Prolarval Stage Research Articles

Chiral thioacetyl derivatives of proline as novel potential agents for beluga reproduction

The aim of the research was to study antioxidant activity and protective potential of new phenol-containing thioacetyl derivatives of D- and L-prolines as potential agents on beluga sperm and survival in the early stages of ontogeny of bester (Huso x Acipenser ruthenus) development. The study indicated that both enantiomers demonstrated the equal scavenging activity towards DPPH•, ABTS•+, O2•–, H2O2 and NO, and that the activity towards all of these radicals, with the exception of nitric oxide, exceeded the activity of BHT. No significant differences were registered between the enantiomers in the inhibition of long-term lipid peroxidation of beluga sperm in vitro. The enantiomers revealed the opposite effect on the motility duration time of beluga spermatozoa, the rate of their fertilization, and survival at the early stages of bester ontogeny. Application of the L-proline derivative for the beluga sperm treatment resulted in the increase of the total period of movement of beluga sperm cells, the raise of the fertility of sterlet eggs and the survival of bester offspring at the prelarval stage, which led to an increase in the number of produced larvae. The introduction of the D-proline derivative or BHT decreased the motility duration time of beluga sperm, the fertility of sterlet eggs and the survival of offspring at the prolarval stage, which led to a decrease in the number of obtained larvae.

Retinal development in the gilthead seabream Sparus aurata.

The retinal development of the gilthead seabream Sparus aurata has been analysed from late embryonic development to juvenile stages using classical histological and immunohistological methods. Five significant phases were established. Phases 1 and 2 comprise the late embryonic and hatching stages, respectively. The results indicate that during these early stages the retina is composed of a single neuroblastic layer that consists of undifferentiated retinal progenitor cells. Phase 3 (late prolarval stage) is characterized by the emergence of the retinal layers and the appearance of neurochemical profiles in differentiating photoreceptors, amacrine and ganglion cells. Phases 4 and 5 comprise the late larval and juvenile stages. In these stages, all the retinal cell types can be detected immunohistochemically. All the maturational events described are first detected in the central retina and, as development progresses, spread to the rest of the retina following a central-to-peripheral gradient. The results of this study suggest that S. aurata is an altricial teleost species that hatches with a morphologically undifferentiated retina. The most relevant processes involved in retinogenesis occur during the late prolarval stage (phase 3).

Morphological changes during the transition from freshwater to sea water in an amphidromous goby, Sicyopterus lagocephalus (Pallas 1770) (Teleostei)

AbstractThe widely distributed amphidromous goby Sicyopterus lagocephalus needs drastic change of habitat to fulfil its life cycle: adults live and spawn in rivers, where eggs hatch into prolarvae that have to reach the sea to acquire characteristics of planktonic larvae. Postlarvae return to rivers where they recruit and grow to the adult reproductive stage. Here, we describe the prolarval stages, namely from hatching to first contact with sea water, as well as the first marine larval stages. The observations were made under experimental conditions. We described 3 prolarval substages in freshwater (L1a–L1c). Prolarvae present a slight but visible ontogenetic development in freshwater, during which the yolk sac begins to reduce, the pigmentation increases on the body and in the eyes, and the lenses appear, although the eyes are not functional. Prolarvae need to reach the sea in a maximum of 96 h to pursue their development. Their transfer in sea water at a salinity of 36.5 induces important morphological modifications (i.e. yolk sac full absorption, appearance of pectoral fins, migration of the eyes in anterolateral position of the head, opening of mouth and anus), enabling the organisms to adapt to their new environment. This marine stage is divided into two substages: L2a corresponding to the organisation of the morphological structures adapted to the marine environment and L2b during which these morphological structures become functional. Whether it is in freshwater or sea water, the duration of the substages depends on the water temperature, but is similar for all individuals for a given temperature.

Analysis of the morphogenesis and cell proliferation in the retina of a pleuronectiform fish, the turbot psetta maxima (Pleuronectiformes: Teleostei)

The morphogenesis and cell proliferation in the retina of turbot (Psetta maxima, Pleuronectiformes: Teleostei) from embryo through metamorphosis have been examined by using proliferating cell nuclear antigen (PCNA)-immunohistochemistry and general histological procedures. In the embryonic retina, cell proliferation and spatial cell reorganization form the anlage of the pigment epithelium and neural retina. Neurogenesis begins around hatching in the temporal retina, dorsal to the optic nerve exit, and then a wave of cell differentiation spreads to the nasal retina to yield a laminated retina by the end of the prolarval turbot stage. Germinal zones in the differentiated retina persist as a rim at the retinal margin, as well as surrounding the optic fissure in premetamorphic and metamorphic turbot larvae. In these zones, progenitor cells with different morphologies show a similar spatial arrangement, which suggests that they have a similar retinogenic potential. During metamorphosis, asymmetric proliferative activity in turbot germinal zones is associated with a marked expansion of the retinal tissue. Scattered stem cells in the laminated retina, related to the lineage of rod photoreceptors, were also observed both in large premetamorphic larvae and metamorphic turbots. The proliferative activity of these cells increases considerably during metamorphosis, when rod photoreceptors become morphologically differentiated.

Post-zygotic hybrid viability in sympatric species pairs: a case study from European lampreys

Ecological speciation mechanisms are widely assumed to play an important role in the early stages of divergence between incipient species, and this especially true of fishes. In the present study, we tested explicitly for post-zygotic barriers to gene flow between a sympatric, recently diverged lamprey species pair that likely arose through ecological divergence. Experimental in vitro hybridization between anadromous parasitic Lampetra fluviatilis and resident nonparasitic Lampetra planeri resulted in a high proportion of embryos capable of attaining the burrowing pro-larval stage, strongly indicating no post-zygotic barriers to gene flow between these species. A sympatric, locally-adapted resident parasitic form of L. fluviatilis was also found to successfully hybridize with both members of this species pair. The consequences of these findings are discussed in the context of lamprey speciation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 108, 378–383.

Serotonin 1A receptor (5-HT1A) of the sea lamprey: cDNA cloning and expression in the central nervous system

Serotonergic cells are among the earliest neurons to be born in the developing central nervous system and serotonin is known to regulate the development of the nervous system. One of the major targets of the activity of serotonergic cells is the serotonin 1A receptor (5-HT1A), an ancestral archetypical serotonin receptor. In this study, we cloned and characterized the 3D structure of the sea lamprey 5-HT1A, and studied the expression of its transcript in the central nervous system by means of in situ hybridization. In phylogenetic analyses, the sea lamprey 5-HT1A sequence clustered together with 5-HT1A sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. In situ hybridization analysis during prolarval, larval and adult stages showed a widespread expression of the lamprey 5-ht1a transcript. In P1 prolarvae 5-ht1a mRNA expression was observed in diencephalic nuclei, the rhombencephalon and rostral spinal cord. At P2 prolarval stage the 5-ht1a expression extended to other brain areas including telencephalic regions. 5-ht1a expression in larvae was observed throughout almost all the main brain regions with the strongest expression in the olfactory bulbs, lateral pallium, striatum, preoptic region, habenula, prethalamus, thalamus, pretectum, hypothalamus, rhombencephalic reticular area, dorsal column nucleus and rostral spinal cord. In adults, the 5-ht1a transcript was also observed in cells of the subcommissural organ. Comparison of the expression of 5-ht1a between the sea lamprey and other vertebrates reveals a conserved pattern in most of the brain regions, likely reflecting the ancestral vertebrate condition.

The effect of temperature and substrate on the development of the cortisol stress response in the lake sturgeon, Acipenser fulvescens, Rafinesque (1817)

Lake sturgeon, Acipenser fulvescens, are considered threatened or endangered throughout most of their North American Range. Current hatchery rearing for re-stocking programs utilise conventional methods with little to no understanding of the relationship between rearing conditions and the development of the hypothalamic-pituitary-interrenal (HPI) stress axis. In the present study we examined the effects of substrate type and temperature on the development of the HPI stress axis in prolarval and larval lake sturgeon. Lake sturgeon raised over either gravel or no substrate did not consistently show an increase in whole body cortisol at the prolarval stage. However, after the onset of exogenous feeding a consistent increase in whole body cortisol following a stress was evident. Lake sturgeon larvae raised in gravel substrate demonstrated a sustained increase in whole body cortisol for at least 240 min post stress whereas whole body cortisol in larvae raised in no substrate returned to baseline within 240 min post stress. Lake sturgeon larvae raised at 9, 12 and 15°C exhibited markedly different cortisol responses with baseline whole body cortisol being, 38.6 ± 3, 5.67 ± 0.41 and 25.38 ± 2.84 ng.g−1 respectively. Furthermore, the chase induced increases in whole body cortisol at the larval stage were significantly different for each temperature treatment. These experiments demonstrate that physical environment has a significant impact on the development of the HPI stress axis in lake sturgeon.

A vesicular glutamate transporter in lampreys: cDNA cloning and early expression in the nervous system

Vesicular glutamate transporters (VGLUTs) accumulate glutamate into synaptic vesicles of glutamatergic neurons, and thus are considered to define the phenotype of these neurons. Glutamate also appears to play a role in the development of the nervous system of vertebrates. Here we report the characterization of a vesicular glutamate transporter of lamprey (lVGluT), a novel member of the VGluT gene family. Phylogenetic analysis indicates that lVGLUT cannot be assigned to any of the three VGLUT isoforms characterized in teleosts and mammals, suggesting that these classes may have been fixed after the splitting between cyclostomes and gnathostomes. Expression pattern analysis during lamprey embryogenesis and prolarval stages shows that lVGluT expression is restricted to the nervous system. The first structure to express lVGluT was the olfactory epithelium of late embryos. In the brain of early prolarvae, lVGluT was expressed in most of the neuronal populations that generate the early axonal scaffold. lVGluT expression was also observed in neuronal populations of the rhombencephalon and spinal cord and in ganglia of the branchiomeric, octaval and posterior lateral line nerves. In the rhombencephalon, lVGluT expression appears to be spatially restricted in dorsal and ventral longitudinal domains. Comparison of the early expression of VGluT genes between the lamprey and some anamniotan gnathostomes (frog, zebrafish) reveals a conserved expression pattern, likely to reflect ancestral vertebrate characteristics.

The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio)

In this study, we investigated the effects of the radioactive metal uranium (U) on the embryonic development, hatching success, growth rate, and survival of juvenile zebrafish (Danio rerio). We studied the effects of depleted uranium (20–500 μg L −1 of DU), inducing mainly chemical toxicity due to its low specific activity, and the combined effects of chemical and radiological toxicity by using a higher specific activity uranium isotope (20 and 100 μg L −1 of 233U). Results showed that early life stages are significantly affected by uranium exposure through both chemical and combined (chemical and radiological) toxicity. Experiments showed significant effects of U on hatching success starting at the concentration of 250 μg L −1 of DU, causing a 42% delay in median hatching times relative to control. Furthermore, a reduction of growth (decrease in body length and weight) was observed followed by a high mortality of pro-larvae stage (up to 100% at DU concentrations of 250 μg L −1 upon a 15 day exposure). Bioaccumulation measurements highlighted that U was mainly localised in the chorion but penetrated in the embryo inside eggs at a higher concentration. The effects differed depending on the isotopic composition of the uranium: sublethal defects in the tail detachment process were more pronounced for 233U than DU exposure, while the presence of 233U specifically affected embryo development and led to higher mortality rates of the prolarvae. The results from this study showed that the early life stages of zebrafish seems to be more sensitive to uranium contamination than more mature stages, and underline the importance of including pro-larval stages into toxicity tests in order to improve the relevancy for environmental risk assessments.

Observations on the breeding biology ofAmbassis agassiziiSteindachner, 1867 (Teleostei: Ambassidae) from the Murray Darling Basin in New South Wales

Ambassis agassizii (Agassiz's Glassfish) collected from Barren Box Swamp was bred in ponds at the Narrandera Fisheries Centre on ten occasions between 1968 and 1971 when surface water temperatures were 19.0 - 27.0°C, between November and early January, and again in February. Breeding conditions and limited embryological development were determined. A single, demersal, transparent, spherical, telolecithal and slightly adhesive egg was collected on 9.xii.70 at 23.0°C. It possessed a single oil globule; and measured 0.61mm in diameter. Eggs hatched in about a day at temperatures between 20.0 and 29.0°C. The smallest larva collected was 2.59mm, and was about 1.6mm at hatching. The pro-larval stage terminated at under 10 days after hatching. The largest male and female adults were 60.0 and 68.0mm (TL) and 2.51 and 3.88g respectively. Egg counts were 380 to 9966 (mean 3257) with higher counts in larger fish. Ovaries generally had two size ranges of oocytes. Spawning was multiple. The GSI rose to 10.50 and 8.86 ...

Breeding biology, and egg and larval development ofGalaxias rostratusKlunzinger, the Murray Jollytail from inland New South Wales

G. rostratus the Murray Jollytail bred in earthen ponds at the Inland Fisheries Research Station, Narrandera, NSW, when surface and bottom water temperatures were above 10.5oC during August and September. Flooding was unnecessary but there was water flow through the pond. Eggs were demersal, transparent, spherical, telolecithal and slightly adhesive; possessed a cluster of oil globules; varied from 1.35mm to 1.66mm in diameter; and appeared to be scattered randomly on the pond bottom during spawning. Eggs hatched after 8½ to 9½ days. The length of recently-hatched larvae ranged from 5.7mm to 8.1mm. The pro-larval stage terminated at around 5days after hatching. Water temperatures varied between 13.8 and 20.0oC during development. The largest adults collected were 15.0cm and weighed 22.5gm. Marked colour differences between the sexes were not obvious, but sex could be determined by the appearance of gonads through the transparent window anterior to the vent close to the breeding season. Fecundity of the fe...

Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.

Development of the dopamine-immunoreactive system in the central nervous system of the sea lamprey

The development of dopamine-immunoreactive (DAir) populations in the central nervous system of the sea lamprey, a modern representative of the earliest vertebrates, was studied to achieve further understanding of dopaminergic systems in vertebrates. The first DAir cell groups appeared in the spinal cord, the posterior tubercle nucleus and the dorsal hypothalamic nucleus of prolarval stages. In larvae, new DAir cell groups were observed in the caudal preoptic nucleus, the postoptic commissure nucleus, the postinfundibular commissure nucleus and the caudal rhombencephalon. All these DAir cell groups observed in larvae were also DA-positive in adults, which showed only one new DAir cell group found in the ventral hypothalamic nucleus. Occasional DAir cells were observed in the telencephalon, the ventral thalamus and/or the isthmus of large larvae and adults. From medium-sized larvae to adults, the regions most richly innervated by DAir fibers were the neurohypophysis, the striatum, the pretectum and the midbrain tegmentum. Striking differences are observed between lampreys and other vertebrates in regard to the relative time of appearance of DAir cells, which is probably related with the complex life cycle of the sea lamprey.

Embryonic and larval development of Thai Pangas (Pangasius sutchi Fowler, 1937)

The embryonic and larval development of Thai pangas was investigated during peak (May-July 1995) and late spawning (August-October 1995) periods. The fertilized eggs are adhesive and spherical with a yellowish or greenish-brown egg capsule. The yolk sac is yellowish-brown in color and 1.20-1.80 mm in diameter. Nine hours post-fertilization, the first cleavage stage, embryonic shield, head, tail region, neural grooves and somites were evident. The incubation period ranges from 24-36 h at a temperature of 20-30 degrees C. The newly hatched larvae are quite transparent and light yellowish in color with a body length of 2.98-3.10 mm. Eye pigments appear and the heart starts to work within 12-14 h of hatching. In 1-day-old pro-larvae, the mouth becomes well developed; barbules are elongated, prominent and look like tiny threads. The yolk sac is fairly well absorbed and the palatine teeth are fully developed during the 3 day pro-larval stage. At the end of 12 days of larval development, the stomach becomes functional and aerial respiration starts. After 2 weeks, the young fry is well-developed, and is of an adult appearance, that is, measuring up to 13.56 mm in length.

Acute toxicity and cholinesterase inhibition in larval and early juvenile walleye exposed to chlorpyrifos

Application of organophosphorus (OP) insecticides to agricultural fields during the spring often overlaps the period of spawning, egg incubation, and larval stages of fish species such as walleye (Stizostedion vitreum). Because life stage can affect uptake, distribution, and effects of contaminants, our objective was to identify the most sensitive life stage of walleye tochlorpyrifos, a broad-spectrum OP insecticide. Prolarvae (yolk sac, endogenous feeding stage) were least sensitive to chlorpyrifos (median lethal concentration [LC50] = 225-316 microg/L), and postlarvae I (oil globule, exogenous feeding stage) were less sensitive (LC50 = 24-29 microg/L) than postlarvae II (oil globule absent; LC50 = 12-13 microg/L). The differences in sensitivity of the larval stages coincide with stages of gill development. Gill filaments were absent until the end of the prolarval stage, and development of secondary lamellae did not occur until the end of the postlarval I stage. Juvenile fish were less sensitive than postlarval stages, but did not differ significantly among the juvenile ages tested. The LC50s ranged from 37 to 45 microg/L for 30- and 90-d-old juvenile walleye, respectively. Larval walleye survived when cholinesterase (ChE) activity was inhibited by as much as 90%; however, 60- and 90-d-old juvenile walleye did not survive when ChE activity was inhibited more than 71%.

Proliferating cell nuclear antigen (PCNA) immunoreactivity and development of the pineal complex and habenula of the sea lamprey

The development of the pineal complex and the habenula of the sea lamprey was studied with proliferating cell nuclear antigen (PCNA) immunocytochemistry. The pineal organ and the habenula primordia appeared in late embryos, and neuron differentiation began in prolarvae, as indicated by the presence of PCNA-negative cells. The parapineal primordium could not be distinguished until early prolarval stages, and cell differentiation was delayed to the larval period. Although the number of cycling (PCNA-immunoreactive) cells gradually decreased during the larval period in the three organs studied, their patterns of differentiation were different. We conclude that the unusual developmental pattern observed is related with the complex life cycle of lampreys.

Gonadotropin-Releasing Hormone Containing Neurons and Olfactory Fibers During Development: From Lamprey to Mammals

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo–pituitary–gonadal axis in all vertebrates. The vast majority of GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. Several antibodies and lectins that recognize cell surface carbohydrates have been useful for delineating the migratory pathway from the olfactory placodes and vomeronasal organ, through the nasal compartment, and across the cribriform plate into the brain. In rats, α-galactosyl-linked glycoconjugates (immunoreactive with the CC2 monoclonal antibody) are expressed on fibers along the GnRH migration pathway and approximately 10% of the GnRH neuronal population. In lamprey, the α-galactosyl binding lectin, Grifonia simplicifolia-I (GS-1), identifies cells and fibers of the developing olfactory system. In contrast to the CC2 immunoreactive GnRH neurons in rats, the GS-1 does not label a subpopulation of presumptive GnRH neurons in lamprey. Results from these and other experiments suggest that GnRH neurons in developing lamprey do not originate within the olfactory placode, but rather within proliferative zones of the diencephalon. However, the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, suggest that olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey throughout life. By contrast, olfactory fibers are directly relevant to the migration of GnRH neurons from the olfactory placodes in mammalian species. Primary interactions between olfactory fibers and GnRH neurons are likely transient in mammals, and so in later life olfactory modulation of GnRH secretion is likely to be indirect.

Relationship of gonadotropin-releasing hormone (GnRH) neurons to the olfactory system in developing lamprey (Petromyzon marinus).

Gonadotropin releasing-hormone (GnRH) regulates the hypothalamo-pituitary-gonadal axis in vertebrates. The regulation of GnRH is intimately related to information from the olfactory system. Additionally, GnRH neurons are thought to be derived from progenitor cells in medial olfactory placodes. The present experiments were conducted to characterize the earliest development of GnRH neurons in lamprey and to determine their relationship to cells and fibers derived from the olfactory system. Eggs from fertile adult sea lamprey were fertilized in the laboratory, and larvae were maintained for up to 100 days. GnRH neurons were visualized within the lamprey preoptic area and hypothalamus as soon as GnRH was detectable (22 days after fertilization). The number of neurons increased with age through day 100. GnRH neurons were never seen within the olfactory system. The cells and fibers of the olfactory system were identified using the lectin, Grifonia Simplicifolia-1 (GS-1). Overlap between the olfactory and GnRH systems were at the level of fiber projections. GS-1 reactive cells of apparent placodal origin did not enter the region of the preoptic area or hypothalamus that contained GnRH neurons. Recently divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). The positions of BrdU-labeled cells after different survival times suggest a predominant medial-lateral radial neuron migration with a small number in positions suggestive of migration between the olfactory epithelium and the telencephalic lobes. Regardless of survival time, these cells were always found close to their entry point into the brain, suggesting minimal rostral-caudal migration. Based on these results, we hypothesize that GnRH neurons in developing lamprey originate within proliferative zones of the diencephalon and not in the olfactory system. Based on the overlap of olfactory- and GnRH-containing fibers from prolarval stages to metamorphosis, olfactory stimuli may play a major role in the regulation of GnRH secretion in lamprey.

Chondrogenesis of a non-collagen-based cartilage in the sea lamprey,Petromyzon marinus

Chondrogenesis of the trabeculae, non-collagen-based cartilages in prolarval stages of the sea lamprey, Petromyzon marinus, was examined by light and electron microscopy. Chondrogenesis of the trabecular cartilages in prolarval lampreys commenced with the formation of mesenchymal condensations. Two peaks in mesenchymal cell density occurred, one prior to condensation formation and a second immediately before cartilage differentiation. The possibility of inductive influences by epithelio-mesenchymal interactions on the initiation of chondrogenesis is discussed. Bilateral condensations first appeared by day 17 post fertilization ventromedial to the eyes in a band of tightly packed yolk-laden mesenchymal cells that represent neural crest derived tissue. Cartilage differentiation occurred by day 19 post fertilization and was indicated by the presence of matrix-synthesizing organelles and the first ultrastructural appearance in the extracellular matrix of lamprin, a structural protein unique to lamprey cartilage. Lamprin was initially deposited as discrete 15- to 40-nm globules. Subsequently, lamprin appeared as fibrils aggregated into branching and parallel arrays arranged in pericellular, territorial, and interterritorial zones. Lengthening of the trabecular cartilages was primarily by appositional growth at the rostral end. The timing of the appearance of trabecular cartilages in prolarval stages likely reflects the functional importance of these structures for supporting the brain as the lamprey initiates burrowing behaviour.

Scale effects of MS-222 on a marine teleost, porgy Pagrus major

The relationship between the effective concentration ( C E ) of the anaesthetic MS-222 and body mass was examined at 20°C in porgy ranging in size from 0.00022 g (just after hatching) to 320 g (595 days old). C E was defined as the concentration which required 3–5 min to induce deep anaesthesia in the fish. Values of C E (ppm) increased monophasically with increasing body mass ( W in g), following the equation C E = 79 W 0.0549 ( N = 30, r = 0.936 between log C E and log W). The recovery ratio ( R R in %), i.e., the number of fish as a percentage which recovered after being placed back in ordinary seawater, varied depending on the developmental stage. R R was lowest in fish of around 0.01 g (30 days old), corresponding to the transitional stage between the post-larval and juvenile stage, whereas it was highest (nearly 100%) in the pro-larval stage (about 0.00023 g in size and 0–6 days of age).