Sex determination and acclimation response of dwarf snakehead fish Channa limbata from West Java

The Channa limbata fish is a type of tropical freshwater fish of the Channidae family which is relatively small as an aquarium ornamental fish with a distinctive color at the tip of its dorsal fin and has a snake-like head (dwarf snakehead). Natural snakehead fishing activities have threatened its sustainability. Breeding C . limbata fish through cultivation can increase its potential for sustainable use. This study aims to evaluate the acclimation response of wild-type dwarf snakehead fish in captivity and its sexual characteristics as a basis for domestication and hatchery technology. The fish samples used were natural catches from rivers in West Java measuring <100 mm to >150 mm of body length then individually acclimated indoors in an aquarium (35×20×20 cm) for 14 days. Snakehead fish live in shallow, slow-flowing river waters with a temperature of 20.2-21.3°C, TDS 16-24 mg/L at neutral pH, while the rearing water temperature and TDS are higher (temperature: 24.9-27.6°C; TDS: 88-110 mg/L). The fish mortality rate during acclimation reached 25% in fish measuring >150 mm of length on tenth day, while fish measuring <150 mm more adaptive with 100% survival. The male fish measuring 100-150 mm have 13-15 pectoral fin rays while female fish have fewer (13-14). The gonad development level of male C . limbata in nature is slower than female fish measuring 100-150 mm with a gonadosomatic index of ovaries reached 10 times higher than testicular.


INTRODUCTION
The ornamental snakehead fish Channa limbata (Cuvier & Valenciennes, 1831), known locally as the "bogo fish," is a tropical freshwater species from the Channidae family, relatively small in size with a head resembling that of a snake (dwarf-snakehead).The distribution of this snakehead fish species includes Iran, Afghanistan, Pakistan, India, Nepal, Bangladesh, Indonesia, Vietnam, Korea, and China (Khoomsab & Wannasri, 2017).In Indonesia, the distribution of the snakehead fish species encompasses Sumatra, Java, Kalimantan, Bangka, Madura, Bali, Lombok, Flores, Ambon, and Halmahera (Tarigan et al., 2016).This fish is inhabiting shallow waters in low to highlands (>1000 m above sea level) with a pH range of 7-8 and a temperature of 23-27ºC, along with relatively low dissolved oxygen and high CO2 (Kusmini et al., 2016).The small-sized dwarf snakehead fish holds economic value as an aquarium ornamental fish due to its attractive color patterns, especially with its fins displaying blue and orange hues (red-tail snakehead).
Furthermore, the snakehead fish contains albumin and potential amino acids for wound healing post-operation, patients with hypoalbuminemia, and children's development (Rahayu et al., 2016).Continuous natural capture activities of snakehead fish have threatened its sustainability, such as in Singapore and China (He et al., 2019), in Java and Kalimantan (Kusmini et al., 2016).Additionally, environmental damage due to anthropogenic activities, agricultural/ plantation waste, and invasive fish also impact the sustained decline in populations (Gustiano et al., 2013).Managing the genetic resources of natural snakehead fish through breeding activities outside their habitats can reduce dependence on natural capture and enhance their potential as a productive, high-quality, and sustainable ornamental fish commodity.For the future of aquaculture primarily could focus on some truly domesticated species, or diversification of native domesticated species.
Acclimatization is the initial stage of domestication to adapt fish from their natural habitat to controlled maintenance outside their habitat and turn them into a cultivation commodity.The success of the initial domestication stage of wild fish adapting to environmental conditions outside their habitat assure better quality generations.The strategies for fish production consist of five levels of 'domestication' for being the most domesticated (Teletchea & Fontaine, 2014).The first to third levels representing a transitory form of fish production dependent on the availability of the wild resource.
The process of transferring fish from their habitat to captivity can cause mortality due to capture, transportation, and acclimatization in the early stages of domestication, which generally lasts for 10-14 days, referred to as hauling loss or delay mortality syndrome (Hadiroseyani et al., 2016).Maintaining fish in captivity can influence the change of fish behavior, both physically and physiologically (phenotypic plasticity).The ability of fish to respond the condition in the captive environment allows them to complete their entire reproductive cycle naturally.Implementing domestication strategies and generational succession for the development of cultivated ornamental snakehead fish requires a study of its bioecology and an understanding of its sexual system as the basis for appropriate fish breeding techniques.
The continuity of the fish's reproductive cycle is crucial for preserving the species (Niass & Fall, 2015).The reproductive function development of each fish species in nature exhibits a high level of diversity according to its habitat conditions (Susatyo et al., 2022).The differentiation of fish genders occurs in the early stages of development influenced by environmental factors such as changes in water temperature and pH (Budd et al., 2015).Determining the sexual identity of fish can be done based on primary and secondary sexual characteristics observed in measurable macroscopic and microscopic ways.
The regulation system of fish's physiological functions and endocrine functions is influenced by environmental sensory signals and transduction to the hypothalamus-pituitary-gonad axis for the fish's reproductive process (Shin et al., 2014;Tao et al., 2013).Snakehead fish are mouthbreeders, storing fertilized eggs inside the male fish's mouth until they hatch (Khairanti et al., 2023).This research aims to evaluate the acclimation response in the initial domestication of wild C.limbata snakehead fish from West Java and determine their gender based on observations of primary and secondary sexual characteristics.The results of this research serve as a foundation for the breeding activities of C. limbata snakehead fish outside their habitat to produce high-quality and sustainable cultivated fish commodities.

Fish collection
The collection of C. limbata snakehead fish samples was conducted in the watershed area of Cilember, Bogor Regency (West Java).Fish were collected from their natural habitat using fishing equipment and water quality measurements were carried out directly at the fish collection site.The captured fish were individually packed in plastic bags with added oxygen.The fish samples consisted of three size groups, juveniles or small (50-100 mm, S), fingerlings or medium (100-150 mm, M), and breeders or large (>150 mm, L).

Fish acclimatization
Subsequently, the captured fish were transported to the breeding location via land transportation and acclimatized at the Fish Reproduction and Genetics Laboratory, IPB University.Observations on habitat characteristics included the physicochemical properties of water (temperature, pH, dissolved oxygen, total dissolved solids, total organic matter, and the type of aquatic substrate).Survival observations were conducted during the transportation process and post-transportation, involving a 30-minute initial adaptation period in the holding container, followed by a 14-day acclimatization period.Prior to acclimatization, post-transportation, the fish were transferred by submerging the plastic bags containing the fish into a temporary holding aquarium for 30 minutes.
Subsequently, the plastic bags were opened, allowing the fish to spontaneously released and were individually housed for a 14-day acclimatization period.The fish acclimatization process was conducted indoors at room temperature using individual aquariums measuring 35×20×20 cm with a water depth of 15 cm.The fish were not fed for two days to purge any potential diseases present in their digestive tracts.On the third day, the fish were provided with a diet consisting of yellow mealworms (larvae of Tenebrio molitor) or bloodworms.
The amount of food given was adjusted based on the fish's feeding response, gradually increasing the quantity.The ideal amount of feed for juvenile snakehead fish per day is 5% of their biomass weight.Observations of adaptation responses included swimming and feeding behavior, as well as their survival rate.Feeding response assessments were categorized as responsive to feed (++), slow or moderate feeding (+), and unresponsive or no feeding (-) (Herjayanto et al., 2020).Survival rates were monitored daily during the acclimatization period.Water quality parameters such as pH, dissolved oxygen (DO), temperature, and total dissolved solids (TDS) were measured daily for maintenance purposes.

Sex determination
Identification of male and female fish characteristics was conducted based on biometric measurements of the fish's body using Channa genus truss morphometrics (Cuvier & Valenciennes, 1831;Nainggolan et al., 2019) and meristic characters (Siregar et al., 2020).The truss morphometric characters measurement was taken directly on the specimen prior to acclimation.
Morphometric characteristics included 26 truss points along the fish's body and three characteristics related to head shape.Meristic characteristics involved the count of rays in the dorsal fin, pectoral fin, pelvic fin, anal fin, and caudal fin (Figure 1).Observed fish samples were categorized into three length groups: 50-100 mm (small, S), 100-150 mm (medium, M), and larger than 150 mm (large, L).The gender of the samples then inspected for their development of gonad observation as primary characteristics by killing the samples.

Gonad histology
Primary sexual characteristics of male and female fish were observed macroscopically and microscopically in gonad and liver samples to assess the level of reproductive function development based on the gonadosomatic index (GSI) and hepatosomatic index (HSI).The observed gonad samples included ovaries and testes of all size classes (S, M, L).GSI and HSI values represent the percentage ratio of gonad and liver weight to body weight (Gong et al., 2017).Microscopic observation of germ cell development was conducted by analyzing gonadal tissue.Gonad samples were fixed in 10% buffered neutral formalin and then transferred to 70% alcohol, stored at room temperature for 24 hours.Subsequently, gonad samples were embedded in paraffin, sliced using a microtome, stained with Hematoxylin and Eosin (H&E) tissue staining, and observed under a light microscope at 40× magnification to assess germ cell development (Milton et al., 2018).

Data analysis
Statistical analysis of morphometric and meristic characteristics between fish size groups (S, M, L) and between male and female sexes was performed using Student's t-test with a confidence interval of 95%.The acclimation response of fish in captivity and the macroscopic and microscopic assessment of gonads were analyzed descriptively.

Result Truss Morphometrics of C. limbata
Based on the size groups of the wild snakehead fish C. limbata (S, M, L) originating from West Java (Table 1), there is a proportional increase in standard length along with an increase in total length (p<0.05).The significant differences obtained were considered to discriminate the fish into three existence morphological groups according to their size.The C. limbata fish measuring less than 150 mm (S, M) and the group measuring more than 150 mm (L) exhibit differences in characteristic values in eye diameter (13), dorsal fin height (17), anal fin height ( 22), as well as the length and height of the caudal fin (24,26).Morphometric characteristics of the head shape of C. limbata snakehead fish in all length groups (S, M, L) do not show significant differences (Table 2).

Sexual Dimorphism C. limbata
The morphometric characteristics distinguishing gender in C. limbata fish from West Java are identified in the height of the dorsal and pectoral fins.In the group of fish sized 100-150 mm, male fish exhibit longer heights in the dorsal fin ( 17) and pectoral fin (20) compared to females (Table 3).The meristic characteristics of male and female C. limbata fish (Table 4) show  The morphometric character of head shape is a comparison of the measured character value with the standard length (2).The t-test at the 95% confidence interval is significantly different marked with the letters: a (S<M), b (M<L), c (S<L).variations in the number of soft-rayed pectoral fin spines, ranging from 13-15 (male) and 13-14 (female).The dorsal fin rays range from 30-33 with a blue-orange strip at the tip; the base of the dorsal fin is positioned behind the base of the pectoral fin, and its end extends beyond the anal fin.Meanwhile, the ventral fin is located in front of the base of the dorsal fin and is half the length of the pectoral fin.

Gonad development level
The gender of fish is influenced by environmental factors that affect gonadogenesis.The gonad development in male and female snakehead fish within the size group of 100-150 mm exhibits different maturity levels (Table 5, Figure 2).Value of gonadosomatic index (GSI) and hepatosomatic index (HSI) in female fish sized 100-150 mm (M) are higher (1.31 ± 0.40% and 1.00 ± 0.28%) compared to males across all fish size groups.In Channa species, the ovarian development generally shows faster progression than testicular development in fish of the same size (Irmawati et al., 2019).The generative cell development in wild-caught male snakehead fish shows spermatocyte (SC) and spermatid (ST) phases in fish sized 50-100 mm (S), the spermatid and spermatozoa (SZ) in fish sized 100-150 mm (M) and >150 mm (L).In the ovaries of fish sized 100-150 mm (M), there are different egg cells present such as oogonia (OG), early perinuclear oocytes (EPO), late perinuclear oocytes (LPO), and yolk vesicles (VY).

Discussion
There are still very few studies on the biological aspects of the Channa limbata fish as a reference in sustainable management and cultivation (Hoomsab & Wannasri, 2017).This research is in the first stage of initial domestication with adaptation to the limited environment in the cultivation container.Water quality in natural passive: the fish stays still at the bottom of the aquarium; active: the fish swims actively; '-': food is not consumed by fish; '+': food is consumed after 1 minute; '++': fish consumed food before 1 minute.S (50-100 mm), M (100-150 mm), L (>150 mm).habitats of C. limbata is at lower temperature and TDS compared to the water temperature and TDS conditions during the rearing period.The habitat of Channa fish can be found in mountain river areas with cold water and low dissolved content values.Feed response and movement response during rearing show that there is a decrease in response as size increases (Herjayanto et al., 2020).
The results of acclimatization for 14 days showed a decrease in movement response, feeding response and survival as the size of fish increased during rearing.The range of survival rate values during 14 days of maintenance shows the highest value of 100% in fish measuring under 100 mm and the lowest value of 75% in fish measuring over 150 mm.The increase in water temperature in captivity compared to the natural habitat of C. limbata is suspected to cause stress and fish mortality.Differences in environmental conditions between the natural habitat and captive conditions can influence the fish's physiological regulatory system (Tao et al., 2013).Larger fish will attempt anaerobic metabolism to prolong their survival in hypoxic conditions (Urbina & Glover, 2013).
As fish grow larger, their metabolic rate tends to decrease.The larger fishes may have a longer satiety period than the small group so that the large group tend to less responsive to eat and movement for more efficient in utilizing energy maintenance.Sexual dimorphism is an important aspect in the domestication stage, namely continuous reproduction (Teletchea & Fontaine, 2014).Morphometric and meristic characters were tested between sizes of male fish and between males and females at sizes 100-150 mm.Sexual dimorphism in other snakehead fish species is identified through differences in body coloration between male and female C. gachua (Jearranaiprepame, 2017) and variations in the length of the anal fin and eye diameter in C. striata (Arma et al., 2014).
Male snakehead fish commonly have longer dorsal fins than females, with a more dominant red coloration on their bodies (Gustiano et al., 2019;Gustiano et al., 2020), and they show a comparison between body weight and length known as allometric (Vodounnou et al., 2017).The characteristics of tail fin height between male fish samples measuring 50-100 mm and 100-150 mm were significantly different.The morphometric characters that are significantly different between male fish measuring 50-100 mm and male fish measuring >150 are the length of the pelvic fin base, eye diameter, pectoral fin height, anal fin height and tail fin height.This difference value can be used as a reference for changes in the morphometric characters of male C. limbata fish towards adulthood.
Studies related to sexual dimorphism in body morphometrics of fish in Channa gachua found that there were no differences in morphometric characters between the sexes except for the characterization of body color between males and females (Jearranaiprepame, 2017).The plasticity to adapt to the environment factors possibly has an impact on the different of specific morphological on fish population at different habitat (Muslimin et al., 2020).The morphometric difference between the sexes of snakehead fish is in the length of the anal fin and eye diameter (Arma et al. 2014).Snakehead fish have a cylindrical body shape towards the head and tapering towards the tail, and their eye diameter is one-fifth to one-eighth of the length of their head (Gustiano et al., 2013).
Local adaptation responded to environmental conditions can yield morphologically distinct populations.The ecological diversification, especially flow regime, was considerably be involved with the different variations of the whole fish body (Jearranaiprepame, 2017).The GSI value of female bogo fish has a higher value than the GSI value of male bogo fish, but the HSI value does not differ between all sizes of fish and gender of bogo fish.The histology results of fish testicles measuring 50-100 mm show that there are spermatocytes that are round in shape and the cell membrane is clearly visible and spermatids that are smaller in size are darker in color (Al Mahmud et al., 2016), whereas in fish measuring 100-150 mm there are spermatids and spermatozoa.
Spermatozoa are the final stage of sperm differentiation with a dark color and are the smallest cells (Al Mahmud et al. 2016) and in the ovaries there are oogonia (OG), early perinuclear oocytes (EPO), late perinuclear oocytes (LPO), and yolk vesicles (VY).Sperm differentiation includes a series of morphological changes that occur in the seminiferous epithelium (Teves et al., 2020).The development of germ cells in large male snakehead fish (100-150 mm) which have differentiated into spermatozoa shows that they are ready for the fertilization process.

Figure 3 .
Figure 3. Survival rate of dwarf snakehead fish C. limbata during 14 days of acclimation in captivity.

Table 1 .
Truss Morphometric characteristics of C.limbata from West Java.

Table 2 .
Morphometric characteristics of head shape C. limbata from West Java.

Table 3 .
Morphometric characters of secondary sexual characteristics of snakehead fish C. limbata from West Java.

Table 4 .
Meristic characteristics of natural snakehead fish C. limbata from West Java.

Table 5
. Level of sexual development of natural snakehead fish C.limbata from West Java.

Table 6 .
The acclimation response of dwarf snakehead fish C. limbata of different sizes (S, M, L) during the rearing period in captivity (indoor) for 14 days.