# Spring Follow-Up ## Thermal regulation of gene expression in pigmentation of *Vanessa cardui* A research plan for Arvin and Lindsey ### Pupose During JanPlan 2023, we measured the influence of temperature on V. cardui wing pigmentation. As shown by TH Morgan 100+ years ago, butterflies reared with cold exposure have increased melanism. Most of our specimens were raised at 20˚C and 28˚C. Exposure to 10˚C produced very few adults, while a 36˚C exposure was lethal. We then measured expression of *yellow*, *DDC* and *optix* via qPCR at 72h from forewings and hind wings. However, it seems this may be too early. We'd like to repeat and expand this experiment to include less extreme low and high temperature treatments and collect older tissue. ### Goals In order of increasing difficulty 1. Collect more adult specimens raised under different low and high temperature treatments 2. Collect tissue for qPCR at later time points (5d / pre-ommochrome, 8d / melanic) 3. RNA extraction 4. cDNA synthesis 5. qPCR 6. HCR ### Details #### 1. Temperature regimes - [ ] Move all the general purpose / breeding stock to a consistent, moderate temperature, such as 20˚C. - [ ] Watch the general stock daily. Separate new pupae. If a pupa appears in the stock, and the stock was cleared of pupae on the previous day, then you can be confident that that individual is on day 1 pupation. If the age of a pupa cannot be determined (because no one removed pupae on the previous day) then it should be moved to a pupation chamber for breeding stock and it should not participate in this experiment. - [ ] From the general stock, obtain day-1 pupae. Give each a unique identifying number (e.g. DRA230210-001). Record this number in a google sheet and be sure the number labels plasticware attached to this pupa. Move pupae to a new cup and place them at one of the following temperature regimes. | temperature | minimum number | | ----------- | -------------- | | 18˚C | 10 | | 20 | 10 | | 28 | 10 | | 32 | 10 | - [ ] Individuals that, for whatever reason, are not collected for tissue samples (see below) can be allowed to eclose as adults. After they have inflated their wings and are capable of flight, they should be euthanized by being placed at -80˚C for 10 min, then stored at -20˚C. They should remain attached to their ID number, for example in a labeled Petri dish. You can also put the butterfly into a labeled envelope or folded paper triangle, but these should be stored in a box to they are not lost or crushed in the freezer. - [ ] Adult specimens should be photographed. Talk to Dave for details on that! #### 2. Tissue collection - [ ] We want to sample 5 biological replicate individuals at two time points: **5 APF** (days after puparium formation) or just before the appearance of red/orange monochrome pigments and **8 APF** or after melanin has formed, but before the insect has eclosed from the puparium. In the google sheet listing pupae IDs and on the pupa's cup, note the target collection date. - [ ] On the target date, dissect out the pupal imaginal wings. Collect the 4 imaginal wings of each pupa into separate tubes. One forewing and one hindwing should be stored in RNAlater for qPCR. The other forewing and hindwing should be fixed in 0.4% paraformaldehyde overnight in the fridge, then dehydrated into methanol for long-term storage. Label each tube with the specimen ID, forewing/hindwing, and the solution. Store specimens for qPCR at -80˚C. Store specimens for HCR at -20˚C. - [ ] For qPCR, the design of this experiment will require 80 samples. 4 temperatures x 2 time points x 2 wings (fore & hind) x 5 bio reps = 80 samples. #### 3. RNA extraction - [ ] Follow the [protocol for RNA extraction using the Maxwell simplyRNA kit](https://hackmd.io/@EcoEvoDevoLab/rnaextraction#RNA-isolation-using-the-Maxwell-16-automated-purifier). - [ ] Spec the total RNA and store it at -80˚C. #### 4. cDNA synthesis - [ ] Calculate the volume of RNA needed for 1 ug of total RNA. - [ ] Follow the [protocol for cDNA synthesis](https://hackmd.io/jSX5BEixQomGsWoaeLkrEw#cDNA-synthesis) using a poly-T primer. - [ ] Store cDNA at -20˚C. #### 5. qPCR - [ ] Design primer and DL probe sets for new genes involved in pigmentation. *tan*, *ebony*, *yellow-d*? - [ ] Modify and execute the plan used in BI347 for qPCR. #### 6. HCR - [ ] Consider doing the HCR procedure this summer.