Vulgaris Paralarvae Research Articles

Trophic ecology of Octopus vulgaris paralarvae along the Iberian Canary current eastern boundary upwelling system

Our knowledge of the diet of wild octopus paralarvae, Octopus vulgaris, is restricted to the first 2 weeks of its planktonic phase when they are selective hunters found near the coastline. These small paralarvae, bearing only three suckers per arm, are transported by oceanic currents from the coast towards offshore waters, where they complete the planktonic phase over 2 months. Here, we have investigated the trophic ecology of O. vulgaris paralarvae in two contrasting upwelling sub-regions of the Iberian Canary current (ICC) eastern boundary upwelling system and have evaluated dietary change as paralarvae develop (inferred by counting the number of suckers per arm, ranging from three to 15) along the coastal-oceanic gradient during their planktonic phase. Using high-throughput amplicon sequencing, we have characterised the diet of 100 paralarvae collected along the Northwest Iberian Peninsula (n = 65, three to five suckers per arm) and off the west coast of Morocco (n = 35, three to 15 suckers per arm), identifying up to 87 different prey species. The diet of paralarvae varied along the ICC, with crabs (53.4%), siphonophores (12.2%), copepods (12.3%), cnidarians (8.4%) and pteropods (3.7%) accounting for 90% of the variability detected off NW Iberian Peninsula, whereas off W Morocco, crabs (46.2%), copepods (23.1%), cnidarians (12.9%), krill (9.3%) and fishes (4.2%) explained 95.6% of the variability observed using frequency of observance (FOO%) data. Ontogenetic changes in the diet based on groups of paralarvae with similar numbers per arm were evidenced by the decreasing contribution of coastal meroplankton and an increase in oceanic holoplankton, including siphonophores, copepods, pteropods and krill. Trophic niche breadth values ranged from 0.06 to 0.67, with averaged values ranging from 0.23 to 0.33 (generalist = 1 and specialist = 0), suggesting that O. vulgaris paralarvae are selective predators through their ontogenetic transition between coastal and oceanic environments.

Dietary lipids as modulators of fatty acid profile and gene expression patterns on body compartments of Octopus vulgaris paralarvae

The common octopus, Octopus vulgaris, is a promising mollusc species for marine aquaculture diversification due to its high growth rates and commercial value. Yet, the elevated mortalities mainly related to lipid-linked nutritional deficiencies during the planktonic stage (paralarvae), hinder the development of efficient protocols for a complete rearing cycle. Although the effect of dietary lipids, especially fatty acids (FA), has been the subject of intense research, the available information is essentially restricted to their impact on the composition of the whole parlarval organism. In contrast, little is known about the effects of dietary signature and the specific requirements of each anatomical structure through the paralarvae development. In addition, knowledge about the endogenous capacity in each paralarvae body structure for adaptation to different dietary scenarios is necessary. In the present work, a series of experiments were carried out based on newly hatched paralarvae (PH) and paralarvae fed (30 days post-hatch (DPH)) either with marine crustacean zoeae (PZ) or with Artemia metanauplii (PA). At the end of the trials, the paralarvae were dissected into functional (mantle, head, and arms) and digestive (digestive gland (DG)) body compartment and the FA profile, as well as the expression patterns of genes involved in the long chain polyunsaturated FA (LC-PUFA) (stearoyl-CoA desaturase (scd), ωx2 desaturase (ωx2), ωx1 desaturase (ωx1), fatty acyl desaturase (fad), elovl2/5, elovl4), and glycerophospholipid biosynthetic pathways (agpta, lpin, chpt, and dgat) were analysed. Results showed a positive effect of the PZ diet on growth and development of paralarvae as compared to PA, and distinct FA composition for the 3 experimental groups. The digestive gland was associated to 18C FA (18:3n-3, 18:4n3, 18:1n9, and 18:2n6), while n-6 and n-3 LC-PUFA (20:4n6, 20:5n3, 22:5n3, and 22:6n3) were found in a higher proportion in functional body compartments. The expression of LC-PUFA biosynthesis-linked genes increased significantly during development, with the functional body compartments of the PA treatment being up-regulated as compared to PZ, pointing at a putative compensatory mechanism. In addition, a higher amount of transcripts linked to the triggering of the phosphatidylcholine synthesis (chpt) was found in the digestive gland of PA and PZ and arms of PZ; whereas genes related with triacylglycerol (TAG) synthesis (lpin and dgat) were enhanced in the digestive gland of PA and PH. Dietary treatments affected the FA profile and the gene expression patterns in both digestive (more similar FA profile and glycerophospholipid biosynthesis) and functional (different FA profile and LC-PUFA biosynthesis) compartments of the paralarvae. Furthermore, LC-PUFA biosynthesis-related genes in the head and glycerophospholipid target genes in DG could be used as biomarkers of nutritional deficiencies in paralarvae.

Proteogenomic Study of the Effect of an Improved Mixed Diet of Live Preys on the Aquaculture of Octopus vulgaris Paralarvae

The common octopus is the most demanded cephalopod species for human consumption. Despite important advances realized recently, the main bottleneck for commercial production of the common octopus, Octopus vulgaris, is the mass mortality of paralarvae in the first 15–20 days post-hatching (dph), with the main responsible factors still unknown. Thus, the identification of the limiting culture factors is, therefore, crucial for their aquaculture. This study investigates proteomic and transcriptomic responses of octopus paralarvae fed on an improved live preys-mixed diet (M) compared to an Artemia-based (A) reference diet. M diet resulted in the highest paralarvae specific growth rate obtained to date under culture conditions. This is supported by most of the proteins and genes over-expressed in the M group being linked to the cell cycle and replication, production of structural components, and development of the nervous system. Furthermore, the differential nutritional regulation of several genes and proteins seems to indicate that, instead of fatty acids, the preferred fuels for cephalopods would be proteins and carbohydrates. Also, M diet provides a better nutrient balance, which has allowed carrying out this comparative study in paralarvae under optimal conditions at a more advanced stage of growth (20 dph) than in previous studies. Moreover, the paralarvae culture extended up to 40 dph showed for the first time a proper pre-settlement behavior.

Diet composition of wild Loligo vulgaris paralarvae along the West Iberian Peninsula coast

Trophic interactions in cephalopod paralarvae are difficult to evaluate due to the ingestion of liquified prey contents that are not recognizable by visual methods; however, molecular techniques such as next generation sequencing (NGS) can provide better resolution. In this study, we used NGS techniques to determine the diet of Loligo vulgaris paralarvae collected along the West Iberian Peninsula coast. Trophic analyses were conducted on 31 paralarvae ranging from 1.61-6.01 mm in dorsal mantle length, with an estimated age between 2 and 28 d. The digestive system of each paralarvae was dissected and a 300 bp section of the mitochondrial cytochrome c oxidase subunit I gene was sequenced with the MiSeq platform. After quality selection, 3201878 reads were obtained and 130 amplicon sequencing variants were identified as prey (158457 reads). Crustaceans accounted for 88.09% of the total reads, mainly represented by calanoids (73.22%), euphausiids (5.73%) and decapods (2.75%). Distance-based linear modelling showed that diet patterns depended mainly on spatial variables, accounting for 46.92% of the total variation, with calanoids and euphausiids comprising the dominant prey in the diet of paralarvae from SW Galicia, while siphonophores and myopsids constituted the most important prey on the Northern Galician coast.

Global impact of diet and temperature over aquaculture of Octopus vulgaris paralarvae from a transcriptomic approach

Common octopus, Octopus vulgaris, is an economically important cephalopod species. However, its rearing under captivity is currently challenged by massive mortalities previous to their juvenile stage due to nutritional and environmental factors. Dissecting the genetic basis and regulatory mechanism behind this mortality requires genomic background knowledge. A transcriptomic sequencing of 10 dph octopus paralarvae from different experimental conditions was constructed via RNA-seq. A total of 613,767,530 raw reads were filtered and de novo assembled into 363,527 contigs of which 82,513 were annotated in UniProt carrying also their GO and KEGG information. Differential gene expression analysis was carried out on paralarvae reared under different diet regimes and temperatures, also including wild paralarvae. Genes related to lipid metabolism exhibited higher transcriptional levels in individuals whose diet includes crustacean zoeas, which had an impact over their development and immune response capability. High temperature induces acclimation processes at the time that increase metabolic demands and oxidative stress. Wild individuals show an expression profile unexpectedly similar to Artemia fed individuals. Proteomic results support the hypothesis revealed by transcriptional analysis. The comparative study of the O. vulgaris transcriptomic profiles allowed the identification of genes that deserve to be further studied as candidates for biomarkers of development and health. The results obtained here on the transcriptional variations of genes caused by diet and temperature will provide new perspectives in understanding the molecular mechanisms behind nutritional and temperature requirements of common octopus that will open new opportunities to deepen in paralarvae rearing requirements.

Physical-biological interactions in the ocean: case studies from the northern Canary Current Upwelling Ecosystem

Physical-biological interactions in the ocean: case studies from the northern Canary Current Upwelling Ecosystem

Effect of Artemia inherent fatty acid metabolism on the bioavailability of essential fatty acids for Octopus vulgaris paralarvae development

The aim of the present study was to determine the effect of Artemia metanauplii endogenous fatty acid (FA) metabolism in the actual availability of dietary essential fatty acids (EFA) for Octopus vulgaris paralarvae development. To this end, both Artemia metanauplii inherent radiolabelled FA metabolism and the Octopus vulgaris paralarvae FA metabolism, after being fed with Artemia metanauplii incubated with radiolabelled FAs, were determined. Metanauplii were incubated in tissue culture plates during 12 h, with 0.3 μM of a [1-14C]FA, including either 18:3n-3, 20:4n-6 (ARA), 20:5n-3 (EPA) or 22:6n-3 (DHA), added individually to separate wells as their potassium salts bound to bovine serum albumin. A total of 3600 one-day-old paralarvae were reared up to 24 h in order to ensure the ingestion of a minimum amount of the labelled Artemia. Paralarvae rearing was performed in 4 L cylinder conical tanks at a density of 25 paralarvae L−1. Labelled Artemia metanauplii were added to each paralarvae rearing tank according to a specific [1-14C]FA treatment. Artemia in vivo metabolism results showed a preferential catabolism of DHA, which was translated into (1) the lower incorporation of this FA into Artemia total lipids (TL); (2) the highest amount of de novo synthesis of shorter chain-length FAs, as a result of the β-oxidation of the original DHA substrate. The registered amounts of radiolabelled substrates incorporated into O. vulgaris paralarvae TL fed with labelled Artemia metanauplii were extremely low. Nonetheless, certain amount of intact [1-14C]ARA and [1-14C]EPA was recovered into octopus paralarvae TL and particularly into polar lipid classes, suggesting the possibility of using Artemia as a vehicle to provide ARA and EPA to octopus paralarvae without interfering their bioavailability for the de novo synthesis of phospholipids. On the other hand, and despite of the high amount of [1-14C]18:3n-3 incorporated into Artemia TL, the FAs with the highest esterification rate into Artemia TAG (18:3n-3 and DHA) were also the lowest incorporated into paralarvae TL. Therefore, the present results suggest that O. vulgaris paralarvae may have a potentially low capacity to metabolise dietary TAG, and so, Artemia may not be the most appropriate vehicle to provide DHA to paralarvae.

Light conditions and heterogeneity in illumination affect growth and survival of Octopus vulgaris paralarvae reared in the hatchery

In order to succeed in common octopus (Octopus vulgaris) aquaculture, it is necessary to overcome the massive paralarval mortalities, which are likely related to zootechnical and/or nutritional aspects. Among the zootechnical aspects, the effect of light could be vital for the development of paralarvae given their sophisticated visual system. O. vulgaris paralarvae display vertical behavior in the wild, being in deeper waters during the day and close to the surface at night. For this reason, the present study traces a collection of light assays in captivity, to test ecologically-driven hypotheses on the growth and survival of octopus paralarvae.The first assay tested a factorial combination of light colors (blue vs white) and intensity (low ~13 lx (36 W/m2), medium ~87 lx (151 W/m2) and high ~265 lx (422 W/m2)). The results pointed to a significant negative impact of low light intensities (13 and 87 lx) on paralarval growth. The second assay was designed to contrast light colors (blue vs white), different levels of intensities (300 lx (478 W/m2) vs 600 lx (1077 W/m2)) and partial coverage of the tanks using a shade mesh, named as “shadow zone” (half-covered vs non-covered tanks). In half-covered tanks, survival was significantly improved, with the best results coming from half-covered tanks with blue light and 600 lx, but no differences in dry weight were observed. The third experiment tested a factor called “light source position” with two levels: “Control” when the light incidence angle was 0° with respect to the normal direction (i.e. perpendicular to the water surface), and “Oblique” when the light incidence angle was 60° with respect to the normal direction. In this experiment, survival significantly improved under oblique light but no differences in dry weight were detected. The last experiment was an unifactorial design combining “light source position” and “tank partial coverage” with three treatments: i) control light with uncovered tanks, ii) oblique light with uncovered tanks, iii) control light with semi-covered tanks. Oblique light with uncovered tanks was associated with a higher survival rate but without statistical significance, probably due to data variability. It can be concluded that light intensity tended to affect paralarval dry weight, whereas the existence of a shadow zone or oblique light are more related with an improvement of survival rates.

Comparative study of age estimation in wild and cultured Octopus vulgaris paralarvae: effect of temperature and diet

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 598:247-259 (2018) - DOI: https://doi.org/10.3354/meps12218 Comparative study of age estimation in wild and cultured Octopus vulgaris paralarvae: effect of temperature and diet C. Perales-Raya1,*, M. Nande2, A. Roura3, A. Bartolomé1, C. Gestal3, J. J. Otero2, P. García-Fernández3, E. Almansa1 1Instituto Español de Oceanografía, Centro Oceanográfico de Canarias, Santa Cruz de Tenerife 38180, Spain 2Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Vigo 362390, Spain 3Instituto de Investigaciones Marinas Consejo Superior de Investigaciones Científicas, Vigo 36208, Spain *Corresponding author: catalina.perales@ca.ieo.es *New email address since May 14, 2019: catalina.perales@ieo.es Advance View was available online September 5, 2017; subsequently updated September 12, 2017 ABSTRACT: The common octopus Octopus vulgaris is a highly valuable species worldwide, but to understand its population dynamics and requirements under culture conditions, it is crucial to improve our knowledge about its planktonic stages. Previous studies validating daily beak growth increments in these stages allowed age estimation and comparison of wild and cultured paralarvae. We aimed to improve age estimations in captivity, addressing the effect of temperature and diet, to obtain an accurate estimation of age in wild specimens collected from the coast to the open ocean off NW Spain and Morocco. We analysed the beak growth increments of reared paralarvae at 14 and 21°C with 2 different crustacean prey taxa (Artemia and spider crab Maja brachydactyla zoeae) over 30 d. Daily increment deposition at 21°C was confirmed, whereas <1 increment d-1 was recorded at 14°C. The width of the reading area grew accordingly with age; therefore, this beak region may be suitable for age estimation. A general linear model (GLM) analysis showed that temperature and the interaction of age × temperature significantly influenced increment deposition, whereas diet did not. The number of growth rings recorded in wild paralarvae beaks ranged from 0-8 on the coast, 7-11 on the continental shelf and 2-28 in the open ocean. Corrected age estimates of wild paralarvae were obtained with the GLM using the mean temperatures recorded in the wild, supporting the hypothesis that O. vulgaris leave the coastal area and develop in the open ocean transported by upwelling filaments. KEY WORDS: Octopus vulgaris · Cephalopod · Paralarvae · Early life · Ageing · Beaks · Microstructure · Prey · Growth Full text in pdf format PreviousNextCite this article as: Perales-Raya C, Nande M, Roura A, Bartolomé A and others (2018) Comparative study of age estimation in wild and cultured Octopus vulgaris paralarvae: effect of temperature and diet. Mar Ecol Prog Ser 598:247-259. https://doi.org/10.3354/meps12218 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 598. Online publication date: June 28, 2018 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2018 Inter-Research.

Characterization of the skin mucus in the common octopus Octopus vulgaris (Cuvier) reared paralarvae

The Octopus vulgaris farming is impaired by the high mortality of the paralarvae during the first month of life. Several factors have been investigated in this regard, but no data exist on the body surface mucus, which represents the interface with the outside environment. This study included morphometric analysis and glycoconjugates characterization of skin mucus in reared Octopus vulgaris paralarvae during the first month of life. Four types of mucous cells were distinguished: mucous 1 (m1) and mucous 2 (m2) cells were scattered in the mantle epidermis, mucous 3 (m3) and mucous 4 (m4) in the epithelium surrounding the sucker. Except for the presence of fucosylated and neutral glycoconjugates in all mucous cells, each cell type expressed a characteristic glycopattern. m2 and m4 contained also suphate and acid non-sulphate glycans, m3 lacked sulphate glycoproteins. Lectin histochemistry showed that mantle mucous cells (m1, m2) expressed GlcNAc and lactosamine terminating glycans. m2 also contained GalNAc terminal or penultimate to sialic acid. m3 was distinguished by mannosylated glycans terminating with lactosamine and m4 by α2,6 sialoglycans. Glycoproteins terminating with lactosamine, Galβ1,3GalNAc, and α1,6-linked fucose were a common feature of paralarvae surface layer. Morphometry revealed a significant decrease of m1 and m2 abundance during the first month of life, afterwards the reared paralarvae died. Since the glycopattern did not change during the investigated period, the mantle mucous cells abundance could be related to the Octopus vulgaris paralarvae survival.

Prey Capture, Ingestion, and Digestion Dynamics of Octopus vulgaris Paralarvae Fed Live Zooplankton.

Octopus vulgaris is a species of great interest in research areas such as neurobiology, ethology, and ecology but also a candidate species for aquaculture as a food resource and for alleviating the fishing pressure on its wild populations. This study aimed to characterize the predatory behavior of O. vulgaris paralarvae and to quantify their digestive activity. Those processes were affordable using the video-recording analysis of 3 days post-hatching (dph), mantle-transparent paralarvae feeding on 18 types of live zooplanktonic prey. We show for the first time in a live cephalopod that octopus paralarvae attack, immobilize, drill, and ingest live cladocerans and copepods with 100% efficiency, which decreases dramatically to 60% on decapod prey (Pisidia longicornis). The majority (85%) of successful attacks targeted the prey cephalothorax while unsuccessful attacks either targeted the dorsal cephalothorax or involved prey defensive strategies (e.g., juvenile crab megalopae) or prey protected by thick carapaces (e.g., gammaridae amphipods). After immobilization, the beak, the buccal mass and the radula were involved in exoskeleton penetration and content ingestion. Ingestion time of prey content was rapid for copepods and cladocerans (73.13 ± 23.34 s) but much slower for decapod zoeae and euphausiids (152.49 ± 29.40 s). Total contact time with prey was always <5 min. Contrary to the conventional view of crop filling dynamics observed in adult O. vulgaris, food accumulated first in the stomach of paralarvae and the crop filled after the stomach volume plateaued. Peristaltic crop contractions (~18/min) moved food into the stomach (contractions ~30/min) from where it passed to the caecum. Pigmented food particles were seen to enter the digestive gland, 312 ± 32 s after the crop reached its maximum volume. Digestive tract contents passed into the terminal intestine by peristalsis (contraction frequency ~50/min) and defaecation was accompanied by an increased frequency of mantle contractions. Current results provide novel insights into both, O. vulgaris paralarvae—live prey capture strategies and the physiological mechanisms following ingestion, providing key information required to develop an effective rearing protocol for O. vulgaris paralarvae.

Feeding Relationship between Octopus vulgaris (Cuvier, 1797) Early Life-Cycle Stages and Their Prey in the Western Iberian Upwelling System: Correlation of Reciprocal Lipid and Fatty Acid Contents.

Under the influence of the Western Iberian upwelling system, the Iberian Atlantic coast holds important hatcheries and recruitment areas for Octopus vulgaris. Recently identified as an octopus hatchery, the Ría de Vigo harbors an important mesozooplankton community that supports O. vulgaris paralarvae during the first days of their planktonic stage. This study represents a preliminary approach to determine the nutritional link between wild O. vulgaris hatchlings, paralarvae and their zooplankton prey in the Ría de Vigo, by analyzing their lipid class content and fatty acid profiles. The results show that octopus hatchlings are richer in structural lipids as phospholipids and cholesterol, while the zooplankton is richer in reserve lipids like triacylglycerol and waxes. Zooplankton samples are also particularly rich in C18:1n9 and 22:6n3 (DHA), that seem to be successfully incorporated by O. vulgaris paralarvae thus resulting in a distinct fatty acid profile to that of the hatchlings. On the other hand, content in C20:4n6 (ARA) is maintained high through development, even though the zooplankton is apparently poorer in this essential fatty acid, confirming its importance for the development of O. vulgaris paralarvae. The content in monounsaturated fatty acids, particularly C18:1n7, and the DHA: EPA ratio are suggested as trophic markers of the diet of O. vulgaris paralarvae.

3D Reconstruction of the Digestive System in Octopus vulgaris Cuvier, 1797 Embryos and Paralarvae during the First Month of Life.

Octopus vulgaris aquaculture is limited due to poor biological knowledge of the paralarval stages (e.g., digestive system functionality), their nutritional requirements (e.g., adequate live diet) and standardization of rearing techniques. These factors are important in explaining the high mortality rate observed in this developmental stage under culture conditions. For a better understanding of nutrition biology of this species, we investigated the 3D microanatomy of the digestive tract of the embryo and paralarvae during the first month of life. O. vulgaris paralarvae digestive system is similar to that in the adult. The “descending branch” has a dorsal position and is formed by the buccal mass, oesophagus and crop. Ventrally, the “ascending branch” is formed by the intestine and the anus. The digestive gland, the posterior salivary glands and the inner yolk sac (in the case of the embryo and hatched paralarvae) are located between the “ascending” and “descending” branches. In the curve of the U-shaped digestive tract, a caecum and the stomach can be found. The reconstructions reveal that anatomically the digestive system is already complete when the paralarvae hatch. The reconstruction of the buccal mass at different post-hatching days has demonstrated that all the necessary structures for food intake are present. However, the radula surface in contact with the pharynx is very small on the first day of life. Although the digestive system has all the structures to feed, the digestive gland and radula take longer to reach full functionality. We have established four development periods: embryonic, early post-hatching, late post-hatching and juvenile-adult. The differentiation between these periods was done by type of feeding (endogenous or exogenous), the state of maturation and hence functionality of the digestive gland, type of growth (linear, no net, or exponential), and measurement of the arm lengths with respect to the mantle length. 3D reconstruction represents a new tool to study the morphology and functionality of the cephalopod digestive system during the first days of life.

You Are What You Eat: A Genomic Analysis of the Gut Microbiome of Captive and Wild Octopus vulgaris Paralarvae and Their Zooplankton Prey.

The common octopus (Octopus vulgaris) is an attractive species for aquaculture, however, several challenges inhibit sustainable commercial production. Little is known about the early paralarval stages in the wild, including diet and intestinal microbiota, which likely play a significant role in development and vitality of this important life stage. High throughput sequencing was used to characterize the gastrointestinal microbiome of wild O. vulgaris paralarvae collected from two different upwelling regions off the coast of North West Spain (n = 41) and Morocco (n = 35). These were compared to that of paralarvae reared with Artemia for up to 25 days in captivity (n = 29). In addition, the gastrointestinal microbiome of zooplankton prey (crabs, copepod and krill) was also analyzed to determine if the microbial communities present in wild paralarvae are derived from their diet. Paralarvae reared in captivity with Artemia showed a depletion of bacterial diversity, particularly after day 5, when almost half the bacterial species present on day 0 were lost and two bacterial families (Mycoplasmataceae and Vibrionaceae) dominated the microbial community. In contrast, bacterial diversity increased in wild paralarvae as they developed in the oceanic realm of both upwelling systems, likely due to the exposure of new bacterial communities via ingestion of a wide diversity of prey. Remarkably, the bacterial diversity of recently hatched paralarvae in captivity was similar to that of wild paralarvae and zooplankton, thus suggesting a marked effect of the diet in both the microbial community species diversity and evenness. This study provides a comprehensive overview of the bacterial communities inhabiting the gastrointestinal tract of O. vulgaris paralarvae, and reveals new research lines to challenge the current bottlenecks preventing sustainable octopus aquaculture.

Diet Composition and Variability of Wild Octopus vulgaris and Alloteuthis media (Cephalopoda) Paralarvae: a Metagenomic Approach.

The high mortality of cephalopod early stages is the main bottleneck to grow them from paralarvae to adults in culture conditions, probably because the inadequacy of the diet that results in malnutrition. Since visual analysis of digestive tract contents of paralarvae provides little evidence of diet composition, the use of molecular tools, particularly next generation sequencing (NGS) platforms, offers an alternative to understand prey preferences and nutrient requirements of wild paralarvae. In this work, we aimed to determine the diet of paralarvae of the loliginid squid Alloteuthis media and to enhance the knowledge of the diet of recently hatched Octopus vulgaris paralarvae collected in different areas and seasons in an upwelling area (NW Spain). DNA from the dissected digestive glands of 32 A. media and 64 O. vulgaris paralarvae was amplified with universal primers for the mitochondrial gene COI, and specific primers targeting the mitochondrial gene 16S gene of arthropods and the mitochondrial gene 16S of Chordata. Following high-throughput DNA sequencing with the MiSeq run (Illumina), up to 4,124,464 reads were obtained and 234,090 reads of prey were successfully identified in 96.87 and 81.25% of octopus and squid paralarvae, respectively. Overall, we identified 122 Molecular Taxonomic Units (MOTUs) belonging to several taxa of decapods, copepods, euphausiids, amphipods, echinoderms, molluscs, and hydroids. Redundancy analysis (RDA) showed seasonal and spatial variability in the diet of O. vulgaris and spatial variability in A. media diet. General Additive Models (GAM) of the most frequently detected prey families of O. vulgaris revealed seasonal variability of the presence of copepods (family Paracalanidae) and ophiuroids (family Euryalidae), spatial variability in presence of crabs (family Pilumnidae) and preference in small individual octopus paralarvae for cladocerans (family Sididae) and ophiuroids. No statistically significant variation in the occurrences of the most frequently identified families was revealed in A. media. Overall, these results provide new clues about dietary preferences of wild cephalopod paralarvae, thus opening up new scenarios for research on trophic ecology and digestive physiology under controlled conditions.

Assessment of stress and nutritional biomarkers in cultured Octopus vulgaris paralarvae: Effects of geographical origin and dietary regime

The common octopus (Octopus vulgaris) is a promising species for aquaculture diversification, but massive mortality during the first life-cycle stages (paralarvae) is the main bottleneck for its commercial production in captivity. The aim of this study was to assess stress and nutritional condition biomarkers (HSP70, ROS enzymes and lipid peroxidation) (RNA/DNA, RNA/protein, protein/DNA and protein) in O. vulgaris paralarvae from different geographical origins and fed with Artemia enriched with marine phospholipids or microalgae (control group). To this end paralarvae were cultured for 30days, in three different centres in Spain (Tarragona-Mediterranean area, Tenerife-Central Atlantic area and Vigo-North Atlantic area), under the same protocol, and fed on Artemia enriched with marine phospholipids (LC60) (Marine Lecithin LC 60®, PhosphoTech Laboratoires) or microalgae (control group). Dry weight and most biomarkers analysed in hatchlings showed significant differences related to their origin (centre). Fifteen day old paralarvae presented significant differences in specific growth rate (SGR) associated with their dietary regime, and also showed differences in biomarkers associated both with their geographical origin and dietary regime. The results suggest that the SGR of paralarvae were positively influenced by LC60, promoting growth and in agreement with the results of nutritional condition biomarkers (nucleic acids ratios). The antioxidant defences against oxidative damage were also boosted in the LC60 paralarvae group, possibly as a result of the elevated content in highly polyunsaturated fatty acids. In addition, the partial correlations found between biomarkers varied according to diet. However, no positive effect of LC60 on survival was observed. The high variability found among geographical origins, despite the use of the same rearing protocol, highlights the need to clarify the sources of such variability. Statement of relevanceStress status varies among geographical origins and diets.

Fatty acid composition and age estimation of wild Octopus vulgaris paralarvae

The fatty acid (FA) profile of wild Octopus vulgaris paralarvae of estimated age was individually analyzed for the very first time in order to establish a reference for comparison in rearing and nutritional studies. Age of each paralarvae was estimated by analysing daily increments on lateral hood surface of beaks. Wild paralarvae age ranged between 6 and 8days and their FA composition resembled that from hatchlings produced under culture conditions. However, when compared with the FA composition of up to 20days old cultured paralarvae described in the bibliography, some striking differences were found. Results showed higher levels of docosahexaenoic acid (22:6n−3, DHA), lower contents of 18:1n−9, 18:1n−7 and 18:2n−6 and negligible levels of 18:3n−3 in wild paralarvae, when collated to reared one. These results seem to indicate that preys/diets supplied to cultured paralarvae fail to resemble paralarval natural composition and as a result do not fulfil their FA requirement. The individual applied technique developed in this study will allow to refine the study of wild paralarvae along its development, as well as to compare wild and cultured paralarvae of similar age. Statement of relevanceArtemia does not fulfil paralarval fatty acid requirements.

Selection of reliable reference genes for RT-qPCR studies in Octopus vulgaris paralarvae during development and immune-stimulation

The common octopus, Octopus vulgaris is a new candidate species for aquaculture. However, rearing of octopus paralarvae is hampered by high mortality and poor growth rates that impede its entire culture. The study of genes involved in the octopus development and immune response capability could help to understand the key of paralarvae survival and thus, to complete the octopus life cycle. Quantitative real-time PCR (RT-qPCR) is the most frequently tool used to quantify the gene expression because of specificity and sensitivity. However, reliability of RT-qPCR requires the selection of appropriate normalization genes whose expression must be stable across the different experimental conditions of the study. Hence, the aim of the present work is to evaluate the stability of six candidate genes: β-actin (ACT), elongation factor 1-α (EF), ubiquitin (UBI), β-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GADPH) and ribosomal RNA 18 (18S) in order to select the best reference gene. The stability of gene expression was analyzed using geNorm, NormFinder and Bestkeeper, in octopus paralarvae of seven developmental stages (embryo, paralarvae of 0, 10, 15, 20, 30 and 34days) and paralarvae of 20days after challenge with Vibrio lentus and Vibrio splendidus. The results were validated by measuring the expression of PGRP, a stimuli-specific gene. Our results showed UBI, EF and 18S as the most suitable reference genes during development of octopus paralarvae, and UBI, ACT and 18S for bacterial infection. These results provide a basis for further studies exploring molecular mechanism of their development and innate immune defense.

Life strategies of cephalopod paralarvae in a coastal upwelling system (NW Iberian Peninsula): insights from zooplankton community and spatio‐temporal analyses

AbstractThe early life stages of cephalopods ‐ octopods, squids, sepiolids and ommastrephids ‐, are uncommon in zooplankton samples and little is known about their life strategies. Accordingly, cephalopod paralarvae were examined in the upwelling ecosystem of the Ría de Vigo (NW Spain) at night from 2008 to 2010. Multivariate analyses and generalized linear models (GLMs) were used to explore relationships between cephalopod paralarvae and the zooplankton communities that they inhabited in 2008. In addition, the foraging strategy and prey preferences of Octopus vulgaris paralarvae within these communities were determined. Multivariate and GLM results showed a strong association of cephalopod paralarvae with coastal and frontal zooplankton communities. Octopus paralarvae were shown to be specialist predators with a strong preference for decapod zoeae in each of the communities examined. Using the three years of sampling, GLM analyses of paralarval spatio‐temporal variations in relation with the upwelling strength showed a positive relationship with upwelling intensity for O. vulgaris and sepiolids, as well as contrasting temporal, horizontal and vertical distributions for the different paralarvae analysed. Under strong upwelling events, Octopus paralarvae were more abundant in surface waters, whereas the abundance of loliginids and sepiolids was higher in the water column. This vertical behaviour in conjunction with the physical conditions of the Western Iberian Upwelling ecosystem suggests the coexistence of two different life strategies: a coastal strategy displayed by loliginid and sepiolid paralarvae that are retained over the shelf, and an oceanic strategy displayed by O. vulgaris paralarvae that are dispersed far from the shelf.

Effect of temperature on energetic demands during the last stages of embryonic development and early life of Octopus vulgaris (Cuvier, 1797) paralarvae

This study describes the effect of seasonal average temperatures (14 and 18°C) in the Ria of Vigo, on the utilization of external yolk over the last five Naef stages of development (XV–XX) for Octopus vulgaris embryos. Also, the transference of the outer yolk to the inner yolk sac, and its use during embryonic development and early life by O. vulgaris paralarvae. Temperature had a marked effect on embryonic development, except during stages XV–XIX (until the second inversion) where development time was the same (14 days), regardless of temperature. There were no significant differences in outer yolk decrease between consecutive Naef stages at 14°C and 18°C. Contrary, significant differences at all Naef stages from XV to XIX (both, with or without outer yolk) were observed for inner yolk between temperatures. A higher accumulation of inner yolk in embryos at 14°C was observed, due to lower yolk consumption. Paralarvae incubated at both temperatures were maintained independently at starvation during 4 days. At 18°C, a reduced accumulation of inner yolk, especially during Naef stage XIX, was observed. In 24 h old paralarvae, there was already significant higher inner yolk content at 14°C than at 18°C. Unfed paralarvae at 18°C lost weight faster than those at 14°C, due to higher energetic requirements. Finally, from these results, we propose a paralarvae rearing protocol during the first days after hatching and during the last five Naef stage (XV–XX) at lower temperatures, since the energy requirements are lower during the initial maturation stage.