Summer 2025 Lab Goals

Group Goals for Week of June 9

Is anyone feeling confident enough with microinjection to try CRISPR? (via egg microinjection) <– I would love to try! - Jackie

Microbiome controls: decision time! Where do we stand on antibiotic treatments? Spike-in?
Micro-CT
- Install software on lab computer
- Check Mallory's scans from this spring
- Run new scans on Mallory's stained bugs

Abby's Goals

May 30, 2025

dsRNA purification
dsRNA spec readings
dsRNA gel
lab meeting (1:00 pm, Olin 335)
weekly lab goals set up
"Expression Analysis in a Dispersal-Fecundity Polyphenism Identifies Growth Regulators and Effectors" reading
"Distinct developmental mechanisms influence sexual dimorphisms in the milkweed bug Oncopeltus fasciatus"
bug care sign up
microinjection

June 2, 2025

June 3, 2025

June 4, 2025

June 5, 2025

June 6, 2025

June 9, 2025

June 10, 2025

June 11, 2025

June 12, 2025

June 13, 2025

June 16, 2025

June 17, 2025

June 18, 2025

June 20, 2025

June 23, 2025

June 24, 2025

June 25, 2025

Jackie's Goals (6/16-6/20)

Molecular Bio Stuff

Ofas-traA and Ofas-traB
- Monitor treatment groups for adulthood
- Harvest DNA (or RNA?) for intersex individuals
- Sex the bugs with pictures and update results spreadsheet
- Begin treatment groups for double injection (3rd and 4th)
Jaedera
- Inject with ATG6
  - PCR of ATG6 754F-920R
  - RNA synthesis of ATG6 754F-920R
  - Mix injection buffer of ATG6 754F-920R
- Inject with GFP

Bug Stuff

Bug Care on appropriate days:
- Monday
- Wednesday

Other

Continue with readings
Catch up on lab notebook
Read up on Prof Cota's Project
Talk to Prof Angelini about CRISPR
Ask Dave about the new part of the sex determination project
MicroCT Training
Crayfish Care (Wednesday)

Jillian's Goals 6/23-6/27

Asher's Goals

David T's goals (6/2 - 6/7)

Wet lab stuff

Bug care for Jadera
Run Oncopeltus DNA extract on 0.8% gel
Practice injecting buffer into Oncopeltus - 20 bugs; might last ~1hr
Prepare T7 templates and dsRNA for GFP (Jackie did this) and AmpR (haven't found primers yet)
Pilot HMW DNA extraction using the NEB Monarch kit from
- Oncopeltus
- Jadera
- Boisea trivitta stored in the freezer – search the -80˚C freezer for stored Btri
- Christina's samples (do this tonight!)
Measure jadera conc. on Anna Forsman's Qubit (also tonight)

Readings

find antiobiotic treatment with bugs-specific antibiotic, conc., and timing (time and duration). Related insects (in Hemiptera): Nilaparvata lugems (brown rice …??), Triatoma ssp (kissing bug), Rhodnius prolixus, Riptortus …(bean bug; scarce research), Halyomorph halys (brown … stinkbugs), Oncopeltus (have a look at/pick up from Jackie's research)
Read grant proposal
(re)read "Manipulation of insulin signaling" paper
Read "Distinct developmental mechanisms" paper
read literature (from Dave) on g-blocks
Read Michael Yorsz's Honors Thesis on Jadera
Literature search: are there sources for "positive controls" or "spike in" in other microbiome studies?

Micro CT stuff

Install micro CT software on the lab's PC
Repeat micro CT processing steps on recent bug scans (assess their quality)
Scan the bugs specimens Mallory prepped for micro CT

Other

Review goals for the week and update my goals

Later potential goals

Everyone

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)

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), however there are tissue-specific differences in the requirements of these genes (Just et al. 2023). 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 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)

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 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 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 to secure bugs in a flight mill (Attisano et al, 2015)
CT scanning of bugs with different flight capabilities to characterize muscle.
RNAi targeting genes related to muscle development or atrophy
- beclin/atg6 (underway)
- Frizzled
- twinfilin
- Akt
- others?
HCR to look at muscle histology and gene expression
Whole genome sequencing and annotation. We have funding from Maine INBRE to sequence the genome and transcriptomes from Jadera sanguinolenta, a non-polyphenic soapberry bug species, and the box elder bug Boisea trivittata, which appear in Waterville every September. We have Jsan in the freezer, but I will attempt to find fresh ones in the wilds of south Florida in July.
Partial genome sequencing. Meanwhile, we can use the Cota lab's minION sequencer. This involves extraction high-molecular weight DNA.
- We can test this first using O. fasciatus and J. haematloma.

Reaction norms

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). 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 ). 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. 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.
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 from Fawcett et al. (2018) 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) 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 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 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)), 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)

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, which provides community reporting of bumblebee data.

In 2023, DNA was extracted from Jadera specimens collected in Florida in 2019. This June we need to send those samples for microbiome sequencing! And later preform the analysis.
- Consider a "spike-in" to calibrate and validate bacterial counts and classifications.
Bumble bee field work
- Sample from campus (outside Eustis) weekly (collection limits: 3 individuals per species and caste). Photograph all bees.
- Sample Allen Island
- other location? islands?
Update outreach website
- Pull data continuously from GBIF
- 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

`#BombusScalingProject` (Bumblebees)

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)

This project is very close to publication!

Verify Mallory's mouthpart meaurements
Update the analysis
Write it all up!

Beetles (Functional genomics of animal weapons)

(Background reading)

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
DNA extraction for RADseq
- Confirm specimens with Grace
RADseq data analysis
Functional genetic tests via juvenile-stage RNAi 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
- 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)

Dave's goals

Writing and Analysis

High priority

Revise & resubmit Mark's manuscript (BombusLandscape)
Submit Jadera genome paper to BMC Genomics (?)
Submit Jane's bumblebee scaling manuscript to AmNat
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.

Misc

Overhaul BioB website
- Change language throughout the site from "citizen science" to "community science"
- Direct community science data to iNaturalist (?)
- 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 package and vignettes
Develop a "computer vision" method to phenotype the venation of the bugs' distal wing membrane.

David T's summer goals

(work on this)

Summer 2025 Lab Goals

Group Goals for Week of June 9

Abby's Goals

May 30, 2025

June 2, 2025

June 3, 2025

June 4, 2025

June 5, 2025

June 6, 2025

June 9, 2025

June 10, 2025

June 11, 2025

June 12, 2025

June 13, 2025

June 16, 2025

June 17, 2025

June 18, 2025

June 20, 2025

June 23, 2025

June 24, 2025

June 25, 2025

Jackie's Goals (6/16-6/20)

Molecular Bio Stuff

Bug Stuff

Other

Jillian's Goals 6/23-6/27

Asher's Goals

David T's goals (6/2 - 6/7)

Wet lab stuff

Readings

Micro CT stuff

Other

Later potential goals

Everyone

Projects

#OfasSexDetProject (Milkweed bug sex determination)

#MuscleProject (Soapberry bug muscle development and atrophy)

Reaction norms

Wolbachia

CRISPR in bugs

Microbiomes

#BombusScalingProject (Bumblebees)

Beetles (Functional genomics of animal weapons)

Outreach

Misc

Dave's goals

Writing and Analysis

High priority

Misc

David T's summer goals

Read more

Micro-CT scanning

RNA Synthesis

Reading List

My first HackMD note (change me!)

`#OfasSexDetProject` (Milkweed bug sex determination)

`#MuscleProject` (Soapberry bug muscle development and atrophy)

`#BombusScalingProject` (Bumblebees)