Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in our Ca2+ imaging experiments, relatively low doses of OA caused increased Ca2+ levels in PTTH neurons, whereas higher doses led to decreased Ca2+ levels. We also demonstrated that a low dose of OA was conveyed through OA β-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.

Larval metamorphosis is inhibited by methimazole and propylthiouracil that reveals possible hormonal action in the mussel Mytilus coruscus

Larval metamorphosis in bivalves is a key event for the larva-to-juvenile transformation. Previously we have identified a thyroid hormone receptor (TR) gene that is crucial for larvae to acquire “competence” for the metamorphic transition in the mussel Mytilus courscus (Mc). The mechanisms of thyroid signaling in bivalves are still largely unknown. In the present study, we molecularly characterized the full-length of two iodothyronine deiodinase genes (McDx and McDy). Phylogenetic analysis revealed that deiodinases of molluscs (McDy, CgDx and CgDy) and vertebrates (D2 and D3) shared a node representing an immediate common ancestor, which resembled vertebrates D1 and might suggest that McDy acquired specialized function from vertebrates D1. Anti-thyroid compounds, methimazole (MMI) and propylthiouracil (PTU), were used to investigate their effects on larval metamorphosis and juvenile development in M. coruscus. Both MMI and PTU significantly reduced larval metamorphosis in response to the metamorphosis inducer epinephrine. MMI led to shell growth retardation in a concentration-dependent manner in juveniles of M. coruscus after 4 weeks of exposure, whereas PTU had no effect on juvenile growth. It is hypothesized that exposure to MMI and PTU reduced the ability of pediveliger larvae for the metamorphic transition to respond to the inducer. The effect of MMI and PTU on larval metamorphosis and development is most likely through a hormonal signal in the mussel M. coruscus, with the implications for exploring the origins and evolution of metamorphosis.

A Vital Staining Practice That Discerns Ancestry within Groups of Settling Larvae of a Brooding Coral

Xenogeneic and allogeneic encounters following aggregated and clustered settlements of coral larvae (planulae) may carry important ecological consequences in shaping coral reefs’ communities. However, larval settlement behaviors and settlement location choices in the presence of conspecifics or heterospecifics have not been examined in detail, due to a lack of experimental tools. One potential approach is the employment of vital staining of planulae with dyes that do not impair larval metamorphosis processes, are stable for prolonged periods, and do not diffuse to un-labeled counterpart planulae. For these purposes, we examined the use of neutral red (NR) dye, as an identification marker, on the planulae of Stylophora pistillata, a Red Sea branching coral species. To examine possible NR impacts on larval settlement in the presence of conspecific planulae, we followed the settlement ratios of kin, non-kin, and mixed assemblages, as a proxy for metamorphosis success. We found no differences in settlement rates of stained vs. unstained larvae, lack of stain diffusion to other larvae and that NR stain is maintained for more than a week under a still water regimen. Thus, staining with NR may serve as a useful experimental tool, opening new opportunities in studying larval settlement patterns in sessile marine organisms.

Effects of Symbiodiniaceae Phylotypes in Clades A–E on Progeny Performance of Two Giant Clams (Tridacna squamosa and T. crocea) During Early History Life Stages in the South China Sea

Unlike most bivalves, giant clams (tridacnids) harbor symbiotic microalgae (zooxanthellae) in their other fleshy bodies. The effects of mixed populations of zooxanthellae on larval metamorphosis has been reported in several papers, but there have been very few studies on the effects of single zooxanthella species on the establishment of symbiosis in giant clams. In this study, we obtained five pure zooxanthella species (clades A3, B1, C1, D1, E1) from antler coral by molecular identification, and analyzed their effects on the larval metamorphosis and progeny performance of two giant clams, Tridacna squamosa and T. crocea, in the South China Sea. Clam larvae with all five zooxanthella species underwent larval settlement and metamorphosis, and formed the zooxanthellal tubular system. There was some variation in metamorphic rate and time to metamorphosis between clams with different zooxanthella species, but no significant differences in size at metamorphosis. After metamorphosis, larvae with all zooxanthella types continued to develop normally. Mantle color was consistent within clam species and zooxanthella species had no effect on mantle color. However, clam progeny with clade E1 zooxanthellae were smaller than progeny with the other four zooxanthella clades (A3, B1, C1, and D1). Survival rate was over 90% for all progeny and there were no significant differences in survival between progeny with Symbiodinium clades A–E during the entire culture process. Two-way ANOVA analysis revealed that giant clam species was the main factor influencing progeny growth, with some variation in growth attributable to zooxanthella type. Our results provide new information on both the symbiotic relationship between giant clams and zooxanthellae and the mantle coloration of giant clams, and will be useful in giant clam seed production and aquaculture.

First evidence of virus-like particles in the bacterial symbionts of Bryozoa

Bacteriophage communities associated with humans and vertebrate animals have been extensively studied, but the data on phages living in invertebrates remain scarce. In fact, they have never been reported for most animal phyla. Our ultrastructural study showed for the first time a variety of virus-like particles (VLPs) and supposed virus-related structures inside symbiotic bacteria in two marine species from the phylum Bryozoa, the cheilostomes Bugula neritina and Paralicornia sinuosa. We also documented the effect of VLPs on bacterial hosts: we explain different bacterial ‘ultrastructural types’ detected in bryozoan tissues as stages in the gradual destruction of prokaryotic cells caused by viral multiplication during the lytic cycle. We speculate that viruses destroying bacteria regulate symbiont numbers in the bryozoan hosts, a phenomenon known in some insects. We develop two hypotheses explaining exo- and endogenous circulation of the viruses during the life-cycle of B. neritina. Finally, we compare unusual ‘sea-urchin’-like structures found in the collapsed bacteria in P. sinuosa with so-called metamorphosis associated contractile structures (MACs) formed in the cells of the marine bacterium Pseudoalteromonas luteoviolacea which are known to trigger larval metamorphosis in a polychaete worm.

Proteome of larval metamorphosis induced by epinephrine in the Fujian oyster Crassostrea angulata

Background The Fujian oyster Crassostrea angulata is an economically important species that has typical settlement and metamorphosis stages. The development of the oyster involves complex morphological and physiological changes, the molecular mechanisms of which are as yet unclear. Results In this study, changes in proteins were investigated during larval settlement and metamorphosis of Crassostrea angulata using epinephrine induction. Protein abundance and identity were characterized using label-free quantitative proteomics, tandem mass spectrometry (MS/ MS), and Mascot methods. The results showed that more than 50% (764 out of 1471) of the quantified proteins were characterized as differentially expressed. Notably, more than two-thirds of the differentially expressed proteins were down-regulated in epinephrine-induced larvae. The results showed that “metabolic process” was closely related to the development of settlement and metamorphosis; 5 × 10− 4 M epinephrine induced direct metamorphosis of larvae and was non-toxic. Calmodulin and MAPK pathways were involved in the regulation of settlement of the oyster. Expression levels of immune-related proteins increased during metamorphosis. Hepatic lectin-like proteins, cadherins, calmodulin, calreticulin, and cytoskeletal proteins were involved in metamorphosis. The nervous system may be remodeled in larval metamorphosis induced by epinephrine. Expression levels of proteins that were enriched in the epinephrine signaling pathway may reflect the developmental stage of the larvae, that may reflect whether or not larvae were directly involved in metamorphosis when the larvae were treated with epinephrine. Conclusion The study provides insight into proteins that function in energy metabolism, immune responses, settlement and metamorphosis, and shell formation in C. angulata. The results contribute valuable information for further research on larval settlement and metamorphosis. Graphical abstract