# 2023 Lab Goals

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

Updated 20 September 2023

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## Projects

Below is a high-level list of the lab's goals for this fall. 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)

([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Scaling-and-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, Grace, Oliver and Thinley made a huge number of DNA extractions and imaged beetle. We need to fill in any gaps in that sampling, finish extractions and imaging, then send samples for sequencing and conduct the analysis. We can also test 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
- Confirm specimens with Grace
- 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?
- Sequence the beetle genome using the minION (?)

### Bugs (Functional genomics of nutritional response)

([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Soapberry-bugs))

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. We also work with a more common insect, [the milkweed bug *Oncopeltus fasciatus*](https://hackmd.io/@EcoEvoDevoLab/rearingmilkweedbugs#General-information), which does not have wing morphs.

#### Developmental stuff

- Routine [bug care](https://hackmd.io/@EcoEvoDevoLab/rearingsoapberrybugs) to maintain stocks!
- Test methods for individual bug rearing (in small plastic cups with filter paper). If this works, then we can make a series of individual ontogenetic allometry measurements based on nutrition, temperature, light regime, tactile experience, etc.

##### [Reaction norms](https://en.wikipedia.org/wiki/Reaction_norm)

- *Jadera* lines new to the lab
- Repeat reaction norm study from [Fawcett et al. (2018 )](https://www.nature.com/articles/s41467-018-04102-1#Fig2). This simply involves raising bugs, keeping good notes and taking their pictures.
- 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?
- Use existing bug image data to test if there's a correlation between wing morph and pigmentation in either the body or wings
- Examine *Jadera* hind wing shapes? Would involve directing wings from dead bugs, imaging them and geometric morphometric analysis.
- Do number of trophic eggs differ by morph? Test by making controlled crosses of *Jhae*: count eggs and hatchlings.
- Test roles of other factors on morph determination
- day length, temperature, spatial dispersion of food
- How 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) to model beak length to examine what influence morph and nutrition have. e.g. ~ ecotype; ~ ecotype + morph; ~ ecotype + food; ~ ecotype + morph + food. Absolute and normalized 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. This may be manageable with individual rearing. Then genotype!

##### Flight muscle

- Thermogravimetric analysis (TGA) to quantify protein and lipid in thorax and abdomen of bugs of different sex, morph and species.
- Bisect and image thoracic interior and gonads
- 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))

##### *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 delivery 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.

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

RNAi is a method to test gene function by [knocking down expression](https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/).

- *Ofas* *tra* to test "master regulator" of somatic sex determination in milkweed bugs ([more background on sex determination](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Sex-determination))
- A lot of effort went into this question this summer. This fall, let's expand RNAi sample sizes and look at gene expression using RT-qPCR.
- *Ofas* *dsx* "at all life stages": parental, repeated juvenile injections
- *Jhae* EGF pathway components (potentially a story extending the study by [Fawcett et al. (2018)](https://www.nature.com/articles/s41467-018-04102-1) on genetic control of nutrition-dependent wing shape)
- *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) in bugs

- 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.
- Try injecting a *Cas9* expression plasmid (e.g. Addgene [p(bhsp68-Cas9)](https://www.addgene.org/65959/)), like people do in *Ciona*?
- 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?

#### Genomics

- Long read data from Christina's minION sequencer. Use the QiaGen [Genomic-tip 20/G](https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiagen-genomic-tips) extraction method for high-molecular weight DNA.
- *J. haematloma*: valid the existing HiC genome assembly
- more *J. haematloma* from different populations to look for structural variants
- *J. sanguinolenta*: before these bugs go extinct in the lab, sequence some to allow genome comparison to *J. haematoloma*.
- Also bumble bee genomes (haploid males!)

### Microbiomes

([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Bee-microbiota))

There are several projects involving microbiome samples and/or analysis: (**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.

- Last summer, DNA was extracted from *Jadera* specimens collected in Florida in 2019. Next, send those samples for microbiome sequencing and preform the analysis!
- 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 in October
- other location? islands?
- 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/)

### Bumble Bees

Allometric scaling of anatomical traits is wide-spread among organisms. Extremes in static allometry often reflect histories of intense selection, such as in sexually competitive weapons. Extreme allometric structures have been shown to have greater static scaling coefficients than other traits and to have increased variance among individuals. We wish to explore this pattern more widely, wondering if certain traits might be subject to different evolutionary forces leading to negative allometries and reduced variance. Bumble bees show an incredible degree of size variation within and between individuals and species. While this variation may allow individuals to specialize in foraging on different flowers suited to their morphology, species differ in their degree of specialization, and individuals may be required to exploit similar floral resources regardless of their size. Therefore, we wish to test a hypothesis that more specialist bumble bee species will display lower allometric scaling of mouthparts and lower variance compared to reference traits (legs and wings).

([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Bumble-Bees))

This project is very close to publication! We just need to increase sample sizes. Bees are already available in the freezers!

- Get bees from the freezers!
- [Image](https://hackmd.io/@EcoEvoDevoLab/photomicrography) them.
- Dissect out the gut and store it for [DNA extraction](https://hackmd.io/@EcoEvoDevoLab/dnaextraction) and [microbiome sequencing](https://hackmd.io/@EcoEvoDevoLab/qiime2tutorial).
- [Mount the mouthparts, legs and wings on slides](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab#Entomology).
- [Image](https://hackmd.io/@EcoEvoDevoLab/photomicrography) those slides.
- Consult with Dave on the analysis

### Butterflies

([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Vanessa))

- [*Vanessa* care](https://hackmd.io/@EcoEvoDevoLab/rearingvanessacardui) to maintain stock
- Fill out the sampling matrix from BI347: rearing at specific temps, wing disc collection for RNA extraction and HCR
- CRISPR: *Vanessa*
- *vermillion* (*v*) - positive control
- Also try the [DIPA method](https://doi.org/10.1016/j.crmeth.2022.100215) of female injection
- other wing pigmentation genes?

### Outreach

- [Blogging](https://bugsinourbackyard.org/blog)
- Consider editing Dave's BI163/BI164 slides and video transcripts into BioB blog posts

### Misc

- Create a database inventory of the contents of boxes in our freezers (-20 and -80)

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## Dave's goals

### Writing and Analysis

#### High priority

- Submit **[*Jadera* genome paper](https://news.colby.edu/story/data-science-makes-genome-sequencing-possible/)** to *BMC Genomics* (?)
- Try WGCNA on the [morphDE](https://github.com/aphanotus/morphDE) dataset
- Add manual annotation of genes to the EnTAP annotation (GFF?)
- Semantic space (PCoA) for enriched annotation terms?
- Map enriched annotation terms back to genes
- Are particualar genes duplicated? in tandem?
- Repeat my mapping of genes to areas of biased expression (by sex, morph, food regime (?)) in the genome using the updated EnTAP+manual annotations
- Submit Jane's **bumblebee scaling manuscript** to *AmNat*
- Did Oliver produce new data in Spring 2023? (Be sure Charis & Oliver are authors!)
- Submit Mark's **bumblebee microbiome/Crithidia manuscript** to *Mol Ecol*
- Submit a minimally edited version to BioRxiv for 8 days, then to *Mol Ecol*!
- Revise & resubmit Will's manuscript (**BeeSeq**) to *Mol Ecol*
- Try enrichment analysis without separating up and down DEGs
- Try enrichment tests for only GO and KEGG terms related to immunity (planned contrasts?)
- Make figures similar to first manuscript: MA plot highlighting biased terms, plot candidate genes by dose
- Add a few more steps to microbiome analysis following [this tutorial](https://grunwaldlab.github.io/analysis_of_microbiome_community_data_in_r/index.html).

#### Lower priority

- Develop Michael’s thesis for publication
- Wait to add e.g. TGA data?
- Check for correlations between *Jadera* wing shape and beak length?

### Misc

- Overhaul *[BioB](https://bugsinourbackyard.org/)* website
- Change language throughout the site from "citizen science" to "community science"
- Direct community 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 a "computer vision" method to phenotype the venation of the bugs' distal wing membrane.

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