Assessing the size-dependent effects of microplastics on zebrafish larvae through fish lateral line system and gut damage.

This study evaluated the size-dependent effects of high-density polyethylene (HDPE) fragments in zebrafish. Larvae were exposed to HDPE microplastic (MP) in three sizes, small (14.12 µm), medium (80.32 µm), and large (120.97 µm), at 20 mg/L. Size-dependent effects in terms of MP intake, subsequent gut damage, and behavioral changes were observed. The results showed that HDPE exposure did not affect the survivability of zebrafish larvae but caused two significant changes. First, exposure to large MPs caused the most serious damage to hair cells and mechanosensory receptors in the fish's lateral line system. Second, exposure to MPs < 100 µm resulted in their ingestion by larvae, thereby causing morphological changes in the gastrointestinal tract. All larvae exposed to MPs showed behavioral pattern changes associated with size differences. This study improves our understanding of the effects of MPs on aquatic organisms and highlights the need to implement efficient strategies for plastic waste management.

Vortex phase matching as a strategy for schooling in robots and in fish.

It has long been proposed that flying and swimming animals could exploit neighbour-induced flows. Despite this it is still not clear whether, and if so how, schooling fish coordinate their movement to benefit from the vortices shed by others. To address this we developed bio-mimetic fish-like robots which allow us to measure directly the energy consumption associated with swimming together in pairs (the most common natural configuration in schooling fish). We find that followers, in any relative position to a near-neighbour, could obtain hydrodynamic benefits if they exhibit a tailbeat phase difference that varies linearly with front-back distance, a strategy we term 'vortex phase matching'. Experiments with pairs of freely-swimming fish reveal that followers exhibit this strategy, and that doing so requires neither a functioning visual nor lateral line system. Our results are consistent with the hypothesis that fish typically, but not exclusively, use vortex phase matching to save energy.

Exploring sample cross-contamination in fish epidermal mucus.

Cross-contamination of epidermal mucus was assessed at three sampling locations on the bodies of Pacific halibut Hippoglossus stenolepis by inducing contact between fish coated with labelled synthetic mucus and non-treated fish. Results indicate a positive relationship between sampling site exposure and sample contamination and that mucous sample cross-contamination can be mitigated by sampling in a location protected from external contact.

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

Localization of anosmin-1a and anosmin-1b in the inner ear and neuromasts of zebrafish.

Anosmin-1, encoded by the KAL-1 gene, is the protein defective in the X-linked form of Kallmann syndrome. This human developmental disorder is characterized by defects in cell migration and axon target selection. Anosmin-1 is an extracellular matrix protein that plays a role, in vitro, in processes such as cell adhesion, neurite outgrowth, axon guidance, and axon branching. The zebrafish possesses two orthologues of the KAL-1 gene: kal1a and kal1b, which encode anosmin-1a and anosmin-1b, respectively. Previous in situ hybridization studies have shown that kal1a and kal1b mRNAs are expressed in undetermined cells of the inner ear but not in neuromast cells. Using specific antibodies against anosmin-1a and anosmin-1b, we report here that both proteins are expressed in sensory hair cells of the inner ear cristae ampullaris and the lateral line neuromasts. Accumulation of these proteins was observed mainly at the level of the hair bundle and also at the cell membrane. In neuromast hair cells, immunogold scanning electronmicroscopy demonstrated that anosmin-1a and anosmin-1b were present at the surface of the stereociliary bundle. In addition, anosmin-1a, but not anosmin-1b, was detected on the track of the ampullary nerve. This is the first report of anosmin-1 expression in sensory hair cells of the inner ear and lateral line, and along the ampullary nerve track.