Possible Reproductive Isolation between Groups of Polydora websteri within the Same Estuary Caused by Seasonal Offset in Breeding Emma Hargreaves, Old Town High School Introduction Oyster aquaculture in Maine, and around the world, has been greatly affected by the blister-creating worm Polydora websteri. P. websteri is in the Spionidae family of polycheate worms (Blake 1). Spionids are all sedentary worms that create tubes and feed using palps (Figure 1) (Blake and Arnofsky 57). In oysters, this creates blackish blisters on the inside shell, and this blemish makes the affected oyster a less-valuable product because of its poor aesthetic appeal (Figure 2). But there isn’t enough known about this pest to fully understand its ecology, and such knowledge could inform efforts to control it. P. websteri larvae have been found in Maine estuaries from April to August, and have been sighted as being especially numerous during May and June (Blake 13). But Blake only collected egg capsules from March to July, leaving one to wonder whether or not Maine’s P. websteri population could still breed in the winter. Blake has found that Maine and New England populations of Polydora concharum and Polydora quadrilobata (two closely related species in the same genus) breed during the winter (Blake 32-38). And in a later publication, Blake and Arnofsky theorize that there could be multiple reasons for this; one being that the “Maine populations could be relics or isolates of a species adapted to a more northern, subarctic climate, where spring/summer reproduction would occur at the same temperatures found along the coast of Maine during winter/spring” (Blake and Arnofsky 66). If it is possible for this to happen to P. concharum and P. quadrilobata, then could it be possible for P. websteri? Knowing the answer could help to understand P. websteri and how it interacts with its environment. This idea of a seemingly singular species becoming different from itself is not without precedent. Stanley Rice found that P. cornuta taken from California, the Carolinas, and Maine were actually three non-interbreeding populations (Rice 1). In a telephone interview with Stanley Rice, the author of many papers, including “The Polydora cornuta Complex,” he speculated the same is also possible for P. websteri. Blake and Arnofsky claim that poecilogony, an occurrence of varying modes of development in young worms across a geographic range, is unusually common in Spionidae (Blake and Arnofsky 78). Furthermore, this kind of divergence could be the beginnings of the genetic isolation that leads to different species. They also point out that

Figure 1. Adult P. websteri (pinkish worm) and egg capsules (yellow spherical clusters) inside of an opened burrow. Photo credit: Sara Lindsay and Paul Rawson

Figure 2. Shucked oyster shell with light shining through. The dark blisters are P. websteri burrows filled with sediment, and U-shaped trails through the burrows are P. websteri still inside. 1

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further investigation would be needed to “fully understand the reproductive plasticity exhibited by Spionids” (Blake and Arnofsky 78). All of this raises an interesting research question: If some P. websteri are still breeding in the winter, could they become reproductively isolated from summer breeders without geographic separation, even within the same estuary? In this research project, I set out to demonstrate that some P. websteri, even a small proportion, are reproductively active in the winter. I hypothesize that a seasonal offset in the timing of reproduction could prevent subpopulations from interbreeding; and I propose further investigation into the possibility that this seasonal offset in subgroups of P. websteri could genetically isolate these subgroups, even if they inhabit the same oyster.

Methods In order to test the hypothesis, Ian Ellis delivered twenty oysters from the Weskeag River Estuary (Maine, USA) on February 3, 2016 (Figures 3 and 4). On that day the estuary water temperature was 3°C (Figure 5). The oysters were held in a controlled facility at the University of Maine where their water temperature was gradually raised

Figure 3. A “ winter” Weskeag River oyster in our lab a few weeks into the experiment. The tubes visible on the edges house hundreds of worms in close proximity. Could seasonal offset of reproduction be preventing some of these worms from breeding?

from 3°C to 8°C between February 3 and February 15, when we decided to keep half at winter-time temperatures (the “winter oysters”) while warming up the other half to summer estuary temperatures (the “summer oysters”). The winter oysters were then stored in a 5°C refrigerator, aerated with an air pump and air stone, and fed Shellfish Diet 1800 from Reed Mariculture. The summer oysters were removed from the refrigerator and underwent a quick, non-lethal warm up managed with insulation and ice packs. These ice packs were changed over periods of time that were gradually lengthened, and parts of the insulation were slowly removed. This process lasted for ten days. It resulted in the oyster’s water rising from 5 to 16 °C at a rate of about one degree per day. They were then kept at room temperature (between 15 and 17 °C) for the duration of the experiment. The winter oysters were kept at 5 °C during the same time period and over the whole experiment using a combination of refrigeration, and ice packs with coolers when they needed to be moved to and from the lab (Figure 6). The oysters were then investigated for signs of P. websteri life and reproduction. This was done by shucking an oyster and opening up visible P. websteri burrows with a scalpel. Once opened, observations were made with a dissecting microscope (with a 45X magnification) for: evidence of a

Figure 4. Close up of the crowded worm tube openings visible on the edges of an oyster. Openings are indicated with blue arrows. Picture taken about a week after the oyster was removed from 3 °C estuary.

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Figure 6. The “winter” oysters in the tank where they were kept for the duration of the experiment. In this photo, they are in a refrigerator. Throughout the experiment when the oysters needed to be moved to the “lab” they were transferred in coolers in order to keep their temperature steady. Figure 5. A picture of floating oyster crates in the Bagaduce River. We used oysters from the Weskeag in our experiment, but this photo of an oyster operation in the winter is instructive. The water temperature was 0.6 °C on the early March day when this photo was taken, and the river was skimmed with ice.

First of all, the delay between taking the oysters out of the estuary and actually testing them for worm activity could have had unknown, adverse effects on the results. The oysters spent weeks in limbo, and may have therefore been exposed to temperature fluxes inconsistent with the natural seasonal cycle of an estuary. Also, about half way

worm’s presence; if the worm was alive; if there were eggs inside the worm or beside the worm in the burrow and/or if the worm had spermatophores inside (Figure 7). These observations were first conducted on at least half of the P. websteri burrows visible in nine winter oysters, and then the same process was repeated with six “summer” oysters that had been kept at room temperature. The percentage of burrows with a presence of eggs, percent of worms with spermatophores, and the livelihood of worms were compared between the summer and winter populations to demonstrate that winter breeding P. websteri are more than an anomaly, and may even be comparably active to the summer breeders.

Results and Discussion These data show that there is little to no meaningful difference between the summer and winter oyster’s activity (Figure 8a, 8b). Forty-eight percent of the winter oysters’ opened P. websteri burrows were active, and about 36% of the summer oyster’s opened burrows were active. Only one burrow from winter burrows showed obvious signs of active reproduction (eggs) and only one burrow from the summer oysters showed obvious signs of reproduction (spermatophores). However, there were many experimental limitations because of the high school lab set-up and the the tools we had access to.

Figure 7. An oyster in the middle of being examined for reproductively active P. websteri. The “top” of the burrow has been removed with a scalpel, and the burrows themselves (circled in red) will be combed through under a microscope.

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microscopes with better computers for our photography equipment. The quick warmup oysters, or “summer” oysters, would have instead been oysters from the same estuary during the summer, opened and examined just as quickly as the winter oysters. With more accurate data from the winter and summer oysters, the comparison would be a better measure of seasonal reproductive activity. Ideal results from an ideal experiment supporting the hypothesis would show irrefutable evidence of P. websteri’s active reproduction in the winter, and demonstrate A that their presence is statistically significant (Figure 9). A finding that wintering P. websteri are still reproductively active, and that this activity could be comparable to that of summer P. websteri, opens the door for further investigation. Two more experiments would be invaluable. Stanley Rice, in 2007, demonstrated that Polydora cornuta are actually a complex that includes reproductively isolated and genetically distinct populations (Rice 1). Rice collected P. cornuta from Florida, California, Maine, and New Zealand and introduced sperm from P. cornuta of one place B to a population of P. cornuta of another place (Rice 2-3). For example, spermatophores Figure 8. The graphs show the data from both the “summer” (A) and “winter” (B) from a mature California P. cornuta would oysters. Inactive burrows are gray, active burrows (burrows with a worm inside when be introduced to a mature female P. cornuta they are opened) are red, and spermatophore presence is green, and egg presence from Maine. Rice found, using these tactics, is yellow. In the winter sample of oysters 48% of the burrows were active and only that Maine/California and Maine/Florida one out of all examined burrows had eggs. No spermatophores were observed. In the crosses resulted in a less than 7% fertiliza“summer” sample of oysters, 36% of the burrows were active and only one out of all tion rate, and that California/Florida crosses examined burrows had spermatophores present. No eggs were observed. could reach fertilization far more often, but their hybrid embryos could not make it past the embryonic stage (Rice 6). We hypoththrough the investigation into the winter oysters, the niesize that P. websteri may become similarly reproductively trate concentration in their tank got dangerously high. isolated inside of the same estuary, because of the possibilAfter this event, worm activity was noticeably lesser, ity for a seasonal offset in reproduction cycles. Therefore, and we will never know just how much this affected the Rice’s experiment would need to be recreated using reproP. websteri in the winter oysters. Lastly, our microscopes ductively active P. websteri populations from both summer and tools for burrow opening were not ideal. In order to and winter P. websteri from the same estuary. see the tiny, usually hidden eggs and spermatophores of P. Stanley Rice also completed a genetic test in which websteri we would have needed higher magnification, and mitochondrial DNA of P. cornuta were extracted and exmore precise tools to open burrows. amined for inconsistencies (Rice 6-7). This work demonAn ideal version of this experiment would be openstrated that the populations of P. cornuta in North America ing the winter oysters at the estuary as soon as they came actually had at least three distinct lineages (Rice 1). out of the water, and examining them with higher power

Introduction to Scientific Research 2015–2016, Old Town High School

Figure 9. This graph is what the “winter” oyster’s data might look like in an ideal experiment, if it was successful. There is irrefutable evidence of reproductive activity, and the amount is substantial enough to assume that the activity is not an anomaly.

Conclusion Similar experiments performed with winter-breeding and summer-breeding P. websteri could test the hypothesis that these subpopulations do not interbreed, are genetically distinct, and could define where these distinctions lie. Such testing is necessary to fully understand P. websteri from a scientist’s point of view and from a grower’s point of view. In an interview, Dr. Paul Rawson, professor at the University of Maine’s School of Marine Sciences, says he and his students are attempting to test whether “the worms burrowing in oysters at different sites from the Gulf of Mexico to the Gulf of Maine are the same species.” They want to compare gene sequences from P. websteri across this geographic range to get an idea of gene flow among the P. websteri populations. I propose that it would be scientifically interesting to test for gene flow among P. websteri within the same oyster. Jesse Leach, a longtime oyster grower on the Bagaduce River (Penobscot, Maine) has witnessed a variety of reproductive flexibility in another polychaete, the sandworm. According to Leach, this worm deposits its eggs in March when estuary temperatures have not warmed much beyond winter temperatures. He thinks these worms show reproductive plasticity (James Blake’s term) because the timing of the eggs’ hatching appears to depend on the

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arrival of “the right conditions.” He says that he wouldn’t be surprised if P. websteri had the reproductive flexibility for there to be a seasonal offset in breeding. In general, he says investigation into these worms is “very interesting and very important. There’s so much we don’t know about worms, especially P. websteri.” The possibility of a seasonal offset in P. websteri’s breeding and the genetic isolation among subpopulations this could create is important to the scientific community and the ever-growing aquaculture community. This ecological knowledge is needed to deal with this small species that has such a big impact.

References Zajac, Roman. “Population ecology of Polydora ligni (Polychaeta: Spionidae).” Marine Ecology Progress Series Nov. 1991: 197206. Print. Blake, James. “ Reproduction and Larval Development of Polydora from Northern New England (Poycheata: Spionidea).” Dec. 1969: 1-18. Print. Blake, James & Arnofsky, Pamela. “Reproduction and Larval Development of the Spioniform Polycheata with Application to Systematics and Phylogeny.” Reproductive Strategies and Developmental Patterns in Annelids 1999: 57-106. Print. Rice, Stanley. “The Polydora Cornuta Complex Contains Populations that are Reproductively Isolated and Genetically Distinct.” The American Microscopical Society Inc. Journal compilation 2007: 1-20. Print. Rice, Stanley. Personal interview. 27 November 2015 Leach, Jesse. Personal interview. 3 May 2016 Rawson, Paul. Personal interview. 4 May 2016