Pua genetics
Daily kilo (observational data) taken by Kumuola staff and partners indicate that wild pua (juvenile mullet species) recruit into the loko iʻa of Waiāhole and Kapalaho year-round (see Figure 1, below). The brackish waters of these loko iʻa support two mullet species, the native ʻamaʻama (Mugil cephalus) and the non-native invasive kanda (Osteomugil engeli), which are very similar morphologically at sizes below ~6cm (check out this identification chart provided by the Hawaiʻi Department of Aquatic Resources). Increasing densities of kanda within the loko iʻa raise concern that competition for resources with the more desired ʻamaʻama could soon become a problem if kanda populations continue to grow.
In a collaboration with Kamehameha Schools-Kumuola, the University of Hawaiʻi at Hilo, and Hawaiʻi Pacific University, a methodology was developed during the summer of 2019 to identify individual pua as either kanda or ʻamaʻama through use of genetic barcoding techniques. Briefly, genetic material is extracted from a small piece of pua tissue, and using fish-specific primers and a process called polymerase chain reaction (PCR), a region of a gene about 500 base-pairs long is copied thousands of times (for an in-depth explanation on PCR, check out this video). These 500 base-pair “snips” of genetic material can then be analyzed at UH-Mānoa to read the base pairs of nucleotides as either adenine, thymine, cytosine or guanine (ATCG for short). The ATCG nucleotide code is a genetic fingerprint of the organism. This specific genetic fingerprint can then be run against an electronic database containing the fingerprints of all living things, searching for the unique genetic fingerprint of the pua from which the genetic material was extracted, and identifying it as either a kanda or an ʻamaʻama. In this technique, it takes about two weeks to go from pua tissue to species identification.
The technique above was further refined at the PCR stage through the development of custom primers to snip the genetic material of ʻamaʻama and kanda to different base-pair lengths (kanda genetic material was cut to ~400 base-pairs, ʻamaʻama genetic material was cut to ~600 base-pairs). The approximately 200 base-pair difference between the kanda and ʻamaʻama “snips” results in the ʻamaʻama genetic material being heavier (having more mass) than the kanda genetic material. This difference in mass can then be visualized in a process known as gel electrophoresis. In gel electrophoresis, the genetic material from unknown pua are mixed with a dye and placed into a small well in a gel. Electric current is applied to the gel and the genetic material is pulled through the matrix over a period of ~20 minutes. The lighter material moves through the gel much faster than the heavier material. After 20 minutes, genetic material from kanda are further ahead in the gel than the genetic material from ʻamaʻama, which allows us to identify species without sending the material to the lab at UH-Mānoa for nucleotide sequencing (see a time-lapse of genetic material running through an electrophoresis gel here). This technique allows us to go from pua tissue to species identification in about 3 hours!
COLLABORATORS
Starting in January of 2019, kiaʻi (stewards) at Kumuola collected a single pua from schools of recruiting wild pua that numbered 10 or greater in size and froze those pua for genetic identification. During the 2019 fall semester, Kamehameha Hawaiʻi AP Biology students extracted the genetic material from these pua and used the custom PCR primers developed over the previous summer to identify the pua as either kanda or ʻamaʻama. Students then informed our recruitment graph with the specific species that was recruiting on those days. The results of their work built the first picture kiaʻi have had on the species-specific recruitment into the loko iʻa of Waiāhole and Kapalaho. The graph below was built using data provided by both the kiaʻi of Kumuola and Kamehameha students, and shows the magnitude of the kanda invasion into the loko iʻa between January and December of 2019 (see Figures 2 & 3, below). This research remains ongoing and we are excited to explore these patterns in more detail with successive biology classes. Stay tuned!
For more information about this research or to partner with us, please contact Luke Mead at lumead@ksbe.edu.
Figure 1. Graph of pua recruitment into Waiāhole mā in 2019.
Figure 2. Graph of species-specific pua recruitment into Waiāhole mā in 2019; green bars = pua ʻamaʻama recruitment, red bars = pua kanda recruitment, purple bars = both pua ʻamaʻama and pua kanda recruitment, orange bars = data unclear.
After approximately 2-3 months in the open ocean, pua (juvenile fish) recruit into the loko iʻa of Waiāhole through a mākāhā (sluice gate) that connects the loko iʻa to the coastal waters. Here we see ~80 juvenile mullet species entering Waiāhole in early spring of 2020. We are using genetic barcoding techniques to identify these recruiting mullet as either our native ʻamaʻama or the invasive kanda to better understand the recruitment patterns of our native and non-native species in east Hawaiʻi.
Figure 3. Graph of averaged species-specific pua recruitment data during 2019. January – April was peak pua ʻamaʻama recruitment, May – December was peak pua kanda recruitment.
Learner Insights
Kayla.mp4“I feel like the reason I like this project so much was because it had an application beyond the classroom and I feel like there's so many opportunities for that to happen just in the existing lesson plans that teachers have and the existing projects that already go on. So I think it's just a matter of finding ways that it can connect to impact more than just our learning in the classroom...
It gives students a sense of it's not just for a grade or an arbitrary thing right now--it's something that's actually going to impact other people and that's what happens in the real world when you do these kinds of projects and do this type of work--it's going to go beyond yourself and your grade.” - Kayla
“I was able to kilo, observe the different species and analyze [them], just like our kupuna would and interpret the data and make decisions based off of our results.
I feel like [this experience] connects to our ‘Ōiwi Edge because we're applying native concepts of observing the fish and then we're also incorporating modern principles, ideas and technology--like the DNA analysis--to use the results and apply it to the real world and solve problems like invasive Kanda.” - Carlos
“I was kind of surprised with the process to get DNA. I didn't realize how many steps it took and how long you took. It took like the whole class to get the DNA. Three classes actually. It took a long time.”
“This lab was different from other labs because usually we're just testing scientific concepts. We take the data and we don't do much with it. But with this [lab] the data is actually going to be used for something, so, it's more important.” - Zander
“We just finished learning about genetics in class and so we're able to tie that knowledge into what we're doing in the lab. We're kind of confined to this school or educational space when we do labs and when we learn, so it was actually kind of scary for me at first knowing that what we did here would have an actual impact in the real world. It really shows that what we learn here has applications outside. I think too, it's also confidence boosting to know that we can do this!" - Travis
“...I feel like it's more of the action piece after now, like what we were going to do with the data in terms of how we're going to care for the loko iʻa? Because right now it's functioning, but not necessarily to the extent that we want it to be and not necessarily with the fish that we want in it. So if it's able to be maintained then that kind of fulfills our purpose.” - Kayla
KumuGlennon.mp4“I was looking for the authenticity of the experience and link it to what they're learning in Biology and AP Biology and that's what we got! Layne, Luke and Trisha were very good about making it clear to the [students] that this data is real data. It's not like they're validating data that you have already got--actually, this is their primary research data that they are going to base their future action on.
From my perspective as a teacher, having gone through this, I know how I'm now going to tie this all back into my curriculum or the AP curriculum, so it was a win-win!" - Kumu Glennon
"This was a fabulous opportunity for them to practice these techniques, be involved with something that is authentic, and real life."
For more information about this research or to partner with us, please contact Luke Mead at lumead@ksbe.edu.
