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!
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.
On 21 July 2022, most of the rearing containers have been sampled and only 5 remain.
Maria is tackling individual length and weight measurments, photographing, labeling and preserving of silversides.
The old swim flume that has seen its fair share of experimental work, is being put to good use once again.
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.
On 16 July 2022, Maria looks concentrated while taking a picture of a reared silverside.
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.
9 dph larvae from the North Carolina Batch fertilization, waiting to be counted on 9 June 2022
Visually stunning comparison of 9 dph silverside larvae reared at 20C from North Carolina batches vs. Massachusetts batches
Digital length measurements of a small subsample of silverside larvae from the 26C treatment (crosses unknown)
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
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.
The US east coast map illustrates our ambitious sampling plan.
On 12th May, fog envelopes the Chesapeake Bridge on our drive south to Morehead City, NC.
Maria, Hannes, Lucas, and Jessi getting ready to beach seine the Morehead City site
Our 100ft beach seine is being laid out on the Morehead City site.
On May 13th, Atlantic silversides caught in Morehead City swim in a bucket.
Lucas checking whether the fish are properly prepared for transport.
Ripe adult silversides are being transported in large coolers, with proper aeration and water changes underway.
Maria driving through the night. The long trip back up north is especially taxing.
On May 14th, Jessi and Tara pull our seine net up the beach on Obear Park, Beverly, MA.
Maria bringing a new sampling bucket to Jessi and Tara (background) seining.
Seining at low tide in Obear Park is made more difficult by ankle deep mud.
On May 15th, at UConn's Rankin Seawater lab, Nina and Jessi strategize about designing crosses.
On May 15th, Hannes, Jessi, and Nina spawn individual silversides.
Jessi squeezing a silverside female for eggs in UConn's Rankin Lab.
A 24 hours old silverside embryo developing at 26C.
On May 15th, Jessi lays out individual crosses to be reared in the circle tanks in UConn's Rankin Lab.
Nina and Maria extract DNA from male and female spawners to determine a specific regions homo- vs. heterozygosity.
Screens with attached embryos are being suspended in buckets for development under two different temperatures.
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
A silverside larva 6 days post hatch produced from NC spawners. The stomach is full of brine shrimp nauplii, pigmentation just started.
2 November 2021. We are happy report that the ICES Journal of Marine Science just published the last major experimental paper on Atlantic silverside CO2-sensitivity from our lab. Callie Concannon and co-authors report on two complementary, long-term rearing trials in 2015/16 and 2018/19, where silverside juveniles or newly fertilized embryos were reared under contrasting temperature and CO2 conditions to maturity. This revealed negative effects of high CO2 conditions on female fecundity, but only at the warm, not the cold temperature treatments (Fig. below). Our study and its data are novel, because they were generated by the first whole-life CO2 rearing experiment of a fish and are the first empirical fecundity effects shown for a broadcast-spawning fish species.
The paper is also special to us, because its publication marks the erstwhile conclusion of our yearlong, NSF-funded efforts (OCE#1536165) to understand the CO2 sensitivity and its mechanisms in this important forage fish and long-standing model in fish ecology and evolution. The project ran from 2015 - 2020, produced 15 publications, 2 book chapters, and over 40 presentations, while furthering the careers of a post-doc, a PhD student, 5 Master students and over 10 undergraduates.
19 November 2020. We are happy to announce that the Journal of Experimental Biology just published the latest paper on CO2 effects in the early life stages of Atlantic silversides! For her PhD research at Stony Brook University, Teresa meticulously measured oxygen consumption in developing silverside embryos and newly hatched larvae exposed to contrasting oxygen and CO2 conditions throughout multiple experiments in 2017 and 2018. Her work shows that the metabolism of embryos but not larvae is sensitive to elevated CO2 conditions, leading to higher metabolic rates at normoxic levels, but reduced metabolic rates under low oxygen conditions, compared to controls. These basic empirical data confirm the emerging picture that CO2 effects in marine fish manifest largely if at all during early ontogeny, i.e., during the embryo stages. Well done, Teresa, and congratulations to your first lead-author paper!
Conceptual diagram of the relationship between PO2 and RMR of M. menidia embryos in ambient and elevated PCO2. Hypothesized shifts in the relationship between embryonic RMR and PO2 are shown for elevated (orange) versus ambient (blue) PCO2. Our results (measured at the PO2 levels marked by black dots) suggest that PCO2 can influence both the critical oxygen partial pressure (Pcrit, gray lines) and the oxygen-independent RMR. At higher PO2 levels, RMR increases with PCO2, potentially owing to increased metabolic demand. As PO2 decreases, embryonic RMR reaches Pcrit and becomes oxygen dependent at a higher PO2 level in acidified than in ambient PCO2 conditions. Low intracellular red blood cell pH caused by high PCO2 can be expected to reduce hemoglobin–O2 affinity (Bohr effect) and make embryonic RMR less hypoxia resistant, which could manifest as an increase in Pcrit for embryos in elevated PCO2.
27 July 2020. Big and proud congratulations to Chris Murray, who published his last big chunk of data from his PhD research on the effects of marine climate change on coastal marine fish. The publication in PLOS One synthesized 3 years of multiple, long-term experiments on Atlantic silversides (Menidia menidia) demonstrating consistent negative growth effects on high CO2 conditions. However, sometimes it takes more than just looking at means and standard deviations to elucidate these effects. Hence, in this paper, shift functions analyzing the different percentiles of distributions are employed.
As humans continue to send large quantities of carbon into the atmosphere, much of that carbon is absorbed by the ocean, and UConn researchers have found high CO2 concentrations in water can make fish grow smaller.
Researchers Christopher Murray PhD ’19, now at the University of Washington, and UConn Associate Professor of Marine Sciences Hannes Baumann have published their findings in the Public Library of Science (PLoS One).
“The ocean takes up quite a bit of CO2. Estimates are that it takes up about one-third to one-half of all CO2 emissions to date,” says Murray. “It does a fantastic job of buffering the atmosphere but the consequence is ocean acidification.”
Life relies on chemical reactions and even a slight change in pH can impede the normal physiological functions of some marine organisms; therefore, the ocean’s buffering effect may be good for land-dwellers, but not so good for ocean inhabitants.
Baumann explains that in the study of ocean acidification (or OA), researchers have tended to assume fish are too mobile and tolerant of heightened CO2 levels to be adversely impacted.
“Fish are really active, robust animals with fantastic acid/base regulatory capacity,” says Murray. “So when OA was emerging as a major ocean stressor, the assumption was that fish are going to be OK, [since] they are not like bivalves or sea urchins or some of the other animals showing early sensitivities.”
The research needed for drawing such conclusions requires long-term studies that measure potential differences between test conditions. With fish, this is no easy task, says Baumann, largely due to logistical difficulties in rearing fish in laboratory settings.
“For instance, many previous experiments may not have seen the adverse effects on fish growth, because they incidentally have given fish larvae too much food. This is often done to keep these fragile little larvae alive, but the problem is that fish may eat their way out of trouble — they overcompensate – so you come away from your experiment thinking that fish growth is no different under future ocean conditions,” says Baumann.
In other words, if fish are consuming more calories because their bodies are working harder to cope with stressors like high CO2 levels, a large food ration would mask any growth deficits.
Additionally, previous studies that concluded fish are not impacted by high CO2 levels involved long-lived species of commercial interest. Baumann and Murray overcame this hurdle by using a small, shorter-lived fish called the Atlantic silverside so they could study the fish across its life cycle. They conducted several independent experiments over the course of three years. The fish were reared under controlled conditions from the moment the eggs were fertilized until they were about 4 months old to see if there were cumulative effects of living in higher CO2 conditions.
Murray explains, “We tested two CO2 levels, present-day levels and the maximum level of CO2 we would see in the ocean in 300 years under a worst-case emissions scenario. The caveat to that is that silversides spawn and develop as larvae and early juveniles in coastal systems that are prone to biochemical swings in CO2 and therefore the fish are well-adapted to these swings.”
The maximum CO2 level applied in the experiments is one aspect that makes this research novel, says Murray,
“That is another important difference between our study and other studies that focus on long-term effects; almost all studies to date have used a lower CO2 level that corresponds with predictions for the global ocean at the end of this century, while we applied this maximum level. So it is not surprising that other studies that used longer-lived animals during relatively short durations have not really found any effects. We used levels that are relevant for the environment where our experimental species actually occurs.”
Baumann and Murray hypothesized that there would be small, yet cumulative, effects to measure. They also expected fish living in sub-ideal temperatures would experience more stress related to the high CO2 concentrations and that female fish would experience the greatest growth deficits.
The researchers also used the opportunity to study if there were sex-determination impacts on the population in the varying CO2 conditions. Sex-determination in Atlantic silversides depends on temperature, but the influence of seawater pH is unknown. In some freshwater fish, low pH conditions produce more males in the population. However, they did not find any evidence of the high CO2 levels impacting sex differentiation in the population. And the growth males and females appeared to be equally affected by high CO2.
“What we found is a pretty consistent response in that if you rear these fish under ideal conditions and feed them pretty controlled amounts of food, not over-feeding them, high CO2 conditions do reduce their growth in measurable amounts,” says Murray.
They found a growth deficit of between five and ten percent, which Murray says amounts to only a few millimeters overall, but the results are consistent. The fish living at less ideal temperatures and more CO2 experienced greater reductions in growth.
Murray concludes that by addressing potential shortcomings of previous studies, the data are clear: “Previous studies have probably underestimated the effects on fish growth. What our paper is demonstrating is that indeed if you expose these fish to high CO2 for a significant part of their life cycle, there is a measurable reduction in their growth. This is the most important finding of the paper.”
This work was funded by the National Science Foundation grant number OCE #1536165. You can follow the researchers on Twitter @baumannlab1 and @CMurray187.
12 December 2019. We are happy to announce that Marine Ecology Progress Series just published our latest paper on Atlantic silversides, but this time not an experimental but a field study! During her time in our lab, Julie Pringle investigated the otolith microstructure of young-of-year silversides, finding intriguing patterns about differential growth in males and females that likely result in sex-selective survival during their growing season. Congratulations, Julie, well done!
This graph shows reconstructed hatch distributions of male and female Atlantic silversides sampled in fall 2015. Counting daily otolith increments, young-of-year fish caught in October could be reliably aged, whereas those from November and December where likely underaged because water temperatures had already decreased below their growth threshold. This graph compbines previous knowledge, environmental monitoring and results of otolith microstructure analysis.
From the abstract:
“We examined the utility of otolith microstructure analysis in young-of-year (YoY) Atlantic silversides Menidia menidia, an important annual forage fish species along the North American Atlantic coast. We first compared the known hatch window of a local population (Long Island Sound, USA) to otolith-derived hatch distributions, finding that YoY collected in October were reliably aged whereas survivors from November and December were progressively under- aged, likely due to the onset of winter ring formation. In all collections, males outnumbered fe- males, and both sexes had bimodal size distributions. However, while small and large females were almost evenly represented (~60 and ~40%, respectively), over 94% of all males belonged to the small size group. We then examined increment widths as proxies for somatic growth, which suggested that bimodal size distributions resulted from 2 distinct slow- and fast-growing YoY phe- notypes. Length back-calculations of October YoY confirmed this, because fast- and slow-growing phenotypes arose within common bi-weekly hatch intervals. We concluded that the partial sexual size dimorphism in this population resulted largely from sex-specific growth differences and not primarily from earlier female than male hatch dates, as predicted by the well-studied phenome- non of temperature-dependent sex determination (TSD) in this species. Furthermore, observed sex ratios were considerably less male-biased than reconstructed thermal histories and published laboratory TSD values predicted. Assuming that selective mortality is generally biased against slower growing individuals, this process would predominantly remove male silversides from the population and explain the more balanced sex ratios at the end of the growing season.”
9 December 2019. During the COP25 summit in Madrid, the International Union for Conservation of Nature (IUCN) released its latest comprehensive report titled “Ocean deoxygenation: everyone’s problem” that compiles the current evidence for the ongoing, man-made decline in the oceans oxygen levels. The 588 page, 11 chapter wake-up call to these detrimental changes was produced by leading experts in the field. We are happy to announce that Hannes is one of the many authors of this document, co-authoring chapter 6 “Multiple stressors – forces that combine to worsen deoxygenation and its effects“.
From the executive summary:
“The equilibrium state of the ocean-atmosphere system has been perturbed these last few decades with the ocean becoming a source of oxygen for the atmosphere even though its oxygen inventory is only ~0.6% of that of the atmosphere. Different analyses conclude that the global ocean oxygen content has decreased by 1-2% since the middle of the 20th century. Global warming is expected to have contributed to this decrease, directly because the solubility of oxygen in warmer waters decreases, and indirectly through changes in the physical and biogeochemical dynamics.”
From the summary of chapter 6:
Human activities have altered not only the oxygen content of the coastal and open ocean, but also a variety of other physical, chemical and biological conditions that can have negative effects on physiological and ecological processes. As a result, marine systems are under intense and increasing pressure from multiple stressors.
The combined effects of ‘stressors’ can be greater than, less than, or different from the sum of each stressor alone, and there are large uncertainties surrounding their combined effects.
Warming, acidification, disease, and fisheries mortality are important common stressors that can have negative effects in combination with low oxygen.
Warming, deoxygenation, and acidification commonly co-occur because they share common causes. Increasing carbon dioxide (CO2) emissions simultaneously warm, deoxygenate, and acidify marine systems, and nutrient pollution increases the severity of deoxygenation and acidification.
A better understanding of the effects of multiple stressors on ocean ecosystems should improve the development of effective strategies to reduce the problem of deoxygenation and aid in identifying adaptive strategies to protect species and processes threatened by oxygen decline.
Callie presenting her poster to other graduate students
November 8, 2019. Callie Concannon joined other graduate students of the Department of Marine Sciences to present her thesis research at the Graduate Climate Conference in Woods Hole, MA. She presented a poster entitled “Long-term CO2 and temperature effects on fecundity and oocyte recruitment in the Atlantic silverside”
Her preliminary findings can be summarized as:
Warmer, more acidic environments impact reproductive output in the Atlantic silverside
The participants of the Graduate Climate Change conference in November 2019
1 August 2019. We are overjoyed that our paper on genetic changes in experimental silverside populations subjected to strong size-selective fishing has just been published by Science!
Over recent decades, many commercially harvested fish have grown slower and matured earlier, which can translate into lower yields. Scientists have long suspected that rapid evolutionary change in fish caused by intense harvest pressure is the culprit.
Now, for the first time, researchers have unraveled genome-wide changes that prompted by fisheries – changes that previously had been invisible, according to a study published in Science by a team of researchers including Hannes Baumann, UConn assistant professor of Marine Sciences, who collaborated with researchers at Cornell University, the University of Oregon, the National Marine Fisheries Service, and Stanford University.
In unprecedented detail, the study shows sweeping genetic changes and how quickly those changes occur in fish populations extensively harvested by humans, says Baumann.
“Most people think of evolution as a very slow process that unfolds over millennial time scales, but evolution can, in fact, happen very quickly,” said lead author Nina Overgaard Therkildsen, Cornell assistant professor of conservation genomics in the Department of Natural Resources.
Observed shifts in adult size. Trends across generations in mean length at harvest (standardized as the difference from the mean of the control populations in each generation) ± the standard deviations in up-selected (blue shades), down-selected (yellow and orange shades), and control populations (green shades).
The all-pervasive human meddling in our planet’s affairs undeniably reached the genetic make-up of its organisms.
— Hannes Baumann, UConn.
In heavily exploited fish stocks, fishing almost always targets the largest individuals. “Slower-growing fish will be smaller and escape the nets better, thereby having a higher chance of passing their genes on to the next generations. This way, fishing can cause rapid evolutionary change in growth rates and other traits,” said Therkildsen. “We see many indications of this effect in wild fish stocks, but no one has known what the underlying genetic changes were.”
Therkildsen and her colleagues took advantage of an influential experiment published back in 2002. Six populations of Atlantic silversides, a fish that grows no bigger than 6 inches in length, had been subjected to intense harvesting in the lab. In two populations, the largest individuals were removed; in another two populations, the smallest individuals were removed; and in the final two populations, the fishing was random with respect to size.
After only four generations, these different harvest regimes had led to evolution of an almost two-fold difference in adult size between the groups. Therkildsen and her team sequenced the full genome of almost 900 of these fish to examine the DNA-level changes responsible for these striking shifts.
The team identified hundreds of different genes across the genome that changed consistently between populations selected for fast and slow growth. They also observed large linked-blocks of genes that changed in concert, dramatically shifting the frequencies of hundreds of genes all at the same time.
Surprisingly, these large shifts only happened in some of the populations, according to the new paper. This means that there were multiple genomic solutions for the fish in this experiment to get either larger or smaller.
“Some of these changes are easier to reverse than others, so to predict the impacts of fisheries-induced evolution, it is not enough to track growth rates alone, we need to monitor changes at the genomic level,” said Therkildsen.
When the experiment was originally conducted nearly two decades ago by co-authors David Conover, professor of biology at the University of Oregon, and Stephan Munch of the National Marine Fisheries Service, the tools to study the genomic basis of the rapid fisheries-induced evolution they observed were not available. Fortunately, Conover and Munch had the foresight to store the samples in a freezer, making it possible to now return – armed with modern DNA sequencing tools – and reveal the underlying genomic shifts.
Research like this can assess human impacts, and improve humanity’s understanding of “the speed, consequences and reversibility of complex adaptations as we continue to sculpt the evolutionary trajectories of the species around us,” Therkildsen said.
“What’s most fascinating about this is that life can find different genetic ways to achieve the same result. In this study, two experimental populations evolved smaller body size in response to the selective removal of the largest fish, which is what most trawl fisheries do. However, only by looking at the genetic level we demonstrated that these two experimental populations evolved via two completely different genetic paths,” says Baumann.
The good news for the Atlantic silversides is that the fisheries selection was able to tap into the large reservoir of genetic variation that exists across the natural range of this species from Florida into Canada, said Therkildsen: “That genetic bank fueled rapid adaptation in the face of strong fishing pressure. Similar responses may occur in response to climate-induced shifts in other species with large genetic variability.”
“Scientists have coined the term Anthropocene in recognition of the all-pervasive human alteration of the earth’s climate, oceans, and land. No matter how ‘pristine’ a piece of nature may look to us at first glance, examine it thoroughly enough and you will find a trace of human in it. Take a cup of water from the middle of Pacific Ocean and a handful of sand from a ‘pristine’ beach – and you will find little plastic particles under the microscope,” says Baumann. “The parallel to this study is that the all-pervasive human meddling in our planet’s affairs now undeniably reached the genetic make-up of its organisms. Today’s fishes may superficially look the same as always, but their genes are not. They bear witness to human alteration.”
In addition to Baumann, Therkildsen, Conover, and Munch, co-authors included former Cornell postdoctoral researcher Aryn P. Wilder, now a researcher at San Diego Zoo Institute for Conservation Research; and Stephen R. Palumbi, Stanford University.
This work was funded by the National Science Foundation.
