Jun 20, 2024
Protocols for entomotoxicological developmental study of Phormia regina (Megnin)
- 1Department of Anthropology, University of Tennessee - Knoxville;
- 2Department of Entomology and Plant Pathology, University of Tennessee
Protocol Citation: Hayden S. McKee-Zech, Charity Owings 2024. Protocols for entomotoxicological developmental study of Phormia regina (Megnin). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjnrpngk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2024
Last Modified: June 20, 2024
Protocol Integer ID: 97615
Keywords: Phormia regina, entomotoxicology, development, blow fly, development, growth rate, morphine, lorazepam, drugs, pharmaceuticals
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Abstract
This protocol was developed specifically for examining the impact of drug exposure on forensically important phenotypes (i.e., developmental duration, size, survivorship) of larval blow flies. This protocol was implemented using a laboratory colony of the black blow fly, Phormia regina (Meigen), a common and abundant blow fly in the eastern and southeastern United States. The end goal of this protocol is the generation of drug-specific developmental datasets that can be used for estimating larval age, which can be used in death investigations to then infer a minimum postmortem interval.
Attachments
Image Attribution
Image depicting experimental housing created by Graycen C. McKee
Experimental image included in this publication were taken by Hayden S. McKee-Zech
Materials
Phormia regina fly colony
32oz plastic deli cups
3oz plastic cups
1oz plastic cups with lids
rubber bands
6"x6" paper napkins
aluminum trays with min of 3"sides
autoclaved sand
sharpie marker
colored tape to differentiate treatments
drugs of choice/decomposition fluid
lean pork
kemwipes
chicken liver blood castoff
paintbrushes
tally counter
flour sieve
plastic tub
labels
pencil
electric tea kettle
distilled water
plastic bin 12"x9"
EtOH 70%
cryovials
liquid nitrogen
20 ml scintillation vial
freezer (0C)
freezer (-80)
bullet blender - cup and base 1 per treatment
micropipette and tips
stereomicroscope + camera
microscope scale calibration
Safety warnings
Refer to your university guidelines for working with potentially hazardous material and Blood Borne Pathogen (BBP) protocol. This work was performed in a Biohazard Safety Level (BSL)-2 laboratory.
Ethics statement
The use of invertebrates in research activities does not require IACUC oversight.
Before start
1) Growth chamber or laboratory space with a fixed recorded temperature and light:dark cycle is needed.
25C temperature
70% RH
12:12 L:D cycle
2) Printing labels for each step of the experiment that can be filled in prior to starting each step with speed the process along immensely.
Entomotoxicology laboratory experiments have limitations:
1) Inject a single drug into excised tissue after death, neglecting metabolization and thus resulting in a scenario where drug interactions are not comparable to those that would be observed in real-life.
We suggest utilizing treatments that examine a single drug, a single metabolite of that drug, mixture of the parent drug and metabolite, as well as, a mixture of different drugs.
2) These experiments are closed systems and do not allow new energy or matter to enter the system, thereby presenting a single predictable, inevitable solution. An open system, such as field experiments, consistently gain and lose energy and matter.
We suggest performing toxicological screening on decomposition fluid of humans and using this fluid
the same as you would a drug mixture to preform experiments.
3) Postmortem redistribution - diffusion, neoformation and metabolism
This is an important factor to consider but a true limitation of a development study of this nature.
Metabolomic analysis may help elucidate these interactions in experiments.
Colony Establishment
Colony Establishment
Fly colonies should be established using local populations of blow flies. Adult flies can be baited with aged organ or muscle tissue, or they can be collected from a vertebrate carcass, refuse, or feces. Flies should be collected with an aerial sweep net, sorted to the desired species, and placed in a BugDorm or other enclosure specifically for insects. Flies can be maintained at ambient conditions in a laboratory and given sugar, water, and cultured buttermilk ad libitum. To ensure sufficient genetic diversity and to minimize the risk for genetic drift within the colony, wild fly collections should span multiple timepoints and the total founding generation (G0) should be ~200 individuals.
Egg collection
Egg collection
Fly colonies should be presented with a fresh protein source daily for ~1 week prior to the start of experiments. For this experiment, we exposed colonies to a kimwipe soaked in chicken blood in a 3 oz bath cup each day.
24 - 48 hours prior to the start of experiments, provide the colony with ~5g chicken liver and a kimwipe soaked with chicken blood in a 3 oz cup.
Check for eggs every 3 hours
Once eggs are observed, remove them from the cage and record the time.
For each observation period, place eggs on ~10g lean pork muscle and incubate under ambient conditions.
Check for egg hatch after 8 hours and then hourly thereafter.
Rearing substrate set-up
Rearing substrate set-up
Weigh out 200g of lean pork.
Blend pork in treatment specific blenders for 45 seconds.
Add treatment specific drug to lean pork.
Blend pork in treatment specific blenders for 1 minute.
Divide blended pork into 4 individual pre-weighed 3oz bath cups with 50g of drugged pork in in each.
Note
Record the exact weight of the 3oz bath cup and the drugged pork using a balance.
Replicate set-up
Replicate set-up
Use a camel-hair paintbrush and a hand counter to add 100 individual larvae onto each replicate. Record the time when complete.
Place the 3oz bath cup in 3" of sterile sand within a 32oz deli cup and cover with a paper napkin and 2 rubber bands.
Note
See image of experimental housing under description
Experimental housing example
Data collection - Larvae
Data collection - Larvae
Observe the development of larvae every 8 hours and record results until wandering larvae are observed
We sampled at:
12:00 AM
8:00 AM
4:00 PM
Record behavior of larvae in each replicate at every observation. Do not disturb the pork, only record what you can visibly see. Record any visible larval masses, dead or presumed dead larvae observed in the cup and movement/behavior of larvae.
Once larvae are observed dispersing from the resource, remove feeding cup and sieve sand through a flour sieve (40mm mesh) into a cleaned plastic bin.
Record the number of wandering larvae present.
Collect 50% of the wandering larvae present in a labeled 20ml scintillation vial, add boiling water to kill, then strain off water and add 70-85% ethanol.
Note
Two labels should be included with preserved samples: one written on the vial cap and one written on paper within the vials. The following information should be present on the labels:
Treatment:
Replicate:
Date:
Collection time:
Label the cryovial with the same information from previous vial.Take a 10% sub-sample of wandering larvae for metabolomic analysis via UHPLC-HRMS.
Freeze samples at -80 °C using liquid nitrogen in a cryovial.
Note
Label the cryovial with the same information from previous vial.
Return sieved sand and feeding cup to the replicate
Replace paper napkin and rubber bands
Wash plastic 12"x9" bin used for sand cast off with distilled water and dry before sampling the next replicate/treatment
Data collection - Diet
Data collection - Diet
After all larvae have dispersed away from the pork, take a final mass of the pork using an analytical balance and record the date and time.
Freeze remaining pork for metabolomic analysis via UHPLC-HRMS.
Data collection - Pupae
Data collection - Pupae
Once pupae are observed during the sieving process, use soft forceps to place each individual pupa into 1oz plastic cups with lids and appropriate labels.
Note
Label each lid with:
Treatment:
Replicate:
Date:
Sampling collection time:
After collecting all pupae from all treatments during the sampling period, immediately weigh each individual pupa to the nearest microgram (0 µg ).
Note
Pupa must be weighed shortly after forming as the duration of pupation may be shorter than anticipated due to the drug treatment.
Check for adult eclosion every 8 hours and record results observed
Record the emergence date and time for each individual.
Data collection - Adults
Data collection - Adults
2h
Allow flies to completely sclerotize after eclosion.
Note
This may take a few hours.
Freeze adult flies at -20C.
Record mortality of pupae.
Record sex of the adult flies. Blow flies exhibit sexual dimorphism that can be easily seen with the naked eye or a dissecting microscope. The eyes of males touch, or are spaced very close together. Females typically have a large gap between their eyes.
Record any abnormalities in fly morphology (e.g., wings not formed).
Dry flies at 60 °C 02:00:00
2h
Weigh each individual adult to the nearest microgram (0 µg )with a microbalance and record.
Data Collection - Larval Length
Data Collection - Larval Length
Calibrate stereomicroscope and camera using the appropriate scale
Place all larvae from a scintillation vial under the microscope and take a photo
Digitally measure each larvae to the nearest mm and record
Protocol references
These protocols are loosely based on the methods and materials outlined in:
Byrd, J. H., & Allen, J. C. (2001). The development of the black blow fly, Phormia regina (Meigen). Forensic Science International, 120(1-2), 79-88.