Background: 20-Hydroxyecdysone (20E) is the most ubiquitous ecdysteroid (Ecd) and plays critical roles during the life cycle of arthropods. To elucidate the metabolism pathway of 20E in the economically important species, Scylla paramamosain, we conducted a comprehensive exploration of the genes involved in the 20E metabolism pathway. Methods: A comprehensive exploration of genes involved in the 20E metabolism pathway was conducted, including gene annotation, local blast using the Drosophila ortholog as query, and TreeFam ortholog genes identification. Bioinformatics and expression profiling of the identified genes were performed to assess their roles in the 20E metabolism of green mud crabs. Results: This experiment indicated that, except for CYP306a1 and CYP314a1, all other ortholog genes involved in the Drosophila 20E metabolism can be found in the mud crab, suggesting that the function of these two genes might be replaced by other CYP genes or the “active” Ecd in mud crabs was not the 20E. All genes had the typical features of each gene family, clustered with the specific clade in the phylogenetic trees. In addition, all the identified genes had the highest expression level in the Y-organ, and sex-biased gene expression was observed in these genes. Conclusions: This study provided some valuable insights into the metabolism and diversity of ecdysteroids in crustaceans.

The conserved role of juvenile hormone (JH) signals in preventing larvae from precocious metamorphosis has been confirmed in insects. Crustaceans have different metamorphosis types from insects; we previously proved that methyl farnesoate (MF) can prohibit larvae metamorphosis in mud crabs, but the molecular signal of this process still needs to be elucidated. In this study, methoprene-tolerant (Met) of Scylla paramamosain was obtained and characterized, which we named Sp-Met. Sp-Met contains a 3360 bp ORF that encodes 1119 amino acids; the predicted protein sequences of Sp-Met include one bHLH, two PAS domains, one PAC domain, and several long unusual Gln repeats at the C-terminal. AlphaFold2 was used to predict the 3D structure of Sp-Met and the JH binding domain of Met. Furthermore, the binding properties between Sp-Met and MF were analyzed using CD-DOCK2, revealing a putative high affinity between the receptor and ligand. In silico site-directed mutagenesis suggested that insect Mets may have evolved to exhibit a higher affinity for both MF or JH III compared to the Mets of crustaceans. In addition, we found that the expression of Sp-Met was significantly higher in female reproductive tissues than in males but lower in most of the other examined tissues. During larval development, the expression variation in Sp-Met and Sp-Kr-h1 was consistent with the immersion effect of MF. The most interesting finding is that knockdown of Sp-Met blocked the inhibitory effect of MF on metamorphosis in the fifth zoea stage and induced pre-metamorphosis phenotypes in the fourth zoea stage. The knockdown of Sp-Met significantly reduced the expression of Sp-Kr-h1 and two ecdysone signaling genes, Sp-EcR and Sp-E93. However, only the reduction in Sp-Kr-h1 could be rescued by MF treatment. In summary, this study provides the first evidence that MF inhibits crustacean larval metamorphosis through Met and that the MF-Met→Kr-h1 signal pathway is conserved in mud crabs. Additionally, the crosstalk between MF and ecdysteroid signaling may have evolved differently in mud crabs compared to insects.

Background: Snipe eels (family Nemichthyidae) are a group of pelagic fishes with unique specializations; yet, species within this study are not well-studied due to a lack of molecular data. As typical mesopelagic-to-bathypelagic fishes, snipe eels exhibit extreme body elongation, reduced skeletal ossification, and highly specialized beak-like jaws that facilitate survival in deep-sea midwater environments. Methods: The complete mitochondrial genome of the deep-sea eel Nemichthys curvirostris (Anguilliformes: Nemichthyidae) was sequenced and annotated, representing the first mitogenomic resource for this species. The phylogenetic position of N. curvirostris was also explored. Results: The circular genome of N. curvirostris was determined to be 16,911 bp in length and contained 37 genes, including 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a single control region, with an overall A + T bias of 56.67%. The maximum-likelihood phylogeny inferred from concatenated mitochondrial protein-coding genes recovered a well-supported monophyletic Nemichthys clade, with N. curvirostris positioned as the sister taxon to N. scolopaceus. The genera Avocettina and Labichthys were recovered as sister taxa, and Nemichthys clustered within a broader clade alongside them. The COX1 haplotype phylogeny showed that the two public database sequences (HQ563894.1 and MN123435.1) appeared as long, isolated branches outside the main N. curvirostris lineage, with COX1 genetic distances from typical N. curvirostris haplotypes reaching 12–13%, far exceeding the expected range of intraspecific variation. Conclusions: This mitogenome provides a valuable molecular resource for phylogenetic, evolutionary, and population genetic studies of deep-sea Anguilliformes.