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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Results

Social structure of Dyopedos bispinis on the masts

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Among 1057 masts identified in underwater photos, 757 structures were inhabited (72%) and sorted into the following groups (Table 1): (1) masts occupied by one or several juveniles (195 masts, 26% inhabited); (2) masts occupied by a single adult (or subadult) female, often with several juveniles (402 masts, 53%); (3) masts occupied by 1 adult or subadult male, rarely with juveniles (30 masts, 4%); (4) masts occupied by two adults (female and male), often with juveniles (125 masts, 17%); (5) masts occupied by three adults (5 masts, less than 1%), typically two females and one male.

During dives, masts occupied by three or more (up to 23) adults were also observed (Table 2), and in one case, all 15 females inhabiting one mast were ovigerous. Such masts were rarely observed, but they were unusually long, measuring approximately 15-20 cm, and were easily detected. Ordinary masts are typically not more than 10-12 cm in length.

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Table 1. Habitancy of typical Dyopedos bispinis masts. Inhabited masts were divided into five groups according number of adult individuals on each mast, and the numbers and proportions of masts in each group are presented in the table columns. Abbreviations: f – adult female, juv – juvenile, m – adult male, * - juveniles are occasionally present.

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Table 2. Habitancy of collective Dyopedos bispinis masts. Amphipods inhabiting each mast (N1-N5) were divided into three groups: “adult females”, “adult males” and “juveniles”; their numbers are presented in the table columns.

Substrata for mast building and formation of the basal section

Dyopedos bispinis masts can be attached to solid substrata (stones, shells of dead or living molluscs and brachiopods) or to other fouling organisms including hydroids, bryozoans, ascidians and sponges; masts rarely occur on soft ground (sand and mud). Examination of the collected masts showed that the most frequent substrata are other organisms, particularly hydroids (Gonothyraea loveni (Allman, 1859), Ectopleura larynx (Ellis & Solander, 1786), Eudendrium sp., Sertularia mirabilis (Verrill, 1873)), bryozoans (Eucratea loricata (Linnaeus, 1758), Flustra sp., and Scrupocellaria sp.), sponges (presumably Leucosolenia complicata (Montagu, 1814)) and, occasionally, amphipod tubes (Crassicorophium bonellii).

On solid substrates, the bases of the masts are disk-like or weakly thickened, but on bush-like hydroid and bryozoan colonies, the masts are attached to the tips of branches (Fig. 2A-C). These masts typically coat a section of the colonies heading downward (H+M), often reaching the base, so that they appear to have been built directly on the solid substrate, without the hydroid or bryozoan.

When more than one mast attaches to a hydroid colony, a ramified mast occasionally results (arrows, Fig. 2A, D). Large masts typically have several supports; i.e., they are composed of several independent hydroid or bryozoan branches combined or even several smaller masts (m, m1, m2, m3) that are attached to different individual branches (Fig. 2A, B).

Some hydroids coated with mast remain alive, and their hydranths are free of masts and have tentacles (Fig. 2A, C). Other branches are completely immured (3, Fig. 2A).

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Fig. 1. Internal structure of a Dyopedos bispinis mast. A – drawing of a Dyopedos bispinis mast. B1, C1 and D1 - scanning electron microscopy (SEM) micrographs of mast cross sections at different levels; B2, C2 and D2 – their schematic representations. E and F – SEM micrographs of masts with damaged cortexes, showing that the cortex always comprises amphipod silk layers and, sometimes, detritus layers. Abbreviations: ASL, ASL-1, and ASL-2 – silk layers of the laminated cortex; C – central cylinder of the mast; C-1 m – the main cylinder; C-2, C-3, C-4 – additional cylinders; DL-1 – detritus layer of the laminated cortex; f – Dyopedos bispinis female; m – Dyopedos bispinis male; P – laminated cortex of the mast; P-1 – laminated cortex of the main cylinder; P-2 – laminated cortex of an additional cylinder; P-c – laminated cortex covering all cylinders.

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Fig. 2. Attachment of Dyopedos bispinis masts to hydroids and bryozoans, multi-supporting and branching masts. А – schematic drawing of two masts attached to a hydroid colony, showing that hydroid branches can be immured by Dyopedos bispinis to varying degrees; B and C – light microscopy (LM) photos showing mast attachment to bryozoans (B) and hydroids (C); D - underwater photo of a branching mast. Abbreviations: 1 – free hydroid branch; 2 – partially immured hydroid branch continuing as a single-supporting mast; 3 – completely immured hydroid branch continuing as a multi-supporting mast; arrows – branching sites of masts; arrowhead – filamentous algae included in mast cortex; fh – free hydranth; H – free section of hydroid branch; ih – partially immured hydranth; m, m1, m2 and m3 – basal masts forming multi-supporting mast; M – mast itself; H+M – basal section of mast built around a hydroid branch; bry – bryozoan branches.

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Fig. 3. Internal structure of a Dyopedos bispinis mast including diatoms and multicellular algae. А – drawing of a semi-thin mast cross-section; B – enlarged detail of A, LM micrograph; C and D – LM micrographs of semi-thin sections illustrating algae included in the central mast cylinder; E and F – SEM micrographs of mast surface showing the diatom Thalassionema nitzschioides; G – SEM micrograph of the inside of the mast laminated cortex showing the presence of numerous pelagic and benthic diatoms. Arrowheads – mucous pads connecting T. nitzschioides cells; as – silk threads; bda – likely benthic diatoms; C – central cylinder of the mast; da – diatoms; ma – multicellular algae; P – laminated cortex of the mast; pda – likely pelagic diatoms; Tn – diatom Thalassionema nitzschioides.

Structure of masts

Mast forms

Dyopedos bispinis structures are representative of the masts of typical dulichiids (Fig. 1A); they are usually 2-6 cm long and rarely reach 16-20 cm. Except for the smallest, masts have variable diameters but reach a maximum at the base and a minimum nearer the tip. The diameter in the middle of the masts varies between 0.02 (smallest) and 0.1 cm.

Internal mast structure

Masts comprise a relatively homogeneous central cylinder (C, diameter 70-460 µm) and a laminated cortex (P, from 440 µm to 20 µm thick and thinner), but the boundary between these structures is occasionally fuzzy (Fig. 1A-D and Fig. 3A). The size of the cortex varies greatly and might account for approximately 0 to 70% of the mast diameter. Along the mast length, the diameter of the cylinder varies only slightly (190-270 µm), but the cortex gradually thins toward the tip of the mast (Fig. 1A-D).

The lower portion of the mast often contains several central cylinders (several smaller masts, C1-C4, Fig. 1A, D), and one cylinder is typically thicker than the others (C-1 m). Each cylinder is coated with its own cortex (P-1, P-2), but together they share one common coat (P-c).

The central cylinder of the mast is composed of closely packed detritus; we did not detect any cementing mucus. The cortex of the masts contains amphipod silk (as) and detritus. A thin cortex (up to 20 µm) might be almost entirely composed of silk (Fig. 1A-B, Fig., 4A), but when the cortex is even slightly thicker, the proportion of detritus is much larger than that of silk (Fig. 1A, C, F). The detritus is represented by isolated or groups of particles surrounded by silk (Fig. 3A-B) or detritus layers (DL) alternating with silk layers (ASL, Fig. 1F). Under mechanical impact, the cortex of the dried mast exfoliates and becomes separate layers of approximately 5 µm in thickness (Fig. 1E).

Numerous diatoms are present within masts (da, bda, pda, Tn, Fig. 3D-E). Some are pelagic (pda, Tn), and others are large and likely benthic species (bda). Diatoms have not been observed on the mast surface outside the silk layer.

Fine mast structure

The mast surface is covered with a layer of silk threads, and the thickness of the silk layer varies (Fig. 4A-C). The silk threads are oriented in all directions and are sometimes almost parallel to the mast axis. Frequently, regular shifts in the parallel threads (approximately 5 µm) are observed (arrowheads, Fig. 4B-C). Furthermore, TEM photos suggest that the silk layer contains sublayers (L1-L6) that likely differ based on their angle of orientation (various directions or perpendicular to the mast axis), thickness and density (Fig. 4E).

Silk threads are 0.1-0.3 µm in diameter, and threads of varying widths (t1, t2) might be observed on the same mast (Fig. 4D). However, we did not observe threads with changing diameters in the photos. The surfaces of the analysed mast tips were covered with thinner threads (0.1 µm) (arrows, Fig. 4A), whereas the remainder of the mast was coated with 0.3-µm threads (arrowheads, Fig. 4B-C).

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Fig. 4. Ultrastructure of Dyopedos bispinis mast surface. А, B, C and D – SEM micrographs of the mast surface near the distal tip (A) and other parts of the mast (B, C and D) showing that silk threads were thinner near the mast tip (arrows, A) than elsewhere (arrowheads, B and C), that threads of varying widths are sometimes presented (t1 and t2, D), that detritus can be visible through the silk layer (B) or not (C) and that regular parallel silk threads are sometimes observable (black and white arrowheads, B and C); E – transmission electron microscopy (TEM) micrograph of an ultra-thin mast cross section showing numerous amphipod silk layers (L1-L6) on the mast surface. Abbreviations: arrows – thin threads; arrowheads – regular parallel thick silk threads; С – central cylinder in mast cross-section; L1-L6 – layers of silk threads (L1 and L3 – threads, likely lying perpendicular to the mast axis; other – at an angle); P – mast cortex in cross section; t1 and t2 – threads of varying widths.

Silk gland morphology

Silk gland general morphology ( Fig. 5A )

Silk glands have been detected in pereopods 3-4 of adult females, males and juveniles (including the youngest ones), and the secretory units (D1, D2) are situated in pereopod segments 2-5 (basis to carpus). The ducts are grouped into a single bundle (DB) leading to the dactylus (segment 7), where this bundle connect to a common chamber (Ch) opening at the dactylus tip (arrow, Fig. 5A). The ducts are lined with epicuticle (2, Fig. 6E, G, I), whereas the chamber has a normal cuticle comprising endo-, exo- and epiuticle.

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Fig. 5. Dyopedos bispinis pereopodal silk-producing glands, schematic reconstructions from serial sagittal, semi-thin sections. A – Distribution of glands in female Dyopedos bispinis 3-d or 4-th pereopod, showing two gland groups, proximal (D1) and distal (D2), and their ducts falling into a common chamber in the tip of the leg; B – duct system of the D1 glands in the basis of pereopods 3 or 4, female. Abbreviations: arrow – common opening of all pereopodal glands; arrowhead – point of coalescence of two D1 main ducts; Ch – dactylar chamber; D1 – proximal glands (D1); D2 – distal glands (D2); DB – duct bundle; LD – lateral duct of D1 gland; m – pereopodal muscles; MD – main duct of D1 gland. Arabic numerals – number of pereopod segments; 2 – basis; 3 – ischium; 4 – merus; 5 – carpus; 6 – propodus; 7 – dactylus.

There are two distinct gland groups in each pereopod 3 or 4: proximal (D1) and distal (D2) (Fig. 5A). The D1 secretory granules stain more intensively with the mix of toluidine and methylene blue and are electron-dense (sg, Fig. 6, 7) compared with the D2 granules (sg, Fig. 8).

Each female pereopod contains approximately 70 secretory cells.

Proximal glands (D1) ( Fig. 6 , 7 )
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Fig. 6. Structure of Dyopedos bispinis proximal pereopodal glands (D1). A – 2D schematic reconstruction of D1 gland fragment from serial longitudinal semi-thin sections; B – fragment of D1 gland, longitudinal semi-thin section, LM micrograph; C – D1 gland, semi-thin cross section; D – fragment of secretory cell cytoplasm (TEM photo) showing secretory granules; E – D1 gland, schematic drawing of ultra-thin cross section; F, G, H, I and J – enlarged details indicated in E (TEM photo) showing axon terminal adjacent to secretory cell (F); boundaries between secretory, duct and lining cells (G); fragment of accumulation site (H); fine structure of main and lateral ducts (I); and differences in ultrastructure of secretory, lining and duct cells (J). Abbreviations: black arrowheads – microtubules in duct and nerve cells; white arrowheads – vesicles in nervous cell; as – accumulation site; c – surface cuticle of pereopod; cz – central area of secretory cell with abundant secretory granules; d – ductules in secretory cell; dw – duct wall, including duct cell and lining; er – endoplasmic reticulum; h – haemocoel; hyp – hypoderm; LD – lateral duct; m – pereopod muscles; MD – main duct; mt – mitochondria; nu - nucleus of secretory cell; pz - peripheral area of secretory cell cytoplasm with rare secretory granules; s – secretion in duct lumen; SC – secretory cell; sg – secretory granules in secretory cells; sg-1, sg-2 - secretory granules, differing in electrone dense; ? – fragments of glandular tissue damaged during fixation. Arabic numerals – details of duct wall; 1 – boundary between secretory and duct cells; 2 – epicuticle lining the main and lateral ducts; 3 – grainy electron-dense layer of duct cell; 4 – electron-lucent layer of duct cell; 5 and 6 – boundary between duct and secretory cells, comprising one (5) or two (6) membranes, respectively; 7 – boundary between secretory and lining cells; 8 – boundary between lining and duct cells damaged during fixation.

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Fig. 7. Cellular membrane invaginations in D1 secretory cells. A and B – schematic representations of two consecutive serial ultra-thin sections (with a 3-5 µm interval) through the D1 gland, showing that the invaginations (i) are likely flat and perpendicular to the section plane; C – TEM micrograph illustrating deep secretory cell invaginations filled with lining cell extensions; D - TEM micrograph of the widening at the tip of an invagination; E and F - TEM micrographs showing different forms of lining represented as multiple overlapping cytoplasmic extensions (E) or as a continuous cytoplasmic layer (F). Abbreviations: arrowheads – overlapping lining cytoplasmic extensions; as – accumulation site; c – cuticle; er – endoplasmic reticulum; h – haemocoel; hyp – hypoderm; i – cellular membrane invaginations of secretory cells; LC – lining cell; mt – mitochondria; nu – nucelus of secretory cell; SC, SC-1 and SC-2 – secretory cells; sg – secretory granules; ? – fragments of tissue damaged during fixation or embedding.

D1 gland location

Female D1 secretory units are only located in the pereopod basis (segment 2), and in males, these structures were also found in the merus (segment 4). The D1 secretory ducts lead to the dactylus (segment 7) (Fig. 5A).

D1 gland structural plan

D1 secretory units comprise approximately 60 secretory cells (SC) lying along 5-6 main ducts (MD, Fig. 5B, Fig. 6A-C), and short (5-8 µm) lateral canals (LD) individually branch away from the main duct to each secretory cell (Fig. 6A-C, E). Each lateral duct terminates into an intracellular globular accumulation site (as) that is 7-9 µm in diameter (Fig. 6A-C, E) and contains numerous cavities. TEM revealed that the accumulation site is composed of ramifying ductules (d) radiating from the end of the lateral duct (Fig. 6E, G-H).

Secretory cells do not contact the haemocoel and are covered with a lining (LC) composed of a strongly flattened cellular layer (Fig. 6E, J and Fig. 7).

D1 gland duct structure

Ductules within the accumulation site are situated directly in the secretory cell, and the lumens are separated from the cytoplasm by a thin (approximately 0.001 µm) electron-dense layer (Fig. 6E, G-H). It is not clear whether this layer comprises only membrane or if it also contains epicuticle; microvilli are lacking.

The main and lateral ducts are surrounded by a nonstaining sheath (duct wall, dw), which has a variable thickness of up to 3 µm (Fig. 6A-C). TEM (Fig 6E, G, I-J) revealed that the duct wall of each D1 gland comprises two cells: “internal” and “external”. The internal cell (which is really a duct cell, DC, Fig. 6E) contacts the duct lumen and the external cell and adjoins the secretory cell near the distal end of the lateral duct. The “external cell” (lining cell, LC) completely covers the “internal cell” and contacts secretory cells and the haemocoel; it is included in the gland lining (Fig. 6E, J). The cytoplasm of the duct (“internal”) and lining (“external”) cell is separated by two membranes (6, Fig. 6E, I), but the longitudinal sections of the lateral duct showed only one membrane between these structures (5, Fig. 6E, G). Thus, the structures what we call “duct” and “lining cells” are possibly components of a single complicated cell.

Duct cells (inner part of the duct wall) comprise the next layers (Fig. 6E, I): (1) the innermost thin electron-dense (0.0015-0.003 µm) layer, which likely includes the membrane and epicuticle; (2) a grainy electron-dense layer (0.1-0.3 µm), likely comprising actin filaments; (3) a wide electron-lucent layer (3-4 µm) containing microtubules, parallel to the duct axis, and occasionally with membrane structures; and (4) an outer cellular membrane. These layers are continuous and closed, and the radial connection between the inner and outer cellular membranes (mesaxon) is absent. The cell cytoplasm contains microtubules and different membrane structures (Fig. 6E, I, J).

The intracellular location of the main ducts remains in the subsequent appendage segments.

Secretory product was observed in the lumens of the intracellular ductules, lateral and main ducts (s, Fig. 6B-C, E, G-I). In each lumen, this product is aggregated into compact bodies, and its electron density and structure are similar to those of the secretory granules in secretory cells (sg, Fig. 6D, H and Fig. 7C-D).

D1 secretory cell structure

Secretory cells (Fig. 6A-C) vary in form and size (cell diameter of 5-35 µm) and are mononuclear. The semilunar nucleus (nu) is located near the centre of the cell, opposite the lateral duct (Fig. 6A-C). Secretory cells contain numerous secretory granules (sg, Fig. 6B, E, H) situated densely in the area adjacent to the accumulation site (cz) and more sparsely at the periphery (pz, Fig. 6B). Mitochondria (mt) and granular endoplasmic reticulum (er) are situated in the spaces between the granules (Fig. 6D, J and Fig. 7C).

Secretory granules (sg, Fig. 6B, D, H, and Fig. 7C) have permanent circular forms (approximately 0.6-1.0 µm in diameter) and stain intensely with a mix of toluidine and methylene blue. The granular material in the thin sections is electron dense and can be heterogeneous to varying degrees (sg-1, sg-2 in Fig. 6D, and sg in Fig. 7C).

The secretory cell membrane forms invaginations (i) filled with extensions of lining cell cytoplasm (Fig. 7A-D), and these invaginations can be significantly deep, reaching the accumulation site. The invaginations are narrow (0.05-0.15 µm) but widen at some sites (Fig. 7C, D), particularly at the end/tip of the invagination (Fig. 7D). Additionally, the invaginations occasionally branch (Fig. 7B). The pattern of invagination distribution was observed in two consecutive serial sections (with a 3-5-µm interval) perpendicular to the lateral duct planes (Fig. 7A-B). Therefore, we propose that these invaginations have a flattened form and are located in the plane of the lateral duct.

D1 lining structure

The lining cells (LC), located between the secretory cells and the haemocoel (h) (Fig. 6C, E, J, and Fig. 7), are strongly flattened (approximately 0.2-0.4 µm in thickness) but expand to surround the gland ducts (Fig. 6C, E) or to hold the nucleus or mitochondria. These cells form extensions into (see above) and between the secretory cells.

In some sections, the lining appears as a continuous cytoplasmic layer (Fig. 7F), while in other sections, the lining comprises multiple overlapping cytoplasmic extensions (arrowheads, Fig. 7E). The lining cytoplasm contains microtubule bundles, membrane vesicles or elongate structures (Fig. 7C, E-F). Near the accumulation site between the secretory and duct cells, we detected a structure (Fig. 6F) containing numerous microtubules (black arrowheads) and electron-dense round structures 0.006-0.1 µm in diameter (white arrowheads) that is likely axon terminal.

Distal glands (D2) ( Fig. 8 )

D2 gland location

The D2 secretory cells in adult females are located in the 2-5 appendage segments (basis-carpus) (Fig. 5A, and Fig. 8A) in three distinct cell groups along the bundle of D1 and D2 ducts. The largest group contains 10-11 cells and begins in the basis (segment 2) and ends at the merus (segment 4). Two other groups, comprising 1-3 cells, are situated in the carpus (segment 5) (Fig. 5A and Fig. 8A).

D2 glands structure

Inside each D2 secretory cell, there is an accumulation site (as) with a duct (D, resembling a D1 gland lateral duct) leading away (Fig. 8A). This duct is lost in the duct bundle (DB), and it is unclear whether it falls into any other ducts or remains separate. The accumulation site, as in the D1 glands, comprises numerous radiating ductules (d, Fig. 8E). The gland duct has a separate wall, likely containing a duct cell cytoplasm, but the wall ultrastructure has not been investigated.

The secretory cells (SC) are 25-35 µm in size, uninuclear, and filled with secretory granules (sg), and in the cell periphery, the granules are situated less densely than near the accumulation site. The space between the granules was observed to be heterogeneous, suggesting that this space is filled with poorly preserved endoplasmic reticulum (er, Fig. 8C-D). Secretory granules are occasionally round, but when densely situated, the shape changes (Fig. 8D). These cells lightly stain with toluidine blue and contain loose electron-lucent material. Within one cell, the secretory granules vary in their electron density and the regularity in which the material is arranged (sg1-3, Fig. 8D). We did not observe any regularity in the different granules distributed throughout the cell. Cell membrane invaginations were not observed in D2 secretory cells.

FIG2

Fig. 8. Structure of distal pereopodal glands of Dyopedos bispinis (D2). A – reconstruction drawings from semi-thin serial sections; B – D - TEM micrographs. A - D2 gland distribution; B – periphery of D2 secretory cell directly adjoining the hypoderm; C - periphery of two D2 secretory cells and the boundary between them; D – different types of secretory granules in D2 cell cytoplasm; E – accumulation site of D2 secretory cell. Abbreviations: as – accumulation site; b - boundary between two D2 secretory cells; c – cuticle; D – duct; d – ductules forming the accumulation site; DB – bundle of D1 and D2 gland ducts; er – endoplasmic reticulum; SC – secretory cell; sg, sg-1, sg-2 and sg-3 – different types of secretory granules. Arabic numerals – number of pereopod segments; 2 – basis; 3 – ischium; 4 – merus; 5 – carpus.