Observational evidence of egg guarding in wild European squid.

It is accepted that loliginids, like other squid, deposit their eggs in crevices on the seabed and then abandon them. In this work, we present observational evidence of egg guarding behavior in wild European squid, Loligo vulgaris. While monitoring a squid spawning crevice at night in Spain, a large mass of squid eggs was located and filmed 17 times during 42 days, until hatching. A male and a female of L. vulgaris were filmed in front of the crevice. The same male was filmed guarding the eggs on consecutive days. In the presence of the divers, male and female alternated their approaches to the crevice repeatedly touching and flushing the egg clusters. This guarding behavior differs from the reproductive habits assumed for the European squid and could represent the first evidence of egg guarding by a male in cephalopods.

Occurrence of Seahorses Hippocampus spp. in the Southernmost Part of Western Europe: A New Maximum Depth Record.

Achieving sustainable resource use is a priority to meet future challenges. The Gulf of Cádiz, located in the Atlantic waters of southern Europe, is home to a significant fishing fleet due to the richness and diversity of its ecosystems. Managing this area is complex due to the diversity of variables, including social, ecological, and oceanographic factors. Therefore, multidisciplinary approaches are proposed for implementing conservation strategies. One strategy for defining area-based management measures is through the use of flagship species, such as seahorses. These emblematic animals can assist in defining such measures. However, there is currently scarce information on the occurrence of seahorses in the Gulf of Cádiz. In this study, we present the first occurrence data of two species of the genus Hippocampus (H. hippocampus and H. guttulatus) in this area. The Gulf of Cádiz is not only described as the southernmost region of their distribution in continental Europe, but it is also a significant landmark for the genus Hippocampus, as one adult was captured at a depth of up to 101 m. Five management areas based on differentiated benthic habitats are proposed. We believe that our study has the potential to significantly improve conservation of seahorses and induce a positive impact on the ecosystem.

Screening of Microalgae for Bioactivity with Antiviral, Antibacterial, Anti-Inflammatory and Anti-Cancer Assays.

Marine microalgae are a rich reservoir of natural compounds, including bioactives. Nonetheless, these organisms remain fairly unexplored despite their potential biotechnological applications. Culture collections with diverse taxonomic groups and lifestyles are a good source to unlock this potential and discover new molecules for multiple applications such as the treatment of human pathologies or the production of aquaculture species. In the present work extracts from thirty-three strains (including twenty dinoflagellates, four diatoms and nine strains from seven other algal classes), cultivated under identical conditions, were examined for their antiviral, antibacterial, anti-inflammatory and anti-cancer activities. Among these, antiviral and anti-inflammatory activities were detected in a few strains while the antibacterial tests showed positive results in most assays. In turn, most trials did not show any anti-cancer activity. Significant differences were observed between species within the same class, in particular dinoflagellates, which were better represented in this study. These preliminary findings pave the way for an in-depth characterization of the extracts with highest signals in each test, the identification of the compounds responsible for the biological activities found and a further screening of the CCVIEO culture collection.

The best defense is a good offense: Anti-predator behavior of the common octopus (Octopus vulgaris) against conger eel attacks.

We present the description of defensive behavior in wild Octopus vulgaris against conger eel (Conger conger) attacks based on three video sequences recorded by recreational SCUBA divers in the eastern Atlantic off the coast of Galicia (NW Spain) and in the Cantabrian Sea (NW Spain). These records document common traits in defensive behavior: (1) the octopuses enveloped the conger eel's head to obscure its view; (2) they covered the eel's gills in an attempt to suffocate it; (3) they released ink; (4) the octopuses lost some appendages because of the fight. In the third video, the octopus did not exhibit the defensive behavior described in the first two videos due to an inability to utilize its arms in defense, and the conger eel's success in capturing octopuses is discussed. Additionally, both the cost that the octopus could face by losing some arms during the fight and whether the experience it acquires can be an advantage for future encounters are analyzed. The defensive behavior exhibited by octopuses in this study highlights their ability to survive in a hostile environment and serves as an example of the extensive repertoire of anti-predator strategies employed by these cephalopods.

Corrigendum: The significance of cephalopod beaks as a research tool: An update.

[This corrects the article DOI: 10.3389/fphys.2022.1038064.].

Mapping dinoflagellate blooms (Noctiluca and Alexandrium) in aquaculture production areas in the NW Iberian Peninsula with the Sentinel-2/3 satellites.

The Galician Rías (northwestern Spain) are periodically affected by harmful algal blooms (HABs), mostly dinoflagellates, which pose a challenge to aquaculture activities due to the accumulation of biotoxins in shellfish. Typically, reddish blooms in the Rías are associated with non-toxic species like Noctiluca scintillans, with a few exceptions such as Alexandrium minutum, a producer of paralytic shellfish toxins (PST). Here, a useful approach is presented for monitoring reddish blooms through satellite imagery based on three case studies, two of them belonged to monospecific blooms of red Noctiluca scintillans, and the third to a bloom of Alexandrium spp. dominated by A. tamarense. In every case, a propulsive index was evaluated using Sentinel-2A/B satellites, which provide high spatial and spectral resolutions, combined with adequate atmospheric and sunglint correction by using the ACOLITE and C2RCC processors. This approach offers a simple and feasible method to accurately and timely map blooms of red N. scintillans and Alexandrium spp. in the study area, useful to detect the distribution of reddish blooms with synoptic observations for monitoring and aquaculture management purposes. Conversely, Sentinel-3A/B satellites with a relatively coarser spatial resolution, lacking adequate visualization and mapping of the extent of small blooms, did not accurately detect bloom footprints in the coastal bay region, although this sensor displays a set of suitable multispectral bands.

The significance of cephalopod beaks as a research tool: An update.

The use of cephalopod beaks in ecological and population dynamics studies has allowed major advances of our knowledge on the role of cephalopods in marine ecosystems in the last 60 years. Since the 1960's, with the pioneering research by Malcolm Clarke and colleagues, cephalopod beaks (also named jaws or mandibles) have been described to species level and their measurements have been shown to be related to cephalopod body size and mass, which permitted important information to be obtained on numerous biological and ecological aspects of cephalopods in marine ecosystems. In the last decade, a range of new techniques has been applied to cephalopod beaks, permitting new kinds of insight into cephalopod biology and ecology. The workshop on cephalopod beaks of the Cephalopod International Advisory Council Conference (Sesimbra, Portugal) in 2022 aimed to review the most recent scientific developments in this field and to identify future challenges, particularly in relation to taxonomy, age, growth, chemical composition (i.e., DNA, proteomics, stable isotopes, trace elements) and physical (i.e., structural) analyses. In terms of taxonomy, new techniques (e.g., 3D geometric morphometrics) for identifying cephalopods from their beaks are being developed with promising results, although the need for experts and reference collections of cephalopod beaks will continue. The use of beak microstructure for age and growth studies has been validated. Stable isotope analyses on beaks have proven to be an excellent technique to get valuable information on the ecology of cephalopods (namely habitat and trophic position). Trace element analyses is also possible using beaks, where concentrations are significantly lower than in other tissues (e.g., muscle, digestive gland, gills). Extracting DNA from beaks was only possible in one study so far. Protein analyses can also be made using cephalopod beaks. Future challenges in research using cephalopod beaks are also discussed.

Turnover Rates and Diet-Tissue Discrimination Factors of Nitrogen and Carbon Stable Isotopes in Seahorse Hippocampus reidi Juveniles Following a Laboratory Diet Shift.

The initial development of seahorse juveniles is characterized by low digestion capabilities. Stable isotope analysis is an effective tool in studies of trophic food webs and animal feeding patterns. The present study provides new insights for the understanding of growth and food assimilation in early developing seahorses following a laboratory diet switch. The study was performed in the early life stages of the seahorse Hippocampus reidi by assessing the influence of diet shift on changes and turnovers in carbon (δ13C) and nitrogen (δ15N) stable isotope in juveniles. Newborn seahorses were fed for 60 days following two feeding schedules (A6 and A11) based initially on copepods Acartia tonsa and subsequently on Artemia nauplii (since days 6 and 11, respectively). After the prey shift, we determined δ13C and δ15N turnover rates as functions of change in either body mass (fitting model G) and days of development (fitting model D), contributions of metabolism and growth to those turnover rates, and diet-tissue discrimination factors. Survival, final dry weight, and final standard length for diet A11 were higher compared to diet A6. The shift from copepods to Artemia led to fast initial enrichments in δ13C and δ15N. Afterwards, the enrichment was gradually reduced until the isotopic equilibrium with the diet was reached. In most cases, both fitting models performed similarly. The isotopic analysis revealed that 100% of tissue turnover was attributed to growth in diet A11, whereas 19-25% was linked to metabolism in diet A6. Diet-tissue discrimination factors were estimated for the first time in seahorse juveniles, resulting in higher estimates for diet A11 (2.9 ± 0.7‱ for δ13C; 2.5 ± 0.2‱ for δ15N) than in diet A6 (1.8 ± 0.1‱ for δ13C; 1.9 ± 0.1‱ for δ15N). This study highlights the relevance of feeding on copepods and their effect on isotopic patterns and discrimination factors in seahorse juveniles after a dietary shift. Regarding the application of the results achieved in relation to the feeding schedules in the rearing of H. reidi, a long period of feeding on copepods during the first days of development is highly recommended.

First evidence of ingestion and retention of microplastics in seahorses (Hippocampus reidi) using copepods (Acartia tonsa) as transfer vectors.

Microplastics (MPs) are a major concern for marine ecosystems since they can be ingested by a wide range of marine species and transmitted through the food web. However, the potential hazardous impact of MPs in fishes, especially in early developing stages, is relatively unknown. In the present study, we assessed for the first time the ingestion and retention of MPs in early developing seahorses Hippocampus reidi. Seahorses are vulnerable species that may also be affected by both the direct ingestion of MPs through their preys and the accidental ingestion of MPs particles present in the water (i.e., seahorses ingest the prey by suction). We used copepods as both preys for seahorse juveniles and transfer vectors of MPs. Fed or starved copepods previously exposed to polyethylene microspheres (1-5 µm in diameter; 10 and 100 µg L-1) for 60 min at 26 °C showed fast evacuation of microspheres. The presence of MPs in copepods was significantly higher in previously fasted copepods compared to fed copepods. Seahorse juveniles fed on copepods pre-exposed to MPs, accumulated MPs in the gut proportionally to the concentration of MPs in copepods. A lower concentration of MPs in seahorses was observed at the longer exposure time (60 min), especially in fish fed with fasted copepods. However, after longer exposure, MPs were mainly accumulated close to the anus both individually or forming aggregates. Further studies should be performed to assess secondary effects of MPs ingestion in seahorses since they are considered a flagship species for marine conservation.

Species-specific heavy metal concentrations of tuna species: the case of Thunnus alalunga and Katsuwonus pelamis in the Western Mediterranean.

Albacore Thunnus alalunga and skipjack tuna Katsuwonus pelamis are highly migratory species that are usually caught together in the Western Mediterranean. These species are top predators that are highly affected by the biomagnification process through the trophic chain. Bioaccumulation pattern of the main metal pollutants (mercury, Hg; lead, Pb; and cadmium, Cd) were analyzed in muscle tissues of 52 individuals (26 T. alalunga and 26 K. pelamis) of these highly consumed species in order to address two objectives: (1) compare the species-specific bioaccumulation between these large-pelagic species, and (2) assess the healthy properties of such valuable resources based on the trace metal limits established by the European Commission Regulation (ECR). Both generalized linear mixed models and redundancy analysis indicated a differential bioaccumulation between these two tuna species. While T. alalunga accumulates higher concentrations of Hg (0.1996 ± 0.0602 mg·kg-1 weight wet-ww), K. pelamis accumulates higher concentrations of Cd (0.0076 ± 0.0049 mg·kg-1 ww) and Pb (0.0031 ± 0.0017 mg·kg-1 ww). Size and trophic ecology support the differences detected in the bioaccumulation pattern. Heavy metal concentrations were below the tolerable limits considered by ECR (1, 0.1, and 0.3 mg·kg-1 ww for Hg, Cd, and Pb, respectively).

A multidisciplinary approach to identify priority areas for the monitoring of a vulnerable family of fishes in Spanish Marine National Parks.

BACKGROUND: Syngnathid fishes (Actinopterygii, Syngnathidae) are flagship species strongly associated with seaweed and seagrass habitats. Seahorses and pipefishes are highly vulnerable to anthropogenic and environmental disturbances, but most species are currently Data Deficient according to the IUCN (2019), requiring more biological and ecological research. This study provides the first insights into syngnathid populations in the two marine Spanish National Parks (PNIA-Atlantic- and PNAC-Mediterranean). Fishes were collected periodically, marked, morphologically identified, analysed for size, weight, sex and sexual maturity, and sampled for stable isotope and genetic identification. Due the scarcity of previous information, habitat characteristics were also assessed in PNIA. RESULTS: Syngnathid diversity and abundance were low, with two species identified in PNIA (Hippocampus guttulatus and Syngnathus acus) and four in PNAC (S. abaster, S. acus, S. typhle and Nerophis maculatus). Syngnathids from both National Parks (NP) differed isotopically, with much lower δ15N in PNAC than in PNIA. The dominant species were S. abaster in PNAC and S. acus in PNIA. Syngnathids preferred less exposed sites in macroalgal assemblages in PNIA and Cymodocea meadows in PNAC. The occurrence of very large specimens, the absence of small-medium sizes and the isotopic comparison with a nearby population suggest that the population of Syngnathus acus (the dominant syngnathid in PNIA) mainly comprised breeders that migrate seasonally. Mitochondrial cytochrome b sequence variants were detected for H. guttulatus, S. acus, and S. abaster, and a novel 16S rDNA haplotype was obtained in N. maculatus. Our data suggest the presence of a cryptic divergent mitochondrial lineage of Syngnathus abaster species in PNAC. CONCLUSIONS: This is the first multidisciplinary approach to the study of syngnathids in Spanish marine NPs. Habitat preferences and population characteristics in both NPs differed. Further studies are needed to assess the occurrence of a species complex for S. abaster, discarding potential misidentifications of genus Syngnathus in PNAC, and evaluate migratory events in PNIA. We propose several preferential sites in both NPs for future monitoring of syngnathid populations and some recommendations for their conservation.

Effects of Tissue Preservation on Carbon and Nitrogen Stable Isotope Signatures in Syngnathid Fishes and Prey.

Isotopic stable analysis (SIA) is a powerful tool in the assessment of different types of ecological and physiological studies. For that, different preservation methods for sampled materials are commonly used prior to isotopic analysis. The effects of various preservation methods (freezing, ethanol and formaldehyde) were analyzed for C:N, and δ13C and δ15N signals on a variety of tissues including dorsal fins (three seahorse and two pipefish species), seahorse newborns (three seahorses species), and prey (copepods and different stages of Artemia) commonly used to feed the fishes under rearing conditions. The aims of the study were: (i) to evaluate isotopic effects of chemical preservation methods across different types of organisms and tissues, using frozen samples as controls, and (ii) to construct the first conversion models available in syngnathid fishes. The chemical preservation in ethanol and, to a lesser extent, in formaldehyde significantly affected δ13C values, whereas the effects on δ15N signatures were negligible. Due to their low lipid content, the isotopic signals in fish fins was almost unaffected, supporting the suitability of dorsal fins as the most convenient material in isotopic studies on vulnerable species such as syngnathids. The regression equations provided resulted convenient for the successful conversion of δ13C between preservation treatments. Our results indicate that the normalization of δ15N signatures in preserved samples is unnecessary. The conversion models should be applicable in isotopic field studies, laboratory experiments, and specimens of historical collections.

Influence of the myotome zone and sex on the muscle cellularity and fillet texture of diploid and triploid turbots Scophthalmus maximus L.

The muscle and textural parameters were analyzed in four myotome zones (epaxial upper, hipoaxial upper, epaxial bottom, and hipoaxial bottom) in seven diploids (D) and seven triploids (T) turbot specimens. Diploid specimens showed the highest values of the size and number of white fibers in the epaxial zones, being such values higher in female than male specimens. In triploid specimens, the highest fibers sizes were found in the upper zones (epaxial and hipoaxial), whereas the lowest number and density of fibers were found in the epaxial upper zone. In this latter group (T), the lowest fibers sizes were found in female specimens, whereas the rest of the parameters were usually higher in female than male specimens. When comparing both groups, the hypertrophy was higher in T than D in all zones. In both ploidy groups, the highest textural values were usually observed in the upper epaxial fillet, being slightly higher in female than male specimens. The values of standard length, total weight, gonad weight, gonadosomatic index and gutted weight were higher in female than male specimens in both groups (D and T).

Notes on the Cultivation of Two Mixotrophic Dinophysis Species and Their Ciliate Prey Mesodinium rubrum.

Kleptoplastic mixotrophic species of the genus Dinophysis are cultured by feeding with the ciliate Mesodinium rubrum, itself a kleptoplastic mixotroph, that in turn feeds on cryptophytes of the Teleaulax/Plagioselmis/Geminigera (TPG) clade. Optimal culture media for phototrophic growth of D. acuminata and D. acuta from the Galician Rías (northwest Spain) and culture media and cryptophyte prey for M.rubrum from Huelva (southwest Spain) used to feed Dinophysis, were investigated. Phototrophic growth rates and yields were maximal when D. acuminata and D. acuta were grown in ammonia-containing K(-Si) medium versus f/2(-Si) or L1(-Si) media. Dinophysis acuminata cultures were scaled up to 18 L in a photobioreactor. Large differences in cell toxin quota were observed in the same Dinophysis strains under different experimental conditions. Yields and duration of exponential growth were maximal for M. rubrum from Huelva when fed Teleaulax amphioxeia from the same region, versus T. amphioxeia from the Galician Rías or T. minuta and Plagioselmis prolonga. Limitations for mass cultivation of northern Dinophysis strains with southern M. rubrum were overcome using more favorable (1:20) Dinophysis: Mesodinium ratios. These subtleties highlight the ciliate strain-specific response to prey and its importance to mass production of M. rubrum and Dinophysis cultures.

Effect of the early temperature on the growth of larvae and postlarvae turbot, Scophthalmus maximus L.: muscle structural and ultrastructural study.

Turbot specimens were kept at three temperatures (T s ): warm (W) (21-22 °C), ambient (A) (17-18 °C) and cold (C) (13-14 °C) during the larval and early postlarval stages. At 90 days posthatching (dph), all of them were transferred to ambient T until 190 dph. At 2-3 dph, the specimens showed a monolayer of red muscle and immature white fibres; external or dermomyotome cells (presumptive myogenic cells) were observed on the surface of the red muscle. In the following stages, many myogenic cells and presumptive myogenic precursors were observed within the myotome, presumably derived of the dermomyotome. When comparing the growth at the same age (2, 10, 25, 37 dph), the body length and the muscle growth were positively influenced by the warm T, being the hyperplasia the muscle parameter more significantly influenced. The development rate was also positively correlated with the high T: the beginning of the metamorphosis took place at 15, 23 and 25 dph at W, A and C temperatures, respectively, with the highest body length values at ambient temperature. The metamorphosis finished at 25, 30 and 37 dph at W, A and C temperatures, respectively, with the highest body length values at warm temperature. However, the muscle cellularity was similar in all the groups at the end of the metamorphosis. At 90 and 190 dph, the largest body length was observed at W temperature. However, the muscle cellularity was similar between A and W; the number of fibres was similar in all the groups at 190 dph, which shows the beginning of a compensatory muscle growth in A and C, mainly in A.

Cannibalistic behavior of octopus (Octopus vulgaris) in the wild.

The first description of cannibalism in wild adult Octopus vulgaris is presented from 3 observations made in the Ría de Vigo (NW Spain), which were filmed by scuba divers. These records document common traits in cannibalistic behavior: (a) it was intercohort cannibalism; (b) attacks were made by both males and females; (c) in 2 of the records, the prey were transported to the den, which was covered with stones of different sizes; (d) the predator started to eat the tip of the arms of its prey; (e) predation on conspecifics occurred even if there were other abundant prey available (i.e., mussels); and (f) the prey/predator weight ratio in the 3 cases ranged from 20% to 25% body weight. The relationships between this behavior and sex, defense of territory, energy balance, food shortage, competition and predation, as well as how the attacker kills its victim are discussed.

Analysis of qPCR reference gene stability determination methods and a practical approach for efficiency calculation on a turbot (Scophthalmus maximus) gonad dataset.

BACKGROUND: Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry. RESULTS: We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency. CONCLUSION: Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot.