Contributions to Zoology, 86 (2) – 2017Nikolai Y. Neretin; Anna E. Zhadan; Alexander B. Tzetlin: Aspects of mast building and the fine structure of “amphipod silk” glands in Dyopedos bispinis (Amphipoda, Dulichiidae)
Materials and Methods

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Morphology of silk glands and masts

Specimen collection and fixation

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Material was collected from the White Sea (Kandalaksha Gulf, Velikaya Salma Strait 66.55-66.56°N, 33.09-33.12°E, depth 8-15 m) in July 2012 by SCUBA diving. Masts with amphipods were plucked using forceps and carefully placed in glass jars, and pereopods 3-4 were detached from the amphipod body and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and then post-fixed in osmium tetroxide (OsO4 ). Before or after the first glutaraldehyde fixation, an incision was made on the pereopods, and they were dissected into 2-3 fragments using a razor blade (after Kronenberger et al., 2012a,b) for better penetration. The samples were then dehydrated in an ethanol series and embedded in epoxy resin (Epon 812). For transmission electron microscopy (TEM), the masts were fixed as above, and for light microscopy and SEM, they were fixed in 10% formalin in seawater (4% formaldehyde).

Light microscopy

Serial semi-thin sections (0.9 mm) of masts and pereopod fragments were cut using DuPont MT 5000 and LKB-III microtomes followed by staining with a mixture of methylene blue and toluidine blue. The pereopod sections were examined using a Carl Zeiss Axioplan 2 imaging light microscope, and the masts sections were examined using a Leica DM 2500 microscope. Digital images were captured using an AxioCam HRm camera for pereopods and a Leica DFC 290 camera for masts. Serial images were aligned using Amira 5.3.2. Altogether, 4 pereopod and 2 mast fragments were dissected.

The lengths and widths of the collected masts were measured using a ruler and micrometre eyepiece scale; a total of 146 masts were analysed.

Transmission electron microscopy

Ultra-thin sections were cut using a Leica Ultramicrotome, collected on formvar-covered single slot copper grids, stained with uranyl acetate and lead citrate, and examined with a JEOL JEM-1011 electron microscope.

Scanning electron microscopy

The fragments of 4 masts were dehydrated in an ethanol-acetone series, critical point dried with CO2 in a Hitachi HCP-2, mounted on aluminium stubs, and sputter-coated with an Au-Pd mixture using an Eiko IB-3 ion coater. To examine the internal structure, thick mast sections (0.5-2 mm) were cut using razor blades prior to dehydration, and for one mast (approximate length of 70 mm), serial sections were generated (each 5-15 mm). The specimens were examined using a Cam Scan S-2 scanning electron microscope.