Arturo P. Sierra-Beltrán

MORPHOLOGY AND TAXONOMY OF PROROCENTRUM MEXICANUM AND REINSTATEMENT OF PROROCENTRUM RHATHYMUM (DINOPHYCEAE)1

Dinoflagellates collected during red tide events in Bahia Mazatlan, Mexico during the early spring of 1999 and 2000 appeared under LM to belong to Prorocentrum mexicanum Osorio‐Tafall. Observations with SEM of those populations showed marked differences in shape and microornamentation from the related species, Prorocentrum rhathymum Loeblich III, Sherley and Schmidt. In P. mexicanum, the presence and dimensions of poroids, the uneven distribution of trichocyst pores not located in depressions, and the general architecture of the periflagellar region are more closely related to Prorocentrum caribbaeum Faust. Also, P. mexicanum has a three‐horned (sometimes two‐horned) spine and is deeper in the anterior than the posterior region, whereas P. rhathymum has a simple small spine and its sagittal view is oval. Furthermore, the number and distribution of trichocyst pores in the periflagellar area is different between the two species, being located on both valves in P. mexicanum and only on the right valve in P. rhathymum. To date, true P. mexicanum has been described only from plankton sampling, whereas P. rhathymum was frequently mentioned associated with floating detritus (macroalgae) but also forming red tides. Altogether, the evidence presented demonstrates that P. mexicanum (planktonic) and P. rhathymum (epibenthic) are distinct species and are not synonyms, as is often accepted.

Trypsin Synthesis and Storage as Zymogen in the Midgut Gland of the Shrimp Litopenaeus Vannamei

Abstract An immunological approach was used to elucidate whether trypsin is synthesized and stored as trypsinogen in the midgut gland of the shrimp Litopenaeus vannamei. Two peptides were constructed using sequences deduced from known shrimp genes: trypsinogen activation peptide and an internal sequence. These peptides were used as haptens to elicit antibodies in rabbits. Specific antibodies were used to detect trypsinogen by Western blot and in histological sections of the midgut gland. Trypsinogen was found by Western blot and was localized into the midgut gland B cells by using immunohistology. In fed shrimp, trypsinogen associated with food particles was found in the lumen of the midgut gland tubules as well. Our results show that regulation of shrimp trypsin activity is similar to that of frequent feeder species, in which trypsin is stored as a zymogen, waiting for secretion and activation.

Toxic events in the Northwest Pacific coastline of Mexico during 1992–1995: origin and impact

Previously considered as toxin-free, the Baja California Peninsula has witnessed several toxic algal blooms during the past three years. Apparently these ‘red-tide’ phenomenas outbreaks are not linked to any human related activity. This may just reflect better detection and training. Such events may be periodical and natural rather than induced. The most common types of marine toxins have been detected along the coast of the Peninsula and neighboring waters by mouse bioassay and chromatographic techniques. These are: Tetrodotoxin (TTX), Amnesic Shellfish Poison (ASP), Paralytic Shellfish Poisons (PSP), Diarrhetic Shellfish Poisons (DSP) and even Ciguatera (CFP), which are related to the presence of organisms of Prorocentrum sp. and Alexandrium sp. groups, and the diatom Pseudonitzschia sp. among others. There are also some indications about different kinds of TTX in the puffer fish of the region, and reasons to believe that we are facing a quite different pattern in toxic components, since PSP toxic potency (defined as the number of mouse units per gram(MU/g)of shellfish meat) is very high in spite of low dinoflagellates cell density registered. The ecological and social impact of the above has been considerable, with mass deaths of shellfish, seagulls, dolphins and turtles, and even some human casualties. The locally registered toxicity records: PSP found in one single fanshell reaches to 23 000 MU/100 g of tissue as determined by the mouse bioassay and, on a different event, two persons killed after ingesting puffer fish fillet. The largest reservoir of commercial marine organisms in Mexico is precisely the Northwest coast of the country and important plans for building large harbors and develop aquaculture areas are in progress. Therefore, a monitoring program is essential for an adequate management of such resources. Considering the large extension of the Peninsula (about1600 km)and, at this time, the lack of efficient communication means and scarce population, the implementation of such monitoring programs presents a big challenge.

Biotoxins from Freshwater and Marine Harmful Algal Blooms Occurring in Mexico

Mexican coasts and watersheds are among the places with the greatest marine toxin diversity worldwide. Toxins produced by harmful algal blooms (HAB) have severely affected the environment in Mexico, even causing several human casualties. HAB toxins chemically proven to date in Mexican waters include: amnesic shellfish poisoning (ASP), ciguatera fish poisoning (CFP), several cyanobacterial toxins (CTXs), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP), and paralytic shellfish poisoning (PSP). Species producing other toxins (azaspiracids, gymnodimine, pectenotoxins, prorocentrolides, yessotoxins) have also been detected in Mexican waters. HAB-related poisoning is often lost from official statistics by designation as diarrhea with indeterminate origin.

Vitellogenin in black turtle (Chelonia mydas agassizii): Purification, partial characterization, and validation of an enzyme‐linked immunosorbent assay for its detection

Black turtle plasmatic vitellogenin (VTG) was purified from 17beta-estradiol-induced males using ion-exchange chromatography. The isolated protein was identified as VTG by its glycolipoprotein nature and amino acid sequence homology with other vertebrate VTG. It was characterized as a 500-kDa dimer composed of two identical, 200- to 240-kDa monomers. Polyclonal antibodies raised against black turtle VTG showed high titer and specificity, as demonstrated by enzyme-linked immunosorbent assay and Western blot analysis, respectively. The range of the assay was estimated to be between 15 ng/ml and 2 microg/ml, and the inter- and intra-assay coefficients of variation were 9.4 and 7.3%, respectively. Black turtle antibody cross-reacted with VTG of two other sea turtle species, Caretta caretta (loggerhead) and Eretmochelys imbricata (hawksbill), extending the applicability of the assay as part of a sea turtle health assessment program.

Are Prorocentrum hoffmannianum and Prorocentrum belizeanum (DINOPHYCEAE, PROROCENTRALES), the same species? An integration of morphological and molecular data.

The taxonomic assignment of Prorocentrum species is based on morphological characteristics; however, morphological variability has been found for several taxa isolated from different geographical regions. In this study, we evaluated species boundaries of Prorocentrum hoffmannianum and Prorocentrum belizeanum based on morphological and molecular data. A detailed morphological analysis was done, concentrating on the periflagellar architecture. Molecular analyses were performed on partial Small Sub‐Unit (SSU) rDNA, partial Large Sub‐Unit (LSU) rDNA, complete Internal Transcribed Spacer Regions (ITS1‐5.8S‐ITS2), and partial cytochrome b (cob) sequences. We concatenated the SSU‐ITS‐LSU fragments and constructed a phylogenetic tree using Bayesian Inference (BI) and maximum likelihood (ML) methods. Morphological analyses indicated that the main characters, such as cell size and number of depressions per valve, normally used to distinguish P. hoffmannianum from P. belizeanum, overlapped. No clear differences were found in the periflagellar area architecture. Prorocentrum hoffmannianum and P. belizeanum were a highly supported monophyletic clade separated into three subclades, which broadly corresponded to the sample collection regions. Subtle morphological overlaps found in cell shape, size, and ornamentation lead us to conclude that P. hoffmanianum and P. belizeanum might be considered conspecific. The molecular data analyses did not separate P. hoffmannianum and P. belizeanum into two morphospecies, and thus, we considered them to be the P. hoffmannianum species complex because their clades are separated by their geographic origin. These geographic and genetically distinct clades could be referred to as ribotypes: (A) Belize, (B) Florida‐Cuba, (C1) India, and (C2) Australia.

A novel design of a multi-antigenic, multistage and multi-epitope vaccine against Helicobacter pylori: An in silico approach

Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148-158, 188-198), cytotoxin-associated gene A (residue 584-602), neutrophil activating protein (residue 4-28), vacuolating cytotoxin gene A (residue 63-81), H. pylori adhesine A (residue77-99), heat shock protein A (residue 32-54) and gamma glutamyl transpeptidase (residue 271-293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.