Induction Of Metamorphosis Research Articles

Analysis of nitric oxide‐cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.

RNAi‐mediated knockdown of juvenile hormone acid O‐methyltransferase gene causes precocious metamorphosis in the red flour beetle Tribolium castaneum

Juvenile hormone controls the timing of insect metamorphosis. As a final step of juvenile hormone biosynthesis, juvenile hormone acid O-methyltransferase (JHAMT) transfers the methyl group from S-adenosyl-l-methionine to the carboxyl group of farnesoic acid and juvenile hormone acid. The developmental expression profiles of JHAMT mRNA in the silkworm Bombyx mori and the fruitfly Drosophila melanogaster suggest that the suppression of JHAMT transcription is critical for the induction of larval-pupal metamorphosis, but genetic evidence for JHAMT function in vivo is missing. In this study, we identified three methyltransferase genes in the red flour beetle Tribolium castaneum (TcMT1, TcMT2 and TcMT3) that are homologous to JHAMT of Bombyx and Drosophila. Of these three methyltransferase genes, TcMT3 mRNA was present continuously from the embryonic stage to the final larval instar, became undetectable before pupation, and increased again in the adult stage. TcMT3 mRNA was localized in the larval corpora allata. Recombinant TcMT3 protein methylated farnesoic acid and juvenile hormone III acid, but TcMT1 and TcMT2 proteins did not. Furthermore, RNA interference-mediated knockdown of TcMT3 in the larval stage resulted in precocious larval-pupal metamorphosis, whereas knockdown of either TcMT1 or TcMT2 showed no visible effects on metamorphosis. Importantly, precocious metamorphosis caused by TcMT3 RNA interference was rescued by an application of a juvenile hormone mimic, methoprene. Together, these results demonstrate that TcMT3 encodes a functional JHAMT gene that is essential for juvenile hormone biosynthesis and for the maintenance of larval status.

The effect of chemical cues on settlement of pearl oyster Pinctada fucata martensii (Dunker) larvae

The pearl oyster Pinctada fucata martensii is an important aquaculture species in South China Sea and has great commercial value in the pearl culture industry. As difficulties in the commercial culturing of molluscs are mainly associated with larval settlement and metamorphosis, it is important to find a routine, inexpensive and effective technique for the induction of synchronous settlement and metamorphosis of larvae. In the present study, the effects of 11 chemicals on inducing larval settlement of the P. fucata martensii were investigated in the laboratory. The larvae were exposed to the chemicals for 96 h. Among the chemicals tested, K+ (10 and 20 mM), Ca2+ (1 and 50 mM), γ-aminobutyric acid (10−4 M), 3-isobutyl-1-methylxanthine (10−4 M), choline (10−3, 10−4 M), acetylcholine (10−4 M), and serotonin (10−3, 10−4, 10−5 M) induced high percentage of the larvae to settle without acute toxic effects, while Mg2+, NH4+, dopamine, and 3-(3,4-Dihydroxyphenyl)-L-alanine at all the tested concentrations were less effective. Serotonin of 10−4 M resulted in the highest settlement rate, but the chemical is expensive. K+ was slightly less effective, but the chemical is much cheaper, therefore maybe more economical in the commercial production.

Metamorphosis and acquisition of symbiotic algae in planula larvae and primary polyps of Acropora spp.

Coral planulae settle, then metamorphose and form polyps. This study examined the morphological process of metamorphosis from planulae into primary polyps in the scleractinian corals Acropora nobilis and Acropora microphthalma, using the cnidarian neuropeptide Hym-248. These two species release eggs that do not contain Symbiodinium. The mode of acquisition of freshly isolated Symbiodinium (zooxanthellae) (FIZ) by the non-symbiotic polyp was also examined. Non-Hym-248 treated swimming Acropora planulae did not develop blastopore, mesenteries or coelenteron until the induction of metamorphosis 16 days after fertilization. The oral pore was formed by invagination of the epidermal layer after formation of the coelenteron in metamorphosing polyps. At 3 days after settlement and metamorphosis, primary polyps exposed to FIZ established symbioses with the Symbiodinium. Two–four days after exposure to FIZ, the distribution of Symbiodinium was limited to the gastrodermis of the pharynx and basal part of the polyps. Eight–ten days after exposure to FIZ, Symbiodinium were present in gastrodermal cells throughout the polyps.

METAMORPHOSIS AND POSTLARVAL GROWTH OF ABALONE HALIOIS RUFESCENS IN A MEXICAN COMMERCIAL HATCHERY

Metamorphosis induction and postlarval growth of the red abalone (Haliotis rufescens) were evaluated in a commercial farm of Baja California, México. This hatchery settles larvae with gamma-aminobutyric acid (GABA, 1-μM final concentration) and culture postlarvae in 250-L tanks placed in a four-story structure inside a building with artificial illumination. Eight tanks (four at the top and four at the bottom of a culture structure) were sampled during four months after settlement. Upper tanks received more light than the lower tanks (means of 727 and 217 lux, respectively) and had a higher mean water temperature (14.9°C and 14.4°C, respectively). Estimates of metamorphosis induction were highly variable (37% to 99%) and mean values were higher in lower (76%) than in higher (54%) tanks. Potential causes of this unexpected variability are discussed. There was a significant positive linear relationship between metamorphosis induction and early (7-day) postlarval survival. Average postlarval growth rates were also highly variable among tanks (37–63 μm/day) and slightly higher in upper than in lower tanks (60 and 52 μm/day, respectively). During the sampling period, growth variability was positively associated with water temperature changes, especially after the formation of the first respiratory pore when growth increased abruptly.

Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis on different macroalgae

Settlement of mussels is commonly associated with macroalgae. The effects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were filamentous in morphology. Macroalgae that were cylindrical, phylloid, flabellate, palmate and pinnate all showed low (<8%) percentages of post-larvae but four other filamentous macroalgae also had low mussel larval settlement, suggesting that chemical factors may also be involved. Seasonal variation had no effect on inductive activities of C. fastigiata and C. tenerrimum. Treatment of C. fastigiata and C. tenerrimum with formalin, ethanol and heat resulted in the significant decrease or loss of their inductive activities. Survival of algal cells within treated macroalgae also decreased significantly. Treatment of the two macroalgae with antibiotics and GeO2 reduced the numbers of bacteria and diatoms on their surface but did not affect their inductive activities, indicating that the cue was produced by macroalgae and not by coexisting bacteria and diatoms. However, conditioned water and crude extracts of these two macroalgae did not induce larval settlement and metamorphosis. Thus, larvae of M. galloprovincialis settled and metamorphosed on specific filamentous macroalgae. The chemical cues produced by C. fastigiata and C. tenerrimum were susceptible to ethanol and heat treatments and were not recovered in the conditioned water nor in the extracts. The finding that inactive C. tenerrimum can be produced from culturing its apical segments provides a new tool to elucidate the chemical cue(s) from macroalgae through manipulation of their culture conditions.

Molecular cloning and developmental expression of the gene encoding juvenile hormone esterase in the yellow-spotted longicorn beetle, Psacothea hilaris

Juvenile hormone (JH) plays a key role in the regulation of growth, development, diapause and reproduction in insects. The regulation of JH titers in the insect body is therefore crucial throughout postembryonic development. One of the major pathways of JH metabolism is degradation by a highly selective enzyme, juvenile hormone esterase (JHE). We obtained a full-length cDNA encoding JHE in Psacothea hilaris ( PhJHE). The complete PhJHE cDNA sequence is comprised of 1989 bp with an open reading frame of 1785 bp encoding 595 amino acid residues. The deduced protein sequence of PhJHE showed high homology with the Tenebrio molitor JHE (50% amino acid identity) and moderate homology with the Drosophila melanogaster JHE (34%). The PhJHE transcript was expressed mainly in the fat body. PhJHE transcript levels were low until day 3 of the 5th (final) larval instar, then steadily increased reaching a peak on day 13 (the prepupa stage), coinciding well with the peak hemolymph enzyme activity level. Sustained starvation of larvae after a period of feeding stimulated the expression of PhJHE mRNA while feeding the larvae with glucose downregulated its expression. These results are discussed with reference to the induction of precocious metamorphosis in this beetle by starvation.

Induction of Metamorphosis Decreases Nitric Oxide Synthase Gene Expression in Larvae of the Marine Mollusc Ilyanassa obsoleta (Say)

Many marine organisms spend the early part of their lives as larvae suspended in the water column before metamorphosing into benthic reproductive adults. Metamorphosis does not occur until a larva has become competent to respond to appropriate stimuli and after a suitable habitat for the young juvenile has been encountered. The gaseous neurotransmitter nitric oxide is thought to be important in the regulation of metamorphosis by holding the organism in the larval state. We have investigated expression of the neuronal nitric oxide synthase (nNOS) gene in larval and metamorphosing individuals of the marine mud snail Ilyanassa obsoleta. Our results indicate that nNOS is expressed at constant levels throughout larval development. In contrast, expression of nNOS decreases markedly during the first 24 h of metamorphosis. Our observations support previous findings that demonstrate that nitric oxide is present in larvae though competence. The decrease in nNOS gene expression that occurs during metamorphosis corresponds with a previously described reduction in nNOS activity.

Functional role of aspartic proteinase cathepsin D in insect metamorphosis

BackgroundMetamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.ResultsCathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.ConclusionBased on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

The effect of larval age on morphology and gene expression during ascidian metamorphosis

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

HEAT SHOCK INDUCED METAMORPHOSIS OF THE QUEEN CONCH, STROMBUS GIGAS: COMPARISON WITH INDUCTION BY ALGAL ASSOCIATED CUES

Abstract Recent research indicates that abiotic environmental factors, including temperature, may be as important as chemical cues in controlling the induction of metamorphosis of marine invertebrate larvae. In this study, the effects of elevated temperature or heat shock on the induction of metamorphosis in the tropical marine gastropod, Strombus gigas, are examined. Elevations in temperature above culture temperatures (28°C to 29°C) to 37°C to 38°C induced high levels of metamorphosis (77% to 100%), equivalent to those induced by a known algal associated inducer, an extract of Laurencia poitei. The age for competency to metamorphosis and the exposure time needed to induce this process were similar for heat shock and for the algal-associated cue. Understanding the interaction between abiotic and biotic factors during metamorphosis may help to better predict recruitment patterns for this commercially important species.

Lipid class dynamics during larval ontogeny of sea scallops, Placopecten magellanicus, in relation to metamorphic success and response to antibiotics

This study examines ontogenetic changes in lipid class composition in relation to survival and growth of five batches of sea scallops, Placopecten magellanicus, from egg to dissoconch stages. It also investigates the effects of antibiotic addition during metamorphosis to determine whether transient treatment during this critical period is effective in increasing metamorphic success. The top-performing larvae (growth rate: 4.7-5.0 μm day − 1 , dissoconch survival: 32–57%) were characterized by a pronounced increase in absolute (ng larva − 1 ) and relative concentrations (wt.% of total lipid) of triacylglycerol (TAG) during the pre-metamorphic period, followed by utilization during metamorphosis (0.5–1.4 μg day − 1 larva − 1 ). In contrast, the low-performing scallops (growth rate: 3.6–4.5 μm day − 1 , dissoconch survival < 1%) exhibited a constant, low level of TAG. These results strongly suggest that the accumulation of TAG during the pre-metamorphic period is a good predictor of scallop performance as measured by survival to dissoconch stage. Antibiotic treatment enhanced absolute and relative TAG concentrations in pre-metamorphic scallops and resulted in higher post-metamorphic survival and TAG concentration than in non-treated controls. However, the proportion of dissoconch to live larvae at the end of the experiment was significantly lower with antibiotic treatment (8% vs. 20% in controls), thus resulting in comparable yields of dissoconch larvae ∼ 8% irrespective of treatment. A possible negative effect of antibiotic treatment on bacterial induction of settlement and metamorphosis of sea scallop larvae is suggested.

Corticotropin-releasing hormone-mediated metamorphosis in the neotenic axolotl Ambystoma mexicanum: Synergistic involvement of thyroxine and corticoids on brain type II deiodinase

In the present study, morphological changes leading to complete metamorphosis have been induced in the neotenic axolotl Ambystoma mexicanum using a submetamorphic dose of T 4 together with an injection of corticotropin-releasing hormone (CRH). An injection of CRH alone is ineffective in this regard presumably due to a lack of thyrotropic stimulation. Using this low hormone profile for induction of metamorphosis, the deiodinating enzymes D2 and D3 known to be present in amphibians were measured in liver and brain 24 h following an intraperitoneal injection. An injection of T 4 alone did not influence liver nor brain D2 and D3, but dexamethasone (DEX) or CRH alone or in combination with T 4 decreased liver D2 and D3. Brain D2 activity was slightly increased with a higher dose of DEX, though CRH did not have this effect. A profound synergistic effect occurred when T 4 and DEX or CRH were injected together, in the dose range leading to metamorphosis, increasing brain D2 activity more than fivefold. This synergistic effect was not found in the liver. It is concluded that brain T 3 availability may play an important role for the onset of metamorphosis in the neotenic axolotl.

カイコのトランスジェニック技術による早熟変態の誘導

カイコのトランスジェニック技術による早熟変態の誘導

Effects of GABA and epinephrine on the settlement and metamorphosis of the larvae of four species of bivalve molluscs

Competent larvae of different marine bivalve species were treated with GABA and epinephrine at different concentrations and times of exposure to test the ability of the drugs to induce settlement and metamorphosis. GABA induced both settlement and metamorphosis in the mussel Mytilus galloprovincialis, the clams Venerupis pullastra and Ruditapes philippinarum and the oyster Ostrea edulis. Maximum induction of settlement (>39%) was achieved after exposure of V. pullastra larvae to 10 −4 M GABA; this concentration of GABA also induced the highest percentages of metamorphosis in the four species studied. Epinephrine was identified as an active inducer of settlement and metamorphosis in bivalve molluscs. Exposure to 10 −5 M epinephrine induced significant levels of settlement in Mytilus, Venerupis and Ostrea. In contrast, epinephrine failed to induce settlement behaviour in Ruditapes. Maximum induction of metamorphosis was produced by 10 −5 M epinephrine in mussels, clams and oysters; Ruditapes showed the highest percentage of metamorphosis (>78%). This is the first report in which the involvement of GABA in the settlement and metamorphosis of bivalve molluscan larvae is demonstrated. It was also recognised that epinephrine plays a role not only in inducing metamorphosis but also in initiating settlement.

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death.

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.

Induction of larval settlement and metamorphosis in the donkey-ear abalone, Haliotis asinina Linnaeus, by chemical cues

The effects of the chemical inducers, gamma-aminobutyric acid (GABA) and potassium chloride (KCl), on the larval settlement and metamorphosis of the donkey-ear abalone, Haliotis asinina, was investigated. H. asinina larvae (5–6 h post-hatch) were exposed to a range of GABA (0.125–2.00 μM) and KCl (1.00–12.00 mM) concentrations for 72 h. Results of the dose response experiments showed that settlement and metamorphosis vary according to the dose levels of the inducer compounds. Under controlled laboratory conditions, 0.45–0.50 μM and 6.0 mM seemed to be the optima for GABA and KCl, respectively, as these concentrations elicited the greatest number of postlarvae that metamorphosed, settled or survived. However, GABA generally promoted better attachment and metamorphic response as well as survival than KCl in H. asinina postlarvae.

Induction of metamorphosis in larvae of the brooding corals Acropora palifera and Stylophora pistillata

Many coral larvae require surface contact with crustose red algae (CRA) to induce metamorphosis; however, many features of the ecology of pocilloporid corals, such as their ability to colonize primary substrata, suggest that their larvae respond to different cues. We compared the metamorphosis of larvae of the brooding corals Stylophora pistillata (family Pocilloporidae) and Acropora palifera (family Acroporidae) in response to a variety of environmental cues. Acropora palifera metamorphosed only in the presence of three species of CRA. In contrast, S. pistillata metamorphosed in all assays, except those containing the brown alga Lobophora sp. Metamorphosis was highest (80 ± 20%) in unfiltered sea water; however, metamorphosis also occurred in 0.2-μm filtered sea water. These results suggest that S. pistillata larvae respond to both large and small water-borne molecular cues. The lack of a stringent requirement for surface contact with CRA will allow S. pistillata larvae to pre-empt species that require a more developed fouling community to induce metamorphosis and this feature of larval ecology may be the key to understanding the success of many opportunistic benthic species.

Combination of macroalgae-conditioned water and periphytic diatom Navicula ramosissima as an inducer of larval metamorphosis in the sea urchins Anthocidaris crassispina and Pseudocentrotus depressus

The induction of larval metamorphosis in the sea urchins Anthocidaris crassispina and Pseudocentro(us depressus was investigated in the laboratory, using waters conditioned by 15 different macroalgae combined with the periphytic diatom Navicula ramosissima. Larvae of P. depressus did not metamorphose, but larvae of A. crassispina showed a high incidence of metamorphosis, especially in waters conditioned by coralline red algae or brown algae. High inductive activity for larval metamorphosis was detected in Corallina pi/ullfera-conditioned water during a 2.5-year investigation, but the activity was relatively low in February or March and in September, the off-growth seasons of the alga. By contrast, Ulva pertusa-conditioned water did not show metamorphosis-inducing activity except in spring or early summer. These results indicate that during their growth phase, red and brown algae release into their environment active substances that are involved in the larval metamorphosis of A. crassispina.

Detection of Proteins with a High Affinity for Imidazole Insect Growth Regulator, KK-42

For explanation on mode of action of KK-42, an imidazole insect growth regulator, an amine analog of KK-42 (1-(3-aminopropyl)-5-[(E)-2,6-dimethyl-1,5-heptadienyl]imidazole, ADHI) has been designed and prepared as a ligand to detect proteins with high affinity in pharate first larvae of wild silk moth associated with artificial hatching. The mode of action of KK-42 for induction of precocious metamorphosis is speculated to be an inhibition of ecdysteroid biosynthesis at the prothoracic gland (PG). Since the PG in 5th instar larvae of silkworm on day 7 has strong biosynthetic activity and is sensitive to KK-42, a supernatant of a homogenate of the PG was applied to ADHI-Sepharose. After optimization of the condition of affinity chromatography, a single protein was eluted with free KK-42. This protein did not bind to the affinity gel when incubated with KK-42 before the application.