Branched tyramides from males of the harvester ant, Pogonomyrmex badius.

Tyramides are produced in microgram quantities by males of species in the large Myrmicine ant sub-family (> 7000 species). Tyramides are transferred to female sexuals during mating where a specific female sexual evolved enzyme hydrolyzes the tyramides to the biogenic amine, tyramine. Tyramine is a ligand for receptors that rapidly activate reproductive development in the newly mated queen-previously reproductively inhibited by the mother queen. Without this elaborate biogenic amine precursor and co-evolved female sexual derived tyramide hydrolase, the defenseless newly mated queen's worker production would be delayed by up to 6 days, which could be lethal to the new queen. This is one of possibly several ant species separation mechanisms evolved to maintain species integrity. Here we report two methyl-branched tyramides from harvester ant, Pogonomyrmex badius, males, including one highly branched tyramide not previously reported.

The Evolution of Tyramides in Male Fungus-Growing Ants (Formicidae: Myrmicinae: Attini: Attina).

Ants use a variety of semiochemicals for essential activities and have been a source for many novel natural products. While ant taxa produce a wide variety of chemicals, the chemistry and ecology of male ants have remained understudied. Tyramides are a class of compounds that have been found only in males of the Myrmicinae ant subfamily. Tyramides found in the fire ant Solenopsis invicta are transferred to gynes during mating where they are converted to tyramine, leading to rapid reproductive development. To further understand the evolution of tyramide production in male ants, we determined the tyramide composition in males of 15 fungus-growing ant species (Formicidae: Myrmicinae: Attini: Attina) and a Megalomyrmex species (Formicidae: Myrmicinae: Solenopsidini). Thirteen tyramides were identified, four for the first time in natural sources, and their percent composition was mapped to the fungus-growing ant phylogeny.

Novel alkaloids from the fire ant, Solenopsis geminata.

South American fire ants, Solenopsis richteri and Solenopsis invicta, were accidently introduced into the southern USA in the 1900s and 1930s, respectively. The rapid spread and high population densities of S. invicta, and its potent sting, resulted in broad economic impacts and a variety of research efforts. In the 1970s, their venom alkaloids were identified as a complex blend of trans-2-methyl-6-alkyl- and alkenyl-piperidines. Solenopsis geminata is a worldwide tramp species but a native of the southern coastal regions of the USA. It was found to only produce cis- and trans-2-methyl-6-undecyl-piperidines. These alkaloids were considered the Solenopsis ancestral alkaloid profile since they were identified from female sexuals (potential queens) of all Solenopsis species in South and North America. The dramatic modification of alkaloids in Solenopsis invicta was attributed to their response to evolutionary pressure and the lack of change in S. geminata alkaloids due to no response to evolutionary pressure. Here we report the unexpected discovery of 6-undecyl-pyridine, 2-methyl-6-undecyl-pyridine and 2-methyl-6-(1)-undecenyl-pyridine as components of S. geminata worker venom, suggesting that S. geminata like its South American relatives have responded to evolutionary pressures. Our results will stimulate future research on S. geminata populations throughout the tropical/subtropical world.

Male fire ant neurotransmitter precursors trigger reproductive development in females after mating.

Social insect queens have evolved mechanisms to prevent competition from their sexual daughters. For Solenopsis invicta, the fire ant, queens have evolved a primer pheromone that retards reproductive development in their winged reproductive daughters. If these daughters are removed from the influence of the queen, it takes about a week to start reproductive development; however, it starts almost immediately after mating. This dichotomy has been unsuccessfully investigated for several decades. Here we show that male fire ants produce tyramides, derivatives of the biogenic amine tyramine, in their reproductive system. Males transfer tyramides to winged females during mating, where the now newly mated queens enzymatically convert tyramides to tyramine. Tyramine floods the hemolymph, rapidly activating physiological processes associated with reproductive development. Tyramides have been found only in the large Myrmicinae ant sub-family (6,800 species), We suggest that the complex inhibition/disinhibition of reproductive development described here will be applicable to other members of this ant sub-family.

GPCR-Based Bioactive Peptide Screening Using Phage-Displayed Peptides and an Insect Cell System for Insecticide Discovery.

The discovery of new insecticides improves integrated pest management (IPM), but is usually a long high-risk process with a low probability of success. For over two decades, insect neuropeptides (NPs) and their G-protein coupled receptors (GPCRs) have been considered as biological targets for insect pest control, because they are involved in almost all physiological processes associated with insect life stages. A key roadblock to success has been the question of how large volume chemical libraries can be efficiently screened for active compounds. New genomic and proteomic tools have advanced and facilitated the development of new approaches to insecticide discovery. In this study, we report a novel GPCR-based screening technology that uses millions of short peptides randomly generated by bacteriophages, and a method using an insect Sf9 cell expression system. The fire ant is a good model system, since bioactive peptides have been identified for a specific GPCR. The novel small peptides could interfere with the target GPCR-ligand functions. Therefore, we refer to this new mechanism as "receptor interference" (RECEPTORi). The GPCR-based bioactive peptide screening method offers multiple advantages. Libraries of phage-displayed peptides (~109 peptides) are inexpensive. An insect cell-based screening system rapidly leads to target specific GPCR agonists or antagonists in weeks. Delivery of bioactive peptides to target pests can be flexible, such as topical, ingestion, and plant-incorporated protectants. A variety of GPCR targets are available, thus minimizing the development of potential insecticide resistance. This report provides the first proof-of-concept for the development of novel arthropod pest management strategies using neuropeptides, and GPCRs.

Identification and Characterization of GPCRs for Pyrokinin and CAPA Peptides in the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae).

The brown marmorated stink bug, Halyomorpha halys, is an invasive hemipteran that causes significant economic losses to various agricultural products around the world. Recently, the pyrokinin and capa genes that express multiple neuropeptides were described in this species. Here we report six pyrokinin and capa GPCRs including two splice variants, and evaluate their (a) ability to respond to neuropeptides in cell-based assays, and (b) expression levels by RT-PCR. Functional studies revealed that the H. halys pyrokinin receptor-1 (HalhaPK-R1a & b) responded to the pyrokinin 2 (PK2) type peptide. RT-PCR results revealed that these receptors had little or no expression in the tissues tested, including the whole body, central nervous system, midgut, Malpighian tubules, and reproductive organs of males and females. HalhaPK-R2 showed the strongest response to PK2 peptides and a moderate response to pyrokinin 1 (PK1) type peptides (= DH, diapause hormone), and was expressed in all tissues tested. HalhaPK-R3a & b responded to both PK1 and PK2 peptides. Their gene expression was restricted mostly to the central nervous system and Malpighian tubules. All PK receptors were dominantly expressed in the fifth nymph. HalhaCAPA-R responded specifically to CAPA-PVK peptides (PVK1 and PVK2), and was highly expressed in the Malpighian tubules with low to moderate expression in other tissues, and life stages. Of the six GPCRs, HalhaPK-R3b showed the strongest response to PK1. Our experiments associated the following peptide ligands to the six GPCRs: HalhaPK-R1a & b and HalhaPK-R2 are activated by PK2 peptides, HalhaPK-R3a & b are activated by PK1 (= DH) peptides, and HalhaCAPA-R is activated by PVK peptides. These results pave the way for investigations into the biological functions of H. halys PK and CAPA peptides, and possible species-specific management of H. halys.

Phenotypic Effects of PBAN RNAi Using Oral Delivery of dsRNA to Corn Earworm (Lepidoptera: Noctuidae) and Tobacco Budworm Larvae.

Insect neuropeptides represent more than 90% of all insect hormones. The pheromone biosynthesis activating neuropeptide (PBAN)/pyrokinin family is a major group of insect neuropeptides. These neuropeptides regulate a variety of biological functions from embryo to adult in moths including, sex pheromone biosynthesis and diapause. Other functions are yet to be determined. The identification of suitable target genes is most important for the successful application of RNA interference (RNAi) for pest insect control. Insect neuropeptide genes including PBAN are known to have multiple functions and could be a good target for RNAi suppression. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target for two economically important moth species, the corn earworm, Helicoverpa zea (Boddie), and the tobacco budworm, Heliothis virescens (Fabricius). We investigated RNAi effects on immature moths that had ingested the specific double-stranded RNA (dsRNA) starting at the first instar larva through pupation. We report that RNAi treatments resulted in delay of larval growth, interference of pupal development, and mortality in the two pest moths. In addition, we selected small interfering RNAs (siRNAs) to determine if they have negative phenotypic effects similar to their full-length RNAi parents. This is one of the few examples of negative RNAi effects on lepidopteran pests via feeding and suggests possible RNAi-based control of pest moths.

Cuticular Hydrocarbon Profiles Differentiate Tropical Fire Ant Populations (Solenopsis geminata, Hymenoptera: Formicidae).

The cuticular hydrocarbons (CHCs) from hexane rinses of workers from two Florida populations (dark and red forms) of the tropical fire ant, Solenopsis geminata, were separated by silica gel chromatography and identified by GC/MS analysis. Both the dark form and the red form produce similar CHCs with carbon chain lengths ranging from 17 to 35. However, the relative percentages of these CHCs were consistently different between the two color forms. The largest CHC component in the dark form is tricosane, and (Z)-9-tricosene for the red form. There were several significant differences in percent composition. For example, the dark form was characterized by a low tricosene:tricosane ratio (ca. 0.25), whereas this ratio was > 2.5 for the red form. The ratio of tricosene:tricosane can be used as a diagnostic biomarker to delimit the dark and red forms. Cluster analysis showed that the CHCs patterns of dark form colonies are completely separated from the CHC pattern of red form colonies. Differences in social behaviors like nestmate recognition and polygyny between workers from this dark form and the red form await further investigation.

Identification and expression of a new member of the pyrokinin/pban gene family in the sand fly Phlebotomus papatasi.

The major family of neuropeptides (NPs) derived from the pk (pyrokinin)/pban (pheromone biosynthesis activating neuropeptide) gene are defined by a common FXPRL-NH2 or similar sequence at the C-termini. This family of peptides has been found in all insect groups investigated to date and is implicated in regulating various physiological functions, including pheromone biosynthesis and diapause, but other functions are still largely unknown in specific life stages. Here we identify two isoforms of pk/pban cDNA encoding the PBAN domain from the sand fly Phlebotomus papatasi. The two pk/pban isoforms have the same sequence except for a 63 nucleotide difference between the long and short forms, and contain no alternative mRNA splicing site. Two NP homologues, DASGDNGSDSQRTRPPFAPRLamide and SLPFSPRLamide are expected, however, sequence corresponding to the diapause hormone was not found in the P. papatasi pk/pban gene. The PBAN-like amino acid sequence homologue SNKYMTPRL is conserved in the gene, but there is no cleavage site for processing a functional peptide. Characterizing the expression of the isoforms in developmental stages and adults indicates that the short form is differentially transcribed depending on the life stage. The P. papatasi pk/pban gene is the only known pk/pban gene with two transcriptional isoforms and from examination of endoproteolytic cleavage sites is expected to produce fewer peptides than most of the pk/pban genes elucidated to date; only Drosophila melanogaster is simpler with a single NP detected by mass spectroscopy. A phylogenetic analysis showed P. papatasi pk/pban grouped more closely with other nematoceran flies rather than higher flies.

Identification and expression of capa gene in the fire ant, Solenopsis invicta.

Recent genome analyses suggested the absence of a number of neuropeptide genes in ants. One of the apparently missing genes was the capa gene. Capa gene expression in insects is typically associated with the neuroendocrine system of abdominal ganglia; mature CAPA peptides are known to regulate diuresis and visceral muscle contraction. The apparent absence of the capa gene raised questions about possible compensation of these functions. In this study, we re-examined this controversial issue and searched for a potentially unrecognized capa gene in the fire ant, Solenopsis invicta. We employed a combination of data mining and a traditional PCR-based strategy using degenerate primers designed from conserved amino acid sequences of insect capa genes. Our findings demonstrate that ants possess and express a capa gene. As shown by MALDI-TOF mass spectrometry, processed products of the S. invicta capa gene include three CAPA periviscerokinins and low amounts of a pyrokinin which does not have the C-terminal WFGPRLa motif typical of CAPA pyrokinins in other insects. The capa gene was found with two alternative transcripts in the CNS. Within the ventral nerve cord, two capa neurons were immunostained in abdominal neuromeres 2-5, respectively, and projected into ventrally located abdominal perisympathetic organs (PSOs), which are the major hormone release sites of abdominal ganglia. The ventral location of these PSOs is a characteristic feature and was also found in another ant, Atta sexdens.

Identification and expression of PBAN/diapause hormone and GPCRs from Aedes aegypti.

Neuropeptides control various physiological functions and constitute more than 90% of insect hormones. The pheromone biosynthesis activating neuropeptide (PBAN)/pyrokinin family is a major group of insect neuropeptides and is well conserved in Insecta. This family of peptides has at least two closely related G-protein-coupled receptors (GPCRs) activated by PBAN and a diapause hormone (DH). They have been shown to control several biological activities including pheromone production and diapause induction in moths. However, beyond some moth species, the biological function(s) of PBAN/pyrokinin peptides are largely unknown although these peptides are found in all insects. In this study we identified and characterized PBAN/pyrokinin peptides and corresponding GPCRs from the mosquito, Aedes aegypti. Ae. aegypti PBAN mRNA encodes four putative peptides including PBAN and DH, and is expressed in females and males during all life stages. The PBAN receptor (PBAN-R) and the DH receptor (DH-R) were functionally expressed and confirmed through binding assays with PBAN and DH peptides. These receptors are differentially expressed from eggs to adults with the relative gene expression of the PBAN-R significantly lower during the 4th instar larval (L4) and pupal (P1-P2) stages compared to the 2nd and 3rd instar larval stages (L2 and L3). However, DH-R expression level is consistently 4-10 times higher than the PBAN-R in the same period, suggesting that PBAN-R is downregulated in the late larval and pupal stages, whereas DH-R stays upregulated throughout all developmental stages. PBAN/pyrokinin mRNA expression remains high in all stages since it produces PBAN and DH peptides. This study provides the foundation for determining the function(s) of the PBAN/pyrokinin peptides in mosquitoes and establishes data critical to the development of methods for disruption of these hormone actions as a novel strategy for mosquito control.

Mitigating the allergic effects of fire ant envenomation with biologically based population reduction.

PURPOSE OF REVIEW: To describe the current efforts to use biological control agents to reduce fire ant population levels, thus ultimately reducing the number of human sting and allergic reaction incidents. RECENT FINDINGS: Climate change and worldwide fire ant expansion will increase the frequency of human encounters and allergenic events, putting additional pressure on the public health sector. Six species of fire ant decapitating flies are now established in the United States. The microsporidium Kneallhazia solenopsae is well established and new fire ant hosts have been identified. The fire ant virus Solenopsis invicta virus 3 shows good potential for use as an environmentally friendly biopesticide because of its virulence and host specificity. SUMMARY: During separate founding events in the United States, Australia, mainland China, and Taiwan, fire ants native to South America escaped their native pathogens and parasites. Consequently, fire ant populations in these introduced regions pose a serious public health threat to the human populations by envenomation and subsequent allergic reactions. Specific, self-sustaining biological control agents have been discovered, studied, and released into fire ant populations in the United States in an effort to re-establish an ecological/competitive balance, resulting in reduced fire ant densities and human exposure.

Contact toxicities of anuran skin alkaloids against the fire ant (Solenopsis invicta).

Nearly 500 alkaloids, representing over 20 structural classes, have been identified from the skin of neotropical poison frogs (Dendrobatidae). These cutaneous compounds, which are derived from arthropod prey of the frogs, generally are believed to deter predators. We tested the red imported fire ant (Solenopsis invicta) for toxicosis following contact with 20 alkaloids (12 structural classes) identified from dendrobatids or other anurans. Individual ants forced to contact the dried residues of 13 compounds exhibited convulsions and/or reduced ambulation. We estimated the cutaneous concentrations of several compounds based on their reported recoveries from skin extracts of free-ranging frogs and our measurements of the skin surface areas of museum specimens. Pumiliotoxin 251D exhibited contact toxicity below its estimated cutaneous concentration in the Ecuadorian frog, Epipedobates anthonyi, an observation consistent with the hypothesized role of this compound in anuran chemical defense. Our results and those of a previous study of mosquitoes indicate that some anuran skin compounds function defensively as contact toxins against arthropods, permeating their exoskeleton.

Ant trail pheromone biosynthesis is triggered by a neuropeptide hormone.

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour's gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN's role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.

Defensive spiroketals from Asceles glaber (Phasmatodea): absolute configuration and effects on ants and mosquitoes.

Insects are the largest and most diverse group of organisms on earth, with over 1,000,000 species identified to date. Stick insects ("walkingsticks" or "phasmids", Order Phasmatodea) are known for and name-derived from their camouflage that acts as a primary line of defense from predation. However, many species also possess a potent chemical defense spray. Recently we discovered that the spray of Asceles glaber contains spiroketals [a confirmed major component: (2S,6R)-(-)(E)-2-methyl-1,7-dioxaspiro[5.5]undecane, and a tentatively identified minor component: 2-ethyl-1,6-dioxaspiro[4.5]decane] and glucose. In this paper, we: 1) illustrate the identification of spiroketals and glucose in the defense spray of A. glaber by using Nuclear Magnetic Resonance (NMR), Gas Chromatography/Mass Spectrometry (GC/MS), and comparison with a synthetic reference sample; 2) provide the elucidation of the absolute configuration of the major spiroketal in that defense spray; and 3) demonstrate the effect of this compound and its enantiomer on both fire ants (Solenopsis invicta) and mosquitoes (Aedes aegypti).

Aerosol delivery of trail pheromone disrupts the foraging of the red imported fire ant, Solenopsis invicta.

BACKGROUND: The fire ant, Solenopsis invicta, is one of the most aggressive and invasive species in the world. The trail pheromone Z,E-α-farnesene (91% purity) was prepared, and disruption of worker trail orientation was tested using an ethanol-based aerosol formulation presenting a single puff of this compound by airbrush and compressed air. Trail-following behaviour was recorded by overhead webcam and ants digitised before and after presentation of the aerosol treatment at four rates (1.6, 16, 160 and 1600 ng cm(-2)). RESULTS: Ants preferred 110 ng cm(-1) over 11, 1.1 and 0.11 ng cm(-1) for trail following. Within seconds of presentation of 1600 ng cm(-2), the highest dose tested, trail disruption was observed. Disruption was evident as reduced arrival success and reduction in the trail integrity statistic (r(2)), as well as increased deviation from the trail (deg). The distribution of walking track angles was also flattened. CONCLUSIONS: The feasibility of using aerosol for delivery of trail pheromone was demonstrated, but the need for high purity combined with the difficulty of commercial supply makes this technique impractical. However, the commercial production of Z,E-α-farnesene of high purity by industrial biotechnology or from (E)-nerolidol may be possible in future, which would facilitate further development of trail pheromone disruption of S. invicta.

Phenotypic impacts of PBAN RNA interference in an ant, Solenopsis invicta, and a moth, Helicoverpa zea.

Insect neuropeptide hormones represent more than 90% of all insect hormones. The PBAN/pyrokinin family is a major group of insect neuropeptides, and they are expected to be found from all insect groups. These species-specific neuropeptides have been shown to have a variety of functions from embryo to adult. PBAN is well understood in moth species relative to sex pheromone biosynthesis, but other potential functions are yet to be determined. Recently, we focused on defining the PBAN gene and peptides in fire ants in preparation for an investigation of their function(s). RNA interference (RNAi) technology is a convenient tool to investigate unknown physiological functions in insects, and it is now an emerging method for development of novel biologically-based control agents as alternatives to insecticides. This could be a paradigm shift that will avoid many problems associated with conventional chemical insecticides. In this study, we selected the PBAN gene and its neuropeptide products as an RNAi target from two insect groups; a social insect, the fire ant (Solenopsis invicta) and a non-social insect, the corn earworm (Helicoverpa zea). Both insects are economically important pests. We report negative impacts after PBAN dsRNA treatment to suppress PBAN gene transcription during developmental and adult stages of both species, e.g. increased adult and larval mortality, delayed pupal development and decreased sex pheromone production in the moth. This is an important first step in determining the multiple functions of the PBAN gene in these two insects. This work illustrates the variety of phenotypic effects observed after RNAi silencing of the PBAN gene and suggests the possibility of novel biologically-based insect pest control methods.

Molecular Structure and Diversity of PBAN/pyrokinin Family Peptides in Ants.

Neuropeptides are the largest group of insect hormones. They are produced in the central and peripheral nervous systems and affect insect development, reproduction, feeding, and behavior. A variety of neuropeptide families have been identified in insects. One of these families is the PBAN/pyrokinin family defined by a common FXPRLamide or similar amino acid fragment at the C-terminal end. These peptides, found in all insects studied thus far, have been conserved throughout evolution. The most well studied physiological function is regulation of moth sex pheromone biosynthesis through the pheromone biosynthesis activating neuropeptide (PBAN), although several developmental functions have also been reported. Over the past years we have extended knowledge of the PBAN/pyrokinin family of peptides to ants, focusing mainly on the fire ant, Solenopsis invicta. The fire ant is one of the most studied social insects and over the last 60 years a great deal has been learned about many aspects of this ant, including the behaviors and chemistry of pheromone communication. However, virtually nothing is known about the regulation of these pheromone systems. Recently, we demonstrated the presence of PBAN/pyrokinin immunoreactive neurons in the fire ant, and identified and characterized PBAN and additional neuropeptides. We have mapped the fire ant PBAN gene structure and determined the tissue expression level in the central nervous system of the ant. We review here our research to date on the molecular structure and diversity of ant PBAN/pyrokinin peptides in preparation for determining the function of the neuropeptides in ants and other social insects.

Male reproductive fitness and queen polyandry are linked to variation in the supergene Gp-9 in the fire ant Solenopsis invicta.

Supergenes are clusters of tightly linked loci maintained in specific allelic combinations to facilitate co-segregation of genes governing adaptive phenotypes. In species where strong selection potentially operates at different levels (e.g. eusocial Hymenoptera), positive selection acting within a population to maintain specific allelic combinations in supergenes may have unexpected consequences for some individuals, including the preservation of disadvantageous traits. The nuclear gene Gp-9 in the invasive fire ant Solenopsis invicta is part of a non-recombining, polymorphic supergene region associated with polymorphism in social organization as well as traits affecting physiology, fecundity and behaviour. We show that both male reproductive success and facultative polyandry in queens have a simple genetic basis and are dependent on male Gp-9 genotype. Gp-9(b) males are unable to maintain exclusive reproductive control over their mates such that queens mated to Gp-9(b) males remain highly receptive to remating. Queens mated to multiple Gp-9(B) males are rare. This difference appears to be independent of mating plug production in fertile males of each Gp-9 genotype. However, Gp-9(b) males have significantly lower sperm counts than Gp-9(B) males, which could be a cue to females to seek additional mates. Despite the reduced fitness of Gp-9(b) males, polygyne worker-induced selective mortality of sexuals lacking b-like alleles coupled with the overall success of the polygyne social form act to maintain the Gp-9(b) allele within nature. Our findings highlight how strong worker-induced selection acting to maintain the Gp-9(b) allele in the polygyne social form may simultaneously result in reduced reproductive fitness for individual sexual offspring.

Sampling high-altitude and stratified mating flights of red imported fire ant.

With the exception of an airplane equipped with nets, no method has been developed that successfully samples red imported fire ant, Solenopsis invicta Buren, sexuals in mating/dispersal flights throughout their potential altitudinal trajectories. We developed and tested a method for sampling queens and males during mating flights at altitudinal intervals reaching as high as "140 m. Our trapping system uses an electric winch and a 1.2-m spindle bolted to a swiveling platform. The winch dispenses up to 183 m of Kevlar-core, nylon rope and the spindle stores 10 panels (0.9 by 4.6 m each) of nylon tulle impregnated with Tangle-Trap. The panels can be attached to the rope at various intervals and hoisted into the air by using a 3-m-diameter, helium-filled balloon. Raising or lowering all 10 panels takes approximately 15-20 min. This trap also should be useful for altitudinal sampling of other insects of medical importance.