Biology Project Report Carroll Cave Biology Project – 2018 report and 2019 activity to date

Overall Results There is lots of life in the cave. The population of fish, isopods and insects seems fairly stable. No evidence so far of WNS even though nearby caves (Perkins) are positive. Bat census counts have been declining. Might be our counting method. Samples have been taken, but no taxonomic identifications have been done.

Oct 27 – Bat Census Five participants, Carroll Passage to Mountain Room. Checked and photographed all guano gauges. Bat census between Lunch Room and Mountain Room. One cluster of bats was found at about the 500 foot marker. Estimated 500 individuals. Very few Eastern Tri-Color bats. Almost everything we saw was either gray or Indiana.

2019 Activity to date Dr. Ashley is now officially retired from teaching. Bat census scheduled for October 26. No other biology project activity so far in 2019. Except for the bat census trip, nothing is planned.

Joshua Mouser eDNA sampling Dr. Ashley introduced me to Shannon Brewer and one of her graduate students, Joshua Mouser. Joshua is doing a Master’s thesis on eDNA sampling of blind cave fish and crayfish. We arranged three trips in Carroll Cave to collect samples. The trips were in late March, mid-April and mid-May of 2017. We established six collection sites. Three are in Carroll River and three in Thunder River. All are marked with flags.

Joshua Mouser eDNA sampling The sampling process involves filtering a 2 liter sample of water through a special filter. The filter traps any DNA in the water. Skin cells, feces and other material is captured. The samples are later analyzed in the lab using special DNA sequencing methods. No animals are captured for this process. We observed fish at four of the collection sites, but did not capture any of them.

Joshua Mouser eDNA sampling The technique allows for sampling all life forms in the cave without taking specimans. This project will look at only Southern Blind Cave fish and cave-adapted crayfish. It will not look for DNA signatures of other life. Wikipedia has a good overview of the technique. https://en.wikipedia.org/wiki/Environmental_DNA

EDNA Results So Far Joshua collected 220 samples from 20 caves, 9 wells and 12 springs. 18 of the samples are from Carroll Cave. Joshua has successfully amplified eDNA samples from Mud Cave in Caney Mountain Conservation Area. The next slide shows qPCR results from eDNA amplification. QPCR is a method of duplicating DNA so that it shows up better on analysis. More details at https://en.wikipedia.org/wiki/Real- time_polymerase_chain_reaction

Left to right: Positive control, two eDNA samples from Mud Cave, second positive control. This graph shows that the eDNA amplification works. The samples are looking for blind cave crayfish that are found only in Mud Cave.

Tile placement Unglazed terra-cotta tiles placed in the stream. Groups of five. Every tile is numbered. UL2, Flat Rock Falls, Thunder River (2 sets), Black Rock Riffles, Carroll River. Snails are much easier to see. Isopods seem to be unattracted.

WNS Considerations CCC now has an official policy. Use gear decontamination protocols. During the winter – Visit the natural entrance at every opportunity. Check for dead bats. Restrict trips downstream from 2nd Water Barrier. Examine bats for signs of WNS. Photograph but do not collect. Camden County is officially WNS-positive.

Future plans I have not heard from Dr. Ashley since the middle of 2017. I would like to find a microbiologist to do some microbe studies in the cave. We need to do some trips to both ends of Thunder River.