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1 Morphology and ultrastructure of the midgut gland ("hepatopancreas") during
2 the ontogeny of the Atlantic spider crab Maja brachydactyla Balss, 1922
3 (Brachyura, Majidae).
4 Diego Castejón 1,2* , Guiomar Rotllant 2, Javier Alba-Tercedor 3, Maria Font-i-Furnols 4,
5 Enric Ribes 5, Mercè Durfort 5, and Guillermo Guerao 6
6 1IRTA, Centre d’Aqüicultura, Ctra. del Poble Nou Km 5.5. 43540 Sant Carles de la
7 Ràpita, Tarragona (Spain).
8 2CSIC, Institut de Ciències del Mar, Passeig Marítim de la Barceloneta 37-49. 08003
9 Barcelona (Spain).
10 3Departamento de Zoología, Facultad de Ciencias, Universidad de Granada, Campus de
11 Fuentenueva, Av. de Fuente Nueva s/n. 18071 Granada (Spain).
12 4IRTA, Qualitat del Producte, Finca Camps i Armet. 17121 Monells, Girona (Spain).
13 5Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular, Fisiologia i
14 Immunologia, Facultat de Biologia, Universitat de Barcelona, Diagonal 645. 08028
15 Barcelona (Spain).
16 6Independent Researcher. Barcelona (Spain).
17 Short title : Maja brachydactyla midgut gland morphology and ultrastructure
18 *Corresponding author: Diego Castejón. E-mail: [email protected];
19 phone: +34 681 154 398; postal address: CSIC, Institut de Ciències del Mar, Passeig
20 Marítim de la Barceloneta 37 -49. 08003 Barcelona (Spain). 21 Abstract .
22 This study is focused in the study of the midgut gland (MGl) of the Atlantic
23 spider crab Maja brachydactyla Balss 1922 along different life stages (zoea, megalopa,
24 juvenile and adult) employing stereomicroscopy, optical microscopy, electron
25 microscopy , computed tomography and micro -computed tomography (micro -CT) . The
26 MGl of the newly hatched larvae is formed by fourteen blind -end tubules , t he number of
27 tubules start to increase in the late megalopa. The MGl of the adults is composed by
28 tens of thousands blind -end tubules. The most important morphological change
29 observed in the MGl during the larval development is the lengthening of the tubules. In
30 all the life stages the tubules are surrounded by a square -net network of muscle fibers.
31 Fo r first time five cell types are described in the MGl of a decapod during the larval
32 stages . T hese cell types show a similar location , histology and ultrastructure in larvae
33 and adults: embryonary (E -) cells, resorptive (R -) cells, fibrillar (F -) cells, bl ister like
34 (B -) cells, and midget (M -) cells . For first time, microapocrine secretion is observed in a
35 digestive organ of a decapod species. T he function of each type of epithelial cells might
36 be similar in all the life stages since no ultrastructural changes were observed during the
37 ontogeny . The role of each epithelial cell type in larvae and adults is discussed.
38 Keywords : Crustacea; ontogeny; digestive system; micro-CT; microapocrine
39 secretion 40 1. Introduction
41 The midgut gland is the largest digestive organ in Decapoda and it is formed by
42 several blind -end tubules (Felgenhauer, 1992 ; Gibson and Barker, 1979 ; Icely and Nott,
43 1992 ). This organ receives several and different names, but traditionally has been
44 known as "hepatopancreas" (Davie et al., 2015 ; Gibson and Barker, 1979 ; Milne-
45 Edwards, 1834a , b; Schlegel, 1911 ; Van Weel, 1974 ). Cervellione et al. (2017b)
46 concluded that the term “hepatopancreas” is inappropriate because this organ inherently
47 differs from the liver and p ancreas of vertebrates and proposed the term "perigrastric
48 organ" . Although we agree with this conclusion , in this study we preferred to use the
49 term "midgut gland" since it is largely used in former studies and "perigrastric organ"
50 terminology is still under discussion .
51 The epithelium of the tubules that forms the MGl is constituted by different cell
52 types : E-cells (embryonic) responsible of cell division and differentiation, F -cells
53 (fibrillar) with "striated" cytoplasm, R -cells (resorptive) rich in lipid vacuoles and
54 glycogen, B -cells (blister like) characterized by a giant supra -nuclear vacuol e (Al-
55 Mohanna and Nott, 1989 ; Felgenhauer, 1992 ; Icely and Nott, 1992 ; Loizzi, 1971 ), and
56 short M -cells (midget) that cannot reach the organ lumen (Al-Mohanna et al., 1985a ).
57 The morph ology, ultrastructure and role of these cells in adult decapods ha ve been
58 largely d iscussed by several authors during decades (Gibson and Barker, 1979 ; Hu and
59 Leung, 2007 ; Van Weel, 1974 ; Vogt, 1996 ) but they have received significantly less
60 attention during the larval stages . In brachyuran crabs even few er studies are focused on
61 the ontogeny of the midgut gland (Li and Li, 1998 ).
62 In relation to the spider crabs of the genus Maja , the general morphology of the
63 MGl was previously described by Milne-Edwards (1834b) for the adults and by
64 Schlegel (1911) for the first zoeal stage. In these studies a detailed cellular description 65 of this organ is missing as well as the function of the different cell types. Here, we study
66 the MGl of the Atlant ic spider crab Maja brachydactyla Balss 1922 along different life
67 stages (zoea, megalopa, juvenile and adult) employing stereomicroscopy, optical
68 microscopy, electron microscopy , computed tomography and micro -CT . The role of
69 each epithelial cell type in larvae and adults is discussed.
70 2. Material and methods
71 2.1 Broodstock and larval culture systems
72 The adults were caught in Atlantic waters by order to fishery companies
73 (LONXANET, CADEMAR) , transported to the Institut de Recerca i Tecnologia
74 Agroalimentàries facilities (IRTA, Sant Carles de la Ràpita, Tarragona, Spain) , and
75 dissected at their arrival or used as broodstock. The broodstock was kept in 2,000 L
76 cylindrical tanks connected to a r ecirculation unit (renewal rate = 3.5 m 3 h-1)
77 maintaining constant environmental conditions: 18 ± 1 °C, salinity of 35 ± 1,
78 photoperiod = 12 h light photoperiod, and f ed with frozen mussels ( Mytilus sp.). The
79 larvae were recovered 12 hours after hatch ing a nd maintained in 600 mL glass beakers
80 placed inside 360 L tanks (96 x 96 x 40 cm) used as incubation chambers following the
81 next conditions: 21 ± 1 °C, salinity of 35 ± 1, 12 h light photoperiod, and fed with
82 Artemia sp. Kellogg, 1906 nauplii (INVE Aquacul ture Nutrition, Salt Lake UT, USA ).
83 The larvae reached the zoea II 3-4 days after hatching (dah), megalopa 6-7dah and the
84 first juvenile 11 -12 dah. Zoea I was sampled as newly hatched (0 dah).
85 2.2 Gross morphology
86 The morphometric study employed six adult specimens. The adults were
87 maintained in cold conditions during 30 -60 min ( i.e. ice filled bucket placed inside a
88 refrigerator room at around 5 ºC) before dissection. We proceed to retire the carapace of 89 the adults to expose the midgut gland using small pliers. The entire fresh MGl of each
90 specimen was weighed. Then samples of each MGl were taken, weighed and fixed in 4
91 % formaldehyde. The fixed samples were weighed again to calculate the relative weight
92 variation due to the fixative process . S ubsamples with known weight (between 15 and
93 60 mg) were taken to count the number of tips of the blind -end tubules to calculate the
94 density of tubules as number of tubules per mass unity . T his value was extrapolated to
95 calculate the number of tubules per MGl . The weight measures were realized using a
96 Mettler Toledo AE200 balance (range: 0 - 205 g, precision: 0.1mg). The observations
97 were realized under a Nikon SMZ800 stereomicroscope.
98 The larvae were sampled at the start of each stage and immediately fixed in 4 %
99 formaldehyde . The larvae were dissected using teasing needles ; between 4 and 6 MGl s
100 per stage were extracted, photographed and measured employing AnalySIS® software
101 tools (Soft Im aging System, Münster, Germany). The measures were realized as
102 describ ed below (see Suppl. Fig. 1):
103 2.2.1. Measures realized on the ventral sub-lobule (VSL): total length (TL) and
104 maximum width (MW), these measures are important since this central tubule
105 represents the main volume of the organ .
106 2.2.2. Measures of the lateral tubules have been differentiated in external (ET)
107 and internal tubules (IT) in relation to their location. In the case of the ET the following
108 measures were taken: total length (ETl) and width (ETw); while in the case of the IT the
109 measures were: total length (ITl) and width (ITw). The lateral tubules are projected in
110 diverse angles, so to take standardized measures we considered the width as the basal
111 line that separates the lateral tubule from the central tubule and total lengt h as the
112 maximum perpendicular distance from the basal line to the tip of the lateral tubule. 113 2.2.3. Measures of the dorsal sub-lobule (DSL): total length (DSl), width (DSw)
114 and height (DSh).
115 The data obtained were analyzed using R version 3.2.0 ( R Development Core
116 Team, 2015 ). One way ANOVA was performed using as factor one measure value (TL,
117 MW, DSl, DSw, DSh, ETl, ETw, ITl, or ITw) per analysis. Comparisons among life
118 stages (zoea I, zoea II, megalopa, first juvenile) after finding significant differences
119 were performed by Tukey -HSD test. Normality and homogeneity were tested by
120 Shapiro -Wilk and Levene tests. The critical level (α) to re ject the null hypothesis was
121 0.05.
122 2.3 Optical microscopy
123 The material selected were entire specimens of each larval stage and first
124 juvenile , in the case of the adults we re selected small pieces of MG l. The fixative
125 employed for all samples was Davidson's (ethanol absolute: seawater: formaldehyde 37
126 %: glycine: glacial acetic acid in proportion 3: 3: 2: 1: 1). The fixation process required
127 24 h, and then the fixed tissues were preserved in ethanol 70 % . The dehydration and
128 paraffin infiltration was realized using an automatic tissue processor ( Myr, Spain). The
129 blocks were form ed in a paraffin processor ( Myr, Spain ) and a microtome (Leica
130 RM2155) was employed to obtain 2 µm sections . The staining pro cedures were : 1.
131 Hematoxylin -Eosin (HE) , to show the general morphology of the tissue. 2. Periodic
132 Acid –Schiff (PAS) contrasted with Methylene B lue , to reveal the presence of
133 polysaccharides, neutral muco polysaccharides and the structure of the cuticle line. 3.
134 Mallory's trichrome stain ( Acid Fuchsine , Orange G and Aniline Blue stain s), to reveal
135 the structure of the muscular and connective tissues . The stained sections were observed
136 under an optical microscope (Leica LB30T 111/97) . The pictures were taken using a 137 camera (Olympus DP70) and image analyzing system software: DP Controller 2.1.1.83
138 with DP Manager 2.1.1.163 (Olympus Corporation, Germany) .
139 2.4 Electron microscopy (TEM and SEM)
140 For transmission electron microscopy (TEM), the material selected were entire
141 specimens of each larval stage and first juvenile stage , while for scanning electron
142 microscopy (SEM) we dissected the MGl of these stages. In relation to the MGl of the
143 adults, we used small pieces of this organ for the fixation for TEM and SEM. The
144 mate rial was fixed in 2 % paraformaldehyde and 2.5 % glutaraldehyde in cacodylate
145 buffer (0.1 mol L -1 pH 7.4) . The fixation process required around 12 h at 4 °C and
146 constant darkness . Fixed samples were rinsed twice with cacodylate buffer (0.1 mol L -1
147 pH 7.4) , post -fixed in 1 % osmium tetroxide, and then dehydrated in a graded series of
148 acetone. For TEM , the samples were embedded in Spurr’s resin, semi -thin sections were
149 obtained using a Leica UCT ultra -microtome and stained by Bromophenol Blue. Then ,
150 the ultra -thin sections (50 - 70nm) were made using the same ultra -microtome and
151 counterstained with uranyl acetate and lead citrate. Observations were made with a
152 JEOL EM -1010 transmission electron microscope (tungsten filament, 80 kV). For SEM ,
153 the post -fixed sam ples after the critical -point -drying were mounted on SEM stubs with
154 self -adhesive carbon stickers and covered by carbon coating. Observations were made
155 with a JEOL JSM -7001F scanning electron microscope (15 kV). The post -fixative
156 treatment and TEM and SEM observations were realized at CCiTUB (Hospital Clinic,
157 University of Barcelona, Barcelona).
158 2.5 Computed tomography and micro-computed tomography (micro-CT)
159 Two protocols were employed for the computed tomography. In the case of the
160 adults, one male and o ne female were scanned with the computed tomography 161 equipment General Electric HiSpeedZx/I (GE Healthcare, Boston, Massachusetts,
162 USA). The scanning parameters were setup as: helical acquisition, pitch 1, 140 kV, 150
163 mA, displayed field of view 250 mm, 3 m m thickness and reconstruction algorithm
164 STD+. Image analysis was performed with the Centricity Radiology RA600 v.7
165 software (GE Medical Systems Information Technologies, Inc., 8200 W, Tower Ave
166 Milkwaukee, WI, USA). This activity was realized at the Insti tut de Recerca i
167 Tecnologia Agroalimentàries facilities (IRTA) of Monells (Girona, Spain). In the case
168 of the larvae, zoeae were fixed in 2 % paraformaldehyde and 2.5 % glutaraldehyde in
169 cacodylate buffer (0.1 mol L -1 pH 7.4) and megalopae were fixed in ethanol 70 % . The
170 fixed larvae were preserved in isopropanol 100 %. Following , the specimens were
171 transferred to a solution of iodine 1 % in ethanol absolute during 72 h, then submerged
172 in hexamethyldisilazane for 4 h and overnight air dried. To maintain t he animals on the
173 sample holder, different methods have been essayed (Alba-Tercedor and Sáinz-Cantero,
174 2012 ). For tiny samples such as the zoea larvae, the best results were obtained by gluing
175 the animal with cyanoacrylate to the tip of a nylon filament line of 200 µm diameter.
176 The m egalopa were mounted inside of a holed piece of BASOTECT® (melamine resin
177 foam, created by the Chemical Company BASF). All samples were enclosed inside a
178 plastic straw, to avoid any movement due to the forced refrigerating air during the scan
179 process. The Basotect’s material results of very low density, thereafter very transparent
180 to X -Ray , and therefore it can be easily eliminated during the segmentation procedure
181 (Alba-Tercedor and Alba-Alejandre, 2017 ). A SkyScan 1172 high resolution
182 microtomographer (Bruker microCT - formerly Skyscan -, Kontich, Belgium), with a
183 Hamamatsu 80/250 source a nd a VDS 1.3Mp camera was used. The scanning
184 parameters for the zoea were setup as: isotropic voxel size of 1.48 µm per pixel, 49 kV,
185 78 µA, rotation step of 0.3° and 180º of rotation scan. The scanning parameters for the 186 megalopa were setup as: isotropic voxel size of 1.47 µm per pixel, 54 kV, 85 µA,
187 rotation step of 0.5° and 180º of rotation scan. For primary reconstructions and the
188 "cleaning" process to obtain the datasets of cross -sectional images (‘slices’), we used
189 the latest versions of the free Bruker microCT , formerly Skyscan (www.skyscan.be)
190 software (NRecon, DataViewer, CTAnalyser); then volume renderings were obtained
191 using Skyscan’s software CTVox. For a more detailed description of the process see
192 Alba-Tercedor (2014) . This activity was realized at the Department of Zoology of the
193 University of Granada, Spain.
194 3. Results
195 General morphology. The observation of the midgut gland (MGl) of M.
196 brachydactyla by transparency and micro -CT during the larval stages reveal that th is
197 organ is located in the middl e of the cephalothoracic cavity covering partially the
198 pyloric stomach (Fig. 1; Suppl. Fig. 2A-B, D -E) . The gross morphology is similar
199 during the entire larval development , as observed by dissection, micro -CT, and SEM
200 (Figs. 1 ; 2 B-C; 3; Suppl. Fig. 2). The MG l present s two asymmetrical lobules (right and
201 left) , each one subdivided in to a dorsal and a ventral sub -lobules (Fig s. 1; 2 B-C; Suppl.
202 Fig. 1). The dorsal sub -lobule is flattened laterally and composed by a single anterior
203 tubule and a pair of posterior tubule s. The ventral sub -lobule has a "trident -like" shape
204 showing a single posterior tubule and three anterior tubules: external, central and
205 internal (Fig. 2B-C; Suppl. Fig. 2 ). The internal tubules form a basement where rest the
206 pyloric stomach (Fig. 1; Suppl. F ig s. 2C, F ; 3C-D). The MG l of the newly hatched
207 larvae has fourteen tubules, the new tubules start to develop as small buds in the late
208 megalopa ( around the ninth or tenth day of development at 21°C). The MG l growth is
209 allometric : the tubules elongated significantly during the larval development, while the
210 width i ncreases marginal ly in comparison (Table 1, Fig. 3). In this sense, the 211 relationship between length and width increases significantly (right lobule: F 3,16 = 12.46
212 p < 0.001; left lobule: F 3,16 = 24.43 p < 0.001) from two in the newly hatched larvae to
213 four in the newly molted juvenile.
214 The midgut gland of the adults of M. brachydactyla is a large and ubiquitous
215 organ that occupies a great volume of the cephalothoracic cavity (Fig. 2A; Suppl. Fi g.
216 3A-B). The MG l is divided in right and left lobes, each one extended inside the
217 cephalothora cic cavity into anterior, lateral and posterior projections (Fig. 2A). The
218 MG l of the adults is composed by a mean of 46,000 blind -end tubules (ranging from
219 35,000 to 60,000 tubules in specimens with a mean carapace length of 135 ± 8 mm ) at a
220 density of 2.3 ± 0.8 tubules per mg of fresh MG l. The tubules are branched in a coral -
221 like pattern (Fig. 1 D). The tubules of the adults are surrounded by thin muscle fibers
222 with two orientations : circular and longitudinal (Fig. 2E). The intimate relation between
223 epithelial cells and muscle fibers impl ies that the epithelium forms basal folds that fit
224 with the se fiber s (Fig s. 2E; 4 F). The circular muscle fibers form parallel rings separated
225 by intervals of 47.58 ± 8.33 µm along the tubule length , while the longitudinal muscle
226 fibers are parallel to the longitudinal axis at intervals of 9 ± 2 µm . T he longitudinal and
227 circular muscle fibers are connected forming a rectangular network (Fig. 2E). Similar
228 morphology was observed in the larvae although longitudinal musculature is less
229 developed (Suppl. Fig. 4 ).
230 Morphology and ultrastructure. The morphology of the epithelial cells is similar
231 in the larvae and fi rst juveniles, then both stages will be described together and referred
232 as "immature stages ". The MG l of M. brachydactyla is lined by a simple columnar
233 epithelium with microvilli . In the immature stages, t he height of the epithelial cells of a
234 single tubule varies from 13 to 37 µm giving an irregular shape to the lumen (Fig. 4D),
235 while in adults the cell height varies from 25 to 100 µm and the lumen is "X" shape d 236 (Fig. 4C). Five cell types have been identified in immature and adult stages (Figs. 4-5):
237 E-cells (embryonic), R-cells (resorptive), F-cells (fibrillar), B-cells (blister -like) and M-
238 cells (midget). The distribution of these cell types is similar in immature and adult
239 stages (Fig. 4A -B): 1) the distal tip is the "E -cell region" since it is occupied by E -cells
240 that often appear in mitosis (Fig. 4E); 2) the "transitional region" is composed by sparse
241 F-cells and numerous R -cells which cytoplasm is poorly vacuolated ; and 3) the l ast
242 region is the "mature region ", it is dominated by R -cells rich in lipid vacuole s and fully
243 developed B-cells . The M -cells are distributed in the "transitional" and "mature regions"
244 (Fig. 4A, F). T he mentioned cell types will be described in detail belo w:
245 E-cells. The E-cells are hard to find, so our observations are restricted to the
246 observations realized by optical microscopy . The E -cells of the immature and adult
247 stages are similar and located on the distal tip of th e tubules . The E-cell s cytoplasm is
248 homogeneously stained not showing PAS positive inclusions or other relevant
249 characteristic . T he nucleus is big in comparison to the cell width (Figs. 4A -B; 5E).
250 Several E -cells are observed in active mitotic activity (Fig. 4E).
251 R-cells. The R-cells of the immature and adult specimens are columnar cells
252 (Fig. 5). They represent the main cell type of the MG l epithelium (85 ± 7 % and 76 ± 6
253 % of the cell population in immature and adult stages, respectively ). The cytoplasm of
254 the R -cells varies depending o f the position of the R -cell along the tubule length : in the
255 "transitional region" it is homogenously stained and contains a low number of lipid
256 vacuoles, while in the "mature region" the cytoplasm is rich in lipid vacuoles and PAS
257 positive inclusions (Fig. 4A -B). In the adults , many R-cells resemble to a "to wer of lipid
258 vacuoles" (Figs. 4C, F; 5G -H) ; while in the immature stages the R -cells usually contains
259 a single lipid vacuole located below the nucleus (Figs. 4D; 5C). The ultrastructural
260 study reveal s a cell membrane with a polarized organization (Fig. 6): 1) th e apical 261 membrane forms numerous undulated and slender microvilli (Fig. 6B-D); 2) the lateral
262 membranes are more undulated and with larger apical cell -to -cell junctions in the adults
263 than in the immature stages (Fig. 6B, D); and 3) the basal membrane is straight in the
264 immature stages but forms occasional folds in the adults ( Fig. 6E -F). The cell apex of
265 immature stages is rich in mitochondria and rough endoplasmic reticulum (RER) (Fig.
266 6B -C), while in adults contains few organelles and vertical filament s associated with
267 RER -like cisternae (Fig. 6D). The cell basis contains abundant mitochondria and
268 smooth endoplasmic reticulum (SER) forming a branched and tubular net , its electron -
269 density is higher in the adults than in the immature stage s (Fig. 6E -F) . T he SER tubes
270 fuse with the basal membrane (Fig. 6H -I). The Golgi bodies are composed by flattened
271 cisternae in all the life stages (Fig. 6G ). In the adult R -cells ha ve been observed other
272 structures as several kinds of vesic les (single or double me mbrane, content varying from
273 lucent to electron -dense ), multivesicular and multilamellar bodies.
274 F-cells. The F-cells are similar in immature stages and adults. The F-cells are
275 identified by three key features: 1) pyramidal shape, 2) highly stained cytoplasm with
276 fibrillar -like marks, and 3) the biggest nucleus among the epithelial cells (Fig. 5B -C, G -
277 H). Th e F-cell s represent 7 ± 3 % and 10 ± 2 % of the cell population in immature
278 stages and adult s, respectively . The presence of PAS positive inclusions is rare. The
279 organization of the cell membrane is polarized: 1) th e apical membrane forms numerous
280 undulated and slender microvilli (Fig. 7B ); 2) in the adults the lateral membranes are
281 more undulated and form larger cell -to -cell juncti ons than in the immature stages; and
282 3) the basal membrane is generally straight ( Fig. 7D ). The cytoplasm shows a similar
283 aspect in all the life stages : it contains an extraordinary abundance of RER ,
284 mitochondria and Golgi bodies . The cisternae of RER are located almost everywhere
285 showing three main organization s: 1) random -like distribution (Fig. 7C), 2) parallel 286 chains (Figs. 7B), and 3) concentric layers (Fig. 7D). The mitochondria are generally
287 small (Fig. 7B -D). The Golgi bodies have a distinctive mor phology in this cell type
288 since the cisternae are highly expanded and globular (Fig. 7B -C). In immature stages
289 and adult s, the cell basis contains a thin band composed by a tubular SEM and
290 mitochondria (Fig. 7D). This band shows a similar height than the r espective band of
291 the R -cells during the immature stages, but it is around three times thinner in
292 comparison to the band of the R -cells in adults .
293 B-cells . The B-cells show a similar morphology and ultrastructure in all the life
294 stages . Th is cell type represent s 8 ± 5 % and 12 ± 5 % of the cell population in
295 immature stages and adult s, respectively . In the immature stage s, the B -cells are taller
296 than the adjacent epithelial cells (Figs. 4B; 5D) , while in th e adults the B -cells are
297 shorter than the adjacent epithelial cells (Figs. 4A; 5G -H). The key character of the B -
298 cells is the giant central vacuole that occupies the majority of the cell volume . This
299 vacuole is generally rounded , single (not multiple as occur in the R -cells ), and always
300 with a supra -nuclear location (never infra -nuclear as occur with some vacuoles of the R -
301 cells ) (Fig s. 4A -C; 5D, G -H). Th e content of this vacuole is heterogeneous and similarly
302 stained than the lumen of the MGl (Fig s. 4B; 5D ). The ultrastructure of the B-cells is
303 similar in the immature stages and adult s. The cell membrane is polarized: 1) the apical
304 membrane forms short and undulated microvilli (Fig. 8B , D ); and 2) the lateral
305 membranes are smooth, form apical cell -to -cell junctio ns and converge to form a
306 cone /dome shaped cell basis (Fig. 8 B-C). The cytoplasm of the cell apex is electron -
307 dense and filled by numerous and lucent vesicles . It is unclear if these vesicles are
308 originated from the disintegration of the giant central vacuole, or by the contrary are
309 merging into the giant central vacuole: many vesicles are fused with the apical border of
310 the central vacuole, and the diameter of t he vesicles decreases toward the cell apex (Fig. 311 8B). The cell basis contains electron-dense cytoplasm with numerous cisternae of RER,
312 sparse mitochondria, and some Golgi bodies formed by flattened cisternae (Fig. 8C, E).
313 The content of the central vacuole is similar in immature stage s and adult s: it is highly
314 heterogeneous showi ng lucent areas , electron -dense agglomerations and lamellate
315 formations (Fig. 8F). We observed "micro -apocrine secretion" in t he adult B-cells :
316 small lucent vesicles are formed in t he microvilli tip and released in the lumen (MVs,
317 Fig. 8A; numbers, Fig. 8D ).
318 M-cells. The M-cells are the smallest cell type of the epithelium: 11.53 ± 1.50
319 µm in width and 7.14 ± 0.48 µm in height , therefore they never reach the lumen of the
320 MG l. This cell type is more easily identified in the adults (Fig. 4A, F ), than in the
321 immature stage s due to the shorter size of the ir epithelium (Fig. 5B) . The M-cells have
322 been identified in larval stages as zoea (Fig. 5B) and megalopa (Fig. 9 C). They are
323 rounded cells with a central nucleus (Fig s. 4F; 5 B; 9C ). The M -cells are similar in
324 immature stage s and adults at ultrastructural level (Fig. 9B-C) . The M -cells do not show
325 any polarity in cell membrane organization or organelles distribution . The cell
326 membrane is generally smooth and the cytoplasm lucent (Fig. 9B -C) . I n general , the
327 density of organelles is low . The mitochondria are small and dispersed arou nd the
328 nucleus (Fig. 9E-F) . The presence of endoplasmic reticulum is unclear , but several
329 lucent vesicles with different morphologies have been observed . The M -cells contain
330 smooth and rounded "electron -dense vesicles" which diameter oscillates between 0.5
331 and 1.0 µm , the content varies from homogeneous to highly heterogeneous and granular
332 (Fig. 9B -C, F ). The Golgi bodies are scarce and small (Fig. 9D). Few annulated
333 lamellae have been observed roaming on the cytoplasm but physically separated from
334 the nucleus (Fig. 9E).
335 Discussion 336 This study describes for first time the gross morphology and ultrastructure of the
337 midgut gland of a brachyuran species during its development. The gross morphology of
338 the MGl in larval stages of M. brachydactyla was studied using steromicroscopy, SEM
339 and micro -CT. The micro -CT has been showed as a reliable technique to study the
340 anatomy in crustacean larvae since shows the three -dimensional morphology of the
341 internal anatomy (including the MGl and other organs) . During the larval stages t he
342 MG l gross morphology of M. brachydac tyla is composed by fourteen blind -end tubules .
343 Previous studies employing optical microscope observations re ported that the MG l of
344 several brachyuran species has a tubular organization during the larval stages although
345 the number of tubules w as not counted (Jantrarotai and Sawanyatiputi, 2005 ; Nakamura,
346 1990 ; Schlegel, 1911 ; Trask, 1974 ). The larval MGl of M. brachydactyla is organized in
347 dorsal and ventral sub -lobules , each one composed by several tubules. This kind of
348 organization was reported in the larval MG l of the anomuran Paralithodes
349 camtschaticus (Abrunhosa and Kittaka, 1997 ). Since brachyurans and anomurans are
350 phylogenetically close (Scholtz and Richter, 1995 ), the gross morphology of the MGl
351 during the early development might be conserved in these two groups . The development
352 of the MG l of M. brachydactyla might be delayed in comparison to other brachyuran
353 species, since the new tubules start to develop in the late megalopa while in other
354 species the new tubules were r eported in the last zoeal stages (Jantrarotai and
355 Sawanyatiputi, 2005 ; Nakamura, 1990 ). The MGl of M. brachydactyla in the adulthood
356 is a huge and orange organ subdivided into three main lobules (anterior, posterior and
357 dorsal) , in turn composed by thousands of tubules . The computed tomography
358 confirmed the overall disposition of the MGl in the cephalothorax . Similar morphology
359 by direct observation after dissection was report ed in o ther brachyuran species , e.g. the
360 eriphiid Menippe rumphii (Erri Babu et al., 1982 ), the gecarcinucid Spiralothelphusa 361 hydrodroma (as Parathelphusa hydrodromus ) ( Reddy, 1937 ), the portunid Scylla
362 serrata (Barker and Gibson, 1978 ), the ocypodids Ocypode platytarsis (Ramadevi et al.,
363 1990 ) and Uca uruguayensis (Cuartas and Petriella, 2002 ), and the varunids Neohelice
364 granulata and Cyrtograpsus angulatus (Cuartas and Petriella, 2002 ).
365 In M. brachydactyla , the organization of the tubules is similar in larval, juvenile,
366 and adult stages . The main difference is the taller size of the epithelial cells during
367 adulthood. The MGL contains five types of epithelial cells : embryonary (E -) cells,
368 resorptive (R -) cells, fibrillar (F -) cells, blister like (B -) cells, and midget (M -) cells .
369 These cell types were widely reported in several decapod s, including other brachyuran
370 species (Davie et al., 2015 ; Felgenhauer, 1992 ; Gibson and Barker, 1979 ; Icely and
371 Nott, 1992 ). In this study, the five cell types were reported for first time in the larval
372 stages of a given brachyuran species, as it has been summarized in Table 2. The
373 different epithelial cell types observed in the MG l of M. brachydactyla are
374 heterogeneously distributed in different regions along the l ongitudinal axis of the
375 tubule. Th e cell distribution is similar in all the life stages, and coincides to the obser ved
376 distribution in several Decapoda taxa (Al-Mohanna and Nott, 1989 ; Gibson and Barker,
377 1979 ; Hopkin and Nott, 1979 ; Icely and Nott, 1992 ; Loizzi, 1971 ; Pillai, 1960 ). Some
378 previous studies describ ed a degenerative "proximal zone " (Al-Mohanna and Nott,
379 1989 ; Icely and Nott, 1992 ; Loizzi, 1971 ) but it was not been identified in M.
380 brachydactyla , as occurred also in Carcinus maenas (Hopkin and Nott, 1980 ). Those
381 studies suggest that the organization of the MG l is highly conserved among decapods.
382 The epithelial cells of immature stages and adults have similar ultrastructural
383 features. This observation supports that the function of each cell type is similar in all the
384 life stages, and suggest that t he MG l of M. brachydactyla is ready for the enzyme
385 production and food digestion as soon as they hatch. In fact, it has been reported that 386 newly hatched larvae of M. brachydactyla are obligate feeders ( Guerao et al., 2012 ;
387 Rotllant et al., 2010 ) and they are able to synthesize several kinds of digestive enzymes
388 (Andrés et al., 2010 ; Rotllant et al., 2008 ). Moreover, we observed that the MGl of the
389 newly hatched larvae is also rich in lipid vacuoles (Fig. 4D). Since the density of lipid
390 va cuoles of the MG l of the decapods decreases greatly in starving conditions (Anger et
391 al., 1985 ; Cervellione et al., 2017a ; Sánchez-Paz et al., 2007 ; Storch and Anger, 1983 ),
392 we suggest that this lipid storage could act as energy res erve to support survival until
393 the animals catch their first preys.
394 The E-cells of M. brachydactyla in all the life stages are restricted to the distal
395 tip of the tubules and its role might be related with the processes of cell division and
396 differentiation, since we observed several E -cells in mitosis. The E -cells of other
397 decapod species show a similar location and a similar role was proposed (Felgenhauer,
398 1992 ; Gibson and Barker, 1979 ; Icely and Nott, 1992 ; Li and Li, 1998 ; Paquet et al.,
399 1993 ). The ultrastructure of the epithelial cells of the MGl of M. brachydactyla (R-cells,
400 F-cells, B -cells an d M -cells) is also similar in all the life stages : R-cells with apical and
401 basal mitochondria, basal SER, and lipid vacuoles; F -cells rich in mitochondria, RER
402 and Go lgi bodies composed by globular cisternae; B -cells containing a big central
403 vacuole with an heterogeneous content and condensed cytoplasm ; and small M -cells
404 with electron -dense vesicles. Since t he same cells types with a closely similar
405 ultrastructure were reported in several decapod species , we suggest that the function of
406 the epithelial cell s of the MG l of M. brachydactyla might be similar to the suggested for
407 these species: R -cells as absorptive, storage, and osmoregulatory cells; and F -cells
408 involved in the production of enzymes (Al-Mohanna and Nott, 1986 , 1987 , 1989 ; Al-
409 Mohanna et al., 1985a ; Bunt, 1968 ; Felgenhauer, 1992 ; Gibson and Barker, 1979 ; Li
410 and Li, 1998 ; Loizzi, 1971 ; Paquet et al., 1993 ; Vogt, 1994 ). 411 The role of the B-cells is unclear. B-cells has been proposed to be the last stage
412 of the F -cells (Al-Mohanna and Nott, 1986 ; Al-Mohanna et al., 1985b ; Hu and Leung,
413 2007 ; Icely and Nott, 1992 ; Stanier et al., 1968 ). Our observations support this
414 hypothesis since we observed epithelial cells with transitional features between these
415 cell types: a F -cell with a supranuclear B -cell like vacuole (Suppl. Fig. 5). This
416 "transitional cell " was also reported in two previous studies (Loizzi, 1971 ; Stanier et al.,
417 1968 ). We observed several mitochondria partially digested in the vacuole of this
418 transitional cell (Suppl. Fig. 5A, C) . Therefore , we propose that the vacuole of the B-
419 cells contains the digested organelles of the previous F -cell stage, then when the vacuole
420 is released to the MG l lumen this content can be recycled for further processes. We also
421 observed that B -cells produce small vesicles from the microvilli tip. There is the first
422 report of this kind of secretion in a digestive organ of a decapod species, although this
423 kind of se cretion has been widely reported in the midgut tract of the insects receiving
424 name s as "microapocrine mechanism" or "microapocrine route" (Cristofoletti et al.,
425 2001 ; Ferreira et al., 1990 ; Monteiro et al., 2014 ; Silva et al., 2013 ). Sonakowska et al.
426 (2015) mentioned "microapocrine" secretion in the midgut tract of the caridean
427 Neocaridina heteropoda , but according to the published pictures they observed
428 undulated microvilli rather than a secretory activity. In insects , this kind of secretion is
429 associated with the release of digestive enzymes (Bolognesi et al., 2001 ; Cristofoletti et
430 al., 2001 ; Silva et al., 2013 ).
431 The role of the M-cells is still unknown, although different hypotheses has been
432 proposed : 1) storage of proteins or enzymes (Al-Mohanna et al., 1985a ; Nunes et al.,
433 2014 ); 2) endocrine cells as they are located near to blood sinuses ( Vogt, 1994 ); 3)
434 "blood cells" involved in the synthesis of hemocyanin or with a phagocytic role (Paquet
435 et al., 1993 ); and, 4) "basal cells" ( Reddy, 1938 ) or "replacement cells" ( Pillai, 1960 ). 436 Our data support the last hypothesis: M-cells might be in fact basal cells and true point
437 of cell regeneration and growth of the tubules . We observed that the ultrasatructure of
438 M-cells is similar to the basal cells of the midgut tract and midgut caeca of M.
439 brachydactyla (personal observation) and to the basal cells of the midgut tract and
440 midgut caeca of other decapods (Komuro and Yamamoto, 1968 ; Pugh, 1962 ). This
441 hypothesis explain s the formation of new tubules as branches of the older tubules .
442 Acknowledgements
443 Financial support provided by the Spanish Ministry of Economy and
444 Competitiv eness through the INIA project [grant number RTA2011 -00004 -00 -00 ] to
445 G.G and a predoctoral fellowship to D.C. [FPI -INIA] . The authors would like to than k
446 to the technicians at IRTA in Sant C arles de la Ràpita (David Carmona, Glòria Macià,
447 Magda Monllaó , Francesc X. Ingla and Olga Bellot ) and CCiTUB in Hospital Clinic,
448 Barcelona (Adriana Martínez, Almudena García, José Manuel Rebled, Rosa Rivera) for
449 their assistance . We also thank the staff of Bruker SkyScan for their effectiveness and
450 fast support, for their constant improvements to the software and for implementing the
451 new options we requested. In this respect, we are especially indebted to Alexander
452 Sasov, Stephan Boons, Xuan Liu, Phil Sal mon and Vladimir Kharitonov.
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613
614 615 Figure 1. Maja brachydactyla . Micro-CT volume rendering CTVox’s
616 reconstruction images. Megalopa. Entire specimen (A -B): dorsal view (A); lateral view
617 (B). Midgut gland , close view (C -D): dorsal view (C); lateral view (D). Abbreviations:
618 A P, anterior posterior; ANC , abdominal nerve cord; C S, cardiac stomach; H,
619 heart; H T, hindgut tract; M , muscles; M T, midgut tract; PC , posterior caecum; MG l,
620 midgut gland; PS , pyloric stomach; TG , thoracic gangli a.
621 Figure 2. Maja brachydactyla . Midgut gland. Gross morphology. Adult, dorsal
622 view, digital ca mera (A). Zoea I, SEM: dorsal (B) and lateral view (C). Adult, blind -
623 end tubules, SEM (D). Adult, muscle network, SEM (E). Abbreviations: AP, anterior
624 projection; CM, circular muscles; CT, central tubule; DSL, dorsal sub -lobule; ET,
625 external tubule; HT, h indgut tract; IT, internal tubule; LM, longitudinal muscles; LP,
626 lateral projection; MGl, midgut gland; PP, posterior projection; Sto, stomach; VSL,
627 ventral sub -lobule .
628 Figure 3. Maja brachydactyla . Diagrams showing different views (ventral,
629 dorsal and lateral) of the larval midgut gland at the start of different stages (zoea I, zoea
630 II, megalopa and first juvenile).
631 Figure 4. Maja brachydactyla . Midgut gland. Morphology. Optical microscopy.
632 Tubule , longitudinal section: adult stained with Hematoxylin -Eosin (A) , and megalopa
633 stained with Mallory's trichrome (B). Tubule, transversal section, Hematoxylin -Eosin:
634 adult (C), and zoea I (D) . Adult, E -cells, Hematoxylin -Eosin ( E). Adult, M -cells,
635 Mallory's trichrome (F). Abbreviations: BC , B-cells; B M, basement membrane ; CM ,
636 circular muscle s; FC , F-cells; MC , M-cells; MV, microvilli; RC , R-cells.
637 638 Figure 5. Maja brachydactyla . Midgut gland. Epithelial cells. Larval and first
639 juvenile stages, g eneral diagra m (A). Zoea I, Hematoxylin -Eosin (B). Megalopa ,
640 Mallory's trichrome (C). Megalopa, B -cell, Mallory's trichrome (D). Megalopa, E -cells,
641 PAS contrasted with Methylene Blue (E). Adult, general diagram (F) . Adult, epithelial
642 cells: Hematoxylin -Eosin (G) and Mallory's trichrome (H) . Abbreviations: BC , B-cells;
643 BM, basement membrane ; EC , E-cells; FC , F-cells; MC , M-cell; MF, muscle fibers;
644 MV , microvilli; RC , R-cells.
645 Figure 6. Maja brachydactyla . Midgut gland. Ultrastructure of the R-cells. TEM.
646 General diagram (A). Megalopa, general view (B). Cell apex: megalopa (C), and adult
647 (D). Cell basis: megalopa (E), and adult (F). Golgi body, close view (G). Fusion of the
648 SER with the basal membrane (arrows): megalopa (H), and adult (I). Abbreviations: BF,
649 basal folds; BL, basal lamina; CJ, cell -to -cell junctions; FS, filamentous structures; LD,
650 lipid droplets; M, mitochondria; MV, microvilli; N, nucleus; RER, rough endoplasmic
651 reticulum; SER, smooth endoplasmic reticulum .
652 Figure 7. Maja brachydactyla . Midgut gland. Ultrastructure of the F-cells. TEM.
653 General diagram (A). Megalopa, cell apex (B). Adult, supranuclear region (C). Adult,
654 infranuclear region and cell basis ( D). Abbreviations: BL , basal lamina; F S, filamentous
655 structures ; G, Golgi bodies; M , mitochondria; MV , microvilli; N , nucleus; RER , rough
656 endoplasmic reticulum; SER , smooth endoplasmic reticulum.
657
658
659 660 Figure 8. Maja brachydactyla . Midgut gland. Ultrastructure of the B-cells. TEM.
661 General diagram (A). Cell apex, supra -vacuolar region (B). Mega lopa, general view (C).
662 Micro -apocrine secretion (numbered) (D). Golgi body (E). Content of the central
663 vacuole (F). Abbreviations: CV , central vacuole; G , Golgi bodies , M: mitochondria;
664 MV , microvilli; MVs, micro -vesicles; N, nucleus; RER , rough endoplasmic reticulum.
665 Figure 9. Maja brachydactyla . Midgut gland. Ultrastructure of the M-cells.
666 TEM. General diagram (A). General view of the M -cells (B -C): adult (B) and megalopa
667 (C). Golgi body (D). Annulate d lamellae and pores (arrows) (E). Ele ctron -dense vesicle
668 (F). Abbreviations: BL , basal lamina; EV , electron -dense vesicles; G , Golgi bodies; M ,
669 mitochondria; N , nucleus.
670 Suppl. Fig. 1. Maja brachydactyla . Midgut gland. Larvae. Diagrams showing the
671 measures taken for the morphometric study . Abbreviations: DSL, dorsal sub -lobule;
672 DSh, dorsal sub -lobule height; DSl, dorsal sub -lobule length; DSw, dorsal sub -lobule
673 width; ET, external tubule; ETl, external tubule length; ETw, external tubule width; IT,
674 internal tubule; ITl, internal tubule lengt h; ITw, internal tubule width; VSL, ventral sub -
675 lobule; MW, maximum width; TL, total length.
676 Suppl. Fig. 2. Maja brachydactyla . Larvae. Location of the midgut gland.
677 Stereomicroscope. Zoea I, lateral view (A). Zoea II, dorsal view (B). Zoea I, dissected
678 midgut gland and adjacent organs (C). Megalopa, lateral view (D). Megalopa, dorsal
679 view (E). Megalopa, dissected midgut gland and adjacent organs (F). Abbreviations:
680 HT, hindgut tract ; M T, midgut tract ; MG l, midgut gland; S to , stomach.
681 682 Suppl. Fig. 3. Maja brachydactyla . Midgut gland. Tomographic and
683 microtomographic cross section images. Adult tomography, the midgut gland is marked
684 by the dotted line (A -B): anatomical area of the stomach (A) and anatomical area of the
685 heart (B). Larvae, Micro -CT Data Viewe r’s cross section images, the midgut gland is
686 marked by the dotted line (C -D): zoea I, anatomical area of the pyloric stomach (C) and
687 megalopa, anatomical area of the pyloric stomach (D). Abbreviations: BC , branchial
688 chamber; M T, midgut tract; MG l, midgut gland; S to, stomach.
689 Suppl. Fig. 4. Maja brachydactyla . Midgut gland. Larvae. Close view of the
690 musculature. Abbreviations: CL, circular musculature; LM, longitudinal musculature .
691 Suppl. Fig. 5. Maja brachydactyla . Midgut gland. Transitional cell: F-cell with
692 features of B -cell (A -C) . Ge neral view (A) . R ough endoplasmic reticulum (B) . Vacuole
693 content , partially digested mitochondria (C).
694
695
696 Table
Table 1 . Maja brachydactyla . Growth of the midgut gland from hatching to the first juvenile stage. Resume of the statistical analyses realized to study the
growth of the midgut gland during the larval development. The size of each structure (mean ± SD) at the age of newly hatched zoea (ZI 0) and newly
metamorphosed first juvenile (J 0) is showed.
Statistical significance per lobule ZI 0 J0 Structure (µm) (µm) right left
Total length TL 621 ± 60 1120 ± 97 F3,16 = 72.05 p < 0.001 F3,16 = 42.53 p < 0.001
Maximum width MW 235 ± 36 267 ± 38 F3,16 = 1.28 p = 0.31 F3,16 = 0.54 p = 0.66
Dorsal sub -lobule , length DSl 368 ± 39 728 ± 61 F3,15 = 52.87 p < 0.001 F3,15 = 38.60 p < 0.001
Dorsal sub -lobule , width DSw 223 ± 40 397 ± 57 F3,15 = 30.14 p < 0.05 F3,15 = 23.19 p < 0.001
Dorsal sub -lobule , height DSh 204 ± 23 293 ± 30 F3,15 = 12.19 p < 0.001 F3,15 = 6.0 p < 0.01
External tubule , length ETl 118 ± 12 241 ± 21 F3,15 = 58.94 p < 0.001 F3,15 = 87.46 p <0.001
External tubule ,width ETw 139 ± 20 199 ± 21 F3,15 = 4.27 p < 0.05 F3,15 = 15.14 p < 0.001
Internal tubule ,length ITl 87 ± 13 158 ± 41 F3,15 = 4.67 p < 0.05 F3,15 = 8.06 p < 0.01
Internal tubule ,width ITw 131 ± 16 154 ± 25 F3,15 = 4.51 p < 0.05 F3,15 = 3.45 p = 0.044 Table
Table 2 . Midgut gland. Types of epithelial cells identified in the larvae of different decapod species. The question mark (?) indicate the description of cells that resemblances to the marked type.
Species Taxa Larval stages E-cells R-cells F-cells B-cells M-cells Reference:
Hyas araneus (Linnaeus, 1758) Brachyura Zoeae - Megalopa X X X X (Storch and Anger, 1983 )
Maja sp. Lamarck, 1801 Brachyura First zoea X (Schlegel, 1911 )
Metacarcinus anthonyi Rathbun 1897 Brachyura Megalopa X (Trask, 1974 )
Portunus trituberculatus (Miers, 1876) Brachyura Megalopa X X (Nakamura, 1990 )
Scylla olivacea (Herbst, 1796) Brachyura Zoeae X X (Jantrarotai and Sawanyatipu ti, 2005 )
Scylla serrata (Forskål, 1775) Brachyura Zoeae X X X X (Li and Li , 1998 )
Paralithodes camtschaticus (Tilesius, 1815) Anomura Zoeae - Glaucothoe X X X X (Abrunhosa and Kittaka, 1997 )
Porcellana platycheles (Pennant, 1777) Anomura Pre -zoea X X X ? (Williams, 1944 )
Homarus americanus H. Milne Edwards, 1837 Astacidae First zoea X X X X (Anger et al., 1985 ; Biesiot and McDowell, 1995 )
Ibacus ciliatus (Von Siebold, 1824) Achelata Phyllosoma X X X (Mikami et al., 1994 )
Jasus edwardsii (Hutton, 1875) Achelata Puerulus X X X (Nishida et al., 1995 )
Panulirus japonicus (Von Siebold, 1824) Achelata Phyllosoma X X X (Mikami et al., 1994 )
Panulirus ornatus (Fabricius, 1798) Achelata Phyllosoma X X X (Johnston, 2006 ) Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image Figure 9 Click here to download high resolution image Supplementary Figure 1 Click here to download high resolution image Supplementary Figure 2 Click here to download high resolution image Supplementary Figure 3 Click here to download high resolution image Supplementary Figure 4 Click here to download high resolution image Supplementary Figure 5 Click here to download high resolution image