Analysis of VP28 Gene in White Spot Syndrome Virus Infection

Analysis of VP28 Gene in White Spot Syndrome Virus Infection Temporal analysis of VP28 gene in White spot syndrome virus infected fresh water crabs Chiin Nei Chinga, Mansi Parihara, R. Sudhakaranb* aSchool of Bioscience and Technology, VIT University, Vellore – 632 014, Tamil Nadu, India. *bAssistant professor, SBST, VIT University, Vellore – 632 014, Tamil Nadu, India. ABSTRACT Aquaculture is the farming of aquatic organisms such as fish, crustaceans, molluscs and aquatic plants. Disease emergence is a concern in wild fisheries due to environmental pressures, the direct impact of human activities and the risk of pathogens spread from aquaculture. Common viruses are Taura syndrome virus (TSV), white spot syndrome virus (WSSV), and the necrotizing hepato pancreatitis bacterium (NHP-B). The White Spot Syndrome Virus is the most economically devastating viral pathogen to global shrimp aquaculture production and has been proposed to be capable of infecting all decapod crustaceans. WSSV is an enveloped ellipsoid virus, which belongs to the genus Whispovirus of the family Nimaviridae. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. In this study, the proteins of the infected crab were purified using SDS page and then Western Blotting was performed to extract that particular protein. The VP28 protein will appear as specific bands in the blot. Keywords:Â  Aquaculture, White spot syndrome virus, VP28, SDS-Page, Western blotting. 1. INTRODUCTION White spot syndrome virus (WSSV) is one of the major shrimp pathogen that causes a high mortality rate of 90-100% within 3-10 days of infection (Lightner, 1996). Natural WSSV infections have been found in captured and cultured specimens of the mud crab. WSSV is an enveloped ellipsoid virus, which belongs to the genus Whispovirus of the family Nimaviridae. In addition, WSSV can also infect a wide range of hosts including both decapod and non-decapod animals with more than hundred species described to date. So far, the genome from three different WSSV isolates has been sequenced. Sequence analysis showed that WSSV contains approximately 500 putative open reading frames (ORFs), most of which have no homology with any known genes or proteins in public databases. Till now, more than 50 structural and non-structural proteins were identified. Tools such as polymerase chain reaction (PCR) and nested PCR, originally developed by Lo etal., have been widely used and recommended by the Office of International Epizootics (OIE) to be used as standard diagnostic methods for the detection of WSSV. Despite their excellence in specificity and sensitivity, these methods were not suited in some circumstances due to their complications, the requirement of thermal cycler, time-consuming, and labor-intensive. Moreover, the classical agarose gel electrophoresis with ethidium bromide staining, following the visualization under the ultraviolet (UV) transilluminator required to analyze the result of PCR products. Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The blot is a membrane of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins in the gel to move to the membrane where they adhere. The membrane is then a replica of the gel’s protein pattern, and is subsequently stained with an antibody. Therefore, these features could be limited their applications, particularly in the resource-limited areas and non-laboratory environments such as at the pond or station sites. 2. MATERIALS AND METHODS 2.1. Tissue homogenate preparation: Gills, muscle, hepato pancreas and Head soft tissue from the crab infected with WSSV were homogenized in 1:10 suspension with NTE Buffer. It was then freeze and thaw for three times then centrifuged at 5000 rpm for 5min. The supernatant were collected separately in a tube and stored at -20Â °C. This supernatant was used for protein analysis. 2.2. Protein estimation: Lowry’s method was performed for the estimation of protein in order to know its concentrations (Lowry et al., 1951). 2% sodium carbonate in 0.1N sodium hydroxide (Reagent A) 0.5% copper sulphate in 1% potassium sodium tartrate (Reagent B) Alkaline copper solution: Mixed 50 ml of solution A and 1 ml of B prior to use (reagent C) Folin Ciocalteau reagent (Reagent D) Protein standard stock: 50 mg of bovine serum albumin (fraction V) was weighed and dissolved in distilled water and made up to 50 ml (1 mg/ml) Working standard- diluted 1ml of stock solution to 5 ml with distilled water. (200 Â µg of protein/ml). 2.3. SDS-PAGE: SDS-PAGE is currently used to determine the molecular masses, and also to investigate the subunit composition and the domain structure of proteins. TAE Buffer (1X) was poured in tank which maintain the temperature and pH of the gel. Gel was prepared and protein sample is mixed with bromophenol blue which act as a tracking dye. Then it was loaded into the well and electric current of 50V is applied into the tank which is later on increase to 100V when the dye contacts the resolving gel. When the dye reach the end of the resolving gel it was taken out and stain with the help of Coomassie Brilliant Blue R-250 (methanol-50 ml, distilled water 40 ml, glacial acetic acid 10 ml) for 1 hour and de-stain the gel by immersing in the de-staining solution (65 ml of distilled water, methanol 10 ml and 25 ml of glacial acetic acid). 2.4. Western blotting: Transferring of the protein into a membrane The membrane can be either nitrocellulose or PVDF. PVDF requires activation with methanol for one minute and is then rinsed with transfer buffer before preparing the stack. After stacking, the electro transfer is carried out for 90 minutes at a constant current of 150 mA set by maintaining the voltage at maximum. Transfer to the membrane and checked using Ponceau Red staining before the blocking step. (PVDF cannot be stained). After ponceau staining, wash the nitrocellulose membrane with TBST for 1 min at room temperature. Blocking and Antibody incubation Block the membrane for 1 hour at room temperature in blocking buffer (10 % milk in TBST). After blocking, incubate the membrane in primary antibody (1:1000 dilutions) in 2.5% milk in TBST overnight at 4Â °C. Wash the membrane for three washes using TBST for 5 minutes each. Incubate the membrane with secondary antibody (1:2000 dilutions) in 2.5% milk in TBST at room temperature for 1 hour. Wash the membrane in three washes of TBST, 5 minutes each. Add ECL reagent (Solution A and B in 1:1 ratio) on to the membrane and spread it thoroughly on to it using a pipette. Remove the excess reagent and place the membrane on to the x-ray cassette and expose to x ray film in the dark room. An initial 10 sec exposure should indicate the proper exposure time because due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hrs. 3. RESULTS AND DISCUSSION 3.1. SDS-PAGE: Fig.1 SDS-PAGE which shows different bands under white light. Lane 1- Gill; Lane 2- HST; Lane 3- Muscle; Lane 4-Hepatopancreas. The polypeptides of purified virus were resolved by SDS-PAGE using discontinuous buffer system. In 12.5% acrylamide gel, the purified virus yielded different polypeptides base on their molecular mass in which lower molecular weight will elute faster as compared to those having higher molecular weight, which were Visualized on Coomassie brilliant blue R 250 staining. 3.2. Western blotting: Fig 2. Western blotting Viral polypeptides separated by SDS-PAGE were electrophoretically transferred to nitrocellulose membrane (0.22 ÃŽ ¼m) in transfer buffer (192 mM glycine, 25 mM Tris and 20% methanol, pH 8.3), at 0.8 mA/cm2for 1 h. it was later on view by using film to visualize the relative abundance of proteins Discussion By performing SDS-PAGE, (fig.1) we can determined the different molecular weight of polypeptide. The difference in the number of the polypeptide may be attributing due to the difference in concentration of the resolving and other physical conditions. The western blot analysis of proteins of semi-purified white spot syndrome virus separated on 12.5% gel (fig.2) showed different number of immunogenic proteins based on its molecular weight. Out of these, those proteins, which stained more intensely, were considered as major immunogenic proteins. However, further studies are needed to establish its immunogenic nature and feasibility for its use as vaccine. Because electrophoretic separation of proteins is always carried out under denaturing conditions, the problem of solublization, aggregation and co-precipitation of target proteins with adventitious proteins are eliminated. REFERENCES [1] Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951): Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193(1), 265-275. [2] van Hulten, M. C., J. Witteveldt, S. Peters, N. Kloosterboer, R. Tarchini, M. Fiers, H. Sandbrink, R. K. Lankhorst and J. M. Vlak (2001): The white spot syndrome virus DNA genome sequence. Virology, 286(1), 7-22. [3] Seetang-Nun Y, Jaroenram W, Sriurairatana S, Suebsing R, Kiatpathomchai W (2013)Â ­: Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification. Mol Cell Probes, 27(2):71-9. [4] Maurice Pagano (1999): Application of electrophoresis and related methods, such as western blotting and zymography to the study of some proteins and enzymes. Analytica Chimica Acta, 83(1–2): 119–125. [4] Lightner, D. V. (1996): A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society.

How to handle your finance after divorce Essay --

How to handle your finance after divorce Divorce is a complex and heartbreaking episode in anyone's lives. You would not want to add in to your burden with monetary issues.This turn out to be a greater disaster if you were always being dependent on your partner or have money crunch. You can no longer afford to rely on anyone else other than yourself. So, gear up and get your act straight. We know it is difficult and at the same time important to start handling financial responsibility. Here are a few steps to help you to take the first step. Who owns the home In case the home is owned by your ex-spouse sole name, then you might want to consider registering for a share using a 'matrimonial home rights notice' or a 'restriction' notice. Both of these notices will help you in guaranteeing that your ex-partner won't try to sell, transfer or mortgage the property without your knowledge or agreement. Clear your mortgage concerns Explain your stand clearly, talk to the lender and discuss how you plan to manage the mortgage repayments. This also applies in case you are staying in...

Analysis of Hip-Hop and Youth Culture Essay -- Papers

Analysis of Hip-Hop and Youth Culture Throughout the last twenty-five years, a new form of expression has continued to evolve. Hip-Hop, once limited to urban music and dance has become a widespread form of communication exhibited and enjoyed by young people throughout the world. Hip-Hop is no longer limited to rap music and break dancing; today it represents a multi-billion dollar industry that influences everything from fashion to prime- time television programming, professional sports, mass media marketing and advertising. Today Hip-Hop is becoming a way of life, a culture that is intricately woven into every aspect of young people's daily lives. We may not necessarily be consciously aware of it, but what we are saying, what we are wearing and our musical; cinematography choices can be subtly influenced by Hip-Hop culture This Hip-Hop emerging culture has similarities to other cultures that have emerged throughout history. Hip-Hop was initially born of the ability of those early practitioners of rap music, DJ wizardry and street-corner fashion creation to overcome their inability to gain acceptance and recognition by the established music, fashion and entertainment industries. Further galvanising the fledgling culture was the lack of acceptance by adult culture, which refused to recognise these newly emerging forms of expression as legitimate. This was particularly true where many parents were concerned. Needless to say, if parents and other authority figures didn't understand Hip-Hop, didn't like it and, in many instances, admonished young people for embracing it, young people were even more compelled to further immerse t... ...is view is indicative of the mindset of many in Hip-Hop culture. Whether subscribing to the ideology of being "gangsta" or simply being radical in their thinking and views of the world, the shared theme throughout Hip-Hop is being "real". It is perhaps in this "being real" that the culture finds its willingness to be arrogant and unapologetic in its brazen disregard for anyone that does not appreciate what Hip-Hop is. Therein also lies some of the confusion because there is as stated earlier no one consistent theme regarding what Hip-Hop are. From the "bling-bling" of such Hip-Hop artists as 50 Cent, to the cross-over influences of Black Eyed Peas and to the pimpish styling of Snoop Dogg, the themes are as broad and conflicted as the frequent disagreements and altercations that arise between various camps in Hip-Hop.

America: Myth Of Equality Essay -- essays research papers

America: Myth of Equality To many, the Unites States serves as the ideal model of democracy for the modern world. Yet, how truly worthy is America of this status? Although it has been said that, â€Å"Equality is as American as baseball, hot dogs, and apple pie,† one must be extremely critical when analyzing such a statement. By taking a historical perspective to the question of how â€Å"equal† American equality actually is, it is simple to recognize how problematic the â€Å"Land of the Free† mentality can be. The early America’s most prominent thinkers have been sensationalized and given credit for developing a free and equal system. However, one can recognize that their manner of thinking was far from this idea of â€Å"all men are created equal† by critical examination of their literature.   Ã‚  Ã‚  Ã‚  Ã‚  When analyzing equality, a sociological and legal approach must be made. In the case of the United States, the sociological stratification, or division of power, of minority groups had adversely affected the development of the legal system. Especially in the 1600s and 1700s, legal and social equality of those not of the Caucasian persuasion, the less fortunate, and of the female gender was almost nonexistent. This inequality is a direct result of the early American society’s ranking system. Equality was not perceived in the minds of such individuals as John Winthrop, John Adams, and James Madison, and as a result, their significant accomplishments towards the development of the American system have tainted the institution itself.   Ã‚  Ã‚  Ã‚  Ã‚  One of the earliest American social groups was the 17th Century Puritans. This society had an extremely unique and strict manner of thinking that was entirely based on inequality. This is clearly represented by the writings of the Puritan leader John Winthrop. In â€Å"A Model of Christian Charity,† Winthrop outlines the societal rationale, and in turn, the disparity of equality in his society. The gist of the Puritan way of life is that, by the grace of God, certain individuals were empowered with the ability to be enlightened and the capability of achieving much within the society itself. However, by the same reasoning, some individuals were also destined to take the lesser roles in society, and as a result, had no power to move up within the ranks because of this inescapable predetermination. Thi... ...the initial American system. The factions that Madison concerns himself with were the population’s majority, otherwise known as the lesser classes. As a result, the establishment of division of power and checks and balances clauses would give the populace a lesser chance of gaining much authority over the already established aristocracy.   Ã‚  Ã‚  Ã‚  Ã‚   If this is the case, why is America perceived to be relatively fair and equal today? Fortunately the early American politicians did develop framework to allow the Constitution to evolve, and combined with the political movements in both minority and women’s rights of the end of this century, much of this unjust stigma has been eliminated from the system. Still, racial discrepancies in the courts occur more frequently then not, and the social makeup of American politicians continues to follow a predetermined â€Å"mold.† Is this a direct result of the discrepancies formed in the earlier stages of American history? It is hard for anyone to tell. What is indisputable is that the Declaration of Independence’s statement, â€Å"All men are created equal,† was far from the truth during early American history. Word Count: 1,323

Kath Walker’s We Are Gloing

Basically the theme of the poem is the Aboriginals oppression. The fact they have had to give up on their old way of life and let go of the world that used to be theirs. The Aboriginals knew â€Å"We belong here, we are of the old ways† but eventually through white civilisation and development this life is â€Å"Gone and scattered,† everything is gone and now they had to leave. In summary, the theme of this poem is the oppression experienced by the Indigenous population that resulted in a loss of culture and life for the Indigenous now â€Å"gone and scattered. One example of a technique in the poem includes repetition such as that of the word ‘gone. ‘ The word gone typifies the themes of loss and it's repetition enforces this concept. Other techniques include alliteration such as ‘silent and subdued,' similes such as â€Å"Where now the many white men hurry about like ants,† capitalisation of the word Thunder, six stanza structure, first perso n narration etc. Good luck! The obvious technique is slow deliberate repetition. This reinforces the ancient sigh of a people dispossessed. All that is sacred has been violated. A history is being expunged. Alliteration: subdued and silent ( we even faintly hear the shush) Simile: white men hurry about like ants There are metaphors used by the naive speaker in this poem. They are the land, the elements, the fauna, the flora and the history. The warning is all this heart will be gone if the white man has his way. Kath Walker was right. Her warning was not heard. And they are gone! 1. Explain why they are â€Å"silent and subdued†. 2. How are white men represented? Why? 3. What is a bora ring and explain why it is so central to this poem. 4. Explain their reaction in line 8. 5. Lines 9-17 begin a ‘litany’. What is the effect produced? 6. Comment on the significance of metaphors used in the poem. 7. Comment on the structure and form of this poem. 8. Why does Thunder have a capital letter? 9. Comment on the mood and atmosphere created here. 10. Combine comments on its theme, title and conclusion. Answers 1. They were silent and subdued because little remained of their tribe and many strangers were busy at work like ants. 2. The white men were represented as ants because they were busy at work and hurrying around. 3. A bora ring is where an initiation is performed and is hardened earth done by foot which is surrounded by raised embankments in a formation of a circle and it is central because the bora ring is not being respected as a sign says, â€Å"Rubbish maybe tipped here†. 4. Their reaction meant that to the white men they are strangers and are treated like they don’t belong in Australia but the Aboriginals feel that the white men are the strangers and are in their land. 5. It gives an effect of showing their side of really saying that they are the real natives of Australia and not the white men and that now that they are the past. 6. It compares and shows how the Aboriginals feel. 7. It has six stanzas and has no rhyming pattern. 8. The emphasis the word as thunder is really the loud terrifying sound as a result of lightning. 9. They are in the fresh air of Australia near the Bora Ring and the mood is of sadness and subduedness because of how the Bora Ring is being mistreated. 10. ‘We are going’ are the three words in the title, conclusion and theme and portrays how the life of the Aboriginals would be as the Aboriginals are not welcome to their own homeland therefore they are saying that they will leave their old ways and be cast upon a low unwanted class forever.

Digital Fortress Chapter 14

Becker gazed down at the cadaver. Even hours after death, the Asian's face radiated with a pinkish glow of a recent sunburn. The rest of him was a pale yellow-all except the small area of purplish bruising directly over his heart. Probably from the CPR, Becker mused. Too bad it didn't work. He went back to studying the cadaver's hands. They were like nothing Becker had ever seen. Each hand had only three digits, and they were twisted and askew. The disfigurement, however, was not what Becker was looking at. â€Å"Well, I'll be.† The lieutenant grunted from across the room. â€Å"He's Japanese, not Chinese.† Becker looked up. The officer was thumbing through the dead man's passport. â€Å"I'd rather you didn't look at that,† Becker requested. Touch nothing. Read nothing. â€Å"Ensei Tankado†¦ born January-â€Å" â€Å"Please,† Becker said politely. â€Å"Put it back.† The officer stared at the passport a moment longer and then tossed it back on the pile. â€Å"This guy's got a class-3 visa. He could have stayed here for years.† Becker poked at the victim's hand with a pen. â€Å"Maybe he lived here.† â€Å"Nope. Date of entry was last week.† â€Å"Maybe he was moving here,† Becker offered curtly. â€Å"Yeah, maybe. Crummy first week. Sunstroke and a heart attack. Poor bastard.† Becker ignored the officer and studied the hand. â€Å"You're positive he wasn't wearing any jewelry when he died?† The officer looked up, startled. â€Å"Jewelry?† â€Å"Yeah. Take a look at this.† The officer crossed the room. The skin on Tankado's left hand showed traces of sunburn, everywhere except a narrow band of flesh around the smallest finger. Becker pointed to the strip of pale flesh. â€Å"See how this isn't sunburned here? Looks like he was wearing a ring.† The officer seemed surprised. â€Å"A ring?† His voice sounded suddenly perplexed. He studied the corpse's finger. Then he flushed sheepishly. â€Å"My God.† He chuckled. â€Å"The story was true?† Becker had a sudden sinking feeling. â€Å"I beg your pardon?† The officer shook his head in disbelief. â€Å"I would have mentioned it before†¦ but I thought the guy was nuts.† Becker was not smiling. â€Å"What guy?† â€Å"The guy who phoned in the emergency. Some Canadian tourist. Kept talking about a ring. Babbling in the worst damn Spanish I ever heard.† â€Å"He said Mr. Tankado was wearing a ring?† The officer nodded. He pulled out a Ducado cigarette, eyed the no fumar sign, and lit up anyway. â€Å"Guess I should have said something, but the guy sounded totally loco.† Becker frowned. Strathmore's words echoed in his ears. I want everything Ensei Tankado had with him. Everything. Leave nothing. Not even a tiny scrap of paper. â€Å"Where is the ring now?† Becker asked. The officer took a puff. â€Å"Long story.† Something told Becker this was not good news. â€Å"Tell me anyway.†

Daphnia Dissection

Effects of Drugs on Heart rate Michelle Dilgard Undergraduate Student Education Major Biology Concentration Tennessee Technological University Cookeville, TN 38505 December 3, 2006 Table of Contents Page Abstract 1 Key Words 2 Introduction3-6 Methods and Materials7-8 Expected Results and Benefits 9 Results 10-11 Discussion12 Conclusion13 Literature Cited 14-15 Abstract This experiment is designed to find out how drugs affect heart rate. This experiment will use Daphnia in order to monitor the effects certain drugs have on heart rates.I will observe the changes in heart rate of Daphnia when exposed to Caffeine, Ibuprofen, Alcohol, and Nicotine. I will have several separate samples of Daphnia in my study. Each community will be exposed to a different drug and observe how the heart rate of the Daphnia changes accordingly. I believe the Daphnia heart rate will increase when the Daphnia are exposed to Caffeine and Nicotine because both of these drugs are stimulants. I believe the Daphnia heart rate will decrease and slow down when the Daphnia are exposed to Ibuprofen and Alcohol.This experiment will show not only the effects of these drugs on Daphnia but also what they similarly do to the human body when it is exposed. Daphnia are used as a humane alternative to Humans when performing this type of experiment. The results on the Daphnia will be very similar to how the human heart would react if exposed to these drugs. Key Words Nicotine Alcohol Caffeine Ibuprofen Effects of Drugs Daphnia Magna Heart rate Drugs Heart Introduction My experiment deals with the affects of different drugs on the heart rate of daphnia. I will focus on the daphnia’s body itself first.Then I will give information pertaining to the drugs used: Caffeiene, Ibuprofen, Alcohol, and Nicotine. Daphnia are small crustaceans that live in the water. They are commonly called water fleas. Daphnia are freshwater zooplankton and consume phytoplankton and some other zooplankton as well. The daphniaà ¢â‚¬â„¢s bodies are transparent and their internal structures can easily be seen. The heart is the internal organ I focused on within the daphnia. The heart can be easily seen within the body cavity of the daphnia which made it easy to find and easy to count the heartbeat (Villegas-Navarro 2003).Caffeine is a very important drug to consider because 90% of American consumes caffeine on a daily basis. Half of all American consume more than 300 mg of caffeine a day which makes it America’s most consumed drug to date. Caffeine is found in coffee, soda, tea, chocolate, etc. Caffeine is known as trimethylxanthine in the medical community. Caffeine can be used as a cardiac stimulant and also as a mild diuretic. Cardiac stimulants increase the heart rate, and diuretics increase urine production. Caffeine is a very addictive drug and operates just like amphetamines such as cocaine and heroin (Nehlig 1992).Caffeine not only stimulates the heart of humans but also the heart of daphnia ( Foster 1997). Ibuprofen is commonly used to relieve pain, tenderness, swelling and stiffness caused by arthritis. It is also used to relieve mild to moderate pain in the body and reduce fever. Ibuprofen is called a NSAIDs. It works by stopping the body’s production of a substance that causes pain, fever, and inflammation. Ibuprofen is most often used to treat arthritis (Cluevers 2004). Alcohol is often used as a solvent in medical drugs, because of its low toxicity and ability to dissolve non-polar substances.Ethanol is often used as an antiseptic, to disinfect the skin before injections are given. When processed correctly Alcohol is drunk in recreation. Alcohol affects the body as a nervous system depressant (Wong 1997). Nicotine like caffeine is a stimulant. This stimulant is found in cigarettes. Cigarettes contain 8 to 20 milligrams of nicotine but when smoke only 1 mg of nicotine actually enters the body. Nicotine can have two effects on the body, it can relax a perso or it can stimulate a person, this is based on the amount and the regularity of smoking or nicotine intake.Nicotine works by causing a releas of adrenaline into the body. Statement of Problem How do certain drugs affect heart rate? Objective/Hypothesis Statement This experiment is designed to find out how drugs affect heart rate. This experiment will use Daphnia in order to monitor the effects certain drugs have on heart rates. I will observe the changes in heart rate of Daphnia when exposed to Caffeine, Ibuprofen, Alcohol, and Nicotine. I will have several separate samples of Daphnia in my study.Each community will be exposed to a different drug and observe how the heart rate of the Daphnia changes accordingly. i. e. Foster 1997 I hypothesize that the Daphnia heart rate will increase when the Daphnia are exposed to Caffeine and Nicotine because both of these drugs are stimulants. I hypothesize the Daphnia heart rate will decrease and slow down when the Daphnia are exposed to Ibuprofen and Alcohol. The null hypothesis to this is: I hypothesize that drugs will have no effect on the Daphnia’s heart rate. Methods and Materials Data Form |Resting Heart Rate |Heart Rate 1 |Heart Rate 2 |Heart Rate 3 | |Alcohol 1 | | | | | |Alcohol 2 | | | | | |Alcohol 3 | | | | | | | | | | | |Ibuprofen 1 | | | | |Ibuprofen 2 | | | | |Ibuprofen 3 | | | | | | | | | | |Caffeine 1 | | | | | |Caffeine 2 | | | | |Caffeine 3 | | | | | | | | | | | |Nicotine 1 | | | | | |Nicotine 2 | | | | | |Nicotine 3 | | | | | ProcedureI used a modified version of the experiment performed by Jasmine Kamai and Varner Allbrett when they studied the effects of Kava on the heart rate of Daphnia. My procedure is as follows: Before beginning my experiment I must mix the drug solutions to be used. First I will dissolve ibuprofen and caffeine tablets into a solution of water in two different containers, that I will later determine what strength to make each. I will also tear open cigarettes and mix the tobac co with water and let sit for 24 hours after which I will drain the water off into a container which will make up my nicotine solution. For alcohol I will just mix vodka and water to a desired strength in a 4th container. Next I will cipher out a daphnia and drop it onto a slide to be viewed under a microscope.I will record its normal heart rate. Next I will add a drop of the alcohol solution and over the next 5 minutes I will record the changes in heart rate after one minute of exposure, 3 minutes of exposure and then 5 minutes of exposure. After recording my results I will dispose of this Daphnia. I will do this same portion of the experiment with two more daphnia. I will then continue with this procedure while using the other three drugs. I plan to use the lab facilities in the Biology Building on Tennessee Tech’s Campus. Along with most of their lab equipment including: microscope, slides, vials, Petri dishes, droppers and other equipment. Materials Water Depression Slide sCover slips Droppers Daphnia Magna Microscope Watch Petri Dishes Daphnia Anatomy Chart Cigarettes 80 proof vodka No-dos Ibuprofen tablets Expected Results and Benefits After performing this experiment I expect to find that the Daphnia’s heart rates will increase with Nicotine and Caffeine because both of these drugs are stimulants and that the Daphnia’s heart rates will decrease with Alcohol and Ibuprofen solution. This experiment will show not only the effects of these drugs on Daphnia but also what they similarly do to the human body when it is exposed. Daphnia are used as a humane alternative to Humans when performing this type of experiment. Results Normal |w/Caffeine |w/Alcohol | |84 |100 |89 | |80 |102 |85 | |86 |105 |90 | |88 |109 |91 | |82 |109 |88 | |84 |103 |85 | |90 |110 |93 | |86 |106 |89 | |80 |90 |84 | |84 |88 |87 | Heart Rate of Daphnia when exposed to Caffeine and Alcohol Change in Heart Rate of Daphnia when exposed to Caffeine Change in Heart Rate of Daphnia when exposed to Alcohol Discussion After performing my experiment I found that Daphnia Magna heart rate greatly increases when exposed to Caffeine. Caffeine is a stimulant and should produce that affect (Nehlig 1992). Caffeine has similar affects on humans (Foster 1997).The heart rate increased anywhere from 10 to 20 beats per minute. As seen in my data table the heart beats sometimes jumped from a normal heartbeat in the 80s to a heartbeat in the 100s when exposed to caffeine. Gerald Adams also found the same result when he tested his hypothesis. I also found that Daphnia’s heart rate slightly increased with the exposure to Alcohol. The heart rate does not have as drastic a change when exposed to Alcohol as it did when exposed to Caffeine. The heart rates just increased by about 5 to 10 beats per minute. As seen in my data table the heart beats only jumped from a normal heartbeat in the 80s to a heartbeat in the 90s when exposed to Alcohol.This was disproved by sever al different experiments (Wong 1997). Wong found that the heart rate of daphnia decreased. Since alcohol is a depressant it should have lowered the heart rate of daphnia. I feel that the increase in stress on the Daphnia may have called this time of increase. When Daphnia is exposed to Nicotine and Ibuprofen, the heart rate of the daphnia could not be seen as any different. (Sollman 1999). Although Cluevers said that there should be a change in the heart rate (Cluevers 2004). Conclusion In conclusion I have determined that the heart rate of Daphnia can be affected by adding different drugs. Daphnia heart rate increased by ten to twenty beats per minute when exposed to Caffeine.Daphnia heart rate increased by five to ten beats per minute when exposed to Alcohol. Unfortunately the results for Ibuprofen and Nicotine could not be seen. There was not enough evidence to determine how the Heart rate changed when the Daphnia were exposed to Ibuprofen and Nicotine. By doing this experiment i t can be assumed that the some of the same reactions could be found if Humans are exposed to these Drugs. Literature Cited Cluevers, Michael. 2004. Mixture toxicity of the anti-inflammatory drugs diclofenac, ibuprofen, naproxen, and acetylsalicylic acid. Exotoxicology an Environmental Safety 59: 309-315. Foster, Rachel. 1997. A stroboscopic method to investigate the effect of caffeine on Daphnia hear rate.Journal of Biological Education 31: 253-255. Nehlig, A. , J. L. Daval, and G. Debry. 1992. Caffeine and the central nervous system: mechanisms of action, biochemical, metabolic and psychostimulant effects. PubMed. com. Villegas-Navarro, Arturo, Esperanza L. Ross, and Jose L. Reyes. 2003. The heart of Daphnia magna: effects of four cardioactive drugs. Comparative Biochemisty and Physiology Part C Toxicology and Pharmacology 136C: 127-134 Wong, Diana C. L. , Philip B. Dorn, and Eric Y. Chai. 1997. Acute toxicity and structure-activity relationships of nine alcohol ethoxylate surfacta nts to fathead minnow and Daphnia magna. Environmental Toxicology and Chemistry 16: 1970-1976. . Appendix Normal |w/Caffeine |w/Alcohol | |84 |100 |89 | |80 |102 |85 | |86 |105 |90 | |88 |109 |91 | |82 |109 |88 | |84 |103 |85 | |90 |110 |93 | |86 |106 |89 | |80 |90 |84 | |84 |88 |87 |