Little smooth bivalves

Back when I was an undergraduate, I did a project on the taxonomy of freshwater crabs in Lake Kivu, one of the Rift Valley lakes in sub-Saharan Africa. I had plenty of experience describing and differentiating species of African freshwater crabs, so my advisor decided I was ready to take on the project. There was only one problem: all the crabs looked the same. It's not that I was inexperienced - far from it - but for whatever reason, in Lake Kivu, all the species of crabs lost their defining spiky features and became little smooth crabs. I eventually found a few structures that differed between the species and wrote reliable descriptions, but the process was far from easy. 

Then during Covid, when I started foraging in the woods around Massachusetts (partly as an excuse to get out of the house and partly as apocalypse preparation), I came across countless non-descript mushrooms. My identification guide warned against picking "little brown mushrooms" with no distinct features. They could be anything - even poisonous - because they were impossible to tell apart. 

The different morphotypes of scallops in my samples. 
Please let one of these morphotypes be scallop larvae. 

Well, friends, I am having pretty intense deja vu this week. I've started digging into my plankton samples from field trips on scallop boats last summer and fall. In each sample, I need to count the number of scallop larvae - sound pretty simple, doesn't it? Here's the problem: there are tons of bivalve larvae in each sample, and I have no clue which of the little smooth bivalves are scallops. It's Kivu crabs and the Covid mushrooms all over again! 

When my PhD student, Kharis, collected a diverse range of larvae in the Arctic Ocean and needed to tell them apart, we first sorted all the larvae to morphotype, sequenced a few representatives from each morphotype, and then compared the morphological and molecular results. The process worked really well, so I decided to replicate it for my scallop samples. 

My first step was figuring out which species was which. Carefully, I surveyed the array of bivalves in each of my samples and pulled out representatives of each distinct morphotype. I extracted their DNA following Kharis's method. I ran a polymerase chain reaction just like she had done. And...fail. None of the PCRs worked. 

That, ladies and gentlemen, is what an electrophoresis
gel is supposed to look like. 

The problem with teeny, tiny bivalve larvae is that they don't have a lot of DNA. There may not have been enough DNA in the PCRs to make the reaction even work. How could I get more bivalve DNA into the reactions? I was already using about half of the extract in each PCR. Man, it's like I need to put the whole animal in the tube! 

Wait. 

My brain flashed back to a paper I read several years ago. In that study, the scientists were trying to identify bivalve larvae, just like I was. They were using PCR to amplify DNA, just like I was. And they put the larvae directly into the PCR tubes! If someone else had done it and succeeded, maybe I could to. At the very least, it couldn't hurt to try. 

My second round of PCRs went perfectly. I got strong, bright bands - indicating that the DNA I wanted had been copied over and over, increasing its concentration in the reaction tube each time. There were no smears, showing that the reactions were clean. In fact, the larva-in-the-tube method gave me the cleanest PCR products I have ever gotten. 

I sent my samples off for sequencing and await the results. The high-quality PCRs make me confident that I will have clean sequences and be able to tell which of my morphotypes are scallop larvae. 

Today, I won over the little smooth bivalves.