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).