Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signaling by ants. We identified fourteen unique CSP sequences in EST libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirms the high level of Si-CSP1 expression, and also shows expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibit Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids shows that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons.
To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).
Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence. In-gel trypsin digestion followed by MALDI-TOF mass spectrometry and HPLC-ESI/MS/MS demonstrated that the protein gel spot contained only the protein corresponding to the cDNA sequence obtained by PCR. The sequence is similar to apolipophorin-III, an exchangeable lipid-binding protein. Fire ant apolipophorin-III is expressed in the antenna as well as the head, thorax and abdomen.
The antennae of the higher Hymenoptera (ants, bees and wasps) have been presumed to be exclusively sensory appendages, although the antennae of a number of the Parasitica also support a variety of glands. Using both SEM and TEM we show the presence of ectodermal glands in the antennae of workers and queens, but not in the males, of the Imported Fire Ant, Solenopsis invicta Buren. These glands and their associated pores are present on a glabrous proximal region of A9 of the antennae of workers and both A9 and A10 of queens. The pores leading to the bicellular secretory units in the antennae are more numerous on A10 of the queen followed by A9 of workers and in both cases they form a uniform ring around the segment. However, the pores on A9 of the queen are more numerous on the dorsal surface. While a paste-like secretion can sometimes be seen emerging from the pores of workers, this material is commonly seen from the pores of queen antennae. In social Aculeata, antennal glands have previously been found only in males of some vespids. This report, the first for ants and the first for females of social Aculeata, gives evidence for antennal glands in S. invicta.
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