Summer 2025 Lab Goals

# Lab Goals Colby [EcoEvoDevo Lab](https://hackmd.io/@EcoEvoDevoLab/AngeliniLab) Group for January 2026 --- ## Group Goals - Continue routine bug care - [ ] Set up *Jadera* bug cohorts with known bug and seed numbers for reaction norm studies - [ ] repeat reaction norm study from Fawcett et al. (2018) - [ ] set aside hatchlings in cages with a known number of seeds and bugs (count them). Record this information as an experimental treatment in the Experimental Treatment Log. - [ ] Set-up the flight mill ### Asher: Muscle Devo / Micro-CT - [ ] Scan 20 day females - [ ] Scan box elder bugs and milkweed bugs - [ ] Quantify flight muscle using CTAn software ### Hanna: Microbiome - Compositional models: - [ ] batch (plate) effect? (if so control for it in later models) - [ ] season (January vs. July) - [ ] host plant (BV vs GRT) - Taxonomic questions: - [ ] Correlation of *Wolbachia* relative abundance with any other metadata? (especially host plant, morph or sex) - [ ] Any taxa unique to the host plant? --- ## Projects Below is a high-level list of the lab's goals for this summer. This list is meant to be ambitious! We may not finish everything, but we should never be uncertain of what we're working towards! ### `#OfasSexDetProject` (Milkweed bug sex determination) ([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Sex-determination)) The development of female and male phenotypes is a common feature of animals. *transformer* is a key gene in the insect sex determination pathway as it is understood from fruit flies. Our lab group has previously described how genes from the pathway are conserved in their sequence in the milkweed bug *Oncopeltus fasciatus* ([Laslo et al. 2021](https://doi.org/10.1002/jez.b.23125)), however there are tissue-specific differences in the requirements of these genes ([Just et al. 2023](https://doi.org/10.1098/rspb.2022.2083)). In the the milkweed bug genome, there has been a duplication of *transformer* and it has been unclear which copy (if any) might be involved in sex determination. Work done in the last year in our lab shows that both Oncopeltus *tra* paralogs are required for female development. RNAi targetting either genes results in more males than expected by chance and in intersex individuals. **Tasks** - **Increase sample sizes!** Use [RNAi](https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/) to knock down each *tra* paralog, to increase sample sizes, but importantly, freeze day-1 adults, isolate RNA, and use qRT-PCR to validate RNAi known-down and examine gene interactions (e.g. with *dsx* and *fru*). - **Write** it all up. ### `#MuscleProject` (Soapberry bug muscle development and atrophy) ([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Soapberry-bugs)) Muscle is a developmentally plastic tissue that normally undergoes growth and atrophy over the lifetime of individuals. These processes are normally coordinated to integrate environmental information, such as nutrition, and influenced by unique genetic variations within and between species. Our lab is developing the [soapberry bug *Jadera haematoloma*](https://bugsinourbackyard.org/bug-story/) as a novel model for the study of muscle atrophy and growth regulation. This species has the dramatic ability regulate development of wings and flight muscle in response to juvenile nutrition. Some individuals never develop flight muscle, while others do and then naturally atrophy the muscle within the first two weeks of adulthood. This project will compare gene composition and function in soapberry bugs and related species under conditions promoting or inhibiting muscle development. Techniques will include quantitative morphometry, micro-CT scanning, functional genetic tests using RNA interference or CRISPR gene editing, and measurement of gene expression. **Tasks** - **Routine [bug care](https://hackmd.io/@EcoEvoDevoLab/rearingsoapberrybugs)** to maintain stocks! - Dave will visit Florida in mid-late July to look for the non-polyphenic species *Jadera sanguinolenta*. - **Lifetime flight capability**. Throw bugs in the air and keep good notes. - Try [magnetic paint](https://magnamagic.com/) to secure bugs in a flight mill ([Attisano et al, 2015](https://dx.doi.org/10.3791/53377)) - **[CT scanning](https://hackmd.io/@EcoEvoDevoLab/stainforCT)** of bugs with different flight capabilities to characterize muscle. - **[RNAi](https://hackmd.io/@EcoEvoDevoLab/rnasynth#PCR-to-create-a-template-for-RNAi)** targeting genes related to muscle development or atrophy - beclin/atg6 (underway) - Frizzled - twinfilin - Akt - others? - **[HCR](https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2020.00075/full)** to look at muscle histology and gene expression - **Whole genome sequencing and annotation**. - *Jadera sanguinolenta*, a non-polyphenic soapberry bug species - the box elder bug *Boisea trivittata* - Others? Resequence Jhae from BV and GRT populations? (contact Scott Carroll?) ### [Reaction norms](https://en.wikipedia.org/wiki/Reaction_norm) Soapberry bug flight morphs are determined by nutrition in individuals, but that threshold has a genetic basis and varies among local bug populations ([Fawcett et al. 2018](https://www.nature.com/articles/s41467-018-04102-1#Fig2)). Our lab did the original work on that in bug populations which have since gone extinct. We have new lines in the lab that we should examine in the same way. - 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. - *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. (A potential correlation has been suggested.) - Examine *Jadera* hind wing shapes? Would involve directing wings from dead bugs, imaging them and [geometric morphometric analysis](https://www.youtube.com/watch?v=yCkjfHPv98U). - 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! ### *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. ### [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? ### Microbiomes ([Background reading](https://hackmd.io/@EcoEvoDevoLab/EcoEvoDevoLabReadingList#Bee-microbiota)) 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. In 2023, DNA was extracted from *Jadera* specimens. - Finish analysis - Write it all up! ### 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 (?) ### Outreach - [Blogging](https://bugsinourbackyard.org/blog) - Consider editing Dave's BI163/BI164 slides and video transcripts into BioB blog posts --- ## 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 Biology* - If the m-chromosome has higher gene density, what are those genes? RT? or regular genes? - Try enrichment comparison - Look through Jhae genome for duplicates of IlS pathway components, especially *Akt*, *HIF1*. Phylogeny of InRs genes similar to [Smykal et al. 2020 MBE](https://academic.oup.com/mbe/article/37/6/1775/5758269) - 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). - Synteny of Jhae vs. [firebug](https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_039877355.1/) - Upload genome data to NCBI SRA ### 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? - 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. ---