Sean Spagnoli

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

The zebrafishs potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the hosts brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the improvement of the zebrafish as a translational model for human behavior.

Structural equation modeling identifies markers of damage and function in the aging male Fischer 344 rat

The male Fischer 344 rat is an established model to study progressive renal dysfunction that is similar, but not identical, to chronic kidney disease (CKD) in humans. These studies were designed to assess age-dependent alterations in renal structure and function at late-life timepoints, 16-24 months. Elevations in BUN and plasma creatinine were not significant until 24 months, however, elevations in the more sensitive markers of function, plasma cystatin C and proteinuria, were detectable at 16 and 18 months, respectively. Interestingly, cystatin C levels were not corrected by caloric restriction. Urinary Kim-1, a marker of CKD, was elevated as early as 16 months. Klotho gene expression was significantly decreased at 24 months, but not at earlier timepoints. Alterations in renal structure, glomerulosclerosis and tubulointerstitial fibrosis, were noted at 16 months, with little change from 18 to 24 months. Tubulointerstitial inflammation was increased at 16 months, and remained similar from 18 to 24 months. A SEM (structural equation modeling) model of age-related renal dysfunction suggests that proteinuria is a marker of renal damage, while urinary Kim-1 is a marker of both damage and function. Taken together, these results demonstrate that age-dependent nephropathy begins as early as 16 months and progresses rapidly over the next 8 months.

13 – Stress in Fish as Model Organisms

1. Introduction 2. Indicators of Stress in Laboratory Fish 3. Factors Impacting Stress in Laboratory Fish Handling 4. Housing 4.1. Density 4.2. Enrichment 4.3. Light/Dark Cycle 5. Feeding and Stress 6. Sex and Hierarchies 7. Sex Determination and Reversal 8. Stress, Cortisol, and Reproduction 9. Anesthetics 10. Underlying Diseases 11. Consistency 12. Conclusion and Key Unknowns With the advent of the zebrafish, the three-spined stickleback, and the medaka as laboratory animals, the emergence of fish as model organisms has provided a wide variety of potential experimental subjects, simultaneously introducing new challenges to both researchers and aquaculturists. With regard to stress in these fishes, we must shift the emphasis beyond the traditional definitions of production in terms of fecundity and growth and toward the goals of experimental consistency, animal welfare, and model robustness. In order to improve fish as model organisms, aquaculturists and researchers must use each organisms natural history as a template for developing appropriate husbandry practices. In this chapter, we review published data regarding stress in laboratory fishes in order to provide a foundation for building better husbandry protocols for the purposes of improving fish as model organisms.

Primary Intestinal and Vertebral Chordomas in Laboratory Zebrafish (Danio rerio)

Chordomas are uncommon neoplasms arising from notochord remnants, most commonly occurring in the axial skeleton. Extraskeletal soft tissue chordomas are rare primary tumors, and primary alimentary tract chordomas have not been reported. Herein we report 24 cases of spontaneous primary intestinal chordomas in zebrafish, as well as 9 spontaneous vertebral chordomas. Both intestinal and vertebral tumors showed invasive behavior, although more commonly in the latter. In all cases of primary intestinal chordomas, there was no axial or peripheral skeletal or other nonvisceral involvement. Although uncommon, intestinal chordomas represent a unique background lesion in aged zebrafish.

Reversibility of Proliferative Thyroid Lesions Induced by Iodine Deficiency in a Laboratory Zebrafish Colony

A laboratory zebrafish colony developed red masses, predominantly under the jaw, in a significant portion of the population. The masses were diagnosed histopathologically as thyroid follicular hyperplasia, adenoma, or carcinoma in accordance with published morphologic criteria. After switching to a higher iodine brand of salt used to maintain a low level of salinity within the water system and a small diet change, the thyroid lesions regressed dramatically. Within 5 months the masses were no longer grossly visible. At the population level, external evaluations and histopathological assessments of whole-body sections document a regression in the prevalence of thyroid neoplasia and hyperplasia to normal thyroid conformation by 11 months after salt change. These findings suggest that a wide range of proliferative thyroid lesions, including neoplasms, in zebrafish may be hormone-dependent, even following lesion development. In addition, these results suggest that zebrafish have an adaptive ability to absorb iodine from water and food, which should be considered in discussions to standardize diets and when describing environmental parameters in publications.

AHR2 required for normal behavioral responses and proper development of the skeletal and reproductive systems in zebrafish

The aryl hydrocarbon receptor (AHR) is a conserved ligand-activated transcription factor required for proper vertebrate development and homeostasis. The inappropriate activation of AHR by ubiquitous pollutants can lead to adverse effects on wildlife and human health. The zebrafish is a powerful model system that provides a vertebrate data stream that anchors hypothesis at the genetic and cellular levels to observations at the morphological and behavioral level, in a high-throughput format. In order to investigate the endogenous functions of AHR, we generated an AHR2 (homolog of human AHR)-null zebrafish line (ahr2osu1) using the clustered, regulatory interspaced, short palindromic repeats (CRISPR)-Cas9 precision genome editing method. In zebrafish, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated toxicity requires AHR2. The AHR2-null line was resistant to TCDD-induced toxicity, indicating the line can be used to investigate the biological and toxicological functions of AHR2. The AHR2-null zebrafish exhibited decreased survival and fecundity compared to the wild type line. At 36 weeks, histological evaluations of the AHR2-null ovaries revealed a reduction of mature follicles when compared to wild type ovaries, suggesting AHR2 regulates follicle growth in zebrafish. AHR2-null adults had malformed cranial skeletal bones and severely damaged fins. Our data suggests AHR2 regulates some aspect(s) of neuromuscular and/or sensory system development, with impaired behavioral responses observed in larval and adult AHR2-null zebrafish. This study increases our understanding of the endogenous functions of AHR, which may help foster a better understanding of the target organs and molecular mechanisms involved in AHR-mediated toxicities.

Histologic processing artifacts and inter-pathologist variation in measurement of inked margins of canine mast cell tumors

Although quantitative assessment of margins is recommended for describing excision of cutaneous malignancies, there is poor understanding of limitations associated with this technique. We described and quantified histologic artifacts in inked margins and determined the association between artifacts and variance in histologic tumor-free margin (HTFM) measurements based on a novel grading scheme applied to 50 sections of normal canine skin and 56 radial margins taken from 15 different canine mast cell tumors (MCTs). Three broad categories of artifact were 1) tissue deformation at inked edges, 2) ink-associated artifacts, and 3) sectioning-associated artifacts. The most common artifacts in MCT margins were ink-associated artifacts, specifically ink absent from an edge (mean prevalence: 50%) and inappropriate ink coloring (mean: 45%). The prevalence of other artifacts in MCT skin was 4–50%. In MCT margins, frequency-adjusted kappa statistics found fair or better inter-rater reliability for 9 of 10 artifacts; intra-rater reliability was moderate or better in 9 of 10 artifacts. Digital HTFM measurements by 5 blinded pathologists had a median standard deviation (SD) of 1.9 mm (interquartile range: 0.8–3.6 mm; range: 0–6.2 mm). Intraclass correlation coefficients demonstrated good inter-pathologist reliability in HTFM measurement (κ = 0.81). Spearman rank correlation coefficients found negligible correlation between artifacts and HTFM SDs (r ≤ 0.3). These data confirm that although histologic artifacts commonly occur in inked margin specimens, artifacts are not meaningfully associated with variation in HTFM measurements. Investigators can use the grading scheme presented herein to identify artifacts associated with tissue processing.

Transmission of a common intestinal neoplasm in zebrafish by cohabitation

Intestinal neoplasms are common in zebrafish (Danio rerio) research facilities. These tumours are most often seen in older fish and are classified as small cell carcinomas or adenocarcinomas. Affected fish populations always contain subpopulations with preneoplastic lesions, characterized by epithelial hyperplasia or inflammation. Previous observations indicated that these tumours are unlikely caused by diet, water quality or genetic background, suggesting an infectious aetiology. We performed five transmission experiments by exposure of naïve fish to affected donor fish by cohabitation or exposure to tank effluent water. Intestinal lesions were observed in recipient fish in all exposure groups, including transmissions from previous recipient fish, and moribund fish exhibited a higher prevalence of neoplasms. We found a single 16S rRNA sequence, most similar to Mycoplasma penetrans, to be highly enriched in the donors and exposed recipients compared to unexposed control fish. We further tracked the presence of the Mycoplasma sp. using a targeted PCR test on individual dissected intestines or faeces or tank faeces. Original donor and exposed fish populations were positive for Mycoplasma, while corresponding unexposed control fish were negative. This study indicates an infectious aetiology for these transmissible tumours of zebrafish and suggests a possible candidate agent of a Mycoplasma species.

Persistence of Triploid Grass Carp in Devils Lake, Oregon

Abstract Grass carp Ctenopharyngodon idella are sometimes used as a biological tool for managing aquatic vegetation in reservoirs. Sterile, triploid fish were stocked in Devils Lake, Oregon, during 1986, 1987, and 1993 to control aquatic vegetation. We present a case study for using multiple measures on the same fish to determine whether illegal stocking of fertile, diploid grass carp occurred. An investigation into the estimated age of a dead grass carp found in Devils Lake suggested that it was significantly younger than would otherwise be expected, given the only stocking events occurred during 1986, 1987, and 1993. To determine whether illegal stocking or reproduction by presumed sterile grass carp had occurred in Devils Lake, we conducted a study that balanced the needs of lethally sampling grass carp for biological measures with the socially and politically sensitive sentiment of the pro–grass carp citizenry of Devils Lake. These considerations, in combination with a low catch per-unit effort, resul...