Immature trematodes of Lecithochirium sp. (Digenea: Hemiuridae) in the California two-spot octopus (Octopus bimaculatus) from Mexico.

Immature trematodes of Lecithochirium sp. are recorded for the first time as parasites of the California two-spot octopus Octopus bimaculatus from Bahía de los Ángeles, Baja California, Mexico. Thirty-nine O. bimaculatus were examined for trematodes and a total of 100 immature specimens of Lecithochirium sp. were recorded from the crop of seven infected octopuses. Based on these records, O. bimaculatus may act as a second intermediate or paratenic host for these parasites. Partial sequences of the 28S (region D1-D3) ribosomal gene corroborate the identifications based on morphological characters. DNA sequences of the 28S gene from GenBank were analyzed to include the immature samples of Lecithochirium sp. within a hemiurid phylogenetic framework. All immature specimens of Lecithochirium sp. were recovered as monophyletic and Pulmovermis cyanovitellosus was identified as the sister species of Lecithochirium sp. However, due to the lack of molecular data for species of the genus Lecithochirium, these phylogenetic inferences must be taken with caution. Therefore, the morphological and molecular data obtained here provide a foundation for future work to develop a systematic comparison among- and within-species of the genus Lecithochirium. Additionally, the present records of Lecithochirium in O. bimaculus add to the knowledge of the parasite fauna of cephalopods.

Two new species of dicyemid mesozoans (Dicyemida: Dicyemidae) from Octopus maya Voss & Solis-Ramirez (Octopodidae) off Yucatan, Mexico.

Two new dicyemid species are described from the endemic cephalopod Octopus maya Voss & Solis-Ramirez collected off Yucatan, Mexico. The renal sacs of 40 juvenile and adult octopuses from four localities were examined. Dicyema hochbergi n. sp. is a medium-sized species that reaches 2,245 µm in length. The vermiform stages consist of 18-24 peripheral cells, a conical calotte and the extension of the axial cell between the base and middle of the metapolar cells. Infusoriform embryos consist of 39 cells with urn cell containing one germinal cell, two nuclei and solid refringent bodies. Dicyema mexcayae n. sp. is a relatively small species that reaches 1,114 µm in length. The vermiform stages are constituted by 14-16 peripheral cells, an elongate calotte and the axial cell extending forward to the middle of the metapolar cells. The infusoriform embryos consist of 37 cells, two solid refringent bodies and urn cells with two nuclei each. The present study represents the first description of a dicyemid species from O. maya and increases the number of described species from Mexican waters to 11.

Clearance rates of sand-burrowed and laterally pressed unburrowed Pismo clam Tivela stultorum (Mawe 1823) in a laboratory open-flow system.

Pismo clam extraction is currently banned in Mexico to help the recovery of natural populations. Thus, the primary objective of this study was to gain insight on its basic biology and husbandry protocols. Growth and clearance rate (CR) of sand-burrowed and sediment-free, laterally pressed adult Pismo clams were quantified in the laboratory as a function of burrowing condition, flow, temperature, and microalgal concentration using open-flow chambers. After 40 days, clams remained healthy regardless of burrowing condition and showed a hyperbolic CR response pattern to increased flow, with CR directly proportional to flows lower than 1000 ml min-1. Maximal asymptotic CR values (300 to 400 ml min-1 org-1) were observed from 1000 to 2000 ml min-1. No significant CR differences were observed between burrowed and laterally pressed clams, yet microalgal concentration effects were detected, with constant maximal CRs of ∼250 ml min-1 in the range of 50 to 200 cells µl-1 and decline at higher concentrations. Maintenance protocols of laterally pressed organisms were validated in the laboratory with both weight and CR data. To our knowledge, this is the first study providing whole-body physiological data translated into effective husbandry protocols for Pismo clams. This approach represents a fresh perspective to traditional research areas, opening the possibility for continued experimentation under controlled conditions.

Linking phenotypic to genotypic metacestodes from Octopus maya of the Yucatan Peninsula.

Octopus maya is an endemic species of the Yucatan Peninsula. This species sustains the octopus' fishery in the region and is the only cephalopod cultured in Mexico. It is known that O. maya harbor a large richness and abundance of metacestodes that have been tentatively identified by light microscopy alone. Since the larval stages of some orders of marine cestodes lack the taxonomic characteristics shown by the adult stages and on which cestode taxonomy is based, identification down to the species level is often unattainable. Hence, the goal of this study was to characterize the parasites, for the first time, at morphological and molecular levels. A total of 60 octopuses were collected from September to December 2017 from four fishery landing ports in Yucatán: Sisal, Progreso, Dzilam de Bravo, and Rio Lagartos (15 hosts per locality). Morphology of metacestodes was characterized by light and Scanning Electron Microscopy (SEM), while the genes 18S and 28S rDNA were sequenced for molecular characterization. Based on phenotypic characters and molecular data, seven taxa of metacestodes were identified, four of them belonging of order Trypanorhyncha: Eutetrarhynchus sp., Kotorella pronosoma, Nybelinia sp., Prochristianella sp. 1; and the three remaining taxa belonging to the order Onchoproteocephalidea: Acanthobothrium sp., Phoreiobothrium sp., and Prosobothrium sp. This work provides, for the first time, molecular support to the morphological characterization of metacestodes recorded in Octopus maya.

Aggregata polibraxiona n. sp. (Apicomplexa: Aggregatidae) from Octopus bimaculatus Verrill, 1883 (Mollusca: Cephalopoda) from the Gulf of California, Mexico.

The Apicomplexa Aggregata spp. are intracellular parasites of cephalopods that infect the intestinal tract of commercially important species such as Octopus bimaculatus, which sustains the octopus fishery in Baja California (B.C.), Mexico. In this study, Aggregata polibraxiona n. sp. was described from the cecum of O. bimaculatus collected from Bahia de Los Angeles, B. C. Light and electron microscopy revealed that oocysts and sporocysts were spherical to ovoid in shape. Sporulated oocysts (293-835 × 177-688 µm) contained 135-674 sporocysts (12-24 × 11-22 µm). The sporocyst wall was covered by tubular projections (0.55-2.19 µm in length) bifurcated in the top, unevenly distributed, covered by a thin membrane. Each sporocyst contains 11-13 sporozoites (16-26 × 1.20-3 µm). Three partial sequences of the 18S rDNA gene were obtained, and two phylogenetic approaches were performed according to Bayesian inference and Maximum Likelihood. In both phylogenetic reconstructions, the sequences of A. polibraxiona n. sp. were recovered as a monophyletic group within the genus Aggregata and placed as a sister group to Aggregata octopiana Lineage II. Aggregata polibraxiona n. sp. is the first Apicomplexa described from a cephalopod host from Mexico and extends the geographical range of Apicomplexa infecting cephalopods.

Morphological and Molecular Characterization of Aggregata spp. Frenzel 1885 (Apicomplexa: Aggregatidae) in Octopus vulgaris Cuvier 1797 (Mollusca: Cephalopoda) from Central Mediterranean.

Coccidian parasites of the genus Aggregata are known to parasitize cephalopods as definitive hosts, however one of the genus members, A. octopiana, has shown an unresolved phylogeny within the same definitive host, the common octopus (Octopus vulgaris). Our study represents a large-scale investigation aimed at characterizing morphological traits and phylogeny of A. octopiana isolated from O. vulgaris inhabiting three distinct geographic areas of the central Mediterranean: The Adriatic, Ionian and Tyrrhenian Seas. The morphology of sporogonic stages of the parasite in octopus tissues was assessed by light and electron microscopy; molecular characterization has been carried out using the 18S rRNA locus. Our results support the hypothesis that two morphologically and genetically different A. octopiana infect O. vulgaris in the investigated areas of the Mediterranean Sea. Additional nuclear and mitochondrial markers for Aggregata should provide further information and better resolution of its phylogeny.

Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana. BIOLOGICAL SIGNIFICANCE: The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of the octopus immune defense against a parasite infection. Particularly, it is centered in the host-parasite relationship developed between the octopus and the protozoan A. octopiana, which induces severe gastrointestinal injuries in octopus that produce a malabsorption syndrome. The common octopus is a commercially important species with a high potential for aquaculture in semi-open systems, and this pathology reduces the condition of the octopus populations on-growing in open-water systems resulting in important economical loses. This is the first proteomic approach developed on this host-parasite relationship, and therefore, the contribution of this work goes from i) ecological, since this particular relationship is tending to be established as a model of host-parasite interaction in natural populations; ii) evolutionary, due to the characterization of immune molecules that could contribute to understand the functioning of the immune defense in these highly evolved mollusks; and iii) to economical view. The results of this study provide an overview of the octopus hemolymph proteome. Furthermore, proteins influenced by the level of infection and implicated in the octopus cellular response are also showed. Consequently, a set of biomarkers for disease resistance is suggested for further research that could be valuable for the improvement of the octopus culture, taken into account their high economical value, the declining of landings and the need for the diversification of reared species in order to ensure the growth of the aquaculture activity. Although cephalopods are model species for biomedical studies and possess potential in aquaculture, their genomes have not been sequenced yet, which limits the application of genomic data to research important biological processes. Similarly, the octopus proteome, like other non-model organisms, is poorly represented in public databases. Most of the proteins were identified from an octopus' hemocyte RNA-seq database that we have performed, which will be the object of another manuscript in preparation. Therefore, the need to increase molecular data from non-model organisms is herein highlighted. Particularly, here is encouraged to expand the knowledge of the genomic of cephalopods in order to increase successful protein identifications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

The role of DNA methylation on Octopus vulgaris development and their perspectives.

DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquaculture.

De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

BACKGROUND: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. RESULTS: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. CONCLUSION: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against pathogens, which could improve octopus farming in the near future.

Molecular phylogenetic analysis of the coccidian cephalopod parasites Aggregata octopiana and Aggregata eberthi (Apicomplexa: Aggregatidae) from the NE Atlantic coast using 18S rRNA sequences.

The coccidia genus Aggregata is responsible for intestinal coccidiosis in wild and cultivated cephalopods. Two coccidia species, Aggregata octopiana, (infecting the common octopus Octopus vulgaris), and A. eberthi, (infecting the cuttlefish Sepia officinalis), are identified in European waters. Extensive investigation of their morphology resulted in a redescription of A. octopiana in octopuses from the NE Atlantic Coast (NW Spain) thus clarifying confusing descriptions recorded in the past. The present study sequenced the 18S rRNA gene in A. octopiana and A. eberthi from the NE Atlantic coast in order to assess their taxonomic and phylogenetic status. Phylogenetic analyses revealed conspecific genetic differences (2.5%) in 18S rRNA sequences between A. eberthi from the Ria of Vigo (NW Spain) and the Adriatic Sea. Larger congeneric differences (15.9%) were observed between A. octopiana samples from the same two areas, which suggest the existence of two species. Based on previous morphological evidence, host specificity data, and new molecular phylogenetic analyses, we suggest that A. octopiana from the Ria of Vigo is the valid type species.

A new dicyemid from Octopus hubbsorum (Mollusca:Cephalopoda:Octopoda).

A new species of dicyemid mesozoan is described from Octopus hubbsorum Berry, 1953, collected in the south of Bahia de La Paz, Baja California Sur, México. Dicyema guaycurense n. sp. is a medium-size species that reaches about 1,600 µm in length. It occurs in folds of the renal appendages. The vermiform stages are characterized as having 22 peripheral cells, a conical calotte, and an axial cell that extends to the base of the propolar cells. Infusoriform embryos consist of 39 cells; 1 nucleus is present in each urn cell and the refringent bodies are solid. This is the first of a dicyemid species from a host collected in the Gulf of California.