Syllabus - Colby College - January 2024 Instructors Dr. Christina D. Cota cdcota@colby.edu Colby Guest Lecturer/Scientist Dr. Allison Barner akbarner@colby.edu

Applications due 10/16/2024 Apply today! Course description Our current understanding of biology is built on studies of numerous model species, using a shared set of investigative approaches and experimental methods. In this intensive course, students will practice many of these methods in investigations of several invertebrate animal models. We will examine the synthesis and regulation of melanic pigmentation, applying techniques from microscopy, cell biology, embryology and developmental biology, genomics and genetics. Students will be required to design, execute and present the results of their own experiments. The course will be conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, which presents unparalleled technical resources and expertise. Students will also have the opportunity to interact with MBL staff, and learn about the institution's history in the growth of biological knowledge. The setting Course activities center on hands-on examination of live animals and cells. Planned activities will build skills in molecular biology, microscopy, and experimental manipulation of the model systems. We will discuss experimental design, and students will have several opportunities to plan and conduct their own experiments with faculty and staff support. Lab activities will be interspersed with short lectures covering fundamental concepts of comparative biology, cell and developmental biology, and the use of current methods in those fields. We will also read and discuss current literature in these areas. The Marine Biological Laboratory (MBL) is a private international center for biological research and education, located in Woods Hole, Massachusetts, and affiliated with the University of Chicago. In its 133-year history MBL has been the setting for several of the most important discoveries in modern biology. It runs annual summer courses at the graduate level, which are renowned for their rigor and intensity. To date, 58 Nobel prizes winners have been associated with the MBL. The facilities and staff of the MBL offer a unique intellectual environment and educational opportunity for Colby students. The research topic

Colocalization Objectives of the lab: The goal this week is to introduce you to one method for quantifying the kind of visual data you might collect during the course of lab this semester: Use image analysis software, ImageJ/FIJI, to quantify the distribution of colocalization of proteins in transgenic mammalian NIH3T3 cells Identify the potential strengths and weaknesses of using this type of analysis in this system In order to open your images and analyze colocalization you will need to download the following program: ImageJ/FIJI. (https://imagej.net/Fiji#Downloads) *Please download the FIJI software (and not ImageJ) as it comes preloaded with plugins necessary for opening your images).

General information The painted lady butterfly, Vanessa cardui, is a model to study wing patterning and other topics. Vanesa cardui are members of the Lepidoptera, a large and diverse order of insects characterized in part by the presence of scale covered wings. Caterpillars are commercially available throughout the year (NatureGifts Caterpillar Refill Kit), and the entire life cycle can be completed in captivity. Newly laid eggs hatch and develop to adulthood in 5 weeks. This species is also native to New England, so (while we will not plan to release any animals) there is limited ecological risk from accidental release. Importantly, a transcriptome is available for this species and a web portal has been constructed for BLAST search of those sequences. The development of butterfly wings has also been studied in some detail. The Importance of Observation in Science With respect to scientific research, observation refers to a systematic data collection approach that is dependent on our five senses. Observation plays a pivitol role in the scientific method, enabling one to identify questions as well as to form and explore hypotheses. While numerous tools and techniques have been developed to amplify/heighten our senses and thus increase their resolving power (including microscopes, computational tools, etc.), the power of these remains limited by the users ability and desire to apply, monitor, record and interpret the data aquired. Therefore, it is critical that we as scientists work to develop these skills that are so critical to our research. Anatomy of a Butterfly Butterflys, have a three-segmented body comprised of a head, thorax and abdomen. A pair of antennae positioned above the compound eyes protrude from the head. Below the eyes is the mouth or proboscis. Attached to the thorax are three pairs of legs and two pairs of wings (a pair of forewings and a pair of hindwings). Behind the thorax is the abdomen, which contains vital organs including the heart and reproductive organs. https://www.anatomynote.com/animal-anatomy/worms-and-insect/butterfly/butterfly-gross-anatomy/

Syllabus - Colby College - January 2024 Instructors Dr. Christina D. Cota cdcota@colby.edu Dr. Jenny McCarthy-Taylor jmccarth@colby.edu (jmccarthy@mbl.edu)

Thermal regulation of gene expression in pigmentation of Vanessa cardui A research plan for Arvin and Lindsey Pupose During JanPlan 2023, we measured the influence of temperature on V. cardui wing pigmentation. As shown by TH Morgan 100+ years ago, butterflies reared with cold exposure have increased melanism. Most of our specimens were raised at 20˚C and 28˚C. Exposure to 10˚C produced very few adults, while a 36˚C exposure was lethal. We then measured expression of yellow, DDC and optix via qPCR at 72h from forewings and hind wings. However, it seems this may be too early. We'd like to repeat and expand this experiment to include less extreme low and high temperature treatments and collect older tissue. Goals In order of increasing difficulty Collect more adult specimens raised under different low and high temperature treatments

Syllabus - Colby College - January 2023 Instructor Dr. Dave Angelini drangeli@colby.edu Colby Guest Lecturer/Scientist Dr. Christina D. Cota cdcota@colby.edu

BI379 at MBL Lab Microinjection can be used to deliver any solution into insects. In our lab, its most common applications are in RNAi, CRISPR, and drug treatments. This protocol will not cover the preparation of those reagents, only the injection process. We will cover injection of beetle (e.g. Tribolium or Gnathocerus) larvae, and adults, juveniles and eggs of true bugs (e.g. Oncopeltus or Jadera). General considerations Insect care Ethically, we have a duty of care to the animals involved in these experiments to minimize their discomfort. Take good care of the insects in experiments. Be sure you follow through and collect the data you are aiming for, including with adequate replicates to ensure a statistically conclusive answer. Importantly, it also follows that experiments should not needlessly use more individuals than necessary. Injection necessarily involves wounding the animals. Most insects are quite robust, and tolerate injections well. To encourage survival:

Protocol Based on the [Patel Lab](http://www.patellab.net/) Hybridization Chain Reaction (HCR) In Situ Protocol available [here](https://www.protocols.io/view/hybridization-chain-reaction-hcr-in-situ-protocol-bunznvf6). *This protocol largely follows the HCR v3.0 protocol for whole-mount Drosophila embryos (Choi et al. 2018). Background In situ Hybridization chain reaction (HCR) is a modern molecular technique for visualizing gene expression in tissues. The technique utilizes short nucleotide probes that form stable hairpin structures (Metastable Small Conditional RNAs, scRNAs) that are able to polymerize only upon exposure to a target DNA initiator fragment. The introduction of the initiator triggers a cascade of hybridization events that yields a fluorescently labelled polymer that is bound to a sequence of interest. Hybridization Chain Reaction (HCR) mechanism Image source: https://commons.wikimedia.org/wiki/File:Hybridization_Chain_Reaction_(HCR)_mechanism.png

BI397 at MBL Lab By brewbooks from near Seattle, USA - Grand Prismatic Spring, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=51511238 Design of sgRNAs With your lab partner and instructor, choose a gene to target for CRISPR. Ideally, lab groups will chose different genes, with some groups choosing "safe" genes, with a history of successful mosaic gene knockout, and others choosing unproven targets. Obtain the sequence of your target gene. Open a web browers to https://www.ncbi.nlm.nih.gov/protein/

BI397 at MBL Lab Background Experimental manipulations are an important tool to investigate how patterns are created during development. By disrupting specific elements of a developing system, we can infer the normal function of that element from changes in the phenotype. Lose-of-function experiments of this kind can be done by removing cells from an embryo or juvenile, but also by removing the function of genes. Historically, genetic control of development was studied by mutagenesis. In this method, mutations are created at random throughout the genome by chemical mutagens or X-ray damage. Many individuals need to be screened for a phenotype altering the structure or developmental process under study. Then the affected gene must be mapped and characterized at the level of its DNA sequence. That process is laborious and prone to bias at levels of mutation and screening. CRISPR/Cas9 CRISPR/Cas9 gene editing is a method that can be used to permanently alter DNA in live cells at specific targeted sequences. It relies on Cas9, which evolved for viral defense in the bacterium Streptococcus pyogenes. Cas9 is an endonuclease, which can cut the phosphodiester bonds on both strands of DNA, producing a double-strand break. Unlike other endonucleases, Cas9 is directed to cut DNA by a short guide RNA. In Strep. pyogenes this guide consists of two separate RNAs. A tracrRNA has a sequence that is recognized and bound by Cas9. This component is generic. However, a crRNA has a 3$^\prime$ region of base complementary to the tracrRNA and a 5$^\prime$ sequence that targets the Cas9/RNA complex to a specific DNA sequence. In Strep. pyogenes the crRNA sequence is derived from viral genes that have previously infected the cell. In this way the bacterium is able to recognize and destroy new, invading viral DNA (Barrangou 2015).

JanPlan 2022 Protocols CRISPR Background CRISPR/Cas9 Molecular Steps Microinjection of Insect Eggs Quick Links