Abstract
Scanning electron microscopy (SEM) has significantly advanced morphological studies, particularly in the investigation of fish scale structures. This technique has unveiled intricate architectural details that are crucial for fish identification and classification. In this study, macro- and microscopic analyses were employed to examine the scale morphology of Schizothorax plagiostomus , a vulnerable cyprinid fish from Kashmir, focusing on two body regions (key scales, which were taken from the region below dorsal fin and above lateral line; lateral line scales which were taken from the lateral line). The general scale type observed in Schizothorax plagiostomus was cycloid. Two types of shapes viz., polygonal and cordate were reported in this species. The rostral margin of the scales displayed round and waved forms. The scales exhibited a small and round focus which was antero-centrally positioned. The scales featured narrow or wide grooves (radii) categorized into three types: primary, secondary, and tertiary, present across all four scale fields (anterior, posterior, nuand lateral), forming a tetra-sectioned type. Circuli, arranged in circular patterns around focus were present which were densely placed in anterior and lateral field and widely spaced in posterior field. Notably, lepidonts on the circuli and chromatophores on the posterior margin were absent in this species. These scale characteristics and their morphologies offer a valuable tool for the identification, classification, and phylogenetic analysis of various freshwater fish species and genera.
INTRODUCTION
The Kashmir valley is located in the Himalayan Mountain system along the eastern area, between the Pir-Panjal and Zanskar ranges. It has a unique position in harboring rich and diverse types of aquatic habitats, occupying 6% of its total land area (Zutshi and Gopal, 2000). There are lentic and lotic waterbodies in the valley well known around the world. Over the years, extensive research has been conducted on the water bodies of the Kashmir valley, particularly focusing on the rich ichthyofauna inhabiting its lakes and streams. Fishes belonging to the families Cyprinidae, Cobitidae, Siluridae, Poecilidae, Sisoridae and Salmonidae are found in the valley (Bhat et al., 2010). The major ichthyofauna of Kashmir is represented mainly by the Central Asiatic fauna in which genus Schizothorax is predominant (Sunder et al., 1979). The genus Schizothorax is the most diversified Schizothoracine, possessing more than 100 species and subspecies and is represented in Kashmir waters by S. niger , S. curvifrons ,S. esocinus , S. labiatus and S. plagiostomus (Waniet al ., 2018). Schizothorax plagiostomus (locally known as Khont) stands out with its elongated body, projecting snout, and lower jaw with a sharp edge. It has distinctive features like expanded lower lip fold. This fish prefers fast flowing streams in Kashmir (Kullanderet al ., 1999). This is an important food fish of Kashmir Himalayas. The fish has been declared vulnerable by International Union for Conservation of Nature (IUCN) on 25th April 2022. The vulnerable state of this indigenous fish species reflects the broader challenges confronting Kashmir’s water bodies, constantly subjected to ecological changes due to various anthropogenic factors (habitat degradation, overfishing and water pollution). Inadequate attention and insufficient measures exacerbate the risks faced by this fish species; therefore, necessary conservation measures should be taken to conserve this vulnerable species. This highlights the importance of my work where use of SEM can enhance taxonomic precision and contribute valuable insights into the morphological features of various species facing threats. This innovative approach not only aids in accurate identification but also forms a foundation for framing effective conservation strategies. By providing a precise tool for species identification, my work extends its benefits to other vulnerable species globally. Application of scanning electron microscopy (SEM) in fish taxonomy is relatively recent development (Delmater & Courtenary, 1974; Kaur & Dua, 2004; Johal et al ., 2006; Liu et al ., 2008). The scanning electron microscope (SEM) is one of the most versatile instruments available for the examination and analysis of microstructure morphology and chemical composition characterizations (Zhou et al ., 2007). It is a type of electron microscope that uses a focused beam of high-energy electrons to scan the surface of a sample to create detailed images of its topography, composition, and other properties. Scanning electron microscopy (SEM) has significantly advanced morphological studies, particularly in the investigation of fish scale morphology (Echreshavi et al., 2021; Esmaeili et al., 2019; Sadeghi et al., 2021). This instrument has greatly enhanced our understanding of the macro- and microstructural morphology of scales, revealing numerous new features that are now effectively utilized in ichthyological research. Since its invention, SEM has made substantial contributions to the field. Fish scales are dermal elements embedded in the dermis and epidermis of most fish species, appearing as thin or thick plates (Wainwright, 2019). These scales come in various forms and shapes, and historical research has established a framework for categorizing them, which is fundamental for describing their morphology (Wainwright, 2019). The use of scales in fish classification dates back approximately 188 years, when Swiss Zoologist Louis Agassiz (1833-1843) first employed them as a taxonomic tool. He classified fish into four groups based on their scales: Placodermi, Ganoidei, Ctenoidei, and Cycloidei. Since then, numerous ichthyologists have described fish scales and utilized their morphology for taxonomy (Goodrich, 1907, 1909; Lagler, 1947; Lanzing & Higginbotham, 1974; Lippitsch, 1990; Williamson, 1851). Fish scales exhibit various structural features such as type, shape, size, circles, radii, lepidonts, granule configurations and lateral line structures, which have been used in fish identification and classification at species and higher taxonomic levels (Batts, 1964; Hughes, 1981; Kaur and Dua, 2004). Tzeng et al . (1994) emphasized the utility of fish scales in comprehending the life history of a fish, including age composition and growth rate. Various researchers, such as Kobayashi (1953), Dulce-Amor et al . (2010), Esmaeli et al . (2012), and Zubia et al . (2015), have utilized scales to study phylogenetic relationships, systematic classification, and sexual dimorphism. Fish scales have proven to be effective bioindicators of water pollution (Kaur & Dua, 2012). Various pollutants have been identified in the environment as a consequence of urbanization, industrialization, and technological advancements (Kaur & Dua, 2012). Aquatic organisms are biologically sensitive and can respond to changes in water quality, with fish scales being among the first structures to be affected by environmental changes, including pollution. Surface morphology and microstructures of scales have been used to identify various fish groups, including cichlids (Lippitsch, 1990), tripterygiids (Jawad, 2005), cyprinids (Jawad, 2005), synodontids (Jawad & Al-Jufaili, 2007), cyprinodontids/aphaniids (Esmaeili et al., 2019; Esmaeili & Gholami, 2007; Ferrito et al., 2003; Ferritoet al., 2009; Teimori et al., 2017), mugilids (Esmaeiliet al., 2014), and clupeids (Dizaj et al., 2020). Schizothoracids have been subjected to various morphological, biological, and molecular studies (Lin et al., 2010; Nie et al., 2014; Zhou et al., 2015; Zhang et al., 2017; Hussainet al., 2018; Farooq et al., 2019; Gu et al., 2019). However, information on detailed scale morphology and microstructures using SEM of genus Schizothorax have not been provided in literature yet.
This study represents the first attempt to utilize Scanning Electron Microscopy (SEM) to describe the ultrastructural features of the scales of the vulnerable cyprinid fish S. plagiostomus , collected from the Jhelum River in Kashmir. Our objectives are (i) to describe and emphasize the microstructural morphology of the scales, and (ii) to identify potential topographical and structural characteristics of the scales that can be used as taxonomic tool.
Materials and Methods
Sampling site and studied taxa
To study the morphological characteristics and ultrastructural design of scales in S. plagiostomus, a total of 12 fish specimens of same size were collected from River Jhelum (32˚58΄- 35˚38΄N, 73˚23΄-75˚35΄E) using cast net in December 2023. Samples were transported to Fisheries Resource Management (FRM) laboratory of Faculty of Fisheries, SKUAST-K in insulated boxes containing ice packs.
Scale preparation for Scanning Electron Microscopy (SEM) imaging
Fish total length and weight were measured using digital vernier caliper and weighing balance before scale removal. Key scales and lateral line scales (Figure 2) were used in the present study following Raffeallaet al., 2020 and Dey et al., 2014. Key scales are the scales taken from the region below the dorsal fin and above the lateral line. Scales taken from the middle of lateral line are referred to as lateral line scales. In this study, a total of 240 scales were examined which comprised 20 scales from each specimen. Utmost care was taken so that no damage to the scales would occur while removing them from the selected regions with fine forceps. For the preparation of scales, Teimori et al. (2017), Esmaeili et al. (2012) and Lippitsch (1990) were followed. The scales were rinsed in distilled water to remove impurities followed by immersion in 5% potassium hydroxide (KOH) solution for a duration of 40 minutes to remove soft tissues from surface. It was followed by dehydration process using ethanol solutions of varying concentrations (30%, 50%, 70%, and 90%) each for a duration of 20 minutes and dried on filter paper. To prevent curling of scale margins, the scales were delicately positioned between two microslides for a period of 2-3 days. For SEM imaging, the prepared scales were carried to the Central Research Facility Centre, National Institute of Technology (NIT)-Srinagar where scales were attached to metal stubs with double adhesive tape, ensuring that the dorsal surface was oriented upward, while the ventral surface was securely affixed to the tape (Dey et al., 2014). Metal stubs were then gold coated to a thickness of 100 Å in gold coating unit. The samples coated with gold were introduced into the SEM’s holding chamber for in-depth analysis. The scales were observed in the SEM using the secondary electron emission mode, employing an accelerating voltage of 20 kV. Tilt control was set at 0° to precisely position the samples in a horizontal plane. The working distance was maintained at 8 mm.
Scale measurements
Various parameters of scale components including length and width of scale, inter-radial distance of circuli, width of circuli, inter-circular space, width of radii, and more, were measured. These measurements were extracted from the SEM micrographs, utilizing reference bars present in the images (Dey et al., 2014). Length of scale (LS) was calculated as anterior-posterior length or the maximum longitudinal diameter of the scale (Figure 3a), width of scale (WS) as maximum transverse diameter of the scale (Figure 3b), anterior focus length (AFL) was measured from anterior margin of scale to focus (Figure 3c), location of focus or Focus index(Fi) was calculated as the ratio of anterior focus length (AFL) to length of scale (LS) (Dey et al., 2014; Teimori et al., 2017; Jufaili et al., 2021).If the value of Fi <0.20, focus is said to be anterior in position; if Fi = 0.21-0.40, focus is said to be antero-central in position; if Fi = 0.41-0.60, focus is said to be central in position; if Fi = 0.61-0.81, focus is said to be postero-central in position and if Fi> 0.81, focus is said to be posterior in position (Brager, 2016; Sabbah et al., 2020; Echreshavi et al., 2021). Morphological descriptive analysis was carried out using R studio version 4.2.2 (2023).