Late Metamorphosis Research Articles

Limited contribution of photoenzymatic DNA repair in mitigating carry-over effects from larval UVB exposure: Implications for frog recruitment

Solar ultraviolet-B (UVB) radiation has increased due to stratospheric ozone depletion, climate and ecosystem changes and is a driver of amphibian population declines. Photoenzymatic repair (PER) is a critical mechanism for limiting UVB lethality in amphibian larvae. However, the link between PER and the UVB-induced effects remains understudied through long-term investigations in vivo. Here, we assessed how larval PER determines the lethal and sublethal effects induced by environmentally relevant acute UVB exposure until the juvenile phase in the Neotropical frog Odontophrynus americanus. We conducted laboratory-based controlled experiments in which tadpoles were or were not exposed to UVB and subsequently were exposed to light (for PER activation) or dark treatments. Results showed that the rates of mortality and apoptosis observed in post-UVB dark treatment are effectively limited in post-UVB light treatment, indicating PER (and not dark repair, i.e. nucleotide excision repair) is critical to limit the immediate genotoxic impact of UVB-induced pyrimidine dimers. Nonetheless, even tadpoles that survived UVB exposure using PER showed sublethal complications that extended to the juvenile phase. Tadpole responses included alterations in morphology, chromosomal instability, increased skin susceptibility to fungal proliferation, as well as increased generation of reactive oxygen species. The short-term effects were carried over to later stages of life because metamorphosis time increased and juveniles were smaller. No body abnormalities were visualized in tadpoles, metamorphs, and juveniles, suggesting that O. americanus is UVB-resistant concerning these responses. This study reveals that even frog species equipped with an effective PER are not immune to carry-over effects from early UVB exposure, which are of great ecological relevance as late metamorphosis and smaller juveniles may impact individual performance and adult recruitment to breeding. Future ecological risk assessments and conservation and management efforts for amphibian species should exercise caution when linking PER effectiveness to UVB resistance.

Unravelling the functional development of vertebrate pathways controlling gaze.

Animals constantly redirect their gaze away or towards relevant targets and, besides these goal-oriented responses, stabilizing movements clamp the visual scene avoiding image blurring. The vestibulo-ocular (VOR) and the optokinetic reflexes are the main contributors to gaze stabilization, whereas the optic tectum integrates multisensory information and generates orienting/evasive gaze movements in all vertebrates. Lampreys show a unique stepwise development of the visual system whose understanding provides important insights into the evolution and development of vertebrate vision. Although the developmental emergence of the visual components, and the retinofugal pathways have been described, the functional development of the visual system and the development of the downstream pathways controlling gaze are still unknown. Here, we show that VOR followed by light-evoked eye movements are the first to appear already in larvae, despite their burrowed lifestyle. However, the circuits controlling goal-oriented responses emerge later, in larvae in non-parasitic lampreys but during late metamorphosis in parasitic lampreys. The appearance of stabilizing responses earlier than goal-oriented in the lamprey development shows a stepwise transition from simpler to more complex visual systems, offering a unique opportunity to isolate the functioning of their underlying circuits.

Effects of six diets on the growth and survival rates of postlarvae of red abalone (Haliotis rufescens) and its hybrid (H. rufescens ♀ × H. fulgens ♂)

One of the main bottlenecks in abalone aquaculture is maintaining individuals during the weaning stage, when the natural diet changes from diatoms (postlarvae) to macroalgae (juvenile). During this period, abalone pass through profound morphological and physiological changes, which suitable nutrient requirements must sustain. An inadequate diet can result in adverse effects such as late metamorphosis, starvation, slow growth and even death during this phase. Two strategies have been proposed to increase the growth and survival rates of weaning abalone: (i) extending feeding with benthic diatoms and (ii) abalone hybridization via interspecific crosses. To evaluate the efficiency of the two strategies, we assessed the growth and survival rates of postlarvae of pure red abalone (Haliotis rufescens) and a hybrid obtained by crossing red abalone females with green abalone (H. fulgens) males. Both crosses were supplied with six different diets consisting of either one macroalgae mono-diet (Macrocystis pyrifera or Ulva ohnoi) or a mixture with Navicula incerta. Overall, cross-specific diets achieved better growth rates, suggesting that each cross may need specific food items (nutrients) during weaning. Moreover, pure red abalone generally showed the highest growth rates, while the hybrid abalone showed the highest survival rates with most tested diets. Hence, hybrids appear to be better at withstanding stressful conditions, and their use in aquaculture could reduce losses and increase commercial production.

Reproductive cycle and gonadal output of the Lessepsian jellyfish Cassiopea andromeda in NW Sicily (Central Mediterranean Sea).

Knowledge of the reproductive strategy is a key prerequisite to predict population dynamics and potential invasiveness of both native and non-indigenous outbreak-forming species. In 2014 the Lessepsian upside-down jellyfish Cassiopea andromeda reached the harbor of Palermo (NW Sicily, Thyrrenian Sea), to date its established westernmost outpost in the Mediterranean Sea. To predict C. andromeda reproductive success in its novel habitat, gonad histology was carried out to record the number and size of mature and immature oocytes. Both male and female simultaneously presented gametes at all stages of development suggesting an asynchronous, yet apparently continuous, reproduction strategy. Indeed, oogenesis was observed throughout the year from pre-vitellogenic, vitellogenetic, and late-vitellogenetic to mature oocytes suggesting multiple reproductive events, as known in other Mediterranean Rhizostomeae. Oocytes were found from May to December, with two seasonal peaks of abundance (late spring = 392 and autumn = 272), suggesting imminent spawning events. Further, jellyfish size varied significantly throughout the year, with maximum diameter (up to 24 cm) in summer, and minimum diameter (6 cm) in winter. Small-sized jellyfish in winter belong to the new cohort, most probably arising from intense summer strobilation of polyps. Late spring fertilization, planula development, and metamorphosis, followed by polyp strobilation in the summer months, may explain the late appearance of a new jellyfish cohort, likely coincident with that recorded throughout winter.

Early development ofProchilodus nigricansSpix & Agassiz 1829 (Characiformes: Prochilodontidae) in captivity

The initial ontogeny of black prochilodus (Prochilodus nigricans Spix & Agassiz 1829) was described through morphological, meristic and morphometric characters. The biological material used was from captivity. 41 eggs, 151 larvae and 10 juveniles were analysed. The eggs were spherical, transparent, nonadhesive and pelagic, with a wide perivitelline space and an average diameter of 2.62 mm. Hatching occurs between 17 and 18 h after fertilization. The standard length of larvae varied from 3.28 to 26.73 mm and that of juveniles from 28.99 to 42.90 mm. The larvae showed an elongated and fusiform body, a convex dorsal profile, a long intestine, extending beyond the median region of the body, a terminal mouth and fleshy lips. The complete sequence of fin rays formation was caudal, dorsal (I,11), anal (I,11), pelvic (9) and pectoral (I,16–17). The number of myomeres ranged from 44 to 46, with mode 45 (n = 122). Growth analyses indicated early and late developmental metamorphosis. P. nigricans presents embryonic and larval development characteristic of fish with telolecithal eggs, in which the newly hatched larva does not yet have all the organic systems fully formed, is limited to a few organs and is still dependent on the yolk for its nutrition.

Tissue and salinity specific Na+/Cl\u2212 cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus)

Two orthologues of the gene encoding the Na+-Cl− cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl− cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

Neuroligin-1 Is a Mediator of Methylmercury Neuromuscular Toxicity.

Methylmercury (MeHg) is a developmental toxicant capable of eliciting neurocognitive and neuromuscular deficits in children with in utero exposure. Previous research in Drosophila melanogaster uncovered that developmental MeHg exposure simultaneously targets the developing musculature and innervating motor neuron in the embryo, along with identifying Drosophila neuroligin 1 (nlg1) as a gene associated with developmental MeHg sensitivity. Nlg1 and its transsynaptic partner neurexin 1 (Nrx1) are critical for axonal arborization and NMJ maturation. We investigated the effects of MeHg exposure on indirect flight muscle (IFM) morphogenesis, innervation, and function via flight assays and monitored the expression of NMJ-associated genes to characterize the role of Nlg1 mediating the neuromuscular toxicity of MeHg. Developmental MeHg exposure reduced the innervation of the IFMs, which corresponded with reduced flight ability. In addition, nlg1 expression was selectively reduced during early metamorphosis, whereas a subsequent increase was observed in other NMJ-associated genes, including nrx1, in late metamorphosis. Developmental MeHg exposure also resulted in persistent reduced expression of most nlg and nrx genes during the first 11 days of adulthood. Transgenic modulation of nlg1 and nrx1 revealed that developing muscle is particularly sensitive to nlg1 levels, especially during the 20-36-h window of metamorphosis with reduced nlg1 expression resulting in adult flight deficits. Muscle-specific overexpression of nlg1 partially rescued MeHg-induced deficits in eclosion and flight. We identified Nlg1 as a muscle-specific, NMJ structural component that can mediate MeHg neuromuscular toxicity resulting from early life exposure.

Effects of thiourea on the skull of Triturus newts during ontogeny.

BackgroundIn amphibians, thyroid hormone (TH) has a profound role in cranial development, especially in ossification of the late-appearing bones and remodeling of the skull. In the present study, we explored the influence of TH deficiency on bone ossification and resulting skull shape during the ontogeny of Triturus newt hybrid larvae obtained from interspecific crosses between T. ivanbureschi and T. macedonicus.MethodsLarvae were treated with two concentrations of thiourea (an endocrine disruptor that chemically inhibits synthesis of TH) during the midlarval and late larval periods. Morphological differences of the cranium were assessed at the end of the midlarval period (ontogenetic stage 62) and the metamorphic stage after treatment during the late larval period.ResultsThere was no difference in the ossification level and shape of the skull between the experimental groups (control and two treatment concentrations) at stage 62. During the late larval period and metamorphosis, TH deficit had a significant impact on the level of bone ossification and skull shape with no differences between the two treatment concentrations of thiourea. The most pronounced differences in bone development were: the palatopterygoid failed to disintegrate into the palatal and pterygoid portions, retardation was observed in development of the maxilla, nasal and prefrontal bones and larval organization of the vomer was retained in thiourea-treated larvae.ConclusionsThis implies that deficiency of TH caused retardation in development and arrested metamorphic cranium skeletal reorganization, which resulted in divergent cranial shape compared to the control group. Our results confirmed that skull remodeling and ossification of late-appearing bones is TH–dependent, as in other studied Urodela species. Also, our results indicate that TH plays an important role in the establishment of skull shape during the ontogeny of Triturus newts, especially during the late larval period and metamorphosis, when TH concentrations reach their maximum.

Transcriptomic analysis of sea star development through metamorphosis to the highly derived pentameral body plan with a focus on neural transcription factors.

The Echinodermata is characterized by a secondarily evolved pentameral body plan. While the evolutionary origin of this body plan has been the subject of debate, the molecular mechanisms underlying its development are poorly understood. We assembled a de novo developmental transcriptome from the embryo through metamorphosis in the sea star Parvulastra exigua. We use the asteroid model as it represents the basal-type echinoderm body architecture. Global variation in gene expression distinguished the gastrula profile and showed that metamorphic and juvenile stages were more similar to each other than to the pre-metamorphic stages, pointing to the marked changes that occur during metamorphosis. Differential expression and gene ontology (GO) analyses revealed dynamic changes in gene expression throughout development and the transition to pentamery. Many GO terms enriched during late metamorphosis were related to neurogenesis and signalling. Neural transcription factor genes exhibited clusters with distinct expression patterns. A suite of these genes was up-regulated during metamorphosis (e.g. Pax6, Eya, Hey, NeuroD, FoxD, Mbx, and Otp). In situ hybridization showed expression of neural genes in the CNS and sensory structures. Our results provide a foundation to understand the metamorphic transition in echinoderms and the genes involved in development and evolution of pentamery.

Functional characterization and osmoregulatory role of the Na+-K+-2Cl- cotransporter in the gill of sea lamprey (Petromyzon marinus), a basal vertebrate.

The present study provides molecular and functional characterization of Na+-K+-2Cl- cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey Na-K-Cl cotransporter 1 (NKCC1), which we show groups strongly with and occupies a basal position among other vertebrate NKCC1 sequences. In postmetamorphic juvenile lamprey, nkcc1 mRNA was present in many tissues but was fivefold higher in the gill than any other examined tissue, and NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+-K+-ATPase (NKA/Atp1a1) were significantly upregulated (20- to 200-fold) during late metamorphosis in fresh water, coinciding with the development of salinity tolerance, and were upregulated an additional twofold after acclimation to seawater (SW). Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes coexpressing NKA, which develop during metamorphosis in preparation for SW entry. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality as well as reduced muscle water content after 24 h in SW; there were no effects of bumetanide in freshwater-acclimated lamprey. This work provides the first functional characterization of NKCC1 as a mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.

The poverty of adult morphology: Bioacoustics, genetics, and internal tadpole morphology reveal a new species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia.

Ikakogi is a behaviorally and morphologically intriguing genus of glassfrog. Using tadpole morphology, vocalizations, and DNA, a new species is described from the Sierra Nevada de Santa Marta (SNSM), an isolated mountain range in northern Colombia. The new taxon is the second known species of the genus Ikakogi and is morphologically identical to I. tayrona (except for some larval characters) but differs by its genetic distance (14.8% in mitochondrial encoded cytochrome b MT-CYB; ca. 371 bp) and by the dominant frequency of its advertisement call (2928–3273 Hz in contrast to 2650–2870 Hz in I. tayrona). They also differ in the number of lateral buccal floor papillae, and the position of the buccal roof arena papillae. Additionally, the new species is differentiated from all other species of Centrolenidae by the following traits: tympanum visible, vomerine teeth absent, humeral spines present in adult males, bones in life white with pale green in epiphyses, minute punctuations present on green skin dorsum, and flanks with lateral row of small, enameled dots that extend from below eye to just posterior to arm insertion. We describe the external and internal larval morphology of the new species and we redescribe the larval morphology of Ikakogi tayrona on the basis of field collected specimens representing several stages of development from early to late metamorphosis. We discuss the relevance of larval morphology for the taxonomy and systematics of Ikakogi and other centrolenid genera. Finally, we document intraspecific larval variation in meristic characters and ontogenetic changes in eye size, coloration, and labial tooth-rows formulas, and compare tadpoles of related species. Ikakogi tayrona has been proposed as the sister taxon of all other Centrolenidae; our observations and new species description offers insights about the ancestral character-states of adults, egg clutches, and larval features in this lineage of frogs.

External Asymmetry and Pectoral Fin Loss in the Bamboo Sole (Heteromycteris japonica): Small-Sized Sole with Potential as a Pleuronectiformes Experimental Model.

Pleuronectiform fish develop marked external asymmetry in eye location and skin color at metamorphosis. The bamboo sole, Heteromycteris japonica, also exhibits loss of the pectoral fins at metamorphosis. Because of its small body size, short generation time, and long spawning season, we focused on the bamboo sole as an experimental model to investigate metamorphic asymmetry and pectoral fin loss during development. In the present study, we utilized a small-scale culture system to evaluate bamboo sole larvae and larval development, and a microinjection system for fertilized eggs. The culture system described here uses an 18 L culture tank for rotifers (the first diet for larvae) and 5 L plastic beakers for larval culture. Under this system, most larvae completed metamorphosis, including one-eye migration and pigmentation of the ocular side, by 23 days postfertilization (dpf) at 25°C. Larvae at density of 120-150 per liter were grown from hatching to 23 dpf with a survival ratio of 60-75% per beaker. Pectoral fins, including coracoid and disk cartilage, formed but were completely lost in late metamorphosis without formation of proximal radials and fin rays. The microinjection system designed here is adequate for the bamboo sole and allows injection of 100 one-cell-stage embryos per day. We expect that the culture and microinjection systems described here will facilitate the use of the bamboo sole as an experimental model organism in developmental biology.

Effects of Echinostoma trivolvis metacercariae infection during development and metamorphosis of the wood frog (Lithobates sylvaticus)

Many organisms face energetic trade-offs between defense against parasites and other host processes that may determine overall consequences of infection. These trade-offs may be particularly evident during unfavorable environmental conditions or energetically demanding life history stages. Amphibian metamorphosis, an ecologically important developmental period, is associated with drastic morphological and physiological changes and substantial energetic costs. Effects of the trematode parasite Echinostoma trivolvis have been documented during early amphibian development, but effects during later development and metamorphosis are largely unknown. Using a laboratory experiment, we examined the energetic costs of late development and metamorphosis coupled with E. trivolvis infection in wood frogs, Lithobates [=Rana] sylvaticus. Echinostoma infection intensity did not differ between tadpoles examined prior to and after completing metamorphosis, suggesting that metacercariae were retained through metamorphosis. Infection with E. trivolvis contributed to a slower growth rate and longer development period prior to the initiation of metamorphosis. In contrast, E. trivolvis infection did not affect energy expenditure during late development or metamorphosis. Possible explanations for these results include the presence of parasites not interfering with pronephros degradation during metamorphosis or the mesonephros compensating for any parasite damage. Overall, the energetic costs of metamorphosis for wood frogs were comparable to other species with similar life history traits, but differed from a species with a much shorter duration of metamorphic climax. Our findings contribute to understanding the possible role of energetic trade-offs between parasite defense and host processes by considering parasite infection with simultaneous energetic demands during a sensitive period of development.

A model of muscle atrophy based on live microscopy of muscle remodelling inDrosophilametamorphosis

Genes controlling muscle size and survival play important roles in muscle wasting diseases. In Drosophila melanogaster metamorphosis, larval abdominal muscles undergo two developmental fates. While a doomed population is eliminated by cell death, another persistent group is remodelled and survives into adulthood. To identify and characterize genes involved in the development of remodelled muscles, we devised a workflow consisting of in vivo imaging, targeted gene perturbation and quantitative image analysis. We show that inhibition of TOR signalling and activation of autophagy promote developmental muscle atrophy in early, while TOR and yorkie activation are required for muscle growth in late pupation. We discovered changes in the localization of myonuclei during remodelling that involve anti-polar migration leading to central clustering followed by polar migration resulting in localization along the midline. We demonstrate that the Cathepsin L orthologue Cp1 is required for myonuclear clustering in mid, while autophagy contributes to central positioning of nuclei in late metamorphosis. In conclusion, studying muscle remodelling in metamorphosis can provide new insights into the cell biology of muscle wasting.

Hoxgenes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum

The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, we have compared these processes in the flour beetle Tribolium castaneum, which develops ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. On the other hand, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, we did not obtain homeotic transformations of embryonic appendages in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and metamorphosis of the hemimetabolous life cycle.

Hylid Tadpoles from the Caribbean Island of Hispaniola: Ontogeny, Description and Comparison of External Morphology

Abstract The island of Hispaniola (Dominican Republic and Haiti) is home to four endemic species of treefrogs: Hypsiboas heilprini, Osteopilus dominicensis, O. pulchrilineatus and O. vastus. These species occur sympatrically over large areas below 1,850 m elevation and are commonly encountered together. We redescribe the external larval morphology of O. dominicensis, O. vastus, and Hybsiboas heilprini, and describe the tadpole of O. pulchrilineatus for the first time based on laboratory-reared specimens representing stages from early—late metamorphosis. Some tadpoles were raised to adults in order to confirm species identity. We document intraspecific variation in labial tooth-rows formulas, report ontogenetic changes in coloration, compare tadpoles of related species, and provide an identification key to the hylid tadpoles of Hispaniola. Hispaniolan hylid tadpoles inhabit fast-running streams or pools. Whereas the lotic larvae of O. vastus and H. heilprini exhibit characteristics commonly associated with...

Insight on the development of neuroventilatory GABA‐mediated inhibition in bullfrog tadpoles

GABA is a main inhibitory neurotransmitter in the adult vertebrate CNS. In mammals, activation of GABA‐A receptors transitions from depolarization to hyperpolarization early in development; GABA acting via GABA‐A receptors matures from an excitatory to an inhibitory neurotransmitter. In bullfrogs this transition was previously associated with late metamorphosis. To identify precisely the timing of this development, we quantified neuroventilatory responses to GABA (0.1 mM) in tadpoles across all metamorphic stages. Neuroventilatory responses to GABA were evaluated via whole‐nerve recordings from isolated brainstems. Throughout development GABA elicited a marked neuroventilatory depression. The results were confirmed at higher GABA concentrations (0.25 & 0.5 mM). Our data suggest that GABAergic inhibition develops earlier than previously reported for bullfrogs. Alternatively, the GABA‐A receptor‐mediated excitation associated with early development could be masked by signaling through GABA‐B receptors, which are consistently inhibitory and play a greater role in GABAergic signaling in early tadpole development. To resolve this issue, studies using receptor‐specific agonists are underway.

Stratification and synaptogenesis in the mushroom body of the honeybee, Apis mellifera

Stratification is a basic anatomical feature of central brain in both vertebrates and many invertebrates. The aim of this study was to investigate the relationship between stratification and synaptogenesis in the developing mushroom bodies of the honeybee. During metamorphosis, the vertical lobe of mushroom body shows progressive stratification with three thick primary strata and more secondary strata and laminae. Three primary strata are formed at the metamorphic stage P1, before the youngest generation of the mushroom body intrinsic neurons, Kenyon cells, is produced. Thus, the primary strata within the lobe are unlikely to represent three major subpopulations of the Kenyon cells sequentially produced in the mushroom bodies. Formation of laminae starts at the stage P2 and culminates at the end of metamorphosis. The laminae appear within the lobe rather than being added sequentially from the ingrowth stratum. Alternating dark and light lamina (lamina doublets) are formed in the vertical lobe in late metamorphosis (stages P6-P9), but they are not visible in adults. The pattern of stratification is not continuous along the vertical lobe at the same developmental stage, and resorting of axons of the Kenyon cells is likely to occur within dark laminae. In the developing vertical lobe, dark laminae show lower synaptic density and exhibit an ultra structure that is indicative for a delay in synaptogenesis relative to the primary strata. A local transient block of synaptogenesis within the dark laminae may provide correct targeting of Kenyon cells by extrinsic mushroom body neurons.

Widespread transcriptional changes pre‐empt the critical pelagic–benthic transition in the vetigastropod Haliotis asinina

Larval settlement is a vital transition period for marine invertebrates and can have far-reaching effects on the ecology and evolution of a species. To explore the molecular mechanisms of this critical process in a nonmodel organism, the abalone Haliotis asinina, we employed cDNA microarrays. By comparing gene expression profiles through mid- to late larval development and metamorphosis, we identified 144 genes as candidates for a role in competence and/or metamorphosis. Gene characterization indicates ~60% of these are significantly similar to known genes from other taxa, while ~40% are novel. A high 49.3% of genes are gastropod or abalone specific, but none appears to be Lophotrochozoan specific, even though metamorphosis is thought to have had a separate origin in this group. Differentially expressed larval and postlarval genes can be clustered into five categories that reveal strikingly different temporal transcriptional patterns occurring during this phase of development. Some gene activation is contingent upon exogenous cues and correlates with initiation of settlement. Importantly, there is also extensive gene activity associated with the endogenous attainment of competence, which occurs before, and independent of, the exogenous induction of settlement. Our results show that as the haliotid veliger larva matures, it requires coordinated regulation of temporally different batteries of genes involved in a wide range of physiological and developmental processes associated with benthic colonization. Although the signalling pathways operating at metamorphosis may be conserved across the animal kingdom, it appears they regulate the expression of novel genes specific to abalone, gastropods and molluscs during H. asinina metamorphosis.

Ontogenetic differentiation of swimming performance in Gilthead seabream ( Sparus aurata, Linnaeus 1758) during metamorphosis

The critical swimming speed (U crit) of gilthead seabream ( Sparus aurata, Linnaeus 1875) was studied in two ontogenetic phases, early (13.7-18.7 mm total length, TL) and late metamorphosis (20.4-34.3 mm TL, after the full development of fin meristics and during squamation ontogeny), under four exercise temperatures (15, 20, 25 and 28 °C). Both the exercise temperature and the ontogenetic stage had a significant effect on the relative U crit (RU crit) of S. aurata, with the fish of early metamorphosis phase (E group) presenting significantly higher RU crit than those of the late metamorphosis stage (L group). This ontogenetic shift in swimming performance was accompanied by significant ontogenetic shifts of body shape and of muscle anatomy. Compared to the L group, S. aurata of the E group were characterized by a streamline body shape and significantly higher relative contribution of the slow-red muscle to the cross-sectional area of the body (31.0 ± 1.3% vs 12.0 ± 1.2% in the L group).