Unveiling the Genetic Secrets of Calanus

Skjerstadfjord in Nordland. Stretching out from the narrow outlet at Saltstraumen it widens and deepens eastward toward Fauske and Rognan, forming the inner part of the Salten Fjord. This fjord is widely known for its rich stocks of popular fish species, such as halibut, saithe and sea trout.

The foundation for this fish richness lies in the tiny copepod Calanus. This small creature, no more than a few millimeters long, feeds on phytoplankton and stores energy in the form of liquid lipids, oils, making it excellent food for small fish larvae.
Here, in Skjerstadfjord, several species of Calanus are found. Not only the well-known C. finmarchicus, better known as raudåte, but also its Arctic relative, C. glacialis.

"The two species are extremely similar and have overlapping distributions. With an increasingly warmer climate, these overlapping areas will become even larger", says researcher Marvin Choquet.

Researchers at Nord University have discovered that the two species are so similar when in the same environment, that genetic methods must be used to distinguish them.

The question now is whether species that thrive in warmer waters, such as C. finmarchicus, will spread northward and possibly displace species that thrive best at low temperatures. Closely related species may also begin to interbreed with each other and give rise to hybrids.

Should C. finmarchicus displace C. glacialis in the Arctic, it could have dramatic consequences for the marine ecosystems.

A Perfect Site for Crossbreeding

But how can one know if a tiny, almost transparent crustacean, so small it must be observed under a microscope, is a hybrid? This question has occupied the research group of Professor Galice Hoarau for the last decade, particularly after reports of findings of hybrids of the two copepod species along the coast of East Canada.

"The question of hybridization led to quite a heated debate", says Hoarau.

For when the researchers in Bodø began searching for hybrids, they found none. Even though they checked zooplankton samples collected across the entire North Atlantic. This led the researchers in Bodø to turn their attention to Skjerstadfjord.

"We had found both species in the fjord, in large numbers throughout the year. We also knew that they would reproduce simultaneously. That made the fjord a perfect place to look", says Marvin Choquet.

Samples from Mistfjorden north of Bodø were also included. But the result was the same. Even though they collected samples every month for an entire year, they did not find a single hybrid. How could this be?

The answer turned out to lie in which genetic markers were used.

"The hybrid findings in Canada were based on the use of microsatellites", explains Choquet.

So what in the world are microsatellites? Here, a bit of basic genetics knowledge might be helpful. Our genes (and those of the copepods) consist of DNA, which is made up of four different molecules, called nucleotides, containing one of the bases Adenine, Thymine, Cytosine, or Guanine. These are abbreviated A, T, C, and G and can be likened to a sort of "alphabet of genes." The four bases are lined up along the DNA strand and can be read as the genetic code or as genetic sequences.

Huge Genome

When researchers use genetic markers, they look for very specific sequences on the DNA strand that can be used as characteristics, e.g., of a particular species. Microsatellites are such a marker and consist of sequences that are repeated a certain number of times. A kind of glitch in the record. In one species, the sequence ATC, as an example, might lie three times in a row, while in another species, it is repeated five times. A cross of the two species would then contain both of these two variants.

"But Calanus has a very complex DNA with many repetitions. This poses challenges when sequencing and analyzing it, with the risk of looking at the wrong regions", explains Choquet.

In other words, one might think that one is looking at microsatellite No. 1 from species A, which is different from microsatellite 1 in species B, and think one has a hybrid. But what one is actually looking at is a microsatellite from a completely different part of the DNA strand.

"That is why we developed six molecular markers, InDels, designed and tested to be used on Calanus", tells Choquet.

InDels are small sequences that over the course of evolution have been added (Insertion) or removed from (Deletion) specific areas on the DNA strand.

"The method was designed and tested specifically to look for Calanus hybrids. We analyzed samples from the entire North Atlantic and the Arctic Ocean, without uncovering hybrids. This suggested that the earlier findings were a result of methodological errors related to these microsatellites", says Choquet.

The findings from Bodø were nevertheless not enough to convince all researchers around the world that the species did not hybridize. This led the researchers at Nord University to take an even closer approach.

"We decided to use the really big hammer", tells Hoarau.

Instead of looking for a few, specific areas on the copepod's DNA, they would now look at the entire genome.

"The challenge is that Calanus has a very large genome. In every single cell, there is so much DNA that it is almost unmanageable to analyze all of it. Calanus has between two and four times as much genetic information as humans", says Choquet.

Only Apparently Similar

The solution was to look for "Single Nucleotide Polymorphisms," abbreviated as SNP and pronounced "snips." A "snip" is a variant where only a single nucleotide, i.e., one of the letters, is swapped out. To get a good selection of such "snips," one picks out random pieces from many different regions of the DNA.

"This method allows us to analyze thousands of markers from the entire DNA, not just from a small part of it, as is done with microsatellites and InDels," says Choquet.

By looking for such "snips" from areas of the DNA that were overlapping for C. finmarchicus and C. glacialis, they found entirely unique DNA profiles for the two species.

"Our analyses show no signs of hybridization, which suggests that it is unlikely that the two species can crossbreed," says Choquet.

If you take a water sample from Skjerstadfjorden and put it under the magnifying glass, you will likely be able to spot specimens of both C. finmarchicus and C. glacialis. But no hybrids.

The two species, which look exactly alike on the outside, are so genetically different that they likely cannot produce viable offspring should they mate with each other.

"It could be due to subtle differences in sexual organs or in pheromones, about which we still know very little," says Choquet.

That the two species do not hybridize makes it easier for researchers to study the effects of climate changes on ecosystems.

"Now that we know they do not hybridize, we can move forward and study whether each species has one or more populations within their distribution areas," Choquet asserts.

Or, to return to the copepods in Skjerstadfjorden: Do the individuals of C. glacialis and C. finmarchicus live isolated inside the fjord, sheltered from their conspecifics out in the open sea? Or is there an exchange where individuals from other areas come and mix their genes with the "fjord dwellers"? Do the "fjord dwellers" migrate out into the Norwegian Sea and spread their genes there? This knowledge is crucial for understanding how stable an ecosystem is.

"Getting an overview of the genetic interaction within a species, across its range, is the first step in understanding the extent to which the species can withstand environmental changes," Choquet firmly states.