A hydrodynamic antenna: novel lateral line design in the tail of myliobatid stingrays

Eagle rays, cownose rays, and manta rays (family Myliobatidae) have a slender tail that can be longer than the animal’s body length, but its function and structure are unknown. Using histology, immunohistochemistry, and 3D imaging with micro-CT scans, we describe the anatomy and function of the tail of Rhinoptera bonasus, the cownose ray. The tail is an extension of the vertebral column with unique morphological specializations. Along the tail after the barb, vertebral centra are absent, and neural and hemal arches fuse and form a solid mineralized structure that we termed caudal synarcual which imparts passive stiffness to the tail, reducing bending. Two lateral lines extend along both sides of the tail. The lateral lines are composed of a main canal connected to an extensive network of tubules, which extends and ramifies toward the dorsal and ventral tail surfaces and opens to the surrounding water via small pores. A remarkable continuous neuromast is located within each main canal. The complex lateral line sensory system in the myliobatid ray tail supports the hypothesis that the tail of myliobatids functions like a hydrodynamic sensory antenna extending posterior to the body and may play an important role in their behavioral and functional ecology.

Methods
Data published in this data set was obtained throught a combination of different methods. They were applied as follows: 

1. Histology:

Histology was used to identify the different tissues and their arrangement in the tail, sections were stained with hematoxylin-eosin (HE) combined with Orange G or with Mallory trichrome. Histology-stained samples were imaged using a Keyence digital microscope VHX-600 (Keyence Corporation) and a Leica light microscope (Leica Microsystems GmbH). Images were analyzed using Fiji 2.0.0.

2. Immunofluorescence imaged with Confocal and Fluorescence Microscope:

Immunofluorescence staining was used to identify nervous tissue within the tail by staining the sections with ß-III-tubulin antibody. For antigen retrieval, samples were incubated in 10 mM sodium citrate buffer with 0.05% Tween 20 (pH 6) for 6 hours at 60ºC. Samples were permeabilized in PBS with 0.025% Triton X-100 and blocked with 5% bovine serum albumin in PBS (BSA; Sigma Aldrich) for 2 hours at room temperature. The primary antibody β-III-tubulin (1:500; Abcam, Cat# ab18207) was used as a neuronal marker due to its previously demonstrated reactivity with catshark tissues. Samples were incubated with the primary antibody in 1% BSA in PBS overnight and then incubated for 1 hour at room temperature with the secondary antibody goat anti-mouse IgG H&L Alexa Fluor 488 (1:500; Abcam, Cat# ab150113) diluted in 1% BSA and then rinsed in PBS. Some tissue structures displayed an autofluorescence signal at 488nm. To avoid interference with the antibody signal, samples were stained with 0.1% Sudan Black B (Sigma Aldrich) following protocols established. Samples were washed in PBS to remove excess stain and mounted with SlowFadeTM Diamond antifade mountant with DAPI (ThermoFisher). This mountant with DAPI added additional autofluorescence signal when the samples were excited with a 405 nm laser, especially in the dermis. However, this did not affect the visualization of the structures of interest.

Images of sections stained with immunofluorescence were acquired using a Zeiss LSM 980 confocal microscope (Carl Zeiss GmbH) and a Leica DM R fluorescence microscope (Leica Microsystems GmbH). 

3. Micro-CT scans

High-resolution µCT scans were conducted to image in 3D the tissues forming the tail, enabling fine-scale morphological analyses. Samples fixed in 4% PFA were washed in PBS and transferred into increasing ethanol concentrations (10%, 30%, and 50%) up to 70% ethanol. High-resolution µCT images were obtained using a Bruker SkyScan 1273 (70 kV and 114 uA). Image pixel size ranged between 4.9 and 13 µm. Samples were post-stained in 0.3% phosphotungstic acid (PTA, AMRESCO) for over a month, changing PTA solution every two weeks to ensure a complete staining. After staining was complete, PTA-stained samples were transferred to 70% ethanol and re-scanned.