Comparative bioinformatic analysis and biochemical characterization of digestive proteinases from Pacific whiting (Merluccius productus).

The hake fishery plays a crucial role due to its significant economic impact. The genus Merluccius includes 12 extant species found along the coasts of the Americas, Europe, and Africa. However, research on their digestive physiology and the enzymes involved in digestion, including proteases, remains limited. Proteases play a key role in protein digestion, a vital process for all living organisms. This study focused on screening the genomes of eight Merluccius spp. for eight specific proteases previously identified in Merluccius polli. Additionally, the study conducted biochemical analyses of proteases found in the stomach and intestine of Pacific whiting fish (Merluccius productus), comparing the results with the genomic findings. The analysis revealed that proteases across Merluccius spp. are conserved, although with slight variations, particularly in chymotrypsin and aspartic proteases. Biochemical characterization of M. productus identified at least three main proteases in the stomach, active at acidic pH, and at least seven proteases in the intestine, active at alkaline pH, as determined by electrophoresis. Further investigation, including specific inhibition studies, determination of molecular mass, and assessment of pH and temperature preferences for catalysis, revealed that one of the stomach proteases functioning at acidic pH likely belongs to the acid peptidase class, likely pepsin. Similarly, analysis of proteases active at alkaline pH indicated the presence of a chymotrypsin and a trypsin, consistent with genomic findings in M. productus. These results are important as they provide insights into the digestive physiology of Merluccius spp., contributing to a better understanding of their nutritional needs.

Antimicrobial and anticarcinogenic activity of bioactive peptides derived from abalone viscera (Haliotis fulgens and Haliotis corrugata).

Bioactive peptides have been studied in several sources due to their valuable potential in the pharmaceutical and food industries. Abalone viscera, which are normally discarded as byproducts, are a rich source of protein. Thus, the aim of this study was to explore the potential bioactivity of peptides derived from abalone viscera (Haliotis fulgens and Haliotis corrugata) after hydrolysis with a commercial mixture of enzymes. The hydrolysates obtained were fractionated using gel filtration chromatography. The resulting hydrolysate fractions were investigated for their antimicrobial and cytotoxic activities, including the expression of gelatinases mmp-2 and mmp-9 in human prostate cancer cell lines (PC3). Results showed antimicrobial activity for protein fractions of H. corrugata against Proteus mirabilis and Pseudomona aeuroginosa (66.2-116.25 kDa), Bacillus subtilis (6.5-21.5 kDa), and Aspergillus niger (97.4-116.25 kDa), while H. fulgens peptide fractions (200-31 kDa) displayed activity against six bacterial strains, and fractions from 116.25 to 21.5 kDa had effects on the fungus A. niger, Alternaria alternata, and Aspergillus flavus. Additionally, protein fractions displayed cytotoxic activity, inhibiting 30.4-53.8% of PC3 cellular growth. Selected fractions decreased the PMA-induced and not-induced expressions of mmp-2 and mmp-9 in PC3 cells. Abalone viscera, as byproducts, can be used as a potential source of antimicrobial and anticancer peptides.

An acidic protein, Hf15, from Haliotis fulgens involved in biomineralization.

Biomineralization leads to the hardening of mineralized materials, such as the shell of Mollusk, to fulfill a wide range of functions, such as (but not limited to) skeletal support, protection of the soft tissues, navigation, etc. The study of the proteins responsible for this process, shell matrix proteins (SMPs), allows addressing questions related to structure-function relationship and to the mechanism of mineral formation, which is limited in gastropod species. In this study, a low molecular weight protein was isolated from the insoluble fraction after decalcification with acetic acid of the shell of Haliotis fulgens and, named Hf15. The unglycosylated protein has a theoretical molecular weight of 15 kDa, it possesses calcium and chiting binding properties. Hf15 can precipitate calcium carbonate in vitro in presence of different salts. Analysis by LC-MS of the five peptide sequences of Hf15 generated by trypsinization revealed that two peptides displayed homology to an uncharacterized protein 3-like from Haliotis rufescens, Haliotis asinia and H. sorenseni. The results obtained indicated that Hf15 is a novel SMP involved in shell mineralization in Haliotis fulgens.

Review: Post-translational modifications of marine shell matrix proteins.

Shell matrix proteins (SMPs) are key components for the Mollusk shell biomineralization. SMPs function has been hypothesized in several proteins by bioinformatics analysis, and through in vitro crystallization assays. However, studies of the post-translational modifications (PTMs) of SMPs, which contribute to their structure and the function, are limited. This review provides the current status of the SMPs with the most common PTMs described (glycosylation, phosphorylation, and disulfide bond formation) and their role in shell biomineralization. Also, recent studies based on recombinant production of SMPs are discussed. Finally, recommendations for the study of SMPs and their PTMs are provided. The review showed that PTMs are widely distributed in SMPs, and their presence on SMPs may contribute to the modulation of their activity in some SMPs, contributing to the crystal growth formation and differentiation through different mechanisms, however, in a few cases the lack of the PTMs do not alter their inherent function.

A shell matrix protein of Pinctada mazatlanica produces nacre platelets in vitro.

Nacre is the main component of the pearl oyster shells and it is synthesized by specialized soluble and insoluble shell matrix proteins. Insoluble proteins from the decalcification of the shell are the less studied proteins due to the technical problems to isolate them from the organic matrix. In this study, an insoluble shell matrix protein from Pinctada mazatlanica, pearlin (Pmaz-pearlin), was successfully cloned from the mantle tissue, and the native protein isolated from the shell was functionally characterized. The full coding sequence of Pmaz-pearlin mRNA consists of 423 base pairs, which encode to a 16.3 kDa pearlin. Analysis of the deduced amino acid sequence revealed that Pmaz-pearlin contained four acidic regions, an NG repeat domain, and Cys conserved residues, the latter potentially forms four disulfide bridges which might stabilize the protein structure. The isolated protein from the shell is a glycoprotein of ~ 16.74 kDa which can produce aragonite and calcite crystals in vitro. Our results show that Pmaz-pearlin is a well-conserved protein involved in nacre layer growth, which produces calcite crystals in the presence of CaCl2, aragonite crystal polymorphs with a hexagonal structure in the presence of MgCl2, and needle-like crystal structure polymorphs in the presence of CaCO3 The identity of the crystals was confirmed using RAMAN analyses.

Biochemical characterization and in vitro biological activities of the epithelial cell extracts from Hypanus dipterurus spine.

Ocean organisms live in competitive environments that demand the production of poisons and toxins. In some cases, these substances have been used in the pharmaceutical industry for human disease treatments. Most fish poisons generally have potent cytolytic activity, probably through cardiovascular and neuromuscular effects. In the case of marine stingrays, the injuries made by their tail venom apparatus are caused by the mechanical penetration of their sting and a subsequent venom release. This study focused on the evaluation of substances with cytotoxic activity in the epithelium that covers the venom apparatus from the marine stingray Hypanus dipterurus. To demonstrate the above, the hemolytic, proteolytic and cytotoxic capacities of H. dipterurus epithelium substances were determined. Discs impregnated with epithelial extract were used on blood agar plates. The proteolytic activity was analyzed using casein as substrate and for gelatin the liquefaction activity test. To determine the cytotoxicity degree of the extracts, the proliferation and cell viability MTT bioassay was implemented on human cervical carcinoma cells (HeLa). The results showed that no hemolytic or proteolytic activity existed against casein associated with the epithelial extract, but gelatin hydrolysis and cytotoxic activity against the HeLa cell line were observed. This study concludes that the substances found in the epithelium covering the H. dipterurus stingray venom apparatus are a mixture of various proteins, among which, glycosylated anionic proteins represent a potential source of molecules with cytotoxic and hydrolytic activity.

Epibiont hydroids on beachcast Sargassum in the Mexican Caribbean.

Massive accumulations of pelagic species of Sargassum have generated recent social, economic and ecological problems along Caribbean shores. In the Mexican Caribbean, these events have prompted the study of diverse biological and ecological aspects of these macroalgae. However, studies on their associated biota, including Hydrozoa, remain scarce. This research provides important species observations in an area where data is lacking. The occurrence and percent cover of hydroids on Sargassum thalli collected on the beach at Puerto Morelos, Quintana Roo, Mexico from April 2018 to March 2019 was studied. Three pelagic species and morphotypes of Sargassum from this area were analyzed: Sargassum fluitans III, S. natans I and S. natans VIII, as well as a benthic species, S. polyceratium var. ovatum. A total of 14 taxa of hydroids, belonging to the superorders "Anthoathecata" and Leptothecata, were identified. In our study, more hydroid taxa were observed on axes of the different species of Sargassum than on leaves or aerocysts. In general, the greatest species richness of hydroids was observed from February to April. Results show that live hydrozoans attached to pelagic Sargassum are transported into the area. This should be considered in future management measures that address the recurring coastal abundance of Sargassum and its associated biota in the Caribbean region.

Comparative and functional analysis of desaturase FADS1 (∆5) and FADS2 (∆6) orthologues of marine organisms.

Fatty acid desaturases are key enzymes involved in unsaturated fatty acid biosynthesis, which insert double bonds at specific positions of fatty acids, playing a pivotal role in unsaturated fatty acid synthesis required for membrane lipid fluidity. The ∆5 and ∆6 desaturases are responsible for producing long chain-polyunsaturated fatty acids (LC-PUFA) through their precursors α-linolenic acid and linoleic acid in organisms lacking or with very low ability to synthesize LC-PUFA by themselves. Extensive studies of fatty acid desaturases are available in model organisms, such as humans and mouse; however, the diversity of these genes in the marine biodiversity is less known. This study performed an exhaustive analysis to identify the ∆5 and ∆6 desaturases in the available marine genomes in databases, as well as transcriptomes and EST databases, and their coding sequences were compared to the well-characterized ∆5 and ∆6 desaturases from humans. The FADS1 and FADS2 genetic structures are well conserved among all the organisms analyzed. A common amino acid pattern was identified to discriminate between ∆5 and ∆6 desaturases. The analysis of the conserved motif involved in catalysis showed that 20% of the desaturases, ∆5 and ∆6, have lost motifs required for catalysis. Additionally, bifunctional ∆5/∆6 desaturases were able to be identified by amino acid sequence patterns found in previously described enzymes. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these marine organism enzymes.

Ps19, a novel chitin binding protein from Pteria sterna capable to mineralize aragonite plates in vitro.

Mollusk shell is composed of two CaCO3 polymorphs (calcite and aragonite) and an organic matrix that consists of acetic acid- or ethylenediaminetetraacetic acid (EDTA)-soluble and insoluble proteins and other biomolecules (polysaccharides, ß-chitin). However, the shell matrix proteins involved in nacre formation are not fully known. Thus, the aim of this study was to identify and characterize a novel protein from the acetic acid-insoluble fraction from the shell of Pteria sterna, named in this study as Ps19, to have a better understanding of the biomineralization process. Ps19 biochemical characterization showed that it is a glycoprotein that exhibits calcium- and chitin-binding capabilities. Additionally, it is capable of inducing aragonite plate crystallization in vitro. Ps19 partial peptide sequence showed similarity with other known shell matrix proteins, but it displayed similarity with proteins from Crassostrea gigas, Mizuhopecten yessoensis, Biomphalaria glabrata, Alpysia californica, Lottia gigantea and Elysia chlorotica. The results obtained indicated that Ps19 might play an important role in nacre growth of mollusk shells.

Biochemical and molecular characterization of N66 from the shell of Pinctada mazatlanica.

Mollusk shell mineralization is a tightly controlled process made by shell matrix proteins (SMPs). However, the study of SMPs has been limited to a few model species. In this study, the N66 mRNA of the pearl oyster Pinctada mazatlanica was cloned and functionally characterized. The full sequence of the N66 mRNA comprises 1,766 base pairs, and encodes one N66 protein. A sequence analysis revealed that N66 contained two carbonic anhydrase (CA) domains, a NG domain and several glycosylation sites. The sequence showed similarity to the CA VII but also with its homolog protein nacrein. The native N66 protein was isolated from the shell and identified by mass spectrometry, the peptide sequence matched to the nucleotide sequence obtained. Native N66 is a glycoprotein with a molecular mass of 60-66 kDa which displays CA activity and calcium carbonate precipitation ability in presence of different salts. Also, a recombinant form of N66 was produced in Escherichia coli, and functionally characterized. The recombinant N66 displayed higher CA activity and crystallization capability than the native N66, suggesting that the lack of posttranslational modifications in the recombinant N66 might modulate its activity.

Purification and functional analysis of the shell matrix protein N66 from the shell of the pearl oyster Pteria sterna.

Mollusk biomineralization is a process controlled by a complex interplay of proteins, ions and external regulators. In spite of several studies, there is a lack of knowledge of who (molecules involved), how (mechanism) and why (evolution and adaptation) mollusk are designed as we know them. In this study, a shell matrix protein, N66, has been purified and characterized biochemically from the shell of Pteria sterna. Two protein bands with carbohydrates associated were separated with a molecular weight of ~60 and 64 kDa. It has carbonic anhydrase activity and it is able to form crystal polymorphs of calcium carbonate in vitro. The mRNA N66 was obtained from the mantle tissue of Pteria sterna and the deduced amino acid sequence contained a carbonic anhydrase (CA) domain and a Asn/Gly-rich domain (aa243-439). The CA domain contained three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu166-Thr525), being thus similar to the human isoform hCAVII. Also, to test whether the posttranslational modifications present on the native N66 affects the CA activity and its crystallization capability in vitro, a recombinant N66 was overexpressed in Escherichia coli and functionally characterized. Our results show that recombinant N66 has higher CA activity and produce larger size crystals in vitro than the native N66 protein, suggesting that intrinsic properties of the native N66, such as glycosylations and/or phosphorylations, might regulate its activity.

Ecdysis triggering hormone receptors regulate male courtship behavior via antennal lobe interneurons in Drosophila.

Ecdysis triggering hormone receptors (ETHR) regulate the behavioral sequence necessary for cuticle shedding. Recent reports have documented functions for ETHR signaling in adult Drosophila melanogaster. In this study, we report that ETHR silencing in local interneurons of the antennal lobes and fruitless neurons leads to sharply increased rates of male-male courtship. RNAseq analysis of ETHR knockdown flies reveals differential expression of genes involved in axon guidance, courtship behavior and chemosensory functions. Our findings indicate an important role for ETHR in regulation of Drosophila courtship behavior through chemosensory processing in the antennal lobe.

Omics approaches to study juvenile hormone synthesis.

The juvenile hormones (JHs) are a family of insect acyclic sesquiterpenoids produced by the corpora allata (CA), a pair of endocrine glands connected to the brain. They are involved in the regulation of development, reproduction, behavior, caste determination, diapause, stress response, and numerous polyphenisms. In the post-genomics era, comprehensive analyses using functional 'omics' technologies such as transcriptomics, proteomics and metabolomics have increased our understanding of the activity of the minute CA. This review attempts to summarize some of the 'omics' studies that have contributed to further understand JH synthesis in insects, with an emphasis on our own research on the mosquito Aedes aegypti.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes.

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

How micronutrients influence the physiology of mosquitoes.

Micronutrients or non-energetic nutrients (NEN) are needed in reduced amounts, but are essential for many mosquito physiological processes that influence biological traits from vector competence to reproductive capacity. The NEN include amino acids (AA), vitamins, salts, metals and sterols. Free AA plays critical roles controlling most physiological processes, from digestion to reproduction. Particularly proline connects metabolic pathways in energy production, flight physiology and ammonia detoxification. Metal, in particular iron and calcium, salts, sterol and vitamin homeostasis are critical for cell signaling, respiration, metabolism and reproduction. Micronutrient homeostasis influence the symbiotic relationships with microorganisms, having important implications in mosquitoes' nutrition, physiology and behavior, as well as in mosquito immunity and vector competence.

Hormonal Signaling Cascade during an Early-Adult Critical Period Required for Courtship Memory Retention in Drosophila.

Formation and expression of memories are critical for context-dependent decision making. In Drosophila, a courting male rejected by a mated female subsequently courts less avidly when paired with a virgin female, a behavioral modification attributed to "courtship memory." Here we show the critical role of hormonal state for maintenance of courtship memory. Ecdysis-triggering hormone (ETH) is essential for courtship memory through regulation of juvenile hormone (JH) levels in adult males. Reduction of JH levels via silencing of ETH signaling genes impairs short-term courtship memory, a phenotype rescuable by the JH analog methoprene. JH-deficit-induced memory impairment involves rapid decay rather than failure of memory acquisition. A critical period governs memory performance during the first 3 days of adulthood. Using sex-peptide-expressing "pseudo-mated" trainers, we find that robust courtship memory elicited in the absence of aversive chemical mating cues also is dependent on ETH-JH signaling. Finally, we find that JH acts through dopaminergic neurons and conclude that an ETH-JH-dopamine signaling cascade is required during a critical period for promotion of social-context-dependent memory.

Endocrine network essential for reproductive success in Drosophila melanogaster.

Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei.

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Modeling the flux of metabolites in the juvenile hormone biosynthesis pathway using generalized additive models and ordinary differential equations.

Juvenile hormone (JH) regulates development and reproductive maturation in insects. The corpora allata (CA) from female adult mosquitoes synthesize fluctuating levels of JH, which have been linked to the ovarian development and are influenced by nutritional signals. The rate of JH biosynthesis is controlled by the rate of flux of isoprenoids in the pathway, which is the outcome of a complex interplay of changes in precursor pools and enzyme levels. A comprehensive study of the changes in enzymatic activities and precursor pool sizes have been previously reported for the mosquito Aedes aegypti JH biosynthesis pathway. In the present studies, we used two different quantitative approaches to describe and predict how changes in the individual metabolic reactions in the pathway affect JH synthesis. First, we constructed generalized additive models (GAMs) that described the association between changes in specific metabolite concentrations with changes in enzymatic activities and substrate concentrations. Changes in substrate concentrations explained 50% or more of the model deviances in 7 of the 13 metabolic steps analyzed. Addition of information on enzymatic activities almost always improved the fitness of GAMs built solely based on substrate concentrations. GAMs were validated using experimental data that were not included when the model was built. In addition, a system of ordinary differential equations (ODE) was developed to describe the instantaneous changes in metabolites as a function of the levels of enzymatic catalytic activities. The results demonstrated the ability of the models to predict changes in the flux of metabolites in the JH pathway, and can be used in the future to design and validate experimental manipulations of JH synthesis.

Correction: Methyl Farnesoate Plays a Dual Role in Regulating Drosophila Metamorphosis.

[This corrects the article DOI: 10.1371/journal.pgen.1005038.].