𝐖𝐈𝐋𝐃𝐄𝐒𝐓 𝐃𝐑𝐄𝐀𝐌𝐒 | 𝐆𝐢𝐥𝐦𝐨𝐫𝐞 𝐆𝐢𝐫𝐥𝐬

53| You're Not Sorry.

53 | You're Not Sorry.

| Sage's POV |

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"That was cruel." Rory said.

"That was astonishing." I countered.

"I know," mom smiled.

"Look how hard he worked on that sign and everything." She pointed out. "Look at the handwriting, it's so precise, so determined. It's focused Luke."

"That's Jess's handwriting." I pointed out.

"Really?" Mom said, turning to glance at me. "How do you know Jess's handwriting?"

I shrugged. "We give each other books a lot. He likes to write in them then we give them back. It's like a system."

"He vandalizes your books?" Mom gasped. I laughed, "no, no I let him. Plus, I even do it to some of his. It's just little notes in the margins. Thoughts, feelings."

"What, like play basketball, eat a sandwich, stuff like that." Rory teased.

"No, dummy. Stuff. Like margin stuff. People like George Orwell and Woody Allen wrote in margins." I replied, picking at my hands. "Act in Annie Hall and become a literary editor of the Tribune?" Mom asked, dumbfounded.

"Forget it," I rolled my eyes.

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"So, class, our project for the next five weeks, is this. Can we improve how we detect and track living things, making it easier and more accessible with environmental DNA research?"

"Primarily, your study will focus on how the development of convenient and effective eDNA detection methods result in accurate detection of multiple species, accounting for obstacles such as the sparseness of target DNA being excreted. You will use these methods to investigate the North American river otter in the Northeast. Your research should hope to address both the lack of data on river otter populations using traditional methodology, as well as the insufficient eDNA biodiversity monitoring research with semi-aquatic and terrestrial species." Mr. Finch explained.

"The goal is threefold: firstly, you will optimize the GoFish nested PCR technique and demonstrate its validity in non-marine mammal testing: secondly, you will use your protocol to find the population density of river otters in various rivers in New York, Connecticut, and Rhode Island. And thirdly, you will test eDNA analysis methods on other semi-aquatic and non-aquatic mammals, such as beaver, muskrat, and raccoon. Ultimately, your goal is to demonstrate the use of a cutting-edge technique to give us more precise identification to have a clear understanding of the wildlife."

"Now, you will flip to page two hundred and nine of your texbook, and use the rest of our period to read. You will begin your research tonight, at home."

I did as he said, taking a deep breath.

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Later that night, eighteen pages into my notebook, I sat at my desk, reading though my research.

Research using eDNA began in 2008, with the goal of detecting the presence or absence of different target aquatic species.

First using fish species and amphibians in freshwater wetlands and rivers (Ficetola, Miaud, Pompanon, and Taberlet) Aquatic organisms were used as target species because of their ability to shed more eDNA than other species, so a higher chance of positive detection arose with the use of amphibious species.

Ficetola et al tested environmental samples using a primer for a frog species, Rana catesbelana, to validate the first designed methods using eDNA in the field of population detection

Their goal was to determine how to make eDNA collection more cost effective, time efficient, and precise in species detection. Thereafter, eDNA studies began to expand with the use of different species as well as varied PCR procedures such as quantitative PCR and nested PCR.

In 2016, Adrian Kalchhauser and Burkhardt Holm used an eDNA assay to map the distribution of a group of invasive species, Ponto-Caspain gobies. This research provided necessary data for the eventual control of these invasive organisms, mapping the overpopulation of the species.

Furthering the methods used in Adrian Kalchhauser and Burkhardt Holm's study and past eDNA research, Weltz et al mapped the distribution of the Maugean skate, an endangered fish species in Tasmania. contributing towards the reintroduction and further protection of the species. With the goal of method support, development, and advancement, eDNA research has evolved and expanded in its applications as a biodiversity monitoring tool.

Altogether, these studies support the major aim of current eDNA research: to modify and strengthen methods of DNA detection from samples.

Studies using eDNA analysis differ by altering species type, environment, location, population size, primer design, or techniques (Goldberg) Each study contains unique research.

However, the majority of eDNA studies use a general six step procedure: sample collection, filtration, primer design, extraction/isolation, amplification, and analysis.

The GoFish nested PCR methodology, developed by Stoeckle et al uses broad range and species specific amplifications in multiple rounds to create a more precise conglomerate of data.

At this point, it has only been applied in a single study, using marine organisms.

Being faster and more cost and time efficient than the other considered methods, GoFish amplification is used in this study, in a way with the use of non marine mammals. The method is able to streamline both broad range and species specific primer design in an expeditious manner, possibly holding the potential to become a primary method in eDNA research (Stoeckle. Mishu, & Charlop-Powers)

Most eDNA studies are conducted using eDNA collected from bodies of water, such as rivers, because it is dispersed more, and easier to filter, test and analyze than collections from terrestrial environments.

Theless, non aquatic and semi aquatic populations have overall been understudied utilizing eDNA research, but hold as much importance in contributing to the understanding of the overall biodiversity of water bodies.

And that's just the overview.

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