Scanning Electron Microscopy of Scales and its Taxonomic ...

Microsc. Microanal. 20, 1188–1197, 2014 doi:10.1017/S1431927614001019

© MICROSCOPY SOCIETY OF AMERICA 2014

Scanning Electron Microscopy of Scales and its Taxonomic Application in the Fish Genus Channa Sudip Dey,1,* Shyama P. Biswas,2 Samujjwal Dey,3 and Shankar P. Bhattacharyya4 1 Electron Microscope Division, Sophisticated Analytical Instrument Facility, North Eastern Hill University, Shillong-793022, Meghalaya, India 2 Department of Life Sciences, Dibrugarh University, 786004 Dibrugarh, Assam, India 3 Department of Zoology, St. Edmund’s College, 793001 Shillong, Meghalaya, India 4 Department of Zoology, G. C. College, 788001 Silchar, Assam, India

Abstract: Scanning electron microscopy (SEM) of scales in six species of the fish genus Channa revealed certain features relevant to taxonomic significance. The location of focus, inter-radial distance and width of circuli, intercircular space, width of radii, shape and size of lepidonts, etc. were found to be different in different species. The importance of SEM of scales in poorly understood taxonomy and phylogeny of the fish genus Channa is discussed with the help of relevant literature. Further, the role of SEM of fish scales for taxonomic applications is discussed in detail. Key words: SEM, Channa, scale, taxonomy, micro structure, fish

I NTRODUCTION Microscopy of scales appears to be of remarkable value in fish taxonomy. During the 20th century, with the advancement of optical microscopy, scale morphology assumed significance in fish systematics (Lagler, 1947; Kobayashi, 1951, 1952). The application of scanning electron microscopy (SEM) in fish taxonomy, however, is a relatively recent development (Delmater & Courtenary, 1974; Kaur & Dua, 2004; Johal et al., 2006; Liu et al., 2008). Delmater & Courtenary (1974) are probably among the few earlier workers who used SEM in relation to surface micro-structural features of scales. Lanzing & Higginbotham (1974) made SEM studies on lepidonts and epidermal surface patterns in the scales of Tilapia mossambica (Peters). Liu et al. (2008) studied the structure and composition of teleost scales from the snake head Channa argus. In India, a country with rich ichthyo-faunistic diversity, pioneering work on the use of SEM of scales in relation to fish taxonomy was carried out by Johal & Agarwal (1997), Kaur & Dua (2004), and Johal et al. (2006). Surface micro structure of fish scales is useful in identification of major groups of fish and even up to species level (Johal et al., 2006). It has been observed that numerous unknown and unexplored details of surface micro-structural features of fish scales can be of remarkable value in identification and classification of fish (Kaur & Dua, 2004). Thickness, arrangement, and relative spacing of circuli around the focus (Casselman et al., 1986) and the texture, shape, orientation, and attachment of lepidonts (Hollander, 1986; Johal & Dhiman, 2007; Negi et al., 2010; Esmaeili & Gholami, 2011) of the scale are known to be important in Received December 20, 2013; accepted April 29, 2014 *Corresponding author. [email protected]

fish taxonomy. Ibanez et al. (2007) used geometric morphometric methods to determine if scale morphology can be used to discriminate between genera, species, geographic variants, etc. of mullet (Mugilidae). From the aforementioned studies, it is clear that SEM of certain components of fish scales has the potential for differentiating between genera, families, and geographic variants of some fish. The genus Channa, belonging to the family Channidae, consists of 29 species, out of which 23 are found in India. Fishes belonging to the genus are economically important for their ornamental value and also for their demand as edible fish. However, no report on SEM of surface micro structure of scales of this group of fish in relation to taxonomic application is available in the literature, although the taxonomy and phylogeny of the channid fishes of India are poorly understood (Lakra et al., 2011) because of confusing morphological and meristic characters. The taxonomy of the genus Channa is incomplete, and a comprehensive revision of the family has not been performed. A phylogenetic study has also indicated the likelihood of the existence of more undescribed species of Channids in south East Asia (Adamson et al. 2010). With this background, a SEM study was undertaken on scales of six species of the fish genus Channa viz, Channa punctatus, Channa gachua, Channa marulius, Channa striata, Channa stewartii, and Channa bleheri to examine the importance of micro-structural characteristics of scales in the taxonomy of this group of fish. Some important information on six species of the genus Channa used in the present study are as follows: (i) C. punctatus (Bloch, 1973): It is commonly known as the spotted snake head. Its body is cylindrical and the color varies with habitats. Generally the body color is

Scanning Electron Microscopy of Scales and its Taxonomic Application

(ii)

(iii)

(iv)

(v)

(vi)

yellowish to brown on back and lighter below. Maximum length of the body is 30 cm. Its distribution is in India and adjoining countries including China. C. gachua (Hamilton, 1822): It is commonly known as the dwarf snake head. It has a maximum length of 20 cm. It is a colorful fish and is distributed in Asian countries from Pakistan to Indonesia. C. marulius (Hamilton, 1822): It is commonly known as the bull’s eye snake head or great snake head. Its maximum length is 183 cm. It is native to South Asia. C. striata (Bloch, 1793): It is commonly known as the striped snake head. Its maximum length is 100 cm. It is distributed in Asia from Pakistan to Thailand and South China. C. stewartii (Playfair, 1857): It is commonly known as the Assamese snake head. Its maximum length is 25 cm. It is distributed in Eastern Himalayan region (India and Nepal). C. bleheri (Vierke, 1991): It is commonly known as the rainbow snake head. Its standard length is 15 cm. It is distributed in India and other Asian countries.

M ATERIALS AND M ETHODS

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SEM After cleaning, scales were dehydrated in an ascending grade of ethanol. To avoid curling, after 70% ethanol, the scales were kept between two microscope slides for 2–3 days (Esmaeili & Gholami, 2011). Scales were dried on filter paper (Lippitsch, 1990), and cleaned and dried scales were then secured horizontally to a brass stub (5 mm diameter × 30 mm height) by double adhesive tape. The dorsal surface of the scale faced upward and the ventral surface was adhered to the tape. A conductive coating of gold was applied to the scales with the help of a Fine coat Ion sputter, JFC-1100 (Jeol) by maintaining a relatively low vacuum in the sputtering chamber. The dried and gold-coated scales were viewed in a JSM-6360 (Jeol) SEM using the secondary electron emission mode at an accelerating voltage of 20 Kv. Tilt control was set at 0° to place the samples exactly at horizontal plane. Working distance was 8 mm. While viewing, each scale was oriented in such a way that the rows of circuli examined were in the same plane. This ensured that differences, if any, in the shapes of some micro structures, such as lepidonts, in the scales of different fish species were due to their uniqueness and not due to differences in their orientation.

Fish Materials

Measurements

Six fish species of the genus Channa, viz, C. punctatus, C. bleheri, C. gachua, C. marulius, C. striata, and C. stewartii were used in the present study. The fishes were collected from different water bodies of Dibrugarh, Assam, India during July–August, 2012 as a personal collection and were also purchased from the fishermen from the consumer markets in and around Dibrugarh, Assam, India. Mature fish in the age class of 6 months to 1 year were used for the fish species studied. The lengths of C. punctatus, C. bleheri, C. gachua, C. marulius, C. striata, and C. stewartii used in the present study were about 11.6, 15.2, 12.5, 46, 25, and 20 cm, respectively. The weights of C. punctatus, C. bleheri, C. gachua, C. marulius, C. striata, and C. stewartii were 11.9, 30.8, 23.2, 980, 100, and 105 gm, respectively. They were identified at the Zoological Survey of India, NER region, Shillong. The fish samples after collection were fixed in 5% neutral formalin. Four individual fishes of each species were used in the study to ensure reproducibility of microstructural data. Ten scales each from four individuals of each fish species were excised from the body between the dorsal fin and the lateral line with the help of fine forceps. Since scales from the region below the dorsal fin and above the lateral line are known to exhibit all the characteristic features, they have been designated as “key scales” (Esmaeili & Gholami, 2011), which is why scales from this region were used for comparative studies in the six species of Channa. Utmost care was taken so that no damage to the scales occurred while removing them from the body. After their removal, the scales were rinsed with distilled water and treated with mild potassium hydroxide solution (Esmaeili & Gholami, 2011). This was followed by preparing the samples for SEM.

Statistical Analysis

The body length of each fish sample was measured with a measuring tape before scales were excised. Measurements of different parameters of various scale components such as inter-radial distance of circuli, width of circuli, inter-circular space, width of radii, etc. were made from the scanning electron micrographs of the scale by taking the bars in the micrographs as references. Ten scales each from four individuals representing each species were used for the measurement. For recording the data, four different regions of each scale were used. Thus, the average data generated were from 160 measurements of each component. The ranges of values and averages were also recorded. For determining the location of focus, its distance from the posterior margin, divided by the total scale length, was recorded.

Students’ t-test was used to determine differences in various parameters such as location of focus, inter-radial distance, inter-circular space and width of circuli, spacing of lepidonts, width of radii, etc. among the different species of the genus Channa. The data are presented as an average with standard deviation along with the number of counts and level of significance.

RESULTS General Morphology The scales of all six fish species of the genus Channa exhibit a nearly rectangular shape (Fig. 1). However, variations were observed in length and breadth of the scale in different species.

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Figure 1. Low magnification scanning electron microscopic image of scales of (a) Channa punctatus (b) Channa bleheri (c) Channa gachua (d) Channa marulius (e) Channa striata (f) Channa stewartii. A, anterior part; B, basal part; F, focus; R, radii; C, circuli. Scale bar is 500 µm.

The scales of a particular species on different parts of the body showed the same morphological features. As in other fishes (Esmaeili & Gholami, 2011), the scales present below the dorsal fin and above the lateral line are found to be the largest. The scales on other parts of the body of all six fish species of the genus Channa are relatively smaller in size.

Fields of the Scale The scales of all the six fish species can be divided into four fields. These are: anterior or rostral (A), posterior or caudal or basal (B), and two lateral fields (L) (Figs. 1a–1f).

Focus Each scale has a focus (Figs. 1a–1f). During ontogenesis, this is the part of the scale that develops first. The focus divides

the scale into anterior, posterior, caudal or basal, and lateral fields. The location and shape of the focus show some differences in different species of Channa (Figs. 1a–1f; Table 1). The focus is more or less circular in the species of Channa examined, except in C. striata and C. stewartii where they are pear shaped or nearly tetragonal, respectively. Location of the focus relative to the posterior edge of the scale in C. punctatus, C. bleheri, C. gachua, and C. stewartii is about 0.451, 0.441, 0.452, and 0.471 µm, respectively. In scales of C. marulius and C. striata location of the focus are about 0.632 and 0.588 µm, respectively. Thus, location of the focus in all members of the gachua group (Vishwanath & Geetakumari, 2009) is more or less similar. Location of the focus in the members of the marulius group (Vishwanath & Geetakumari, 2009) is almost the same, but is distinctly different from that of the gachua group.

Table 1.

Structural Details of Some Scale Components in Six Species of the Fish Genus Channa.

Characters 1 Focus (a) Shape (b) Location (distance from proximal border of scale base relative to total scale length) (n = 160) 2 Anterior circuli (a) Inter-radial distance (n = 160) (b) Average width (n = 160)

(b) Fragmentation pattern (c) Surface features (d) Dentition 4 Lepidonts (a) Shape (b) Spacing (n = 160) (c) Length (d) Breadth 5 Radii (a) Number (n = 160) (b) Width (n = 160)

C. bleheri

C. gachua

C. marulius

C. striata

C. stewartii

Nearly circular 0.451 ± 0.01 µm (Range: 0.441–0.461 µm)

Nearly circular 0.441 ± 0.01 µm (Range: 0.431–0.451 µm)

Nearly circular 0.452 ± 0.01 µm (Range: 0.442–0.462 µm

Circular 0.632 ± 0.01 µm (Range: 0.622–0.642 µm)

Pear shaped 0.588 ± 0.01 µm (Range: 0.578–0.598 µm)

Nearly tetragonal 0.471 ± 0.01 µm (Range: 0.461–0.481)

158 ± 40 µm (Range: 110–245) 6.6 ± 0.1 µm (Range: 6.5–6.7) 8.8 ± 3 µm (Range: 5.8–11.8)

185.2 ± 15 µm (Range: 170.2–200.2) 10.8 ± 0.8 µm (Range: 10–11.6) 14.96 ± 4.0 µm (Range:10.96–18.96)

115 ± 5 µm (Range: 110–120) 8 ± 0.3 µm (Range: 7.7–8.3) 10.8 ± 1.6 µm (Range: 8.4–11.6)

105 ± 5 µm (Range: 100–110) 8.3 ± 0.1 µm (Range: 8.2–8.3) 16.3 ± 0.1 µm (Range: 16.2–16.4)

100 ± 9 µm (Range: 91–109) 8.3 ± 0.1 µm (Range: 8.2–8.3) 8.3 ± 0.1 µm (Range: 8.2–8.4)

145 ± 9 µm (Range: 136–154) 6.6 ± 1.5 µm (Range:5.1–8.1) 11.9 ± 3.6 µm (Range: 8.3–15.5)

23.76 ± 0.6 µm (Range: 23.1–24.3) Medium Rough Absent

30.76 ± 6.2 µm (Range: 26.93–39.33) Less Smooth Absent

23.3 ± 0.4 µm (Range: 22.9–23.7) Medium Smooth Few

23.6 ± 0.2 µm (Range: 23.4–23.8) Medium Smooth Intense

15.38 ± 0.2 µm (Range: 15.18–15.58 Medium Smooth Intense

23.3 ± 0.2 µm (Range: 23.1–23.5) Intense Smooth Intense

Conical 2.1 ± 0.7 µm (Range: 1.5–2.8) 2.4 ± 0.2 1.5 ± 0.1

Conical 1.7 ± 0.4 µm (Range: 0.9–1.7) 3.0 ± 0.2 1.7 ± 0.1

Conical 1.0 ± 0.1 µm (Range: 0.9–1.1) 3.1 ± 0.2 1.2 ± 0.1

Conical 1.1 ± 0.2 µm (Range: 09–1.3) 3.8 ± 0.1 2.0 ± 0.2

Conical 1.4 ± 0.2 µm (Range: 1.2–1.6) 2.5 ± 0.1 2.7 ± 0.1

Conical 1.1 ± 0.1 µm (Range: 1.0–1.2) 3.5 ± 0.2 2.4 ± 0.1

15 19.15 ± 4 µm

12 7.5 ± 0.2 µm

17 7.7 ± 0.2 µm

24 3.8 ± 0.2 µm

16 4.1 ± 0.2 µm

18 13.8 ± 0.4 µm

The differences of values in various parameters of scale components among different species are significant at 0.05. n, number of measurement.


(c) Average inter-circular space (n = 160) 3 Lateral circuli (a) Inter-circular distance (n = 160)

C. punctatus

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Figure 2. Enlarged scanning electron microscopic image of circuli on the scales of (a) Channa punctatus (b) Channa bleheri (c) Channa gachua (d) Channa marulius (e) Channa striata (f) Channa stewartii. C, circuli; R, radii. Scale bar is 100 µm.

Circuli Lines of growth or ridges that start appearing from the focus are the circuli (Figs. 2a–2f). Between the circuli there are spaces, called inter-circular spaces. Circuli are distinct and compact in the anterior part and separated widely in the lateral part. Circuli are separated from one another by radii. Variations are observed in the inter-radial distance circuli, width, circuli, and inter-circular space in the anterior part of the scale in different species of Channa studied (Figs. 2a–2f, 3a–3f; Table 1). The inter-radial distance of ciculii are distinctly different in the scale of each species examined. However, in the members of the marulius group its value is similar and it is much less than those of the members of the gachua group. Width of the circuli are also similar in members of the marulius group and is much different from members of the gachua group. Inter-circular spaces in the anterior circuli also show distinctness in the scale of each species. The largest inter-circular space was recorded from the anterior scale of

C. marulius and the smallest from the scale of C. striata. Lateral circuli also show variations in inter-circular space, fragmentation pattern, surface features, and dentition in different species of Channa examined (Fig. 4; Table 1).

Lepidonts Lepidonts are the teeth-like structures present on the edge of circuli. Their arrangements are different in different species (Figs. 3, 5). Spacing of lepidonts is also different in different species and shapes are more or less conical in all the species, but there are differences in their lengths and widths (Table 1).

Radii The deep and narrow grooves that run radially toward the focus and provide partition between adjacent circuli are called radii. Radii are well developed in scales of all six species of the fish genus Channa examined in the current study. The width


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Figure 3. Scanning electron microscopic micrographs showing the arrangement of lepidonts in the scale of (a) Channa punctatus (b) Channa bleheri (c) Channa gachua (d) Channa marulius (e) Channa striata (f) Channa stewartii. Scale bar is 10 µm.

and number of radii show variations among different species (Figs. 2a–2f; Table 1).

Surface Architecture of Basal Part of Scale The basal part of the scale shows distinct differences in the six species of the fish genus Channa. (i) C. punctatus: In C. punctatus, the basal part of the scale shows well-demarcated plates within which fingerprint-like patterns exist. The presence of mucous pores at different locations is also evident (Fig. 6a). The number of pores was 7 ± 1/mm2 and the diameter 2.3 ± 0.1 µm. (ii) C. bleheri: In this species, well-demarcated plates are present in the basal part of the scale. However, the fingerprint-like patterns are less pronounced here, and many of the plates are found to be devoid of the pattern. Mucous pores of varying diameter are present (Fig. 6b).

The average diameter was 3.07 ± 0.2 µm and the density of pores 10 ± 1/ mm2. (iii) C. gachua: In C. gachua, the basal portion of the scale shows well-demarcated plates within which distinct fingerprint-like patterns are visible. However, the lines of the fingerprint-like patterns in this species show distinct cross bands, which are not present in any other species of Channa examined in the current study (Fig. 6c). The mucous pores could not be detected here. (iv) C. marulius: In this species of Channa, neither the distinct plates nor the fingerprint-like patterns are seen. The entire surface of the scale base is characterized by the presence of minute round structures and some fibers scattered through out the surface. A few mucous pores are also present (Fig. 6d). The diameter of the pore was 08 ± 0.01 µm and the density 2 ± 0.1/mm2. (v) C. striata: In Channa striata, distinct plates and fingerprint-like patterns are not seen. As in C. marulius,

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Figure 4. Scanning electron microscopic micrographs of lateral circulii in the scale of (a) Channa punctatus, (b) Channa bleheri, (c) Channa gachua, (d) Channa marulius, (e) Channa striata and (f) Channa stewartii. Scale bar: (a) 100 µm and (b–f) 50 µm.

the entire surface shows the presence of minute round structures and fine fibers. A few mucous pores of varying size are also present. The average diameter of the pores was 8.2 ± 0.5 µm and 3 ± 0.1/mm2. Some of the fibrous structures showed a tendency to orient in distinct plate-like configurations (Fig. 6e). (vi) C. stewartii: In this species of Channa, the presence of plates and distinct fingerprint-like patterns is evident. However, some of the plates are well demarcated, while others do not show clear demarcation with the adjacent plate. Mucous pores of varying size are present through out the surface (Fig. 6f). The average diameter of the pores was 2.7 ± 0.8 µm and density 18 ± 0.1/mm2. Differences in the average diameter and density of the mucous pores in different fish species examined are significant at p < 0.05.

DISCUSSION Micro-structural components of fish scales, which are considered to be of taxonomic significance, are the focus, chromatophores, radii, circuli, lepidonts, and region below focus. Some of these parameters are prominent in some groups of fish, whereas they are less prominent in others. In the current study, the focus is present in different locations in the six species of the genus Channa. This is in agreement with an earlier report on differences in location of the focus such as at the central position or above center (Kaur & Dua, 2004). The present observation on closeness in location of the focus in C. punctatus, C. bleheri, C. gachua, and C. stewartii, which is significantly different from those of C. marulius and C. striata, is in agreement with grouping of the former four species in the gachua group and the later two in marulius group (Viswanath & Geetakumari, 2009).


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Figure 5. Enlarged scanning electron microscopic micrographs of lepidonts in the scale of (a) Channa punctatus (b) Channa bleheri (c) Channa gachua (d) Channa marulius (e) Channa striata (f) Channa stewartii. Scale bar: (a) 2 µm and (b–f) 5 µm.

The focus of the scale is also important in the elaborate and excellent study on scale surface ultra structure in the cyprinid fish Rutilus frisii (Esmaeili & Gholami, 2011) in relation to taxonomic applications. The line of growth from the focus, i.e., circuli is very prominent in all six species of Channa studied in the present work. Differences in inter-circular space, inter-radial distance of circuli, and width of circuli in different species of Channa recorded in the present study appear to be of taxonomic significance. In this context, it is to be noted that although the importance of circuli in fish taxonomy is amply highlighted in the existing literature (Johal et al., 2006; Esmaeili & Gholami, 2011), differences in inter-radial distance and width of circuli was observed for the first time in the six species of the genus Channa in the present study. Closeness in the inter-radial distance of circuli and the width

of radii in the scales of C. marulius and C. striata (marulius group) and the differences of the same from the species included in the gachua group (Viswanath & Geetakumari, 2009) suggests the importance of inter-radial distance as well as the width of radii in taxonomy of the fish genus Channa. The inter-circular space, which is different in different species of the genus Channa in the present study, was reported to be of taxonomic significance in other fish species as well (Kaur & Dua, 2004; Johal et al., 2006; Esmaeili & Gholami, 2011). Lepidonts, the teeth-like structures located on the margin of circuli are also distinct features of the scales of the six species of the genus Channa. Differences in the size and spacing of lepidonts in the scales of all six species studied suggest that these parameters are of taxonomic significance for the genus Channa and also for the family Channidae.

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Figure 6. Scanning electron microscopic micrographs of surface architecture at the base of the scale of (a) Channa punctatus (b) Channa bleheri (c) Channa gachua (d) Channa marulius (e) Channa striata (f) Channa stewartii. Scale bar: (a) 5 µm, (b) 10 µm, and (c–f) 5 µm.

In this context, it is worthwhile to mention that shape and size of lepidonts on the circuli crest have been considered to be of high taxonomic significance in other fishes (Kaur & Dua, 2004). The fixed number of radii on the “key scales” of the six species of Channa also appears to be taxonomically significant. This is supported by the fact that in greater lizard fish, the number of radii was reported to be constant (three) and this consistency in radii number makes it a good taxonomic criterion for identification of greater lizard fish (Javad & Al-Jufaili, 2007). The present study suggests that the same may be true for the genus Channa. However, further studies are required to confirm whether this is true for species of Channa other than those examined in the present study. The distinct differences in inter-circular distance, smoothness or roughness of circuli surface, the extent of fragmentation pattern of circuli, and presence, absence or differences in density of lepidonts in the circuli on the lateral aspects of scales

in all six species of Channa appear to be important criteria that can be explored for taxonomic applications. Characteristic orderly architectural patterns on the surface of the basal portion of the scale, their distinct differences among the species possessing them, irregularities in the pattern in some species, and complete absence of the same in others, suggest that the said parameter can be of high taxonomic value in the fish genus Channa. On the basis of morphological and osteological comparisons, Vishwanath & Geetakumari (2009) placed nine species of the genus Channa into two groups, namely the gachua group and the marulius group. The gachua group includes Channa amphibeus, Channa aurantimaculata, Channa barca, C. bleheri, C. gachua, C. punctatus, and C. stewartii. The marulius group includes C. marulius and C. striata. Morphologically, members of the marulius group were found to be distinct from those of the gachua group (Vishwanath & Geetakumari, 2009) on the basis of certain distinguishing


characters. In the present study on SEM of scales, similar distinction between the two groups was observed in certain parameters of scale components such as location of focus, inter-radial distance of anterior circuli, average width of circuli, pattern of dentition in lateral circuli, width of radii, and surface architecture of the basal parts of scales. The presence of well-demarcated plates along with fingerprint-like patterns in all species of Channa included in the gachua group, and their complete absence in C. marulius and C. striata, of the marulius group (Vishwanath & Geetakumari, 2009) suggests that surface architecture of the scale base can be considered as important criteria for taxonomic applications. The significant differences in the average diameter and density of the mucous pores in the basal parts of scales in different fish species examined, suggest that these characters also may be explored to find if they have any taxonomic value. Distinct differences as well as similarities observed in certain micro-structural features of scales in some members of the gachua group suggest that there is a need to examine whether or not some sub groups can be formed within the gachua group. Thus, it is clear that SEM of scales may provide additional information and important support to classical taxonomy in the fish genus, Channa.

CONCLUSION The current SEM study on scales in six species of the genus Channa reveals that certain components of the scale, such as focus, circuli, lepidonts, radii, and surface architecture of the basal region are different in different species. The aforementioned structures can be resolved precisely with the help of SEM. It is thus concluded that there are ample opportunities in using SEM of fish scale micro structures in determining species specificity in fish for taxonomical applications and other important applications (Dey et al., 1994, 2008; Psenicka et al., 2008; Ravneet et al., 2009).

ACKNOWLEDGMENTS The authors are thankful to the Head, SAIF, North-Eastern Hill University, Shillong, India for encouragement and to the Zoological Survey of India, NE region, Shillong for identification of fish.

REFERENCES CASSELMAN, J.M., CROSSMAN, E.J., IHSSEN, P.E., REIST, J.D. & BROOKE, H.E. (1986). Identification of Muskellunge Northern Pike and their hybrids. Am Fish Soc Spec Pub 15, 14–46. DELMATER, E.D. & COURTENARY, W.R. (1974). Fish scale as seen by scanning electron microscopy. Biol Sci 37, 141–149. DEY, S., DKHAR, B. & BHATTACHARJEE, C.R. (1994). The optical significance of site-specific elemental distribution in a fresh water fish lens. Cytobios 78, 91–98. DEY, S., KHARBULI, S.M., CHAKRABORTY, R., BHATTACHARYYA, S.P. & GOSWAMI, U.C. (2008). Toxic effect of environmental acid stress on the sperm of a hill stream fish Devario aequipinnatus: A scanning electron microscopic evaluation. Microsc Res Tech 72, 76–78.

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ESMAEILI, H.R. & GHOLAMI, Z. (2011). Scanning electron microscopy of the scale morphology in cyprinid fish, Rutilus frisii Kutum Kamenskii, 1901 (Actinopterygii: Cyprinidae). Iranian J Fish Sci 10, 155–166. HOLLANDER, H.R. (1986). Micro analysis of scale of Poeciliid fishes. Copeia 1, 86–91. IBANEZ, A.L., COWX, I.G. & HIGGIN, O.P. (2007). Geometric morph metric analysis of fish scales for identifying genera, species and local populations within Mugilidae. Can J Fish Aq Sci 64, 1091–1100. JAVAD, L.A. & AL-JUFAILI, S.M. (2007). Scale morphology of greater lizard fish Saurida tumbil (Bloch, 1795) (Pisces: Synodontidae). J Fish Biol 70, 1185–1212. JOHAL, M.S. & AGARWAL, T. (1997). Scale structure of Oreochromis mossambicus (Peters). Res Bull 47, 41–49. JOHAL, M.S., ESMAEILI, H.R. & SHARMA, M.L. (2006). Scale structure of a cobitid fish, Cobitis linea (Heckel, 1849) using different modes of SEM. Curr Sci 9, 1464–1466. JOHAL, M.S. & DHIMAN, M. (2007). Ultra structure of fish scales as a tool in fish identification up to species level of genus Puntius Hamilton-Buchanon. Res J Sci 57, 73–81. KAUR, N. & DUA, A. (2004). Species specificity as evidenced by scanning electron microscopy of fish scales. Curr Sci 87, 692–696. KOBAYASHI, H. (1951). On the value of the scale character as material for the study of affinity in fishes. Jpn J Ichthyol 1, 226–237. KOBAYASHI, H. (1952). Comparative studies of the scales in Japanese fresh water fishes with special reference to phylogeny and evolution. I. Introduction. Jpn J Ichthyol 2, 183–191. LAGLER, K.F. (1947). Lepidological studies.1. Scale characters of the families of Great lakes. Trans Amer Microsc Soc 66, 149–171. LAKRA, W.S., GOSWAMI, M., GOPALAKRISHNAN, A., SINGH, D.P., SINGH, A. & NAGPURE, N.S. (2011). Genetic relatedness among fish species of genus Channa using mitochondrial DNA genes. Biochem Systemat Ecol (doi10.1016/j.bse.2010.12. 012). LANZING, W.J.R. & HIGGINBOTHAM, D.R. (1974). Scanning microscopy of surface structures of Tilapia massambica (Peters) scales. J Fish Biol 6, 307–310. LIPPITSCH, E. (1990). Scale morphology and squamation pattern in cichilids (Teleostei, Perciformes): A comparative study. J Fish Biol 37, 265–291. LIU, C.-H & SHEN, S.-C. (1991). Lepidology of the mugilid fishes. Journal of Taiwan Museum 44, 321–357. NEGI, R.K., JOHAL, M.S. & RAWAL, Y.K. (2010). Ultra structure of the scale of hill stream fish, Schistura monotanus (Mc Clelland) and its phylogenetic significance. The Bioscan 5, 395–397. PSENICKA, M., RODINA, M., FLAJSHANS, M., KASPAR, V. & LINHART, O. (2008). Structural abnormalities of common carp Cyprinus carpio spermatozoa. Fish Physiol Biochem 35, 591–597. RAVNEET, K., SHARMA, M.L. & KANG, H.P.S. (2009). High resolution scanning electron microscope examination of the fish scale: Inspiration for novel biomaterials. J Biomim Biomater Tissue Eng 4, 13–20. VISHWANATH, W. & GEETAKUMARI, K.H. (2009). Diagnosis and interrelationships of fishes of the genus Channa Scopoli (Teleostei: Channidae) of north eastern India. J Threatened Texa 1(2), 97–105.