Connor Leong’s research while affiliated with Oregon State University and other places

Zebrafish have become an essential model organism in screening for developmental neurotoxic chemicals and their molecular targets. The success of zebrafish as a screening model is partially due to their physical characteristics including their relatively simple nervous system, rapid development, experimental tractability, and genetic diversity combined with technical advantages that allow for the generation of large amounts of high-dimensional behavioral data. These data are complex and require advanced machine learning and statistical techniques to comprehensively analyze and capture spatiotemporal responses. To accomplish this goal, we have trained semi-supervised deep autoencoders using behavior data from unexposed larval zebrafish to extract quintessential “normal” behavior. Following training, our network was evaluated using data from larvae shown to have significant changes in behavior (using a traditional statistical framework) following exposure to toxicants that include nanomaterials, aromatics, per- and polyfluoroalkyl substances (PFAS), and other environmental contaminants. Further, our model identified new chemicals (Perfluoro-n-octadecanoic acid, 8-Chloroperfluorooctylphosphonic acid, and Nonafluoropentanamide) as capable of inducing abnormal behavior at multiple chemical-concentrations pairs not captured using distance moved alone. Leveraging this deep learning model will allow for better characterization of the different exposure-induced behavioral phenotypes, facilitate improved genetic and neurobehavioral analysis in mechanistic determination studies and provide a robust framework for analyzing complex behaviors found in higher-order model systems.

Intestinal helminth parasite (IHP) infection induces alterations in the composition of microbial communities across vertebrates, although how gut microbiota may facilitate or hinder parasite infection remains poorly defined. In this work, we utilized a zebrafish model to investigate the relationship between gut microbiota, gut metabolites, and IHP infection. We found that extreme disparity in zebrafish parasite infection burden is linked to the composition of the gut microbiome and that changes in the gut microbiome are associated with variation in a class of endogenously produced signaling compounds, N-acylethanolamines, that are known to be involved in parasite infection. Using a statistical mediation analysis, we uncovered a set of gut microbes whose relative abundance explains the association between gut metabolites and infection outcomes. Experimental investigation of one of the compounds in this analysis reveals salicylaldehyde, which is putatively produced by the gut microbe Pelomonas, as a potent anthelmintic with activity against Pseudocapillaria tomentosa egg hatching, both in vitro and in vivo. Collectively, our findings underscore the importance of the gut microbiome as a mediating agent in parasitic infection and highlight specific gut metabolites as tools for the advancement of novel therapeutic interventions against IHP infection. IMPORTANCE Intestinal helminth parasites (IHPs) impact human health globally and interfere with animal health and agricultural productivity. While anthelmintics are critical to controlling parasite infections, their efficacy is increasingly compromised by drug resistance. Recent investigations suggest the gut microbiome might mediate helminth infection dynamics. So, identifying how gut microbes interact with parasites could yield new therapeutic targets for infection prevention and management. We conducted a study using a zebrafish model of parasitic infection to identify routes by which gut microbes might impact helminth infection outcomes. Our research linked the gut microbiome to both parasite infection and to metabolites in the gut to understand how microbes could alter parasite infection. We identified a metabolite in the gut, salicylaldehyde, that is putatively produced by a gut microbe and that inhibits parasitic egg growth. Our results also point to a class of compounds, N-acyl-ethanolamines, which are affected by changes in the gut microbiome and are linked to parasite infection. Collectively, our results indicate the gut microbiome may be a source of novel anthelmintics that can be harnessed to control IHPs.

Intestinal helminth parasite (IHP) infection induces alterations in the composition of microbial communities across vertebrates, although how gut microbiota may facilitate or hinder parasite infection remains poorly defined. In this work we utilized a zebrafish model to investigate the relationship between gut microbiota, gut metabolites, and IHP infection. We found that extreme disparity in zebrafish parasite infection burden is linked to the composition of the gut microbiome, and that changes in the gut microbiome are associated with variation in a class of endogenously-produced signaling compounds, N-acylethanolamines, that are known to be involved in parasite infection. Using a statistical mediation analysis, we uncovered a set of gut microbes whose relative abundance explains the association between gut metabolites and infection outcomes. Experimental investigation of one of the compounds in this analysis reveals salicylaldehyde, which is putatively produced by the gut microbe Pelomonas, as a potent anthelmintic with activity against Pseudocapillaria tomentosa egg hatching, both in vitro and in vivo. Collectively, our findings underscore the importance of the gut microbiome as a mediating agent in parasitic infection and highlights specific gut metabolites as tools for the advancement of novel therapeutic interventions against IHP infection.

Phenol isopropylated phosphates (IPP) are an additive organophosphate flame retardant (OPFR) which has been extensively used in furniture, electronics, automobiles, plastics, and childrens products to slow down the spread of fire. The processing and distribution of IPP- containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP-induced developmental toxicity using zebrafish embryos. We first conducted range finding experiments with embryonic zebrafish exposures to 0- 200 micromolar IPP from 6 to 120 h post fertilization and found significant morphological impacts like pericardial edema, yolk sac edema and spinal curvature at higher concentrations. For behavioral readouts, we performed larval photomotor response (LPR) assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Following this, relying on secondary analyses of our whole embryo mRNA-seq data, we conducted- 1) retinoic acid receptor (RAR) signaling assay and 2) DNA methylation assays. In vitro assay for RA receptors indicate that IPP significantly inhibits RAR-alpha;, but not RAR-beta; and RAR-gamma;. Whole-mount immunohistochemistry for 5-methylcytosine and global DNA methylation assay showed significant IPP-induced hypermethylation in situ. We conducted IPP co-exposure studies with a methylome modifier 5-azacytidine (Aza-c a methylation inhibitor) or retinoic acid signaling activators to assess if LPR phenotypes were mitigated by co-exposures. Data showed that Aza-c co-exposures partially reversed IPP-induced LPR hypoactivity and DNA hypermethylation, co-exposure with retinoic acid as well as AM580 (an RAR alpha; activator) were not able to reverse IPP-induced hypoactivity. Finally, based on RNA-seq data, we hypothesized that IPP affects the development of brain and eyes. Firstly, we performed global DNA methylation in brain and eyes, but did not find any significant effects. Then, we conducted mRNA sequencing on dissected brains and eyes, and found 2 and 135 differentially expressed genes, respectively. Gene ontology revealed that IPP affect phototransduction, voltage gated ion channels, synaptic and neurotransmitter signaling. Collectively, our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response, potentially through methylomic regulation. Finally, we observed that IPP affects gene expression within the developing eye, establishing synaptic transmission, vision and muscle contraction as a potential causative factor for LPR responses.

A novel microsporidium was observed in wild swamp guppies Micropoecilia picta from Levera Pond within Levera National Park Grenada, West Indies. Initial observations indicated similarity with Pseudoloma neurophilia , an important pathogen in zebrafish Danio rerio . P. neurophilia exhibit broad host specifity, including members of the family Poecillidae, and both parasites infect the central nervous system. However, spore morphology and molecular phylogeny based on rDNA showed that the swamp guppy microsporidium (SGM) is distinct from P. neurophilia and related microsporidia ( Microsporidium cerebralis and M. luceopercae ). Spores of the SGM were smaller than others in the clade (3.6 µm long). Differences were also noted in histology; the SGM formed large aggregates of spores within neural tissues along with a high incidence of numerous smaller aggregates and single spores within the surface tissue along the ventricular spaces that extended submeninx, whereas P. neurophilia and M. cerebralis infect deep into the neuropile and cause associated lesions. Analysis of small subunit ribosomal DNA sequences showed that the SGM was <93% similar to these related microsporidia. Nevertheless, one of 2 commonly used PCR tests for P. neurophilia cross reacted with tissues infected with SGM. These data suggest that there could be other related microsporidia capable of infecting zebrafish and other laboratory fishes that are not being detected by these highly specific assays. Consequently, exclusive use of these PCR tests may not accurately diagnose other related microsporidia infecting animals in laboratory and ornamental fish facilities.

3,3’,5.5’-Tetrabromobisphenol A (TBBPA) is a widely used brominated flame-retardant utilized in the production of electronic devices and plastic paints. The objective of this study is to use zebrafish as a model and determine the effects of TBBPA exposure on early embryogenesis. We initiated TBBPA exposures (0, 10, 20 and 40μM) at 0.75 h post fertilization (hpf) and monitored early developmental events such as cleavage, blastula and epiboly that encompass maternal-to-zygotic transition (MZT) and zygotic genome activation (ZGA). Our data revealed that TBBPA exposures induced onset of developmental delays by 3 hpf (blastula). By 5.5 hpf (epiboly), TBBPA-exposed (10-20 μM) embryos showed concentration-dependent developmental lag by up to 3 stages or 100% mortality at 40 μM. Embryos exposed to sublethal TBBPA concentrations from 0.75-6 hpf and raised in clean water to 120 hpf showed altered larval photomotor response (LPR), suggesting a compromised developmental health. To examine the genetic basis of TBBPA-induced delays, we conducted mRNA-sequencing on embryos exposed to 0 or 40 μM TBBPA from 0.75 hpf to 2, 3.5 or 4.5 hpf. Read count data showed that while TBBPA exposures had no overall impacts on maternal or maternal-zygotic genes, collective read counts for zygotically activated genes were lower in TBBPA treatment at 4.5 hpf compared to time-matched controls, suggesting that TBBPA delays ZGA. Gene ontology assessments for both time- and stage-matched differentially expressed genes revealed TBBPA-induced inhibition of chromatin assembly- a process regulated by histone modifications. Since acetylation is the primary histone modification system operant during early ZGA, we immunostained embryos with an H3K27Ac antibody and demonstrated reduced acetylation in TBBPA-exposed embryos. Leveraging in silico molecular docking studies and in vitro assays, we also showed that TBBPA potentially binds to P300- a protein that catalyzes acetylation- and inhibits P300 activity. Finally, we co-exposed embryos to 20 μM TBBPA and 50 μM n-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) -a histone acetyltransferase activator that promotes histone acetylation- and showed that TBBPA-CTPB co or pre-exposures significantly reversed TBBPA-only developmental delays, suggesting that TBBPA-induced phenotypes are indeed driven by repression of histone acetylation. Collectively, our work demonstrates that TBBPA disrupts ZGA and early developmental morphology, potentially by inhibiting histone acetylation. Future studies will focus on mechanisms of TBBPA-induced chromatin modifications.

Zebrafish have become an essential tool in screening for developmental neurotoxic chemicals and their molecular targets. The success of zebrafish as a screening model is partially due to their physical characteristics including their relatively simple nervous system, rapid development, experimental tractability, and genetic diversity combined with technical advantages that allow for the generation of large amounts of high-dimensional behavioral data. These data are complex and require advanced machine learning and statistical techniques to comprehensively analyze and capture spatiotemporal responses. To accomplish this goal, we have trained semi-supervised deep autoencoders using behavior data from unexposed larval zebrafish to extract quintessential “normal” behavior. Following training, our network was evaluated using data from larvae shown to have significant changes in behavior (using a traditional statistical framework) following exposure to toxicants that include nanomaterials, aromatics, per- and polyfluoroalkyl substances (PFAS), and other environmental contaminants. Further, our model identified new chemicals (Perfluoro-n-octadecanoic acid, 8-Chloroperfluorooctylphosphonic acid, and Nonafluoropentanamide) as capable of inducing abnormal behavior at multiple chemical-concentrations pairs not captured using distance moved alone. Leveraging this deep learning model will allow for better characterization of the different exposure-induced behavioral phenotypes, facilitate improved genetic and neurobehavioral analysis in mechanistic determination studies and provide a robust framework for analyzing complex behaviors found in higher-order model systems.

The intestinal nematode Pseudocapillaria tomentosa in zebrafish (Danio rerio) causes profound intestinal lesions, emaciation and death and is a promoter of a common intestinal cancer in zebrafish. This nematode has been detected in zebrafish from about 15% of the laboratories. Adult worms are readily detected about 3 weeks after exposure by either histology or wet mount preparations of the intestine, and larval worms are inconsistently observed in fish before this time. A quantitative PCR (qPCR) test was recently developed to detect the worm in fish and water, and here we determined that the test on zebrafish intestines was effective for earlier detection. Four lines of zebrafish (AB, TU, 5D and Casper) were experimentally infected and evaluated by wet mounts and qPCR at 8, 15-, 22-, 31- and 44-day post-exposure (dpe). At the first two time points, only 8% of the wet mounts from exposed fish were identified as infected, while the same intestines screened by qPCR showed 78% positivity, with low and consistent cycle threshold (Ct) values at these times. Wet mounts at later time points showed a high prevalence of infection, but this was still surpassed by qPCR.

Ubiquitous anthropogenic contaminants of concern, per- and polyfluoroalkyl substances (PFAS) are frequently detected in the environment and human populations around the world. Diet is a predominate route of human exposure, and PFAS are frequently measured in food. Manufacturing trends have shifted from legacy PFAS to shorter-chain alternatives that are suggested to be safer, such as perfluorohexanoic acid (PFHxA). However, the current amount of data to support safety assessments of these alternatives is not yet sufficient. The present study investigated the effects of a 42-day dietary exposure to 1, 10, or 100 ng/g PFHxA in juvenile zebrafish. The zebrafish model was leveraged to interrogate morphometrics, fecundity, and numerous behavior endpoints across multiple generations. Dietary PFHxA exposure did not result in measurable body burden and did not affect growth, fecundity, adult social perception behavior, or associative learning. PFHxA exposure did induce abnormal adult anxiety behaviors in the F0 generation that persisted transgenerationally in the F1 and F2. Abnormal larval and juvenile behavior was observed in the F1 generation, but not in the F2. PFHxA juvenile dietary exposure induced subtle and multigenerational behavior effects that warrant further investigation of this and other alternative short-chain PFAS.