A comparative study of protein and enzymatic activity in venoms of some common wasps (Hymenoptera: Vespidae) from São Paulo State

The Hymenoptera Aculeata venoms, with few exceptions, have been poorly studied and characterized. Nevertheless, they have raised increasing interest due to their medical importance, since accidents with these insects are fairly frequent in Brazil and may cause severe allergic reactions. The objectives of the present work were the quantitative characterization of the main allergenic enzymes present in the venom of the species Polybia paulista, Polybia ignobilis, Polistes simillimus, and Agelaia pallipes pallipes through biochemical assays for the determination of total protein content, as well as the level of the enzymatic activity of phospholipase, hyaluronidase, acid phosphatase and esterase. These results, in addition to providing biochemical knowledge about the venom of the species in question, also supply studies that allow phylogenetic inferences among them.

Dominance hierarchy in different stages of development in colonies of the primitively eusocial wasp Mischocyttarus cassununga (Hymenoptera, Vespidae)

Seven colonies of Mischocyttarus cassununga were studied under field conditions at Universidade Federal de Juiz de Fora, MG, in southeastern Brazil: in pre-emergence, post-emergence and decline stages, during 145.8 hours. Dominance interactions among the females were quantified to verify the dynamics of succession in the social hierarchy of the colonies. Early pre-emergence colonies present more intense aggressive interactions than late pre-emergence ones, because the females are engaged in securing the role of main egg layer in the nest. In post-emergence (pre-male) colonies the dominance hierarchy is more defined and the frequency of dominance and subordination behaviors were lower than in pre-emergence stages: most of the agonistic behaviors are restricted to the first ranked females (potentially queens) and the subordinate individuals play the role of workers in the nests. In the post-emergence, post-male, and decline stages the hierarchy is still maintained by the aggressive behaviors of the 1st-ranked female but because the presence of males and future nest foundresses these interactions are not well defined in a linear way.

Method for the evaluation of insecticidal activity over time in Atta sexdens rubropilosa workers (Hymenoptera: Formicidae)

The active ingredients used in the formulation of toxic baits for leaf-cutting ants (Hymenoptera: Formicidae) should possess a delayed action defined as an insecticidal activity whereby worker mortality is ≤15% at 24 hours and ≥90% at 21 days. Serious shortcomings have occurred in the search for new active ingredients, such as the initial selection of fenoxycarb, copper oxychloride and diflubenzuron, compounds considered very promising but whose inefficiency was verified only later, indicating methodological problems. In view of this situation, we developed a classification method for insecticidal activity over time using workers of the leaf-cutting ant Atta sexdens rubropilosa Forel. The insecticides used were fipronil, sulfluramid GX071HB and sulfluramid GX439, vehicled in an attractive pasty formulation prepared based on citrus pulp. The results obtained were consistent from a toxicological viewpoint and agreed with the literature in terms of the control of colonies. Sulfluramids were found to possess a delayed action at a broad range of concentrations, in agreement with the fact that these substances are highly effective in the control of all leaf-cutting ant species. The smaller range of concentrations of fipronil with delayed action is probably related to its lower efficacy for species more difficult to control such as Atta capiguara (Forti et al. 2003). We discuss the importance of relating behavioral particularities to the specific feeding habits of leaf-cutting ants, with methodological adequacy of the assessment of insecticides aimed at toxic baits.

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.

Development of worker ovaries in Brazilian Africanized honeybees (Hymenoptera: Apidae)

The present paper compares the worker ovaries development in queen right and orphaned colonies of Brazilian Africanized honeybee. The results show that in both colonies some workers starting 20 days after emergence, present some kind of ovary development. As the workers from queen right colony get older the degree of the ovary development increases. Vitellogenesis in worker ovaries may occur in queen right and orphaned colonies, but its frequency is lower in queen right colonies.

Toxicity of sesame extracts to leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Extracts of different sesame plant (Sesamum indicum, Linnaeus) organs were tested through ingestion and contact experiments to investigate their toxicity to Atta sexdens rubropilosa (Forel) workers. Dichloromethane extracts of seeds were toxic to the leaf-cutting ants and the factor responsible for the toxicity does not show seasonal occurrence.

Toxicity of sesame seed to leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Extracts of the ripe seeds of the sesame plant (Sesamum indicum, Linnaeus) were tested through contact experiments to investigate their toxicity to Atta sexdens rubropilosa workers. Dichloromethane extract of seeds was toxic to the ants and the factor responsible for this effect was distributed through the ethyl acetate fraction. This fraction was divided into four sub fractions composed of: A) triglycerides, B) monoglycerides + diglycerides + triglycerides, C) diglycerides + sesamoline + sesamine and D) sesamine. However, when these sub fractions were separated, no toxicity was observed. Therefore, in order to determine why the activity was lost, the concentration of each sub fraction was duplicated, and the possible combinations among them were also tested. We concluded that the toxicity to the ants is due mainly to a mixture of triglycerides, and sesamoline or the combination of sesamoline + sesamine can be a synergistic factor in this fraction.

Oral toxicity of chemical substances found in Dimorphandra mollis (Caesalpiniaceae) against honeybees (Apis mellifera) (Hymenoptera: Apidae)

Dimorphandra mollis is a characteristic plant from Brazilian saP vanna-like vegetation. The pollen of this species could be toxic to bees and the objective of the present investigation was to study the toxicity of methanolic extracts obtained from the flowers, peduncles and stem bark of D. mollis to Apis mellifera workers. For the study, the extracts were incorporated into the diet of the bees for later evaluation of mortality rates. The substances isolated: neoisoastilbin, catechin, astilbin and tannins were tested on adult workers and only catechin did not cause toxic effects. The data obtained in the toxicity bioassays were analyzed statistically by Log Rank test and all methanolic extracts showed significant (p<0.0001) toxic effects. Astilbin is also the major component of pollen grains, and is probably responsible for honeybee mortality during blooming periods.

Astilbin toxicity to leaf-cutting ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

The toxicity of astilbin, isolated from Dimorphandra mollis, was tested in laboratory ingestion bioassays for the leaf-cutting ant Atta sexdens rubropilosa Forel. Worker ants that were fed an artificial diet daily to which astilbin was added had a higher mortality rate than the controls. The substance astilbin was incorporated into the diet with and without the utilization of a solvent. Results for both methodologies utilized were similar and indicate that astilbin reduces the median survival of ants. All concentrations tested showed toxic effects against ant workers, and the statistical comparison of survivorship rates from control and treated groups was significantly different, confirming the insecticidal properties of the substance astilbin.

Comparative study of the fat body in some genera of the attini tribe (Hymenoptera: Formicidae)

The Cyphomyrmex rimosus Spinola and Mycetarotes parallelus Emery species of ants, considered basal, and Acromyrmex disciger Mayr and Atta laevigata Smith, considered derived, have fat bodies specially distributed on their gaster, around their organs and right below the cuticle. The fat body is formed by trophocytes, which are characterized by their pronounced vacuolization of the cytoplasm and the irregular morphology of their nuclei caused by the pressure exerted by cytoplasmic vacuoles. In C. rimosus, the nuclei are more regular, presenting an oval or a star form. In A. disciger and A. laevigata the nuclei present chromatin in a cord form, while in C. rimosus and M. paralellus the chromatin is uniformly distributed in the nucleoplasm, very condensed in the latter species. The parietal trophocytes of A. disciger show cytoplasm with a smaller quantity and smaller sizes of vacuoles compared to ones from the perivisceral region, the opposite is observed in C. rimosus. In A. laevigata and M. parallelus there were no differences observed in their cytoplasm between both regions of cells. In the trophocytes of C. rimosus, A. disciger, A. laevigata, there was a reticular aspect of the cytoplasm observed in the region between vacuoles, not seen on M. parallelus. Another cellular type, oenocyte, was found associated with the fat body cells, and is dispersed between trophocytes with an inner contact to them, but no membrane fusion with them. The oenocytes have a spherical form and are smaller than the trophocytes; they have acidophilic cytoplasm with a small quantity of small vacuoles, and round nuclei.

Temporal polyethism and survivorship of workers of Agelaia pallipes (Hymenoptera, Vespidae, Epiponini)

In this study, known-age workers of Agelaia pallipes (Olivier) were introduced into a colony maintained in the laboratory and the effects of age polyethism on the worker life span were observed. As in other Neotropical swarm-founding wasps, the results showed that younger workers remained on different places inside the nest performing activities related to the brood and nest maintenance, while risky tasks such as foraging were performed by older workers. Furthermore, the start of foraging activities had a significant effect on worker mortality.

Toxicity of Cedrela fissilis to Atta sexdens rubropilosa (Hymenoptera: Formicidae) and its symbiotic fungus

Crude extracts from roots, stems, branches, fruits and leaves of Cedrela fissilis were tested to verify their toxicity to Atta sexdens rubropilosa workers and to their symbiotic fungus Leucoagaricus gongylophorus. The workers that were fed daily on an artificial diet to which crude extracts from this plant were added had a higher mortality rate than the controls, especially for the hexane, dichloromethane and methanol crude extracts from roots (RH, RD and RM) and from leaves (LH, LD and LM). Fungal growth was inhibited by the hexane (RH) and dichloromethane crude extract from roots (RD). The RH, RD and FD crude extracts were fractioned and their fractions were tested. All the fractions tested presented toxicity to the ants and some fractions (RH-H, RH-D, RD-4 and RD-5) completely inhibited fungus development. The possibility of controlling these insects in the future using Cedrela fissilis compounds that can simultaneously target both organisms is discussed.

Enzymatic variability among venoms from different subspecies of Apis mellifera (Hymenoptera: Apidae)

The enzymatic variability was analyzed in venom extracts from bees reared in different colonies of the Africanized, A. m. ligustica and A. m. carnica subspecies. The implications of this variation focused on the biochemistry differentiation and immunogenicity of these venoms. The results showed the existence of a huge variability among the subspecies as well as among the colonies for three out of the six tested components - hyaluronidase, acid phosphatase and proteases - suggesting the utilization of these features as possible biochemical markers. Furthermore, although not statistically significant, it was found that the Africanized bee venom presented slightly higher levels of protein content and esterase activity, when compared to the other subspecies. If the esterase plays a role in the pain intensity caused by the sting, as suggested elsewhere, this might suggest a reason for a bigger algogenicity of this venom in relation to that of European bees. On the other hand, A. m. ligustica bees presented the highest levels of proteolytic and acid phosphatase activities, whose functions are not enlightened in Hymenoptera venoms. The A. m. carnica workers presented the highest hyaluronidase and the lowest acid phosphatase activity levels. The extremely variable results among colonies of the same subspecies and among subspecies, for the tested venom components, justify the absence of correlation between allergic reactions and tests with pooled venom.

Study of the venom glands in Ectatomma quadridens (Hymenoptera: Formicidae). Evolutionary hypothesis in subfamily ponerinae

The venom glands of worker ants of the species Ectatomma quadridens morphologically resemble an elongated sac or reservoir ending in a narrower portion that has the function of releasing the secretion to the exterior. Two external secretory filaments are individually inserted into the proximal portion of the gland and end inside the convoluted gland. The venom gland of workers of E. quadridens is, therefore, morphologically subdivided into four distinct portions: a) sac-shaped reservoir measuring approximately 1mm in length; b) excretory duct, proximal portion of the reservoir that joins the gland to the sting apparatus; c) convoluted gland, final portion of the external secretory filaments located inside the reservoir; and d) two secretory filaments measuring about 2 mm in length; their free extremities end blindly and are individually inserted into the reservoir wall at the proximal region of the venom gland. The histological data showed that the filaments and the convoluted gland are composed of cubic cells of secretory function. The reservoir consists of a simple cubical epithelium externally surrounded by muscle fibers. A thick cuticle internally coats the epithelium of the reservoir. The application of histochemical tests allowed us to establish that the final secretion of the venom gland of Ectatomma quadridens is of glycoproteic nature. This secretion undergoes several modifications at the secretory filaments, at the convoluted gland, and in the reservoir before reaching the excretory duct, the point at which the secretion is released in its final composition, namely the venom. Based on the differences among various Ponerinae species we propose a hypothesis suggesting a probable evolutionary process that the venom glands of members of this subfamily might have undergone.

Comparative study of the histology, histochemistry and ultramorphology of the proventriculus in the cephalotini tribe (Hymenoptera: Formicidae)

The use of optical microscopy and scanning electron microscopy propitiated the comparative examination of the nature of the histology, histochemistry and ultramorphology of the proventriculus bulb of Cephalotes atratus, C. clypeatus and C. pusillus. This portion of the digestive tract possesses highly sclerotized cuticular projections which act in the selection of victuals. A layer of amorphous material with mucous characteristics is present over the cuticle projections. That layer seems to optimize the efficiency of the proventriculus in the selection of food for social function in the colony (trophallaxis). More details of the characteristics of this structure are described in this study.