Insect Physiology

Studying the relationship between stored resources and diapause preparation by characterizing lipids accumulated by UZ and BE O. nubilalis in diapause and non-diapause conditions during the wandering stage.

Affiliation: University of Florida, Department of Entomology and Nematology

Date started: 20180101

Date end: 20180601

Literature

Table of contents (for 201 entries)

Page 1: 2018-01-01. UF Respirometry

20171128 ECB Life History Experimental Design

Respirometry:

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry

Hourly CO2 production

20180101_CO2_Hour

20180101_CO2_Hour

Hourly CO2 production weighted by mass

20180101_CO2_Hour_Mass

20180101_CO2_Hour_Mass

Page 2: 2018-01-05. Committee meeting following Research Proposal | Respirometry

Attendee’s: Dan Hahn, John Beck, Rob Meagher James’ take on proposal presentation Prior to delivering the talk at 0930 on Jan 5th 2018, I arrived in the seminar room at 0730a and tested the equipment and delivered a mock talk. I started from the begining (read: introduction/title slide) and ended with the thank you slide as though I was delivering it to an audience. During that time I felt confident and assured that I was doing well to hit all the marks. Specifically, I introduced information linearly, gave time for pauses, and engaged the audience (read: empty chairs) in eye contact. When the time came for the proposal presentation with the live audience I was a bit rattled. During the talk I tried to present the information as linearly as possible. However there were time that I feel I lost my train of thought. I think the loss of thought was a consequence of having not been as prepared as I could have been.

Committee comments following proposal Rob:

  1. Literature - Rob is willing to assist me in reviewing literature that could assist me in better understand how ECB fits into its varied global ecology.
  2. Confidence - Rob, in agreement with John and Dan, suggests that the question session after a seminar is the perfect time to speculate. Specifically, if someone asks me a question I may not necessarily know the answer to it is perfectly fine for me to say, “I do not know the answer, but here is what I think….”
  3. Admin - I need to email Rob and schedule meeting times with him for the semester.

John:

  1. Presentation tips: John would like my presentations to include expected questions from the audience. This could contexualize the Q&A session after my talk and possibly ensure I am prepared for said question session.

Dan:

  1. Big picture - Dan would like to more growth from me and my research as it relates to a larger picture or perspective. Specifically, how does this work fit into the larger body of knowledge, why does my research matter (think IPM, big physiology questions, and evolution). When it comes to presenting my research to the scientific community or any community I need to make sure I am answering the question, “How does this work fill a gap in scientific knowledge?”.
  2. Synthesis - The questions I was asked after my presentation suggest I am uncomfortable or less confident with synthesizing data or information on the fly (for lack of a better phrase). Dan suggests I read literature more broadly to better understand the relationship between my study system and its ecological position. Also I would benefit from continuing my education in evolution.
  3. Admin - With Rob as a resource, I will be starting weekly or biweekly literature meetings with R. Meagher and Dan would like the contents of those meetings shared using GitHub.

Respirometry:

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180105_CO2_Hour

20180105_CO2_Hour

Hourly CO2 production weighted by mass

20180105_CO2_Hour_Mass

20180105_CO2_Hour_Mass

Page 3: 2018-01-09. USDA with undergrads | UF Respirometry | Meeting with Andrew

USDA with Jeremiah

Jeremiah:

  1. Talked about schedule - Wednesday and Friday from 1pm until 5pm should be a go
  2. Discussed in detail the project we are doing and how it related to national food security, insect pest management, diapause research, etc. In short, I reviewed the “ECB” and “Experimental” sections of my proposal. Jeremiah asked alot of interesting questions and eexpresses an interest in insect chemical ecology, agricultural engineering (genetic/biological control), push and pull ag-systems, etc.
  3. Discussed with Jeremiah the possibility of completing an independent project, scholarship opportunities (which I will look into), and the possibility of remaining at the USDA for the long term.
  4. Gave Jeremiah a introduction to general lab safety: fire exits, emergency information, decontamination stations, and PPE locations.
  5. Discussed in general the lipid extraction procedure.

Erin:

  1. Laid out lipid samples for Erin to practice the FAME protocol
  2. Erin arrived at the USDA around 1pm and was able to complete the entire procedure (11 samples) by 1439.
  3. I did not get the opportunity to interact with her because I was on campus doing respirometry

Meeting with Andrew

  • Introduced me to software necessary to create and modify lab protocols
  • Installed and tested software
  • Discussed the outline and approch to creating protocols using R markdown
  • Discussed the goal and necessity of using version control software to track revisions and ensure my science is reproducible.

Respirometry:

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180109_CO2_Hour

20180109_CO2_Hour

Hourly CO2 production weighted by mass

20180109_CO2_Hour_Mass

20180109_CO2_Hour_Mass

Page 4: 2018-01-10 USDA

Work Day (short due to A/C service and doctor appt)

  1. Checked larvae and added diet to containers as needed
  2. Contacted Caitlin concerning LC fault via email
  3. Using the FAMEs produced by Erin on 20180109
    • prepared an instrument blank
    • created a sequence template
    • ran all 12 vials and ended with a sleep method to save gas

Page 5: 2018-01-11 USDA with Undergraduate Erin

####Tasks and To-Do’s

  1. Check FID run and determnination the status of the Hexanes
  • instrument blanks compared to an “ovenblank” and there were some peaks that occured in both the chromatograms around 20k in intensity.
  • Oven turned up to 240C in an attempt to remove the latent peaks from 10am until 2pm. Oven blank ran immediately following column baking.
    • comparison between solvent blank and oven blank
    • oven blank removed many of the extra peaks
    • further investigation needs to be conducted to determine the source of the rocky baseline during solvent blank runs.
  • Ran Pentane, Hexanes, and Dichloromethane to test instrument baseline.
  1. Check on larvae
  2. Run TLC plate on vials in the freezer
    • Push to Friday
  3. Input and organize data on GitHub
  4. Schedule a date and time to meet with Rob
    • Push to Friday
  5. Email Nancy with a ENSO club reminder: missed
  6. Class
    • practice loading data into sas and begin group assignment
  7. Read 2 broad articles about diapause metabolism
    • push to Friday

Working with Erin

  1. Update FAME protocol
    • add instruction for thawing, aliquoiting, and storing samples and standards while speedvac cools down
    • add volume and mass to samples and standards.
  2. Shadowed Erin during FAME derivitization to ensure she was equiped and capable of running the FAME method without supervision.
    • Positives:
      • She walked me through the entire process and asked questions about the theories and mechanisms that she was confused about.
      • Her dexterity is impressive
      • She explained to me that she was confortable with the FAME process and that she is ready to move on to working on “real samples”
      • she is willing to run 14 reaction vials during one analysis.
        • 8 samples, 3 reaction blanks, and 3 triglyceride standards plus 1 FAME standard and 1 instrument blank
    • Needs Improvement:
      • Handling pipeters
        • avoid inverting them when transferring solutions
      • When quenching the reaction
        • while keeping the vial inplace, acid needs to be added directly to the reaction.
      • Working in the hood
        • be careful not to place equipment and tools directly onto the LabMat
      • Operating the SpeedVac
        • explained operation procedure for the SpeedVac
    • Future directions:
      • During her next work day she and I will go through the process of running samples on the FID.
      • Erin is interested in a career in agriculture and indicated to me that she would like to volunteer for the USDA until she graduates.
      • When needed, we will go through the process to properly dilute acids and bases used for the FAME reaction

Page 6: 2018-01-12 USDA | UF Seminar, Respirometry

USDA 0800-1000

  • GC FID:
    • compared chromatograms
    • ran more solvent and oven blanks
    • conducted inlet maintenance
    • altered FAME method to make the inlet temp the same as the post run temp
    • determmined how to sleep the instrument
      • before performing a run select the sleep method
      • organize the sequence
      • run the sequence with your method of choice
      • after the sequence is completed, the GC will default back to the sleep method
  • Larvae maintenance

UF 1030-1345:

  • Seminar: vector borne diseases
    1. interesting stuff however it became a bit technical and some if the talk was lost on me.
      • he discussed how his research focuses on “curing” mosquitoes of malaria
      • he discussed how recently infected mosquitoes seek out the extrafloral nectar of this one pest weed species
      • he discussed how the treatment he is developing works in two ways
        • first the vaccine (an antibody treatment that blocks the membrane protein that binds the malaria parasite) is micronized and injected into the lymph tissue of the patient
          • on the same dose date a bundle of antigen is injected that slowly releases antigen into the blood stream for upto 6 months. This slow release thing acts as a booster

Respirometry:

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180112_CO2_Hour

20180112_CO2_Hour

Hourly CO2 production weighted by mass

20180112_CO2_Hour_Mass

20180112_CO2_Hour_Mass

Page 7: 20180115 USDA

  • Larvae
    • Fed larvae as needed
    • Prepped 32 larvae for respirometry
    • Trafficked those larvae to UF
  • TLC
    • Procedure and results below
Plate # Lane Treatment
1 1 T1 - Rep 1
1 2 T1 - Rep 2
1 3 T2 - Rep 1
1 4 T2 - Rep 2
1 S Tripal Std
1 5 T3 - Rep 1
1 6 T3 - Rep 2
1 7 T4 - Rep 1
1 8 T4 - Rep 2
2 1 T5 - Rep 1
2 2 T5 - Rep 2
2 3 T6 - Rep 1
2 4 T6 - Rep 2
2 S Tripal Std
2 5 T7 - Rep 1
2 6 T7 - Rep 2
2 7 T8 - Rep 1
2 8 EMPTY

#### Plate 1 20180117_P1_Reusable #### Plate 2 20180117_P2_Reusable

Page 8: 20180116 USDA Larvae Prep

  • Class
    • We alluded to how to deal with factorial data.
    • Specifically is the levels are quantative then you need to perform a regression
    • Durinng the lab portion we had the opportunity to:
      • Read in data, practice dealing with errors, opening different types of data files, reading in data using macros, setting up a file system using macros
      • Assignment 2 was due at 5p
  • Respirometry
    • BE12:
      • performed respirometry on remaining samples
    • UZ12:
      • respirometry performed on the 6 larvae that entered the 5 instar
    • UZ16:
      • respirometry performed on 25 larvae that entered the 5 instar
  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Hourly CO2 production

20180116_CO2_Hour

20180116_CO2_Hour

Hourly CO2 production weighted by mass

20180116_CO2_Hour_Mass

20180116_CO2_Hour_Mass

  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180116_CO2_HourUZ

Hourly CO2 production weighted by mass

20180116_CO2_Hour_MassUZ

20180116_CO2_Hour_MassUZ

Page 9: 20180117 UF Respirometry | USDA

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180117_CO2_HourUZ

Hourly CO2 production weighted by mass

20180117_CO2_Hour_MassUZ

20180117_CO2_Hour_MassUZ

  • USDA: Instructing Jeremiah
    • Preparing solutions to specific concentrations
    • Diluting solutions to specific concentrations for testing
    • Introduction to GC-FID, Sequence templates, and operating GC-FID
      • see attached protocol for more details 20180117_mscalcurv

Page 10: 20180118 USDA with JLM and EVL

  • Jeremiah
    • Recapitulate solutions prep, dilution, and GC-FID run.
    • Jeremiah completed the dilutions however he could not run the samples due to timing
  • Erin
    • Instruction on GC-FID
      • Ran prepared FAMEs on FID
      • Discussed: Operation, sequence templating, method selection, rinsing solvents, method runs also discussed FID output and chromatogram integration, retention times, and chromatagram comparisons

Page 11: 20180119 USDA | UF Seminar and Respirometry

  • USDA Output from Jeremiah’s cal curve run #1 Due to the r-squared and the low value for the smallest concentration, I will have him re run all of his dilutions to rule out some accident 20180124_JLM_mscalcurv

  • Seminar (see image)
    • Stinging nematodes in an agricultural setting
      • Population fluctuations of nematodes as a function of microbe populations

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180119_CO2_Hour

20180119_CO2_Hour

Hourly CO2 production weighted by mass

20180119_CO2_Hour_Mass

20180119_CO2_Hour_Mass

  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180119_CO2_HourUZ

Hourly CO2 production weighted by mass

20180119_CO2_Hour_MassUZ

20180119_CO2_Hour_MassUZ

Page 12: 20180120 Respirometry

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180120_CO2_Hour

20180120_CO2_Hour

Hourly CO2 production weighted by mass

20180120_CO2_Hour_Mass

20180120_CO2_Hour_Mass

  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180120_CO2_HourUZ

Hourly CO2 production weighted by mass

20180120_CO2_Hour_MassUZ

20180120_CO2_Hour_MassUZ

Page 13: 20180121 Respirometry

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180121_CO2_HourUZ

Hourly CO2 production weighted by mass

20180121_CO2_Hour_MassUZ

20180121_CO2_Hour_MassUZ

Page 14: 20180122 Respirometry | USDA shutdown

  • USDA Shutdown
    • turn down equipment and postpone assays insects moved to UF incubators

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production

20180122_CO2_Hour

20180122_CO2_Hour

Hourly CO2 production weighted by mass

20180122_CO2_Hour_Mass

20180122_CO2_Hour_Mass

  • UZ12:
    • respirometry performed on the 6 larvae that entered the 5 instar
  • UZ16:
    • respirometry performed on 25 larvae that entered the 5 instar #### Hourly CO2 production 20180122_CO2_HourUZ

Hourly CO2 production weighted by mass

20180122_CO2_Hour_MassUZ

20180122_CO2_Hour_MassUZ

Page 15: 20180123 USDA Notebook updates | Analyse JLM Cal Curve | Graph Data

20180125: 1. Output from JLM cal curve run #2 20180125_JLM_mscalcurv

  • Notebook updates
    • electronic notebook updated

Page 16: Notebook update: 20180124 thru 20180204

  • 20180124:
  • 20180125: #### Respirometry
  • 20180126: #### Respirometry
  • 20180128: #### Respirometry
  • 20180129: #### Respirometry
  • 20180130:
  • 20180131: #### Respirometry
  • 20180201:
  • 20180202: #### Respirometry 20180203: #### Respirometry 20180204: #### Respirometry ——

Page 17: 20180204 UF Respirometry | Meeting with Dan

Admin

  1. Rob and I will meet on Monday at 1pm to discuss literature.
  1. Expect a lit dump before the end of the week
  1. Run acetone rinses on GC-FID
  2. Split UZ and BE cups
  3. Meet with Dan at 1230p
  1. Discuss more reps and previous data

Meeting

  1. Respirometry data looks interesting and we should be thinking about ways to make these figures more impactful
  2. With the larvae in BE conditions: relocate less than half of the larvae into “long-day” conditions and monitor for 7 to 10 days
  3. Continue to monitor UZ until day 35 and re-asses with Dan
  • There seems to be an interesting effect of UZ metabolic activity at the begining of the respirometry. Try to apply a meaning to that.
  1. Remove pupa and adults from the graph, compare the means between larvae only

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production weighted by mass

20180204_CO2_Hour_Mass

20180204_CO2_Hour_Mass

  • UZ12:
    • performed respirometry on remaining samples

Hourly CO2 production weighted by mass

20180204_CO2_Hour_MassUZ

20180204_CO2_Hour_MassUZ

Page 18: 20180206 USDA admin and notebook update | with ELM

  • Notebook update
    1. Updated electronic notebook
    2. Animal husbandry
      1. Split UZ and BE rearing cups and place in separate into long day and short day conditions
      2. Ensure larvae are fed and the cup microclimate is humid
    3. Prep FAME standard and find more storage vials
    4. GC-FID carry over analysis DB Wax Rinse Test
  • With ELM
    1. ELM tasks
      1. get rid of working containers and start new ones
      2. Complete FAME of samples in 20170701 box plus, blanks, and TAG standards.
      3. Store samples
      4. Update data on GitHub and local HD
      5. discussed with Erin the future of her time here at the USDA (extractions and data analysis)

Page 19: 20180207 USDA | Husbandry, Standard preparations, Data analysis, Column Carry-over | with JLM

1. Husbandry * check larvae for health, humidity, and growth 2. Standards Prep * FAME standard in hexanes * did not prep standard: waiting until column carry-over is resolved. 3. Data analysis * Work through D. Hahn’s comments on data presentation * add mixed model analysis * Begin working on a .ppt for Monday

2. With JLM * 8 samples + 3 Blanks * this will be the sample block for 10 runs to acquire a baseline of experimental background with JLM running the extractions * start time: 1315 prep * extraction begins: 1422 * extraction ends: 1703

3. Column Carry-over * Setup: Samples willl be flanked with blanks to determine the degree to which analytes are remaining on the column, or if the inlet, or septa are causing a response. * Sample setup:

vial type
1 DCM
2 DCM
3 DCM
4 DCM
5 Sample
6 DCM
7 DCM
8 DCM
9 DCM
10 Sample
11 Sample
12 Sample
13 Sample
14 Sample
15 DCM
16 DCM
17 DCM
18 DCM

Results TBD

Page 20: 20180208 USDA | Husbandry, Column Carry-over, with EVL and JLM | UF Husbandry

USDA

  • Husbandry
    1. feed larvae
    2. check growth
  • Column Carry-over
    1. Inspect chromatograms
      • Q1: What is the response of the FID with an injection and without an injection 20180208_carryover_ovenblankdcm1dcm4_5to15 20180208_carryover_ovenblankdcm1dcm4_15to25
      • Q2: How does the response of the FID change after the injection of 4 DCM injections 20180208_carryover_dcm_dcm_dcm_dcm_5to15 20180208_carryover_dcm_dcm_dcm_dcm_15to25
      • Q3: How does the response of the FID change before and after a sample? 20180208_carryover_dcm_sample_dcm_5to15 20180208_carryover_dcm_sample_dcm_15to25
      • Q4: How does the FID response change after a batch of 5 samples? 20180208_carryover_batch_beforedcm_beforedcm_afterdcm_afterdcm_5to15 20180208_carryover_batch_beforedcm_beforedcm_afterdcm_afterdcm_15to25
    2. Run a sequence where each sample is flanked by a DCM blank and compare the column before and after each run.
  • with EVL
    1. Derivatized July samples, prepped samples for GC-FID, entered data, prepped samples on the SpeedVac
  • with JLM
    1. Extracted lipids from August samples by himself, completed samples, entered data, and discussed pros and cons. #### UF
  • Husbandry

    1. check humidity

Page 21: 20180209 USDA | Husbandry, Prep samples for the GC carryover testing | UF Respirometry, New hire lunch, discussion with Chao about data

USDA

  1. Husbandry:
    • Feed larvae, check conditions

  2. Carry-over testing:

    vial type
    1 DCM
    2 DCM
    3 DCM
    4 DCM
    5 Sample
    6 DCM
    7 DCM
    8 DCM
    9 DCM
    10 Sample
    11 Sample
    12 Sample
    13 Sample
    14 Sample
    15 DCM
    16 DCM
    17 DCM
    18 DCM
    19 Sample
    20 DCM
    21 Sample
    22 DCM
    23 Sample
    24 DCM
    25 Sample
    26 DCM
    27 Sample
    28 DCM
    29 DCM
    30 DCM
    31 DCM

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on remaining samples

Hourly CO2 production weighted by mass

20180204_CO2_Hour_Mass

20180204_CO2_Hour_Mass

Page 22: 20180212 USDA | Husbandry, Carry-over testing | UF Respirometry, Lab Meeting

USDA

Husbandry

  • CHecked Larvae for health and checked incubators for environmental conditions

Carry-over Testing

  • After talking to Nausheena I will change the rinse solvent from acetone to dichloromethane. Nausheena thinks the abundance of peaks could be the result of a dirty syringe. He advice is to rinse with DCM and Hexanes this would eliminate the need to run blanks between each sample. See run sequence below

    vial type
    1 Hex
    2 Hex
    3 Hex
    4 Hex
    5 Sample
    6 Hex
    7 Hex
    8 Hex
    9 Hex
    10 Sample
    11 Sample
    12 Sample
    13 Sample
    14 Sample
    15 Hex
    16 Hex
    17 Hex
    18 Hex
    19 Sample
    20 Hex
    21 Sample
    22 Hex
    23 Sample
    24 Hex
    25 Sample
    26 Hex
    27 Sample
    28 Hex
    29 Hex
    30 Hex
    31 Hex

UF

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on samples

Hourly CO2 production weighted by mass

  • UZ12:
    • performed respirometry on remaining samples

Hourly CO2 production weighted by mass

Page 23: 20180213 USDA | Husbandry, Carry-over testing, w/EVL, SpeedVac Samples, chat with Paul

Husbandry

  • Checked Larvae for health and checked incubators for environmental conditions

Carry Over Testing

  • After talking to Nausheena, again, I have two options:
    1. Capture and report the response of the column to solvent by running a series of solvent blanks and average the peak area
    2. Clip the end of the column that enters the inlet area to (theortically) remove the contamination that is stuck in the tip of the column.
vial type
1 Hex
2 Hex
3 Hex
4 Hex
5 Hex
6 Hex
7 Hex
8 Hex
9 Hex
10 Hex
11 Hex
12 Hex
13 Hex
14 Hex
15 Hex
16 Hex
17 Hex
18 Hex
19 Hex
20 Hex

w/ EVL

  1. FAMEs run and samples prepped
  2. data uploaded to data folder #### SpeedVac samples
  3. Lean mass dried for 4 days: 20180208-20180213
  4. Lean mass recorded #### chat with Paul
  5. Lab tour w/ Paul
    • observed white eye Plodia interpunctella adult females modified with CRSPR tech
    • observed spodoptera cell lines for wolbacia rearing
    • observed Spodoptera frugiperda larva modified with CRSPR tech
  6. Take away: develop a project outline that integrates each level of model available (tissue cultures, lab colonies, field populations).

    • Ecology, IPM, Insect Chemical Ecol. biological control, etc

Page 24: 20180214 USDA | Column carry-over, Vial prep, Husbandry, w/ JLM

Column Carry Over

  • Column cutting
    1. Tools: Inlet tool, spanner, opaque inlet, cotton gloves, magnifying galsses, column cutting tool
    2. Remove column from inlet
      • Turn off inlet, remove inlet liner, and insert opaque liner with opaque tool
      • Unscrew column bolt at inlet, remove ferrel, and cut off old septa
      • Push new septa onto column and replace ferrel onto column.
      • Cut approximately 6-inches to 12-inches of column and ENSURE cut is not chipped or jagged
    3. Replace column into inlet
      • Loosely screw ferrel (and column) into inlet
      • Push column into inlet until column tool line reaches the appropriate level
      • Tighten ferrel into inlet slowly, ensuring the column tool level is maintained.
      • Replace insert liner and run samples to test column. GC FID Column Cutting
  • Solvent blanks run on GCFID after maint
vial type
1 Hex
2 Hex
3 Hex
4 Hex
5 Hex
6 Hex
7 Hex
8 Hex
9 Hex
10 Hex
11 Hex
12 Hex
13 Hex
14 Hex
15 Hex
16 Hex
17 Hex
18 Hex
19 Hex
20 Hex

Vial Prep

  1. Vials rinsed with water, added liquinox, and placed in sonicator
  2. Vials rinsed with water after sonicator in preparation for cleaning #### Husbandry
  3. Checked Larvae for health and checked incubators for environmental conditions #### w/ JLM
  4. JLM will extract larval samples, with blanks, and enter data in triplicate
  5. washed septa

Page 25: 20180215 USDA | Full Inlet Maint, Husbandry, w/JLM, w/EVL

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions #### w/ JLM
  2. JLM will extract larval samples, with blanks
  3. Enter data in triplicate #### w/ EVL
  4. FAMEs run and samples prepped
  5. Increase sample volumn from 100ul to 400ul
  6. data uploaded to data folder #### Full Inlet Maint. ##### Tools: Inlet tool, spanner, opaque inlet, cotton gloves, magnifying galsses, column cutting tool, cleaning solvents, q-tips, gold seal
    1. Break Down injection port
      • Turn off inlet
      • Remove inlet liner and septa
    2. Break down inlet-column interface
      • Unscrew column bolt at inlet, remove ferrel, and cut column to remove old septa
      • Push new septa onto column and replace ferrel onto column.
      • Prepare to cut approximately 1-2 loops of column and ENSURE cut is not chipped or jagged
      • unseat glass wool insulator inside the oven
      • unscrew inlet bolt away fom inlet and remove gold seal
        • sonicate bolt in methanol for 30 mins and allow bolt to dry
    3. Rinse Inlet and cut column
      • Rinse inlet with three solvents using q-tips
        • DCM -> Acetone -> Methanol
      • Move bolt into oven to dry
      • With new septa, bolt and ferrel in place, cut column using cutting tool
      • Retrieve dry bolt
    4. Replace column into inlet
      • Properly place gold seal and return inlet bolt to inlet
      • Replace inlet insulator
      • Loosely screw ferrel (and column) into inlet
      • Push column into inlet until column tool line reaches the appropriate level
      • Tighten ferrel and bolt into inlet slowly, ensuring the column tool level is maintained.
      • Replace insert liner, septa, and o-ring
      • Bake column @ 20-40 degrees below max for 30minutes
      • Run ovenblank to test baseline

Page 26: 20180216 USDA | Post Inlet Maint, Husbandry | UF Meeting, Respirometry, PopBio

Post Inlet Maint

  • Baked column to test response
  • Ran an oven blank to test response
  • Run solvent-only samples to equilibrate column.
  • Run 40-solvent only runs with caps
vial 1 2 3 4 5 6 7 8 9 10
sample Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex
vial 11 12 13 14 15 16 17 18 19
sample Hex Hex Hex Hex Hex Hex Hex Hex Hex

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • UZ12:
    • performed respirometry on samples

Hourly CO2 production weighted by mass

Meeting with Dan

  1. Line plots need a broken stick regression
  2. Fit CO2 production as a function of mass on a single plot
  3. Perform a repeated measures analysis on co2 production by: larva, mass, day
  4. use markers to differentiate pupation
  • Next steps
  • 20180217_Diapause%20Sampling%20Plan
    1. Run 2 replicates of time course sampling of BE strain in long and short day conditions
    2. Run 1 replicate of time course sampling on UZ under both treatments
    3. Sample 20 larvae at each time point (8 points total)
    • Given distribution of larvae stages sampling will occur as follows:
    Sample # 1 2 3 4 5 6 7 8
    Day Post 5th 1 3 7 9 15 20 30 45
    1. For each larva observe: wet mass, lean mass, lipid mass, TAG mass

Page 27: 20180217 USDA | Post Inlet Maint, Husbandry, Drying lipids

Post Inlet Maint

  • Checked solvent runs from previous day for stability
  • Baked column to test response
  • Ran an oven blank to test response
  • Run solvent-only samples to equilibrate column.
vial 1 2 3 4 5 6 7 8 9 10
Sample Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex
vial 11 12 13 14 15 16 17 18 19 20
Sample Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex
vial 21 22 23 24 25 26 27 28 29 30
Sample Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex
vial 31 32 33 34 35 36 37 38 39 40
Sample Hex Hex Hex Hex Hex Hex Hex Hex Hex Hex

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Drying Lipids

  1. Extracted lipids placed on speedvac to dry

Page 28: 20180219 USDA | Husbandry, Diet prep | UF (PM) Meeting, Respirometry, Setup BE for respir

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions #### Diet Prep ECB%20Diet%20F9478B
  2. measure out:
  • water, diet meal, agar
  1. Mix water and agar using a stir plate and stir bar
  2. Heat water/agar mix to 100C
  3. Cool mixture to ~50C while stirring
  4. Using a blender, blend together diet meal and mixture for 3mins.
  5. Quickly pour diet into a storage container #### BE Respirometry Setup
  • 32 BE larvae in long day and short day conditions placed in 32-well trays
  • Each larvae will be assayed for metabolic activity as proxied by CO2 production #### Lab Meeting
  • Leigh discussed the advanced PhD program funded by the NSF in Antarctica #### Respirometry
  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • UZ12:
    • performed respirometry on samples

Hourly CO2 production weighted by mass

Page 29: 20180220 USDA | w/EVL and LC Extraction, BE assay setup, Husbandry, Set SpeedVac samples

<<<<<<< HEAD #### BE assay setup * setup BE larvae into assay trays @ USDA and UF * freeze dry 20 Day 1 samples from long day condition #### Husbandry 1. Checked Larvae for health and checked incubators for environmental conditions #### SpeedVac * Load lipid samples into speedVac in preparation for dilutions PM #### POSTPONED: w/EVL & LC ======= #### BE Respirometry Setup * 25 BE larvae in long day conditions placed in a 32-well tray * Each larvae will be assayed for metabolic activity as proxied by CO2 production #### Husbandry 1. Checked Larvae for health and checked incubators for environmental conditions #### SpeedVac ON HOLD * Load lipid samples into speedVac in preparation for dilutions #### w/EVL & LC On HOLD >>>>>>> 9467477a87516e4328d0d88aadbe67ed7c43dfa9 * EVL: Data entry, vial rinsing * EVL and LC: Teach Extraction technique to EVL and LC * 3 samples and 3 blanks

Page 30: 20180221 USDA | Husbandry, BE assay setup, w/JLM, Lipid Dilutions

Lipid Dilutions

  1. AM: Lipids on speedVac to remove solvent
  2. PM:
    • Weigh dry lipids
    • Dilute DRY lipids with 4ml DCM
    • Pass diluted lipids through a milli-q syringe filter
    • Store at -20C #### BE assay setup
  • setup BE larvae into assay trays @ USDA #### Husbandry
  1. Checked Larvae for health and checked incubators for environmental conditions #### Respirometry
  2. Larvae weighed and loaded into a 5mL syringe
  3. Syringe air purged of CO2, and hydrated
  4. Larvae held for ~1hr in syringe
  5. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE16:
    • performed respirometry on samples

w/ JLM ON HOLD

1. JLM will extract larval samples, with blanks, and enter data in triplicate

Page 31: 20180222 USDA | w/EVL, BE assay setup, Husbandry

w/ EVL

  1. FAMEs run and samples prepped
  2. Increase sample volumn from 100ul to 400ul
  3. data uploaded to data folder

BE assay setup

  • setup BE larvae into assay trays @ USDA
  • annotated 5th instar
  • Used liquid N2 to freeze larvae and stored them in -80C

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Page 32: 20180223 USDA | BE assay setup, Husbandry | UF Respirometry

BE assay setup

  • setup BE larvae into assay trays @ USDA
  • annotated 5th instar
  • Used liquid N2 to freeze larvae and stored them in -80C

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE16:
    • performed respirometry on samples
  • BE12:
    • performed respirometry on samples

Page 33: 20180226 USDA | Husbandry, BE larvae assay | UF Respirometry

BE assay setup

  • setup BE larvae into assay trays @ USDA
  • annotated 5th instar

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions
  • Larvae trays innoculated by latent mold: some larvae lost

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE16:
    • performed respirometry on samples

Page 34: 20180227 USDA | BE assay larvae (weights, freeze-dry, sampling date update) Teach EVL, clean SpeedVac

BE assay setup

  • setup BE larvae into assay trays @ USDA
  • annotated 5th instar
  • larvae annotated for 5th instar, sampled for continuous sample, and data entered

Teach EVL

  • Practice samples to learn extraction process
  • Reviewed:
    • preparation, sample location, equipment, and answered questions
      • EVL showed high aptiitude and technical ability
      • During next visit, she will conduct extraction on actual samples

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:

    • performed respirometry on samples

Page 35: 20180228 USDA | w/JLM, dilutions | UF Respirometry

BE assay setup

  • setup BE12 20180116
  • larvae into assay trays @ USDA
  • annotated 5th instar
  • larvae annotated for 5th instar, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried in preparation for dilution

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on samples

Page 36: 20180302 UF | Respirometry USDA | w/EVL dilutions

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on samples

Dilutions

  • Lipid extracts dried
  • Dried lipids weighed to obtain lipid mass
  • EXACTLY 4ml of DCM added to each lipid sample
  • Diluted lipids filtered using PVDF syringe filter
  • Diluted and filtered lipid samples stored at -20C

Page 37: 20180306 USDA | Husbandry, BE sampling, add/remove Freeze dry samples UF | Respirometry, Meeting Prep

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.
  • BE12:
    • performed respirometry on samples

Bio-assay

  • annotated for life stage
  • larvae sampled for continuous growth, and data entered

Freeze dried samples

  • larvae added and removed from freeze drier
  • samples annotated for mass (dry and wet mass)

Meeting Prep with Dan

  • slide presentation preparation

Page 38: 20180309 UF | Meeting Notes

Meeting Notes

  • Understand how the strain effect ould be masking some effect
    • Mass as a covariate or a fixed factor determining the accumulation of lipid mass
Dan’s Input
  • Frequency plot of pupa stage by time needed
  • Repeated measures analysis of respir data
    • maybe taking into consideration splines to determine the slope of change in CO2 over days
  • Plot each larva for CO2 by day
  • Plot each larva Pupa eclosion by day
  • Don’t forget the big question when sliding…?

Page 39: 20180314 USDA | Bio-assay, double extraction w/ JLM, and volunteer admin

Bio-assay setup

  • annotated for life stage
  • larvae sampled for continuous growth, and data entered

Double Extraction w/ JLM

  1. JLM will extract larval samples, with blanks
  2. Enter data in triplicate

Volunteer admin

  • Emailed Enrique an invitation to interview for the volunteer position

Page 40: 20180319 UF | Respirometry

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Correcting the baseline

  1. View -> “li6222”: save
  2. Create -> new channel
  3. drag marker to cover the peak -> area -> OK -> execute -> exit 4 repeat steps for each peak

Page 41: 20180321 USDA | Husbandry, Bioassay prep, Meeting w/Dan, w/JLM

Bio-assay setup

  • setup BE12 20180116
  • larvae into assay trays @ USDA
  • annotated 5th instar
  • larvae annotated for 5th instar, sampled for continuous sample, and data entered

w/ JLM

  1. JLM will extract larval samples, with blanks
  2. Enter data in triplicate

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Page 42: 20180322 UF | Meeting notes

  • cumulative pupa plot data

  • feeding: census pop each day using a tracker population to detemrine the proper day to sample for wandering.

Page 43: 20180323 UF | ENSO prep USDA | dilutions prep, bioassay

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried in preparation for dilution

Page 44: 20180326 UF | Experimental Design write-up, USDA | ACS abstract, LC prep

####Experimental design * Treatments: Long day (16:8) Short Day (12:12) * UZ: 1 replicate (possibly two) BE: 2 replicates * Goal of 20 larvae to be sampled at each time point * Wandering stage determined using “Poop Test” * Long Day: 2 samples * Short Day: 6 samples * Measures: wet mass, lean mass, lipid mass, TAG mass

Page 45: 20180327 USDA | LC prep, Bioassay, diet prep, lit meeting with Rob UF | Lab meeting, respirometry

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Page 46: 20180329 USDA | w/JLM, Full inlet maintenance

Full Inlet Maint.

Tools: Inlet tool, spanner, opaque inlet, cotton gloves, magnifying galsses, column cutting tool, cleaning solvents, q-tips, gold seal
  1. Break Down injection port
     * Turn off inlet 
     * Remove inlet liner and septa

  2. Break down inlet-column interface
     * Unscrew column bolt at inlet, remove ferrel, and cut column to remove old septa
     * Push new septa onto column and replace ferrel onto column.
     * Prepare to cut approximately 1-2 loops of column and ENSURE cut is not chipped or jagged
     * unseat glass wool insulator inside the oven
     * unscrew inlet bolt away fom inlet and remove gold seal
        * sonicate bolt in methanol for 30 mins and allow bolt to dry
        
  3. Rinse Inlet and cut column
     * Rinse inlet with three solvents using q-tips
        * DCM -> Acetone -> Methanol
     * Move bolt into oven to dry
     * With new septa, bolt and ferrel in place, cut column using cutting tool 
     * Retrieve dry bolt
     
  4. Replace column into inlet
     * Properly place gold seal and return inlet bolt to inlet
     * Replace inlet insulator
     * Loosely screw ferrel (and column) into inlet
     * Push column into inlet until column tool line reaches the appropriate level
     * Tighten ferrel and bolt into inlet slowly, ensuring the column tool level is maintained. 
     * Replace insert liner, septa, and o-ring
     * Bake column @ 20-40 degrees below max for 30minutes
     * Run ovenblank to test baseline

Page 47: 20180330 USDA | Bioassay, Abstract UF | respirometry, seminar

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Page 48: 20180331 USDA | Dilutions prep, Bioassay prep, bioassay, LC prep and blank run

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried in preparation for dilution

LC Prep

  1. Turn on pump, remove line to column, prime, purge, and pump LC with ACN (30m)
  2. Attach column, pump LC with ACN (30m)
  3. Prime, purge, flush and pump LC with target solvent (30mins)

Page 49: 20180401 USDA | Dilutions, Bioassay, lab maint. LC prep

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

LC Prep

  1. Prime, purge, flush, and pump LC with target solvent (15m)
  2. Run blank sequence using intended solvent gradient

Dilutions

  • Lipid extracts dried
  • Dried lipids weighed to obtain lipid mass
  • EXACTLY 4ml of DCM added to each lipid sample
  • Diluted lipids filtered using PVDF syringe filter
  • Diluted and filtered lipid samples stored at -20C

Page 50: 20180402 UF | Respirometry, lab meeting USDA | Bioassay, LC prep

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

LC Prep

  1. Prime, purge, flush, and pump LC with target solvent (15m)
  2. Run blank sequence using intended solvent gradient

Page 51: 20180403 USDA | Bioassay, LC batches and runs

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

LC Batches and Runs

  • Batches prepared w/QC and cal curve
  • sequence template constructed
  • LC prepared and sequence run initialized

Page 52: 20180404 USDA | Bioassay and setup, df updates, meeting

Bio-assay setup

  • setup BE 0314
  • larvae into assay trays @ USDA
  • annotated 5th instar

  • larvae annotated for 5th instar, sampled for continuous sample, and data entered

Page 53: 20180405 USDA | Lab maintenance, bioassay, Shirk lab instrumentation demo

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Demo

  • Cell sorting instrument:
    • sorts cells using GFP, RFP tags
    • optimized to sort single cells, two at a time

Page 54: 20180406 UF | Respirometry, ENSO meeting prep/execute

Respirometry

  1. Larvae weighed and loaded into a 5mL syringe
  2. Syringe air purged of CO2, and hydrated
  3. Larvae held for ~1hr in syringe
  4. Using a LiCor meter, the syringe air is analyzed for CO2 production.

Page 55: 20180407 UF | BE adult cage husbandry, BugFest table USDA | Bioassay

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Husbandry

1. Checked ECB adults for health and checked incubators for environmental conditions

Page 56: 20180408 USDA | df updates, Bioassay, dilutions prep

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried in preparation for dilution

Page 57: 20180409 USDA | Bioassay, Lit Meeting w/ RLM, Lab meeting w/QCC, Journal club

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Page 58: 20180410 USDA | LC Runs, Bioassay, Lipid dilutions, LC pre-column config

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried
  • Dried lipids weighed to obtain lipid mass
  • EXACTLY 4ml of DCM added to each lipid sample
  • Diluted lipids filtered using PVDF syringe filter
  • Diluted and filtered lipid samples stored at -20C

Pre-Column

  • LC pre-column prepared and connected to the LC run flow

LC Batches and Runs

  • Batches prepared w/QC and cal curve
  • sequence template constructed

Page 59: 20180411 USDA | Prep LC runs, Bioassay prep and analysis, df updates, lit search for RLM, w/ JLM

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

LC prep runs

  • Prepared LC batch run “Blank” sequence initialized

Page 60: 20180414 USDA | Bioassay

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Page 61: 20180415 USDA | Bioassay, LC runs batch 3

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

LC Runs

  • Prepared LC batch sequence initialized

Page 62: 20180416 USDA | Dilutions, Bioassay, Batch prep, Mass data collection

Bioassay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried
  • Dried lipids weighed to obtain lipid mass
  • EXACTLY 4ml of DCM added to each lipid sample
  • Diluted lipids filtered using PVDF syringe filter
  • Diluted and filtered lipid samples stored at -20C

Page 63: 20180417 USDA | Dillutions prep, bioassay, husbandry, HB interview, admin

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions

Dilutions

  • Lipid extracts dried in preparation for dilution

Page 64: 20180418 USDA | Bioassay, Dilutions, colony tracker, meetings prep, exp design, project update

Bio-assay

  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried
  • Dried lipids weighed to obtain lipid mass
  • EXACTLY 4ml of DCM added to each lipid sample
  • Diluted lipids filtered using PVDF syringe filter
  • Diluted and filtered lipid samples stored at -20C

Page 65: 20180419 USDA | Bioassay, Dilutions prep, Husbandry, w/JLM

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions #### Bio-assay
  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Dilutions

  • Lipid extracts dried in preparation for dilution

JLM

  • completed two runs of extraction protocol and became ill. 

  • 4 remaining runs will be completed 4/24/18

Page 66: 20180420 USDA | Bioassay, Husbandry

Husbandry

  1. Checked Larvae for health and checked incubators for environmental conditions #### Bio-assay
  • larvae annotated for 5th instar, wandering status, sampled for continuous sample, and data entered

Page 67: 20180421 USDA | Bioassay, dilutions, read & write

Bioassay

Dilutions

#### read & write

Page 68: 20180426 USDA | Meeting w/Rob Prep, Thesis writing, student project meeting w/Dan

Reviewing ECB literature

Setting up Thesis in LaTex

Discussion with Dan about the goal of the student project

* Experimental design
* Predictions
* Projected contributions: thesis and students

Page 69: 20180427 USDA | Bioassay, dilutions, cleaning glasswear

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Dilutions

CSR Meeting: Department opening

Page 70: 20180502 USDA | Bioassay, Extraction, ENSO prep, Reading,

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Extraction

* 19 samples and 3 blanks

Prep talk for ENSO Meeting

Reading for J-club, RLM, and thesis

Page 71: 20180503 USDA | Bioassay, Extraction

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Extraction

* 16 samples and 3 blanks

Page 72: 20180504 USDA | Bioassay, Dilutions, ENSO Meeting

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

ENSO meeting

* guest speaker and meeting  administration

Page 73: 20180505 USDA | Bioassay, Glasswear, reading & writing

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

REading and writing

* reading for thesis, j-club, RLM, etc

Page 74: 20180509 USDA | Bioassay, Extraction

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Extraction

* 16 samples and 3 blanks

Page 75: 20180511 USDA | Bioassay, AM-Extraction, PM-Extraction, ELSD run prep

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

AM-Extraction

* 16 samples and 3 blanks

PM-Extraction

* 13 samples and 3 blanks

Page 76: 20180512 USDA | Bioassay, Extraction

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Extraction

* 13 samples and 3 blanks

Page 77: 20180526 USDA | Bioassay

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Page 78: 20180527 USDA | Bioassay

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Page 79: 20180528 USDA | Bioassay

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Page 80: 20180531 USDA | ELSD prep/ Run, Bioassay

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

ELSD

* prep elsd: prime, flush, purge lines and filters
* Run sequence on ELSD

Page 81: 20180601 USDA | Bioassay, OEL meeting, DAH meeting

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Meetings

* OEL meeting about PhD possibilities
* DAH meeting about PhD ideas

Page 82: 20180606 USDA | Bioassay, Standards preparations

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Standards Preparations

* Tripal: 0.00106g/ml
* Trihepta: 0.00108g/ml
* Trister: 0.00122g/ml

Page 83: 20180608 USDA | Bioassay, Read for students, Emails, Read, Write

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Read for students

* edit and return abstracts

Emails

* DAH for meeting time
* Rob for meeting time

REad

* Rob meeing
* J-club

Write

* formatting and thesis writing

Page 84: 20180627 USDA | Bioassay, Extraction

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Extraction

* 16 samples

Page 85: 20180628 USDA | Bioassay, Dilution

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Dilution

* 30 samples

Page 86: 20180629 USDA | Bioassay, Dilution

Bioassay

* preparing for new assay
* maint of larvae in assay, population tracking

Dilution

* 30 samples

Page 87: 20180703 USDA | Dilution, Larvae on corn

Dilution

* 30 samples

Larvae on corn

* Larvae fed corn PRN, hatchers

Page 88: 20180711 USDA | Student, Prep for absence, AAR with JLM and EVL, Write for EVL, Emails, REad, Run sequence

Students

* Prep trays with corn 
* Prep FAME dilutions

Prep for absence

* Maggs note
* Instructions to JLM
* Instructions to EVL
* Instructions to NB and DDR

Edits to EVL

* Notes and comments to EVL on abstract

Emails

* Email Bonnie, Anna, Pathways, Shawn, Hans

Read

* J-club, PhD, Thesis

ELSD

* Prep ELSD: purge, prime, flush
* Run sequence

Template:

Primary Literature Template

Author: —
Title —
Model —
Question? —
Goal —
Methods —
Quote —
Note —

Discussion

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THESIS:

(20180326: A Brief History)

Author: Title Model Question? Goal Methods
Thomas, Y. et. al GENETIC ISOLATION BETWEEN TWO SYMPATRIC HOST-PLANT RACES OF THE EUROPEAN CORN BORER, OSTRINIA NUBILALIS HÜBNER. I. SEX PHEROMONE, MOTH EMERGENCE TIMING, AND PARASITISM O. nubilalis Investiging the factors involved in the genetic isolation of these two races at a field site near Paris, France. “…question is whether the variations in pheromone composition and voltinism are connected to the genetic divergence of sympatric host-plant populations.” Determing effects of assortative mating: life history differences in emergence. Pheromone comparison between host strains. Parasitism rates compared. Relationship between pheromone response, emergence, and the Tpi locus
Introduction
Quote “…parasitoid wasp, Macrocentrus cingulum, that killed more than 50% of the larvae overwintering on mugwort”
Note THis difference in parasitism in the mugwort host is intersting. Review some lit
Quote “…Sympatric speciation was considered implausible because the conditions required were thought by many to be very unlikely to occurin nature, notably due to the selection/recombination antag-onism identified by Felsenstein”
Note argument agains sympatric specation
Quote “…habitat- or host-plant-based assortative mating”
Note argument for sympatric speciation
Quote “…In Europe, its main hosts are maize (Zea mays) and mugwort (Artemisia vulgaris; Thompson and Parker 1928).”
Note host plant variety and life history

|Quote|“…The second polymorphic trait involved in reproductive iso- lation is the number of generations per year (Stengel and Schubert 1982; Glover et al. 1992). Populations of ECB are classified into genetically univoltine (U) population with a compulsory diapause and genetically bivoltine (B) popula- tions with a facultative diapause.”| |—|—| |Note|ecologically cumpulsory|

Quote “For all three sets of stalks for the maize race, we found no deviation from Hardy-Weinberg expectations at any individual locus or across all loci (Table 4). Conversely, several deviations from Hardy-Weinberg equilibrium were found within the mugwort race (Table 4).”
Note HW-genetic variation in the pop is constant w/o disturbance
Quote “The Tpi locus showed a significant deficit of heterozygotes in overwintering larvae (fˆ? 0.248, P ? 0.035) and in moths emerging in laboratory conditions (fˆ ? 0.449, P ? 0.040), but not in moths emerging in natural conditions (fˆ? 0.079, P? 0.663). Moths emerging in natural conditions displayed a major deficit of heterozygotes at both the Mpi (fˆ ? 0.484, P ? 0.001) and the Pgm (fˆ ? 0.414, P ? 0.001) loci, responsible for the significant deficit in heterozygotes detected across all loci (fˆ ? 0.215, P ? 0.001; Table 4).”
Note mugwort: less Tpi heteros in lab, less mpi and pgm heteros in nature
Discussion
Sex Pheromone Composition
" Males respond maximally to the pheromone blend of their own race (Priesner and Baltensweiler 1987)." “…In laboratory conditions, Z males do not respond to the E blend and very few E males respond to the Z blend (Glover et al. 1990).” “… Finally, assor-tative mating between the two races may be further enhanced because of the differences in calling periodicity between the E and Z ECB.”
Voltinsim
“…we concluded that the popu-lation collected on maize was bivoltine Z (bivoltine using Z pheromone), whereas the population sampled on mugwort was mainly, although not exclusively, bivoltine E (bivoltine using E pheromone).”

(20180409: A Brief History)

Author: Title Model Question? Goal Methods
Velasquez, M. et al. Reproductive Isolation Between Two Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) Collected in Corn and Rice Fields From Central Colombia Spodoptera frugiperda What are the pre and post zygotic factors at play maintaining the rice and corn strains The aim of this study was to test for prezygotic (temporal isolation) and postzygotic isolation be- tween Colombian corn and rice strains of S. frugiperda and to compare patterns of isolation with the con- trasting results obtained using different populations from the United States. Strain ID via MspI restriction enzyme analysis - Prezygotic isolation via mating timing using same day virgins - Postzygotic isolation measured via preovipo timing, number of eggs, hatch time, larva mortality, number of adults, number of males, longeviety of adults, and pupa weight
Summary
Prezygotic: Time of mating was similar between strains. Other tests between strains have revealed that pheromone blend may be more important in maintaining strains.
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Postzygotic: differrenfces found in egg masses, larvae, females, pupa devcelopment time
Author: Title Model Question? Goal Methods
Roelofs, W. et al. Three European corn borer populations in New York based on sex pheromones and voltinism -Ostrinia nubilalis The degree to which biotypes and pheromone strains contribute to the existance of distinct ECB populations in upstate NY Determine the correlation between the biotypes (voltinism) and pheromone strains (races). Female Pheromone analysis via soaking in Skelly B and GC analysis of eggs collected during each flight peak (June-bi, July-un, and Aug-bi)
Summary
Eden site analysis: two flight peaks in june and aug with Z-pheromone in 1983. In 1984, 3 flight peaks: 5 june cage-collected and 8-field collected females were Z strain except 1 female.
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Geneva site analysis 1983: overwintered larva collected from May to June, same plots were caged and showed adult emergence in June. Larvae reared and Eggs collected from adults indicate E strain. On July 5, two females Z strain emerged. Geneva 1984: June 7-18 flight, E strain females or hybrids. June 23 flight Z strain females
Meeting Notes: Email Rob By Tuesday COB

What is the deal with host race emphasis in europe? What is the US equvalent to mugwort or family similarity? Read Maleusa paper 2005. Showers, mating areas for ECB paper? Read Showers 1975 and Roelolfs 1977. Web of Science to find authors…..Do it! Read Bethenod 2005 paper.

(20180423: Voltinism vs Host Strains)

Author: Title Model Question? Goal Methods
2011 Folcher et. al Changes in Parasitoid Communities Over Time and Space: A Historical Case Study of the Maize Pest O. nubilalis O. nubilalis, O. scapulalis, and associated parasitoids We document here changes in the larval parasitoid communities of Ostrinia nubilalis — the main pest of maize — and its sibling species O. scapulalis, based on two historical datasets, one collected from 1921 1928 and the other from 2001–2005. Each of these datasets encompasses several years and large geographical areas and was based on several thousands/millions of host larvae. We provide here an analysis of the complete dataset for the larval ECB parasitoid community obtained from 2001 to 2005 by the French Ministry of Agriculture. We compared this dataset with the USDA dataset for 1921 1928, to determine (i) whether and to what extent the larval parasitoid communities of O. nubilalis and O. scapulalis had changed over a 80-year period marked by intense environmental modifications, (ii) whether these changes were of similar magnitude for the two main groups of parasitoids, tachinids and hymenopterans and (iii) whether the expansion of O. nubilalis into the range of O. scapulalis driven by human activity was accompanied by changes in diversity and, more specifically, by any phenomenon of parasite release, “spill over” or “spill back”.
Results
Quote Parasitoids following their hosts into new areas may eventually parasitise native hosts in the newly colonized habitats, a situation known as parasite ‘‘spill over’’ [42,43]. Finally, parasitoids already established in areas into which the species with the expanding range moves may switch host to the invader from resident host species related to the invader, triggering a parasite ‘‘spill back’
Note spill over: host range expansion = parasitoid range expansion. could lead to parasitoid preferring native hosts to invader host —- spill back: native parasioids prefering invader hosts to native hosts
Quote “Meanwhile, population genetics and ecological studies showed that the Ostrinia larvae feeding on mugwort actually belonged to O. scapulalis, a sibling species of O. nubilalis [56]. Although interfertile and similar morphologically, these two species are genetically differentiated from each other and feed on different host plants: mugwort, hop (Humulus lupulus L.) and hemp (Cannabis sativa L.) for O. scapulalis and maize for O. nubilalis (see [56] for a review). For unknown reasons, O. scapulalis is mostly restricted to northern France [53,57]. A few larvae of this species have been detected on mugwort in central France [53,54] but it does not infest this weed in southern France [53,57].”
Note these two ostrinia “species” have different ranges and although the hosts are available throughout the distribution of both “species”

|Quote|This difference in H9 resulted mostly from the decrease in PR over time documented above.| |—|—| |Note|H’ = diversity index — PR = parasitism rates|

|Quote| “The mean PR per site in the newly colonized regions was half that in the ancestral regions (Table 3). PR per site in the intermediate regions was twice that in ancestral regions and four times that in newly colonized regions For all parasitoids considered together, the mean SR per site in the newly colonized regions was one half to one third that in the intermediate and ancestral regions, respectively”| |—|—| |Note|SR = species richness, |

Discussion
Overview
"…differences in abundance, composition and richness between these two periods of time probably result from real and durable changes within the parasitoid communities rather than biased and/or limited sampling.
Changes in parasitoid community over time
for O. nubilalis, parasitism rates have declined but species richness has not changes. For O. scapulalis, parasitism rates have NOT declined but species richness has declined. THe decline in SR could be due to the range expansion of one braconid wasp and the loss of one species
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O. nubilalis and O. Scapulalis infestation densities have not changed between the 1920s and 2000s
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Lower species richness in O. nubilalis could be due to the introduction of herbicides and pesticides in managed agro-ecosystems in which they live.
Changes in ECB range and parasite community
Parasitism rate reduction in ECB regions could be due to the enemy free space provided by maize introduction
Author: Title Model Question? Goal Methods
Bailey, R. Dispersal Propensity and Settling Preferences of European Corn Borers in Maize Field Borders O. nubilalis present in the study sites. We were interested in the turnover and dispersal behaviour of adults once they have emerged from a maize field and moved into herbaceous borders of the cultivated fields, i.e. places in which they frequently rest during the day and mate
at night (Pleasants & Bitzer 1999). To examined the influence of a number of factors on this proportion, including habitat preferences, sex and, for an extended data set covering 3 years and five flight periods, a number of weather variab For that purpose, we released ECB adults into such borders and estimated the proportion staying in the close vicinity of their point of release over 1 year. We have investigated further the dispersal behaviour of ECB using sweep nets for recapture, in an attempt to recapture exhaustively all released males and females present in the study sites.
Exp Design
Exp 1: sex and habitat preference with different adjacent crops

Exp 2: sex and habitat preference with same corn as adjacent crop AND unmarked adults

Exp 3: dispersal of virgins and immigrants during first annual flight

Exp 4: during first flight. number of young virgin females mated during a single night

Exp 5: During second flight. number of young virgin females mated during a single night |

Quote Recapture rates were lower in field borders close to a maize than a sunflower field, and lower after night than
Note exp 1: maize adjacent decreased recapture, sex is not significant
Quote in Fig. 1. They were 34-5% (30-0-39 1%) after 12 h day, 7-7% (3-12-4%) after 12 h night, 2-7% (1-4-2%) after 24 h, < 1% after 36 h and < 0-2% after 48 h (Fig. 1)
Note exp 2: recapture higher during the day = more dispursal at night

recapture higher where immigrants were, attraction?|

|Quote|0-244, respe the significant (F4,28 = 9"10, P < 10-4) Pj between experiments had been accounted for, a significant effect of temperature (F1,28 = 6 P, decreased when night temperature in wind speed (FI,28 = 4-50, P = 0-043; Pj increased with increasing wind speed) was detected| |—|—| |Note|temp has sig effect on dispursal|

Discussion

Dispursal Behav
adults that did leave the study area could have done so by means of long range dispersaL
Implications for Bt resistance management
BECAUSE these insecdts have such a short dispersal range, the use of refuge areas to decrease the occurance of resistance may need to be approached in a different way
Author: Title Model Question? Goal Methods
1975 Showers, W.B. et. al Ecotypes of the European corn borer in North America O. nubilalis Could strains based on environmental tolerance or ecotypes develop in the future given current biological variation The papers purpose is to kinda develop a framework to apply to emerging ecotypes of ECB as a function of temperature and photoperiod Exp 1: Diapause response of transplanted populations: larvae were collected, reared, and mated from the “collection” sites and tested in the field at the “test” sites. Exp 2: Survival and diapause response of indeginous ECB pops

Discussion

Survuval
1- Heat treatment increased spore innoculation and reduced survival. 2- As seasonal temps increase with decreasing latitude, ECB survival decreases. 3- corn at tiftin may have been superior
Diapause
1- This study attemps to show the interation between photoperiod and temperature as in the diapause response as a function of ecotype. 2- the interaction between photoperiod and temperature does vary as temperatre and photoperiod icidence varies across latitude. NOTE this variation in diapause responce also varies with ecotype. 3 - this ecotype specificty couold be confounded by the fact that there are more than one strain within each collection. THe larvae that were collected from each location were collected during diapause. Depending on when these larvae were collected in the life stage of the insects lives there could be a unique mixture of different strains of ECB in each collection site which was not conotrolled for in the present experiment

(20180521: Pheromone Strains/Species Complex, US Host Strains)

Author: 2007 CALCAGNO, V. et. al
Title Sympatric host races of the European corn borer: adaptation to host plants and hybrid performance
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Model ECB
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Goal “…We conducted two independent experiments, in field and greenhouse conditions, to determine whether the two host races are locally adapted to their host species”
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Methods Larvae from the maize race and the mugwort race were introduced on the two host species. Two larval densities (number of larvae introduced per plant) were used: 10 and 30. This defined four (two host races · two larval densities) infestation treatments. Each treatment was applied to 25 randomly chosen plants within both host species. At the end of summer, the number of surviving individuals was recorded for each plant. Larvae were weighed and dissected for sex determination.
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Title Genetic Variation and Inheritance of Diapause Induction in Two Distinct Voltine Ecotypes of Ostrinia nubilalis (Lepidoptera: Crambidae)
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Title Parallel evolution of behaviour during independent host- shifts following maize introduction into Asia and Europe
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Title Flight Tunnel Responses of Z Strain European Corn Borer Females to Corn and Hemp Plants
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Title Assortative Mating in Sympatric Host Races of the European Corn Borer
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Title Aggregation and Dispersal Behavior of Marked and Released European Corn Borer (Lepidoptera: Crambidae) Adults
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Lab Meetings

(20180409: The role of PDF (pigment dispursing factor) in the regulation of ecdysone)

Author: Title Biological System Model Experiments Methods
Masatoshi, I. et al. Pigment Dispersing Factor Regulates Ecdysone Biosynthesis via Bombyx Neuropeptide G Protein Coupled Receptor-B2 in the Prothoracic Glands of Bombyx mori Ecdysteriod production Bombyx mori —

(20180409: Understanding the biosynthetic pathway of ecdysone production starting with the receptor)

Author: Title Model Question? Goal Methods
Iga, M et al. Pigment Dispersing Factor Regulates Ecdysone Biosynthesis via Bombyx Neuropeptide G Protein Coupled Receptor-B2 in the Prothoracic Glands of Bombyx mori Bombyx mori What are the receptors involved in the biogenesis of ecdysone Tissue cultures - Small molecule inhibitors - Transcriptome library production via RNA-seq tags - cDNA library production via Q-PCR - Phylogenic tree production via MUSCLE analysis - Receptor testing via production of HEK293 cell lines of BNGR-B2 or pME18s receptors - Ecdysone titer quant via LC-MS/MS - Receptor ID via western blot
Summary
In an effort to expose novel regulatory pathways important in the production of ecdysone, these researchers began their search starting with receptors known to be produced by the biogenic tissues that produced ecdysone and worked forward to find corresponding ligands. They discover that a novel g-coupled receptor (BNGR-B2) in the biogenic pathway and its ligand, PDF (a neuropeptide).
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Screening: RNA seq tags were aligned to Bombyx transcriptome. the rate of expression in the PG tissue was compared to the rate of brain tissue and 3 candidates were selcted: torso, BNGR-A34 and BNGR-B2
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Character of BNGR-B2: larvae were starved and RT-pcr was done on PG across several time point. The flux of this receptor was correlated with ecdysone titers (previous lit titers)
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Character of BNGR-B2: the amino acid sequence is similar to the PDHR and DH31 (homologues). This info was used to select ligand canidates. BNGR-B2 and an empty protein receptor were expressed in a cell line and those two ligands were tested against each line.
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Effect of ligand on PG tissue: PG tissue cultured with each ligand and ecdysone titers quantified. PDF was able to flux titers. PDF dose response was tested. PG tissues across several larval stage timepoints was tested. When PG’s were dosed with PDF or PTTH with and without calcium, Ecdysone titer production quantified.
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Signaling path of PDF mediated ecdysone biogenesis: because of the flux of cAMP given PDF, PDF is likely associated with a cAMP response element and is possibly controlling transcription of some factor inmportant to ecdysone biogenesis-NO joy when tested. BUT PKA translation inhibitor did effect ecdysone synthesis.
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PDF mediated phosphorylation: Previous lit says MAPK and TOR paths are indicted in the biosynthesis of ecdysone.
Discussion
During V4, PG tissues can respond to PTTH because the receptor is available (but during this time PTTH titer is low). Calcium is necessary for the induction of ecdysone synth via PTTH but not PDF. When calcium is depleted, cAMP titers are not effected. The transcription inhibitor molecules in the presence of PDF did not inhibit the transcription of ecdysone transcription WHILE translation inhibition did!

Meeting notes

Qinwen’s work: The role of PDF in diapause duration and the degree to which PDF is expressed differentially in BE and UZ
Note to self:
  • Make a quick reference slide to describe the life history and the strain differences. Make it pretty!
  • When writing the dissertation possibly using direct formatting could be useful to have a direct comparison in the literature.

Journal Club

20180212: Nutrition and reporductive consequences

Author: Title Biological System Model Experiments Methods
Barrett et al Separate and combined effects of nutrition during juvenile and sexual development on female life-history trajectories: the thrifty phenotype in a cockroach Naipphoeta cinerea Tracking the effect of diet quality on longeviety, ovary size, body length, Diet switching effects on juvenile and sexual maturation: observed survivorship, by diet quality ——-

20180219: Environmental effects on thermal tolerance

Author: Title Biological Study Model Experiment Methods
Deutsch et al. Impacts of climate warming on terrestrial ectotherms across latitudes Understanding the relationship between insect thermal biology and the effects of increasing temerpatures 38 terreterial insect taxa Integration of thermal performance among taxa Thermal performance curves
thermal%20performance%20curve

thermal%20performance%20curve

Summary
A major concern in biology is the question about how will plants and animals respond as temperatures continue to increase. Understanding how animals respond to temperature will help us to predict how habitats might be affected by changes in animals diversity. As it relates to agriculture, understanding the relationship between an insect pest and the environmental temperatures it experiences will be an important factor in how we manage insect pests. One way to frame the relationship between insects and environmental temperatures is to consider them categorically as winners or losers and thermal tolerance is a quantifiable scale use to measure the response of said insect to environmental temperatures.
Losers: increasing environemtal temperatures for these insects exceed their thermal maximum and eventually cause mortality.
Winners: increasing environmental temperatures for these insects would increase within the thermal limits and could even reach their thermal optimum.
* Winners could have wider thermal breadths and tolerate warmer temperatures.
* Winners, whose thermal environment is currently below their thermal optimum, could experience increased performance as temperatures increase towards their thermal optimum.
Deutsch et al. (2008) tracked and compared population size between 38 representative insect species from temperate and tropical latitudes. For those representative species across temperate latitudes, the thermal breadth of these insects tended to be wider and the thermal conditions experienced in these locations, on average, tended to be further away from their critical thermal maximum compared to the representative taxa from tropical latitudes.
In the tropics, environmental temperatures vary little relative to temperatures in temperate regions and insects in tropical regions experience temperatures that tend to be closer to their optimum temperature relative to temperate insects whose environment tends to be cooler than optimum. This suggests that tropical insects already live near their thermal limits and thus could quickly become losers as climate warms.
Figures
1: “fitness” is described as a function of population growth by temperature (an archived measure)
  1. current fitness of temperate taxa
  2. current fitness of tropical taxa
  3. predicted fitness with change in temperature given by latitude.| |2: Predicted temp increase effects on thermal performance
  4. increased temperature and thermal limit
  5. increased temperature and thermal safety margin| |3: Predicted impact of incereased temp on ectotherms
  6. extrapolation of thermal performance for insects by latitude
  7. extrapolation of complete insect data globally
  8. extrapolation of warming data for non insect taxa by latitude|

20180312: Spider courtship and signal alignment

Author: Title Model Question? Goal Methods
Echeverri, et al. Control of signaling alignment during the dynamic courtship display of a jumping spider Habronattus pyrrithrix (Jumping Spider) Receiving signals can be effected by angle and direction of perception. How do animals establish and maintain signal alignment? To characterize the spatial dynamics of visual courtship Recorded relative positions of actors during courtship: movement, orientation of males and females when female movement was fixed and free
Summary
Visual signals and systems often have strong directional biasis and selection should lead to behaviors that increase alignment:

1-Tendancy for angle dependancy implies spatial limits: change in visual signal due to distance 2-Intrinsic sensitivity biasis of signals due to system preceiving the signal: view field and signal reception limits|

Introduction:
1-Both males and females studied during courtship 
  a: during displays males behavior is stereotypical and icludes ornaments depending on distance from the receiver
  b: these movements are obsecured depending on viewing angle due to the visual limits of female color sight and the location of the color signals on the male

Questions: 1-To what extent is alignment established and maintained during courtship 2-What are the contributions of each actor to alignment|

Methods
1- spiders from arizona and kept at constant temp, RH, and LD. Food 2x body mass. Court trials held 3 months 4 months and 8 months after capture

2- Court trial a: was 10mins, 8 pairs of wild adults were trialed and results compared to later cohorts b: n=14 for stationary trials, n=12 for turning trials, continuous court=60s display|

Results
1- Analysis of manipulation experiments reveals dynamic interx during courtship between actors 
2- Live Males and Live Females:
  a: Live Females: Males at long range did little orientation in the direction of the female and males at short range orientated face-towards female
  b: Model Females: Males at long range orientated face-towards female as did short range males|
Discussion

Echeverri%20fig2 Echeverri%20fig3

20180326:

Author: Title Model Question? Goal Methods
Simoes, et al. Natural vegetation management to conserve biodiversity and soil water in olive orchards Natural Vegatation Tillage (NVT) vs Natural Vegetation Mowing (NVM) in Olive Orchards Tillage in olive orchards can cause control weeds, imporving tree health, but is there an alternative? Determine the degree to which olive yield is effected under mowing and tillage regimens Observed plant community composition and cover, soil water content and resistance to penetration, and olive yield over an 8-year period, in a Mediterranean rainfed olive orchard.
Summary Hypothesis: Instead of tilling, mowing to control the natural vegetation of olive orchard in spring can increase biodiversity and improve soil quality, without negative effects in production
Olive production did not differ between treatments in any of the years. Soil water was higher in NVM, at both soil depths, particularly in mid-summer and after the first autumn rains (1 to 2%). Soil resistance to penetration was 1 Mpa higher in NVM than in NVT. As compared to conventional tillage, natural vegetation cover mowed in spring seems to be an effective management practice to improve the overall rainfed olive orchard biodiversity and soil quality, without affecting production
Methods
Plots: 5-ha rainfed olive orchard, Planted in 1991, experiment period: 2000-2007.
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CRD divided into two treatments: NVT and NVM. Mowed and tilled plant debris left on site. Forb specific Herbicide sprayed 2003. Plant abundance and composition surveyed in the spring. Yield assessed for each tree within the plot
Results
Fig 1:
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Fig 2
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Fig 3
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Fig 4
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Table 1
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Fig 5
Discussion
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20180523:

Author: Pelz-Stelinski et al
Title Patterns of habitat use by the Asian citrus psyllid, Diaphorina citri, as influenced by abiotic and biotic growing conditions
Model Asian citrus psyllid
Goals/ Question? Whether biotic or abiotic characteristics could be used to predict pop abundance and how ag intensity explained distributionduring the winter
Methods Examining the abundance of ACP in groves under 4 different types of management regimens: conventional, intermittent, unmanaged, and organic
Result: “…only conventional management of groves reduced D. citri populations during winter periods.”
Quote
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20180530: RA

Author: Tarsa, C., McMillan, A., Warren, R. J.
Title Plant pathogenic fungi decrease in soil inhabited by seed-dispersing ants
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Model Aphaenogaster spp.
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Question? If fungal protection is a potential benefit of ant-mediated dispersal, we expected that ant-occupied soil (soils inhabited by ants for colony nesting) would contain fewer phytopathogenic fungi than proximate soils without ant nests.
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Goal investigated published Aphaenogaster soil fungi data and conducted our own metataxonomic analysis of ant-occupied and unoccupied soils to identify phytopathogenic fungi and investigate whether ant colony presence corresponds with reduced plant fungal pathogens in the soil
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Introduction/Importance The obvious benefit of animal- mediated dispersal is movement—it allows the colonization of new habitat. A somewhat cryptic benefit is the escape from negative density-dependent conditions near parental plants, where intraspecific competitors, as well as predators and pathogens
— Finally, directed dispersal suggests that animals may not necessarily save seeds from adverse con- ditions, but deliver them into sites or microhabitats more favorable for survival
— In the tropics, ants “clean” seeds by removing fruit tissue from the seed coat (Guimaraes and Cogni 2002). Seeds cleaned by ants suffer less fungal attack and have improved germination rates than non-cleaned seeds and seeds cleaned by hand
— metapleural glands, which are paired organs on the sides of ants that secrete a waxy, acidic anti-microbial substance…. Whereas metapleural and mandibular compounds specifically inhibit entomopathogenic bacteria and fungi, they also inhibit many additional bacterial and fungal spe- cies with no known ant or insect pathogenicity
Notes
Quote We used Cohen’s d effect size to quantify the size of the differ- ence between treatment levels (ant occupied and control)
Note The use of effect size
Quote Eighty-four percent of all fungal species were more fre-quent in unoccupied than occupied, and 53% of those were plant pathogens
Note without ants fungtal richness inccreases by 84% with 54% of that total being plant pathogens
Quote Our soil replicates were limited, and by taking field soil samples, we could not control for length of occupation, as the anti- microbial effects may be cumulative. Moreover, it is possible that the ants selected microhabitats to colonize that already included low phytopathogenic fungi
Note great point, occupation duration could change fungal community
— an argument for ant choice in habitat location
Quote Gall wasps and Stick insects
Note two other taxa that could be benefiting from the anti-fungal protection from ants

Discussion

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Books

Author: Title
Chown, S.L. and Nicolson, S. Insect Physiological Ecology: Mechanisms and Patterns (DOI:10.1093/acprof:oso/9780198515494.001.0001)
Summary/Abstract
This book provides a modern, synthetic overview of interactions between insects and their environments from a physiological perspective that integrates information across a range of approaches and scales. It shows that evolved physiological responses at the individual level are translated into coherent physiological and ecological patterns at larger, even global scales. This is done by examining in detail the ways in which insects obtain resources from the environment, process these resources in various ways, and turn the results into energy which allows them to regulate their internal environment as well as cope with environmental extremes of temperature and water availability. The book demonstrates that physiological responses are not only characterized by substantial temporal variation, but also show coherent variation across several spatial scales. At the largest, global scale, there appears to be substantial variation associated with the hemisphere in which insects are found. Such variation has profound implications for patterns of biodiversity as well as responses to climate change, and these implications are explicitly discussed. The book provides a novel integration of the understanding gained from broad-scale field studies of many species and the more narrowly focused laboratory investigations of model organisms. In so doing, it reflects the growing realization that an integration of mechanistic and large-scale comparative physiology can result in unexpected insights into the diversity of insects.
Chapters
Chapter 1: Introduction

Chapter 2: Nutritional Physiological Ecology Chapter 3: Metabolism and Gas Exchange Chapter 4: Water Balance Physiology Chapter 5: Lethal Temperature Limits Chapter 6: Thermoregulation Chapter 7: Conclusion|