Misadventures in Research

By Yonara Garcia
English edit: Lídia Paes Leme and Katyanne Shoemaker

Illustration by: Caia Colla 

   As I was finishing writing my thesis, I started to consider all of the challenges that I went through in the two years of my Masters education in order to deliver such a perfectly rounded piece of work. My feelings were mixed because I was obviously happy to finish this step in my life, but I realized that this document didn’t contain even a third of all the misadventures that got me to this point. In my opinion, the thesis was missing a chapter; there should have been a chapter on “the making of” the research, just to explain how much went wrong and what it really takes to deliver good work. 

   When starting a Masters course, you must submit a proposal, which includes your research objectives, the hypothesis, and how you plan to answer that hypothesis. Wow! It was so easy so far! You just follow a previously described method and you understand what your results will look like and how they should be treated. This is all based on previously done work on a similar subject, which of course you know all about after reviewing the relevant literature. Two years to finish this project? No problem! …or at least that is what I thought. For me, it was not this easy, so I am going to tell you a little bit about my many misadventures during my masters.

   My work was a behavioral study of marine planktonic organisms in a 3D system. To build the system, I got together with a crew of post-graduate students who would also be using the experimental tank. This is where the soap opera began. We believed that we could build our system based on previous studies, but we quickly noticed that several components were not correct. There were issues with the magnification, color of LED lights, and the shape and positioning on the table. Everything had to be disassembled and reassembled to incorporate the necessary changes. The entire system had to be rebuilt 4 times because with each assembly we noticed new flaws. After months of arranging and rearranging pieces, and with the help of specialists in the area of optics, we finally reached a working system. 

   Ok, that took a few more months than expected, but now I could finally perform my tests, generate results, and graduate, right? Wrong. My work required filming the trajectories my target organism takes in the water column. However, the software for the two cameras we had would only film for 20 seconds at a time, which was not a long enough time span to get a valid representation of swimming behavior. We increased the computer’s memory, but that was not the issue. Thankfully, a student in our lab was proficient with computer science, and he became a key contributor to this project’s success. The filming software was completely replaced with software he developed. This new program didn’t have a time limit, however it could not utilize two cameras at once, so two computers had to be used. Having the two computers meant we could be introducing human error in timing; no matter how hard I tried, I cannot click the mouse at the exact same time on two computers. The solution for this problem was to use two microcontrollers that were activated by a potentiometer. Finally, we had a working system with a program that could be modified according to our needs!

   Onto the experiments! One of the primary challenges of working with living organisms is that you depend on them to be present in a certain collection spot at a given time of year. Unfortunately for me, by the time the experimental system was set up, we could not find enough individuals to run the experiments. It took several months of daily sampling to have enough individuals to perform all of my experiments, but I finally finished. 

   After video collection, the next step of the project was to use a computer program to find coordinates and relevant numerical data on the trajectories of the organisms. Given the topic of this post, it may not be a surprise to say something went wrong in this part too. As it turned out, the program that was originally going to be used could not compare the long videos that we fought so hard to attain. Once more, we turned to our computer science hero, and he developed software that could give us the organisms’ trajectories independent of video size. Let me take a moment to point out that software development is not an easy task; it took several iterations to get it to the point we needed it.

   With data in hand, I could finally analyze them and get my results. Data analysis is never easy, but given what I had already gone through, the challenges seemed minor in comparison. I had no idea how far off that initial Master’s plan would end up being, or that I would face so many challenges. I also didn’t expect how much this project would shape me. I had to be more than a biologist for this work; I learned how to solder, make electrical connections, understand physics, be a computer technician, and learn a little about programming. 

   Beyond all of the research obstacles, you still have to live your own life. This may be the most complicated part of the whole project. I often felt defeated and like I couldn’t carry on as I was faced with problem after problem. I know many others have lived through much bigger issues with their graduate research, but no matter the size of the problem, it shakes you to the core, and it can often be debilitating. 

   But, if you can push through these setbacks and fears of failure, you will eventually reach the end with a huge sense of accomplishment, as I did. It is important for me to share these misadventures in research with you to show what it actually takes to do research—it involves many tries and more wrongs than rights, but in the end, you publish a beautiful piece of well-crafted work. Even with all of the pressures and obstacles I faced, I still love what I do. Through all of the challenges, I grow more certain that I made the right choice.

   What challenges have you faced in your research? Comment below to share a little of your story with us!

Honey, I’m pregnant!

By Raquel Moreira Saraiva and Yonara Garcia

Image (right): Flickr

Today we are going to talk about the Super Dad of the animal kingdom, the seahorse! This peculiar organism is considered a Super Dad for a good reason: the males become pregnant! That's it! Seahorses stand out in the animal kingdom because the males are responsible for all parental care after fertilization: they carry the pups during gestation, experience the "birth pangs," and finally give birth! Recent research also shows that seahorse daddies have even more similarities to human mommies than we thought! But before we talk about those peculiarities, let's get to know a little bit about seahorses in general.

Two "pregnant" seahorses.

Image: Flickr

Seahorses are bony fishes (teleosts) belonging to the genus Hippocampus and the syngnathidae family (Syngnathidae). This family has the unique developmental characteristic of viviparity, where embryonic development occurs within the body (the same as humans), which in this case, is the paternal body. There are more than 50 species of seahorses distributed throughout the world in tropical and temperate regions. Of these, three species occur on the Brazilian coast: Hippocampus reidi, Hippocampus erectus, and Hippocampus patagonicus, present in the marine and estuarine environment.

Representatives of the three species of seahorses that occur in Brazil: Hippocampus reidi, Hippocampus erectus, and Hippocampus patagonicus, respectively.

Images:  Projeto Hippocampus

These fish move vertically through wave movements of their dorsal fins, which vibrate rapidly. This type of vertical locomotion slows them down to the point of being considered one of the slowest fish in the oceans. Seahorses are predators, with a diet based on plankton, crustaceans, and small animals that are sucked through their tubular snout. They are also skillful at camouflaging themselves: if they feel threatened, they can change color and develop skin projections that mimic algae or coral polyps. Additionally, they can become rigid and immobile, fixing themselves on algae and corals through their prehensile tail. But these disguises are not infallible: crabs, some carnivorous fish (e.g. tuna), penguins, sea birds, and even humans predate upon adult seahorses (to learn more about plankton, read our post O que você sabe sobre o plâncton?).

Seahorses mimic the environment when they feel threatened.

Images: Flickr

Most seahorses are monogamous, so that both the male and the female of a formed pair repel other partners who try to interfere with the relationship. For mating, they perform a type of dance in which they synchronize their movements, turning around one another with interlaced tails. Male pregnancy has interesting implications for the classic sex roles in mating. In most species, males compete for access to females, so it is common to see the evolution of secondary sex characteristics * in males. According to researcher Adam Jones of the University of Texas, in the case of seahorses, females exhibit a competitive behavior that is typically characteristic of males. In addition, males appear "demanding" in relation to the choice of their partners, an attribute commonly observed in females.

Illustration: Joana Ho

Now let's get down to business: how can males in this group get pregnant? The male seahorse has a specialized brood pouch where the female places her oocytes (reproductive cells). When it is ready to mate, the male signals the female by filling the pouch with water. The female, in turn, swims and presses against it, placing her ovipositor into a dilated hole in the male's pouch. After the oocytes are transferred, the hole closes, and the male fertilizes them. Thus begins the development of the babies (called fry) inside the body of the male.

The gestation period of this group varies greatly, according to the species and the water temperature, and can occur between ten days to six weeks. In tropical regions, seahorses have a gestation period of around 12 days. They reproduce throughout the year, and from the first year of life, a couple is able to produce more than 1000 larvae per gestation.

Seahorse giving birth.

Source: https://www.youtube.com/watch?v=sgoinj5XGR0

The challenges of pregnancy are the same for all animals, including ensuring the adequate supply of oxygen and nutrients to the embryos. Recent studies have shown that several animal taxa have overcome these challenges in a similar way. Seahorse embryos, like many other viviparous animals, acquire many nutrients from the vitellus of the mother’s egg, which is equivalent to the egg yolks of chickens. Researcher Dr. Camilla Whittington and colleagues at the School of Biological Sciences, University of Sydney, Australia, have shown in studies published in Molecular Biology and Evolution that additional nutrients, such as calcium and some lipids, are secreted by the fathers from the brood pouch and absorbed by the embryos. In addition, the dad’s pouch also maintains the complex challenges of gas exchange, excreta removal, and providing immunological protection to the young!

Pregnancy is accompanied by many morphophysiological adaptations, such as the remodeling of the brood pouch, transport of nutrients and residues, gas exchange, osmoregulation, and immunological protection of embryos. Another curiosity discovered by researchers is that the genetics related to these adaptations are very similar to the genetic expression of the internal reproduction of mammals, reptiles, and other fish. It is surprising that, even in animals with very distant evolutionary histories, the genetic tools for reproduction have developed remarkably similar to each other, even between viviparous aplacental (seahorses) and placental (mammalian) animals (Caspermeyer, 2015; Whittington et al., 2015).

Seahorse populations are declining worldwide. In addition to their limited locomotion capacity, the destruction of their habitat and incidental and targeted fisheries have threatened the lives of these fish. There is high demand for live specimens among ornamental fish enthusiests. Dehydrated, they are used as ingredients of homemade and industrialized drugs and as decoration, which leaves them even more vulnerable. The purchase of these fish, even alive, encourages their capture and trade, in addition to contributing to the ecological imbalance. Genetic, physiological, and ecological studies of these animals help not only to understand their biology and the evolutionary steps that led to the inversion in sexual behavior, but also contribute knowledge to the management of these species. The best option is to leave seahorses in their natural habitat, reduce exploitation, and take care of the environments in which they live, including coral reefs and mangroves. This way you can get to know these fish better while helping in their preservation.

Dehydrated seahorses being sold at a market in Hong Kong. In Asia they are much appreciated in cooking and as raw material for the manufacture of medicines.

Image: Britt-Arnhild’s House in the Woods

*secondary characters: characteristics that develop during the sexual maturity of animals, but which, unlike the sexual organs, are not part of the reproductive system.

To learn more about the subject:

Projeto Hippocampus - Iniciativa do Laboratório de Aquicultura Marinha - LABAQUAC para educação ambiental e estudos de conservação de cavalos-marinhos. www.projetohippocampus.org

Caspermeyer, J. Unraveling the Genetic Basis of Seahorse Male Pregnancy Mol Biol Evol (2015) 32 (12): 3278 first published online November 17, 2015 doi:10.1093/molbev/msv238

Jones, AG & Avise, JC. Mating Systems and Sexual Selection in Male-Pregnant Pipefishes and Seahorses: Insights from Microsatellite-Based Studies of Maternity J Hered, 2001.

Rosa IL, Oliveira TPR, Osório FM, Moraes LE, Castro ALC, Barros GML & Alves RRN. Fisheries and trade of seahorses in Brazil: historical perspective, current trends, and future directions. Biodivers Conserv, 2011.

Silveira, R. B. Dinâmica populacional do cavalo-marinho hippocampus reidi no manguezal de Maracaípe, Ipojuca, Pernambuco, Brasil. (2005).

Whittington CM, Griffith OW, Qi W, Thompson MB & Wilson AB. Seahorse brood pouch transcriptome reveals common genes associated with vertebrate pregnancy.Molecular Biology and Evolution, 2015.

Ocean fertilization and climate change

By Yonara Garcia

Have you heard of geoengineering? It’s a tool becoming increasingly used, but is often controversial because, in some cases, the result can be completely unexpected!

Today we’ll talk about a polemic experiment carried out in July 2012 by Russ George, an American businessman who dumped approximately 100 tons of iron sulphate in the Pacific Ocean as part of a geoengineering project off the west coast of Canada (http://www.nature.com/news/ocean-fertilization-project-off-canada-sparks-furore-1.11631).

Ocean fertilization by iron sulfate. Source: http://officerofthewatch.com/2012/11/05/canada-iron-fertilization-incident/

Iron is considered an essential element, often limiting, for phytoplankton growth. Phytoplankton perform photosynthesis, a process in which sunlight is used as an energy source and absorbs carbon dioxide (CO2) and water to produce organic matter in the form of carbohydrates. Phytoplankton cells are formed from these carbohydrates with the addition of other substances such as proteins, amino acids, and other molecules.

In 1980, oceanographer John Martin proposed that certain regions of the ocean (the areas called HNLC - High Nutrient, Low Chlorophyll), although rich in nutrients, would be poor in primary production due to lack of iron. Thus, the addition of iron should increase the production of phytoplankton and hence affect the carbon cycle, reducing CO2 levels in the atmosphere. His famous phrase “Give me half a tanker full of iron and I’ll give you an Ice Age” caused great excitement because he believed that if certain areas of the ocean were fertilized, the effects of global warming could be reversed, cooling the Earth.

Thus arose the idea that the American businessman put into practice. Russ and his team released a certain amount of iron into the sea, believing it would promote photosynthetic activity and thus increase the efficiency of the carbon sequestration processes in the ocean. Just like the process to fertilize a crop for it to go grow faster! This issue has generated much controversy because it conflicts with ethical and political questions about the effects that an intervention like this would bring to a complex ecosystem. We still know relatively little about the ocean. To better understand why the idea of this project is so controversial, let’s first talk about some important processes in the “wonderful world ocean.”

Have you ever heard of “physical pump”? Or a “biological pump”? No, it’s not a kind of weapon of war to decimate an enemy population. The physical pump is the process related to the solubility of CO2 in the ocean (solubility = maximum amount of a substance that can be dissolved in a liquid). The biological pump takes into account what happens to the CO2 after it is dissolved in the ocean, when a fraction of dissolved carbon is absorbed through photosynthesis, in the surface layers of the ocean, and transported to the bottom. The diagram below explains how carbon is transported in the ocean.

Carbon movement in the ocean system. 1) Using solar energy, carbon dioxide is fixed by phytoplankton in the photic zone (where there is light). 2) Part of this organic matter is consumed by zooplankton and some heterotrophic microorganisms. 3) Other organic matter is exported from the photic zone toward the mesopelagic zone (about 1000 m deep), and a fraction of this organic matter is remineralized while the rest goes to the bottom of the ocean, where it will take thousands of years to return to the surface. Adapted from United States Joint Global Ocean Flux Study.

CO2 is a gas capable of dissolving in the surface of the oceans. This solubility mechanism is related to the concentration of this gas in the atmosphere and the water temperature: the more CO2 in the atmosphere and the lower the temperature, the greater the amount of gas dissolved in the ocean surface. Once dissolved in water, the CO2 passes to a further stage of the cycle, where it can be absorbed by photosynthetic marine organisms.

Part of the organic matter formed during photosynthesis is used in cellular respiration and released back into the seawater as CO2. The other fraction, which was used in the formation of the cell, is consumed by zooplankton (primary consumers in marine food webs - read more here) and/or transported by gravity to the bottom of the ocean through  “marine snow,” particles made up of food debris and fecal pellets coming from feeding zooplankton, shells, and dead microorganisms. This carbon transfer process to the deep ocean decreases the amount of carbon in the photic zone (zone that receives enough sunlight for photosynthesis to occur), sequestering (removing) billions of tons of carbon from the atmosphere each year. Some studies have estimated that the biological pump is responsible for removing about 5-15 gigatons of carbon per year (Henson et al., 2011).

Marine Phytoplankton. Source: http://www.smithsonianmag.com/science-nature/vanishing-marine-algae-can-be-monitored-from-a-boat-with-your-smartphone-2785190/?no-ist

You can probably imagine how important this removal is when looking at the large amount of carbon that our industrial activities, cars, and planes have emitted into the atmosphere over the last few years. It is important to remember that the much discussed global warming, among other issues, is largely caused by an excess of carbon in the atmosphere. According to the IPCC (Intergovernmental Panel on Climate Change) 2014,  in 2010 alone, 49 gigatons of carbon were released into the atmosphere by human activities. And that is precisely why these experiments with iron have gained so much popularity.

Sounds simple, right? Okay, solved the problem of global warming! Let's fertilize the oceans! But it is not so simple. Interfering in natural ecosystems is an extremely sensitive subject, which can cause incalculable and irreparable damage.

Some researchers performed similar experiments as the American businessman and concluded that despite the fertilization increasing the rate of photosynthesis, it can trigger changes in ocean chemistry by changing the operation of the entire system. For example, increased photosynthetic rates by phytoplankton are directly proportional to the amount of dimethylsulfide (DMS - volatile sulfur in reduced form) secreted by these microalgae in water, which is vaporized and form condensation particles in the air (i.e. more photosynthesis by the phytoplankton = more dimethylsufide into the air). In the atmosphere, these particles facilitate the formation of clouds, which would be great, because with the increased formation of clouds there is increased reflection of solar radiation and thus greater cooling of the planet. However, not all types of clouds have the property to cool the planet. Recent studies suggest that other climatic factors may also affect the distribution and properties of clouds, which could increase the temperature of the planet. Furthermore, it was observed that fertilization also increases the production of nitrous oxide (N2O), a molecule that heats 320 times more than CO2.

Another study, published in April 2014 in Geophysical Research Letters, showed that more than 66% of the carbon sequestered by the ocean returns to the atmosphere in 100 years. That is, the biological pump may lessen the temperature of the Earth, sequestering carbon from the atmosphere, but we do not know what will happen when this carbon returns. Controversial enough for you?

Image obtained by NASA, satellite view of a phytoplankton bloom.

Thus, although the processes that occur in the ocean are responsible for reducing the concentration of CO2 in the atmosphere, altering the system may not be the best solution because there are many chemical, physical, and biological processes that are not fully understood. While we did not reach a more integrated understanding of these processes, the reduction of CO2 emissions would be much more efficient and safer than trying to remedy a problem by manipulating a process so complex and poorly understood.

Literature:

http://www.nature.com/ngeo/journal/v6/n9/full/ngeo1921.html

http://www.nature.com/nature/journal/v446/n7139/full/nature05700.html

https://www.ipcc.ch/publications_and_data/ar4/wg1/en/ch7s7-3.html

http://onlinelibrary.wiley.com/doi/10.1002/2013GL058799/full

https://www.ipcc.ch/pdf/assessment-report/ar5/syr/AR5_SYR_FINAL_SPM.pdf

Henson, S. A., R. Sanders, E. Madsen, P. J. Morris, F. Le Moigne, and G. D. Quartly (2011), A reduced estimate of the strength of the ocean's biological carbon pump, Geophysical Research Letters, 38