New bacilli-based probiotic for aquaculture: efficacy study on Macrobrachium rosenbergii

The data on the study of the effectiveness of a new probiotic based on strains B. toyonensis B-13249 and B. pumilus B-13250 when growing shrimps are presented. The experiments were carried out on a private farm in Kazakhstan. The control group of shrimps, which did not receive the probiotic, completed the molting process on the 28th day of the experiment, and the experimental group – 10 days earlier. It was also found that the probiotic has a positive effect on the hydrochemistry of water in recirculating aquaculture system (RAS). The concentration of ammonium (NH3/NH4 +), nitrite (NO2) and nitrate (NO3-) nitrogen in the experimental group did not reach the limit value, in contrast to the control. Based on the obtained results, this probiotic can be recommended for greening aquaculture. The dosage of the probiotic when applied with brine shrimp as feed for shrimp is 1 g/100 g of dry cysts. The dosage of the probiotic when the RAS is added to the water is 5 g/600 l of circulating water once a week.

An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a novel role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a negative regulator of molting. Whereas loss of adm-2 led to strong suppression of molting defects in partial loss-of-function nekl mutants, overexpression of ADM-2 induced molting defects in wild-type animals. CRISPR genome editing implicated the Zn-binding motif within the metalloprotease domain as critical for mediating the effects of ADM-2 on molting. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We also identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of LRP-1, ADM-2 overexpression caused a reduction in LRP-1 abundance, suggesting that ADM-2 may function as a sheddase for LRP-1. We propose that loss of adm-2 suppresses molting defects in nekl mutants by eliminating a negative regulator of LRP-1, thereby compensating for defects in the efficiency of LRP-1 and sterol uptake. Our findings emphasize the importance of endocytic trafficking for both the internalization of factors that promote molting and the removal of proteins that would otherwise be deleterious to the molting process.

Understanding molt control switches: Transcriptomic and expression analysis of the genes involved in ecdysteroidogenesis and cholesterol uptake pathways in the Y-organ of the blue crab, Callinectes sapidus

The crustacean molting process is regulated by an interplay of hormones produced by the eyestalk ganglia and Y-organs (YO). Molt-inhibiting hormone and crustacean hyperglycemic hormone released by the sinus gland of the eyestalk ganglia (EG) inhibit the synthesis and secretion of ecdysteroid by the YO, hence regulating hemolymph levels during the molt cycle. The purpose of this study is to investigate the ecdysteroidogenesis pathway, specifically genes linked to changes in ecdysteroid levels occurring at early premolt (ePM). To this end, a reference transcriptome based on YO, EG, and hepatopancreas was de novo assembled. Two genes (cholesterol 7-desaturase Neverland and cytochrome p450 307a1-like Spook) involved in ecdysteroidogenesis were identified from the YO transcriptome using sequence comparisons and transcript abundance. Two other candidates, Hormone receptor 4 and probable cytochrome p450 49a1 potentially involved in ecdysteroidogenesis were also identified. Since cholesterol is the ecdysteroid precursor, a putative cholesterol carrier (Apolipoprotein D-like) was also examined to understand if cholesterol uptake coincided with the increase in the ecdysteroid levels at the ePM stage. The expression level changes of the five candidate genes in the YO were compared between intermolt (IM) and induced ePM (iePM) stages using transcriptomic analysis. Expression analysis using qPCR were carried out at IM, iePM, and normal ePM. The increase in Spook and Neverland expression in the YO at the ePM was accompanied by a concomitant rise in ecdysteroid levels. The data obtained from iePM stage were congruent with those obtained from the normal ePM stage of intact control animals. The present findings support the role of Halloween genes in the ecdysteroidogenesis and molt cycle in the blue crab, Callinectes sapidus.

A Zinc Metalloprotease nas-33 Is Required for Molting and Survival in Parasitic Nematode Haemonchus contortus

Molting is of great importance for the survival and development of nematodes. Nematode astacins (NAS), a large family of zinc metalloproteases, have been proposed as novel anthelmintic targets due to their multiple roles in biological processes of parasitic nematodes. In this study, we report a well conserved nas-33 gene in nematodes of clade V and elucidate how this gene is involved in the molting process of the free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. A predominant transcription of nas-33 is detected in the larval stages of these worms, particularly in the molting process. Knockdown of this gene results in marked molecular changes of genes involved in cuticle synthesis and ecdysis, compromised shedding of the old cuticle, and reduced worm viability in H. contortus. The crucial role of nas-33 in molting is closely associated with a G protein beta subunit (GPB-1). Suppression of both nas-33 and gpb-1 blocks shedding of the old cuticle, compromises the connection between the cuticle and hypodermis, and leads to an increased number of sick and dead worms, indicating essentiality of this module in nematode development and survival. These findings reveal the functional role of nas-33 in nematode molting process and identify astacins as novel anthelmintic targets for parasitic nematodes of socioeconomic significance.

Genome-Wide Characterization and Identification of Long Non-Coding RNAs during the Molting Process of a Spider Mite, Panonychus citri

Molting is essential for arthropods to grow. As one of the important arthropod pests in agriculture, key spider mite species (Tetranychus and Panonychus) can normally molt three times from the larva to adult stage within a week. This physiological strategy results in the short lifecycle of spider mites and difficulties in their control in the field. Long non-coding RNAs (lncRNAs) regulate transcriptional editing, cellular function, and biological processes. Thus, analysis of the lncRNAs in the spider mite molting process may provide new insights into their roles in the molting mechanism. For this purpose, we used high-throughput RNA-seq to examine the expression dynamics of lncRNAs and mRNAs in the molting process of different development stages in Panonychus citri. We identified 9199 lncRNAs from 18 transcriptomes. Analysis of the lncRNAs suggested that they were shorter and had fewer exons and transcripts than mRNAs. Among these, 356 lncRNAs were differentially expressed during three molting processes: late larva to early protonymph, late protonymph to early deutonymph, and late deutonymph to early adult. A time series profile analysis of differentially expressed lncRNAs showed that 77 lncRNAs were clustered into two dynamic expression profiles (Pattern a and Pattern c), implying that lncRNAs were involved in the molting process of spider mites. Furthermore, the lncRNA–mRNA co-expression networks showed that several differentially expressed hub lncRNAs were predicted to be functionally associated with typical molting-related proteins, such as cuticle protein and chitin biosynthesis. These data reveal the potential regulatory function of lncRNAs in the molting process and provide datasets for further analysis of lncRNAs and mRNAs in spider mites.

Comparative Analysis of the Intermolt and Postmolt Hepatopancreas Transcriptomes Provides Insight into the Mechanisms of Procambarus clarkii Molting Process

In the present study, we used RNA-Seq to investigate the expression changes in the transcriptomes of two molting stages (postmolt (M) and intermolt (NM)) of the red swamp crayfish and identified differentially expressed genes. The transcriptomes of the two molting stages were de novo assembled into 139,100 unigenes with a mean length of 675.59 bp. The results were searched against the NCBI, NR, KEGG, Swissprot, and KOG databases, to annotate gene descriptions, associate them with gene ontology terms, and assign them to pathways. Furthermore, using the DESeq R package, differentially expressed genes were evaluated. The analysis revealed that 2347 genes were significantly (p > 0.05) differentially expressed in the two molting stages. Several genes and other factors involved in several molecular events critical for the molting process, such as energy requirements, hormonal regulation, immune response, and exoskeleton formation were identified and evaluated by correlation and KEGG analysis. The expression profiles of transcripts detected via RNA-Seq were validated by real-time PCR assay of eight genes. The information presented here provides a transient view of the hepatopancreas transcripts available in the postmolt and intermolt stage of crayfish, hormonal regulation, immune response, and skeletal-related activities during the postmolt stage and the intermolt stage.

Insights into the L3 to L4 developmental program through proteomics

The establishment of infection with the lymphatic dwelling filarial parasites is dependent on the infectivity and subsequent development of the infective larvae (L3) within the human host to later stages (L4, adults) that require several developmental molts. The molecular mechanisms underlying the developmental processes in parasitic nematodes are not clearly defined. We report the proteomic profiles throughout the entire L3 to L4 molt using an established in vitro molting process for the human pathogen B. malayi. A total of 3466 proteins of B. malayi and 54 from Wolbachia were detected at one or more time points. Based on the proteomic profiling, the L3 to L4 molting proteome can be broadly divided into an early, middle and late phase. Enrichment of proteins, protein families and functional categories between each time point or between phases primarily relate to energy metabolism, immune evasion through secreted proteins, protein modification, and extracellular matrix-related processes involved in the development of new cuticle. Comparative analyses with somatic proteomes and transcriptomes highlighted the differential usage of cysteine proteinases (CPLs), BmCPL-1, -4 and -5 in the L3-L4 molt compared to the adults and microfilariae. Inhibition of the CPLs effectively blocked the in-vitro L3 to L4 molt. Overall, only 4 Wolbachia proteins (Wbm0495, Wbm0793, Wbm0635, and Wbm0786) were detected across all time points and suggest that they play an inconsequential role in the early developmental process