But as our climate changes, the availability of water is also changing, leaving animals with limited or unreliable supplies of this critical resource.

However, a new Arizona State University study published in the Royal Society journal Proceedings of the Royal Society B: Biological Sciences shows for the first time that animals may be able to use their own muscles to get water when it’s not available. Researchers from ASU and the Centre d’Etudes Biologiques de Chizé in France teamed up on the project and the findings were published June 27.

“We know about the importance of fat reserves to fuel the energetic costs of reproduction. But what about water? Our study shows that during reproduction, muscle metabolism is linked to the water requirements of developing offspring. Fat is only about 10 percent water, whereas muscle is closer to 75 percent, so burning muscle will release extra water,” said George Brusch IV, lead investigator for the project and doctoral student at Arizona State University. “From an evolutionary perspective, the concept of capital breeding — or using stored resources to fuel reproduction — is currently restricted to energetic needs. We propose that this should extend to a broader, multi-resource strategy that also includes water allocation.”

The researchers looked into this concept by studying the effects of water deprivation on the reproductive efforts of female Children’s pythons, a medium-sized snake that reproduces during the dry-season in Australia, where natural water sources are extremely limited. They found that muscles play an important role in providing water to the body when none is available.

“Female Children’s pythons can change how they use internal resources based on limitations in the environment,” said Brusch. “Understanding exactly how animals cope with resource restrictions will help scientists predict whether the animals might be impacted by future climate change, where, in many regions, rain is expected to be less reliable,” said Brusch, a doctoral candidate in the School of Life Sciences biology PhD program.

During the study, the researchers paired pregnant Children’s pythons with similarly sized non-reproductive females. During a three-week period when the snakes were pregnant, only half of the pairs had access to water. Reproductive females, both in the lab and the wild, don’t eat during pregnancy and rely on internal reserves such as fat and muscle for their energy needs.

The scientists then measured the by-products of burning fat and muscle such as ketones and uric acid, as well as muscle size and impact on the snakes’ eggs and clutch sizes. The animals without water burned more muscle than fat to meet their water requirements. In addition, the animals without water laid a similar number of eggs per clutch, but their eggs weighed less and the shells were thinner.

Brittany Kaminsky, an ASU student researcher on the team, just graduated from the School of Life Sciences with a Bachelor of Science degree in neurobiology, physiology and behavior. Kaminsky, who will attend U.C. Davis School of Veterinary Medicine in the fall, coordinated much of the project.

“I think what was most interesting about our results is that dehydration can play complex roles in reproduction, such that it affects other body systems. I feel, at the organism level, a single ailment can manifest in a variety of ways, and I think this is valuable information that I will carry with me into my veterinary career,” said Kaminsky.

During the project, Kaminsky helped pair the females into the reproductive and non-reproductive groups by mass and snout-vent length. She also monitored the reproductive progress of the females by ultrasound and learned how to collect blood samples, process and store them.

“Few of my peers have had the opportunity to train a team of other undergraduates or learn some of the technical skills I developed throughout the course of the study, such as cardiocentesis and ultrasound,” said Kaminsky. “Dr. Dale DeNardo and George were both there when I needed guidance, but they also gave me a lot of space to learn on my own and grow as a scientist. I couldn’t have asked for better mentors.”

Most animals need steady access to water in order to survive, especially during reproduction, and the authors suggest that using muscle as a water store may be a widespread phenomenon. “Our enhanced knowledge regarding the relationship between hydration and reproductive investment will also enable us to better understand global responses to water limitations and change the way scientists approach reproductive investment in ecological contexts, which, in the past, frequently ignore water and focus solely on energetic resources,” said Brusch.

Propionate, a byproduct of metabolism by a group of bacteria called the Bacteroides, inhibits the growth of Salmonella in the intestinal tract of mice, according to the researchers. The finding may help to explain why some people are better able to fight infection by Salmonella and other intestinal pathogens and lead to the development of better treatment strategies.

A paper describing the work will be published July 26 in Cell Host and Microbe.

The researchers determined that propionate doesn’t trigger the immune response to thwart the pathogen. Instead, the molecule prolongs the time it takes the pathogen to start dividing by increasing its internal acidity.

Salmonella infections often cause diarrhea, fever and abdominal cramps. Most people recover within four to seven days. However, the illness may be severe enough to require hospitalization for some patients.

Salmonella causes about 1.2 million illnesses, 23,000 hospitalizations and 450 deaths nationwide each year, according to the Centers for Disease Control and Prevention. Most cases are caused by contaminated food.

Different responses to exposure

“Humans differ in their response to exposure to bacterial infections. Some people get infected and some don’t, some get sick and others stay healthy, and some spread the infection while others clear it,” said Denise Monack, PhD, professor of microbiology and immunology and the senior author of the paper. “It has been a real mystery to understand why we see these differences among people. Our finding may shed some light on this phenomenon.”

For years, scientists have been using different strains of mice to determine how various genes might influence susceptibility to infection by intestinal pathogens. But this is the first time that researchers have looked at how the variability of gut bacteria in these mice might contribute to their different responses to pathogens.

“The gut microbiota is an incredibly complex ecosystem. Trillions of bacteria, viruses and fungi form complex interactions with the host and each other in a densely packed, heterogeneous environment,” said Amanda Jacobson, the paper’s lead author and a graduate student in microbiology and immunology. “Because of this, it is very difficult to identify the unique molecules from specific bacteria in the gut that are responsible for specific characteristics like resistance to pathogens.”

From mice to men

The scientists started with an observation that has been recognized in the field for years: Two inbred strains of mice harbor different levels of Salmonella in their guts after being infected with the pathogen. “The biggest challenge was to determine why this was happening,” Jacobson said.

First, they determined that the differences in Salmonella growth could be attributed to the natural composition of bacteria in the intestines of each mouse strain. They did this by performing fecal transplants, which involved giving mice antibiotics to kill off their usual composition of gut bacteria and then replacing the microbial community with the feces of other mice, some of whom were resistant to Salmonella infection. Then, the researchers determined which microbes were responsible for increased resistance to Salmonella infection by using machine-learning tools to identify which groups of bacteria were different between the strains.

They identified a specific group of bacteria, the Bacteroides, which was more abundant in mice transplanted with the microbiota that was protective against Salmonella. Bacteroides produce short-chain fatty acids such as formate, acetate, butyrate and propionate during metabolism, and levels of propionate were threefold higher in mice that were protected against Salmonella growth. Then, the researchers sought to figure out whether propionate protected against Salmonella by boosting the immune system like other short-chain fatty acids do.

The scientists examined their Salmonella model for the potential impact of propionate on the immune system but found that the molecule had a more direct effect on the growth of Salmonella. Propionate acts on Salmonella by dramatically decreasing its intracellular pH and thus increasing the time it takes for the bacterium to start dividing and growing, the study found.

“Collectively, our results show that when concentrations of propionate, which is produced by Bacteroides, in the gut are high, Salmonella are unable to raise their internal pH to facilitate cellular functions required for growth,” Jacobson said. “Of course, we would want to know how translatable this is to humans.”

Reducing the impact of salmonella

“The next steps will include determining the basic biology of the small molecule propionate and how it works on a molecular level,” Jacobson said. In addition, the researchers will work to identify additional molecules made by intestinal microbes that affect the ability of bacterial pathogens like Salmonella to infect and “bloom” in the gut. They are also trying to determine how various diets affect the ability of these bacterial pathogens to infect and grow in the gut and then shed into the environment. “These findings will have a big impact on controlling disease transmission,” Monack said.

The findings could also influence treatment strategies. Treating Salmonella infections sometimes require the use of antibiotics, which may make Salmonella-induced illness or food poisoning worse since they also kill off the “good” bacteria that keep the intestine healthy, according to Monack. Using propionate to treat these infections could overcome this limitation. “Reducing the use of antibiotics is an added benefit because overuse of antibiotics leads to increased incidence of antibiotic-resistant microbes,” Monack said.

Other Stanford co-authors of the paper are postdoctoral scholar Manohary Rajendram, PhD; graduate students Lilian Lam, Fiona Tamburini, Will Van Treuren, Kali Pruss, Jared Honeycutt and Kyler Lugo; Trung Pham, MD, instructor of pediatrics and infectious diseases; life science researcher Russel Stabler; Donna Bouley, DVM, PhD, professor emeritus of comparative medicine; José Vilches-Moure, PhD, assistant professor of comparative medicine; senior research scientist Mark Smith, PhD; Justin Sonnenburg, PhD, associate professor of microbiology and immunology; Ami Bhatt, MD, PhD, assistant professor of medicine and of genetics; and KC Huang, PhD, associate professor of bioengineering and of microbiology and immunology.

Bhatt, Huang, Monack, Sonnenburg and Vilches-Moure are members of Stanford Bio-X. Bhatt, Huang, Monack and Sonnenburg are faculty fellows at Stanford ChEM-H. Bhatt, Bouley and Vilches-Moure are members of the Stanford Cancer Institute. Bhatt and Monack are members of the Stanford Child Health Research Institute. Vilches-Moure is a member of the Stanford Cardiovascular Institute. Bouley is an affiliate of the Stanford Woods Institute for the Environment. Sonnenburg and Huang are Chan Zuckerberg Biohub Investigators.

The study was funded by the National Institutes of Health (grants R01DK085025, T32GM007276, R01AI116059 and F32AI133917), the Paul Allen Stanford Discovery Center on Systems Modeling of Infection and the National Science Foundation.

Stanford’s Department of Microbiology and Immunology also supported the work.

The remaining marine wilderness is unequally distributed and found primarily in the Arctic, in the Antarctic, or around remote Pacific Island nations. In coastal regions, there is almost no marine wilderness left at all.

“We were astonished by just how little marine wilderness remains,” says Kendall Jones of the University of Queensland, Australia, and the Wildlife Conservation Society. “The ocean is immense, covering over 70 percent of our planet, but we’ve managed to significantly impact almost all of this vast ecosystem.”

On land, rapid declines in wilderness have been well documented. But much less was known about the status of marine wilderness. Wilderness areas are crucial for marine biodiversity.

“Pristine wilderness areas hold massive levels of biodiversity and endemic species and are some of the last places of Earth where big populations of apex predators are still found,” Jones says.

In the new study, Jones and his colleagues used the most comprehensive global data available for 19 human stressors, including commercial shipping, fertilizer and sediment runoff, and several types of fishing in the ocean and their cumulative impact. They systematically mapped marine wilderness globally by identifying areas with very little impact (lowest 10% percent) from 15 anthropogenic stressors and also a very low combined cumulative impact from these stressors.

In order to capture differences in human influence by ocean regions, the researchers repeated their analysis within each of 16 ocean realms. They found wide variation in the degree of human impacts. For instance, more than 16 million square kilometers of wilderness remains in the Warm Indo-Pacific, accounting for 8.6 percent of the ocean. But it’s even worse in Temperate Southern Africa, where less than 2,000 square kilometers of marine wilderness remains — less than 1 percent of the ocean.

The study also shows that less than 5 percent of global marine wilderness is currently protected. Most of this is in offshore ecosystems, with very little protected wilderness found in high-biodiversity areas such as coral reefs.

“This means the vast majority of marine wilderness could be lost at any time, as improvements in technology allow us to fish deeper and ship farther than ever before,” Jones says. “Thanks to a warming climate, even some places that were once safe due to year-round ice cover can now be fished.”

The findings highlight an urgent need for action to protect what remains of marine wilderness, the researchers say. Such an effort requires international environmental agreements to recognize the unique value of marine wilderness and sets targets for its retention.

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