Larval development stages
The developmental stages of the larvae were classified as pro-larvae (hatching to complete yolk absorption) and post-larvae (yolk absorption to miniature adult shape) (Table 1), as recommended by
Hubbs (
1943). Upon hatching, the fertilized eggs emerged into a relatively undeveloped pro-larval stage, measuring 3.434±0.16 mm in length, with a protuberant yolk sac measuring 0.797±0.075 mm
3. Although smaller in length compared to
Macrognathus aculeatus (4-5 mm)
(Sahoo et al., 2007), the newly hatched larvae had well-differentiated brains, otic capsules, U-shaped jaws, elongated pectoral fin buds and intense melanophores without any pattern on the body (Fig 1a, b). From 6-48 hph, the larvae underwent significant morphological changes, including the appearance of melanophore stripes on the body (Fig 1c-1g). Complete eye pigmentation was observed at 60 hph, coinciding with the typical time for exogenous feeding
(Pepe-Victoriano et al., 2021). At 54 hph, a pair of olfactory pits, a few sharp teeth, and widening of the intestine with an anal pore were noticed (Fig 1h–1j). By 60 hph, the larvae had distinct lenses in their eyes, functional opercula, rayed pectoral fins, prominent swim bladder and melanophore stripes (Fig 2a) and a depleted yolk-sac (90.8% or above) (Fig 3), with the yolk sac being fully absorbed between 60-72 hph. A study by
Pongjanyakul et al., (2020) on
Macrognathus siamensis larvae also reported complete yolk absorption at 72 hph. The open mouth, dentition for grasping and holding live prey, opening of anal aperture, wide rectum and formation of a pouch-like intestine (Fig 1l) in the present study suggested that the exogenous feed should be available on the third day of hatching. The timing of exogenous feeding varies depending on the fish species, yolk-sac utilization and larval development rates. In several fish species, the onset of exogenous feeding occurred between 2 and 4 dph
(Pradhan et al., 2012; Saxena et al., 2019; Kumar et al., 2021; Reyes-Mero et al., 2022).
The post-larval stage of development varies among species in terms of size, shape, fin size, pigmentation, shape and time of organ formation. In the present study, the post-larvae undertook significant changes in the head, eyes, jaws, mouth, fins and body pigmentation. The upper jaw extension was noticed at 14 dph and the formation of erectile dorsal spines at 21 dph (Fig 2b), two striking features of Mastacembelids. At four weeks of age, the larvae displayed a V-shaped rostrum, an inferior mouth and prominent black stripes on the anal and dorsal fins (Fig 2c). The jaw length continued to increase until the rostrum formation- a trunk-like appendage at 45 dph (Fig 2d). At this stage, the larvae underwent metamorphosis into the fry stage (81%), resembling miniature adults with the beginning of benthic and burrowing habits. In several species, metamorphosis from larvae to juveniles occurred between 32-50 dph
(Anil et al., 2018; Anzeer et al., 2019). In fish, the metamorphosis is reported to be accompanied by changes in habitat, body proportions, fin differentiation, pigmentation patterns and scale formation
(Kendall et al., 1984; Urho, 2002). In the present study, a denser juvenile-adult body with patterns of white dots along the body girth forming a series of rings was noticed during 45–91 dph (Fig 2d). The environmental temperature plays a crucial role in larval development and growth. Larvae at higher temperatures tend to deplete their yolk reserves more quickly than those at lower temperatures
(McMahon et al., 2023). The temperature during the study (29.3±1.4°C) was found to be optimal for the larval development of most tropical fish species.
Yolk reserves and utilization
Fish larvae contain two kinds of energy reserves: yolk and oil globule (
Bjelland and Skiftesvik, 2006). In the case of
M. pancalus, the newly hatched larvae had a large elliptical-shaped yolk sac with a mean length of 1.329±0.044 mm and a volume of 0.797±0.075 mm3. Additionally, there was an oil globule located at the anterior tip of the yolk sac, with an average diameter of 0.440±0.018 mm and a volume of 0.045±0.005 mm3. The yolk-sac was utilized at a faster rate compared to the oil-globule exhaustion rate (Fig 3), with 41.8% of the initial yolk sac being significantly utilized within 12 hours. On the other hand, there was no significant decrease (8.9%) in oil globule reserves during this period (Fig 3). Over time, the yolk-sac reserves were reduced by 79% at 36 hph and were completely exhausted within 72 hph. In silver perch larvae, similar observations of faster yolk-sac utilization compared to oil-globule exhaustion were documented by
Sulaeman and Fotedar (2017).
Larval feeding and cannibalism
The first feeding marks a critical period for larval survival, growth and development. Larval feeding with live zooplankton and boiled egg yolk was documented in lesser spiny eel (Das and Kalita, 2003;
Sahoo et al., 2009). However, boiled egg yolk resulted in 100% mortality of
M. pancalus larvae on the 17
th day
(Afroz et al., 2014). Kumar et al., (2021) reported successful rearing of stinging catfish larvae until 22 dph with mixed zooplankton without dependence upon
Artemia nauplii. In
M. aculeatus larvae,
Sahoo et al., (2009) reported that the larvae fed mixed zooplankton attained a mean final TL and BW of 21.33 mm and 32.22 mg. In contrast, our study recorded a higher mean TL (28.8 mm) and BW (100 mg) after 30 days of the rearing. The most commonly cultured freshwater rotifer was used for the first feeding, but we observed higher cannibalism between 5-14 dph, as indicated by the disappearance of larvae.
Serajuddin and Ali (2005) reported the carnivorous and predatory habits of wild
M. pancalus specimens, with young individuals feeding on annelids as their basic food. In our study, the development of dorsal spines and the appearance of the inferior mouth at 21 dph with
Tubifex worm as basic food resulted in a decrease in cannibalism rate.
Larvae behaviour
The newly hatched larvae were inactive and remained attached to the sides and bottom of the tank, taking shelter under the roots of aquatic plants. Due to their large yolks, they exhibited upside-down, irregular movements when disturbed. By 60 hph, they displayed intermittent swimming activity with pauses.
Sahoo et al., (2009) observed similar hiding behavior in
M. aculeatus, which lasted for 7-8 days. Fast swimming in
M. pancalus was initiated between 5-7 dph, which coincided with the disappearance of the vertical black stripes. The low specific gravities in pro-larvae, due to the presence of a protuberant yolk sac and oil globule, probably needed to remain under the roots of floating weeds. However, with the absorption of the yolk sac and oil globule, the specific gravity of the larvae increased and they moved with the water column. This coincided with the formation of the swim bladder, indicating the ontogenetic change in the buoyancy (Table 1). In burbot
Lota lota larvae,
Palińska-Żarska et al., (2014) recommended a low water depth (up to 10 cm) until the moment of the swim bladder inflation. The presence of a swim bladder in the early larval stages of barred spiny eel is likely necessary for its benthopelagic life.
Larval growth, allometric analysis and condition factor
The mean TL values at 21 and 28 dph were significantly higher than the values until 14 dph (p
<0.05) (Fig 4a). During the pro-larval phase, a significantly higher SGR of 12.81±0.8%/day for TL was noticed, compared to 6.91±0.6%/day at 14 dph (p
<0.05). Thereafter, SGR increased to 7.44±0.6% at 28 dph, followed by a significant decrease, reaching 3.57±0.3% at 91 dph. Weekly observations showed a significantly higher SGR of 8.91±0.9% in the first week than the second week (4.92±0.3%), followed by a significant increase (p
<0.05) in the third (8.33±0.6%) and fourth (7.75±0.5%) weeks (Fig 4b). Several authors reported the rapid growth phase of fish larvae immediately following hatching, followed by a period of slow growth during yolk depletion. In the Pacific fat sleeper
Dormitator latifrons, the larvae exhibited the fastest growth during the initial 24 hph, with approximately 52% of yolk absorption
(Reyes-Mero et al., 2022). In silver perch, the SGR of TL reached approximately 12% per day during the first 4 dph, followed by a slow growth rate of 4.8% only, from 4 to 10 dph (
Sulaeman and Fotedar, 2017).
The LWR, log BW = 2.5628 log TL-1.7094 indicated negative allometric growth (b=2.56, R²=0.997, p
<0.05). The larvae appeared slender as they grew, becoming faster and more agile, with increasing TL suited to their predatory nature. However, the estimated b value fell within the expected range of 2.5-3.5 (
Froese, 2006). The condition factor is a crucial indicator of the well-being and nutritional status of fish
(Rao et al., 2024). It is used to monitor feeding intensity, age and growth rates in fish. Generally, when the condition factor is close to or equal to 1, it indicates a satisfactory fitness level for fish species
(Jisr et al., 2018). The lower ‘K
A’ value of less than 1.0 and declining values observed in this study are likely associated with their transition to an anguilliform/ eel-like shape in the early life stages of
M. pancalus. Furthermore, K
A values decreased sharply, reaching the lowest value at 20-30 mm TL and 21-28 dph (Fig 5a, b), followed by an increase in the K
A values and SGR, indicating the suitability of feeding
Tubifex worms to
M. pancalus larvae.
