Physiology and Cell Biology Abstracts

Phototaxis in the brittle star Amphiodia occidentalis

Kelsey Gil, 499

Light sensing abilities are well documented within the Echinoderms. The class Ophiuroidea is replete with examples of visual acuity, some associated with specialized light focusing structures. Phototactic preference and specialized light focusing structures are previously undocumented in the family Amphiuridae. Here I test the ability of Amphiodia occidentalis to sense a dark target in a circular arena, as a measure of visual acuity. A frequency distribution diagram showed no selection for, or away from the dark target. A subsequent K-S test showed random selection of location in the arena. These results indicate that A. occidentalis may not have a preference for the dark target, or perhaps that light sensing abilities are absent in this species.


Toxicity of Silver Nanomaterials

Dana Snell, 499

Nanoparticles (NPs) have at least one dimension less than 100nm, resulting in large surface area to volume ratios that give nanoparticles unique physicochemical characteristics relative to their bulk material counterparts. Silver (Ag) NPs have been used in medical devices and water treatment because of their bactericidal properties. However, exposure to Ag NPs has also caused toxicity in fish and invertebrates causing concern over their widespread use and development. Toxicity may be due to released silver ions, which are known inhibitors of carbonic anhydrase and Na+,K+-ATPase, or to the nanoparticles themselves. Furthermore, exposure to Ag NPs has caused developmental abnormalities in zebrafish and interfered with sodium regulation in trout. Distinguishing between ion effects and NP effects is important for the development of safe NPs and establishing environmental regulations. For this project, we are studying the toxic effects of Ag nanomaterials on zebrafish embryos. The nanoparticles were synthesized on the surface of chabazite or Engelhard titanium silicate, both zeolite minerals, and will be used in water treatment. The NPs have been characterized with transmission electron microscopy and dynamic light scattering. Dialysis experiments have been performed to determine whether toxic effects are due to the ion or the nanomaterial. We will measure the effects of exposures at various concentrations on a variety of morphological defects and the expression of metallothionein, which is up-regulated in response to heavy metals, to establish median lethal concentrations.


Acute exposure effects of aqueous metal nanoparticle exposures on RBL-2H3 degranulation

Van Ortega, Ph.D.

The emergence of nanomaterials has promised a revolution of the medical treatment, consumer product and technological fields, but has also raised concerns over the potential for their unique properties to adversely affect biological systems. Numerous cytotoxic studies have reported effects on cellular stress and apoptotic pathways, however there still remains a paucity of information on the potential for NPs to affect immune cell viability and function. In this study, we examined the potential for NPs to impact the viability of a rat basophilic leukemia (RBL) immune cell-line, and to modulate their cellular functions. RBLs are representative granulocytes that release intracellular granules to combat foreign antigens in a process known as degranulation. Since NPs can differ in their physico-chemical properties, their behavioural characteristics in solutions are typically assessed together with toxicity measurements. Various techniques were used to characterize three aqueous
metal-oxide polyacrylic acid encapsulated NPs (TiO2, CeO2, nanocapsule). Immunotoxicity was measured by exposing RBLs to NP concentrations up to 200mg/L over 48h and were analyzed for cellular viability and proliferation using flow cytometry and propidium iodide as a fluorescent marker for cell death. Results showed dose-dependent decreases in viability with increasing NP concentrations and exposure times. To examine the influence of NPs on RBL degranulation we dosed RBLs at both sub-lethal and lethal concentrations and showed that degranulation was inhibited at all doses including those that did not decrease cell viability. Degranulation was also fully abrogated when the IgE antibody, used to initiate degranulation, was also exposed to NPs. Antibody binding affinity assays showed decreased Fcε receptor binding when exposed to NPs, which may provide a mechanism for decreased NP inhibitory effects on degranulation.


The role of ube4b in zebrafish muscle development

Katharine Pelletier, 499

Myopathies are human disorders that affect muscle tissue, resulting in non-functioning or degenerating muscles. In one category of myopathies, normal contractile units (sarcomeres) are never formed in the embryo. Such muscle formation requires precise assembly and organization of the contractile proteins myosin and actin within the sarcomere. Work on simpler model systems such as C. elegans has shown that integration of myosin protein into ‘thick filaments’ is dependent on proper protein folding, aided by the co-chaperone factor Unc45. In turn, unc45 protein levels are tightly controlled by a second factor, Ube4b. Invertebrate models have shortcomings that often make it difficult to translate to human applications. Danio rerio, more commonly known as the zebrafish, provides a vertebrate system in which it is easier to study this interaction and its role in muscle development. Our data supports the use of zebrafish as a model with which to study the Ube4b and Unc45 interaction. Preliminary data has indicated that a knockdown of Ube4b has a conserved role in the development of muscle tissue.


Examination of Innate Immunity receptor-mediated Control of Phagocytosis in Fish

Dustin Lillico, M.Sc.

Innate immune cell responses such as phagocytosis represent key effector mechanisms that protect a host against pathogens. Among vertebrates, these responses are controlled by immunoregulatory receptors, which are expressed on a wide variety of immune cell-types. One example are the Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs), which share structural features with innate immune receptors in mammals and are believed to play vital roles in the control and coordination of fish immune cell responses. Two IpLITR-types that are being characterized in the lab are termed IpLITR1.1b and IpLITR2.6b that are defined as inhibitory and stimulatory receptors. Specfically, IpLITR1.1b encodes an intracellular cytoplasmic tail containing ITIMs (immunoreceptor tyrosine containing inhibitory motifs), while IpLITR2.6b represents a stimulatory receptor containing an ITAM (immunorecpetor tyrosine containing activation motif). Predictably, IpLITR2.6b functions as a stimulatory receptor by engaging kinase signaling pathways and inducing phagocytosis. However, IpLITR1.1b that was previously shown to facilitate phosphatase-dependent inhibitory functions has now also recently been shown to promote phagocytosis. This surprising result reveals that certain immunoregulatory receptors may have dual functionality. My research is focused on exploring the mechanisms of IpLITR-mediated phagocytosis and examination of the ‘unconventional’ ability of a prototypical inhibitory receptors induction of phagocytosis. Recent data using flow cytometric phagocytosis assays and pharmacological blockers will be presented.


Morphology and Histology of the Pacific Hagfish

Alyssa Weinrauch, 499

Hagfish are an ideal model for acid/base and ion regulation studies of vertebrates because they are the only vertebrate osmoconformer. This means body fluids are isosmotic with the surrounding seawater, apart from a few divalent cations (Ca2+, Mg2+, SO42-), which are regulated at lower concentrations. Furthermore, hagfish permit quick retrieval of homeostasis when faced with changing plasma pH levels and along with lamprey, they are the only remaining agnathans, or jawless fishes. This leaves them at the base of the vertebrate phylogenic tree, allowing for a unique opportunity to observe the early evolutionary standing of fishes. Examination of these primitive fishes not only provides information about agnathans, but also provides insight to the evolution of modern teleost physiology. In-depth analysis of these primitive systems could provide mechanistic and evolutionary understanding of physiological processes, like acid/base regulation, through comparisons of the simplified agnathan system with more complex teleost systems. Currently, there is no comprehensive atlas of anatomy or histology of the hagfish to support specific research presently being conducted on hagfish physiological systems. Therefore, this study provides such an atlas of the Pacific Hagfish (Eptatretus stoutii) by analyzing both gross morphology and histology of each tissue type.


Nucleosides and nucleic acids as novel chemosensory stimuli for fish

Angela Shamchuk, M.Sc.

In fish and other animals, chemosensory stimuli are often biomolecules, such as bile salts, amino acids and nucleotides, which are components of excreted waste. While it is known these compounds evoke both physiological and behavioral responses, it is unknown if breakdown products of these compounds do the same. This study aimed to determine if the breakdown products of nucleotides, nucleosides (adenosine and guanosine) and nucleic acids (adenine and hypoxanthine), evoke an olfactory response in goldfish and a behavioral response in zebrafish. Olfactory responses to candidate stimuli were measured by recording electro-olfactograms (EOGs) from the olfactory epithelium of mature goldfish. Swimming behavior was recorded and analyzed for change post candidate stimuli addition in zebrafish 5 and 7 days post fertilization. To date results suggest that goldfish can detect the compounds via olfaction and do so using multiple receptors. A change in swimming behavior was also seen in larval zebrafish at both 5 and 7 days post fertilization following introduction of all candidate stimuli. Our findings suggest nucleosides and nucleic acids act as chemosensory stimuli for fish and further investigation of both physiological and behavioral responses to these compounds may help to elucidate their function in the natural environment.


The effects of aging and exercise on the whole-organism performance of zebrafish (Danio rerio)

Matthew Gilbert, M.Sc.

A rapidly aging global population has motivated the development and use of zebrafish as a model for human aging. Many sub-organismal parallels with human aging have been identified in zebrafish, however, little has been done to relate them to the functionality of the whole organism. Furthermore, the effectiveness of exercise as a means to mitigate the effects of aging has not been studied in zebrafish. We investigated the independent and interactive effects of aging and intermittent exercise on swimming performance, mechanics and behaviour. Young, middle-aged and old fish (10, 18 and 26 months) were exercised to exhaustion in sustained and sprint swimming tests once a week for four weeks in a swim-tunnel. Both sustained and sprint swimming performance decreased significantly with increased age. Swimming performance improved with training in young and middle-aged fish but not in old fish. Tail-beat amplitude however, increased for all age groups with training. Fish turning frequency, a measure of swimming behaviour, decreased with age but showed no change with exercise.. Together, our results show that as with humans and mammalian models, zebrafish exhibit a decline in whole-organism performance and physiological plasticity with age.



Phosphate uptake and regulation in the pacific hagfish Eptatretus stoutii

Erik Folkerts, 499

Hagfish are a unique and scientifically important organism to study due to their basal position on the evolutionary tree and subsequent different behavioural and physiological processes. One area of physiological study currently of scientific interest is cell molecule transport systems. Recently, studies have shown hagfish to be able to transport certain molecules, such as amino acids, across skin and gill epithelium. That has stemmed further research to discover a novel epithelial phosphate transporter, the only phosphate transporter known to exist outside the intestine in an animal, in hagfish. My project has been to isolate this phosphate transporter, and prepare future experiments to further study the transport kinetics and qualities of this transporter in a novel system.


Using zebrafish to study the interplay between Prion Protein (PrP) and Amyloid precursor protein (APP)

Patricia Leighton, Ph.D.

Zebrafish have two homologues of both proteins (APPa and APPb; Prp1 and Prp2). We are also developing zebrafish Alzheimer disease models that express human Aβ peptides, neurotoxic cleavage products of APP. We identified a genetic interaction between APPa and PrP1 using morpholino gene knockdown (Kaiser et al., 2012). We have also engineered and characterized PrP2 zinc finger nuclease (ZFN) mutant fish (Fleisch et al., Accepted). We aim to engineer PrP1 and APPa mutant zebrafish to examine their genetic interactions and influence on behavioural assays of synaptic development. Our Prp2 maternal zygotic mutant larvae exhibit lower baseline activity than wild type larvae. Both Prp2 maternal zygotic and zygotic larvae are more susceptible to pentylenetetrazole, a seizure-inducing drug, than wildtype larvae. We have created a Prp1 ZFN that is capable of inducing germline mutations and identified a potential mutant. We are also working to create transgenic zebrafish lines that express Aβ fragments. Knowledge of the normal biological functions of APP and PrP are crucial for rational drug design. Further, zebrafish Alzheimer disease models will facilitate high throughput screening of therapeutics and diagnostics.


Revising the Role of Titin in Muscle Formation

Jordan Hills, 499

The contractile apparatus of muscle is a complex protein array called the sarcomere. The mechanisms of sarcomere function are well-studied, but the process of sarcomerogenesis during muscle development is not well characterized. Textbook models of sarcomere formation involve use of the super-protein titin as a molecular scaffold. In our recent analysis of the mechanisms of muscle formation in zebrafish, we have been examining mutants in which normal muscle is not formed. Of particular interest is the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces defective titin protein, yet molecular evidence suggests that normal muscle patterning is still initiated in these mutants. This evidence is in contrast with previous models of early sarcomere patterning and supports a titin-independent model of sarcomerogenesis. Our findings are consistent with the symptoms of titin myopathies such as tibial muscular dystrophy, which exhibit a late onset.


Time course of the acute response of Pacific dogfish (Squalus suckleyi) to low salinity

Sam Guffey, M.Sc.

Dogfish can briefly enter estuaries, but cannot survive there long-term. The acute response of Pacific dogfish (Squalus suckleyi) to low salinity was tested by exposing sharks to 60% SW and repeatedly sampling blood and water over 48 hours. We quantified the temporal trends in blood pH, plasma osmolality, total CO2, Na+, Cl-, K+, and urea concentrations, and the rates of urea efflux and O2 consumption. The rate of O2 consumption increased around 6 h, peaking after 12 h 58% above control O2 consumption After 9 h, plasma [Cl-] stabilized 9% below initial levels, while plasma [Na+] decreased more than 20% and did not stabilize within 48 h. Plasma [urea] dropped by 15% between 4 and 6 h, and continued to decrease. The rate of urea efflux increased over time, peaking after 36 h 73% above the initial rate. This high-resolution study reveals that dogfish exposed to 60% SW for 48 h can maintain homeostasis with respect to some physiological variables, but not others.