A T-maze test is an experimental approach that is used in congenital research. However, the food reward-based protocol for the T-maze test in fish has low efficiency and a long training period. The aim of this study is to facilitate the T-maze conditions by using a combination of the principles of passive avoidance and a spatial memory test. In our modified T-maze settings, electric shock punishment (1–2 V, 0.3–0.5 mA) is given at the left arm, with a green cue at the right arm. Also, the depth of both arms of the T-maze was increased. The parameters measured in our T-maze design were latency, freezing time, and time spent in different areas of the T-maze. We validated the utility of our modified T-maze protocol by showing the consistent finding of memory impairment in ZnCl₂−treated fish, which has been previously detected with the passive avoidance test. In addition, we also tested the spatial memory performance of leptin a (lepa) mutants which displayed an obesity phenotype. The results showed that although the learning and memory performance for lepa KO fish were similar to control fish, they displayed a higher freezing behavior during the training phase. In conclusion, we have established a modified T-maze protocol that can be used to evaluate the anxiety, learning, and memory capacity of adult zebrafish within three days, for the first time.

Lead and lead-derived compounds have been extensively utilized in industry, and their chronic toxicity towards aquatic animals has not been thoroughly addressed at a behavioral level. In this study, we assessed the risk of exposure to lead at a waterborne environmental concentration in adult zebrafish by behavioral and biochemical analyses. Nine tests, including three-dimension (3D) locomotion, novel tank exploration, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and a short-term memory test, were performed to assess the behavior of adult zebrafish after the exposure to 50 ppb PbCl₂ for one month. The brain tissues were dissected and subjected to biochemical assays to measure the relative expression of stress biomarkers and neurotransmitters to elucidate the underlying mechanisms for behavioral alterations. The results of the behavioral tests showed that chronic exposure to lead could elevate the stress and anxiety levels characterized by elevated freezing and reduced exploratory behaviors. The chronic exposure to PbCl₂ at a low concentration also induced a sharp reduction of aggressiveness and short-term memory. However, no significant change was found in predator avoidance, social interaction, shoaling, or color preference. The biochemical assays showed elevated cortisol and reduced serotonin and melatonin levels in the brain, thus, altering the behavior of the PbCl₂-exposed zebrafish. In general, this study determined the potential ecotoxicity of long-term lead exposure in adult zebrafish through multiple behavioral assessments. The significant findings were that even at a low concentration, long-term exposure to lead could impair the memory and cause a decrease in the aggressiveness and exploratory activities of zebrafish, which may reduce their survival fitness.

Donepezil (DPZ) is an acetylcholinesterase inhibitor used for the clinical treatment of mild cognitive impairment. However, DPZ has been reported to have adverse effects, including causing abnormal cardiac rhythm, insomnia, vomiting, and muscle cramps. However, the existence of these effects in subjects without Dementia is unknown. In this study, we use zebrafish to conduct a deeper analysis of the potential adverse effects of DPZ on the short-term memory and behaviors of normal zebrafish by performing multiple behavioral and biochemical assays. Adult zebrafish were exposed to 1 ppm and 2.5 ppm of DPZ. From the results, DPZ caused a slight improvement in the short-term memory of zebrafish and induced significant elevation in aggressiveness, while the novel tank and shoaling tests revealed anxiolytic-like behavior to be caused by DPZ. Furthermore, zebrafish circadian locomotor activity displayed a higher reduction of locomotion and abnormal movement orientation in both low- and high-dose groups, compared to the control group. Biomarker assays revealed that these alterations were associated with an elevation of oxytocin and a reduction of cortisol levels in the brain. Moreover, the significant increases in reactive oxygen species (ROS) and malondialdehyde (MDA) levels in muscle tissue suggest DPZ exposure induced muscle tissue oxidative stress and muscle weakness, which may underlie the locomotor activity impairment. In conclusion, we show, for the first time, that chronic waterborne exposure to DPZ can severely induce adverse effects on normal zebrafish in a dose-dependent manner. These unexpected adverse effects on behavioral alteration should be carefully addressed in future studies considering DPZ conducted on zebrafish or other animals.

As a nicotinoid neurotoxic insecticide, imidacloprid (IMI) works by disrupting nerve transmission via nicotinic acetylcholine receptor (nAChR). Although IMI is specifically targeting insects, nontarget animals such as the freshwater shrimp, Neocaridina denticulata, could also be affected, thus causing adverse effects on the aquatic environment. To investigate IMI toxicity on nontarget organisms like N. denticulata, their physiology (locomotor activity, heartbeat, and gill ventilation) and biochemical factors (oxidative stress, energy metabolism) after IMI exposure were examined. IMI exposure at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 ppm) to shrimp after 24, 48, 72 h led to dramatic reduction of locomotor activity even at low concentrations. Meanwhile, IMI exposure after 92 h caused reduced heartbeat and gill ventilation at high concentrations. Biochemical assays were performed to investigate oxidative stress and energy metabolism. Interestingly, locomotion immobilization and cardiac activity were rescued after acetylcholine administration. Through molecular docking, IMI demonstrated high binding affinity to nAChR. Thus, locomotor activity and heartbeat in shrimp after IMI exposure may be caused by nAChR blockade and not alterations caused by oxidative stress and energy metabolism. To summarize, N. denticulata serves as an excellent and sensitive aquatic invertebrate model to conduct pesticide toxicity assays that encompass physiologic and biochemical examinations.

Oxygen-binding proteins, such as myoglobin, hemoglobin, neuroglobin, and cytoglobin, play a role in oxygen binding and delivery to tissues. In icefish, the loss of myoglobin and hemoglobin genes has been reported to be an adaptive evolution event. This interesting finding prompted us to exam oxygen-binding protein expression in diverse fish species. Taking advantage of substantial RNAseq data deposited in the NCBI (National Center for Biotechnology Information) database, we adopted a meta-transcriptomic approach to explore and compare four oxygen-binding protein gene expression levels in the skeletal muscle of 25 diverse fish species for the first time. RNAseq data were downloaded from the NCBI Sequence Read Archive (SRA) database, and de novo assembly was performed to generate transcript contigs. The genes encoding oxygen-binding proteins were then identified by the BLAST search, and the relative expression level of oxygen-binding protein genes was normalized by the RPKM (Reads per Kilobase Million) method. By performing expression profiling, hierarchy clustering, and principal component analysis, pacu and loach fish were noticed by their high myoglobin expression levels in skeletal muscle tissues among 25 diverse fish species. In conclusion, we demonstrated that meta-transcriptomic analysis of RNAseq data is an informative approach to compare the oxygen-binding protein expression and putative gene expansion event in fish.