TITLE: Protocols for entomotoxicological developmental study of Phormia regina (Megnin)
AUTHORS: Hayden S. McKee-Zech, Charity Owings

[DESCRIPTION]
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.

[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. 


[STEPS]
SECTION: Egg collection
2. 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.
SECTION: Egg collection
3. 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.
SECTION: Egg collection
4. Check for eggs every 3 hours
SECTION: Egg collection
5. Once eggs are observed, remove them from the cage and record the time.
SECTION: Rearing substrate set-up
8. Weigh out 200g of lean pork. 
SECTION: Egg collection
6. For each observation period, place eggs on ~10g lean pork muscle and incubate under ambient conditions. 
SECTION: Rearing substrate set-up
10. Add treatment specific drug to lean pork.  
SECTION: Rearing substrate set-up
9. Blend pork in treatment specific blenders for 45 seconds. 
SECTION: Rearing substrate set-up
11. Blend pork in treatment specific blenders for 1 minute. 
SECTION: Rearing substrate set-up
12. Divide blended pork into 4 individual pre-weighed 3oz bath cups with 50g of drugged pork in in each. 

 

SECTION: Replicate set-up
13. Use a camel-hair paintbrush and a hand counter to add 100 individual larvae onto each replicate. Record the time when complete. 
SECTION: Replicate set-up
14. 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. 
 


 

SECTION: Egg collection
7. Check for egg hatch after 8 hours and then hourly thereafter.
SECTION: Data collection - Larvae
15. 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 
17. 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. 
SECTION: Data collection - Pupae
21. 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.
 

SECTION: Data collection - Adults
22. 
Allow flies to completely sclerotize after eclosion.

 

17.1. Record the number of wandering larvae present.
17.2. 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.
 



SECTION: Data collection - Pupae
21.1. After collecting all pupae from all treatments during the sampling period, immediately weigh each individual pupa to the nearest microgram (  ).  

 

SECTION: Data collection - Pupae
21.2. Check for adult eclosion every 8 hours and record results observed

SECTION: Data collection - Pupae
21.3. Record the emergence date and time for each individual. 
SECTION: Data collection - Adults
22.1. Freeze adult flies at -20C.
SECTION: Data collection - Adults
22.3. 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.
SECTION: Data collection - Adults
22.2. Record mortality of pupae.
SECTION: Data collection - Diet
20. 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. 
SECTION: Data collection - Diet
20.1. Freeze remaining pork for metabolomic analysis via UHPLC-HRMS. 
17.3. 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. 

 



18. Return sieved sand and feeding cup to the replicate
18.1. Replace paper napkin and rubber bands 
19. Wash plastic 12"x9" bin used for sand cast off with distilled water and dry before sampling the next replicate/treatment
SECTION: Data Collection - Larval Length
24. Calibrate stereomicroscope and camera using the appropriate scale 
SECTION: Data Collection - Larval Length
25. Place all larvae from a scintillation vial under the microscope and take a photo
SECTION: Data Collection - Larval Length
26. Digitally measure each larvae to the nearest mm and record 
SECTION: Colony Establishment
1. 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. 
SECTION: Data collection - Larvae
16. 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. 
SECTION: Data collection - Adults
23.2. Weigh each individual adult to the nearest microgram (  )with a microbalance and record.  
SECTION: Data collection - Adults
23.1. Dry flies at 60 °C 120 min 

SECTION: Data collection - Adults
23. Record any abnormalities in fly morphology (e.g., wings not formed).