Author: Hannes Baumann

NSF awards our collaborative sand lance grant!

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24 June 2023. We are overjoyed to be able to announce today that NSF's Division of Integrative Organismal Systems has awarded our proposed research to better understand sand lance CO2 sensitivity!

With a sense of pride and humility we will take on this intriguing case, follow it down some rabbit holes, while keeping in mind the big picture. This fall, our collaborative team will begin its renewed work, now on both congeneric sandlance species (Ammodytes dubius, A. americanus).

We already have two talented PhD students recruited to the task, Lucas Jones and Emma Siegfried. With curiosity and anticipation, we look forward to the next years of eco-evolutionary research on some of the most important forage fish species on the Northwest Atlantic Shelf.

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Earlier work showed that sand lance embryos are unusually sensitive to high CO2

NSF-ORCC (Organismal Response to Climate Change): Collaborative Research: Mechanisms underpinning the unusual, high CO2 sensitivity of sand lances, key forage fishes on the Northwest Atlantic Shelf (#2307813, 2023-2026, $1,050,000)

The research team: Hannes Baumann (lead-PI, UConn), Zofia Baumann (UConn), David Wiley (NOAA), Nina Therkildsen (Cornell), Chris Murray (WHOI), Neel Aluru (WHOI)

*** Why are sand lance so sensitive to future high CO2 conditions in the ocean? ***

Public Award Abstract
Ocean warming and acidification are direct, predictable consequences of man-made climate change with likely vast but still insufficiently understood consequences for marine life.

So far, most tested fish species appear only mildly sensitive to ocean acidification, but sand lances are an exception. Sand lances are small, eel-like, schooling fishes of enormous importance as food for marine fish, seabirds and mammals in temperate to polar ecosystems, and recent research conclusively demonstrated that many sand lance embryos have trouble developing and hatching under predicted future ocean conditions.

This project uses modern experimental and molecular tools to understand exactly WHY sand lance embryos are so unusually sensitive and which genes and enzymes are responsible for this. Genes will also reveal whether some specific genotypes are less sensitive to warming and acidification, which can then be used to predict whether the species could evolve to be more tolerant over time.

Another important objective is to test a closely related sand lance species to find out, whether the high climate sensitivity might be of general concern in this important group of forage fishes. This project combines innovative ecological, evolutionary, and genomic research to help society anticipate looming marine ecosystem changes in the 21st century, while equipping the next generation of scientists with the needed tools and expertise to succeed in the challenges ahead.

The project also creates opportunities for high school students from underprivileged Connecticut schools to accompany the team on sand lance sampling trips to Stellwagen Bank National Marine Sanctuary.

American sand lance (Ammodytes americanus) swimming in surface waters of Wells Harbor, ME in November 2021

Technical Award Abstract
Two recent studies on Northern sand lance (Ammodytes dubius), a key forage fish on offshore sand banks across the Northwest Atlantic shelf (NWA), have robustly demonstrated that predicted future CO2 conditions induce some of the most severe reductions in embryo survival and hatching success seen yet among tested fish species. This project has four objectives for revealing the mechanisms underpinning this unusual, high CO2-sensitivity as well as the ubiquity and genetic basis of this phenomenon.

[1] For the first time, we will rear A. dubius offspring produced from wild spawners to late larval stages at factorial CO2 × temperature conditions to test whether sand lance larvae are as CO2-sensitive as embryos.

[2] For the first time, we will use transcriptomic tools (RNAseq, RT-qPCR) to elucidate mechanisms causing ‘CO2-impaired hatching’, focusing specifically on hatching enzymes, to better understand a newly discovered mortality mechanism due to high CO2 in fishes.

[3] Modern genomic approaches (low-coverage whole genome sequencing; allele frequency shifts, relatedness analyses) will reveal whether high CO2-sensitivity has a genetic basis in sand lance and could therefore evolve.

[4] And for the first time, we will extend CO2 × temperature experiments to a congener, the American sand lance (A. americanus), which provides an important scientific contrast between nearshore vs. offshore species CO2-sensitivities and will yield critical insights whether high CO2-sensitivity is a wider concern within the sand lance family.

Feeling the pulse of Mumford Cove

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23 March 2023. For almost 8 years now, the Evolutionary Fish Ecology Lab has conducted research in nearby Mumford Cove, a small, eelgrass covered embayment on eastern Long Island Sound. Using a set of battery-powered probes we have continuously measured temperature, pH, oxygen, salinity, and depth in 30 min intervals in the Cove - almost 120,000 times. This ongoing effort is not funded by any grant or institution; instead, it has been sustained over all these years by the firm belief in the prescient, if undervalued societal service of monitoring, an activity without short-term reward but important long-term benefits in understanding how ecosystems change on short and long time-scales. To commemorate the effort, we simply thought that it is time to show you some data, some pictures, and draw some early, cautious conclusions about the very interesting case of Mumford Cove. Have a look!

Fig01---Mumford-Cove-sketch
Fig.1: Schematic section of the upper part of Mumford Cove, showing the deployed probe (blue) between the bottom anchor (grey) and a subsurface float (orange), marked by a surface float (white). The probe sits in the deepest part of the Cove (Channel), at constant 50 cm distance to the bottom, but variable water level above (red histogram).

"Future generations will certainly have better theories, tools, models, and computers, but they will still depend on the data and measurements taken here and now."

Aerial01
Aerial view of Mumford Cove (Picture: Jamie Vaudrey)

ICES JMS publishes sand lance population structure paper!

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5 December 2022. We are proud to announce that the ICES Journal of Marine Science just published our latest sand lance study! The work spearheaded by Lucas Jones and subject of his Masters thesis research has brought together a large, international group of collaborators to better understand the genetic relationships between disparate sand lance populations across their large geographical range. This is an Open Access publication that will hopefully be of use to researchers studying sand lance everywhere.

Ammodytes dubius
Sand lance, a vital forage fish, may be facing new challenges as the oceans warm. Researchers have found two genetically distinct populations of Northern sand lance which may help inform conservation and management decisions. These sand lance were caught off the coast of Greenland (Photo courtesy of Thomas Pederson)

Press release. By Elaina Hancock

Genetic Barriers, a Warming Ocean, and the Uncertain Future for an Important Forage Fish

In the vast oceans, one would assume their inhabitants can travel far and wide and, as a result, populations of a species would mix freely. But this doesn’t appear to be the case for a vital forage fish called the sand lance.

Sand lance are small schooling fish impressively rich in lipids, which makes them a fantastic and significant food source for at least 70 different species ranging from whales and sharks to seabirds, says UConn Associate Professor of Marine Sciences Hannes Baumann.

The Northern sand lance can be found from the waters off New Jersey all the way north to Greenland. Researchers, including Baumann and Ph.D. student Lucas Jones, were interested to see if sand lance constitute a massive, homogenous population, or whether there are genetically distinct groups. Their findings are published in the ICES Journal of Marine Science.

Baumann explains these are important questions to answer when considering conservation and sustainable management of the species, especially since the regions where sand lance live are warming faster than many areas of the planet due to climate change.

Sampling fish from such a broad range is no small task, but two years ago, Baumann and Jones began reaching out to other researchers to see if they had tissue samples to spare. Baumann credits the work to the international group of colleagues who contributed samples including co-authors from Canada and Greenland, and who helped sequence and analyze the data including co-authors from Cornell University.

In all, Baumann, Jones, and the team were able to sequence and analyze nearly 300 samples from a variety of locations across the sand lance’s range using a technique called low-coverage whole genome sequencing. They also sequenced the first reference genome for sand lance.

In a nutshell, Baumann says they found an area on the Scotian Shelf, off the coast of Nova Scotia, where a genetic break occurs. The researchers distinguished two distinct groups, one north and one south of the divide, with parts of the genome differing quite dramatically – namely on chromosomes 21 and 24. Without obvious physical barriers like a mountain range separating the groups, Baumann says it’s logical to ask how these differences are possible.

“That is the scientific conundrum,” says Baumann, and the answer, it appears, lies in the currents.

“When fish from the north reproduce and drift south, they are genetically less adapted to warmer southern waters, even if it’s five or six degrees warmer in the winter, they are just not surviving,” Baumann says. “These populations may be linked by the ocean currents, but the realized connectivity is basically zero.”

Separation of 3 sand lance species based nuclear and mitochondrial DNA (Jones et al. ICES JMS 2022)

Fig02---PCA+haplo_data
(A) Principal component analysis of SNP-based, individual-level covariance matrices with superimposed FST values for each interspecies comparison. The asterisk denotes 5 likely misidentified A. americanus specimens from the Northern GSL (B) Mitochondrial haplotype network of all analyzed specimens, with the number mutations separating congeners.

This finding is a first for the sand lance, but it has been shown in other species such as lobsters, cod, and scallops, and this research adds further evidence to an apparent temperature divide at the Scotian Shelf, and helps demonstrate that temperature is an important factor in survival.

“Example after example shows that the ocean is not as homogeneous a place as expected, and there are all kinds of things that prevent that constant mixing,”Baumann says. “We found another striking example of that.”

When researchers find adaptation in an environment where mixing is continuous, like in the ocean, Baumann says, the question is how it is possible that groups stay different, even though they are constantly encountering other genotypes. That is where powerful genomic methods, like the ones used in this paper, come in handy.

“Parts of the genome in many species have what we call a ‘genetic inversion,’ which means that the genes on the chromosome from one parent have a certain order and the genes on the same chromosome that come from the other parent that code for the same thing, and they’re the same area, but they’re flipped,” Baumann says.

These inversions mean recombination cannot occur; therefore, the genes are passed down through the generations and play an important role in adaptation.

“We discovered on chromosomes 21 and 24 there are whole regions that are completely different and that is like the trademark signature of what we call an inversion because there’s no recombination going on.”

Baumann says that knowing there are genetic and ecological barriers on the Scotian Shelf is important, because with climate change, this barrier may move north and while that may be good news for southern fish, it’s bad news for the fish currently there.

The researchers were also a little relieved in finding two clusters, because had there been many smaller clusters, it could make management and conservation more challenging, especially considering scenarios like the construction of offshore wind parks. Areas potentially well situated for wind turbines can also be habitats for sand lance, and construction disrupts habitats. If there were many, smaller population clusters, a single construction project could pose the risk of completely wiping out a cluster, whereas with more widely dispersed populations, though the local population may be temporarily disturbed, it will not be long before they are able to re-establish after construction is completed.

Baumann plans to focus further research on studying the genetic basis of the thermal divide.

“We want to make sure that this fish is productive and resilient, despite climate change, so we should make sure these areas where they are occurring are protected,” Bauman says. “These decisions should include experts to ensure if there’s an area that is very critical to sand lance, that any disturbance is temporary.”

It isn’t an unsolvable conflict, but it is something that we need to do, says Baumann, who also notes that it is possible that sand lance north of the thermal divide are already suffering more from warming because the region is warming faster.

“It could be that these two clusters have different vulnerabilities to climate change,” he says. “We don’t know that yet but that’s something that should be pursued.”

Fishing for silversides … in Chile!

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A two-week stint to south-central Chile ends with a successful proof-of-concept that planned research on a Chilean silverside species will be highly feasible, opening avenues for a budding US-Chilean collaboration.

Tumbes---panorama
Fishing boats in Caleta Tumbes near Concepcion, Chile

Concepcion (Chile), 12 October 2022. Two incredible weeks of adventure and scientific exploration for new and potentially groundbreaking science are coming to a close. In preparation for next year’s sabbatical, Hannes has met and made friends with colleagues at the Universidad the Concepción in southern Chile, travelled some 2,000 miles along the stunning Chilean coast, scoured local fish markets and accompanied artisanal fishermen on their nightly pursuits. The goal: finding a small fish that looks all too familiar – a silverside!

The Chilean silverside (Odontesthes regia), locally known as ‘pejerrey’, looks eerily similar to the Atlantic silverside (Menidia menidia), the model that has already inspired decades of eco-evolutionary research across many labs including ours. And like its northern hemisphere cousin, Chilean silversides occur over an astounding geographical range along the South American Pacific coast, all the way from Puerto Montt (42°S) to southern Peru (10°S)! There, average coastal temperatures change predictably with latitude and therefore provide a natural climate gradient in space that could serve as an analogue to climate change in time. Whether and how Chilean silversides show similar local adaptations to their latitudinal gradient is a big question – and next year’s sabbatical will start to provide some important answers.

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Pejerrey are usually caught with gillnets as here in the picture

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A Chilean silverside embryo of a few days post fertilization. Eyes beginning to pigment and a prominent yolk artery provides nutrients for growth

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A Chilean silverside embryo close to hatch

To prepare, Hannes spent two weeks in September and October 2022 in Chile. Hosted by the ever-enthusiastic Prof. Mauricio Urbina from the zoology department and thanks to a visiting grant from the university, we were ready to start exploring. Our specific goal for this trip was to find spawning-ripe pejerrey in two of the planned four locations along the coast.

The luck was on our side and the timing of the visit turned out to be perfect. On a nightly fishing trip with the artisanal fisherman Juan Figueroa from the small village of Tumbes near Concepción, we caught running ripe males and females, observed naturally deposited egg masses in nearshore waters, and were able to subsequently document the temperature-dependent development of newly fertilized embryos.

egg-masses

Dichato-panorama
Left: During spawning season, pejerrey deposit enormous masses of eggs on vegetation in shallow water. Right: The Marine Station of the Universidad de Concepcion in Dichato

On an epic road-trip up the coast all the way to Coquimbo, Hannes and graduate student Rocio Barrios stopped at many villages and local fish markets, gathering information and finally securing precious samples of spawning-ripe pejerrey from a fisherman at the Coquimbo fish market. Transporting the embryos was a success, too, thereby paving the way for the proposed research plan next year.

The real, big common garden experiments will take place from September – December 2023 at the Dichato Marine Station near Concepción, a small but recently renovated station with excellent facilities for our purposes.

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On the road during our trip to Coquimbo

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Beautiful spring at the scenic Coliumo Bay near Concepcion

While at the University, Hannes also gave a seminar talk to the students and faculty explaining his excitement and plans for coming to Chile, which received great interest, curiosity, and students expressing interest to play a part in this.

    Baumann, H. 2022. Principles of local adaptation across environmental gradients (or: why I’m so darn interested in studying Chilean silversides). Invited seminar talk. University de Concepción, 29 Sep 2022

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A gillnet used to fish for Chilean silversides on the beach of Tumbes near Concepcion

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A newly hatched Chilean silverside measuring already an astounding 9 mm TL

Hannes & Max at the 45th Larval Fish Conference in San Diego

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2 September 2022. After two grueling years of pandemic restrictions, Zoom conferences and meetings, the Baumann lab was as elated as anyone else to attend the first in-person conference again! Hannes & Max went to beautiful La Jolla in San Diego to participate in this years 45th Larval Fish Conference (Aug 29 - Sep 2). The stunning setting of the Scripps campus amidst the sound of the Pacific Ocean breeze provided the right kind of backdrop to again mingle with colleagues, meet fellow graduate students for scientific and just fun discussions, while an eclectic number of talks across the spectrum of Larval Fish and Larval Biology renewed the inspiration for our science. Hannes gave a keynote about our recently published work on sand lance CO2 sensitivity, while Max presented his first conference talk about juvenile Black Sea Bass growth and energy allocation.

A big, heartfelt thanks to Noelle Bowlin (NOAA) and her team for pulling off this remarkable conference during these still uncertain, post-COVID times!

LFC45-break
On 29 August 2022, LFC45 participants mingle in front of the Pacific Ocean at the Scripps Auditorium (people from left to right: Lee Fuiman, Teresa Schwemmer, Max Zavell, Chris Chambers, Tom Hurst, Darren Johnson, Jeremy Miller).

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On 30 August, Hannes talks about potential mechanisms of sand lance CO2 sensitivity

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At the end of a day full of science, two conference participants enjoy the sunset over the Pacific Ocean

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Participants of the 45th Larval Fish / Larval Biology Conference at the Scripps Campus on August 31st 2022

  • Baumann H. 2022. Why are sand lance embryos so sensitive to future high CO2 oceans? Keynote at the 45th Larval Fish Conference, San Diego 29 Aug - 1 Sep 2022
  • Zavell, M., Mouland, M., Schultz, E., and Baumann H. 2022. Overwinter growth and energy allocation of Black Sea Bass juveniles from Long Island Sound. 45th Larval Fish Conference, San Diego 29 Aug - 1 Sep 2022

NCMA2022 experiment update: Phenotyping 1,147 fish

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Groton, 24 July 2022. A remarkable week ends with a sense of pride, accomplishment and a good dose of exhaustion. After more than two months of rearing, the silversides from our crosses in May have grown big, and at least for the warm, 26C treatment, they reached their final, intended size (~40 mm). Our rearing job came to a successful end, and now post-doctoral researcher Jessica Rick and PhD-student Maria Akopyan came back to the Rankin Lab equipped with hundreds of tubes and lots of RNAlater to finalize the sampling. This involved measuring the critical sustained swim speed of every individual fish, followed by meticulous measurements, photographs, and eventually an individual tube for preservation in RNAlater. Over the course of 9 days (and over 10h of work each day), the team measured 1,147 fish, which ranged in size between 15 and 50 mm. A big hat tip to the hard-working geneticists!

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On 16 July 2022, Maria (left) and Jessi (right) are working meticulously through their individual sampling protocol of silversides reared for two months at 26C. Every rearing container contains about 40 fish.

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On 21 July 2022, most of the rearing containers have been sampled and only 5 remain.

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Maria is tackling individual length and weight measurments, photographing, labeling and preserving of silversides.

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The old swim flume that has seen its fair share of experimental work, is being put to good use once again.

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To calibrate the dial of the swim flume to flow speeds, we use dye and video recording.

Now, our rearing job still continues until the crosses in the 20C treatment reach their final size, when the second session of phenotyping will need to happen. And of course, there iare so many more steps to follow. Stay tuned.

Update02_End_of_26C_04
On 16 July 2022, Maria looks concentrated while taking a picture of a reared silverside.

World Ocean’s Day at 3rd grade Groton Elementary School

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7 June 2022. This is World Ocean Week and many Marine Science students and faculty do their bit to increase outreach to our community. Hannes had the privilege of dropping by the 3rd graders of the Catherine Kolnaski Magnet School, talking about what Marine Scientists do, which ocean critters eat another and "What was the weirdest fish you ever caught?" Oh, and "Are you really sure that the Megalodon [Charchardon megalodon] is no longer alive?" Thank you to Mr. Moon, Mrs. Laudone for the opportunity to come visit the school!

WorldOceanDay01-CKMS7JUN22

WorldOceanDay02-CKMS7JUN22

NCMA2022 experiment update: first genetic material sampled

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18 June 2022. We are happy to report that our genomic silverside experiment has progressed from "Can we really pull this off?" to "We think we just might" over the past weeks. The silverside larvae of these different crosses show stunning size variability, between populations and temperatures. We already obtained two early life mortality estimates and lots of genetic material, including a full set of crosses reared at 26C and ad libitum food, reaching 20 mm in roughly 4 weeks post hatch. Fingers crossed for the rest of the rearing time.

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9 dph larvae from the North Carolina Batch fertilization, waiting to be counted on 9 June 2022

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Visually stunning comparison of 9 dph silverside larvae reared at 20C from North Carolina batches vs. Massachusetts batches

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Digital length measurements of a small subsample of silverside larvae from the 26C treatment (crosses unknown)

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Silverside juveniles subsampled on 16JUN 2022 from bucket 3 of tank D (D#), housing the Massachusetts Batch (B4 of 7) in the 26C treatment, counted via ImageJ, preserved in RNAlater

John and Hannes travel to Bermuda to install a new CO2 system

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BIOS-Explorer
The Bermuda Institute of Ocean Sciences (BIOS) with its flagship the R/V 'Atlantic Explorer' in May 2022

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John Hamilton (right) and Hannes Baumann (left), the UConn team for the BIOS CO2 project

29 May 2022. When in a few months researchers and students at the Bermuda Institute of Ocean Science (BIOS) begin using their new outdoor mesocosm facility, they can now manipulate and control the CO2 levels in as many of 9 flow-through basins. The important new capacity of the system will allow realistic ocean warming and acidification experiments and has been the product of a wonderful collaboration between BIOS researcher Dr. Yvonne Sawall and our UConn Marine Sciences team consisting of John Hamilton and Hannes Baumann.

The newly developed system shares some of the design ideas with ALFiRiS, the factorial rearing system we developed and used over the past years at UConn's Rankin Seawater Lab. For example, we again developed and installed a central pH measurement hub that sequentially collects water samples via pumps from each of 12 independent basins, which is advantageous, because it only relies on a single, high-end pH sensor, therefore making measurements always comparable. Similarly, we are using LabView software (National Instruments) to switch pumps on and off and log, display, and graph the pH conditions in real time for researchers to have confidence in their chosen environmental parameters.

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A view over Mullet Bay from Slip Point Lane in St.George/Bermuda

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A downward view of BIOS' outdoor mesocosm facility, still in the middle of the major refurbishment

While most of the planning and design work was done remotely via frequent online meetings, Hannes and John worked with Yvonne during the past week at the BIOS station on installing and testing the systems major components. Working mostly out in the open under a warm and clear Bermudan sky was a particular treat of this assignment. Big shout-out, too, to facilities manager Kevin Hollis for his tireless onsite help!

Despite setbacks in form of supply chain delays and an unfortunate last moment COVID infection preventing team member Lucas Jones from traveling to Bermuda, soon the new outdoor mesocosm facility at BIOS will become operational and allow new and advanced kinds of experimental research on global change biology.

Staying at the Mary and James Buttler suite at BIOS was a particular treat

ElectronicsBox
The electronic box designed & assembled by John controls the sampling pumps

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On May 24th, John is explaining the workings of the software to Yvonne and Roderick

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On May 26, Yvonne measures pH in a mock-up of the CO2 header tanks for the mesocosm facility

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On May 28, our work is done and we enjoy the evening on the dock of Yvonne's place in St.George

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'Winky' is the queen of BIOS

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The R/V Atlantic Explorer is the flagship of BIOS and the main operation platform for the BATS time series

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John takes a picture of a Royal Poinciana (Delonix regia, Fabacea, Caesalpinioideae), a particularly stunning tree at the BIOS and all over Bermuda

Another crazy road trip for genetic silverside research

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15 May 2022. A full, blood red moon rises over Pine Island this Sunday evening. The sight makes not just humans swoon – its pull extents underwater to all kinds of critters that take it as cue for reproduction. Critters just like the Atlantic Silverside, which once again we pursue this season to extract more of its genomic 'secrets'.

Specifically, it is this weekend that we embark on yet another ambitious road trip to find and sample spawning-ripe silversides from two very far apart places: Morehead City, North Carolina and Beverly, Massachusetts. The goal: transport spawners live from each population to UConn's Rankin Seawater lab and produce calculated crosses that will allow studying the role of genomic inversions in local adaptation.

The crew this time are Maria Akopyan and Jessica Rick from Cornell University, along with Lucas Jones and Hannes Baumann from UConn. Big shout-out to Tara Duffy for her help with beach seining at Beverly, MA. During the spawning event on May 15th, Nina Therkildsen also joined the efforts. The design and experiment are part of Jessi's successful NSF post-doctoral fellowship proposal, which the whole UConn-Cornell silverside team supports.

Click through the pictures below to retrace the steps of an exhausting but so far successful effort. Fingers crossed that all goes well during the next weeks, when the fish need to hatch, survive and grow, so they can be assessed for their traits.

Map-trip
The US east coast map illustrates our ambitious sampling plan.

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On 12th May, fog envelopes the Chesapeake Bridge on our drive south to Morehead City, NC.

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Maria, Hannes, Lucas, and Jessi getting ready to beach seine the Morehead City site

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Our 100ft beach seine is being laid out on the Morehead City site.

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On May 13th, Atlantic silversides caught in Morehead City swim in a bucket.

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Lucas checking whether the fish are properly prepared for transport.

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Ripe adult silversides are being transported in large coolers, with proper aeration and water changes underway.

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Maria driving through the night. The long trip back up north is especially taxing.

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On May 14th, Jessi and Tara pull our seine net up the beach on Obear Park, Beverly, MA.

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Maria bringing a new sampling bucket to Jessi and Tara (background) seining.

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Seining at low tide in Obear Park is made more difficult by ankle deep mud.

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On May 15th, at UConn's Rankin Seawater lab, Nina and Jessi strategize about designing crosses.

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On May 15th, Hannes, Jessi, and Nina spawn individual silversides.

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Jessi squeezing a silverside female for eggs in UConn's Rankin Lab.

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A 24 hours old silverside embryo developing at 26C.

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On May 15th, Jessi lays out individual crosses to be reared in the circle tanks in UConn's Rankin Lab.

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Nina and Maria extract DNA from male and female spawners to determine a specific regions homo- vs. heterozygosity.

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Screens with attached embryos are being suspended in buckets for development under two different temperatures.

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A specific capture probe (TARMS gel) allows the quick determination whether adult spawners were homo- or heterozygous for specific inversions on chromosomes 11, 18, or 24

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A silverside larva 6 days post hatch produced from NC spawners. The stomach is full of brine shrimp nauplii, pigmentation just started.