BML scientists study many aspects of animal biology including how animals respond to changing environmental conditions. Natural and anthropogenic environmental stresses such as temperature, hypoxia, salinity, and dehydration can be quantified by alterations in circulating hormone levels, expression of stress proteins, embryo/larval development and growth. Physiology research at BML has a focus on comparative physiology and biochemistry and reproductive and developmental physiology.
Crustacean Physiology: Stress Hormone
Ernie Chang’s laboratory has worked on the characterization of the crustacean hyperglycemic hormone (CHH). As the name indicates, this neuropeptide hormone regulates blood glucose. This hormone is a member of a novel arthropod-specific neurohormonal family. Members of this peptide family control a number of important physiological processes. The Chang laboratory developed a sensitive ELISA to quantify the hormone and have examined expression of the CHH gene. With these molecular tools, they have examined alterations in the circulating levels of this hormone in lobsters during various environmental stresses, including season of capture, method of live storage, depth of capture, hauling rate, and infectious diseases. These various collaborative studies involved colleagues from the Univ. of Maine, Univ. of Montpellier (France), Univ. of Denmark, Univ. of Birmingham (U.K.), and Mahidol Univ. (Thailand).
Crustacean Physiology: Regulation of the Molting Gland
The crustacean molting gland is normally inhibited by a peptide hormone called the molt-inhibiting hormone (MIH). It is related to CHH. The Chang laboratory has conducted a number of studies aimed at elucidating the mode of action of MIH upon the molting gland. In collaboration with Dr. Don Mykles (Colorado State Univ.), they have hypothesized that cyclic nucleotides are involved in the signal transduction of MIH regulation of the molting gland. The Chang laboratory cloned a peptide with both MIH and CHH activity in the land crab and characterized its action on circulating glucose and production of the steroid molting hormone. This provided them with a necessary tool for our subsequent studies on the regulation of the molting gland.
The Mykles and Chang laboratories have also cloned nitric oxide synthase and three guanylyl cyclases and characterized their expression in various crab tissues. The expression of these regulatory enzymes, which are involved in the synthesis of cGMP, are altered with varying amounts of circulating ecdysteroids (molting hormones). This observation is consistent with the role of cGMP as a second messenger for MIH. This project is supported by the National Science Foundation.
Claw muscle in crabs undergoes atrophy in response to elevated levels of molting hormones (ecdysteroids) while thoracic muscle undergoes atrophy in response to unweighting (deliberate loss of limbs, leaving the thoracic muscles intact). The signaling pathways that regulate muscle atrophy in crustaceans are largely unknown. Myostatin (Mstn) is a negative regulator of muscle growth in mammals, and the Mykles and Chang laboratories characterized a Mstn-like cDNA that is expressed in muscle of the land crab.
They also cloned a cDNA encoding a Mstn-like gene from the lobster, Homarus americanus. Sequence analysis and three-dimensional modeling of the lobster Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Quantitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of the transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation on lobster Mstn expression in the claw and abdominal muscles. Their data indicate that the transcription of lobster Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced muscle atrophy.
These data are of interest for basic studies of crustacean physiology, but also for their potential biomedical applications. For example, their studies could provide insight into degenerative diseases such as muscular dystrophy, which involves abnormal degeneration of skeletal muscle. Also, studies on crustacean muscle growth are of interest to the aquaculture and fisheries industries. These studies were also conducted in collaboration with Dr. Don Mykles (Colorado State Univ.)
The Jim Clegg laboratory has worked on the biochemical and biophysical mechanisms that allow certain invertebrates to live in what we consider to be highly stressful environments. Some of these animals can be said to be “extremophiles” because they thrive under conditions that kill most animals. These conditions include extremes of temperature and pressure, desiccation, anoxia, various forms of radiation and very high salinity.
The favorite animal extremophile of the Clegg lab is the primitive crustacean known as Artemia, also called the brine shrimp. Although their interest in them concerns basic science, they are also of considerable commercial importance to the aquacultural and aquarium industries. The encysted embryos of Artemia, also called cysts, are arguably the most stress resistant of all animal life history stages and the motile (swimming) forms are among the most salt tolerant aquatic animals. Clegg and others working in this area have identified several proteins that act as “molecular chaperones” that protect macromolecules in the embryos from damage and destruction due to these various severe stresses. These molecular chaperones are clearly the products of evolution that enable these embryos to be extremophiles. Their goals include an understanding of the mechanisms by means of which this protection occurs.
Among Clegg lab collaborators are two Canadians, Tom MacRae (Dalhousie University) and Alden Warner (University of Windsor), Ralph Schill (University of Stuttgart, Germany), Wei-Jun Yang (Zhejiang University, Hangzhou, China) and Lynda Beladjal, Johan Mertens and Patrick Sorgeloos, all at Ghent University in Belgium. These highly respected scientists, and their students and colleagues are in the process of studying various aspects of animal extremophiles.
BML researchers (the Cherr laboratory) are working to addresses molecules and physiological mechanisms involved in fertilization and early development. This research has focused on fertilization biology using plants, invertebrates, lower vertebrates, and mammals. While significant portions of this research are directly related to how environmental parameters impact successful fertilization and development, much of this body of work focuses on developing a basic understanding of mechanisms associated with sperm-egg interactions and the novel or the conserved aspects of the cell biology.
An example of focus is the study of the mechanisms of sperm motility initiation in Pacific herring from the San Francisco Bay estuary and the egg-derived ligand that signals the intracellular ionic changes and subsequent motility initiation. These sperm have evolved a unique mode of motility initiation in that they surprisingly remain immotile in the environment for hours until they contact an egg, at which time an extracellular glycoprotein stimulates an ionic cascade resulting in flagellar motion. These ionic events are dependent on lowered salinity (in particular Na+) that is typical in the winter months in the SF estuary. Research in the Cherr laboratory showed that herring sperm motility initiation was due to activation of a reverse Na+/Ca++ exchanger (Proceedings of the National Academy of Sciences USA).
More recently with colleagues from Japan, the Cherr lab published the model of sperm-egg interaction in Pacific herring that resolves initial mechanistic differences observed between the BML group and those of colleagues from Japan. For much of this physiological research, the BML Fluorescence Imaging Facility has been used. This facility is open to students, postdoctoral scholars, and researchers at BML, as well as those from UC Davis departments. The facility was equipped in part from a multi-user NSF major equipment proposal (Cherr, PI) for our scanning laser confocal microscope (with seawater immersion objectives and temperature controlled stage), while other NSF support was used for high-speed fluorescence imaging systems including an upright fixed stage microscope with microinjection capabilities, a stereo fluorescence system designed for computer controlled high content screening, a fluorometer with live cell capabilities, etc.
The Cherr lab has been working on defensins in reproductive biology. Defensins (including ß-defensins) are part of the innate immune system that is understood to be the key defense against disease in invertebrates; homologous peptides exist in invertebrates as “antimicrobial peptides”. In the Cherr lab collaboration over the past 9 years with Professor Charles Bevins (an internationally recognized expert on defensins) at the UC Davis Medical School as well as others, they have discovered a polymorphism in the DEFB126 gene, a dinucleotide deletion in the coding region of the DEFB126 mRNA that results in a null mutation. A beta-defensin (DEFB126) comprises the sperm glycocalyx, and provides a “cloaking device” for sperm so they are not detected in the female tract. This glycocalyx must be “peeled” away from sperm as they capacitate just prior to interaction with the oocyte. Interestingly, once DEFB126 comes off of sperm, it can be added back to sperm and it reattaches and returns sperm to a functional state they were in prior to its release. In 2011, we published a groundbreaking study on the consequences and high frequency in men worldwide (~25%) of this non-stop deletion mutation in the human gene encoding DEFB126 in Science Translational Medicine, which was listed in the Top 100 Scientific Discoveries of 2011 of Discover magazine.
Marine Plant Physiology
Marine plant physiology informs ocean health and marine conservation: seaweed, cordgrass, and seagrass physiology are sensitive indicators of environmental stress. Ecosystem function depends on marine species diversity and genetic diversity. BML has one of very few labs equipped for marine macrophyte physiological studies. The Williams lab performs physiological research on marine macrophytes (seagrass, cordgrass, seaweeds) to investigate coastal marine plant response to stress. Research in field and lab validate that reflectance and fluorescence provide early warning signs of effects of herbicides in coastal marshes and plant photochemical responses. Plant photosynthesis and nitrogen acquisition are ecosystem functions critical to providing food web support. Physiological research at BML has advanced the understanding of the relationship between a) seaweed species diversity and ecosystem function and b) eelgrass genetic diversity and the ecosystem functions it provides.