Colby EcoEvoDevo Lab Group Group Goals for Week of June 9 [ ] Training on qPCR (Dave will call a lab meeting for group training, probably Tuesday) [ ] Self-directed training on use of R and Rstudio via Data CarpentryWe'll follow-up on this with a meeting for Q&A (probably Wednesday) Continue:[ ] Freezer inventory [ ] Routine bug care [ ] Set up Jadera bug cohorts with known bug and seed numbers for reaction norm studies [ ] Enter new Jadera adults into flight testing protocol [ ] dsRNA prep (espcially for GFP and/or AmpR control)[ ] Also, positive controls (pigmentation genes)

EcoEvoDevoLab Updated 17 June 2025 Screenshot 2024-11-20 at 12.00.10 PM Image from microphotonics.com Colby's micro-CT scanner is a Bruker SkyScan 1173. Manuals for its use are available on its accompanying desktop computer. Start by reading Method Note MCT-052 How to Set-up a Scan

EcoEvoDevo Lab By brewbooks from near Seattle, USA - Grand Prismatic Spring, CC BY-SA 2.0, https://commons.wikimedia.org/w/index.php?curid=51511238 General considerations RNA synthesis allows RNAi and CRISPR. The preparation of dsRNA or sgRNA from a template DNA sequence has 4 steps. First, a region within the template DNA is amplified by PCR using primers that add the T7 RNA polymerase promoter sequence to one or both ends. Second, that PCR product is used as template in an in vitro transcription reaction. After annealing, the new RNA is purified. Finally, the RNA concentration is quantified and prepared for microinjection. Working with RNA Single-strand RNA is prone to degradation by RNase, which can be ubiquitous in the environment. As you prepare RNA, take precautions that will preserve your efforts.

EcoEvoDevo Lab This reading list is meant as a resource for new lab members -- or anyone! Soapberry bugs Bug Story - Bugs in our Backyard's general audience background on soapberry bug natural history Fawcett et al. 2018 Nature Communications - This paper from our lab describes soapberry bugs as an experimental system for the investigation of polyphenism. It establishes nutrition of the determinging factor in wing polyphenism, links it to insulin signaling, and describes differences among J. haematoloma populations. Also uses GMM to characterized wing shape. Angelini et al. 2022 Int Comp Biol - Transcriptome comparisons in the bugs by sex, morph and nutrition Jadera haematoloma genome - Quick summary of the soapberry bug genome project Carrol & Boyd 1992 Evolution - Scott Carroll's textbook example host shift in Jadera

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 The Angelini lab group at Colby College. Current Lab Goals Reading List Standard operating procedures (SOPs) General Info for Students in the Lab (including how to get involved and lab safety)

Based on a recently published study in crickets, we decided to look at the influence of atg6 (also known as beclin-1) on flight muscle development and atrophy in soapberry bugs. plot.flight.test.results Some conclusions so far My first impression is that atg6 RNAi is having an effect. It looks like those bugs are able to fly better, sooner (panels B, D in the attached figure) and that they spend more time really flying (panel D: “level4 career”). We need more bugs! Especially dsGFP treatment bugs, and preferably long-winged bugs, since they’re the ones who fly. Right now sample sizes for many contrasts are still too small to make firm conclusions. Having daily information is great, but it may be overkill. I think it’s okay if individuals are skipped occasionally, for weekends etc. There appears to be no circadian effect, but again sample sizes aren’t huge. Perhaps we should increase the concentration of dsRNAs?

EcoEvoDevoLab Updated 20 November 2024 Screenshot 2024-11-20 at 12.00.10 PM Image from microphotonics.com This protocol is intended to visualize thoracic muscle in true bugs. It is based on the reccommendations of Metscher (2009), who demonstrated the procedure on Sisyra sp. (Neuroptera).

TL;DR The Ascidiella assembly is decent but covers perhaps 6% of conserved genes. Molgula needs more (and probably better quality) nanopore data. Work flow graph LR; A["nanopore data\n(FAST5)"]--guppy-->B["filtered reads\n(FASTQ)"]; B--flye-->C["assembled contigs\n(FASTA)"]; C--BUSCO-->Q1["quality assessment\n(text)"]; C--repeatmodeler-->R["repeat predictions\n(FASTA)"];

EcoEvoDevoLab Updated 8 March 2022 Butterflies caught in the wild or raised in the lab can be studied for variations in wing shape and pattern. First, it is necessary to photograph them under consistent conditions. Relaxing dry butterfly specimens Butterflies collected dead in the field or from lab stocks typically dry in orientations that don't allow good photography. Thankfully, they can be "relaxed" by exposure to high humidity and repositioned before imaging. There is a danger that the high humidity will encourage mold growth. So it is best to do this procedure no longer than over-night. Ideally, you can practice with low-value specimens, before trying to relax rare specimens. Find a suitable chamber large enough to tightly enclose butterfly specimens and liquid water, without the two touching. I have had success with an old plastic croissant tray sold at Walmart.

EcoEvoDevoLab Updated 9 September 2023 Angelini lab alumni, Abbe Labella '10 (AU) and Ariel Aspiras '12 (MS, AU). The lab is yours. It exists to allow you to do research that provides you experience in the practice of science, exposure to details of biological knowledge, and furthers humanity's understanding of development, genetics and evolution. Importantly, these goals should be met in a way that is ethical, safe and fun! As a member of the lab, you are free to be there at any time. Conducting experiments should always take priority, but as long as space allows, you are welcome to use the lab to study or just hang around while a friend is working. If you're in the lab outside regular business hours, don't work alone. Bring a friend. You should also call campus security to let them know you're there.

Main page Enterotype Analysis Before attempting this analysis, be sure you are comfortable working in linux on a HPC cluster. Review the linux tutorial if necessary. Some other useful resources for microbiome analysis will be the Qiime2 Moving Pictures Tutorial (versions: 2021.4, 2023.5) and my analysis of Lyndell's cownose ray gut contents. A note on using screen Anytime you'll run a command in linux that might take a while to complete or otherwise be problematic, it's useful to run screen. Starting a screen is like creating a new instance of your linux session, nested within the original one. To start it just type screen. The screen will clear, and you'll see the same prompt. Here you can run some command and while it's running you can return to the original session. Type [control]-[A] and then [D] to "disconnect" from the current screen. Now you're back where you started. To recover the screen again, enter screen -r. If you have multiple screens running at the same time, you'll be prompted to specify the one you want.

Dave says stuff wc -l

EcoEvoDevoLab Updated 8 June 2023 Protocol This protocol is intended to obtain DNA suitable for long-read sequencing. It is based on a protocol recommend by Oxford Nanopore. Because DNA is a very long polymer, isolating very large fragements can be difficult. Care should be taken not be vortex samples or to pass the DNA quickly through pipette tips, all of which can shear long DNAs. Live tissue is also the best source. Materials

EcoEvoDevoLab Updated 1 July 2022 Project goal For RADseq genotyping, we need DNA samples from individual beetles containing at least 50 μl of high molecular weight (>10kb) DNA at a concentration of 20 ng/μl. Final sample concentrations must be 20 ng/μl and normalized across samples. Extracted samples catalogued here Flour beetles are tough samples. They are small and contain relatively little DNA. Their exoskeletons are tough, and they contain aromatic chemicals that may inhibit DNA extraction. This protocol has been modified from the Qiagen DNeasy Blood & Tissue Kit protocol for optimal extractions from whole flour beetles by Devin O'Brien, Chloe Schiff and Dave Angelini.

EcoEvoDevo Lab Updated 27 June 2022 Based on the protocol from New England Biolabs for their Cas9 reagent (catalog number M0386). Exercise RNase precautions. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626505/ :::info Use a molar ratio of Cas9 and sgRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here.

EcoEvoDevoLab Updated 24 September 2021 Photo by Dave Angelini, Flickr General information The red-shouldered soapberry bug, Jadera haematoloma is a scentless plant bug (order Heteroptera; family Rhopalidae). Rhopalids are mostly tropical bugs. Species of the genus Jadera live in the New World tropics where they feed on plants of the soapberry family (Sapindaceae). The only other common rhopalid in the United States is the box elder bug, Boisea trivittata, which feeds on box elder or maple seeds. Jadera haematoloma is a native of south Florida old growth, hardwood dry forests—what the locals call "hammock". This environment once covered the Florida Keys and areas in and around the Everglades, and it can still be found in some locations. Here Jadera feed on balloon vine (Cardiospermum ssp.), which grows on other plants in clearings, empty lots, and along roadsides where light is good.

EcoEvoDevoLab Updated 26 October 2022 Photo by Matthew Beziat, Flickr (CC BY-NC 2.0) General information Oncopeltus fasciatus are seed bugs (order Heteroptera; family Lygaeidae). Species of Oncopeltus are common in the Caribbean, Central and northern South America. Oncopeltus fasciatus lives in the southern and temperate US, and individuals may colonize colder northern states in the summer. Some over-winter in diapause as adults. In the wild, they live and feed on milkweed (Asclepias sp.), and the bugs gain distasteful, toxic compounds called cardenolides from the plant. Their bright red/orange and black colors are aposematic, advertising their toxicity to would-be predators. To enhance this effect, the bugs congregate in semi-social groups. Heteroptera, like Oncopeltus, are hemimetabolous, meaning that there is no dramatic metamorphosis, as in other insects like beetles, moths, bees and flies. Instead, juveniles (also called nymphs) resemble adults in their overall body plan, but lack wings or genitalia. There are five juvenile instars (stages separated by molts of the cuticle) before adulthood. Each instar can be distinguished by its relative size and pattern of pigmentation (see below).

EcoEvoDevo Lab Updated 7 July 2022 By Victuallers - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=35119980 Measuring concentration and DNA storage Use the NanoDrop spec to measure the concentration of DNA. Record this information in your notebook and on the side of the tube. Also note the purity of the sample. Pure DNA should have an A260/A280 ratio ~ 1.8 and an A260/A230 ratio > 1.8 Store DNA at 4˚C for less than a week or at -20˚C for longer periods.

EcoEvoDevo Lab Updated 24 September 2021 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.