Histological and Histochemical Investigation of the Compound Eye of the Whiteleg Shrimp (Litopenaeus Vannamei)
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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 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.