BIOL112 Lab Manual Fall 2024.pdf

How to Be Successful in Lab 1. Read the assigned laboratory exercise before coming to class. This is key for the first 5 weeks of lab when you are conducting the pre-designed exercises. Your instructor will assume that you have read the lab exercise and therefore will n...

How to Be Successful in Lab 1. Read the assigned laboratory exercise before coming to class. This is key for the first 5 weeks of lab when you are conducting the pre-designed exercises. Your instructor will assume that you have read the lab exercise and therefore will not spend a great deal of time reviewing the topics. There will be a quiz on this material at the beginning of each of the first 5 labs. 2. Participate in the laboratory exercises. The best way for most students to learn is to actually do hands-on work with a subject. Science is one of the few fields in college where hands-on exercises are part of the curriculum, so take advantage of these opportunities. You will better understand the concepts presented in class if you fully participate in lab. In addition, participation in the exercises is part of your laboratory grade. 3. Work as a team and share ideas. At the beginning of the semester, you will be placed into lab groups and be expected to work together. This means that you will collaborate on ideas and conduct experiments together. In addition, you will each be assigned specific roles in your group that you must complete. This means that one person cannot do all the work while the other students watch. All of you need to be contributing members of the team and participate (see #2). You will be assessed on the contribution that you, individually, make to your team. 4. Do your own work. While it is important that you work as a team, in the end you are responsible for the material in your lab notebook as well as your understanding of the concepts. So share ideas and discuss with your lab group, but entries in your lab manual and notebook, as well as most written assignments, must be your own. 5. Take the lab seriously. We have redesigned the laboratory experience to make it an authentic research experience for students in which you will be discovering new information in biology (something that no one else yet knows!). Our goal is to introduce you to real science rather than simply have you conduct cookbook experiments that have been done hundreds of times to arrive at a predetermined result. However, this requires a great deal of effort on your part and you will be expected to contribute in a meaningful fashion. 4  Format of the BIOL 112 Lab PREMISE An introductory biology lab course may consist of a number of cookbook-type exercises followed by a limited amount of experimental design. This approach is useful in that it reviews topics that were covered in lecture and provides students with an opportunity to interact with the material again. It also teaches basic techniques and aspects of experimental design. However, this is not a representation of real science and does not allow you to create new scientific knowledge. Instead, you are simply repeating experiments that have already been done thousands of times in universities around the world. The structure of our BIOL 112 lab is designed to provide many of the same benefits of the traditional laboratory experience but also give you a more realistic experience of how science is actually done. You will still be given the opportunity to review lecture material and learn new techniques, but you will also be given an authentic opportunity to design experiments and collect novel data. OVERVIEW OF LAB STRUCTURE Labs will be held for three hours. Each of the first five labs will start with a quiz, therefore, you must read the lab manual before lab in preparation for the quiz. These labs will focus on learning specific techniques such as microscopy, dilutions, using a spectrophotometer, etc. When learning these techniques, you will gain experience with the model organism for the course, the marine planktonic copepod Tigriopus californicus. You will also be drafting sections of a paper for the experiments that you will conduct during the latter portion of the semester. The lab manual contains background information on copepods, as well as areas of potential research in which the answers to basic questions are still unknown. Working in groups, you will choose an area of research, develop a testable hypothesis, and design a series of experiments to test this hypothesis. After the first five weeks, you will begin your own experiments with T. californicus. You will first present your progress to the class, including any problems that you are having and any conclusions that you are able to make at that point in your experiments. Every student will have the opportunity to present this information individually. Following the presentations, you will continue to work on your experiments. The instructor will act as your research mentor, providing guidance to each group. Your instructor will also check your lab notebook each week and assign a grade. If necessary, students can perform some of their research during in other Biol 112 lab sections, and the instructor of that section will also mentor these students. 5  ROLES FOR STUDENTS Students will work in groups to design and carry out experiments and present their findings at the end of the semester. Each student will work on all aspects of the research project, however, each student in the group will be the lead on one aspect of the project as detailed below. All assignments are to be done individually, except for those designated as group assignments. The grade for the laboratory portion of the course will include both individual and group grades. The roles for the semester are outlined below, and each group will be allowed to determine which student takes on which role. 1. Principal Investigator a. Organize and take the lead on background research b. Present the introduction during the Group Presentation 2. Protocol Expert a. Organize and take the lead on protocols b. Present the methods during the Group Presentation 3. Data Expert a. Organize and take the lead on the collection and presentation of data b. Present the results during the Group Presentation 4. Analysis Expert a. Organize and take the lead on analyzing data, summarize existing research as it relates to the new data, and develop conclusions b. Present the conclusions during the Group Presentation 6  Group Work is Essential for Success Stages of Group Development You can’t expect a new team to perform perfectly and efficiently when it first comes together. Team formation takes time, and teams often go through recognizable strategies as they change from being collections of strangers to becoming united groups with common goals. Bruce Tuckman’s Forming, Storming, Norming and Performing model (1965) describes these stages. When you understand it, your new team can become effective more quickly. Stage 1: Forming In this initiation phase, most team members act very nice, positive, and polite. Some are anxious, as they haven’t fully understood what the team will do. Others are simply excited about the task ahead. The roles and responsibilities of each team member are not very clear yet. Rules of behavior seem to be to keep things simple and to avoid controversy. Serious topics and feelings are avoided.The major task functions concern orientation. Members attempt to become oriented to the tasks as well as to one another. Discussion centers around defining the scope of the task, how to approach it, and similar concerns. This stage can last for a week or two, as students start to work together, and as they make an effort to get to know their new teammates. To grow from this stage to the next, each member must relinquish the comfort of non-threatening topics and risk the possibility of conflict. Stage 2: Storming Moving into this phase, people stop acting so “nice” and start pushing against the boundaries established in the Forming Stage. Various ideas compete, often fiercely, for consideration. This is the stage where some teams begin to break down. Storming often starts where there is a conflict between team members' natural working styles. People may work in different ways for all sorts of reasons but, if differing working styles cause unforeseen problems, individual members of the team may become frustrated. Some individuals may jockey for position as their roles are clarified. Others may feel overwhelmed by their workload, or they could be uncomfortable with how the project is progressing. Team members who stick with the task at hand may experience stress, particularly as they don't have the support of established processes or strong relationships with their peers. Questions may arise about who is responsible for what, what the rules are, what the reward system is, and what the criteria for evaluation are. Because of the discomfort generated during this stage, some members may remain completely silent while others attempt to dominate. 7  In order to progress to the next stage, group members must move from a "testing and proving" mentality to a problem-solving mentality. The most important trait in helping groups to move on to the next stage seems to be the ability to listen. Stage 3: Norming Gradually, most teams begin to agree on ways of working together and on acceptable behaviors, or norms of the group. Students start to resolve their differences, appreciate each others’ strengths, and respect the informal leaders (if any) who have risen up in the team. Once you and your team members know one another better, you may socialize together, begin to feel more comfortable asking each other for help and providing constructive feedback. You develop a stronger commitment to the team goal, and start to see good progress towards it. There is sometimes a prolonged overlap between storming and norming, because, as new tasks come up, the team may lapse back into behavior from the storming stage. Norming is often the stage at which members decide just how seriously they are going to take their design project work. As such, it is important for members who want successful outcomes to recognize that simply ignoring unacceptable behavior or poor work products will not be productive. For many teams, the norms of behavior that are established during the norming stage become the basis for behavior for the remainder of the lab project. Stage 4: Performing The team is now able to function as a unit. Hard work consistently leads to the achievement of successful projects. The team gets the job done smoothly and effectively without inappropriate conflict or the need for external supervision. Team members have a clear understanding of what is required of them at a task level. They are competent, autonomous, and able to handle the decision-making process. A “can do” attitude is visible. Offers to assist one another are made. Adapted from: Tuckman, B. (1965) Developmental Sequence in Small Groups. Psychological Bulletin, 63, 384-399. Tuckman, B. & Jensen, M. (1977) Stages of Small Group Development. Group and Organizational Studies, 2, 419-427. mindtools.com/pages/article/newldr_86.htm Caputi, M. 2018. Design 15 Class Workbook. 8  CONSTRUCTIVE AND DESTRUCTIVE GROUP BEHAVIORS Constructive Group Behaviors Cooperating: Is interested in the views and perspectives of the other group members and is willing to adapt for the good of the group. Clarifying: Makes issues clear for the group by listening, summarizing and focusing discussions. Inspiring: Enlivens the group, encourages participation and progress. Harmonizing: Encourages group cohesion and collaboration. For example, uses humor as a relief after a particularly difficult discussion. Risk Taking: Is willing to risk possible personal loss or embarrassment for the group or project success. Process Checking: Questions the group on process issues such as agenda, time frames, discussion topics, decision methods, use of information, etc. Destructive Group Behaviors Dominating: Takes much of meeting time expressing self views and opinions. Tries to take control by use of power, time, etc. Rushing: Encourages the group to move on before task is complete. Gets “tired” of listening to others and working as a group. Withdrawing: Removes self from discussions or decision-making. Refuses to participate. Discounting: Disregards or minimizes group or individual ideas or suggestions. Severe discounting behavior includes insults, which are often in the form of jokes. Digressing: Rambles, tells stories, and takes group away from primary purpose. Blocking: Impedes group progress by obstructing all ideas and suggestions. “That will never work because…” Adapted from: Brunt (1993). Facilitation Skills for Quality Improvement. Quality Enhancement Strategies. 1008 Fish Hatchery Road. Madison. WI 53715 9  What to do if your team struggles Great teams pull together. When successful, they share the rewards. When they fail, they share the consequences. Most people are willing to accept these terms if it seems everyone contributes equally. When individual commitment and participation are out of balance, the whole teamwork model begins to fall apart. In these situations, there are at least FOUR Constructive Actions you can follow: 1. Read the Stages of Group Development (a few pages back in this lab manual). Have a team meeting to read the Stages of Group Development together. Sometimes a member simply needs some insight or encouragement to get on board with making a firm commitment to good teamwork. 2. Discuss expectations. This is a time when you or someone else can talk about the importance of everyone doing his or her fair share. You can also discuss ideas on how your team can work this out in practice. If the team is already established, this discussion can take the form of a team checkup. Everyone should be offered the chance to voice an opinion about how things are going from his or her perspective. 3. Talk directly to the member. If someone is not holding up their end of the team, it seems reasonable that you would say something to them. Respectfully tell your team member what you’re noticing about the imbalance in contribution. Share how you’re feeling about this and how it impacts you. Invite the member to talk with you about how to create a better situation. Be prepared for the member to respond with some version of “I don’t see the problem”. 4. Complete a Team Member Issue Form (see below). If the team has followed all three Constructive Actions above, and the issue with the team member persists, it’s time to escalate the issue to the next level by submitting a Team Member Issue Form (TMIF) to your laboratory instructor who can provide some intervention. The instructor will review the form and take appropriate action for the issue in order to hold the member accountable for not pulling their weight while attempting to restore harmony and balance to the team. Adapted from: tomlaforce.com/theslackerproblem Caputi, M. 2018. Design 15 Class Workbook. 10  Team Member Issue Form Escalating the Issue to the Next Level If an issue with a team member persists, it’s time to escalate the issue to the next level by submitting a TMIF to the laboratory instructor who can provide some intervention. Complete the first section below before submitting the TMIF. Three Constructive Actions to take before submitting the TMIF 1. Did the team read the Stages of Group Development? Yes No Date: Outcome: 2. Did the team discuss expectations? Yes No Date: Outcome: 3. Did the team talk directly with the team member? Yes No Date: Outcome: After completing your good-faith effort above, complete the second section below. Turn in the TMIF with the particular assignment that was involved with the issues (if applicable). The issue is with (print full name of team member): Describe the issue: By initialing your response, you are agreeing to abide by the Honor Code that: We pledge on our honor that we have completed this TMIF with honesty and integrity. Initials: Initials: Initials: 11  Lab 1: An Introduction to Copepods and Tigriopus californicus Objectives: At the end of this lab students will be able to: 1. Observe morphology and behavior of Tigriopus californicus. 2. Identify male and female copepods. 3. Develop research question. 4. Identify instances of plagiarism and learn to properly paraphrase and cite primary literature. WHAT IS PLANKTON? The research system that we will be using in the course is a planktonic organism known as a copepod. Plankton are actually a wide group of organisms that are defined by their habit rather than by their actual species. The traditional definition of plankton is an aquatic organism that cannot swim against the current. This means that they are typically small organisms, often in the microscopic range, and they cannot make any progress against the currents in their body of water. Plankton occur in both freshwater and saltwater environments, but we will be examining saltwater (marine) plankton in this course. Plankton are also classified into two large groups (phytoplankton and zooplankton, as shown below) based on the way they obtain their food. Phytoplankton are those plankton that can photosynthesize and therefore are considered autotrophs. Some of the more common phytoplankton are algae and diatoms. These are the basis of the food chain in the world’s oceans and therefore play a critical role in the survival of life on this planet. Remember that photosynthesis is the process of using solar energy to create carbon-containing compounds, such as glucose. There are so many phytoplankton in the oceans that they create 50% of the carbon-containing compounds on the planet!! Zooplankton, on the other hand, are the members of the plankton community that obtain their energy by eating other plankton. Some of them eat phytoplankton, and some of them eat other zooplankton. There are a wide variety of these organisms including things such as copepods, which spend their whole life as plankton, and others, such as crab larvae, who are only plankton when they are young. Zooplankton serve as a food source for a wide range of larger organisms such as fish, jellyfish, and even blue whales. Images from http://octopus.gma.org/onlocation/globecactiv.html 12 WHAT ARE COPEPODS? Copepods are a very diverse group with >11,000 described species (including free-living and parasitic forms); by biomass, they are one of the largest groups of animals on earth. The particular 13 species of plankton that we will be studying this semester is Tigriopus californicus (Baker, 1912), which is a member of the zooplankton and a copepod in the Family Harpacticidae. To understand what this means, we must first consider what copepods are. They are small members of the Phylum Arthropoda in the Subphylum Crustacea (see below for phylogenetic relationships). Hopefully you remember the classification scheme from high school (Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species). If we follow the taxonomic scheme from top to bottom (below), we can see that it provides us with the biological classification of T. californicus. Domain: Eukarya Kingdom: Animalia Phylum: Arthropoda Subphylum: Crustacea Class: Hexanauplia Order: Harpacticoida Family Harpacticidae Genus: Tigriopus Species Tigriopus californicus Copepods are in the domain Eukarya, which means that they are comprised of eukaryotic cells, just like humans. They are in the Kingdom Animalia, which clearly makes them animals. The Phylum Arthropoda actually translates to “jointed-foot,” and arthropods do, in fact, have legs with joints. The arthropods are the largest group of organisms on the planet, and includes insects, crabs, lobsters, spiders, ticks, mites, centipedes, millipedes, beetles, horseshoe crabs and many more. Their uniting characteristic is their exoskeleton, which is the external skeleton that makes crab shells so hard to crack. The copepods you will be examining this semester are arthropods, and have many joints on their legs as well as an external skeleton. If we continue down the classification scheme, we see that our copepods are in the subphylum Crustacea – these are the arthropods with especially hard outer shells. The Class Hexanauplia are those crustaceans that are typically smaller in size and feed with modified head appendages called maxillae. We are now to the Order Harpacticoida. All species in this order are copepods, and the majority live on the bottom of the ocean or lake. However, our species, T. californicus, lives up in the water column. The harpacticoids are grouped together because they have short first antennae and a major joint halfway down their body, where they are quite flexible. If we continue to look at the taxonomic classification, we will see that currently our species is listed in the Family Harpacticidae, but this is still under debate and at the moment, most members of this family live in freshwater. Let’s put all of the pieces together: so far, we have a small animal with a hard exoskeleton, jointed legs, eats with its head appendages, and has short first antennae and a major joint in the body. If you look at the images that follow, this is clearly a good description. 14  Phylogeny of Animalia Phylogeny of the Arthropoda (Telford et al. 2015) (Regier et al. 2010) Find where copepods are found within the phylogeny of animals. You should be able to explain how they are part of the arthropods and to what superphyletic grouping they belong. Hint: they must molt to grow! Current phylogeny of copepod orders based on molecular data (from Khodami et al., 2017). Where does Tigriopus californicus belong? What is the sister taxon to its order? 15  Adult Male T. californicus Adult Female T. californicus with egg sac (Images by G.R. Fisher) T. CALIFORNICUS HABITAT As the name implies, members of the species T. californicus are commonly found along the coast of California, and are actually found all along the western coast of the United States from the Baja Peninsula to southern Alaska. They are most commonly inhabitants of tide pools along the shoreline. A tide pool is a body of water that remains when the tide has receded. Most tide pools are found on rocky shores, where the rocks form small depressions that hold water (see image below). Tide pools are actually a very stressful environment for aquatic species, which are left stranded in them when the tide goes out. Because tide pools contain a relatively small volume of water, the temperature of the tide pool fluctuates more quickly than the ocean, which can be a stressor for the organisms in the pool. Salinity can also change with evaporation or a sudden rainstorm. Of course, when the tide returns, the temperature and the salinity can change quite quickly again! Many aquatic organisms have evolved the ability to deal with slow changes in environmental conditions, but these quick changes can be more challenging. In addition to the natural stressors in a tide pool, human-caused issues occur as well. Run-off of pesticides and fertilizer from nearby agriculture can change the chemical composition of the tide pools, as can oil spills and any pollutants on the beach. As you might imagine, tide pool organisms must be very resilient and able to deal with a wide range of environmental stressors. 16  (Image from http://www.wunderground.com/wximage/quickeye/1611) LIFE CYCLE The life cycle of T. californicus is quite complex and involves a number of different stages. If we start at the beginning, we see that females will carry eggs attached to their bodies in an external egg sac. A life cycle stage called the nauplius will hatch from the egg, looking very different from the This is a general life cycle diagram for calanoid copepods, a different order of copepods. (Image from http://www.imas.utas.edu.au/zooplankton/image-key/copepoda ) adult copepod. The nauplius will then undergo metamorphosis for a total of 6 different naupliar stages (N-I through N-VI). Each stage lasts about 1-2 days, and each subsequent stage is larger than the previous one, and has an additional segment or two added to the abdomen (it is unclear how many are added at each specific stage). At the end of the last naupliar stage, the N-VI nauplius will metamorphose into the next life cycle stage, the copepodid. This stage looks much more like the adult, but will continue to mature until it reaches the 6th and final stage, which is the adult copepod. Each stage can last from 2-4 days, but this appears to depend on the environmental conditions, and 17 there is little detailed information available in the literature on the timing of each stage (Powlik et al. 1997). As the copepodid matures from one stage to the next, more appendages appear and more body segments are added. By the 5th copepodid stage, it becomes possible to determine the sex of the individual, although Coull (1982) argued that this was distinguishable during the C-IV stage. In particular, males have larger antennules and a larger body size than do females. While much is known about similar species of copepods, we are still lacking a complete description of each stage of the life cycle of T. californicus, including the specifics of the size and changes that occur during each phase. MATING BEHAVIOR Tigriopus californicus exhibits a mating style that is common in the arthropods. Because arthropods have a hard external skeleton, internal fertilization can be an issue. In particular, it can be difficult to deliver sperm to the internal body of the female when she is covered in armor. One solution to this problem is to wait until the female molts. During molting, she will shed the hard exoskeleton, and the new one underneath will be soft for a period of time. For a male, the best strategy then is to find a female that has recently molted. However, such females can be difficult to locate, and it would be a waste of time and energy to constantly swim from one female to another in hopes that she is ready to molt. Instead, males will find a female who is in the later copepodid stages and hold on to her until she is ready to molt. For this reason, when you find copepods in the tide pools, you will find a significant number of coupled pairs with the male grasping the back of the female, as seen in the image below. In many Male grasping immature female cases, it can be nearly in preparation for mating when impossible to find a she molts to the adult stage. solitary male in a Image by Dr. Terue Kihara from population. http://photoity.com/creepy-crawlies-nikon- small-world-competition-2014/ Once the female molts, she will mate with the male, and this will be her only mate for life. She will store the sperm and produce multiple clutches of eggs over her lifetime, but all the sperm will be from the original male. In contrast, once the male releases the female, he will move on and seek another mate. Burton (1985) found that the males will mate with an average of 2.5 females in 72 hours. There are some interesting questions that arise from this type of mating behavior. How does the male choose a particular female? What are the costs of holding on to the female? Is there competition between males for specific mates? We do know that for some species of copepods, the 18 males will choose the oldest females they can find (this reduces the wait time, and he can then mate more often during his lifespan). In addition, males of a similar species of copepod are able to recognize sisters and will avoid mating with them if given a choice. Preliminary evidence from Dr. Fisher’s lab at the University of Northern Colorado has indicated that male T. californicus will choose an older female and will avoid his sibling and mate with a non-sibling. However, these are just preliminary data with small samples sizes. FEEDING AND DIET One of the most basic questions about any animal is what do they eat? Strangely enough, this question still remains for T. californicus. This is odd because many aquarists actually raise copepods to feed to their fish. What, then, do they feed them? Many aquarists feed their copepods basic fish food and this seems to be quite successful. While this may work for rearing animals to feed to fish, this is clearly not what they eat in their natural environment. Because many researchers work on various species of copepods similar to T. californicus, a quick search of the literature will find that each research lab seems to feed their copepods a slightly different food. Most use algae or bacteria (or both), but there is not a standard amount or species recommended for rearing these animals in the lab. Copepods, especially T. californicus, have a reputation as generalist feeders, in that they seem to survive on many different food sources. However, there is a paucity of information on which foods result in the best survival rates, growth rates, egg production, etc. Students who have taken this course previously researched diet and found some interesting data listed below. However, many questions are still left to be investigated. FINDING MORE INFORMATION This lab manual was designed to introduce you to the basics of copepods and their lifestyle. With your own research question(s) in mind, you will now need to conduct your own literature search to see what research is out there. On the blackboard site for this lab course, you will find a folder entitled “copepod resources”. This folder contains quite a few articles to get you started, but is by no means all the information that is available. You and your group are expected to use this information as a starting point to conduct your literature searches. SO MANY QUESTIONS!!!! For this semester, your group will need to choose a research question that you will spend the majority of the class trying to answer. It is important that you choose your question carefully and consider whether you will be able to gather data to answer this question within the time frame you are given and with the available supplies. Below is listed a summary of the types of data collected by students in past semesters. It is very important that you do NOT repeat their exact studies, but instead you can ask questions based on the results that they found. These are just some guidelines to get you started as you develop YOUR UNIQUE question to be answered. Below are a few of the potential areas where more information about T. californicus is needed. 19  1. Diet: The overall results from previous students indicate survival rates are highest when fed the algae Tetraselmis. This algae has been compared to Nannochloropsis, Chlorella, Isochrysis and alternative food sources such as protein powder and fish food, as well as with live versus dead food. a. Questions that remain. i. What is the effect of other algal types? Supplementing diet with amino acids or fats? ii. Do copepods readily ingest microplastics? What is their gut residence time? Note: microplastics (plastic debris