Experimental assessment of cardiovascular physiology in the chick embryo.

High resolution assessment of cardiac functional parameters is crucial in translational animal research. The chick embryo is a historically well-used in vivo model for cardiovascular research due to its many practical advantages, and the conserved form and function of the chick and human cardiogenesis programs. This review aims to provide an overview of several different technical approaches for chick embryo cardiac assessment. Doppler echocardiography, optical coherence tomography, micromagnetic resonance imaging, microparticle image velocimetry, real-time pressure monitoring, and associated issues with the techniques will be discussed. Alongside this discussion, we also highlight recent advances in cardiac function measurements in chick embryos.

Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia.

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment.

The Coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron variants with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this study, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated, and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD, was generated and further characterized. These mAbs were shown by ELISA to be specific for each immunogen/antigen. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P2-20 is shown to be favorable for ELISA, immunoblotting, and immunohistochemistry (IHC), however, not live virus neutralization. In contrast, mAb CU-28-24 is most effective at live virus neutralization as well as ELISA and IHC. Moreover, mAb CU-28-24 is active against rRBD proteins from Omicron variants BA.2 and BA.4.5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the immunoglobulin genes has been sequenced using Next Generation Sequencing, which allows the expression of respective recombinant proteins, thereby eliminating the need for long-term hybridoma maintenance. The synthetic peptides and hybridomas/mAbs and quantitative antigen-binding data are under the intellectual property management of the Clemson University Research Foundation, and the three CDRs have been submitted as an invention disclosure for further patenting and commercialization.

Use of Polyvinyl Alcohol-Porcine Small Intestine Submucosa Stent in the Prevention of Anastomotic Leaks in the Porcine Colon.

INTRODUCTION: Gastrointestinal anastomoses are performed millions of times per year worldwide. The major complication they share is anastomotic leak. We describe the development and initial safety/efficacy of a novel luminal stent which aims to address this clinical issue. MATERIALS AND METHODS: The stent was created out of two materials, a polyvinyl alcohol core and outer layer of acellular porcine small intestine submucosa. Ten healthy pigs underwent laparotomy, a portion of the colon was transected, and the stent was placed within the colonic lumen at the site of resection. Pigs were sacrificed at the end of postoperative week 2, and postoperative week 4. A portion of the descending colon was resected, and tissue samples from the anastomosis, intentional defect scar, and normal bowel overlying the stent were sent for histopathologic examination. RESULTS: All ten animals survived the study. None developed any clinical signs of obstruction, infection, leakage, fistula, wound complications, or bleeding. No evidence of colonic leak or luminal stenosis/stricture was noted. CONCLUSIONS: The results of this study show that a polyvinyl alcohol/acellular porcine small intestine submucosa stent sewn underneath a colonic anastomosis with a 2 cm intentional defect will result in no anastomotic complications. There were also no complications from placing this stent in any pigs. Additional studies with a control group should be conducted to see if this same stent can be built in different diameters, lengths, and configurations to prevent leaks in other organs. These encouraging results will hopefully lead to decreased leaks and the need for temporary ostomies in humans.

Prenatal hypoxia affects scaling of blood pressure and arterial wall mechanics in the common snapping turtle, Chelydra serpentina.

In reptiles, exposure to hypoxia during embryonic development affects several cardiovascular parameters. These modifications may impose different mechanical stress to the arterial system, and we speculated that the arterial wall of major outflow vessels would be modified accordingly. Since non-crocodilian reptiles possess a partially divided ventricle, ensuing similar systemic and pulmonary systolic pressures, we investigated how morphological and mechanical properties of segments from the left aortic arch (LAo) and the proximal and distal segments of the left pulmonary artery (LPAp and LPAd, respectively) change as body mass (Mb) increases. Eggs from common snapping turtles, Chelydra serpentina, were incubated under normoxia (21% O2; N21) or hypoxia (10% O2; H10), hatched and maintained in normoxia thereafter. Turtles (0.11-6.85 kg) were cannulated to measure arterial pressures, and an injection of adrenaline was used to increase pressures. Portions of the LAo, LPAp and LPAd were fixed under physiological hydrostatic pressures for histology and mechanical assessment. Arterial pressures increased with Mb for N21 but not for H10. Although mechanical and functional characteristics from the LPAp and LPAd were similar between N21 and H10, wall thickness from LAo did not change with Mb in the H10 group, thus wall stress increased in larger turtles. This indicates that larger H10 turtles probably experience an elevated probability of arterial wall rupture without concomitant changes in the cardiovascular system to prevent it. Finally, collagen content of the LPAp and LAo was smaller than in LPAd, suggesting a more distensible arterial wall could attenuate higher pressures from larger turtles.

Embryotoxicity and Physiological Compensation in Chicken Embryos Exposed to Crude Oil.

Terrestrial, marine, or aquatic oil spills can directly or indirectly contaminate bird eggs. We hypothesized that chicken embryos exposed to crude oil can physiologically compensate to mitigate the potentially toxic effect of lower doses of oil. Embryos exposed to 0, 1, 3, or 5 µL of oil on embryonic days 4 and 10 were initially analyzed for mortality. All oil doses decreased day 4 embryo survival, but only the 2 highest oil doses lowered survival when applied on day 10. Thus, day 15 embryos treated with 1, 3, and 5 µL of source oil on day 10 had arterialized blood analyzed. The hematological variables hematocrit, red blood cell concentration ([RBC]), and hemoglobin concentration increased in response to 1 µL, were unchanged by 3 µL, and decreased by 5 µL of oil treatment. No changes occurred in arterialized blood gas variables (partial pressure of O2 [PO2 ], pH, bicarbonate concentration) for 1 and 3 µL embryos, but 5 µL of oil decreased PO2 and caused metabolic acidosis. Increased blood lactate in embryos treated with 3 and 5 µL of oil was correlated with decreased hematocrit and [RBC] and increased body mass, the latter likely reflecting edema. We conclude that embryos in middle development physiologically compensated for negative effects of lower doses of crude oil but that higher doses of oil were harmful to the embryos at all developmental stages. Environ Toxicol Chem 2021;40:2347-2358. © 2021 SETAC.

Assessing Pressure-Volume Relationship in Developing Heart of Zebrafish In-Vivo.

During embryogenesis, the developing heart transforms from a linear peristaltic tube into a multi-chambered pulsatile pump with blood flow-regulating valves. In this work, we report how hemodynamic parameters evolve during the heart's development, leading to its rhythmic pumping and blood flow regulation as a functioning organ. We measured the time course of intra-ventricular pressure from zebrafish embryos at 3, 4, and 5 days post fertilization (dpf) using the servo null method. We also measured the ventricular volume and monitored the opening/closing activity of the AV and VB valves using 4D selective plane illumination microscopy (SPIM). Our results revealed significant increases in peak systolic pressure, stroke volume and work, cardiac output, and power generation, and a total peripheral resistance decrease from zebrafish at 4, 5 dpf versus 3 dpf. These data illustrate that the early-stage zebrafish heart's increasing efficiency is synchronous with the expected changes in valve development, chamber morphology and increasing vascular network complexity. Such physiological measurements in tractable laboratory model organisms are critical for understanding how gene variants may affect phenotype. As the zebrafish emerges as a leading biomedical model organism, the ability to effectively measure its physiology is critical to its translational relevance.

Arterial wall thickening normalizes arterial wall tension with growth in American alligators, Alligator mississippiensis.

Arterial wall tension increases with luminal radius and arterial pressure. Hence, as body mass (Mb) increases, associated increases in radius induces larger tension. Thus, it could be predicted that high tension would increase the potential for rupture of the arterial wall. Studies on mammals have focused on systemic arteries and have shown that arterial wall thickness increases with Mb and normalizes tension. Reptiles are good models to study scaling because some species exhibit large body size range associated with growth, thus, allowing for ontogenetic comparisons. We used post hatch American alligators, Alligator mississippiensis, ranging from 0.12 to 6.80 kg (~ 60-fold) to investigate how both the right aortic arch (RAo) and the left pulmonary artery (LPA) change with Mb. We tested two possibilities: (i) wall thickness increases with Mb and normalizes wall tension, such that stress (stress = tension/thickness) remains unchanged; (ii) collagen content scales with Mb and increases arterial strength. We measured heart rate and systolic and mean pressures from both systemic and pulmonary circulations in anesthetized animals. Once stabilized alligators were injected with adrenaline to induce a physiologically relevant increase in pressure. Heart rate decreased and systemic pressures increased with Mb; pulmonary pressures remained unchanged. Both the RAo and LPA were fixed under physiological hydrostatic pressures and displayed larger radius, wall tension and thickness as Mb increased, thus, stress was independent from Mb; relative collagen content was unchanged. We conclude that increased wall thickness normalizes tension and reduces the chances of arterial walls rupturing in large alligators.

Effects of crude oil vapors on the cardiovascular flow of embryonic Gulf killifish.

Direct contact with toxicants in crude oil during embryogenesis causes cardiovascular defects, but the effects of exposure to airborne volatile organic compounds released from spilled oil are not well understood. The effects of crude oil-derived airborne toxicants on peripheral blood flow were examined in Gulf killifish (Fundulus grandis) since this model completes embryogenesis in the air. Particle image velocimetry was used to measure in vivo blood flow in intersegmental arteries of control and oil-exposed embryos. Significant effects in oil-exposed embryos included increased pulse rate, reduced mean blood flow speed and volumetric flow rate, and decreased pulsatility, demonstrating that normal-appearing oil-exposed embryos retain underlying cardiovascular defects. Further, hematocrit moderately increased in oil-exposed embryos. This study highlights the potential for fine-scale physiological measurement techniques to better understand the sub-lethal effects of oil exposure and demonstrates the efficacy of Gulf killifish as a unique teleost model for aerial toxicant exposure studies.

Underlying Ossification Phenotype in a Murine Model of Metastatic Synovial Sarcoma.

Synovial sarcoma, an uncommon cancer, typically affects young adults. Survival rates range from 36% to 76%, decreasing significantly when metastases are present. Synovial sarcomas form in soft tissues, often near bones, with about 10% demonstrating ossification in the tumor. The literature is inconclusive on whether the presence of ossification portends a worse prognosis. To this end, we analyzed our genetic mouse models of synovial sarcoma to determine the extent of ossification in the tumors and its relationship with morbidity. We noted higher ossification within our metastatic mouse model of synovial sarcoma. Not only did we observe ossification within the tumors at a frequency of 7%, but an even higher frequency, 72%, of bone reactivity was detected by radiography. An enrichment of bone development genes was associated with primary tumors, even in the absence of an ossification phenotype. In spite of the ossification being intricately linked with the metastatic model, the presence of ossification was not associated with a faster or worse morbidity in the mice. Our conclusion is that both metastasis and ossification are dependent on time, but that they are independent of one another.

Methodology for exposing avian embryos to quantified levels of airborne aromatic compounds associated with crude oil spills.

Oil spills on birds and other organisms have focused primarily on direct effects of oil exposure through ingestion or direct body fouling. Little is known of indirect effects of airborne volatiles from spilled oil, especially on vulnerable developing embryos within the bird egg. Here a technique is described for exposing bird embryos in the egg to quantifiable amounts of airborne volatile toxicants from Deepwater Horizon crude oil. A novel membrane inlet mass spectrometry system was used to measure major classes of airborne oil-derived toxicants and correlate these exposures with biological endpoints. Exposure induced a reduction in platelet number and increase in osmolality of the blood of embryos of the chicken (Gallus gallus). Additionally, expression of cytochrome P4501A, a protein biomarker of oil exposure, occurred in renal, pulmonary, hepatic and vascular tissues. These data confirm that this system for generating and measuring airborne volatiles can be used for future in-depth analysis of the toxicity of volatile organic compounds in birds and potentially other terrestrial organisms.

Natural development of dermal ectopic bone in the american alligator (Alligator mississippiensis) resembles heterotopic ossification disorders in humans.

Heterotopic ossification (HO) occurs when soft tissues are inappropriately converted to bony tissue. Several human diseases result in HO with few reliable treatment options. Animal models that naturally produce dermal ectopic bone (i.e., osteoderms), such as crocodilians, have never been utilized as models for studying these disorders in humans. Here, a histological evaluation and staging criteria for osteoderm development is described for the first time in the American alligator (Alligator mississipiensis). Differential staining and immunohistochemistry of alligator scales depict a progressive change during development, where woven bone forms from the differentiated dermis. Bone formation proceeds via intramembranous ossification, which is initiated in part by endothelial cell precursors that undergo endothelial-to-mesenchymal transition and eventually acquire an osteoblast phenotype. As such, the development of osteoderms in the American alligator bears morphological and mechanistic similarities to HO in humans, presenting a potential model for future study of soft tissue mineralization pathologies and providing insight into the morphological and molecular development of osteoderms in other vertebrate lineages. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:56-76, 2018. © 2017 Wiley Periodicals, Inc.

Biomarkers of Aryl-hydrocarbon Receptor Activity in Gulf Killifish (Fundulus grandis) From Northern Gulf of Mexico Marshes Following the Deepwater Horizon Oil Spill.

Following the Deepwater Horizon oil spill, shorelines throughout the Barataria Basin of the northern Gulf of Mexico in Louisiana were heavily oiled for months with Macondo-252 oil, potentially impacting estuarine species. The Gulf killifish (Fundulus grandis) has been identified as a sentinel species for the study of site-specific effects of crude oil contamination on biological function. In November and December 2010, 4-5 months after the Macondo well was plugged and new oil was no longer spilling into the Gulf waters, Gulf killifish were collected across the Barataria Basin from 14 sites with varying degrees of oiling. Fish collected from oiled sites exhibited biological indications of exposure to oil, including increase in cytochrome P4501A (CYP1A) mRNA transcript and protein abundances in liver tissues. Immunohistochemistry revealed increases in gill, head kidney, and intestinal CYP1A protein at heavily oiled sites. Intestinal CYP1A protein was a sensitive indicator of exposure, indicating that intestinal tissue plays a key role in biotransformation of AHR ligands and that ingestion is a probable route of exposure, warranting additional consideration in future studies.

Incubation relative humidity induces renal morphological and physiological remodeling in the embryo of the chicken (Gallus gallus domesticus).

The metanephric kidneys of the chicken embryo, along with the chorioallantoic membrane, process water and ions to maintain osmoregulatory homeostasis. We hypothesized that changes in relative humidity (RH) and thus osmotic conditions during embryogenesis would alter the developmental trajectory of embryonic kidney function. White leghorn chicken eggs were incubated at one of 25-30% relative humidity, 55-60% relative humidity, and 85-90% relative humidity. Embryos were sampled at days 10, 12, 14, 16, and 18 to examine embryo and kidney mass, glomerular characteristics, body fluid osmolalities, hematological properties, and whole embryo oxygen consumption. Low and especially high RH elevated mortality, which was reflected in a 10-20% lower embryo mass on D18. Low RH altered several glomerular characteristics by day 18, including increased numbers of glomeruli per kidney, increased glomerular perfusion, and increased total glomerular volume, all indicating potentially increased functional kidney capacity. Hematological variables and plasma and amniotic fluid osmolalities remained within normal physiological values. However, the allantoic, amniotic and cloacal fluids had a significant increase in osmolality at most developmental points sampled. Embryonic oxygen consumption increased relative to control at both low and high relative humidities on Day 18, reflecting the increased metabolic costs of osmotic stress. Major differences in both renal structure and performance associated with changes in incubation humidity occurred after establishment of the metanephric kidney and persisted into late development, and likely into the postnatal period. These data indicate that the avian embryo deserves to be further investigated as a promising model for fetal programming of osmoregulatory function, and renal remodeling during osmotic stress.

Is Exposure to Macondo Oil Reflected in the Otolith Chemistry of Marsh-Resident Fish?

Genomic and physiological responses in Gulf killifish (Fundulus grandis) in the northern Gulf of Mexico have confirmed oil exposure of resident marsh fish following the Macondo blowout in 2010. Using these same fish, we evaluated otolith microchemistry as a method for assessing oil exposure history. Laser-ablation inductively-coupled-plasma mass spectrometry was used to analyze the chemical composition of sagittal otoliths to assess whether a trace metal signature could be detected in the otoliths of F. grandis collected from a Macondo-oil impacted site in 2010, post-spill relative to pre-spill, as well as versus fish from areas not impacted by the spill. We found no evidence of increased concentrations of two elements associated with oil contamination (nickel and vanadium) in F. grandis otoliths regardless of Macondo oil exposure history. One potential explanation for this is that Macondo oil is relatively depleted of those metals compared to other crude oils globally. During and after the spill, however, elevated levels of barium, lead, and to a lesser degree, copper were detected in killifish otoliths at the oil-impacted collection site in coastal Louisiana. This may reflect oil contact or other environmental perturbations that occurred concomitant with oiling. For example, increases in barium in otoliths from oil-exposed fish followed (temporally) freshwater diversions in Louisiana in 2010. This implicates (but does not conclusively demonstrate) freshwater diversions from the Mississippi River (with previously recorded higher concentrations of lead and copper), designed to halt the ingress of oil, as a mechanism for elevated elemental uptake in otoliths of Louisiana marsh fishes. These results highlight the potentially complex and indirect effects of the Macondo oil spill and human responses to it on Gulf of Mexico ecosystems, and emphasize the need to consider the multiple stressors acting simultaneously on inshore fish communities.

Cross-resistance in Gulf killifish (Fundulus grandis) populations resistant to dioxin-like compounds.

The Houston Ship Channel (HSC) in Houston, Texas is an aquatic environment with a long history of contamination, including polychlorinated dibenzodioxins (PCDD), polychlorinated dibenzofurans (PCDF), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. Populations of Gulf killifish (Fundulus grandis) from the HSC have adapted to resist developmental cardiac deformities caused by dioxin-like compounds (DLCs). Contaminants in the HSC have acted as a strong selective pressure on resident Gulf killifish populations. Rapid adaptation can lead to fitness costs, some as a direct result of the mechanisms involved in the adaptive process, whereas other adaptations may be more general. To explore potential fitness costs, we evaluated two Gulf killifish populations with documented resistance to DLC-induced cardiac teratogenesis (Patrick Bayou and Vince Bayou), and one previously characterized reference population (Gangs Bayou). We also characterized a previously unstudied population from Galveston Bay as an additional reference population (Smith Point). We tested the sensitivity of F1 larvae from these four populations to two classes of pesticides (pyrethroid (permethrin) and carbamate (carbaryl)) and two model pro-oxidants (tert-butyl hydroquinone (tBHQ) and tert-butyl hydroperoxide (tBOOH)). In addition, we explored their responses to hypoxia and measured resting metabolic rates (M.O2). Both adapted populations were cross-resistant to the toxicity of carbaryl and both pro-oxidants tested. There were no population differences in sensitivity to permethrin. On the other hand, one reference population (Gangs Bayou) was less sensitive to hypoxia, and maintained a lower M.O2 . However, there were no differences in hypoxia tolerance or resting metabolic rate between the second reference and the two adapted populations. This investigation emphasizes the importance of including multiple reference populations to clearly link fitness costs or cross-resistance to pollution adaptation, rather than to unrelated environmental or ecological differences. When compared to previous literature on adapted populations of Fundulus heteroclitus, we see a mixture of similarities and differences, suggesting that F. grandis adapted phenotypes likely involve multiple mechanisms, which may not be completely consistent among adapted populations.

Multitissue molecular, genomic, and developmental effects of the Deepwater Horizon oil spill on resident Gulf killifish (Fundulus grandis).

The Deepwater Horizon oil rig disaster resulted in crude oil contamination along the Gulf coast in sensitive estuaries. Toxicity from exposure to crude oil can affect populations of fish that live or breed in oiled habitats as seen following the Exxon Valdez oil spill. In an ongoing study of the effects of Deepwater Horizon crude oil on fish, Gulf killifish ( Fundulus grandis ) were collected from an oiled site (Grande Terre, LA) and two reference locations (coastal MS and AL) and monitored for measures of exposure to crude oil. Killifish collected from Grande Terre had divergent gene expression in the liver and gill tissue coincident with the arrival of contaminating oil and up-regulation of cytochrome P4501A (CYP1A) protein in gill, liver, intestine, and head kidney for over one year following peak landfall of oil (August 2011) compared to fish collected from reference sites. Furthermore, laboratory exposures of Gulf killifish embryos to field-collected sediments from Grande Terre and Barataria Bay, LA, also resulted in increased CYP1A and developmental abnormalities when exposed to sediments collected from oiled sites compared to exposure to sediments collected from a reference site. These data are predictive of population-level impacts in fish exposed to sediments from oiled locations along the Gulf of Mexico coast.

Genomic and physiological footprint of the Deepwater Horizon oil spill on resident marsh fishes.

The biological consequences of the Deepwater Horizon oil spill are unknown, especially for resident organisms. Here, we report results from a field study tracking the effects of contaminating oil across space and time in resident killifish during the first 4 mo of the spill event. Remote sensing and analytical chemistry identified exposures, which were linked to effects in fish characterized by genome expression and associated gill immunohistochemistry, despite very low concentrations of hydrocarbons remaining in water and tissues. Divergence in genome expression coincides with contaminating oil and is consistent with genome responses that are predictive of exposure to hydrocarbon-like chemicals and indicative of physiological and reproductive impairment. Oil-contaminated waters are also associated with aberrant protein expression in gill tissues of larval and adult fish. These data suggest that heavily weathered crude oil from the spill imparts significant biological impacts in sensitive Louisiana marshes, some of which remain for over 2 mo following initial exposures.

Influence of cortisol on the attachment and metamorphosis of larval Utterbackia imbecillis on bluegill sunfish (Lepomis macrochirus).

The larvae of unionid freshwater mussels (i.e., glochidia) undergo a parasitic stage requiring their attachment to the external epithelia of fish hosts, where they metamorphose into free-living juveniles. We describe the physiological effects in bluegill sunfish (Lepomis macrochirus) of infection with glochidia from the paper pondshell (Utterbackia imbecillis). Glochidia accumulation on bluegill increased dramatically at concentrations of 2000 glochidia liter(-1) and above, reaching a maximum attachment density of about 30 glochidia g(-1) fish at 4000 glochidia liter(-1). Plasma cortisol was the most sensitive indicator of biological effect to glochidial exposure, increasing significantly in hosts exposed to 2000 glochidia liter(-1) or greater. Glochidia were 31% more likely to undergo successful juvenile metamorphosis when attached to bluegill with elevated plasma cortisol, largely due to the enhanced survivorship of these larvae during the first 48 h after infection. We tested the hypothesis that glochidial attachment and juvenile metamorphosis were stimulated directly by plasma cortisol in fish hosts. Bluegill were given an intraperitoneal injection of cortisol, then infected with 1000 glochidia liter(-1) at 48 h after hormone supplementation. Cortisol-injected fish had a 42% increase in the number of attached glochidia g(-1) fish and a 28% increase in larval metamorphosis compared to sham-injected and control fish. We provide evidence that cortisol enhances glochidial metamorphosis on hosts by improving the retention of attached glochidia. This study gives insights into the influence of host physiology on glochidial attachment and juvenile mussel transformation.