H48 Critical t-value= =T.INV.2T(0.05,23)