The ontogenic study of early life stages of culture-bred Nomorhamphus sp. (Zenarchopteridae) from Lindu, Central Sulawesi Studi ontogeni stadia awal hidup pada budidaya Nomorhamphus sp. (Zenarchopteridae) asal Lindu, Sulawesi Tengah

Nomorhamphus sp. is a freshwater fish that has been traded as an ornamental fish. This fish is unique as an endemic species with a halfbeak-like jaw and orange color on the caudal fin. However, this fish culture information needs a further information. Based on this condition, it is necessary to conduct a study as a basis for ornamental fish breeding and growing-out activities through domestication. A crucial problem in this fish is larval rearing, which can be observed through ontogeny studies. The study was conducted on the newly-born larval behavior, morphological development, andropodium development, growth, and survival rate at the early stages, namely larvae to juvenile. The results showed that the newly-born larvae of Nomorhamphus sp. Lindu had a total length of 1.6-1.8 cm. Larvae could swim four hours 22 minutes after birth and feed artemia nauplii with surface feeding type. The initial juvenile stage occurred 25 days of post-birth period with a total length of 2.0-2.2 cm. The water condition of the rearing during the study could support the larval transformation to juvenile. This study is the first report related to the aquaculture success of the early


INTRODUCTION
Nomorhamphus is a tropical freshwater genus from the Zenarchopteridae family. Seventy percent of Nomorhamphus species are found as endemic species in Sulawesi Island (Huylebrouck et al., 2014;Hadiaty, 2018;Kraemer et al., 2019a;Kobayashi et al., 2020). The habitats of this fish are freshwater rivers and lakes (Herjayanto et al., 2019;Kraemer et al., 2019a;Kobayashi et al., 2020), as reported in Lindu Lake, Central Sulawesi (Wicaksono et al., 2022). Morphologically, this fish has a distinct superior jaw, as the lower jaw is longer than the upper jaw, called a halfbeak (Kraemer et al., 2019a;Kobayashi et al., 2020).
This fish also has a unique reproduction strategy through internal fertilization (viviparous), thus male fish have an additional sexual organ called andropodium. This organ functions as a spermatozoan distributor to female genital pore during the mating process (Kraemer et al., 2019b). The uniqueness of endemic species causes Nomorhamphus sp. is collected by hobbyists or foreign ornamental fish collectors. Moreover, the color and shape are also unique as part of the aquascape biota. However, these fish are supplied from natural catch, but limited culture studies information. Wicaksono et al. (2022) have reported the initial characterization of biometrical and morphological approaches of Nomorhamphus sp. from Lindu. Yet, information regarding breeding, larval rearing, and controlled seed maintenance remains unknown. In general, studies that have been conducted in Nomorhamphus species in Central Sulawesi are species description (Kraemer et al., 2019a) and the latest habitat condition (Bandjolu et al., 2021;Kraemer et al., 2019a). Therefore, a cultural approach for Nomorhamphus sp. rearing is necessary to support its sustainable production as an ornamental fish. Early life history observation is highly important to gain basic data for larval rearing management (Dos Santos et al., 2016).
This observation is related to morphological development, namely body shape and behavior during the developmental stage, which can be called an ontogenic study (Baras et al., 2012;Sørensen et al., 2016;Tinguely et al., 2019). The ontogenic study in fish is necessary to provide information for further breeding activities in the nursery sector as part of the aquaculture activities (Baras et al., 2012). Based on the following condition, a study was performed to describe the ontogenic early life period of Nomorhamphus sp. Lindu in the cultural environment. The ontogenic studies are mainly focused on the initial larval development to become juvenile. Observations were performed following the post-birth larval behavior (pbl), morphological development, andropodium development, growth, and survival rate in the culture environment.

Fish Catching
Fish samples were caught in the inlet flow of Lindu Lake at >1,000 asl (1°22'19.20" S 120°01'40.80" E). using a net at 5.5-mm mesh size. The total female fish caught was nine individuals with a total length of 6.6-9.0 cm. After being caught, the fish were maintained by stocking them in an aerated container. During maintenance, the fish remained unfed.
Maintenance was performed for 24 hours. Fish were packed and transported in a closed system, following Hadiroseyani et al. (2016), Herjayanto et al. (2018), and Herjayanto et al. (2020), which were modified at low stocking density. The initial transportation was performed from Lindu Lake to Palu City through a land route. Then, fish were sent through an air route from Palu City to Soekarno-Hatta Airport, followed by a land route from the airport to Laboratory of Aquaculture, University of Sultan Ageng Tirtayasa, Banten. In this location, fish were reared in a controlled tank.

Fish acclimatization
Acclimatization was performed based on the wild fish maintenance method modified by Herjayanto et al. (2018) and Hadiroseyani et al. (2016) by stocking the fish in a packing plastic in the rearing tank for 30 minutes for temperature adaptation. Then the fish was removed from the plastic and allowed to escape slowly into the rearing tank for acclimatization usingaquarium of 100×50×40-cm 3 .

Handling Pregnant-broodstock
The pregnant female broodstock of Nomorhamphus sp. from the wild were separated and reared solitary ( Figure 1). The rearing tank for pregnant female fish used an aerated clear plastic container at 35 cm×25 cm×15.5 cm size. The total pregnant female fish was five individuals with a total length of 7-8 cm. During the rearing, the female broodstocks were fed with live feeds, namely Tubifex sp. and Daphnia sp. three times a day with ad libitum method. Broodstocks were reared until giving birth to the larvae.

Larval rearing
The 40 individuals of Nomorhamphus sp. larvae were reared in an aerated clear plastic container at 35 cm×25 cm×15.5 cm size. The rearing tank was filled with water until 5 L volume. Larval rearing used five larvae that were stocked at 1 larvae/L. Larvae were fed with Artemia nauplii at one day of post-birth (pbl). Rearing was performed for 30 days after giving birth. Behavior, ontogenic morphology, growth (total length, head length, lower jaw length, and fin length), and survival rate were observed at 0, 15, 25, and 30 days of pbl.
The larval swimming behavior was observed from the 10 post-birth larvae. The ontogenic morphology development was documented in a photo to observe the difference between larval and juvenile stadia, as modified by Baras et al. (2012) and Wicaksono et al. (2022). The photo was taken in a glass container at 24 cm×7 cm×15 cm, filled with water at 10 cm height. During the rearing, water quality parameters, namely temperature, dissolved oxygen, and pH were measured. Feed residue and fecal waste were siphonized once in three days.

Data analysis
Data from behavior, ontogenic morphology, growth, i.e., total length, head length, lower jaw length, and anal fin length (Figure 2), and survival rate were analyzed descriptively using Microsoft Excel 2010. The analysis results are presented in figures and tables.

Results
The newborn larvae of Nomorhamphus sp. had a total length of 1.6-1.8 cm (1.69 ± 0.08 cm) (n=3). Larvae could open their mouth with an orange-colored lower jaw. The pectoral, ventral, anal, dorsal, and caudal fins have become definitive organs (Figure 3). The newborn larvae  Based on the behavior of 10 larvae, 60% of the larvae could swim on the water column at 20 minutes of post-birth (pbl), then increased by 90% at 3 hours 6 minutes of pbl. All larvae could swim after 4 hours 22 minutes of pbl. This swimming capability has occurred as the swimming bladder has been filled. The swimming position of the larvae at early life stage was only swimming with their head and water surface in perpendicular position.
As the larvae grew older, they could swim onto the water column horizontally. After horizontal swimming, larvae could chase the Artemia nauplii. During the developmental stage, the Nomorhamphus sp. larvae had a morphological change in their belly shape. Based on the observational results in five days of pbl, larvae had a digestive tract full of Artemia nauplii. Also, the body pigmentation started to spread on the transparent body surface (Figure 4).
The digestive tract of Nomorhamphus sp. Lindu larvae were still visible until 15 days of pbl as the belly was still transparent. After 20 days of pbl, the fish belly became non-transparent, as the Artemia consumed by the larvae was invisible ( Figure 5). The anal fin as a secondary sexual character in male fish (andropodium) was yet visible at 15 and 20 days of pbl. The orange color in the ventral fin and caudal fin edge was visible when the fish reached 25 days of pbl.
The anal fin of Nomorhamphus sp. Lindu, which was modified as andropodium began to emerge at 25 days of pbl ( Figure 6). Therefore, the early stage of juvenile stadia in Nomorhamphus sp. Lindu emerges at 25 days of pbl. The total length of Nomorhamphus sp. juvenile was 2.11 cm with a head length of 0.47 cm, lower jaw length of 0.11 cm, and anal fin length of 0.30 cm (Table 1).  The survival rate of Nomorhamphus sp. at the final rearing period (30 days of pbl) was 75-100% (90.60 ± 11%). Fish death has only occurred at three days of pbl and 11 days of pbl. The water quality during the rearing period could support the larval and juvenile life of Nomorhamphus sp. Based on the water quality parameters measurement, the temperature was maintained at 22.30-25.50°C (24.00 ± 0.62°C), dissolved oxygen at 4.80-8.90 mg/L (6.85 ± 1.19 mg/L), and pH at 7.19-9.41 (8.08 ± 0.58). Figure 5. Ontogenic morphology of Nomorhamphus sp. Lindu larvae at 15 days of pbl (above) and 20 days of pbl (below). Fish size is presented in Table 1. Lower jaw length (cm) 0.08 ± 0.02 0.10 ± 0.00 0.11 ± 0.01 0.14 ± 0.02 Anal fin length (cm) 0.24 ± 0.03 0.29 ± 0.01 0.30 ± 0.00 0.39 ± 0.05 Figure 6. Ontogenic morphology of Nomorhamphus sp. Lindu juvenile at 25 days of pbl. Male (above) and female (below). Fish size is presented in Table 1.

Discussion
The early life stage of fish requires more thorough management as larvae are susceptible to environmental changes and feed availability. Therefore, an ontogenic study of early life stages is important to provide basic data for larval rearing management (Baras et al., 2012;dos Santos et al., 2016). It has been confirmed that Nomorhamphus sp. is viviparous fish, as embryonic development occurs in the female gonad and birth larvae (Kraemer et al., 2019a;Kraemer et al., 2019b;Kobayashi et al., 2020). Based on the newborn Nomorhamphus sp. Lindu larvae observation, larvae were unable to swim and remained in the bottom container.
This condition has occurred as Nomorhamphus sp. Lindu fish are lecithotrophic viviparous fish, as their larvae have yolk as a nutrient source during the developmental stage in the female gonad. This reproductive strategy causes the newborn Nomorhamphus sp. larvae to require more time to develop and swim freely by counting on the yolk energy remaining after birth. Another lecithotrophic viviparous fish is Poecilia vivipara (Poecillidae), which has yolk as a nutrient source until the final stage of embryonic development (Arcanjo et al., 2014). During larval development, transformation emerges in the organ system and body shape (Shahjahan et al., 2013;Herjayanto et al., 2017). The Nomorhamphus sp. larvae gradually performed a jerky motion.
This behavior is an effort for larvae to fill the swimming bladder (Herjayanto et al., 2017). Another viviparous species, P. reticulata, has newborn larvae that can swim, feed, and move away from danger (Shahjahan et al., 2013). In rainbow fish larvae, Iriatherina werneri, the newborn larvae spend most of their time resting in the bottom rearing tank, while the fish in the juvenile stage can swim in the water column. Swimming activity elevates as age increases (Herjayanto et al., 2017). After the swimming bladder has been filled, the Nomorhamphus sp. larvae could swim well to chase Artemia nauplii as their feed.
This external feeding becomes an energy source for larvae to conduct metabolism, grow, and develop. The larval body shape causes changes and development to reach a definitive shape along with increasing age. As they were newly born, the Nomorhamphus sp. larvae had a transparent body. The morphological change started to emerge at 20 days of pbl, as the belly started to become transparent. At 25 days of pbl (initial juvenile stage), the orange color in the ventral fin and caudal fin edge was visible.
Fish sexual dichromatics and dimorphism can be used as secondary sexual characteristics in fish (Wicaksono et al., 2022). In viviparous fish, Xenotoca eiseni, orange color in male fish caudal fin initially emerge at four weeks of pbl or 28 days of pbl (Tinguely et al., 2019). The secondary sexual character observed in Nomorhamphus sp. is andropodium in male fish (Kraemer et al., 2019b). In this study, andropodium was confirmed to emerge at 25 days of pbl. Thus, 25 days of pbl is considered as the initial stage of juvenile in Nomorhamphus sp. Lindu.
The juvenile fish can grow as a young fish then become a mature broodstock. It has been reported that sexual maturity in another viviparous fish, namely P. reticulata, is occurred at 8-10 weeks of pbl or 56-70 days of pbl, then becomes a broodstock at six months of pbl or around 180 days of pbl (Shahjahan et al., 2013). The sexual dimorphism in the anal fin, namely gonopodium, is visible at 28 days of pbl (Ahasan et al., 2014). The modified anal fin in Xenotoca eiseni was initially emerged at four weeks of pbl or 28 days of pbl (Tinguely et al., 2019). The secondary sexual character formation in modified anal fin was affected by the androgen hormone, based on the expression of sonic hedgehog (Shh) gene (Ogino et al., 2004).
The live feed, Artemia nauplii, can be used as a feed for Nomorhamphus sp. Lindu larvae, as this feed is suitable with larval mouth opening. It has been reported that Artemia nauplii had a size of 379-480 μm, commonly used as a live feed for fish culture (Piotrowska et al., 2021). The newborn Nomorhamphus sp. Lindu larvae were incapable of feeding Daphnia due to its size of 0.48-0.81 mm (Ebert, 2022). However, this study did not measure the size of the larval mouth opening, so it is necessary to carry out a more detailed characterization of the ontogeny of the larval mouth opening in future studies. The preliminary observations indicate that Daphnia can be applied at 30 days of pcl.
Another halfbeak species, Zenarchopterus dunckeri, has been reported to change its diet as it grows in nature. In general, juvenile fish feed more on zooplankton and small gastropods, while larger juveniles feed land insects, such as ants and flies (Kanai et al., 2017). In another halfbeak species, Hemiramphus far, is known as an omnivorous fish, as it feeds on fish, isopods, ostracods, Daphnia, shellfish, algae, and unidentified parts of plants and animals (Tabassum et al., 2017). The superior jaw shape of the Nomorhamphus, where the lower jaw is longer than the upper jaw, indicates that this fish is a type of surface feeding, as seen in Z. dunckeri (Hirota et al., 2015), H. far (Tabassum et al., 2017), and other Nomorhamphus as endemic species in Sulawesi (Kraemer et al., 2019a;Kobayashi et al., 2020). The success of maintaining the early life stages in this study provides important information for the culture management of Nomorhamphus sp. Lindu is an ornamental fish.

CONCLUSION
The Nomorhamphus sp. Lindu larvae have a total length of 1.6-1.8 cm and can swim after 4 hours and 22 minutes post-birth (pbl). These larvae feed on Artemia nauplii through the surface feeding method. The juvenile stage has occurred at 25 days of pbl with a total length of 2.0-2.2 cm. The rearing media condition can support the early life stages of Nomorhamphus sp. Lindu.