Hidden species diversity and mito-nuclear discordance within the Mediterranean cone snail, Lautoconus ventricosus.

The Mediterranean cone snail, Lautoconus ventricosus, is currently considered a single species inhabiting the whole Mediterranean basin and the adjacent Atlantic coasts. Yet, no population genetic study has assessed its taxonomic status. Here, we collected 245 individuals from 75 localities throughout the Mediterranean Sea and used cox1 barcodes, complete mitochondrial genomes, and genome skims to test whether L. ventricosus represents a complex of cryptic species. The maximum likelihood phylogeny based on complete mitochondrial genomes recovered six main clades (hereby named blue, brown, green, orange, red, and violet) with sufficient sequence divergence to be considered putative species. On the other hand, phylogenomic analyses based on 437 nuclear genes only recovered four out of the six clades: blue and orange clades were thoroughly mixed and the brown one was not recovered. This mito-nuclear discordance revealed instances of incomplete lineage sorting and introgression, and may have caused important differences in the dating of main cladogenetic events. Species delimitation tests proposed the existence of at least three species: green, violet, and red + blue + orange (i.e., cyan). Green plus cyan (with sympatric distributions) and violet, had West and East Mediterranean distributions, respectively, mostly separated by the Siculo-Tunisian biogeographical barrier. Morphometric analyses of the shell using species hypotheses as factor and shell length as covariate showed that the discrimination power of the studied parameters was only 70.2%, reinforcing the cryptic nature of the uncovered species, and the importance of integrative taxonomic approaches considering morphology, ecology, biogeography, and mitochondrial and nuclear population genetic variation.

Chromosome-level genome of the venomous snail Kalloconus canariensis: a valuable model for venomics and comparative genomics.

BACKGROUND: Genomes are powerful resources to understand the evolutionary mechanisms underpinning the origin and diversification of the venoms of cone snails (Conidae: Caenogastropoda) and could aid in the development of novel drugs. FINDINGS: Here, we used PacBio continuous long reads and Omni-C data to assemble the chromosome-level genome of Kalloconus canariensis, a vermivorous cone endemic to the Canary Islands. The final genome size was 2.87 Gb, with a N50 of 79.75 Mb and 91% of the reads located into the 35 largest scaffolds. Up to 55.80% of the genome was annotated as repetitive regions, being class I of transposable elements (16.65%) predominant. The annotation estimated 34,287 gene models. Comparative analysis of this genome with the 2 cone snail genomes released to date (Dendroconus betulinus and Lautoconus ventricosus) revealed similar genome sizes and organization, although chromosome sizes tended to be shorter in K. canariensis. Phylogenetic relationships within subclass Caenogastropoda were recovered with strong statistical support. The family Conidae was recovered as a clade, with K. canariensis plus L. ventricosus sister to D. betulinus. CONCLUSIONS: Despite the great diversity of cone snails (>900 species) and their venoms (hundreds of peptides per species), only 2 recently reported genomes are available for the group. The high-quality chromosome-level assembly of K. canariensis will be a valuable reference for studying the origin and evolution of conotoxin genes as well as whole-genome duplication events during gastropod evolution.

The genome of the venomous snail Lautoconus ventricosus sheds light on the origin of conotoxin diversity.

BACKGROUND: Venoms are deadly weapons to subdue prey or deter predators that have evolved independently in many animal lineages. The genomes of venomous animals are essential to understand the evolutionary mechanisms involved in the origin and diversification of venoms. RESULTS: Here, we report the chromosome-level genome of the venomous Mediterranean cone snail, Lautoconus ventricosus (Caenogastropoda: Conidae). The total size of the assembly is 3.59 Gb; it has high contiguity (N50 = 93.53 Mb) and 86.6 Mb of the genome assembled into the 35 largest scaffolds or pseudochromosomes. On the basis of venom gland transcriptomes, we annotated 262 complete genes encoding conotoxin precursors, hormones, and other venom-related proteins. These genes were scattered in the different pseudochromosomes and located within repetitive regions. The genes encoding conotoxin precursors were normally structured into 3 exons, which did not necessarily coincide with the 3 structural domains of the corresponding proteins. Additionally, we found evidence in the L. ventricosus genome for a past whole-genome duplication event by means of conserved gene synteny with the Pomacea canaliculata genome, the only one available at the chromosome level within Caenogastropoda. The whole-genome duplication event was further confirmed by the presence of a duplicated hox gene cluster. Key genes for gastropod biology including those encoding proteins related to development, shell formation, and sex were located in the genome. CONCLUSIONS: The new high-quality L. ventricosus genome should become a reference for assembling and analyzing new gastropod genomes and will contribute to future evolutionary genomic studies among venomous animals.

Comparative transcriptomics of the venoms of continental and insular radiations of West African cones.

The transcriptomes of the venom glands of 13 closely related species of vermivorous cones endemic to West Africa from genera Africonus and Varioconus were sequenced and venom repertoires compared within a phylogenetic framework using one Kalloconus species as outgroup. The total number of conotoxin precursors per species varied between 108 and 221. Individuals of the same species shared about one-fourth of the total conotoxin precursors. The number of common sequences was drastically reduced in the pairwise comparisons between closely related species, and the phylogenetical signal was totally eroded at the inter-generic level (no sequence was identified as shared derived), due to the intrinsic high variability of these secreted peptides. A common set of four conotoxin precursor superfamilies (T, O1, O2 and M) was expanded in all studied cone species, and thus, they are considered the basic venom toolkit for hunting and defense in the West African vermivorous cone snails. Maximum-likelihood ancestral character reconstructions inferred shared conotoxin precursors preferentially at internal nodes close to the tips of the phylogeny (between individuals and between closely related species) as well as in the common ancestor of Varioconus. Besides the common toolkit, the two genera showed significantly distinct catalogues of conotoxin precursors in terms of type of superfamilies present and the abundance of members per superfamily, but had similar relative expression levels indicating functional convergence. Differential expression comparisons between vermivorous and piscivorous cones highlighted the importance of the A and S superfamilies for fish hunting and defense.

Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones.

The transcriptome of the venom duct of the Atlantic piscivorous cone species Chelyconus ermineus (Born, 1778) was determined. The venom repertoire of this species includes at least 378 conotoxin precursors, which could be ascribed to 33 known and 22 new (unassigned) protein superfamilies, respectively. Most abundant superfamilies were T, W, O1, M, O2, and Z, accounting for 57% of all detected diversity. A total of three individuals were sequenced showing considerable intraspecific variation: each individual had many exclusive conotoxin precursors, and only 20% of all inferred mature peptides were common to all individuals. Three different regions (distal, medium, and proximal with respect to the venom bulb) of the venom duct were analyzed independently. Diversity (in terms of number of distinct members) of conotoxin precursor superfamilies increased toward the distal region whereas transcripts detected toward the proximal region showed higher expression levels. Only the superfamilies A and I3 showed statistically significant differential expression across regions of the venom duct. Sequences belonging to the alpha (motor cabal) and kappa (lightning-strike cabal) subfamilies of the superfamily A were mainly detected in the proximal region of the venom duct. The mature peptides of the alpha subfamily had the α4/4 cysteine spacing pattern, which has been shown to selectively target muscle nicotinic-acetylcholine receptors, ultimately producing paralysis. This function is performed by mature peptides having a α3/5 cysteine spacing pattern in piscivorous cone species from the Indo-Pacific region, thereby supporting a convergent evolution of piscivory in cones.

Effects of different slipping methods on the mortality of sardine, Sardina pilchardus, after purse-seine capture off the Portuguese Southern coast (Algarve).

The effects of two different slipping methods on the survival, physical and physiological response of sardines, Sardina pilchardus, captured in a purse-seine fishery were investigated in southern Portugal. Sardines were collected and transferred into holding tanks onboard a commercial fishing vessel after being captured, crowded and deliberately released using two slipping procedures: standard and modified. The standard slipping procedure aggregated fish at high densities and made them "roll over" the floatline, while the modified procedure aggregated the fish at moderate densities and enabled them to escape through an opening created by adding weights to the floatline. Both slipping methods were compared with minimally harmed non-slipped sardines (sardines collected from the loose pocket of the purse seine). Survival rates were monitored in captivity over 28 days using three replicates for each treatment. The estimated survival of sardines was 43.6% for the non-slipped fish, 44.7% for the modified slipping and 11.7% for the standard slipping treatments. Scale loss indicated the level of physical impact experienced, with dead fish from the non-slipped and modified slipping technique showing significantly lower scale loss than those fish from the standard slipping treatment within the same period. Of the physiological indicators of stress measured, cortisol, glucose, lactate and osmolality attained peak values during slipping and up to the first hours after introduction to captivity. This work indicates that although delayed mortality after release may be substantial, appropriately modified slipping techniques significantly enhance survival of slipped sardines.

Phylogenetic relationships of cone snails endemic to Cabo Verde based on mitochondrial genomes.

BACKGROUND: Due to their great species and ecological diversity as well as their capacity to produce hundreds of different toxins, cone snails are of interest to evolutionary biologists, pharmacologists and amateur naturalists alike. Taxonomic identification of cone snails still relies mostly on the shape, color, and banding patterns of the shell. However, these phenotypic traits are prone to homoplasy. Therefore, the consistent use of genetic data for species delimitation and phylogenetic inference in this apparently hyperdiverse group is largely wanting. Here, we reconstruct the phylogeny of the cones endemic to Cabo Verde archipelago, a well-known radiation of the group, using mitochondrial (mt) genomes. RESULTS: The reconstructed phylogeny grouped the analyzed species into two main clades, one including Kalloconus from West Africa sister to Trovaoconus from Cabo Verde and the other with a paraphyletic Lautoconus due to the sister group relationship of Africonus from Cabo Verde and Lautoconus ventricosus from Mediterranean Sea and neighboring Atlantic Ocean to the exclusion of Lautoconus endemic to Senegal (plus Lautoconus guanche from Mauritania, Morocco, and Canary Islands). Within Trovaoconus, up to three main lineages could be distinguished. The clade of Africonus included four main lineages (named I to IV), each further subdivided into two monophyletic groups. The reconstructed phylogeny allowed inferring the evolution of the radula in the studied lineages as well as biogeographic patterns. The number of cone species endemic to Cabo Verde was revised under the light of sequence divergence data and the inferred phylogenetic relationships. CONCLUSIONS: The sequence divergence between continental members of the genus Kalloconus and island endemics ascribed to the genus Trovaoconus is low, prompting for synonymization of the latter. The genus Lautoconus is paraphyletic. Lautoconus ventricosus is the closest living sister group of genus Africonus. Diversification of Africonus was in allopatry due to the direct development nature of their larvae and mainly triggered by eustatic sea level changes during the Miocene-Pliocene. Our study confirms the diversity of cone endemic to Cabo Verde but significantly reduces the number of valid species. Applying a sequence divergence threshold, the number of valid species within the sampled Africonus is reduced to half.

Mitogenomic phylogeny of cone snails endemic to Senegal.

Cone snails attain in Senegal one of their highest peaks of species diversity throughout the continental coast of Western Africa. A total of 15 endemic species have been described, all placed in the genus Lautoconus. While there is ample data regarding the morphology of the shell and the radular tooth of these species, virtually nothing is known regarding the genetic diversity and phylogenetic relationships of one of the most endangered groups of cones. In this work, we determined the complete or near-complete (only lacking the control region) mitochondrial (mt) genomes of 17 specimens representing 11 endemic species (Lautoconus belairensis, Lautoconus bruguieresi, Lautoconus cacao, Lautoconus cloveri, Lautoconus cf. echinophilus, Lautoconus guinaicus, Lautoconus hybridus, Lautoconus senegalensis, Lautoconus mercator, Lautoconus taslei, and Lautoconus unifasciatus). We also sequenced the complete mt genome of Lautoconus guanche from the Canary Islands, which has been related to the cones endemic to Senegal. All mt genomes share the same gene arrangement, which conforms to the consensus reported for Conidae, Neogastropoda and Caenogastropoda. Phylogenetic analyses using probabilistic methods recovered three major lineages, whose divergence coincided in time with sea level and ocean current changes as well as temperature fluctuations during the Messinian salinity crisis and the Plio-Pleistocene transition. Furthermore, the three lineages corresponded to distinct types of radular tooth (robust, small, and elongated), suggesting that dietary specialization could be an additional evolutionary driver in the diversification of the cones endemic to Senegal. The reconstructed phylogeny showed several cases of phenotypic convergence (cryptic species) and questions the validity of some species (ecotypes or phenotypic plasticity), both results having important taxonomic and conservation consequences.

Drug allergy and asthma.

The prevalence of drug induced asthma in a series of 347 patients with drug induced adverse effects has been evaluated corresponding to 10% of drug adverse effects always due to NSAIDS.

Drug allergy and pollinosis. A short report.

2 series of patients with pollinosis and drug allergy studied 10 years apart and comprising 115 cases are presented. In both series grass pollens and parietaria are as usual the most common cause of pollinosis but Parietaria was more common in the first series. Beta-lactams were the major cause of drug allergy in the first group, supersed by NSAID in the second group. Rhinitis was the more frequent symptom on pollinosis and urticaria/angioedema on drug allergy.

"In vivo" and "in vitro" tests in the diagnosis of Beta-lactams allergy.

The results of "in vivo" and "in vitro" diagnostic tests in 114 patients reporting an allergic reaction to Beta-lactams are presented. Skin test gave an overall positivity of 85% and determination of specific IgE of 42%. Skin tests have a greater sensitivity but "in vitro" tests are an useful associated diagnostic method.

Alpha-1-antitrypsin deficiency asthma and allergy.

The biochemistry, genetics and pathology of alpha-1-anti-trypsin deficiency are reviewed. The geographical distribution in Europe of more current phenotypes M, SZ is discussed. Two cases of alpha-1- anti-trypsin are presented one homozygotic ZZ non-smoker without any respiratory pathology and one heterozygotic SZ heavy smoker with a severe chronic obstructive pulmonary disease and reversibility to Beta-2-mimetics suggesting asthma. The relationship between alpha-1-antitrypsin and asthma is discussed and general measures of treatment or prevention suggested.

"In vivo" and "in vitro": tests in the diagnosis of trichophyton allergy.

A group of 100 patients with Trichophyton allergy and a control of group of 100 without fungal allergy have been compared in order to evaluate the diagnostic value of skin prick and intradermal tests and assay of Trichophyton specific IgE. The evaluation of sensitivity, specificity efficacy, positive and negative predictive values suggests that skin tests in two steps, prick and intradermal and research of specific IgE must be used to a better diagnostic approach. In cases were allergy is not cleared by antifungal therapy specific immunotherapy is worthwhile to be tried with a great percentage of success.

Clinical efficacy and safety of preseasonal sublingual immunotherapy with grass pollen carbamylated allergoid in rhinitic patients. A double-blind, placebo-controlled study.

BACKGROUND: The aim of this study was to confirm the clinical efficacy and safety of a preseasonal sublingual immunotherapy (SLIT) in a group of allergic patients with seasonal rhinoconjunctivitis with or without mild intermittent or mild persistent asthma. The immunotherapy was administered through the oral mucosa with a monomeric carbamylated allergoid (allergoid SLIT) for grass pollens. A secondary endpoint was to evaluate the effect of the allergoid SLIT on nasal reactivity. METHODS AND RESULTS: A single-center, randomized, double-blind, placebo-controlled study was performed. Patients were selected and randomly allocated to two groups: one group received active treatment (allergoid SLIT) for 2 years and the other received placebo. Both groups received the necessary drug treatment throughout the trial. Thirty-three outpatients (20 men and 13 women, mean age: 30 years; range: 19-43) attending our center were enrolled in the study. Symptoms and medications were scored on diary cards during the pollen season. An allergen nasal challenge was performed at baseline and after 2 years of SLIT to evaluate nasal reactivity. Because the clinical scores were non-normally distributed, the Mann-Whitney and the Chi-square tests for intergroup comparisons and the Wilcoxon test for intragroup comparisons were used. The results were evaluated after 1 and 2 years of treatment. Between the first and second years of treatment, no changes in the scores for the placebo group were found, while for the active vaccine group significant decreases were found in rhinorrhea (p < 0.03), sneezing (p < 0.03), and conjunctivitis (p < 0.02). Symptom scores after nasal challenge decreased (p < 0.03) after 2 years' treatment. Nasal steroid use significantly decreased in the active treatment group during May and June in both the years of treatment (p < 0.02). Only two mild local adverse events were reported in the active group and none was reported in the placebo group. CONCLUSIONS: The results of this study show that the allergoid SLIT is safe and effective in decreasing symptom scores and drug use in rhinitic patients allergic to grass pollen.

Prevalence of drug allergy in an out-patient population.

The prevalence of drug allergy in an out-patient population has been studied, 448 cases were diagnosed mainly to NSAIDS and beta-lactams and local anesthetics, sulfa drugs, quinolones and others. Clinical history and skin tests are sufficient to diagnose most cases of drug allergy.

Trichophyton allergy: review of 89 cases.

A serie of 89 cases (55 men 34 women) of Tricophyton allergy is presented. All the patients had a clinical history and in 86 out of 89 lesions of dermatophytosis urticaria/angioedema was present in 57, eczema in 8, rhinitis in 12, conjunctivitis in 1 and asthma in 12. Skin prick tests to Tricophyton were positive in 53 (59,6%) of the patients and intradermal test in all. Specific IgE was found in 45 (69,3%) out of 70 patients. Antifungal treatment was effective at mean term in 18 cases. Specific immunotherapy was evaluated in 54 with good or very good results in 41 (83,3%). Tricophyton dermatophytosis must be looked for in all the cases of urticaria/angioedema and most cases of respiratory allergy without clear allergy to inhalants.

Skin tests in NSAIDS hypersensitivity.

Prick tests are more specific but less sensitive than ID which can give false positive results. 3 NSAIDS groups can be considered: Aspirin, diclofenac and metamizol with a higher percentage of positivity. For others NSAIDS including the newer coxibs positivity around 50%. Nimesulid and acetaminophen with a lower of positivity. Skin tests can be used in NSAIDS hypersensitivity to confirm clinical history but also to choose, case by case, a drug less prone to give hypersentivity reactions.

Monoid sublingual immunotherapy.

Sublingual monoid immunotherapy with monomeric allergoids has been largely used in Europe in the last few years. An open trial of allergoid in tablets has been done in rhinitic patients allergic to house dust mites, grass pollens and Parietaria with clear improvement in clinics and drug consumption scores. In a second phase a double blind placebo controlled trial of grass pollens allergoids have been done in hay fever patients with significant decrease on the scores of rhinorrea, sneezing and conjunctivitis nasal steroid consumption and clinical score after serial nasal challenges. Monomeric allergoids are an efficace and safe immunotherapy in allergic rhinitis.

Molecular systematics of Hispaniolan pupfishes (Cyprinodontidae: Cyprinodon): implications for the biogeography of insular Caribbean fishes.

We used sequence variation in the mtDNA control-region and ND2 and cyt b genes to assess the systematics and biogeography of the five species of pupfish (Cyprinodon) on Hispaniola. These include four endemics, the relatively large-bodied Cyprinodon bondi, Cyprinodon nichollsi, and Cyprinodon sp., each from a separate lake in southwestern Hispaniola, and Cyprinodon higuey from a coastal lake in eastern Hispaniola. The fifth species consists of coastal populations referable to Cyprinodon variegatus riverendi. The results indicate that Hispaniola has been invaded by at least two forms, first by a late Pliocene progenitor of Cyprinodon variegatus ovinus and the large-bodied Hispaniolan species, and, more recently, by one or more ancestral forms allied with Cyprinodon variegatus variegatus and C. v. riverendi. Levels of divergence indicate that large expanses of open sea have not acted as long-term barriers to inter-island dispersal of cyprinodontiform fishes. This study, together with the molecular systematics of other insular Caribbean fishes, indicates that most insular groups originated from late Neogene dispersal from the mainland. The patterns of mtDNA variation in Cyprinodon showed little congruence with the species/subspecies taxonomy.

Immunoblot studies in allergic patients to hymenoptera venom before and during immunotherapy.

BACKGROUND: Hymenoptera venom immunotherapy (VIT) is immunologically effective in patients with systemic allergic reactions after hymenoptera stings. OBJECTIVE: To evaluate the effect of VIT on specific IgE and IgG4 immunoblotting bands in VIT-treated patients. MATERIAL AND METHODS: Specific IgE and IgG4 immunoblotting bands for hymenoptera venom were performed with ALABLOT in sera of 17 patients (8 allergic to honeybee venom, 8 to wasp and 1 to polistes venom) before and during successful VIT (1 and 3 years). Before immunotherapy, all patients had experienced moderate/severe systemic reactions to a hymenoptera sting, with positive skin tests and venom-specific IgE. During immunotherapy all patients suffered field stings, without any systemic reaction. RESULTS: Before VIT we detected several immunoglobulin-binding bands in different regions, with different individual patterns. After VIT, we observed in some patients (5/8 for honeybee venom, 6/8 for wasp and 1/1 for polistes) complete disappearance of some IgE-binding bands, mainly the 15 kDa region (honeybee) and 23 and 44 kDa regions (wasp and polistes). All patients showed decreased intensity of IgE-binding bands, most pronounced in regions 16, 44 and 52 kDa (honeybee); 44 and 35 kDa bands (wasp) and 23 kDa (polistes). Some patients showed de novo appearance of IgG4-binding bands (4/8 for honeybee and 8/8 for wasp venom), mainly in 52 kDa (honeybee) and in 23 and 44 kDa regions (wasp). All patients showed increased intensity of IgG4 bands that were already present before VIT, more pronounced in 52 and 44 kDa (honeybee) and in 44 and 35 kDa regions (wasp). CONCLUSIONS: During successful VIT there are changes in intensity and number of IgE and IgG4 binding bands, which could reflect the immunological improvement induced by VIT. These changes are more pronounced/frequent in wasp VIT, a fact that could explain the best results usually seen in these patients.