Monthly Archives: June 2018
Bahia Adventures 2018. Part 1
Another installment of adventure in science at Bahia de los Angeles on the Gulf of California. This year, I am once again working with students from Ocean Discovery Institute to exapand on what we have learned about feeding assays and DNA sequencing to develop more insight into the diet of Elysia diomedea, and how kleptoplast photosynthesis makes them taste bad.
Drew and I left San Diego with a truck full of stuff, including the tanks, sump and chiller for a slug setup, as well as a perfectly functioning PCR thermocycler from NIH surplus. Drove through Calexico to Mexicali, caught route 5 south through San Felipe. The road was largely good, but the unpaved section in the middle still not finished.
USD truck parked on the Mexicali side of the border. 6/20/18
We arrived in Bahia about 5:30, and stopped for the usual photo from the hills leading into town. When we arrived at the station, the setup crew was working away, although the station was mostly ready for business.
View of Bahia de Los Angeles from the hills above town. 6/20/18
We spent the rest of the evening starting to get organized. I also found out that we currently do not have a permit for collecting the slugs (all other activities have been approved), so it is not 100% clear that we will be able to do any work.
The next morning, I was eager to get started on assembling the lab space. It looked as though everything survived shipping from Maryland to San Diego and the drive to Bahia. We got some new tables, and strung some cords to safely manage electricity flow to the tanks, chiller, freezer, and other lab equipment.
Lab area ready to be set up. 6/21/18
Then we got to set up the tanks. All the plumbing I had assembled in Maryland connected nicely, but the connectors for the chiller were too small for the ¾” tubing used for the rest of the system. I could swear we double checked that. That meant we needed to head to “Home Depot,” the local construction supply lot. Fortunately, Ric, the Ocean Discovery fellow who is assisting me this summer speaks excellent Spanish, so he could easily explain what we were trying to do.
The very nice owner was pessimistic that he would have the right adaptor, but he found one. Then Ric told him we needed two, and his pessimism increased. He miraculously managed to produce another. However, he had no hose for the connection, so he gave us directions to the other, larger supply lot that we had never heard of. It was indeed larger, and they had lots of hose. We bought 3 meters, to have some extra for a siphon hose, and were on our way.
Tanks set up , plumbed and ready for water. 6/21/18
The tank was quickly assembled, and we hauled buckets of sea water to fill up the two tanks and sump. The water was brown from a dinoflagellate bloom, and looked kind of yucky. We hoped the skimmer woul clear it out. Once the tanks were full, we turned the main pump on for a test and cleaned up the splashed water so that we could look for leaks. It all seemed just right, but water kept on pooling on the floor. Sure enough, there was a crack on the bottom of the sump, presumably from shipping. After some discussion and back and forth, Ric and I drove back to Home Depot to get adhesives for a possible fix. The guy was amused that we were back, and showed us what he had. We bought the multi-purpose adhesive and some silicone sealer just to be sure.
It was time for the first snorkel. I put on my wetsuit and headed into the water in front of the staff house. The door for charging the camera opened promptly after I entered the water and that was the end of that. Oy. Nonetheless I looked at a lot of Codium, and was grunted at and bitten by many very aggressive damsels. Most importantly, I found about 4 small slugs up in front of the house, and called it a day.
Small Elysia diomedea in bay in front of station. 6/23/18
I borrowed Drew’s nearly identical camera for a snorkel on the next day, and went out with Ric in the same area. The visibility was still crummy, but I managed to find a small slug on Codium after a bit, and brought both up to show Ric. Soon enough, he was finding his own, and we found quite a few, taking some good photos. Without a permit, however, the slugs will stay in the bay for now.
Ric with yet another slug. 6/23/18
Elysia diomedea in Bahia. 6/23/18
The lab was set up, there were plentiful slugs in front of the station, and the students would be arriving that night, so it looks pretty good for a successful time in the field.
Lab just about ready to go, with slug tanks (left) extraction table (middle) and PCR table (right). 6/23/18
In Bahia, there is always some drama. In this case, it appeared in the form of permits. It turned out that all of the group’s collecting activities, which included an enormous range of fishes, crustaceans, anemones, and molluscs, were approved, with the exception of my project. For some reason, maybe something I wrote set off alarm bells, the collection of Elysia diomedea and marine algae required approval from a different agency.
Stay tuned…
Elysia Neuroanatomy: Old becomes new again!
Ah, the Good Old Days, when “curiosity-driven” science wasn’t naughty. Of course, one might not want to get too nostalgic when the woman whose work is the subject of this post is referred to as “Miss Lillian Russell” in the running head of the paper. Apparently, gender and marital status were considered relevant in a scientific publication 90 years ago.
The primary goal of the Solar Sea Slug project is to study the neurobiological specializations of photosynthetic molluscs. In order to chart the input and output pathways, we need a good roadmap of the central nervous system (CNS) and the nerves that connect it with the rest of the animal. I had hoped that someone had generated at least a crude diagram, and had found some drawings of the nervous systems of Elysia viridis (Huber, 1993, J. Moll. Stud. 59:381) and E. crispata (Gascoigne, 1972, Trans. R. Soc. Edinburgh 69:137), but had not found much more. I fully expected to map the peripheral nerves by myself, and we had even done some preliminary experiments with fluorescent labels for axons for Slug Club during spring semester.
As is often the case, the breakthrough came as I was burrowing through the references from one of the older papers. Huber’s 1993 paper reviews the structure of the CNS of a wide range of gastropod molluscs, and includes a nice diagram of the central nervous system of E. viridis, but provides no information about the periphery. I had skimmed the section describing the image, but had mostly focused on the figure itself. He made a passing reference to earlier work by Russell (1929, Proc. Zool. Soc. Lond. 14:197), so, for the sake of completeness, I ordered the paper through inter-library loan, making it clear that I would like them to include any photographic plates, which are often in a section separate from the main body of the paper.
When I downloaded the paper about a day later, I was pleasantly shocked. As part of a study of the taxonomy of the ascoglossans (= sacoglossans), Lillian Russell had performed a thorough, detailed analysis of the nervous system, nerves, and internal anatomy of E. viridis and a nudibranch, Aeolidia papillosa. She provided an excellent description of the fusion of about seven ganglia to produce the nerve ring that comprises the CNS, along with multiple excellent drawings of the CNS, the peripheral nerves and internal organs of E. viridis.
Plate 4 of Russell (1929). Detailed map of the ganglionic origins and terminations of peripheral nerves that innervate the body wall.
It is hard to know which image is my favorite, but I am very fond of the drawing below, which shows the ganglia in the head, the nerves that originate from them, and the terminations of the nerves in sensory organs such as the eyes and rhinophores.
Russell (1929) plate 1. Detail of the head, including the nervous system and internal organs. Highlights include the eyes (labeled as such), the cerebral ganglia (labeled 1 and 8a), and the rhinophorial nerve (labeled n. 1).
The drawing below, of the CNS and nerves removed from the animal, is also quite impressive, showing the individual ganglia that make up the CNS, and the origins of the many nerves that carry sensory information in, and motor commands out.
Russell (1929) plate 3. Isolated central nervous system and nerves. The ganglia that comprise the CNS can be seen clearly with the esophagus removed.
Aside from publishing a similar description of E. clarki (which probably only differs in slight details), and leaving a note in her will to let me know about it, I can’t imagine how she could have done any more to help me out. Although I had intended to do the work myself, anatomical description of this quality requires considerable patience and artistic skill. I am not very patient, nor am I a good artist. Thanks to Russell’s hard work, I am free to do my behavioral experiments and electrical recordings, and be in my happy place.
So, why is this useful? In a general sense, it gives a starting place for finding neurons and circuits that generate behavior. If you want to know where sensory information goes in the nervous system, finding the nerve that carries the axons from the relevant sense organ or part of the body is a good start. Same goes for finding the motor neurons that cause muscles in a certain part of the body to contract. As a specific example, if we are trying to find how information about light gets to the brain, we first need to find the nerves that connect to the eyes or (hypothetically) other sense organs that detect light or photosynthetic activity. Then we can record monitor impulse activity in the nerves, or from the specific neurons with axons in those nerves.
This is a nice reminder about a few things.
- Possibly most importantly, we truly are standing on the shoulders of others as we try to move science forward. I hesitate to say “on the shoulders of giants,” because, for all I know, Lillian Russell was of average height.
- You really never know when something you did will be useful to another scientist. Ninety years ago (90!), nobody knew about kleptoplasty, and the study was carried out because nervous system structure was considered to be a good marker for evolutionary relationships. It is not reasonable to argue that every experiment that can be imagined must be performed, but during this time in which research needs to be either “translational” or “transformative” in order to be funded, it would be nice to make a tiny bit of room for work that is simply of high quality.
- It may be necessary to dig deep into the literature to avoid reinventing the wheel. Many of us are guilty of looking over the figures of a paper and skimming the text, rather than reading every word. Time is short and papers can be long. Nonetheless, some of the most relevant information to people in the field is in the body of the text, rather than in figures or tables. Who knows what else I will find as I work my way through the old literature.
The Solar Sea Slug project has progressed by leaps during the past half year or so. In addition to finding the roadmap described above, and establishing a self-sustaining colony of E. clarki, some of the students in the Neurobiology Lab course helped me to work out procedures for recording from neurons in the CNS. There are still a few improvements to be made, such as more easily getting the electrode through the sheath that surrounds the ganglia, but we have a little real data. Below is an action potential that was stimulated by injecting 1 nA of current into a large neuron in the abdominal ganglion (Pierce’s “Parker Cell?”).
Action potential recorded from a large neuron in abdominal ganglion of Elysia clarki. 4/19/18
How are we doing?
Self-sustaining culture:
Neuroanatomical roadmap:
Dissection techniques:
First intracellular recording:
This project is really starting to take off.
First 100% Maryland Eggs
We have now come full cycle. Eggs from slugs collected in the Keys have hatched, and the little ones have settled, grown to adulthood and have now produced their own eggs. The slugs in Box of Slugs 2.0 have been producing eggs, but, because one wild-collected slug remains, I can’t be 100% sure who laid them.
This egg mass was laid in the 10-gallon growout tank at USG. All of the potential parents came from egg masses collected between 1/19/18 and 1/24/18, and started hatching between 1/31/18 and 2/6/18. So the maximum possible age of the parents is about 4 months, but they could have been a few weeks younger.
Egg mass in 10-gallon growout tank at USG. Found 5/27/18, but could have been deposited 5/26 or 5/27. Photograph 5/27/18.
Importantly, the eggs are almost 100% fertile. The embryos could be seen developing within a few days, and started to look like veligers in less than a week.
A small section of a developing egg mass from 5/27/18, approximately 8 days post deposition. Depending on the viewing angle, it is possible to see a faint velum extending from each side of the head (embryo viewed face-on), or the shell and head structures (side view). Rare unfertilized embryo marked with an asterisk. 6/4/18.
Because I will be away for much of the summer, I will not be able to rear these embryos. I am hoping for many more broods in the future.
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