Oviducal gland transcriptomics of Octopus maya through physiological stages and the negative effects of temperature on fertilization.

Background: Elevated temperatures reduce fertilization and egg-laying rates in the octopus species. However, the molecular mechanisms that control the onset of fertilization and egg-laying in the octopus' oviducal gland are still unclear; and the effect of temperature on the expression of key reproductive genes is unknown. This study aims to better understand the molecular bases of octopus fertilization and egg-laying, and how they are affected by elevated temperatures. Method: RNA-seq of oviducal glands was performed for samples before, during, and after fertilization and their transcriptomic profiles were compared. Also, at the fertilization stage, the optimal and thermal-stress conditions were contrasted. Expression levels of key reproductive genes were validated via RT-qPCR. Results: In mated females before egg-laying, genes required for the synthesis of spermine, spermidine, which may prevent premature fertilization, and the myomodulin neuropeptide were upregulated. Among the genes with higher expression at the fertilization stage, we found those encoding the receptors of serotonin, dopamine, and progesterone; genes involved in the assembly and motility of the sperm flagellum; genes that participate in the interaction between male and female gametes; and genes associated with the synthesis of eggshell mucoproteins. At temperatures above the optimal range for reproduction, mated females reduced the fertilization rate. This response coincided with the upregulation of myomodulin and APGW-amide neuropeptides. Also, genes associated with fertilization like LGALS3, VWC2, and Pcsk1 were downregulated at elevated temperatures. Similarly, in senescent females, genes involved in fertilization were downregulated but those involved in the metabolism of steroid hormones like SRD5A1 were highly expressed.

Sex-specific role of the optic gland in octopus maya: A transcriptomic analysis.

The optic glands (OG) of cephalopods are a source of molecules associated with the control of reproductive traits and lifecycle events such as sexual maturation, reproductive behavior, feeding, parental care, and senescence. However, little is known about the role of the optic gland in Octopus maya adults during mating and egg laying. RNA sequencing, de novo transcriptome assembly, ubiquity and differential expression analysis were performed. First, we analyzed the expression patterns of transcripts commonly associated with OG regulatory functions to describe their possible role once the maturation of the gonad is complete. The transcriptomic profiles of the optic gland of both sexes were compared with emphasis on the signaling pathways involved in the dimorphism of reproductive traits. Results suggest that in the OG of males, the reproductive condition (mated or non-mated) did not affect the general expression profile. In contrast, more differentially expressed genes were observed in females. In mated females, the mRNA metabolic process and the response to norepinephrine were enriched, suggesting a high cellular activity in preparation for the laying of the embryos. Whereas in egg-laying females, energetic and metabolic processes were the most represented, including the oxidation-reduction process. Finally, the gene expression patterns in senescence females suggest a physiological response to starvation as well as upregulation of genes involved retrotransposon activity. In conclusion, more substantial fluctuations in gene expression were observed in the optic glands of the fertilized females compared to the males. Such differences might be associated with the regulation of the egg-laying and the onset of senescence.

Distribution of pathogenic vibrios and Vibrio spp. in the water column and sediment samples from the southern Gulf of Mexico.

This study quantified the distribution of Vibrio spp. by qPCR and pathogenic vibrio species by metagenomics, during 2 oceanographic cruises-XIXIMI-04 and XIXIMI-05 -in the southern Gulf of Mexico (GoMex). A total of 708 samples from various levels of the water column and 22 sediment samples were analyzed, according to a designed net of sampling lines. Sampling was focused on reported water masses with distinctive characteristics, to detect the presence-absence of vibrios. The results indicated that the genus Vibrio was detected along the entire water column and in sediments. Pathogenic vibrios, such as V, campbellii, V. parahaemolyticus, V. vulnificus or V. cholerae were also detected in the water column and in sediments, in both oceanographic cruises. Thus, the ecological conditions of the GoMex permit the growth of Vibrio spp. in deep water environments of the GoMex, despite continuous oil input from natural and anthropogenic sources.

Effects of the recombinant crustacean hyperglycemic hormones rCHH-B1 and rCHH-B2 on the osmo-ionic regulation of the shrimp Litopenaeus vannamei exposed to acute salinity stress.

The Pacific white shrimp Litopenaeus vannamei is a euryhaline organism that copes with salinity fluctuations in the environment; therefore, its osmotic and ionic regulation abilities are vital. Osmoregulation may be controlled by the crustacean hyperglycemic hormone (CHH), a neuropeptide mainly expressed in the eyestalks. In L. vannamei, CHH-B1 and CHH-B2 are CHH isoforms isolated from the eyestalks whose expression is influenced by environmental salinity. It has been suggested that they are involved in the response to salinity stress. To clarify this, we investigated the effect of the recombinant peptides, rCHH-B1 and rCHH-B2, on the osmo-ionic regulation of shrimp acutely exposed to different salinity conditions (8, 26 and 45‰). Both rCHHs promoted differential effects on the osmoregulatory capacity (OC) and the ionoregulatory capacity (IC) for hemolymph Na+ and Cl- during iso-osmotic (26‰) and hyper-osmotic (45‰) transfers. These changes were linked to the changes observed in Na+/K+ ATPase and carbonic anhydrase gene expression in gills, especially under high salinity conditions, suggesting that the hormones may regulate the expression of these genes. Glucose and protein levels measured during acute salinity transfer suggest their roles as sources of metabolic energy for osmotic regulation or as organic osmolytes. These results taken together suggest that both the CHH-B1 and CHH-B2 peptides are important regulators of the physiological response of L. vannamei to acute salinity fluctuations.

Cloning and expression of the recombinant crustacean hyperglycemic hormone isoform B2 (rCHH-B2) and its effects on the metabolism and osmoregulation of the Pacific white shrimp Litopenaeus vannamei.

Crustacean hyperglycemic hormones (CHHs) are multifunctional neuropeptides ubiquitous in crustaceans. In Litopenaeus vannamei, CHH-B2 is a CHH eyestalk isoform whose expression has been shown to vary with enviromental conditions, suggesting its relevance for ecophysiological performance of shrimp, controlling processes related to metabolism and osmo-ionic regulation. To study the involvement of CHH-B2 in these processes, we cloned and expressed a recombinant version with a free C-terminal glycine (rCHH-B2-Gly) in the methylotrophic yeast Pichia pastoris. The rCHH-B2-Gly peptide secreted to the culture medium was purified by RP-HPLC and used for in vivo glucose, triglyceride, and osmoregulation dose-response analyses with juvenile shrimp. The peptide was also amidated at the C-terminus using an α-amidating enzyme to produce rCHH-B2-amide. The shrimp showed a dose-dependent effect of rCHH-B2-Gly to hemolymph glucose and triglyceride levels, inducing maximal increases by injecting 500 and 1000pmol of hormone, respectively. Additionally, 10pmol of hormone was sufficient to reduce the hypo-osmoregulatory capacity of shrimp at 35‰. These findings suggest that CHH-B2 has regulatory roles in carbohydrate and lipid metabolism, and a potential involvement in osmoregulation of L. vannamei. Injection of 100pmol of rCHH-B2-amide increased glucose and triglyceride levels by 15 and 28%, respectively in comparison with rCHH-B2-Gly, suggesting an important role for the C-terminal amidation. Additionally, an in silico structural analysis done with the CHH-B1 and rCHH-B2-Gly peptides suggests that the C-terminal region may be relevant for the activity of the L. vannamei isoforms and explain the functional divergence from other crustacean CHH/CHH-like peptides.

Effect of silver nanoparticles on the metabolic rate, hematological response, and survival of juvenile white shrimp Litopenaeus vannamei.

White spot syndrome virus (WSSV) is highly lethal and contagious in shrimps; its outbreaks causes an economic crisis for aquaculture. Several attempts have been made to treat this disease; however, to date, there is no effective cure. Because of their antimicrobial activities, silver nanoparticles (AgNPs) are the most studied nanomaterial. Although the antiviral properties of AgNPs have been studied, their antiviral effect against viral infection in aquaculture has not been reported. The AgNPs tested herein are coated with polyvinylpyrrolidone (PVP) and possess multiple international certifications for their use in veterinary and human applications. The aim of this work was to evaluate the survival rate of juvenile white shrimps (Litopenaeus vannamei) after the intramuscular administration of AgNPs. For this, different concentrations of metallic AgNPs and PVP alone were injected into the organisms. After 96 h of administration, shrimp survival was more than 90% for all treatments. The oxygen consumption routine rate and total hemocyte count remained unaltered after AgNP injection, reflecting no stress caused. We evaluated whether AgNPs had an antiviral effect in shrimps infected with WSSV. The results revealed that the survival rate of WSSV-infected shrimps after AgNP administration was 80%, whereas the survival rate of untreated organisms was only 10% 96 h after infection. These results open up the possibility to explore the potential use of AgNPs as antiviral agents for the treatment of diseases in aquaculture organisms, particularly the WSSV in shrimp culture.

Hyperglycemic activity of the recombinant crustacean hyperglycemic hormone B1 isoform (CHH-B1) of the Pacific white shrimp Litopenaeus vannamei.

Crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide produced by the X-organ/sinus gland (XO/SG) complex in the crustacean eyestalk. CHH plays a principal role in the control of glucose metabolism. The CHH-B1 isoform is produced in the eyestalk of Litopenaeus vannamei by alternative splicing of the chhB gene and its cDNA sequence has revealed that this isoform has a non-amidated C-terminal residue (CHH-like peptide). In this work, a recombinant CHH-B1 (rCHH-B1) with a sequence identical to the native hormone was expressed in the methylotrophic yeast Pichia pastoris X-33 and purified from the culture medium by RP-HPLC. The identity of the purified rCHH-B1 was confirmed by N-terminal sequencing and by using an anti-CHH-B1 polyclonal antibody. An in vivo assay showed that the hyperglycemic effect was dependant of the dosage of rCHH-B1, and the maximal hyperglycemic response was obtained with 250pmol treatment. These results suggest that the amino acid sequence of the C-terminus and its correct structure are both important for the hyperglycemic activity of naturally occurring non-amidated CHH peptides, such as CHH-B1. CHH-B1 appears to be the first reported CHH-like peptide with significant hyperglycemic activity produced in the sinus gland of a penaeid shrimp.

Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock.

BACKGROUND: In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab')2 fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy. Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured. RESULTS: Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab')2 antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose100 (LD100) LPS administration. TNF blockade with either VNAR domains or F(ab')2 fragments were associated with lower mortality (60% and 75%, respectively) compared to LD100. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS. In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab')2 fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group. CONCLUSIONS: Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.

Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris

Crustacean hyperglycemic hormone (CHH) is the most abundant and best studied member of the CHH/MIH/GIH neuropeptide hormone family. CHH plays a major role in controlling glucose levels in the hemolymph, and it also has significance in regulating molting, reproduction, and osmoregulation. In contrast, molt-inhibiting hormone (MIH) is responsible for maintaining animals in an intermolt stage. In this study, Liv-MIH-1 cDNA, which encodes a mature neuropeptide from the eyestalk of white shrimp, Litopenaeus vannamei, was expressed in methylotrophic yeast (Pichia pastoris KM71) under the control of an alcohol oxidase promoter. Recombinant Liv-MIH-1 was secreted into the culture medium using the á-factor prepro-sequence without Glu-Ala repeats. The expected protein, which had an apparent molecular mass of 12.1 kDa, was detected by Tricine-SDS-PAGE analysis and confirmed by Western blot. Pure recombinant Liv-MIH-1 was obtained by affinity chromatography, and N-terminal sequence analysis confirmed expression of the protein. Biological assays for CHH and MIH activity were also performed. Purified recombinant Liv-MIH-1 showed the ability to elevate the glucose level of hemolymph of L. vannamei, but molting was unaffected. Since these results are in agreement with the high structural and phylogenetic similarity that has been observed between Liv-MIH-1 and other CHH neuropeptides we propose to rename the protein Liv-CHH-SG1.