Age, Growth, and Reproductive Biology of Euchiloglanis davidi in the Middle and Lower Yalong River, China

Abstract

In the present study, we investigated the age, growth, and reproductive characteristics of Euchiloglanis davidi, a species of Sisoridae fishes, from the Yalong River, China. Fish samples were collected from the tributaries of the middle and lower Yalong River in May 2013 and December 2015. Their standard length ranged from 67 to 190 mm, and the total weight ranged from 3.42 to 75.06 g. The age and growth pattern of E. davidi were determined using the vertebra. Furthermore, the sampled fish were categorized into 10 age groups (1–10 years old): the maximum age was 10 years and 9 years for male and female fish, respectively. The relationship between the standard length and weight was expressed as W = 6.744 × 10⁻⁵ L^2.653, and the t-test showed allometric growth. The growth of E. davidi was defined by the von Bertalanffy growth function, described as L_t = 260.8(1−e^{−0.104(t+1.942)}), W_t = 173.5(1−e^{−0.104(t+1.942)})^2.653, respectively. The growth inflection point was observed at 7.44 years, with corresponding length and weight values of 162.50 mm and 49.46 g. The fecundity ranged from 105 to 352 eggs, with an average of 205 eggs. The relative fecundity of weight (F_W) ranged from 2.90 to 8.98 eggs/g, and the relative fecundity of length (F_L) ranged from 0.71 to 2.11 eggs/mm. Altogether, these characteristics of slow growth and low fecundity demonstrated the vulnerability of E. davidi to exploitation in the middle and lower reaches of the Yalong River.

1. Introduction

Euchiloglanis davidi, a Sisoridae fish species, is widely distributed in the main streams and tributaries of the Jinsha River and Min River, China, at an altitude of 1800 to 3600 m [1]. It is a cold-water fish and is endemic to the upper reaches of the Yangtze River. It is known to frequently inhabit fast-flowing tributaries of rivers and mountain streams, with boulders and cobbles in the riverbed. The fish shows creeping movement with its flattened abdomen and the ventral surface of the mouth and thorax attached to the rocks. E. davidi feeds primarily on aquatic insects and their larva and attached algae [2]. Commercially, it is one of the crucial species in Sichuan Province, because of its high content of nutritional and medicinal products. However, over-exploitation of natural resources, hydroelectric development, and indiscriminate habitat degradation have severely declined the population of E. davidi. Because of its dwindling numbers, the species has been listed as the key protected wildlife of Sichuan Province and the national secondary key protected wildlife [3].

The current research on E. davidi is largely concentrated on its taxonomy, morphology, phylogeny, and biology. The genus Euchiloglanis was first reported by Chu [4], following which Zhou et al. [5] described its two species, namely E. davidi and E. kishinouyei. Guo et al. [6] and Zou et al. [7] studied the phylogeny of the genus Euchiloglanis using morphological characteristics, 16S RNA sequencing, and mitochondrial genome analysis. Luo et al. [8] reported that the skull morphology of E. davidi was highly specialized for adaption to its special living environment with rocky substrates. Pan et al. [9] reported that Euchiloglanis is a rich source of nutritious food with a high meat content and suitable composition of amino acids and fatty acids. Similar to other Sisoridae fishes, E. davidi is a small fish, characterized by slow growth and large oocytes, which might increase its vulnerability to overfishing [2,10]. Nevertheless, efforts to develop an effective population management strategy have been hampered by a lack of basic biological information about this fish species. Age and growth constitute important components of fish biology; an accurate determination of fish age can provide a theoretical basis and support to conduct biological studies on its reproduction, feeding habits, and mortality [11,12,13]. In addition, studying reproductive biology is essential in understanding a species’ life history and provides a fundamental prerequisite for its management and conservation.

The Yalong River is a typical canyon river, whose basin is located in the south of the Qinghai-Tibet Plateau with 36,000 km² of drainage area. It originates in the southern Bayan Har Mountains and spans a length of 1571 km [14]. It constitutes the largest tributary of the lower Jinsha River and is known for its unique plateau environment, which makes it suitable for E. davidi inhabitance. This area has a subtropical climate with an annual temperature of 7.3 °C and an annual average rainfall of 900 to 1400 mm [15]. Because of a sharp decrease in the elevation (from 4500 m to 900 m) and huge hydropower resources, the Yalong River Basin has emerged as an important hydropower development base in China [14]. However, hydropower development and excessive human interference have resulted in reduced fish resources in the middle and lower Yalong River, characterized by low age and miniaturization, reduced fish abundance and diversity, and even an increased possibility of extinction [16].

The high degree of specialization, reduced wild resources, and low fecundity of E. davidi make their recovery highly challenging after damage. Therefore, it is of utmost importance to conserve the wild resources of E. davidi. The main goals of this study were to (1) describe the annulus characteristics of vertebra and otolith and compare age estimates between the two calcified structures, (2) estimate the age and growth parameters of E. davidi using vertebrae and compare growth characteristics of the species with other Sisoridae fishes, and (3) describe the reproductive characteristics of E. davidi including the male-specific organ genital papilla and study the fecundity, relative fecundity, and the regression analysis of relationships between standard length, total weight, ovary weight, age, and fecundity.

2. Materials and Methods

2.1. Sample Collection

A total of 150 specimens were collected from the Yayi River, Jiulong River, Litang River, and Anning River, which are the tributaries of the middle and lower Yalong River (Figure 1), in May 2013 and December 2015, using trap nets (mesh size of 1.5 mm) and backpack electro-fishing gear. After sampling, fish (Figure 2) were euthanized with tricaine methanesulfonate (TMS, MS-222, Wuhan, China), followed by measuring the standard length (SL) and total length (TL) to the nearest 1 mm and the total weight (W) to the nearest 0.01 g. The sex was identified by observing the genital papilla and gonads macroscopically [17,18]. The gonads were assigned a gross maturity stage based on the macroscopic features [17].

The 2nd–7th vertebrae were removed, stored in labeled packets, and frozen until further processing. The right and left lapillus otoliths were extracted from the vestibular apparatus. The otoliths were rinsed with water, air-dried, and stored in labeled tubes. A total of 35 females with mature stages were selected, and their ovaries were weighed and fixed in 7% formalin for further calculation of fecundity.

2.2. Age Determination

The vertebrae and lapilli were used to estimate the age of E. davidi. The vertebrae were boiled in water for 1 to 2 min, following which the attached muscles and connective tissue were removed with a tweezer and brush. Next, they were immersed in 1% hydrogen peroxide for 24 h. The vertebrae were placed on concave slides and soaked in xylene at an adjusted angle for observation under a dissecting microscope (Zeiss Stemi 508, Oberkochen, Germany) [19].

Each lapillus was fixed on a slide with nail polish, ground manually with wet sandpaper (2000–2500#), and polished with alumina paste (3 µm) until the core and all annuli were visible under a compound microscope (Olympus CX23, Tokyo, Japan) [20].

Blinded to the sample size, sex, and collection date, the age of each vertebra and lapillus was identified by the same reader after an interval of 3 weeks. For a better comparison between two calcified structures, two counts should be consistent. If the two counts were different, then the structures were re-counted and the final count was accepted as the agreed age. The annulus clarity of the calcified structures was scored for readability on a 5-point scale as 1. excellent, 2. good, 3. acceptable, 4. poor, and 5. unreadable [21] (

Table 1. The annulus clearness grades of age materials.

Rank	Readability Level	Annuli Characteristics
1	Excellent	The annulus is clear and obvious, which can accurately identify the age
2	Good	The annulus is basically clear, and can identify the age accurately
3	Average	Some annuli are not clear or cannot be accurately identified
4	Poor	Most annuli cannot be accurately identified and may count as possible
5	Unreadable	Annulus cannot be identified

). In addition, the success rate of age estimation from vertebra and otolith was summed by the percentage of scale 1–4.

2.3. Length–Weight Relationship

The relationship between the length and weight of fish was calculated as W = a L^b, where W is the total weight (W, g), L is the standard length (SL, mm), a is the intercept, and b is the allometric growth coefficient. The allometric index value (b) obtained was compared to the expected value “3” using a t-test for allometry [22].

2.4. Growth Parameters

The growth parameters were calculated using the von Bertalanffy growth equation [23], $L_t=L_{\infty }(1-e^{-k(t-t_0)})$, $W_t=W_{\infty }(1-e^{-k\left(t-t_0\right)}{)}^b$, where t is the age of the fish, L_t and W_t are the standard length (mm) and weight (g), respectively, L_∞ and W_∞ are the theoretical maximum length (mm) and weight (g), respectively, k is the growth coefficient, and t₀ is the hypothetical age at length 0. The growth inflection point was calculated by the equation t = lnb/k + t₀. The growth performance index was calculated using the equation of Munro and Pauly [24] as Ø = log₁₀ k + 2 log₁₀ L_∞; Ø was used to compare the growth parameters obtained in this study with those reported by other Sisoridae fishes.

2.5. Fecundity and Oocyte Diameter

Thirty-five females at mature stage were used to study fecundity. To determine fecundity, all oocytes that had begun vitellogenesis were counted as potentially ripe eggs. The relative fecundity (F) was calculated by the formula F_L = F/L or F_W = F/W, where L is the standard length and W is the total weight. The correlations between absolute fecundity and standard length, weight, ovary weight, and age of E. davidi were assessed using the regression analysis.

Two females were randomly selected and their oocytes were imaged using a dissecting microscope (Zeiss Stemi 508). The diameters of oocytes were measured to the nearest 0.01 mm (Image Pro Plus 6.0).

2.6. Statistical Analyses

Data were processed in Microsoft Excel 2016. Statistical analyses were performed and graphs were generated using SPSS 22.0 and OriginPro 2016 at a significance level of α = 0.05.

3. Results

3.1. Comparison of Calcified Structures

A microscopic view of the vertebrae revealed that translucent bands were separated by distinct opaque bands through the focus to the margin. The vertebrae were characterized by a typical pattern of teleost fishes, with translucent zones alternating with opaque zones that were attributed to fast and slow growth periods. Annuli appeared as a series of concentric zones and parallel to the edge of the centrum. The annulus was clear and could be used to identify the age of the fish in most cases (Figure 3). However, the first and the marginal annuli were highly indistinguishable in several large centrums.

The lapillus otolith core was unclear, and the rings were not evident. The core of the otolith was thick in the middle and thin at the edges, making it difficult to polish. After grinding, the annuli were fuzzy, making it challenging to determine the age.

The annuli on the vertebrae were clearer and were rated as “excellent” and “good” in 47.00% and 27.35% of specimens, respectively (

Table 2. Distribution of scores for calcified structure of E. davidi.

Calcified Structure	Proportion of Readability Scores (%)
	1	2	3	4	5
Vertebrae	47	27.35	8.55	9.40	7.69

). The success rate of the identification of age using vertebrae annuli was 94%, whereas the success rate of age identification using lapillus otoliths was only about 30%. Therefore, the vertebrae were selected to determine the age of E. davidi.

3.2. Length–Frequency Distribution and SL–W Relationship

The standard length of all individuals ranged from 67 to 190 mm (mean length: 107.3 ± 35.6 mm), mainly distributed in the range of 70–100 mm and accounting for 55.33%, followed by 140–170 mm, accounting for 27.33%. The standard length of 65 males ranged from 68 to 190 mm (mean: 95.8 ± 31.3 mm), concentrated at 70–100 mm, accounting for 69.23%. The standard length of 85 females ranged from 67 to 184 mm (mean: 116.1 ± 36.3 mm), largely distributed at 70–100 mm and 140–170 mm, accounting for 44.71% and 42.35%, respectively (Figure 4).

The relationship between the standard length and weight of the total samples was determined using the following power function formula: W = 6.744 × 10⁻⁵ L^2.653 (R² = 0.962, n = 150) (Figure 5). The growth index (b) and the expected value 3 were compared using a t-test, indicating that the growth of E. davidi was negative allometric (t = 8.000, p < 0.05).

3.3. Age and Growth

Out of 150 specimens, age was successfully identified for 141. They were categorized into 10 age groups, i.e., 1–10 years. The dominant age groups were 1 and 2 years, accounting for 20.6% and 27.3% of the total specimens, respectively, followed by 3 and 6 years, accounting for 12% and 10.7%, respectively. The ages of 5 and 7 accounted for 8.5% and 9.2%, respectively. There were very few individuals with ages of 4, 8, 9, and 10 years (Figure 6). The number of samples, standard length, and weight of different age groups are depicted in

Table 3. Number, standard length, and weight in different age groups of E. davidi.

Age	Number	Standard Length (mm)		Weight (g)
		Mean ± S.D.	Range	Mean ± S.D.	Range
1	31	76.6 ± 15.8	67–85	7.0 ± 7.2	3.42–11.16
2	41	80.0 ± 16.0	67–97	7.5 ± 7.2	4.30–11.68
3	18	101.7 ± 18.4	89–126	15.8 ± 8.1	8.57–36.02
4	3	126.3 ± 30.0	117–132	28.0 ± 15.2	17.20–38.36
5	12	145.3 ± 31.5	121–162	36.4 ± 14.7	28.09–44.40
6	16	149.9 ± 36.6	134–170	39.7 ± 17.6	28.98–53.54
7	13	160.9 ± 34.6	145–190	49.1 ± 17.0	36.07–75.06
8	5	160.2 ± 35.7	144–184	45.4 ± 17.5	36.60–59.05
9	1	159	159	46.86	46.86
10	1	179	179	71.38	71.38
Total	141	107.3 ± 35.6	67–190	20.8 ± 17.7	3.42–75.06

.

The von Bertalanffy equation was used to describe the growth of E. davidi. The length growth equation was L_t = 260.8(1−e^{−0.104(t+1.942)}), R² = 0.897 (Figure 7) and the weight growth equation was W_t = 173.5(1−e^{−0.104(t+1.942)})^2.653 The growth inflection point was calculated to occur at 7.44 years, and the corresponding length and weight were 162.50 mm and 49.46 g, respectively. The growth performance index of E. davidi was 3.85.

3.4. Gonad Development and Secondary Sex Characteristics

The testis of mature males displayed branching and hanging on the dorsal wall of the abdominal cavity. The number of blood vessels on the testis surface decreased and the testes were hypertrophic, with milky white color. A small amount of white semen oozed out after squeezing the abdomen. The finger-like protrusions were plump, the sperm-storage vesicles and vas deferens were filled with spermatozoa, and the spermatozoa were evenly distributed with synchronous development (Figure 8a). The males of E. davidi possess a mating organ that is extremely rare in freshwater fishes of the class Osteichthyes. The genital papilla was usually located between the pelvic fins and anal fin, partially hidden in the abdomen; it protruded from the surface of the abdominal skin behind the anus. This organ was retractable, with a duct connected to the external cloaca, through which semen was released (Figure 8b). This male-specific organ allowed the easy distinction of the sexes directly by observing the genital papillae.

The ovary of females was single and cryptomere. During the mature stage, the ovaries were largely filled with yolk, had similarly sized oocytes, which were dark yellow, irregular, or oval shaped, and were sticky (Figure 8c). During the breeding season, the abdomen in the females was highly bulged and protruded (Figure 8d).

3.5. Oocyte Diameter and Sexual Maturity

Measurement of the oocyte diameter of randomly selected females displayed a single-peaked distribution (Figure 9). The oocyte diameters were distributed between 2.87 and 4.51 mm (with an average of 3.59 mm) and concentrated at 3.2–3.8 mm, accounting for 64.67%.

In the samples collected, the youngest sexually mature female was 4 years old, with a length of 132 mm and a weight of 28.34 g. The youngest sexually mature male was 3 years old, with a length of 99 mm and a weight of 22.76 g.

3.6. Fecundity

The standard length of the selected samples (35 individuals) ranged from 135 to 184 mm, whereas the weight ranged from 31.88 to 59.05 g. The absolute fecundity calculated was 105–352 eggs, with an average of 205.34 ± 45.38 eggs. The weight relative fecundity (F_W) was 2.90–8.98 eggs/g, with an average of 5.65 ± 1.38 eggs/g. The length relative fecundity (F_L) was 0.71–2.11 eggs/mm, with an average of 1.34 ± 0.29 eggs/mm. A significant relationship was observed between fecundity and standard length, weight, and ovary weight (p < 0.05); however, the correlation coefficient was low (Figure 10).

4. Discussion

4.1. Comparison of Calcified Structures

Age constitutes an important component of fish biology research. It is a basic parameter for studying growth, reproduction, and population structure, assessing fish resources, and providing basic data for formulating fishery conservation policies [25,26]. The most reliable aging method may vary among the species; thus, the evaluation of the precision and accuracy of calcified structures should be studied [26]. Because Siluriform fishes are scaleless, vertebrae, otoliths, and pectoral fins are used to determine their age. Certain studies have reported vertebrae as the best material for determining age in most Siluriformes [27,28,29,30]. Similarly, studies on Sisoridae fishes, including Euchiloglanis kishinouyei [31], Creteuchiloglanis macropterus [32], Pseudecheneis sulcatus [33], and Glyptosternon maculatum [10], report vertebrae as a highly reliable material to estimate age. In the present study, vertebrae were used to determine the age of E. davidi, with a success rate of 94%. However, inconspicuous opaque and translucent zones and blurred edges in a few individuals (6%) made it difficult to identify their ages. In addition, the age identification from otoliths is inaccurate in E. davidi because it is a small fish with thin, fragile, and small otoliths, which makes it difficult to read annuli after polishing. We found that the otoliths were irregularly shaped and difficult to process, whereas vertebrae were more convenient to handle and resulted in more accurate interpretation. A similar phenomenon was observed in G. maculatum [10] and P. sulcatus [33].

4.2. Growth Characteristics

The b value in the SL–W relationship reflects the growth situation of fish at different developmental stages and in different habitats. The b value is within the expected range of 2.5 to 3.5 [34]. The index b of E. davidi was 2.653, representing allometric growth, indicating that the length growth was faster than the weight growth at this stage. This is consistent with the results obtained for Pareuchiloglanis kamengensis [35] and C. macropterus [32]. In contrast, the b values of E. kishinouyei [31] and G. maculatum [36] were close to 3, reflecting isometric growth (

Table 4. Comparison the growth characteristics of several Sisoridae fishes.

Species	River Drainage	n	Length Range (mm)	Age Range	Growth Parameters					Reference
					L∞ (mm)	k	t0	b	Ø
Glyptosternum maculatum	Yarlung Zangbo River	128	115–270	3–13	342.66 (♀)	0.114 (♀)	0.7488 (♀)	3.1420 (♀)	5.07 (♀)	[10]
		77	147–320	3–13	460.24 (♂)	0.082 (♂)	0.2312 (♂)	3.1474 (♂)	5.32 (♂)
Glyptothorax fukiensis	Jialing River	238	42.9–102.9	1–5	140.20	0.254	0.0696	3.2130	3.70	[28]
Euchiloglanis kishinouyei	Dadu River	383	92–190	3–13	208.42	0.089	1.2000	2.9279	4.63	[31]
Pseudecheneis sulcatus	Yarlung Zangbo River	203	50–144	1–9	189.56	0.118	1.9810	2.9292	4.55	[33]
Euchiloglanis davidi	Yalong River	150	67–190	1–10	260.80	0.104	1.9420	2.6530	3.85	This study

♀, Female; ♂, Male.

). The variability in b values among different species of Sisoridae fishes indicates that species-specific values of b vary according to the fish shape and the diet condition of fish under different environments. Furthermore, the b values of the same species could vary between sexes or during different development periods [37].

Both the growth performance index (Ø) and growth coefficient (k) are key parameters for assessing the growth potential of fish populations; Ø was used to compare the growth performance of the congeneric fish [24]. The growth performance indices (Ø) of E. davidi were smaller than those of other Sisoridae fishes (except for G. fukiensis) (Table 4), revealing that the growth of E. davidi is relatively slower than others. In addition, k is the velocity curvature parameter that determines the velocity of fish reaching its asymptotic length, which reflects its life cycle and growth rate [23]. Furthermore, L_∞ and k have an inverse relationship. A decreasing L_∞ decreases the time to approach L_∞ or increases the curvature k, and vice versa [38]. When compared to several other Sisoridae fishes, E. davidi had the smallest k value and the largest L_∞ (except for E. kishinouyei) (Table 4). Slow-growing fish have k values of 0.05 to 0.10, medium-growing fish have k values of 0.10 to 0.20, and fast-growing fish have k values of 0.20 to 0.50 [37]. E. davidi (k = 0.104) is a slow-growing fish with a long life cycle, similar to the high-elevation fishes E. kishinouyei (k = 0.089) [31] and P. kamengensis (k = 0.073) [35]. P. sulcatus (k = 0.118) [33] is a medium-growing fish. The low-elevation Glyptothorax fukiensis (k = 0.254) [28] is a fast-growing fish (Table 4). Research has indicated that with an increase in altitude and a decrease in water temperature, the lifespan increases but the growth rate decreases in Sisoridae fishes [39]. The differences between the k values of E. davidi and other fishes in the Sisoridae family could be ascribed to several environmental factors, such as altitude, water temperature, and diet conditions, which are adaptations to the different living environments.

4.3. Fecundity

Individual fecundity and oocyte size are often used as indicators to assess the potential reproductive capacity of fishes, which reflects their reproductive strategy. It is determined by a combination of genetics, environmental factors, and nutritional status. Fecundity varies among species and represents the adaptation of populations to environmental changes [40,41]. Studies on the reproductive strategy of fish contribute to the efficient management and utilization of fishery resources [42]. However, the literature on the reproductive biology of Sisoridae fishes is rare, with several reports only on E. kishinouyei [43], G. maculatum [17], P. sulcatus [44,45], and G. fukiensis [46]. Previous studies have reported differences in fecundity among different genera in Sisoridae fishes, which could be related to fish size, oocyte diameter, spawning type, and living conditions. Even the individual fecundity of the same fish in different environments is known to vary [47]. The absolute fecundity of Sisoridae fishes is low, ranging from a few hundred to a few thousand eggs, and the absolute and relative fecundity of E. davidi are both the lowest when compared with other Sisoridae fishes (

Table 5. Comparison of the fecundity of several Sisoridae fishes.

Species	River Drainage	n	Standard Length (mm)	Absolute Fecundity (Eggs)	Relative Fecundity (Eggs/g)	Reference
Glyptosternum maculatum	Yarlung Zangbo River	128	133–243	525–2058	3.24–27.01	[17]
Euchiloglanis kishinouyei	Zumuzu River	69	120–165	123–489	2.80–13.95	[43]
Pseudecheneis sulcatus	Yarlung Zangbo River	52	114–200	247–2886	16.80–77.10	[44]
Pseudecheneis sulcatus	Yarlung Zangbo River	78	92.99–144.49	346–799	20.69–92.80	[45]
Glyptothorax fukiensis	Western Guangdong waters	49	31.9–63.7	45–420	31.70–130.40	[46]
Euchiloglanis davidi	Yalong River	35	135–184	105–352	2.90–8.98	This study

). This could be attributed to the fact that the majority of Sisoridae fishes live in rapids and benthic environments with limited activity range and a lack of food sources.

Relative fecundity can be used to compare the fecundity of different species and understand the reproductive strategies of different populations. A low relative fecundity is usually accompanied by fewer eggs with a larger oocyte diameter and sufficient yolk. Large eggs are more likely to produce relatively larger and well-developed offspring. Fish tend to select the most suitable reproductive strategy as per the current environmental changes to improve the survival rate of their offspring [48]. For instance, E. davidi exhibited lower individual fecundity (2.90–8.98 eggs/g), with a diameter of 3.6 mm of the mature oocyte, suggesting higher survival rates. Lower relative fecundity and larger oocyte diameter are major characteristics of Sisoridae fishes, including E. kishinouyei [43], G. maculatum [17], and P. sulcatus [45]. These traits enhance the survival rate of offspring, maintain the stability of the population structure, and perpetuate the population size during the long-term evolutionary process.

5. Conclusions

We determined the age and studied the growth and reproduction of E. davidi in the middle and lower reaches of the Yalong River. Vertebrae were the most suitable material to estimate the age of E. davidi. Slow growth and low fecundity were found to be the primary biological characteristics of this species. Hence, the findings of this study complement the basic biological information of E. davidi and provide a reference for the subsequent research on the conservation and artificial breeding of this species.