Reproductive biology of the totoaba (Totoaba macdonaldi), an endangered species in the Gulf of California.

The totoaba (Totoaba macdonaldi) is an emblematic species endemic to the Gulf of California, which has been protected since the mid-70 s by Mexican and international laws. For the present study, 153 totoaba individuals were collected from 2011 to 2014, on which gonad histology analyses were carried out. We identified six ovarian phases and eleven oocyte subphases in the gonad development of females; in males, four development phases were identified. The totoaba is an iteroparous species based on the number of breeding opportunities. According to the organization of the ovary and the presence of oocyte subphases, ovarian dynamics can be considered asynchronous, as demonstrated by the multimodal distribution of oocyte diameter. The totoaba is a batch spawner that releases oocytes over several months. The reproductive period spans from February to May, with a reproductive peak in March. Likewise, the gonadosomatic index showed that the reproductive activity of totoaba peaked in March and April. The female:male sex ratio, although variable throughout the sampling period, yielded an overall value of 0.89:1, with non-significant differences between the number of females and males (P > 0.05). The length at first sexual maturity (L50%) was 1 354 mm TL for females and 1 299 mm TL for males. The average fecundity was 2 662 626 (standard error, s.e., 1 021 124) oocytes, and the average relative fecundity was 99 (s.e. 22) oocytes g-1 (ovary-free). The mean and maximum oocyte diameter showed a positive and non-significant (P > 0.05) relationship with the mean sea surface temperature.

Effect of dissolved metabolites of the dinoflagellate Gymnodinium catenatum (Graham, 1943) on the white shrimp Litopenaeus vannamei (Boone, 1931): A histological study.

Harmful algae blooms (HABs) are a conspicuous phenomenon that affect the coastal zone worldwide. Aquaculture industry zones are not excluded from being affected by HAB that cause organism mortality and jeopardize their innocuity due to the contamination by phytotoxins with the concomitant economic losses. Direct ingestion of metabolites from HAB species or organisms contaminated with phycotoxins together with dermal absorption of dissolved metabolites (DM), including toxins, are the two main routes of poisoning. From these poisoning routes, the effect of DM, particularly paralytic shellfish toxins (PST), has been relatively understudied. This intoxication route can be conspicuous and could be involved in many significant mortalities of cultivated marine organisms. In this study, white shrimp juveniles (2.1 g wet weight) of Litopenaeus vannamei were exposed to extracts of 104, 105 and 106 cells/L of the dinoflagellate Gymnodinium catenatum, a PST producer. The experiment ended after 17 h of exposure when shrimps exposed to 106 cells/L extract started to die and the rest of the shrimps, from this and other treatments, did not respond to gentle physical stimulus and their swimming activity was low and erratic. Toxin concentrations were determined using high performance liquid chromatography while qualitative and quantitative histological damages were assessed on the tissues. In general, most toxins were accumulated in the hepatopancreas where more than 90% were found. Other tissues such as intestine, muscle, and gills contained less than 10% of toxins. Compared to the control, the main significative tissue damages were, loss of up to 80% of the nerve cord, 40% of the muscle coverage area, and reduction of the gill lamella width. Also, atrophy in hepatopancreas was observed, manifested by a decrease in the height of B cells, lumen degeneration and thinning of tubules. Some damages were more evident when shrimps were exposed to higher concentrated extracts of G. catenatum, however, not all damages were progressive and proportional to the extract concentration. These data confirm that PST dissolved enter the shrimp, possibly via the gills, and suggest that dissolved metabolites, including PST, may cause tissue damage. Other dissolved metabolites produced by G. catenatum, alone or in synergy, may also be involved. These results also pointed out the importance of dissolved molecules produced for this dinoflagellate and the potential effect on cultured shrimp.

First subcellular localization of the amnesic shellfish toxin, domoic acid, in bivalve tissues: Deciphering the physiological mechanisms involved in its long-retention in the king scallop Pecten maximus.

Domoic acid (DA), the phycotoxin responsible for amnesic shellfish poisoning (ASP), is an excitatory amino acid naturally produced by at least twenty-eight species of the bloom-forming marine diatoms Pseudo-nitzschia spp. Suspension feeders, such as bivalve mollusks, can accumulate and lengthy retain high amounts of DA in their tissues, threatening human health and leading to extensive-prolonged fishery closures, and severe economic losses. This is particularly problematic for the king scallop Pecten maximus, which retains high burdens of DA from months to years compared to other fast-depurator bivalves. Nonetheless, the physiological and cellular processes responsible for this retention are still unknown. In this work, for the first time, a novel immunohistochemical techniques based on the use of an anti-DA antibody was successfully developed and applied for DA-detection in bivalve tissues at a subcellular level. Our results show that in naturally contaminated P. maximus following a Pseudo-nitzschia australis outbreak, DA is visualized mainly within small membrane-bounded vesicles (1 - 2.5 µm) within the digestive gland cells, identified as autophagosomic structures by means of immune-electron microscopy, as well as in the mucus-producing cells, particularly those from gonad ducts and digestive tract. Trapping of DA in autophagososomes may be a key mechanism in the long retention of DA in scallops. These results and the development of DA-immunodetection are essential to provide a better understanding of the fate of DA, and further characterize DA contamination-decontamination kinetics in marine bivalves, as well as the main mechanisms involved in the long retention of this toxin in P. maximus.

Assessment of bacteriophage vB_Pd_PDCC-1 on bacterial dynamics during ontogenetic development of the longfin yellowtail (Seriola rivoliana).

The Seriola genus includes species of worldwide commercial importance due to its rapid growth and easy adaptability to confinement conditions. However, like other fish species, large mortalities occur during their early life stages, where the main problems are caused by opportunistic bacteria. Disease control strategies are thus urgently needed. The present study aimed to evaluate the efficacy of phage vB_Pd_PDCC-1 during the early development of longfin yellowtail (Seriola rivoliana), as well as its effect on microbial communities. This broad-host-range phage was added to the culture every 3 days starting from the egg-stage until 12 days after hatching (DAH) at a concentration of 1.41×1010 plaque-forming units (PFU) per mL and at a multiplicity of infection (MOI) of 1. The results showed positive effects (p<0.05) on egg hatching, survival, growth, and pigmentation area in treated larvae. Moreover, high-throughput sequencing analysis of 16S rRNA genes showed that phage administration did not produce significant changes (p>0.05) in the composition and structure of the associated microbiota. However, sequences affiliated to the Gammaproteobacteria class were displaced by those belonging to the Alphaproteobacteria class over time regardless of the treatment received. At the family level, there was a decrease in Rhodobacteraceae, Pseudoalteromonadaceae, and Flavobacteriaceae in both groups over time. To our best knowledge, this study represents the first attempt to evaluate the effect of a phage as a biological control agent during ontogenetic development of longfin yellowtail larvae. KEY POINTS: • Phages can be used against proliferation of Vibrio in fish cultures. • Seriola includes several important commercial fish species due to its rapid growth. • Phages do not cause significant changes in the associated microbiota.

Reduction of spermatophore melanization in Litopenaeus vannamei shrimp fed Ulva clathrata during a commercial hatchery production.

Male broodstock (Litopenaeus vannamei, 36 ± 7 g, n = 600) reproductive performance, spermatophores and reproductive tract melanization, prostaglandin concentrations and biochemical composition were evaluated after including 3.8% Ulva clathrata meal in the diet (dry base) of a commercial hatchery during 45 days. Males fed Ulva had less melanized spermatophores (120 compared with 233, P < 0.01), less bacteria in the ductus deferens (P < 0.01), more sperm cells in testicles (P < 0.05), and increased courtship activity (839 compared with 689, P < 0.01), with no effect on mortality. Ulva-fed males had more arachidonic acid (20:4n-6) in the spermatophores (P < 0.05) but this did not affect the prostaglandin concentrations in response to diet. Males fed Ulva had more carotenoids in the hepatopancreas (0.08 ± 0.02 compared with 0.01 ± 0.01 mg/g, P < 0.05), and phenolic compounds in hepatopancreas (6.1 ± 0.7 compared with 1.8 ± 0.7 mg eq. phloroglucinol/g, P < 0.05) and muscle (0.4 ± 0.3 compared with 0.2 ± 0.1 mg eq. phloroglucinol/g, P < 0.05). Males fed the Ulva also had a lesser carbohydrate content in the hepatopancreas (P < 0.01) and muscle (P < 0.01). In conclusion, supplementing fresh maturation diets with a small dose of dried Ulva allowed for improvement of reproductive performance and to decrease melanization in spermatophores and the male reproductive tract.

Transcriptomic analysis of Pacific white shrimp (Litopenaeus vannamei, Boone 1931) in response to acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by marine bacteria Vibrio Parahaemolyticus, is a huge problem in shrimp farms. The V. parahaemolyticus infecting material is contained in a plasmid which encodes for the lethal toxins PirABVp, whose primary target tissue is the hepatopancreas, causing sloughing of epithelial cells, necrosis, and massive hemocyte infiltration. To get a better understanding of the hepatopancreas response during AHPND, juvenile shrimp Litopenaeus vannamei were infected by immersion with V. parahaemolyticus. We performed transcriptomic mRNA sequencing of infected shrimp hepatopancreas, at 24 hours post-infection, to identify novel differentially expressed genes a total of 174,098 transcripts were examined of which 915 transcripts were found differentially expressed after comparative transcriptomic analysis: 442 up-regulated and 473 down-regulated transcripts. Gene Ontology term enrichment analysis for up-regulated transcripts includes metabolic process, regulation of programmed cell death, carbohydrate metabolic process, and biological adhesion, whereas for down-regulated transcripts include, microtubule-based process, cell activation, and chitin metabolic process. The analysis of protein- protein network between up and down-regulated genes indicates that the first gene interactions are connected to oxidation-processes and sarcomere organization. Additionally, protein-protein networks analysis identified 20-top highly connected hub nodes. Based on their immunological or metabolic function, ten candidate transcripts were selected to measure their mRNA relative expression levels in AHPND infected shrimp hepatopancreas by RT-qPCR. Our results indicate a close connection between the immune and metabolism systems during AHPND infection. Our RNA-Seq and RT-qPCR data provide the possible immunological and physiological scenario as well as the molecular pathways that take place in the shrimp hepatopancreas in response to an infectious disease.

Effect of Homeopathic Medicines on Intestinal Coccidia and Immune Response Cells in Spotted Rose Snapper (Lutjanus guttatus).

BACKGROUND: Homeopathy has been widely applied in freshwater species but rarely in marine fish. Farm stress destabilises host-pathogen equilibrium, favouring parasites and disease. Coccidian endoparasites cause intestinal infections and cell degeneration. MATERIALS AND METHODS: Naturally parasite-infested juvenile snapper Lutjanus guttatus (n = 430; weight 1.9 ± 0.01 g; length 4.9 ± 0.03 cm) specimens were distributed in group sizes of 43, in 10 fiberglass tanks (100 L) with aeration and continuous water change. Five groups in duplicate were assessed: Passival (PaV); Passival and Phosphoricum acid Similia (PaV-PhA); Passival and Silicea terra Similia (PaV-SiT); Endecto and Infecçoes (End-Inf) and a control (Ethanol) for 45 days. Feed was sprinkled with treatment (5% v/w) and dried to avoid ethanol side-effects. Statistical results were expressed as mean ± standard error of the mean. RESULTS: Intestinal coccidia were recorded histopathologically, with the least incidence attained at T45 with End-Inf treatment (p < 0.001), coinciding with the highest lymphocyte (p = 0.015) count. Fish treated with PaV-PhA recorded a higher percentage of neutrophils (p = 0.015), and those treated with PaV-PhA, PaV-SiT and End-Inf revealed a decrease in lesions, degree of alteration and change in intestine and stomach tissues (p < 0.05). The number of mucous cells in gills was greater (p < 0.001) for End-Inf (43 ± 0.58), PaV-SiT (40 ± 2.89) and PaV-PhA (39 ± 3.46) as compared respectively with T0 and control (19 ± 0.58 and 28 ± 2.31). PaV and PaV-SiT increased carbohydrate reserves in liver (28.4 ± 1.39% and 22.4 ± 0.12%, respectively) compared with T0 and control (3.4 ± 0.32 and 5.6 ± 0.66%). The highest survival rate was 97.7 ± 0.16% in fish treated with PaV and End-Inf. CONCLUSION: Homeopathic treatments had a positive effect on fish health. Besides the accumulation of metabolic reserves in the liver, homeopathic treatment was associated with mucin increase in gills as well as raised production of lymphocytes and neutrophils in blood, which could act as macrophages against intestinal coccidia.

Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei.

Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular) exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies.

Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus.

Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-D-glucosamine, α-D-glucose, α-D-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops.

[Lipid variation in oocytes of the jellyfish Stomolophus meleagris (Scyphozoa: Rhizostomeae) from Las Guasimas Lagoon, Mexico, during gonadal development]. / Variación lipídica en los ovocitos de la medusa Stomolophus meleagris (Scyphozoa: Rhizostomeae), durante el desarrollo gonádico, en la laguna Las Guásimas, Sonora, México.

The jellyfish Stomolophus meleagris has potential for commercial exploitation but there is little information on their reproductive biology. This paper seeks to evaluate some biochemical and demographic characteristics of the species. Samples were taken monthly during 2005 and 2006. Jellyfish collected in 2005 were used to describe the characteristics and quantity of oocyte triglycerides and phospholipids with the Sudan black technique, and to ascertain the degree of gonadal development and sex ratio by the hematoxylin-eosin technique. The 2006 jellyfish were used to determine the size at first maturity and protein and total lipids contents. Four stages of development in both sexes were determined, with a continuous gamete development. The highest percentage of mature organisms was recorded in April. The proportion of sexes was 0.7:1.3. We found higher concentrations of triglycerides than phospholipids in the cytoplasm. There was a positive correlation between triglycerides and the diameter of the oocyte. The size at first maturity for both sexes was 105 mm. The highest protein and lipids contents were obtained in April and March respectively.

Variación lipídica en los ovocitos de la medusa Stomolophus meleagris (Scyphozoa: Rhizostomeae), durante el desarrollo gonádico, en la laguna Las Guásimas, Sonora, México

Lipid variation in oocytes of the jellyfish Stomolophus meleagris (Scyphozoa: Rhizostomeae) from Las Guasimas Lagoon, Mexico, during gonadal development. The jellyfish Stomolophus meleagris has potential for commercial exploitation but there is little information on their reproductive biology. This paper seeks to evaluate some biochemical and demographic characteristics of the species. Samples were taken monthly during 2005 and 2006. Jellyfish collected in 2005 were used to describe the characteristics and quantity of oocyte triglycerides and phospholipids with the Sudan black technique, and to ascertain the degree of gonadal development and sex ratio by the hematoxylin-eosin technique. The 2006 jellyfish were used to determine the size at first maturity and protein and total lipids contents. Four stages of development in both sexes were determined, with a continuous gamete development. The highest percentage of mature organisms was recorded in April. The proportion of sexes was 0.7:1.3. We found higher concentrations of triglycerides than phospholipids in the cytoplasm. There was a positive correlation between triglycerides and the diameter of the oocyte. The size at first maturity for both sexes was 105mm. The highest protein and lipids contents were obtained in April and March respectively. Rev. Biol. Trop. 58 (1): 119-130. Epub 2010 March 01.

La medusa S. meleagris, ha mostrado potencial de explotación pero hay escasa información sobre su biología reproductiva. El presente trabajo pretende conocer el contenido de los triglicéridos y fosfolípidos en los ovocitos durante el desarrollo gonadal, así como la proporción de sexos, talla de primera madurez y la concentración de proteínas y lípidos totales en la medusa. Se realizaron muestreos mensuales durante 2005 y 2006. A las medusas recolectadas en 2005, se les aplicó la técnica del sudán negro, para describir las características del ovocito y la cantidad triglicéridos y fosfolípidos y la técnica de hematoxilina-eosina para conocer el grado de desarrollo gonádico y la proporción de sexos. Las medusas del 2006 se emplearon para determinar la talla de primera madurez y el contenido de proteínas y lípidos totales. Se observaron cuatro fases de desarrollo en ambos sexos, con un desarrollo gamético continúo. El mayor porcentaje de organismos maduros se registró en abril. La proporción de sexos fue de 0.7:1.3. Se encontró mayor concentración de triglicéridos que de fosfolípidos en el citoplasma. Se obtuvo una correlación positiva entre triglicéridos y el diámetro del ovocito. La talla de primera madurez para ambos sexos fue de 105 mm. El mayor contenido de proteínas se obtuvo en abril y para lípidos en marzo.