This protocol uses a novel approach of sampling the chorion from hatched butterfly ovi for genetic analysis. It may be particularly useful when other tissue sampling techniques are impractical, unavailable, or not permitted. This protocol has a relatively low overall cost using easy to acquire supplies and targets residual organic material that eliminates the need to temporarily capture or manipulate and potentially harm living organisms to acquire genetic samples.
The overall protocol is straightforward and easy to learn, but requires dexterity and strict attention to detail, specifically to successfully locate, identify, and remove very small target samples to avoid cross-contamination between sample collection events. Begin by carefully reviewing and gathering the complete list of collection-related supplies needed. If sample collection occurs at multiple locations or by multiple individuals or teams, gather and organize all supplies needed into separate deployable units.
Transport supplies to community scientists or other personnel responsible for field collection. If personnel are not local, carefully pack supplies into a separate cardboard shipping box with a complete shipping label and ship them. Upon receipt of collection supplies including the collection tackle box, carefully review the laminated supply list and conduct a complete inventory.
Notify the project leader if any supplies are missing. Prefill 1.5 milliliter microcentrifuge tubes with 180 microliters of lysis buffer. Apply a pre-printed unique ID label to each microcentrifuge tube and place all tubes in the 64-well microcentrifuge tube storage box.
Firmly secure the lid after loading. Ensure all digital equipments are fully charged and any spare batteries are packed. Partially fill a small cooler with ice for field storage and local transportation of the collected samples.
Pack all collection supplies into the collection tackle box or similar sturdy weatherproof container for safe transport and consolidated storage. Review the non-destructive genetic sample collection protocol and detail before heading into the field. Upon arriving at the field site, use a pencil or all weather pen to record the time of day, date, overall property name, specific field site location name or description, GPS reading if available, and the full name and roles of all the individuals present in the weatherproof field notebook.
Locate a suitable habitat for the presence of a larval host plant to the species-specific target butterfly. Using a smartphone or camera, take detailed photographs of all field sites, sample collection locations, host plants, and other relevant information. Use smartphones or other cameras that record GPS coordinates.
Comprehensively search all the larval host plant parts such as leaves, flower buds, flowers, or developing fruit known to be selected by ova-positing adult female butterflies for hatched eggs. For most Lycaenidae, look for white hatched eggs with a distinct hole in the center resembling a donut from which the neonate larva emerged. Look for unhatched eggs that are darker in color, often green or bluish, and will be completely intact without a hole in the middle.
Once a hatched egg is located, wear nitrile gloves. Using sterile and pointed forceps, grab and gently pull the hatched egg off the host plant and place it directly into a labeled microcentrifuge tube prefilled with lysis buffer. Similarly, other tissue samples can be used and processed.
Ensure that all collected material is fully submerged in the lysis buffer within each 1.5 milliliter microcentrifuge tube. Tap the bottom of the tube on a hard surface to re-submerge any samples if needed. When collecting a new sample, discard used nitrile gloves and replace them with a clean pair.
After each egg collection, carefully clean the forceps tips by dipping or dousing them in a vial of 95%ethanol from a squeeze bottle or using alcohol wipes. Collect at least five samples per host plant patch per site, each sample containing one to 20 eggs, aiming for more than 50 eggs total at a location if available. Collect one to 20 hatched eggs from multiple plants in the same host patch into a single microcentrifuge tube and firmly close the lid.
For butterfly species utilizing larger herbaceous or woody host species, collect multiple eggs from different locations on the same plant if host patches are limited. In the weatherproof field notebook, record the assigned unique ID label along with the type of sample, the approximate number of hatched eggs, and collection information. Place the closed microcentrifuge tube with egg debris sample back in the 64-well microcentrifuge storage box.
Maintain the storage box in a cooler with ice while in the field. Place all other collection supplies in the collection tackle box for safe and consolidated storage and transport between sites. Out of these 160 samples, DNA was successfully extracted from 88 with an average concentration of 1.67 nanograms per microliter and the highest DNA yield was 26.8 nanograms per microliter.
DNA concentrations by tissue type confirmed that the samples containing a single egg debris had only one egg case and samples containing multiple egg cases had at least two cases with the number of cases ranging from two to 20, averaging to 5.3 per sample. Materials for collecting up to 563 samples were sent to eight conservation and community scientists from nine states across the species range in Eastern North America. Materials were sent out over three months during 2021 prior to local peak flight times.
A total of 160 Calephelis iris tissue samples have been received. It is crucial to minimize cross-contamination between the samples, ensure the collected sample is fully submerged in the lysis buffer, and accurately record the unique ID label and collection data for each sample. This protocol enabled broad deployment and use by community scientists to non-destructively sample extant populations of a rare butterfly to help conduct a range wide genetic structure analysis.
