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)

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Materials and Methods

Identifying the social structure on masts

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To examine the territorial behaviour and social structure of Dyopedos bispinis on masts, 285 photos were obtained in July 2012 in various biotopes in the White Sea (Kandalaksha Gulf, Velikaya Salma Strait 66.55-66.56°N, 33.09-33.12°E, depth 8-15 m). Before the photographs were taken, a ruler with 1-mm divisions was placed on the bottom. From the photographs, the numbers of adult males, females and juveniles were counted on each inhabited mast that was in focus (790 masts), and the proportions of masts with one, two, or more than two adult amphipods were calculated. Adult amphipods were distinguished by their length (approximately 5 mm), specific body form (slender body and curved back) and brighter coloration, and adult males were identified by their strongly enlarged second gnathopods. In the cases when the maturity of the amphipods could not be accurately determined, the masts were not included in the analysis.

Over 10 dives, all masts with 3 and more adult residents were collected; the masts were plucked with forceps near the mast base and carefully placed in individual tubes that were immediately closed. In the lab, the specimens were fixed in 96% ethanol, and the amphipods on each mast were counted as described above. Collected such method adult females were distinguished by the presence of oostegites and often by the presence of eggs in the marsupium, males were identified by enlarged second gnathopods. A total of five masts were collected.

Identifying the mast substrata

To identify the substrata of Dyopedos bispinis masts, underwater observations were performed during 10 dives, and the numbers of masts attached to different types of substrata were counted inside a 0.2-m×0.2-m square frame. For a more detailed assessment, all masts inside 2 survey frames were collected in jars using forceps and a trowel and examined by binocular microscopy. Subsequently, the proportions of masts associated with each substratum were defined, and the substratum was defined for approximately 350 whips. When in focus (210 masts), the substrata were also identified from photos (see above).

Morphology of silk glands and masts

Specimen collection and fixation

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.