Morphology of the Mandibular and Intramandibular Glands of Army Ant Workers of Labidus praedator (Smith 1858) and Labidus coecus (Latreille 1802) (Formicidae: Dorylinae).

Social insects are characterized by having a wide diversity of exocrine glands, with highlights for ants with about 85 glands spreading throughout the body. The mandibular and intramandibular glands are associated with the production of pheromones. The army ants (Dorylinae) play an important role in the structure of the invertebrate community because they are efficient predators and provide suitable conditions for various animals following their invasions in the food search. Labidus coecus (Latreille) is an underground-ameliorating ant and Labidus praedator (Smith) is a generalist surface predator which can deplete invertebrate biomass by up to 75%. This work investigated the morphology of the mandibular and intramandibular glands of L. praedator and L. coecus workers. The glands were analyzed by light microscopy, histochemistry, and scanning electron microscopy. The mandibular and intramandibular glands of the two species were classified as class III glands. The data on the morphology of the mandibular glands has revealed that they have characteristics in common with other subfamilies. The intramandibular glands of the two species of Labidus have similar morphology and chemical composition, which indicates that the components of these glands can have the same function despite their different habits.

First observation on the predation of a non-arthropod species by a dung beetle species: The case of Canthon chalybaeus and the snail Bulimulus apodemetes.

We described, for the first time, a case of predation of a non-arthropod species by a dung beetle species. Canthon chalybaeus Blanchard, 1843 kills healthy individuals of the terrestrial snail Bulimulus apodemetes (D'Orbigny, 1835) showing an evident pattern of physical aggressiveness in the attacks using the dentate clypeus and the anterior tibiae. The description of this predatory behaviour was complemented with the analysis of the chemical secretions of the pygidial glands of C. chalybaeus, highlighting those main chemical compounds that, due to their potential toxicity, could contribute to death of the snail. We observed a high frequency of predatory interactions reinforcing the idea that predation in dung beetles is not accidental and although it is opportunistic it involves a series of behavioural sophistications that suggest an evolutionary pattern within Deltochilini that should not only be better studied from a behavioural point of view but also phylogenetically.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Camphor and Borneol as the Male-Produced Sex Pheromone of the Shield Bug, Orsilochides leucoptera (Hemiptera: Scutelleridae).

The ability of stink bugs to release high amounts of strong-smelling and irritating defensive compounds is related to their metathoracic gland (MTG), which is an exocrine gland with defensive, sexual, alarm and aggregation signal functions. Orsilochides leucoptera (Scutelleridae) is a widespread species in the Neotropical region that feeds on plants of the families Malvaceae, Poaceae and Euphorbiaceae. A series of compounds (ketones, alcohols and esters) have been identified in the MTGs among the three species of Scutelleridae whose MTG secretions have been investigated thus far; however, no sex pheromone compounds have been described for any scutellerid species. The aim of this work was to study sex pheromone communication within this family of stink bugs, and identify the compounds present in the MTG of O. leucoptera. Analysis by gas chromatography/mass spectrometry (GC/MS) revealed two male specific compounds identified as (R)-camphor (1) and (R)-borneol (2), which were attractive to females in Y-tube olfactometer bioassays. Also, GC/MS analysis of secretions from MTG of males and females of O. leucoptera, identified eight additional compounds: 2-(E)-hexenal (3), (E)-4-oxo-2-hexenal (4), (E)-hex-2-enyl acetate (5), (R)-linalool (6), (R)-α-terpineol (7), dodecane (8), 1-tridecene (9) and n-tridecane (10). From these, (R)-α-terpineol was detected only in the gland of males, and is probably a biosynthetic intermediate of the pheromone components. Most of the MTG compounds identified in O. leucoptera have been identified in other heteropteran species. Camphor is often a toxic and repellent compound for insects. However, we report it, for the first time, as a sex pheromone component of an insect.

Dissecting Toxicity: The Venom Gland Transcriptome and the Venom Proteome of the Highly Venomous Scorpion Centruroides limpidus (Karsch, 1879).

Venom glands and soluble venom from the Mexican scorpion Centruroides limpidus (Karsch, 1879) were used for transcriptomic and proteomic analyses, respectively. An RNA-seq was performed by high-throughput sequencing with the Illumina platform. Approximately 80 million reads were obtained and assembled into 198,662 putative transcripts, of which 11,058 were annotated by similarity to sequences from available databases. A total of 192 venom-related sequences were identified, including Na+ and K+ channel-acting toxins, enzymes, host defense peptides, and other venom components. The most diverse transcripts were those potentially coding for ion channel-acting toxins, mainly those active on Na+ channels (NaScTx). Sequences corresponding to ß- scorpion toxins active of K+ channels (KScTx) and λ-KScTx are here reported for the first time for a scorpion of the genus Centruroides. Mass fingerprint corroborated that NaScTx are the most abundant components in this venom. Liquid chromatography coupled to mass spectometry (LC-MS/MS) allowed the identification of 46 peptides matching sequences encoded in the transcriptome, confirming their expression in the venom. This study corroborates that, in the venom of toxic buthid scorpions, the more abundant and diverse components are ion channel-acting toxins, mainly NaScTx, while they lack the HDP diversity previously demonstrated for the non-buthid scorpions. The highly abundant and diverse antareases explain the pancreatitis observed after envenomation by this species.

"Sweeter than a rose", at least to Triatoma phyllosoma complex males (Triatominae: Reduviidae).

BACKGROUND: The Triatoma phyllosoma complex of Trypanosoma cruzi vectors (Triatominae: Reduviidae) is distributed in both Neotropical and Nearctic bioregions of Mexico. METHODS: Volatile organic compounds emitted by disturbed Triatoma longipennis, Triatoma pallidipennis and Triatoma phyllosoma, and from their Brindley's and metasternal glands, were identified using solid-phase microextraction coupled with gas chromatography-mass spectrometry. RESULTS: Disturbed bugs and the metasternal glands from T. phyllosoma released or had significantly fewer compounds than T. longipennis and T. pallidipennis. Isobutyric acid was the most abundant compound secreted by disturbed bugs of the three species, while Brindley's glands of all species produced another four compounds: propanoic acid, isobutyric acid, pentyl butanoate, and 2-methyl hexanoic acid. Two novel compounds, both rose oxide isomers, were produced in MGs and released only by disturbed females of all three species, making this the first report in Triatominae of these monoterpenes. The principal compound in MGs of both sexes of T. longipennis and T. phyllosoma was 3-methyl-2-hexanone, while cis-rose oxide was the principal compound in T. pallidipennis females. The major components in male effluvia of T. pallidipennis were 2-decanol and 3-methyl-2-hexanone. CONCLUSION: Discriminant analysis of volatile organic compounds was significant, separating the three species and was consistent with morphological and genetic evidence for species distinctions within the complex.

Bothrops jararaca accessory venom gland is an ancillary source of toxins to the snake.

In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus, the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory gland during venom production and secretion cycle. We showed that the accessory gland expresses and synthesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular endothelial growth factor, serine proteinase, and l-amino acid oxidase. Our data have shown that toxin synthesis in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the snake, and provides inhibitors that could control venom toxicity (and integrity) during storage. SIGNIFICANCE: In this study, we propose that the accessory venom gland acts as an important ancillary source of toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom toxicity (and integrity) during its storage.

Chemical Composition of Alphitobius diaperinus (Coleoptera: Tenebrionidae) Abdominal Glands and the Influence of 1,4-benzoquinones on its Behavior.

This study aimed to identify and quantify the compounds present in the abdominal glands of Alphitobius diaperinus Panzer, 1797 (Coleoptera: Tenebrionidae) and to evaluate the influence of these compounds on its behavior. The extraction of volatiles present in the abdominal glands was made by dissection (10 individuals per sex) and by air entrainment (200 insects per sex), and they were analyzed by gas chromatography-flame ionization detector, gas chromatograph-mass spectrometer, and gas chromatograph-electroantennography detector (GC-EAD). The influence of these volatiles on the behavior of conspecifics was evaluated in a four-arm olfactometer. Twenty-three compounds were identified from male and female abdominal gland extracts, of which six were quinones: the 2-methyl-1,4-benzoquinone and the 2-ethyl-1,4-benzoquinone were the major components, and 1,4 benzoquinone and three hydroquinones were registered for the first time for this species. The GC-EAD analysis using the crude extracts from abdominal glands showed that male and female antennae responded to the three major benzoquinones. For the olfactometer bioassays, both genders were repelled either by the abdominal gland extracts or by synthetic solutions containing the three benzoquinones. The results suggest that the 1,4-benzoquinones play a role as a repellent to A. diaperinus.

Intramandibular glands in different castes of leaf-cutting ant, Atta laevigata (Fr. Smith, 1858) (Formicidae: Attini).

Intramandibular glands have been poorly studied in polymorphic ants, where the differences between castes were unsufficiently scrutinized. Leaf-cutting ants possess one of the most complex systems of communication and labor division, which is polymorphic well as age polyethism, and makes them an ideal model for the study of intramandibular glands. This study has investigated the occurrence of intramandibular glands in female castes and subcastes of Atta laevigata. The mandibles of the queen, medium, and minor workers, and soldiers were submitted to histological, histochemical, ultrastructural, and morphometric analyses. The class-3 gland cells and the epidermal gland with a reservoir were found in all the castes. The queens and soldiers showed a higher number of class-3 gland cells, distributed within the mandible as well as a greater gland size in comparison to the workers. The histochemical tests, periodic acid-Schiff (PAS), mercury-bromophenol, and Nile blue, were similar for the class-3 gland cells and epidermal glands with a reservoir. However, the tests evidenced differences between the castes, with carbohydrates strongly positive in all of them, whereas neutral lipids were found in the queen and soldiers. The protein was weakly positive in the queen, whereas in the soldier, medium, and minor workers these reactions were strongly positive in the intramandibular glands. Our findings in A. laevigata suggest that intramandibular glands are directly involved in labor division and consequently in chemical communication between the castes.

Gene expression analysis by ESTs sequencing of the Brazilian frog Phyllomedusa nordestina skin glands.

The subfamily Phyllomedusinae has attracted a great interest of many researchers mainly due to the high diversity of these frog species and plethora of pharmacological activities frequently observed for their skin secretions. Despite of this fact, mainly for new species, limited information is available regarding the molecular composition of these skin secretions and the cellular components involved in their production. Phyllomedusa nordestina is a recently described Brazilian frog species also popularly known as 'tree-frogs'. Aiming at contributing to the biological knowledge of this species, we show here the gene expression profile of this frog skin secretion using a global ESTs analysis of a cDNA library. The marked aspect of this analysis revealed a significant higher transcriptional level of the opioid peptide dermorphins in P. nordestina skin secretion than in Phyllomedusa hypochondrialis, which is its closest related species, belonging both to the same phylogenetic group. Precursors of bioactive peptides as dermaseptins, phylloseptins, tryptophyllins, and bradykinin-like peptideswere also found in this library. Transcripts encoding proteins related to ordinary cellular functions and pathways were also described. Some of them are chiefly involved in the production of the skin secretion. Taken together, the data reported here constitute a contribution to the characterization of the molecular diversity of gene-encoded polypeptides with potential possibility of pharmacological exploitation. The transcriptional composition of the skin secretion may also help to give the necessary support for the definition of P. nordestina as a new species, which actually relies basically on frog morphological characteristics and geographical distribution.

Chemical composition of metapleural gland secretions of fungus-growing and non-fungus-growing ants.

The metapleural gland is exclusive to ants, and unusual among exocrine glands in having no mechanism for closure and retention of secretion. As yet, no clear conclusion has been reached as to the function of metapleural gland secretion. Metapleural gland secretions were investigated for fungus-growing ants representing the derived attines Trachymyrmex fuscus, Atta laevigata, and Acromyrmex coronatus, the basal attines Apterostigma pilosum and Mycetarotes parallelus, and non-fungus-growing ants of the tribes Ectatommini (Ectatomma brunneum) and Myrmicini (Pogonomyrmex naegeli). Our results showed that the secretions of leaf-cutting ants (A. laevigata and A. coronatus) and the derived attine, T. fuscus, contain a greater variety and larger quantities of volatile compounds than those of myrmicine and ectatommine ants. The most abundant compounds found in the metapleural glands of A. laevigata and A. coronatus were hydroxyacids, and phenylacetic acid (only in A. laevigata). Indole was present in all groups examined, while skatole was found in large quantities only in attines. Ketones and aldehydes are present in the secretion of some attines. Esters are present in the metapleural gland secretion of all species examined, although mainly in A. laevigata, A. coronatus, and T. fuscus. Compared with basal attines and non-fungus-growing ants, the metapleural glands of leaf-cutting ants produce more acidic compounds that may have an antibiotic or antifungal function.

Defensive compounds and male-produced sex pheromone of the stink bug, Agroecus griseus.

Agroecus griseus is a serious corn pest in Brazil. Contents of the dorsal abdominal glands (DAGs) in nymphs, and the metathoracic gland (MTG) in adults of this species were characterized and quantified. Compounds found were similar to those of other Pentatomidae species and included aliphatic hydrocarbons, aldehydes, oxo-alkenals, and esters. However, two compounds were found in the MTG that have not been described previously for this family. Mass spectrometry, infrared spectroscopy, and gas chromatographic analysis using coinjection with authentic standards confirmed the identities of the compounds as enantiopure (S)-2-methylbutyl acetate and 3-methyl-2-butenyl acetate. The five nymphal instars showed significantly different ratios of components, mainly between those of the first and later instars. No significant differences were detected in MTG contents between sexes. Gas chromatography (GC) analysis of aeration extracts of males and females showed the presence of a compound released exclusively by males. Gas chromatography - electroantennographic detection (GC-EAD) assays indicated that the male-specific compound is bioactive in females, suggesting the presence of an attractant pheromone. The mass spectrum and infrared data for this compound matched with methyl 2,6,10-trimethyltridecanoate, a sex pheromone component previously detected in the stink bugs, Euschistus heros and E. obscurus. The synthetic standard coeluted with the natural pheromone on two different GC stationary phases, confirming the proposed structure. Y-tube olfactometer assays showed that the synthetic standard was strongly attractive to females, and GC-EAD tests produced responses with antennae from females similar to those of the natural pheromone.

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis.

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (~37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom.

A transcriptomic view of the proteome variability of newborn and adult Bothrops jararaca snake venoms.

BACKGROUND: Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. METHODS/PRINCIPAL FINDINGS: Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some gender-related differences. CONCLUSIONS/SIGNIFICANCE: We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.

Skin glands, poison and mimicry in dendrobatid and leptodactylid amphibians.

In amphibians, secretions of toxins from specialized skin poison glands play a central role in defense against predators. The production of toxic secretions is often associated with conspicuous color patterns that warn potential predators, as it is the case of many dendrobatid frogs, including Ameerega picta. This species resembles the presumably nontoxic Leptodactylus lineatus. This study tests for mimicry by studying the morphology and distribution of skin glands, components of skin secretion, and defensive behavior. Dorsal skin was studied histologically and histochemically, and skin secretions were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed phase high performance liquid chromatography and assays for proteolytic activity. We found that poison glands in A. picta are filled with nonprotein granules that are rich in carbohydrates, while L. lineatus glands present protein granules. Accordingly, great amounts of proteins, at least some of them enzymes, were found in the poison of L. lineatus but not in that of A. picta. Both species differ greatly on profiles of gland distribution: In L. lineatus, poison glands are organized in clusters whose position coincides with colored elements of the dorsum. These regions are evidenced through a set of displays, suggesting that poison location is announced to predators through skin colors. In contrast, A. picta presents lower densities of glands, distributed homogeneously. This simpler profile suggests a rather qualitative than quantitative investment in chemical defense, in agreement with the high toxicity attributed to dendrobatids in general. Our data suggest that both species are toxic or unpalatable and transmit common warning signals to predators, which represents a case of Müllerian mimicry.

Factors affecting Anastrepha fraterculus female receptivity modulation by accessory gland products.

In the context of the sterile insect technique (SIT), mass-rearing and male irradiation are imperative. Post-teneral treatments such as the addition of protein in adult's male diet and male hormonal treatment are used to improve sexual performance and to accelerate sexual maturation. In this work we investigated the effect of male accessory glands products (AGPs) on female receptivity of the South American fruit fly Anastrepha fraterculus (Wiedemann), and the effect of strain rearing history, male irradiation, male diet and hormonal treatment on AGPs. Injections of aqueous extracts of male accessory glands into the abdomen of females reduced their receptivity. The AGPs from laboratory males were more effective in inhibiting female receptivity, compared to AGPs from wild males, irrespective of females' origin. The AGPs from fertile males were more effective than AGPs from sterile males. The AGPs from protein-fed males were more effective than AGPs from sugar-fed males. Finally, the AGPs of males treated with juvenile hormone were less effective in inhibiting female receptivity than AGPs of untreated males. We conclude that inhibition of sexual receptivity of A. fraterculus mated females is mediated by products in male accessory gland's and the way that these products act vary widely according to the effect of extrinsic factors. We discuss the results in the perspective of the SIT application for A. fraterculus.

Morphology and histochemistry of the intramandibular glands in Attini and Ponerini (Hymenoptera, Formicidae) species.

An understanding of the internal morphology of the ant mandible is important in explaining the relationship between the exocrine system and the behavioral and phylogenetic characteristics of different subfamilies of Formicidae. In this study, we investigated the occurrence of intramandibular glands in ants from the Ponerini (Ponerinae) and Attini (Myrmicinae). These ants possess glands from classes I and III, and secretory epithelial cells with a reservoir. The intramandibular glands show a distinct histology in the studied species, varying in their location, degree of development, and chemical content. Using this information, it is possible to hypothesize that the glands from different tribes produce different substances, which may indicate a variety of functions, depending on the chemical nature of the cellular constituents. A cladistic analysis using the characters of the intramandibular glands separated both tribes, suggesting that structural differences in the intramandibular glands may contribute to future phylogenetic studies of the Formicidae.

Simple and rapid CE-UV method for the assessment of trail pheromone compounds of leaf-cutting ants' venom glands.

Leaf-cutting ants cause large losses in several crops around the world. In the ant species Atta sexdens, each colony comprises up to 5 million individuals. In order to keep a close connection among such a large number of individuals, an efficient chemical communication system is necessary. Among other different substances, these animals use alkylpyrazines to mark their trails and to guide ant workers from the nest to their sources of food. In this study, CE-UV was used to apply a method for qualitative analysis of venom gland components of leaf-cutting ants. Mobility of these compounds proved to be a function of the ionization capability of these bases as well as their volumes. Migration order was thoroughly explained in terms of such parameters. The best analysis conditions were achieved with a BGE composed by 0.8% formic acid plus 20% methanol in water, hydrodynamic injection, and application of external pressure. Such analysis conditions may be easily applied in CE-MS analyses as well. CE-UV analyses proved to be as adequate as GC to analyze such compounds due to system detectability (LD ≅ 0.005 mmol/L), separation efficiency (from 5.07×10(4) to 1.23×10(5) theoretical plates), and resolution (minimum of 2.35). In addition, analysis time was ca. 15 min, which shows another advantage of CE analysis when compared with GC. Although the analytes are found in concentrations as low as 50 ng/venom glands, four putative pyrazine ring moiety substances could be detected in real samples, due to sample stacking and use of a capillary with extended detection cell.

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom.

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.

General biochemical and immunological characterization of the venom from the scorpion Tityus trivittatus of Argentina.

Tityus trivittatus is the Argentinean scorpion reported to cause the majority of human fatalities in the country, however no systematic studies have been conducted with the venom of this species. This communication describes a general biochemical and immunological characterization of the venom obtained from T. trivittatus scorpions collected in the city of Buenos Aires and various provinces of Argentina: Catamarca, Cordoba, Entre Rios, La Rioja, Santa Fe and Santiago del Estero. These are places where human accidents were reported to occur due to this scorpion. For comparative purposes two types of samples were assayed: whole soluble venom obtained by electrical stimulation and supernatant from homogenized venomous glands. Two strains of mice (NIH and CF-1) were used for LD(50) determinations by two distinct routes of administration (intravenously and intraperitoneally). Important variations were found that goes from 0.5 to 12 mg/kg mouse body weight. Samples of soluble venom were always more potent than Telson homogenates. More complex pattern was observed in homogenates compared to soluble venom, as expected. This was supported by gel electrophoretic analysis and high performance liquid chromatographic (HPLC) separations. Additionally, the HPLC profile was enriched in proteins resolved at similar elution times as other known toxins from scorpion venoms studied. Immune enzymatic assays were also conducted comparatively, using four different anti-venoms commercially available for treatment of scorpion stings (Argentinean antidote from INPB, two anti-venoms from Butantan Institute of Brazil and Alacramyn from the Mexican Bioclon Institute). Cross-reactivities were observed and are reported among the various venoms and anti-venoms used. Lung, heart, liver and pancreas pathological modifications were observed on tissues of intoxicated mice. It seems that there are important variations on the venom compositions of the various samples studied and reported here, depending on the geographical area where the scorpions were captured. The results reported here are important for the clinical outcome of human accidents.