histological and histochemical investigation of the compound eye … · examined histological...

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 staining 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 neuron 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

Histological and histochemical investigation of the compound eye of the whiteleg shrimp

(Litopenaeus vannamei)

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

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

Road, South Dist., Taichung, Taiwan.

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

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

Taiwan.

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

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

Fax: +886-4-2285-2186

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Abstract

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 staining 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

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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

neuron 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.

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

glandular cells, neural cluster

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Background

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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examined histological structures after use of five different staining methods that are frequently

used in histology and pathological diagnosis.

Histochemical staining methods are usually applied to identify tissues and chemical

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

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

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

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

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

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

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

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

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

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

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

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

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

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No investigations of compound eyes in penaeid shrimps are available. The information

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

will help to remedy this deficit.

Material and Methods:

Sample preparation

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

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

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

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

any notable abnormalities in appearance.

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

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

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

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

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

subjected to histochemical and immunohistochemical staining.

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Histochemical staining

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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The sections were analyzed under a light microscope, and images were acquired using a

digital camera (Olympus DP70, Japan).

Immunohistochemistry (IHC) staining

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

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

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

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

Results

Histochemical staining

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

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

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

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

Cuticle, cornea, and ommatidia

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The superficial layer of the compound eyes is a thin layer termed cuticle. Below the cuticle

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

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

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

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

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

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

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

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

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

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

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

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

cells (Fig. 2B).

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

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

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

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pigments) are seen to be concentrated in the upper and bottom zones of the rhabdom after H&E

staining (Fig. 3A).

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

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

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

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

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

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

Optic nerve layer

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

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

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

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

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

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

and lamina ganglionaris.

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The cytoplasm of FGCs is PAS-positive (stained pink by Schiff’s solution) (Fig. 3C), and

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

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

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

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

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

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

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

LSP of ommatidia.

Lamina ganglionaris

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

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

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

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

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

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Medulla

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

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

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

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

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

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

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

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

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

Immunohistochemistry (IHC) staining

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

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

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

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

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Other markers, including pan-CK, GFAP, NSE and BDNF, were not detectable in the FGCs (Fig.

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

Discussion

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

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

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

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

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

consistent between different studies.

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

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

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

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

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

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

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epicuticle is largely composed of hydrophobic wax, which retains red coloration (Biebrich

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

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

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

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

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

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

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

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

Screening pigments with different histochemical characteristics were observed in the bottom

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

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

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

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

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

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

the primary and secondary pigments.

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The ONL is composed of abundant nerve fibers and lacunae, and the density of nerve fibers

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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is a type of glycoprotein, and is regarded as a highly sensitive marker for neuroendocrine cells

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

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

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

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

is a strong indication that FGCs are of neuroendocrine origin.

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

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

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

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

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

vannamei.

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

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

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

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

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Conclusion

Using several histochemical staining methods, the histological features and biochemical

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

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

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

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

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Figures-text

Fig. 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.

Fig. 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.

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Fig. 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.

Fig. 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.

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Fig. 5. FGCs stained by IHC markers. (A) vimentin, (B) SYN, (C) pan-CK, (D) GFAP, (E) NSE,

(F) and BDNF.

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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.


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.


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.


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.


Figure 5

FGCs stained by IHC markers.

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


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.


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.


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;


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;


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