Histological and histochemical investigation of the compound eye of the whiteleg shrimp (Litopenaeus vannamei)

Hao-Kai Chang 1 , Cheng-Chung Lin Corresp., 1 , Shih-Ling Hsuan Corresp. 1

1 Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, Taichung, Taichung, Taiwan

Corresponding Authors: Cheng-Chung Lin, Shih-Ling Hsuan Email address: [email protected], [email protected]

The compound eye is the primary visual system in crustaceans. Although the histological structure and histochemical characteristics of compound eyes of some insect and crab species are now well understood, no such studies have been undertaken in the whiteleg shrimp (Litopenaeus vannamei). In this study, eye samples from L. vannamei were fixed and paraffin sections were stained using several histochemical methods. The histological structure of each layer of the compound eye was examined and compared using different histochemical methods. It was found that the compound eye of L. vannamei consisted of cuticle, cornea, ommatidia, optic nerve layer, lamina ganglionaris, and medulla in an outside-in order. The cuticle of L. vannamei eyes was very thin, composed of a single epicuticle layer, as confirmed by Masson’s trichrome stain. The screening pigments produced by screening pigment cells were arranged at the junction of the ommatidia and optic nerve layer; these pigments stained differentially after different histochemical staining methods suggesting the screening pigment cells can be classified into different types. Notably, clusters of foamy glandular cells (FGCs) were observed in the optic nerve layer; these stained positively with periodic acid-Schiff and toluidine blue, and appeared blue after Masson’s trichrome stain. Immunohistochemical (IHC) staining was used to further define the origin and characteristics of FGCs. The IHC analysis showed that FGCs were positive for vimentin and synaptophysin (SYN), suggesting their neuroendocrine nature. In the medulla internalis and medulla terminalis, the neural clusters that surround the neurophil could be divided into three types by differences in morphology: the largest and the smallest cell clusters were clusters and neurosecretory cells, respectively; the middle-sized cell clusters appeared SYN-positive and have not previously been described. Overall, this study is the first to provide a detailed description of the normal features of the compound eye of L. vannamei. The identification of different types of screening pigments in the ommatidia, the endocrine nature of FGcs in the optic nerve layer, and the novel neural clusters between the medulla internalis and medulla terminalis, will be important information for further study into the compound eye of L. vannamei.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 1 Histological and histochemical investigation of the compound eye of the whiteleg shrimp

2 (Litopenaeus vannamei)

3 Hao-Kai Chang1, Cheng-Chung Lin1*, Shih-Ling Hsuan1*

4 1Graduate Institute of Veterinary Pathobiology, National Chung Hsing University, 145 Xingda

5 Road, South Dist., Taichung, Taiwan.

6 *Corresponding author: Shih-Ling Hsuan and Cheng-Chung Lin, Graduate Institute of Veterinary

7 Pathobiology, National Chung Hsing University, 145 Xingda Road, South Dist., Taichung,

8 Taiwan.

9 E-mail address: [email protected] (SL Hsuan), [email protected] (CC Lin)

10 Tel.: +886-4-2284-0368 ext. 28

11 Fax: +886-4-2285-2186

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 12 Abstract

13 The compound eye is the primary visual system in crustaceans. Although the histological

14 structure and histochemical characteristics of compound eyes of some insect and crab species are

15 now well understood, no such studies have been undertaken in the whiteleg shrimp (Litopenaeus

16 vannamei). In this study, eye samples from L. vannamei were fixed and paraffin sections were

17 stained using several histochemical methods. The histological structure of each layer of the

18 compound eye was examined and compared using different histochemical staining methods. It

19 was found that the compound eye of L. vannamei consisted of cuticle, cornea, ommatidia, optic

20 nerve layer, lamina ganglionaris, and medulla in an outside-in order. The cuticle of L. vannamei

21 eyes was very thin, composed of a single epicuticle layer, as confirmed by Masson’s trichrome

22 stain. The screening pigments produced by screening pigment cells were arranged at the junction

23 of the ommatidia and optic nerve layer; these pigments stained differentially after different

24 histochemical staining methods suggesting the screening pigment cells can be classified into

25 different types. Notably, clusters of foamy glandular cells (FGCs) were observed in the optic

26 nerve layer; these stained positively with periodic acid-Schiff and toluidine blue, and appeared

27 blue after Masson’s trichrome stain. Immunohistochemical (IHC) staining was used to further

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 28 define the origin and characteristics of FGCs. The IHC analysis showed that FGCs were positive

29 for vimentin and synaptophysin (SYN), suggesting their neuroendocrine nature. In the medulla

30 internalis and medulla terminalis, the neural clusters that surround the neurophil could be divided

31 into three types by differences in morphology: the largest and the smallest cell clusters were

32 neuron clusters and neurosecretory cells, respectively; the middle-sized cell clusters appeared

33 SYN-positive and have not previously been described. Overall, this study is the first to provide a

34 detailed description of the normal features of the compound eye of L. vannamei. The

35 identification of different types of screening pigments in the ommatidia, the endocrine nature of

36 FGCs in the optic nerve layer, and the novel neural clusters between the medulla internalis and

37 medulla terminalis, will be important information for further study into the compound eye of L.

38 vannamei.

39 Key words: Litopenaeus vannamei, compound eye, histology, screening pigments, foamy-

40 glandular cells, neural cluster

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 41 Background

42 The eyes of animals are classified into two major systems, single-chamber eyes and

43 compound eyes (Buschbeck, 2014). The anatomy of compound eyes is well known in several

44 insect species, but a limited amount of information is available from a small number of

45 crustacean species (Alkaladi and Zeil, 2014; Buschbeck, 2014). Numerous studies have explored

46 the micro-structure and organization of compound eyes in insects with emphasis on the structure

47 of the ommatidia and comparative neurological investigations of retinular cells (Alkaladi and

48 Zeil, 2014; Arikawa et al., 2009; Buschbeck, 2014; Zeil and Hemmi, 2006); comparatively few

49 histological studies have been conducted. In crustaceans, functional studies on the eye are well

50 established, because the eye and eye stalk include the important physical functions of vision and

51 endocrine activity (Luo et al., 2015; Saigusa, 2002; Shui et al., 2014). However, the

52 histochemical characteristics of the various structures in the compound crustacean eye are less

53 well understood. Litopenaeus vannamei is a crustacean species of great economic importance

54 since it is the most commonly farmed member of the shrimp family Penaeidae (Brito et al.,

55 2016). In order to characterize the histochemical features of L. vannamei compound eyes, we

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 56 examined histological structures after use of five different staining methods that are frequently

57 used in histology and pathological diagnosis.

58 Histochemical staining methods are usually applied to identify tissues and chemical

59 properties in animal cells. Toluidine blue is a metachromatic dye that has been widely applied in

60 studies of compound eyes of insects as it can distinguish structures with different chemical

61 characteristics through a simple staining procedure (Jia and Liang, 2015; Narendra et al., 2016).

62 Masson’s trichrome stain and periodic acid–Schiff (PAS) stain are used to differentiate collagen

63 fibers and muscle from other structures (Foot, 1933; Gürses et al., 2014), and to detect

64 polysaccharides and mucosubstances (Wang et al., 2016; Xavier-Júnior et al., 2016), respectively.

65 Luxol fast blue stain is useful for staining nerve fibers and can be combined as a counter-stain

66 with hematoxylin and eosin (H&E) or PAS.

67 In the present study, the origin and physiological characteristics of specific cells in L.

68 vannamei compound eyes were investigated by immunohistochemical (IHC) staining for bio-

69 markers of nerve-associated cells, including pan-cytokeratin (pan-CK), vimentin, glial fibrillary

70 acidic protein (GFAP) (Silva et al., 2015), synaptophysin (SYN) (Rusyn et al., 2014), neuron

71 specific enolase (NSE), and brain-derived neurotrophic factor (BDNF) (Olafsdottir et al., 1997).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 72 No investigations of compound eyes in penaeid shrimps are available. The information

73 obtained here from examination of the histological structures of the L. vannamei compound eye

74 will help to remedy this deficit.

75 Material and Methods:

76 Sample preparation

77 Five live L. vannamei with body lengths of 12–14 cm (Fig. 1A) were obtained from a

78 traditional market in the south district of Taichung, Taiwan, and transported in 22°C, 1.5% salt

79 water to the Graduate Institute of Veterinary Pathobiology, National Chung Hsing University,

80 within 10 minutes. The shrimps used in this study were alive at the start of the study and without

81 any notable abnormalities in appearance.

82 The shrimps were killed by immersion in ice-cold water (shrimp weight : ice weight = 1:2)

83 for 10 minutes. Shrimp eyes and attached eyestalks (Fig. 1A) were removed and fixed in

84 modified Davidson’s solution (Latendresse et al., 2002) for 48 hours at room temperature. To

85 ensure that the eyes were all cut at the same horizontal level, they were placed in 1% agarose gel

86 before being embedded in paraffin. Serial sections were cut from the paraffin blocks and

87 subjected to histochemical and immunohistochemical staining.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 88 Histochemical staining

89 The following histochemical stains were used in this study: H&E, periodic acid Schiff (PAS)

90 (Lillie, 1954), Masson’s trichrome (Meinen et al., 2007; Sakaew et al., 2008), toluidine blue (TB)

91 (Sridharan and Shankar, 2012), and modified luxol fast blue-periodic acid Schiff stains (LFB-

92 PAS). Staining with LFB-PAS was carried out as described previously with some modifications

93 (Sheehan and Hrapchak, 1980). A slide with a section of pig cerebellar tissue was used as a

94 positive control and stained at the same time as the shrimp sections. Slides were deparaffinized

95 with xylene and hydrated with 100% and 95% ethanol. The slides were placed in 65°C-preheated

96 LFB solution and incubated at 65°C overnight, allowed to cool to room temperature (about 23°C)

97 for 10 minutes, and immersed in 95% ethanol to remove the surplus dye. The slides were

98 differentiated in 0.05% lithium carbonate for 20 seconds, and then in 70% ethanol until the white

99 matter and gray matter of the control slide (pig cerebellum) could be clearly distinguished. The

100 slides were then washed and oxidized in 0.5% periodic acid solution for 5 minutes, and stained

101 with Schiff reagent (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes. The sections were

102 counterstained using Mayer’s hematoxylin for 30 seconds (Hotchkiss, 1948).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 103 The sections were analyzed under a light microscope, and images were acquired using a

104 digital camera (Olympus DP70, Japan).

105 Immunohistochemistry (IHC) staining

106 IHC staining was performed by Li-Tzung Biotechnology Inc. (Kaohsiung, Taiwan) using

107 primary antibodies specific to pan-CK, vimentin, SYN, NSE, and BDNF. Binding of the primary

108 antibody was detected with mouse/rabbit probes and a horseradish peroxidase (HRP) labeling kit

109 with 3’,3’-diaminobenzidine (BioTnA, Kaohsiung, Taiwan).

110 Results

111 Histochemical staining

112 The compound eye of L. vannamei can be divided into several layers following routine H&E

113 staining (Fig. 1B). From the outermost cuticle layer inward are cornea, ommatidia, optic nerve

114 layer, lamina ganglionaris, and medulla (Fig. 1B). The fine structure and cells in each layer are

115 described in the following sections and are summarized in Tables 1 and 2.

116 Cuticle, cornea, and ommatidia

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 117 The superficial layer of the compound eyes is a thin layer termed cuticle. Below the cuticle

118 are the ommatidia, composed of cornea, corneal hypodermal cells, crystalline cone, primary

119 pigment cells, and retinular cells. At the base of the ommatidia is the ommatidia-optic nerve layer

120 juncture (Figs. 2A and 2B). After H&E staining, the cornea appears as an eosinophilic,

121 rectangular, and hyaline structure attached tightly to the cuticle (Fig. 2A). After MT staining, the

122 cuticle appears as a very thin, red-to-firebrick layer, whereas the cornea is stained navy blue

123 (Figs. 2C and 2D). The other stains applied here failed to clearly distinguish the cuticle from the

124 cornea (data not shown). The corneal hypodermal cells are rectangular to polygonal in shape with

125 light- cytoplasm and a transparent nucleus in the center. Crystalline cone cells are

126 present below the corneal hypodermal cells and above the eosinophilic crystalline cone and have

127 dark-basophilic dimers with a transparent crystal-like structure at the lateral side. Retinular cells

128 are light-eosinophilic and have a long-rod shape without clear nucleus. Brown pigment granules

129 are distributed around the crystalline cones and retinular cells and are termed primary pigment

130 cells (Fig. 2B).

131 The ommatidia-optic nerve layer juncture lies on the basement membrane and consists of

132 several types of cell (Fig. 3A). The top layer of the ommatidia-optic nerve layer juncture contains

133 proximal retinular cells, which are round to spindle-shaped. Brown pigments (screening

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 134 pigments) are seen to be concentrated in the upper and bottom zones of the rhabdom after H&E

135 staining (Fig. 3A).

136 Interestingly, the appearance of the screening pigments on the top-side of the rhabdom

137 (upper screening pigments, USP) and in the bottom of the rhabdom (lower screening pigments,

138 LSP) varied using different staining methods. USP and LSP were brown with similar densities

139 after H&E staining (Fig. 3A). However, LSP were much darker than USP after TB (Fig. 3B) and

140 PAS staining (Fig. 3C). USP also stained brown with MT (Fig. 3D) and LFB-PAS (Fig. 3E),

141 while LSP were apple-green after LFB-PAS (Fig. 3E) and colorless with MT staining (Fig. 3D).

142 Optic nerve layer

143 The optic nerve layer (ONL) lies below the basement membrane of the ommatidia. In the

144 ONL, a large number of long, silk-like, longitudinal optic nerves are attached to the base of

145 ommatidia and to the top of the lamina ganglionaris (Fig. 3A) with many lacunae (hemocoels).

146 Notably, many foamy-glandular cells (FGCs) were present in the ONL (Fig. 3A). The FGCs have

147 foamy, basophilic, and granular cytoplasm with one to multiple eccentric round nuclei. There are

148 also sporadic brown pigments widely scattered in this layer, especially in the junction of the ONL

149 and lamina ganglionaris.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 150 The cytoplasm of FGCs is PAS-positive (stained pink by Schiff’s solution) (Fig. 3C), and

151 appears dark violet after TB staining (Figs. 3B and 3F) and royal-blue after MT staining (Fig.

152 3D). The PAS positive staining indicates that FGCs are enriched with polysaccharides and

153 mucosubstances (Lillie, 1954). The dark violet staining with TB indicates the contents of FGCs

154 are β-metachromatic (Sridharan and Shankar, 2012).

155 The pigments scattered at the base of the ONL stained brown with H&E and PAS, black to

156 gray with TB, blue with MT, and were colorless after LFB-PAS staining (Fig. 3). The staining

157 characteristics of the pigments in this area were significantly different from those of the USP and

158 LSP of ommatidia.

159 Lamina ganglionaris

160 The lamina ganglionaris consists of glial cells, support cells, optic nerve fibers and hemal

161 sinus. Glial cells were slim, horizontally arranged, and had basophilic, silk-like cytoplasm and a

162 spindle-shaped nucleus after H&E staining (Fig. 3A). After TB staining, the cytoplasm of glial

163 cells was violet and could be seen clearly (Fig. 3B). However, the glial cells were colorless after

164 MT staining (Fig. 3D) and showed a very weak PAS-positive reaction (Figs. 3C and 3E).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 165 Medulla

166 The medulla of compound eyes is mainly composed of optic nerve fibers and is arranged in

167 three parts, the medulla externalis, the medulla internalis, and the medulla terminalis (Fig. 1B).

168 Neural cell clusters surround the medulla internalis and medulla terminalis (Fig. 4A). These

169 neural cell clusters can be classified into three groups based on their morphology and size. The

170 largest cell cluster is composed of of 40–50 μm diameter, containing abundant cytoplasm

171 and a hollow nucleus. The smallest cell clusters have a high nucleus-cytoplasm ratio and are

172 neurosecretory cells. The middle size cell clusters are composed of polygonal cells that have a

173 hollow nucleus and foamy cytoplasm. None of the histochemical staining methods used in this

174 study highlighted any cells or structures in the medulla of the compound eyes (data not shown).

175 Immunohistochemistry (IHC) staining

176 In order to better understand the origin and biochemical characteristics of the FGCs and the

177 middle size cell clusters in the medulla, a panel of 6 IHC markers were used to define their

178 cytoskeletons and neuroendocrine structures. FGCs were positive for vimentin and SYN,

179 indicating that these cells most likely derive from mesenchymal tissue and neuroendocrine cells.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 180 Other markers, including pan-CK, GFAP, NSE and BDNF, were not detectable in the FGCs (Fig.

181 5). The middle size cell clusters in the medulla showed SYN-positive staining (Fig. 4B).

182 Discussion

183 Previous studies have paid little attention to the fine histological structure of the compound

184 eye of L. vannamei; thus, many of the terms used in this study are adapted from studies of other

185 animal species, including crab (Alkaladi and Zeil, 2014; Zeil and Hemmi, 2006), lobster (Shields

186 and Boyd, 2014), and insects (Jia and Liang, 2015; Narendra et al., 2016; Reisenman et al., 2002;

187 Ribi and Zeil, 2015). The terminology for several fine structures or cell types is not always

188 consistent between different studies.

189 In the present study, the outermost layer (the cuticle) of the compound eye of L. vannamei

190 was composed of a single thin epicuticle layer and lacked exocuticle and endocuticle. However,

191 in lobster, the stratification of the cuticle in compound eyes is similar to the somite, which can be

192 divided into epicuticle, exocuticle and endocuticle (Poulaki and Colby, 2008). In our unpublished

193 data, the exocuticle and endocuticle of somite stain blue with MT in L. vannamei and crayfish

194 (Cherax quadricarinatus), because of the presence of abundant collagen fiber. In contrast, the

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 195 epicuticle is largely composed of hydrophobic wax, which retains red coloration (Biebrich

196 Scarlet) due to its low permeability (Sheehan and Hrapchak, 1980).

197 The corneal stroma is primarily composed of collagen fiber (Poulaki and Colby, 2008) and

198 stains blue with MT. The corneal hypodermis of L. vannamei has a rectangular shape, but is

199 triangular in lobster and is arranged in pairs (Shields and Boyd, 2014). The crystalline cells in L.

200 vannamei have a homogeneous dark-basophilic cytoplasm and indistinguishable nuclei, with a

201 hollow crystal-like structure in the lateral side. However, these cells are polygonal with

202 eosinophilic cytoplasm, a distinct basophilic nucleus, and a hollow crystal-like structure in the

203 cell center in lobster (Shields and Boyd, 2014).

204 Screening pigments with different histochemical characteristics were observed in the bottom

205 and top of the rhabdom of L. vannamei. In insects, the primary pigment surrounds the crystalline

206 cone and retinular cells, while the secondary pigment (screening pigment) lies on the basement

207 membrane (Stavenga, 1979), which contains different pigment granules (Hamdorf, 1979;

208 Stavenga, 2002; Stavenga, 1979). However, the primary pigments do not extend from the

209 retinular cells into the basement membrane. Therefore, the different histochemical characteristics

210 of the pigments in the top and bottom of the rhabdom of L. vannamei may differ from those of

211 the primary and secondary pigments.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 212 The ONL is composed of abundant nerve fibers and lacunae, and the density of nerve fibers

213 is much higher in L. vannamei than in lobster (Shields and Boyd, 2014). Although the ONL and

214 lamina ganglionaris were expected to stain sky-blue after LFB-PAS, they were in fact colorless.

215 We suggest that the low pH of the fixed tissues may be responsible for this effect; the fixative

216 used in the present study was a modified Davidson solution. The principle of LFB staining is a

217 simple acid–base reaction between dye and tissues (Klüver and Barrera, 1953; Sheehan D and B,

218 1980); thus, acidification of the tissue might affect the color presentation of the tissues.

219 We found that several FGCs were present in the ONL; this feature has not been described

220 previously in insects or crustaceans. FGCs were PAS-positive, suggesting the presence of

221 mucosubstances (glycol group or alkylamino derivatives) in the cellular content (Gollnick et al.,

222 2013). The dark violet staining of FGCs with TB may indicate that the granules in the cytoplasm

223 are β-metachromatic, and are similar to the endocrine granules in mammalian cells and to the

224 nutritional granules in some (Sridharan and Shankar, 2012). FGCs stained blue with MT,

225 indicating the permeability of the cells was much lower than for other structures in the ONL

226 (Foot, 1933; Meinen et al., 2007).

227 FGCs showed positive staining for vimentin and SYN, indicating that they might originate

228 from mesenchymal tissue, and should be characterized as neuronal or neuroendocrine cells. SYN

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 229 is a type of glycoprotein, and is regarded as a highly sensitive marker for neuroendocrine cells

230 (Albert et al., 2016; Modi et al., 2016; Rusyn et al., 2014). NSE, also known as γ-enolase (Braga

231 et al., 2013; Korse et al., 2012), and GFAP, also known as intermediate filament protein (Wick

232 and Hornick, 2010), are highly specific biomarkers for determinate neurons and neuroendocrine

233 cells. Although staining for NSE and GFAP was negative, the presence of SYN positive staining

234 is a strong indication that FGCs are of neuroendocrine origin.

235 The chemical characteristics of the cells in the medulla of compound eyes of L. vannamei

236 were similar; this may be the reason that the histochemical stains used here did not identify

237 significant differences. The medulla of compound eyes is largely composed of nerve fibers and

238 different types of neural clusters in insects and lobster (Alkaladi and Zeil, 2014; Goldsmith and

239 Philpott, 1957; Jia and Liang, 2015; Shields and Boyd, 2014); a similar structure is present in L.

240 vannamei.

241 The middle-size neuron clusters that we identified between the medulla internalis and

242 medulla terminalis have not been reported previously. These cells show positive SYN staining,

243 and are different to large-size neuron clusters and neurosecretory cells. These differences indicate

244 that the middle-sized neuron cluster may belong to a new-type of neuroendocrine cell.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 245 Conclusion

246 Using several histochemical staining methods, the histological features and biochemical

247 characteristics of the compound eyes of L. vannamei were investigated. Specific structures and

248 histochemical results were reported for the first time, such as the identification of foamy-

249 glandular cells, the differential staining of screening pigments, and the presence of unique cell

250 clusters in the medulla. The physiological functions of these cells require further investigation.

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PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 374 Figures-text

375 Fig. 1. Gross and subgross pictures of the L. vannamei eye. (A) The eye stalk of L. vannamei. (B)

376 The lamination and sub-macrostructure of the compound eye of L. vannamei. The structures

377 shown by H&E staining are: CUT, cuticle; OMA, ommatidia; ONL, optic nerve layer; LG,

378 lamina ganglionaris; ME, medulla externalis; MI, medulla internalis; MT, medulla terminalis.

379 Fig. 2. Histological structure of the ommatidia after H&E or MT staining. (A and B) H&E

380 staining. (C and D) MT staining. The structures shown are: CUT, cuticle; RC, retinular cells; PR,

381 proximal retinula; SP, screening pigments; CH, corneal hypodermis cells; COC, crystalline cone

382 cells; C, crystalline cone; PP, primary pigment.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 383 Fig. 3. Histological structure of the ommatidia-ONL juncture and ONL. (A) Ommatidia-ONL

384 juncture and ONL with H&E staining. (B) The FGCs and glial cells stain positively with TB. (C)

385 FGCs also show PAS-positive staining, (D) and stain blue with MT. (E) The USP stains brown,

386 but the LSP stains apple-green with LFB-PAS. (F) At higher power, many fine granules can be

387 seen in FGCs and are TB-positive.

388 BM, basement membrane; GC, glial cells; HS, hemal sinus; LH, lacuna of hemocoel; LSP, lower

389 screening pigments; SC, support cells; USP, upper screening pigments; *, foamy glandular cells;

390 ✦, pigments in ONL.

391 Fig. 4. The fine histological structure between the medulla internalis and medulla terminalis. (A)

392 Three differently sized cell clusters can be observed. The middle-size cell clusters (arrow) have

393 not been described before. H&E staining. (B) The middle-size cell clusters (arrow) show SYN

394 positive staining. The inset shows the middle-size cell cluster at higher magnification. ART,

395 arteriole; ONF, optic nerve fiber; NS, neurosecretory cells; NC, neuron clusters; MI, medulla

396 internalis.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 397 Fig. 5. FGCs stained by IHC markers. (A) vimentin, (B) SYN, (C) pan-CK, (D) GFAP, (E) NSE,

398 (F) and BDNF.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Figure 1

Gross and subgross pictures of the L. vannamei eye.

(A) The eye stalk of L. vannamei. (B) The lamination and sub-macrostructure of the compound eye of L. vannamei. The structures shown by H&E staining are: CUT, cuticle; OMA, ommatidia; ONL, optic nerve layer; LG, lamina ganglionaris; ME, medulla externalis; MI, medulla internalis; MT, medulla terminalis.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Figure 2

Histological structure of the ommatidia after H&E or MT staining.

(A and B) H&E staining. (C and D) MT staining. The structures shown are: CUT, cuticle; RC, retinular cells; PR, proximal retinula; SP, screening pigments; CH, corneal hypodermis cells; COC, crystalline cone cells; C, crystalline cone; PP, primary pigment.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Figure 3

Histological structure of the ommatidia-ONL juncture and ONL.

(A) Ommatidia-ONL juncture and ONL with H&E staining. (B) The FGCs and glial cells stain positively with TB. (C) FGCs also show PAS-positive staining, (D) and stain blue with MT. (E) The USP stains brown, but the LSP stains apple-green with LFB-PAS. (F) At higher power, many fine granules can be seen in FGCs and are TB-positive. BM, basement membrane; GC, glial cells; HS, hemal sinus; LH, lacuna of hemocoel; LSP, lower screening pigments; SC, support cells; USP, upper screening pigments; *, foamy glandular cells; ✦, pigments in ONL.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Figure 4

The fine histological structure between the medulla internalis and medulla terminalis.

(A) Three differently sized cell clusters can be observed. The middle-size cell clusters (arrow) have not been described before. H&E staining. (B) The middle-size cell clusters (arrow) show SYN positive staining. The inset shows the middle-size cell cluster at higher magnification. ART, arteriole; ONF, optic nerve fiber; NS, neurosecretory cells; NC, neuron clusters; MI, medulla internalis.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Figure 5

FGCs stained by IHC markers.

(A) vimentin, (B) SYN, (C) pan-CK, (D) GFAP, (E) NSE, (F) and BDNF.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Table 1(on next page)

The histochemical staining results of cells in cuticle, ommatidia, and the juncture of ommatidia- ONL.

*, negative or the color of background SYN, synaptophysin; NSE, neuron specific enolase; USP, upper screening pigments; LSP, lower screening pigments.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Table 1. The histochemical staining results of cells in cuticle, ommatidia, and the juncture of ommatidia- ONL.

Structure Cells Histochemical stain method H&E TB MT PAS LFB-PAS Cuticle eosinophilic blue red -* -

Ommatidia

cornea eosinophilic blue navy blue -- cornea hypodermis basophilic blue violet firebrick --

cone cells dark basophilic

crystalline cone dark eosinophilic navy blue dark red --

retinular cells eosinophilic medium purple dark magenta plum plum Juncture of the ommatidia- ONL

USP brown dark gray brown light brown brown

LSP brown dark brown - dark brown apple green

rhabdom eosinophilic blue indigo - light blue

*, negative or the color of background SYN, synaptophysin; NSE, neuron specific enolase; USP, upper screening pigments; LSP, lower screening pigments.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Table 2(on next page)

The histochemical staining results of cells in ONL, lamina ganglionaris, and medulla.

*, negative or the color of background #, the optice nerve is eosinophilic with fine brown granules infiltrated diffusely. a, only the parts of smaller neural cells are positive. b, the collagen fiber that surround the ateriole is blue. c, the hemolymph in the arteriole is positive. USP, upper screening pigments; LSP, lower screening pigments; FGCs, foamy glandular cells; LH, lacuna of hemocoel; NS, neurosecretory cells;

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017 Table. 2 The histochemical staining results of cells in ONL, lamina ganglionaris, and medulla. Structure Cells Histochemical staining method H&E TB MT PAS LFB-PAS ONL optic nerve eosinophilic# blue magenta -

FGCs basophilic dark violet royal blue pink pink

LH eosinophilic blue dark red -- pigment light brown dark gray royal blue brown - Lamina ganglionaris glial cells light basophilic dark violet - -- support cells basophilic blue magenta -- hemal sinus eosinophilic blue dark red -- Medulla nerve fiber eosinophilic blue dark magenta -- neuron cluster eosinophilic blue dark magenta -- NS basophilic blue dark magenta -- arteriole eosinophilic blue blueb --

*, negative or the color of background #, the optice nerve is eosinophilic with fine brown granules infiltrated diffusely. a, only the parts of smaller neural cells are positive. b, the collagen fiber that surround the ateriole is blue. c, the hemolymph in the arteriole is positive. USP, upper screening pigments; LSP, lower screening pigments; FGCs, foamy glandular cells; LH, lacuna of hemocoel; NS, neurosecretory cells;

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3453v1 | CC BY 4.0 Open Access | rec: 8 Dec 2017, publ: 8 Dec 2017