# Summer 2022 Lab Goals

Colby [EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab) Group

Updated 12 May 2022

## Skill/Knowledge Building

### Tasks

- [ ] Read from the background [Reading List](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList) -- Start with the general audience background from *Bugs in our Backyard* on [soapberry bugs](https://bugsinourbackyard.org/bug-story/) and [bumble bees](https://bugsinourbackyard.org/bumblebees/).
- [ ] Check out [*Bugs in our Bacykard*](http://www.bugsinourbackyard.org/), and think about writing contributions to the [blog](https://bugsinourbackyard.org/blog/)
- [x] Get a lab notebook!
- [ ] Let Dave know any dates when you will be away during the summer
- [x] Complete the Moodle lab safety training module
- [ ] Attend the lab safety training session -- TBA
- [ ] Complete the self-directed [R tutorial](https://hackmd.io/@aphanotus/Rtutorial), and the exercises at the end.
- [ ] Get a [HackMD](https://hackmd.io/) user account and try editting some docs. For example, remove your name from this list: Grace, Thinley
- [ ] Check out the [tutorial on the Linux command line interface (CLI)](https://hackmd.io/@EcoEvoDevoLab/linuxbasics)
- [ ] Use the microscope-mounted Motic cameras and software. You should all try this on both set-ups (in the beetle area, and in the insectary)
- [ ] Practice [microinjections](https://hackmd.io/@EcoEvoDevoLab/microinjection)
- [ ] Eat ice cream

### Workshops

This is a list of ideas for lab training sessions we might do over the summer.

- [x] [Intro to the Linux command line](https://hackmd.io/@EcoEvoDevoLab/linuxbasics)
- [ ] Linux for RADseq analysis
- [ ] [Qiime2](https://qiime2.org/) microbiome analysis
- [ ] Making graphs with R ([Intro to R](https://hackmd.io/@aphanotus/Rtutorial))
- [ ] [Statistics in R](https://hackmd.io/@aphanotus/StatsInR)
- [ ] Photography, in general
- [x] Photomicrography
- [x] Bee [Macrophotography](https://hackmd.io/s/r1FcQYF-m)
- [ ] Using Adobe Suite / ImageJ / Outreach
- [ ] Regulation of organ growth
- [ ] [Insect appendage patterning](https://pubmed.ncbi.nlm.nih.gov/16112665/)
- [ ] Animal weapons
- [ ] Insect diversity

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## Summer Projects

Below is a high-level list of the lab's goals for this summer. This list is meant to be ambitous. We may not finish everything, but we should never be uncertain of what we're working towards!

### Beetles (Functional genomics of animal weapons)

Sexually selected weapons are among the most extreme and diverse morphologies in the animal world. Our lab group has conducted a large-scale artificial selection experiment to identify developmental mechanisms regulating weapon growth in the broad-horned flour beetle, *Gnathocerus cornutus*. During the summer of 2022, we plan to generate and analyze genetic mapping data from divergent selection lines, and to preform follow-up experiments on gene expression and gene function using RNA interference (RNAi) in *G. cornutus* and a related beetle, *Tribolium castaneum*.

- [Routine beetle care](https://hackmd.io/@EcoEvoDevoLab/rearingflourbeetles)
- [DNA extraction](https://hackmd.io/@EcoEvoDevoLab/dnaextraction) for RADseq (~500 beetles)
- Confirm specimens with Devin
- RADseq data analysis
- Functional genetic tests via [juvenile-stage RNAi](https://hackmd.io/@EcoEvoDevoLab/microinjection#Injecting-beetle-larvae) in *G. cornutus* and *T. castaneum*
- target pathways:
- growth: *warts*, *fat*, *hpo*
- patterning: *dac*
- sex determination: *dsx*
- Which dsRNAs are already on hand?

### Bugs (Functional genomics of nutritional response)

The [soapberry bugs](https://bugsinourbackyard.org/bug-story/) *Jadera haematoloma* and *J. sanguinolenta* are sibling species native to south Florida. The first species has the dramatic ability regulate development of wings and other organs in response to juvenile nutrition; while the second does not. This project will leverage our knowledge of the *J. haematoloma* genome and take an integrated approach to understanding its developmental plasticity. Studies of insect growth in both species will combine quantitative morphometry with functional genetic tests using RNA interference or CRISPR gene editing.

#### Developmental stuff

- Routine [bug care](https://hackmd.io/@EcoEvoDevoLab/rearingsoapberrybugs)
- Work out methods for lipid and triglyceride assays -- or [muscle mass](https://hackmd.io/@EcoEvoDevoLab/musclemass)? -- Or just count internal lipid bodies?

##### Reaction norms

- *Jadera* lines new to the lab
- Especially *J. sanguinolenta*!
- Compare egg production of any female short-winged *J. sanguinolenta* to their normal long-winged conspecifics.
- *Oncopeltus*: rear them like we do for *Jadera*, check resulting adult [wing shapes](https://www.bugsinourbackyard.org/wp-content/uploads/2020/10/gmm-tutorial.html). Any correlation to nutrition?
- Work out methods for individual *Jadera* bug rearing
- Examine *Jadera* hind wing shapes (?)
- Controlled crosses of *Jhae*: count eggs and hatchlings -- Do number of trophic eggs differ by morph?
- Test roles of other factors on morph determination
- day length, temperature, spatial dispersion of food
- Hoe might these factors (and crowding and seed number) confound one another?
- Maternal effects?
- Using the [data](https://www.nature.com/articles/s41467-018-04102-1#Sec26) from [Fawcett et al. (2018)](https://www.nature.com/articles/s41467-018-04102-1) model beak length to examine what influence moprh and nutrition have. e.g. ~ ecotype; ~ ecotype + morph; ~ ecotype + food; ~ ecotype + morph + food. Absolute and normaluzed beak length. Compare effect sizes.
- Repeat [Dingle et al. (2009)](https://doi.org/10.1111/j.1420-9101.2009.01819.x) artificial selection for *J. haematoloma* beak length (up and down) with the expectation that wing morph frequencies will change too. Start with bugs collected recently from Key Largo. Use the out-crossing strategy. (?) Think carefully about rearing logistics first!

##### Flight muscle

- Along with the reaction norm treatments, test newly emerged adults for flight capability following Michael's procedures (**add link!**).
- Try [magnetic paint](https://magnamagic.com/) to secure bugs in a flight mill ([Attisano et al, 2015](https://dx.doi.org/10.3791/53377))
- Bisect and image thoracic interior and gonads

##### *Wolbachia*

*[Wolbachia](https://en.wikipedia.org/wiki/Wolbachia)* is an obligate endosymbiotic or parasitic bacterium that commonly infects insects. In some species it can influence sex determination (typically inducing female development) or cause mating incompatibility among hosts with different *Wolbachia* strains. Our transcriptome study of *J. haematoloma* identified 13 highly expressed transcripts from *Wolbachia*, 12 of which had significanly higher expression in bugs from GRT populations. *Wolbachia*-derived xenic transcripts had no correlation with wing morph or shape, but there was a weak negative correlation between expression of Wolbachia-derived *rpS11* and adult abdominal width.

- What's the phenotypic effect of *Wolbachia*? -- Treat them with antibiotics for one full generation. If they survive (big if) check reaction norm and GMM. Do this with FR and KL populations. May require optimizing dose and deliver method.
- Is *Wolbachia* present across the *J. haematoloma* range? -- Check for bands using RT-PCR on lab and wild-caught samples from 2019. Target a *Wolbachia*-specific genomic sequence that will allow semi-quantitative counts, e.g. TR75328|c0_g2_i1 (*rpS11*).
- Is *Wolbachia* maternally transmitted? -- Check eggs via RT-PCR too. (Bleach surface.)
- If we develop a good PCR-based screen, check *J. sanguinolenta* and *O. fasciatus* too.

##### [RNAi](https://hackmd.io/@EcoEvoDevoLab/microinjection#Injecting-juvenile-true-bugs)

- *Ofas* *tra*
- *Ofas* *dsx* "at all life stages": parental, repeated juvenile injections
- *Jhae* EGF pathway components
- *Jhae* FoxO RNAi + insulin treatment (?)
- *Ofas* *Dll* RNAi effects on wing shape?
- *Jhae* *Dll* RNAi effects on wing shape? membrane venation?
- Also leg, beak Lengths
- Use Ariel's old *Ofas* specimens, old *Jhae* RNAi specimens & measurements
- *Ofas* RNAi for terminal appendage genes (*al*, *C15*, *EGF*) -- Does it affect the distal tip of stylets? Use new SEM.

#### [CRISPR](https://hackmd.io/@ColbyMBL/crisprbackground)

- CRISPR: *Oncopeltus*
- knockout *vermillion* (*v*)
- knockout an X-linked body pigmentation gene -- do we know of any? Guess based on synteny? Ask Lesley Pick? -- use homozygotes in a cross to wild type to identify the sex of embryos.
- CRISPR: *Jadera*
- knockout *vermillion* (*v*)
- insert an mNeonRed reporter
- Identify a potential endogenous promoter from the genome?
- Or request a construct from a lab that's had success? e.g. Leslie Pick? Marce Lorenzen?

### Microbiomes in space and time (Bumble bees)

(**1**) Lab-based isolation, sequencing and analysis of gut microbiota from soapberry bugs collected from field sites across southern Florida during the winter and summer of 2019. (**2**) Field collection and analysis of bumblebees from sites across Maine, including Allen Island and other off-shore islands. (**3**) Improvement of our outreach [website](https://hobbes.colby.edu/bee.map/), which provides community reporting of bumblebee data.

- Extract DNA from 2019 *Jadera* specimens: image, DNA from gut & body
- Bumble bee [field work](https://hackmd.io/@EcoEvoDevoLab/fieldworkislands)
- Sample from campus (outside Eustis) weekly (collection limits: 3 individuals per species and caste). [Photograph](https://bugsinourbackyard.org/how-to-take-amazing-pictures-of-insects/) all bees.
- Sample [Allen Island](https://hackmd.io/@EcoEvoDevoLab/fieldworkislands) at least once per month
- other location? islands?
- Sample *Bombus* gynes in Fall 2022 and Spring 2023 from specific sites (Colby, Allen Island): How does microbiota change through over-wintering?
- Update [outreach website](https://hobbes.colby.edu/bee.map/)
- Pull data continuously from [GBIF](https://www.gbif.org/)
- Add a tab that explains the background on bees and the research project
- Add the microbome data (Tab with microbiome data map?)
- Link the Bee Map to [BioB](https://bugsinourbackyard.org/)

### Butterflies

The lab is also maintaining a colony of [paitined lady butterflies (*Vanessa cardui*)](https://hackmd.io/@ColbyMBL/ryGIDpl2Y). These insects will be used to teach the [MBL JanPlan course (BI397)](https://hackmd.io/@ColbyMBL/syllabus) next year and by at least one research student this fall.

- [*Vanessa* care](https://hackmd.io/@EcoEvoDevoLab/rearingvanessacardui)
- Try CRISPR [targetting](https://hackmd.io/iekwsEc9QMqHlteYI9sRtQ#Injecting-eggs-of-Vanessa) *vermillion* (*v*)
- Try juvenile stage RNAi targetting *vermillion* (*v*)

### Outreach

- [Blogging](https://bugsinourbackyard.org/blog)
- CAPS: July 13-14

### Misc

- Clean the lab!
- Inventory the lab's pipetters (do we need more?)
- Move out of Johnson Lab space
- Freezer organization
- Organize boxes into racks in -80
- Organize images of -80 boxes
- Photograph -20 freezer boxes

---

## Dave's summer goals

### At the lab bench

- Get *Oncopeltus* L4 & L5 qPCR data for revision of Fini's *ProcB* manuscript

### Writing and Analysis

#### High priority

- Submit Jane's manuscript to *AmNat*
- Submit Mark's manuscript to *Mol Ecol*
- Revise & resubmit Will's manuscript to *Mol Ecol* (try *PNAS*?)
- Just re-do the analysis!
- Submit *Jadera* genome paper to *BMC Genomics* (?)
- Finish genome annotation -- **Get BRAKER working!**
- If necessary, address reviews on the *ICB* manuscript
- Analyze programmatic assessment survey -- Write it up for an UG Bio Ed journal? or *ICB*?

#### Lower priority

- Check for correlations between *Jadera* wing shape and beak length?

### Misc

- Overhaul *[BioB](https://bugsinourbackyard.org/)* website
- Update WordPress theme (twentynineteen?)
- Direct citizen science data to [iNaturalist](https://www.inaturalist.org/pages/managing-projects) (?)
- Background pages on each of the focal CitSci projects: *Jadera*, *Oncopeltus*, *Boisea*, *Bombus*
- Finish the *Bugs in our Backyard* coloring book Spanish edition -- Other translations?
- Submit *Jadera* transcriptomes and genome to NCBI
- Up-date *[borealis](https://github.com/aphanotus/borealis)* package and vignettes
- Develop the [method](https://github.com/aphanotus/tree.comparison#treecomparison) from [Laslo et al.](https://doi.org/10.1002/jez.b.23125) into an R package (?)
- Develop a "computer vision" method to phenotype the venation of the bugs' distal wing membrane.

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