Transcriptome analysis in venom gland of the predatory giant ant Dinoponera quadriceps: insights into the polypeptide toxin arsenal of hymenopterans.

BACKGROUND: Dinoponera quadriceps is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the D. quadriceps venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation. RESULTS: We conducted a transcriptome analysis of a cDNA library from the D. quadriceps venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences. CONCLUSIONS: To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant D. quadriceps. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans.

Patterns of venom synthesis and use in the fire ant, Solenopsis invicta.

In the life of the fire ant, Solenopsis invicta, venom plays several important roles, including prey capture, defense, and anti-microbial action. Although this venom's chemistry, pharmacology, and effects on humans have been extensively studied, its patterns of synthesis and use have not. We determined the ability of different-aged workers to synthesize venom, and measured the amount of venom workers delivered per sting. Newly eclosed workers contained little or no venom in their reservoirs. The rate of synthesis was highest in workers aged 1 day after adult eclosion (1.17 microg venom/day for workers with headwidths of 1 mm), declined by 75% in workers aged 15 days (0.30 microg venom/day), and was negligible thereafter. Inducing ants to sting dummies as surrogates for humans, workers were found to deliver an average of 0.66 nl per sting, amounting to 3.1% of the average individual venom supply. Older workers (foragers) delivered less venom per sting than mid-age workers (reserves) and venom dose from nest-defenders was 55% higher in the spring than in the rest of the year. Thus, fire ant venom synthesis is limited to early life, and injected venom dose appears to be modulated. Economic explanations for the observed venom-use patterns are discussed.

[Recombinant proteins containing amino acid sequences of two ectatomin chains]. / Rekombinantnye belki, soderzhashchie aminokislotnye posledovatel'nosti dvukh tsepei éktatomina.

Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

Production of a recombinant imported fire ant venom allergen, Sol i 2, in native and immunoreactive form.

BACKGROUND: The complementary DNA encoding for the important imported fire ant venom allergen, Sol i 2, has previously been cloned. The binding of human IgE antibodies to Sol i 2 has been demonstrated to be conformation-dependent. METHODS: A couple cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinant Sol i 2 was isolated and characterized. RESULTS: Recombinant (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 micrograms/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was identical to that of the natural molecule. No significant increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal antibody was lost by heating in sodium dodecylsulfate and reduction. CONCLUSION: A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.

Alkaloid venom of European ants in the genus Monomorium. Site of synthesis, identification and quantification.

The venom of the European Ant Monomorium is composed of five alkaloids: three 2.5 transdialkylpyrrolidines and two 2.5 transdialkylpyrrolines. The venom is synthesized by a glandular complex composed of a biramous external filamentous gland, and a single internal gland invaginated into the reservoir. External glands and the proximal two-thirds of the internal gland are composed of glandular units, with one glandular cell and one associated duct cell. Glandular cells contain numerous giant mitochondria, a well-developed smooth endoplasmic reticulum and enigmatic rod-shaped structures. The secretory product is electron-lucent. The distal third of the internal gland is composed of 38-48 large secretory cells issuing directly into the reservoir. Their secretory product presents the same characteristics as the venom accumulated in the reservoir (opacity, affinity to stains and osmium).