Nemertean Larval ID

Collect

Invented by Johannes Müller in 1846, a plankton net is a fine mesh through which water flows and planktonic organisms are trapped. Typically, we use a mesh size of 153 µm, which means that we collect planktonic organisms that are 153 µm or larger. Collecting a plankton sample is as easy as dragging a net through the water and can be achieved dockside or behind a boat. The net concentrates plankton into a small volume, which one can then sort under a dissecting microscope.

Sort & Raise

We collect all nemertean larvae we find in a sample and photograph them live after gently trapping them between a microscope slide and a cover slip supported with small clay feet. After that the larvae are either cryopreserved at -80˚C in a small drop of filtered seawater or raised to more advanced developmental stages. We have been successful raising larvae on a diet of the cryptomonad, Rhodomonas lens (which is why the guts of many larvae look pink in the photos) for many weeks, and have successfully raised the larvae of several species until metamorphosis.   

Identify

Larval and adult stages can be connected in three ways:

1) By obtaining gametes from known adults and rearing larvae in the lab.

We collect adults and obtain gametes. Females release primary oocytes (arrested in prophase I of meiosis), which typically need some time to ‘round up’ and undergo germinal vesicle breakdown in filtered seawater before they can be fertilized with a diluted suspension of sperm (1/1000). We culture early embryos in 150 ml glass dishes set in the tables with flowing sea water (at ambient temperature). Embryos should not be overcrowded (no more than a monolayer on the bottom of the dish). Once embryos become ciliated and begin to swim we thin them to a concentration of one larva per 1 ml of seawater. Once larvae begin to feed we transfer them to a 1-gallon glass jar where they are continuously stirred with a plexiglass paddle. As larvae grow, they are ultimately thinned to a concentration of approximately one larva per 5 ml seawater.

2) Raise wild-caught larvae through metamorphosis and into adulthood.

For many species larvae acquire species-specific characters at later developmental stages or after they metamorphose from their larval body. In these instances, we usually have a single larva which we photograph and feed routinely until it reaches metamorphosis. In one recent case, the morphology of newly metamorphosed juveniles hinted at the identity of the unusual larva, called pilidium recurvatum (see Hiebert et al. 2013).

3) “DNA barcoding”: use DNA sequence data to match larvae to adults.  This third method is feasible for identifying larvae on a large scale, but, obviously, requires having sequences from both larvae and adults.

We use DNA sequence data from two mitochondrial gene regions, commonly used for “DNA barcoding”: cytochrome oxidase I and 16S rNDA. DNA can be extracted from individual larvae using Chelex resin (InstaGene Matrix, BioRad). 16S and COI gene regions are amplified by PCR using “universal” primers (e.g. Palumbi et al. 1991; Folmer et al. 1994). PCR products are purified and sequenced in both directions. Larval sequences are trimmed and proofread, and then compared to sequences from other larval and adult nemerteans. In many cases, larval sequences exactly match adult sequences from the same geographic region or they differ by less than 1%. In such cases we are confident that the larva and adult belong to the same species. We note all instances where sequence divergence (% difference between larval and adult sequences) is higher than 1%.

Practically, we use all three approaches because we tend to find some species more commonly as larvae, while others are more commonly found as adults.