Media Contact: dkenney@mbl.edu; 508-685-3525

Longfin inshore squid (Doryteuthis pealeii) hatchlings. On the left is a control hatchling; note the black and reddish brown chromatophores evenly placed across its mantle, head and tentacles.
In contrast, the embryo on the right was injected with CRISPR-Cas9 targeting a pigmentation gene (Tryptophan 2,3 Dioxygenase) before the first cell division ; it has very few pigmented chromatophores and light pink to red eyes. Credit: Karen Crawford

The team used CRISPR-Cas9 genome editing to knock out a pigmentation gene in squid embryos, which eliminated pigmentation in the eye and in skin cells (chromatophores) with high efficiency.

“This is a critical first step toward the ability to knock out -- and knock in -- genes in cephalopods to address a host of biological questions,” Rosenthal says.

Rosenthal is a senior scientist in the MBL’s Eugene Bell Center for Regenerative Biology and Tissue Engineering. Crawford is a professor of biology at St. Mary’s College of Maryland and a Whitman Center investigator at the MBL.

Cephalopods (squid, octopus and cuttlefish) have the largest brain of all invertebrates, a distributed nervous system capable of instantaneous camouflage and sophisticated behaviors, a unique body plan, and the ability to extensively recode their own genetic information within messenger RNA, along with other distinctive features. These open many avenues for study and have applications in a wide range of fields, from evolution and development, to medicine, robotics, materials science, and artificial intelligence.

The ability to knock out a gene to test its function is an important step in developing cephalopods as genetically tractable organisms for biological research, augmenting the handful of species that currently dominate genetic studies, such as fruit flies, zebrafish, and mice.

It is also a necessary step toward having the capacity to knock in genes that facilitate research, such as genes that encode fluorescent proteins that can be imaged to track neural activity or other dynamic processes.

“CRISPR-Cas9 worked really well in Doryteuthis; it was surprisingly efficient,” Rosenthal says. Much more challenging was delivering the CRISPR-Cas system into the one-celled squid embryo, which is surrounded by an exceedingly tough outer layer, and then raising the embryo through hatching. The team developed micro-scissors to clip the egg’s surface and a beveled quartz needle to deliver the CRISPR-Cas9 reagents through the clip.

Studies with Doryteuthis pealeii have led to foundational advances in neurobiology, beginning with description of the action potential (nerve impulse) in the 1950s, a discovery for which Alan Hodgkin and Andrew Huxley became Nobel Prize laureates in 1963. For decades D. pealeii has drawn neurobiologists from all over the world to the MBL, which collects the squid from local waters.

Recently, Rosenthal and colleagues discovered extensive recoding of mRNA in the nervous system of Doryteuthis and other cephalopods. This research is under development for potential biomedical applications, such as pain management therapy.

D. pealeii is not, however, an ideal species to develop as a genetic research organism. It’s big and takes up a lot of tank space plus, more importantly, no one has been able to culture it through multiple generations in the lab.

For these reasons, the MBL Cephalopod Program’s next goal is to transfer the new knockout technology to a smaller cephalopod species, Euprymna berryi (the hummingbird bobtail squid), which is relatively easy to culture to make genetic strains.

The MBL Cephalopod Program is part of the MBL’s New Research Organisms Initiative, which is widening the palette of genetically tractable organisms available for research – and thus expanding the universe of biological questions that can be asked.

Doryteuthis pealeii, often called the Woods Hole squid. Studies with D. pealeii have led to major advances in neurobiology, including description of the fundamental mechanisms of neurotransmission. The Marine Biological Laboratory collects D. pealeii from local waters for an international community of researchers. Credit: Roger Hanlon

Ring of mosaic squid hatchlings (Doryteuthis pealeii). These embryos were injected with CRISPR-Cas9 at different times before the first cell division, resulting in mosaic embryos with different degrees of knockout. Credit: Karen Crawford

Citation:

Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal (2020) Highly Efficient Knockout of a Squid Pigmentation Gene. Current Biology, DOI: 10.1016/j.cub.2020.06.099

Homepage photo: Squid embryo at hatching with pigmentation gene knocked out. Credit: Karen Crawford

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

For a copy of the paper, contact: Diana Kenney, MBL
dkenney@mbl.edu; 508-685-3525

Acutodesmus deserticola, a desert-derived green algae, grows in liquid culture at MBL. This alga’s cells are tiny but extremely resilient, capable of surviving multiple cycles of desiccation and rehydration in a single day. Credit: E.L. Peredo.

WOODS HOLE, Mass. -- Deserts of the U.S. Southwest are extreme habitats for most plants, but, remarkably, microscopic green algae live there that are extraordinarily tolerant of dehydration. These tiny green algae (many just a few microns in size) live embedded in microbiotic soil crusts, which are characteristic of arid areas and are formed by communities of bacteria, lichens, microalgae, fungi, and even small mosses. After completely drying out, the algae can become active and start photosynthesizing again within seconds of receiving a drop of water.

How are they so resilient? That question is at the core of research by Elena Lopez Peredo and Zoe Cardon of the Marine Biological Laboratory (MBL), published this week in Proceedings of the National Academy of Sciences. Given the intensified droughts and altered precipitation patterns predicted as the global climate warms, understanding the adaptations that facilitate green plant survival in arid environments is pressing.

Working with two particularly resilient species of green microalgae (Acutodesmus deserticola and Flechtneria rotunda), Peredo and Cardon studied up- and down-regulation of gene expression during desiccation, and added a twist. They also analyzed gene expression in a close aquatic relative (Enallax costatus) as it dried out and ultimately died. Surprisingly, all three algae – desiccation tolerant or not – upregulated the expression of groups of genes known to protect even seed plants during drought. But the desiccation-tolerant algae also ramped down expression of genes coding for many other basic cellular processes, seemingly putting the brakes on their metabolism. The aquatic relative did not.

Peredo’s and Cardon’s research suggests this new perspective on desiccation tolerance warrants investigation in green plants more broadly. Upregulation of gene expression coding for protective proteins may be necessary but not sufficient; downregulation of diverse metabolic genes may also be key to survival.

Citation: Elena L. Peredo and Zoe G. Cardon (2020) Shared upregulation and contrasting downregulation of gene expression distinguish desiccation tolerant from intolerant green algae. PNAS, doi: 10.1073/pnas.1906904117

Homepage photo: Desiccation-tolerant green microalgae are frequently found in the microbiotic crusts covering soil in arid areas, such as the deserts of the Southwestern U.S.A. (Credit: Z.G. Cardon).

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

Media Contact: dkenney@mbl.edu; 508-685-3525

Mother rotifer (Brachionus manjavacas) carrying four eggs (right) and newly-hatched daughter (left). Image of live rotifers acquired using polychromatic polarization and phase contrast. Credit: Michael Shribak, Evgeny Ivashkin, Kristin Gribble

WOODS HOLE, Mass. – The offspring of older mothers don’t fare as well as those of younger mothers, in humans and many other species. They aren’t as healthy, or they don’t live as long, or they have fewer offspring themselves. A longstanding puzzle is why evolution would maintain this maternal effect in so many species, since these late-born offspring are less fit to survive and reproduce.

In a new study in rotifers (microscopic invertebrates), scientists tested the evolutionary fitness of older-mother offspring in several real and simulated environments, including the relative luxury of laboratory culture, under threat of predation in the wild, or with reduced food supply. They confirmed that this effect of older maternal age, called maternal effect senescence, does reduce evolutionary fitness of the offspring in all environments, primarily through reduced fertility during their peak reproductive period. They also suggest an evolutionary mechanism for why this may occur.

The study, a collaboration between Kristin Gribble of the Marine Biological Laboratory, Christina Hernández and Michael Neubert of Woods Hole Oceanographic Institution, and Silke van Daalen and Hal Caswell of the University of Amsterdam, is published this week in Proceedings of the National Academy of Sciences.

“This study is unique in that combines laboratory data from our prior work with mathematical modeling to address a longstanding question in the evolution of aging,” Gribble says. “Natural selection should weed out these less-fit offspring of older mothers. So why do we see this phenomenon across so many species?”

To address this, Hernández and collaborators built mathematical models to calculate, for the first time, the strength of natural selection pressure on the survival and fertility of offspring populations as functions of the age of their mothers. They found this pressure, called the selection gradient, declines with maternal age.

“Because the selection pressure decreases as the mothers age, it may not be strong enough to remove these less-fit [offspring] from the population,” Hernandez says.

“Because of this, maternal effect senescence will persist and continue to evolve in the population, even though it results in decreased fitness,” Gribble adds. They don't yet fully understand the genetic mechanisms that cause offspring quality to decrease with maternal age.

The models that the team developed can be applied to a wide range of species to evaluate the fitness consequences of maternal effect senescence. “As long as you have experimental data, as we did, on lifespan and fecundity of offspring from mothers of different ages, you can address this question in many organisms,” Gribble says.

Citation: Hernández, Christina M. et al (2020) A demographic and evolutionary analysis of maternal effect senescence. PNAS: 10.1073/pnas.1919988117

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

Media Contact: dkenney@mbl.edu; 508-685-3525

WOODS HOLE, Mass. – A picture is worth a thousand words –but only when it’s clear what it depicts. And therein lies the rub in making images or videos of microscopic life. While modern microscopes can generate huge amounts of image data from living tissues or cells within a few seconds, extracting meaningful biological information from that data can take hours or even weeks of laborious analysis.

To loosen this major bottleneck, a team led by MBL Fellow Hari Shroff has devised deep-learning and other computational approaches that dramatically reduce image-analysis time by orders of magnitude -- in some cases, matching the speed of data acquisition itself. They report their results this week in Nature Biotechnology.

“It’s like drinking from a firehose without being able to digest what you’re drinking,” says Shroff of the common problem of having too much imaging data and not enough post-processing power. The team’s improvements, which stem from an ongoing collaboration at the Marine Biological Laboratory (MBL), speed up image analysis in three major ways.

First, imaging data off the microscope is typically corrupted by blurring. To lessen the blur, an iterative “deconvolution” process is used. The computer goes back and forth between the blurred image and an estimate of the actual object, until it reaches convergence on a best estimate of the real thing.

Lateral and axial images of 32-hour zebrafish embryo, marking cell boundaries within and outside the lateral line primordium. Credit: H. Vishwasrao and D. Dalle Nogare, National Institutes of Health

By tinkering with the classic algorithm for deconvolution, Shroff and co-authors accelerated deconvolution by more than 10-fold. Their improved algorithm is widely applicable “to almost any fluorescence microscope,” Shroff says. “It’s a strict win, we think. We’ve released the code and other groups are already using it.”

Next, they addressed the problem of 3D registration: aligning and fusing multiple images of an object taken from different angles. “It turns out that it takes much longer to register large datasets, like for light-sheet microscopy, than it does to deconvolve them,” Shroff says. They found several ways to accelerate 3D registration, including moving it to the computer’s graphics processing unit (GPU). This gave them a 10- to more than 100-fold improvement in processing speed over using the computer’s central processing unit (CPU).

“Our improvements in registration and deconvolution mean that for datasets that fit onto a graphics card, image analysis can in principle keep up with the speed of acquisition,” Shroff says. “For bigger datasets, we found a way to efficiently carve them up into chunks, pass each chunk to the GPU, do the registration and deconvolution, and then stitch those pieces back together. That’s very important if you want to image large pieces of tissue, for example, from a marine animal, or if you are clearing an organ to make it transparent to put on the microscope. Some forms of large microscopy are really enabled and sped up by these two advances.”

4 x 2 x 0.5 mm3 volume of brain from fixed and cleared mouse. Progressively higher resolution subvolumes highlight the detailed structures with isotropic submicron resolution. Credit: Y. Su, Y. Wu, H. Vishwasrao and T. Usdin, National Institutes of Health

Lastly, the team used deep learning to accelerate “complex deconvolution” – intractable datasets in which the blur varies significantly in different parts of the image. They trained the computer to recognize the relationship between badly blurred data (the input) and a cleaned, deconvolved image (the output). Then they gave it blurred data it hadn’t seen before. “It worked really well; the trained neural network could produce deconvolved results really fast,” Shroff says. “That’s where we got thousands-fold improvements in deconvolution speed.”

While the deep learning algorithms worked surprisingly well, “it’s with the caveat that they are brittle,” Shroff says. “Meaning, once you’ve trained the neural network to recognize a type of image, say a cell with mitochondria, it will deconvolve those images very well. But if you give it an image that is a bit different, say the cell’s plasma membrane, it produces artifacts. It’s easy to fool the neural network.” An active area of research is creating neural networks that work in a more generalized way.

“Deep learning augments what is possible,” Shroff says. “It’s a good tool for analyzing datasets that would be difficult any other way.”

Lateral and axial images of C. elegans embryo expressing neuronal (green) and pan-nuclear (magenta) markers. Isotropic resolution enables lineage tracing and inspection of neurite outgrowth pre-twitching. Credit: L. Duncan and M. Moyle, Yale School of Medicine

This research stems from an ongoing collaboration in the MBL Whitman Center between MBL Fellows Hari Shroff, senior investigator at the National Institute of Biomedical Imaging and Bioengineering; Patrick La Rivière, professor at the University of Chicago; and Daniel Colón-Ramos, professor at Yale Medical School. First author Min Guo is postdoctoral fellow in Shroff’s lab and a former teaching assistant for Shroff in the MBL’s Optical Microscopy and Imaging in the Biological Sciences course.

Citation: Min Guo et al (2020) Rapid image deconvolution and multiview fusion for optical microscopy. Nature Biotechnology: DOI: 10.1038/s41587-020-0560-x

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

WOODS HOLE, Mass. – Corals are “part animal, part plant, and part rock - and difficult to figure out, despite being studied for centuries,” says Philippe Laissue of University of Essex, a Whitman Scientist at the Marine Biological Laboratory. Many corals are sensitive to bright light, so capturing their dynamics with traditional microscopes is a challenge.

To work around their photosensitivity, Laissue developed a custom light-sheet microscope (the L-SPI) that allows gentle, non-invasive observation of corals and their polyps in detail over eight continuous hours, at high resolution. He and his colleagues, including MBL Associate Scientist and coral biologist Loretta Roberson, published their findings this week in Scientific Reports.

Below is a video explainer of the research:

Credit: Philippe Laissue

 
Coral reefs, made up of millions of tiny units called polyps, are extremely important ecosystems, both for marine life and for humans. They harbor thousands of marine species, providing food and economic support for hundreds of millions of people. They also protect coasts from waves and floods, and hold great potential for pharmaceutical and biotechnological discovery.

But more than half of the world’s coral reefs are in severe decline. Climate change and other human influences are gravely threatening their survival. As ocean temperatures rise, coral bleaching is afflicting reefs worldwide. In coral bleaching, corals expel their symbiotic algae and become more susceptible to death.

“The L-SPI opens a window on the interactions and relationship between the coral host, the symbiotic algae living in their tissues, and the calcium carbonate skeleton they build in real time,” Roberson says. “We can now track the fate of the algae during [coral] bleaching as well as during initiation of the symbiosis.”

Roberson is also using Laissue’s imaging technology to measure damage to corals from “bioeroders” – biological agents like algae and sponges that break down a coral’s skeleton, a problem exacerbated by ocean acidification and increasing water temperatures.
 

Using the L-SPI to visualize the re-infection of an Astrangia polyp (cyan from reflected light) with algae (red from chlorophyll fluorescence), injected by pipette into the mouth of the polyp. Credit: Loretta Roberson

A coral polyp (Astrangia) in the L-SPI sample chamber with tentacles fully extended. Credit: Loretta Roberson

Darkfield imaging is used to reveal the non-autofluorescent coral tissue in the Northern Star coral (Astrangia poculata), a species with no intrinsic fluorescence. Non-fluorescent tissue (cyan) and algae (magenta) are combined in the image. Credit: Philippe Laissue, Scientific Reports, 2020

Laissue has been coming regularly to the MBL for research since obtaining a Whitman Center Fellowship in 2017. He is continuing to collaborate with Roberson and MBL Fellow Amy Gladfelter, and to develop the L-SPI microscope with MBL-CZI Imaging Scientist Abhishek Kumar and MBL Fellow Hari Shroff.

A video of a coral polyp emerging that Laissue imaged with the L-SPI microscope won first place in the 2019 Nikon Small World in Motion. See the video here.

Citation: Laissue, P.P., Roberson, L., Gu, Y. et al. (2020) Long-term imaging of the photosensitive, reef-building coral Acropora muricata using light-sheet illumination. Scientific Reports, DOI: 10.1038/s41598-020-67144-w

Homepage photo: The Northern star coral (Astrangia poculata) is New England's only native hard coral. This one appears white because it does not have any photosynthetic algae inside it. Credit: Philippe Laissue and Loretta Roberson

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.