Download Discovering Pheromones of the Red Imported Fire Ant (Solenopsis

Discovering Pheromones of the Red Imported Fire Ant
(Solenopsis invicta Buren): A Review and Proposed New
Target for Pheromone Disruption1
Robert Renthal
Department of Biology University of Texas at San Antonio, San Antonio, Texas 78249
USA
J. Agric. Urban Entomol. 20(3): 113–121 (July 2003)
ABSTRACT
A method is described for discovering pheromones of the red
imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), using
affinity chromatography with the pheromone-binding proteins of the ant. Proteins in the pheromone- and odorant-binding protein family, which are abundant in moths and other insect species, are not apparent in fire ant antennae.
Two other types of hydrophobic ligand-binding protein—chemosensory protein, and apolipophorin-III—could be important in fire ant olfaction. These
proteins may be useful for identifying pheromones by affinity chromatography.
The availability of fire ant pheromones could be a valuable addition to other
pest management strategies.
KEY WORDS
Hymenoptera, Formicidae, Solenopsis invicta, red imported
fire ant, pheromones, pheromone-binding proteins, chemosensory proteins, encapsulins, apolipophorin-III
Ants use pheromones to identify the colony, signal alarms, mark trails to food,
attract workers to brood and to the queen, and bring males and females together
for mating (Hölldobler & Wilson 1990). Queen pheromones also may be involved
in the maintenance of polygyny (Keller & Ross 1998, Ross & Keller 1998, Krieger
& Ross 2002) and in founding slave-making colonies (Mori et al. 2000). In addition, foraging, feeding, and defending the nest depend on detection of general
odors and tastes and on detection of kairomones (signals from other species).
Clearly, interference with pheromone-based communication in a fire ant colony
would be a useful goal for management of the red imported fire ant (RIFA),
Solenopsis invicta Buren (Hymenoptera: Formicidae).
Pheromones of some insect species have become useful tools in pest management. Two strategies based on pheromones have been used with varying success:
attract and kill, in which a sex pheromone lures insects to insecticide (Charmillot
et al. 2000); and mating disruption (Shani 2000), in which a mating pheromone is
released at the time of mating to mask directional information contained in concentration gradients. Pheromones of relatively large insects can be identified by
simple extraction and chromatographic methods, when the glandular source of
the pheromone is known and the behavioral response is clear. Pheromone iden-
1
Accepted for publication 13 February 2004.
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tification technology has been enhanced by advances such as electroantennographic recording as a detector for gas chromatography (Arn et al. 1975), and solid
phase microextraction techniques (Jones & Oldham 1999). Although many RIFA
pheromones have been identified (Hölldobler & Wilson 1990, Williams et al. 1981,
Vander Meer et al. 1980, Rocca et al. 1983a,b, Glancy et al. 1984, Vander Meer et
al. 1988, Alonso & Vander Meer 1997, Vargo 1997), pheromones have not yet been
useful for control of RIFA or any other pest ant species. This may be the result in
part, of the complexity of chemical signaling by ants. Vargo & Hulsey (2000) found
multiple glandular sources of an ant pheromone. Another difficulty in exploiting
ant pheromones is that some responses to releaser pheromones require primer
pheromones, which complicates the process of pheromone discovery.
My laboratory has been pursuing a strategy of using molecular biology tools to
identify fire ant pheromones by exploiting protein components of the ant’s olfactory system. Insect pheromones are received by sensilla primarily located in the
antenna, where they first are captured at the air/water interface by pheromonebinding proteins (Pelosi & Maida 1995; Fig. 1). These proteins are thought to
increase the solubility of pheromones in the aqueous sensillar lymph, and they
may also be involved in signaling to the pheromone receptors in the olfactory
neuron membrane. Purified ant pheromone-binding proteins could be used to
capture their pheromone targets from extracts of ants, using the technique known
as affinity chromatography. In general, this strategy is far more complicated than
traditional pheromone purification methods. If the complete genome of an insect
species has been sequenced, then it is straightforward to identify potential pheromone-binding protein sequences. However, even for a species such as RIFA, which
lacks a sequenced genome, there may be advantages in attempting to identify
pheromones via their binding proteins, as explained in the following sections.
Pheromone-Binding Protein Families
There are several different pheromone-binding protein families. Although
these proteins have unusually high levels of amino acid sequence divergence, the
families can be distinguished by characteristic amino acid sequence patterns and
three-dimensional structures. The first pheromone/odorant-binding proteins
were identified in vertebrates (Pevsner et al. 1988). These are members of the
lipocalin family of hydrophobic ligand-binding proteins. Lipocalins, with molecular weights in the range of 25 kDa, have sequences containing three distinctive
amino acid sequence motifs, and they fold into a ␤-barrel structure (Flower 1996).
Lipocalins are found in insects, for example as a pigment carrier in moth
hemolymph (Holden et al. 1987), but their role in insect olfaction seems limited.
In a swallowtail butterfly, a lipocalin was found to be involved in carrying lipophilic plant substances to tarsal receptors for locating oviposition sites (Tsuchihara et al. 2000); and in a cockroach, a lipocalin was found in a tergal gland
secretion (Korchi et al. 1999). So far, lipocalins have not been identified in insect
antennae.
Two types of antennal ligand-binding proteins that are unique to insect olfaction have been identified: the pheromone-binding protein/odorant-binding protein
(PBP/OBP) family, and the chemosensory (or sensory appendage) protein family.
The PBP/OBP family consists of proteins with molecular weights in the 12- to
16-kDa range. The amino acid sequences are extremely divergent, but all contain
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Fig. 1. Cross-section of antenna (left panel) showing sensilla and olfactory receptor neuron. Blow-up (right panel) shows pheromone-binding proteins
capturing pheromones and carrying them to the neuronal membrane surface.
a pattern of six cysteines that form three disulfide bonds. The sequence can be
described in the Prosite notation (http://www.expasy.org/prosite/) as: C-x(26,35)C-x(3)-C-x(36,43)-C-x(8,14)-C-x(8)-C. The three-dimensional structure of the
PBP/OBP proteins is known from X-ray crystallography and nuclear magnetic
resonance spectroscopy of the Bombyx mori (Lepidoptera: Bombycidae) sex pheromone-binding protein (Sandler et al. 2000, Horst et al. 2001, Lee et al. 2002). The
protein consists of a cluster of six or seven ␣-helices surrounding a hydrophobic
cavity where the pheromone binds. PBP/OBPs are commonly expressed in antennal sensilla and also in sensilla on mouth parts. In S. invicta, a PBP/OBP family
member is expressed in the thorax of queens (Krieger & Ross 2002). Different
PBP/OBPs are localized to particular subgroups of sensilla (Vogt et al. 1991,
Pikielny et al. 1994). Some sensilla may contain more than one type of PBP/OBP
(Hekmat-Scafe et al. 1997). Thus, their localization resembles the distribution of
olfactory receptor neurons, which appear to specialize in particular odors and may
occur together in the same sensillum with receptor neurons of different specificity. The binding of pheromones or general odors to PBP/OBPs is usually not as
specific as binding to the olfactory receptors on the membranes of the olfactory
neurons. For example, two different species of moth, which use two different
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isomers of the same molecule for the sex pheromone, have the same PBP for both
molecules (Willett & Harrison 1999). Furthermore, a PBP was found to be incapable of distinguishing between the R and S enantiomers of a beetle pheromone,
in contrast to the enantiomer-specific olfactory receptors for this pheromone
(Wojtasek et al. 1998). The dissociation constants for ligands from PBP/OBPs
were reported to be in the micromolar range (Du & Prestwich 1995), or 0.1 to
1 ␮M (Campanacci et al. 2001). This is several orders of magnitude weaker than
ligand dissociation constants found for G-coupled receptors (Caron & Lefkowitz
1976), but similar to the interactions found for vertebrate pheromone-binding
proteins (Vincent et al. 2000). Ligand release appears to be triggered by low pH,
which causes a substantial conformational change in the OBP (Wojtasek & Leal
1999, Horst et al. 2001). The negative surface charge of the receptor membrane
may be sufficient to lower the surface pH to the level, which triggers the ligandreleasing conformational change. Proteins related to the insect PBP/ OBP family
include the B-protein of the tubular accessory gland secretion of Tenebrio molitor
(Coleoptera: Tenebrionidae) (Paesen & Happ 1995), and also Tenebrio
hemolymph protein THP12 (Rothemund et al. 1999).
A second type of ligand binding protein involved in insect olfaction is known as
the chemosensory protein (CSP) or sensory appendage protein (SAP) family (Mameli et al. 1996, Picimbon & Leal 1999, Ishida et al. 2002). These proteins are
expressed in antennae, palps, tarsi, and other tissues. The CSP family has less
divergent amino acid sequences than the PBP/OBP and lipocalin families. Members of the CSP family have a conserved pattern of four cysteines, which form two
disulfide bonds. The sequence can be summarized in Prosite notation as C-x(6,8)C-x(18,19)-C-x(2)-C. The three-dimensional structure, known from X-ray crystallography of CSP from the moth Mamestra brassicae (Lepidoptera: Noctuidae),
shows six ␣-helices with a different spatial arrangement from the PBP/OBP family (Lartigue et al. 2002). Most CSPs are smaller than PBP/OBPs, having molecular weights around 13 kDa. Binding of model compounds indicates ligand
dissociation constants in the micromolar range (Lartigue et al. 2002). A protein
with a sequence similar to the CSP family was isolated from Drosophila melanogaster (Diptera: Drosophilidae) ejaculatory bulb (Bohbot et al. 1998).
Because the PBP/OBPs and the CSPs are found in both olfactory and nonolfactory tissues, Leal (2003) recently suggested using the term “encapsulins” to
refer to these proteins. This term provides a category for classification of newly
discovered proteins that are similar in structure to olfactory proteins but that do
not yet have a known biological function. The encapsulins contain two protein
families with unrelated amino acid sequences. I suggest using the terms “2X
encapsulins” to denote the CSP family and “3X encapsulins” to denote the PBP/
OBP family. The 2X and 3X designations refer to the distinctive sequence patterns in the two families: CXXC in the CSP family, and CXXXC in the PBP/OBP
family (using the amino acid single letter code, where C is cysteine and X is any
amino acid).
Pheromone Discovery
When a receptor is known but the ligand molecules that bind to it are unknown, affinity chromatography can be used to identify the ligands. To identify
unknown pheromones, a purified pheromone-binding protein can be attached to a
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117
solid support, such as agarose (Fig. 2A). An extract containing the pheromones is
passed down a chromatography column packed with the agarose-coupled pheromone-binding protein. Unbound molecules are washed away (Fig. 2B), leaving the
ligands bound to the column. The ligands can be eluted (e.g. with a pH change, or
solvent change) and analyzed by mass spectrometry. Using the pheromonebinding proteins to capture the ligands has the virtue of collecting groups of
isomers, in cases where the pheromone is a complex mixture, since the binding
specificity is not as exact as the neuronal olfactory receptors (Campanacci et al.
2001).
Although the total numbers of pheromone-binding proteins is not known in
any ant species, a BLAST search of the Drosophila genome shows at least twelve
3X encapsulin genes and four 2X encapsulin genes, and the Anopheles gambiae
(Diptera: Culicidae) genome shows at least eighteen 3X encapsulin genes and six
2X encapsulin genes. This contrasts with about 80 olfactory receptors each in
Drosophila and Anopheles. Because of the high sequence diversity of the encapsulins, it is not possible to isolate orthologs from ants using these dipteran sequences to design PCR primers. Instead, it is necessary to isolate the proteins
from the ant antenna to obtain an amino terminal amino acid sequence for designing PCR primers. The full-length sequence is then obtained by PCR from
antennal cDNA. For subsequent ligand-binding studies, the protein is expressed
in bacteria using recombinant DNA techniques. Unfortunately, the RIFA worker
antenna shows a large diversity of low molecular weight proteins, making identification of encapsulins difficult. Of the major low molecular weight proteins with
free amino termini, one has an N-terminal sequence similar to a soft cuticle
protein known in other insect species, and the other may be similar to a CSP first
identified in the Argentine ant, Linepithema humile (Hymenoptera: Formicidae)
(Ishida et al. 2002). We thought that the RIFA male would have a simpler set of
olfactory proteins in the antenna because males have a simpler behavioral repertoire. RIFA male’s only apparent task is to participate in mating flights. The
male antennal morphology is much simpler than that of workers (Renthal et al.
2003), suggesting simpler olfactory processing. To our surprise, we found that the
major low molecular weight male antennal protein with a free amino terminus is
apolipophorin-III (Guntur et al. 2004). This hemolymph protein was first identified in locusts as a lipid transport protein (Narayanaswami & Ryan 2000). Apolipophorin-III (ALP-III) has a molecular weight of 18 kDa and the amino acid
sequences from different species are highly divergent. A single distinguishing
sequence pattern has not yet been identified. The three-dimensional structures of
moth (Wang et al. 1997) and locust (Breiter et al. 1991) ALP-III show a cluster of
five parallel ␣-helices, which are thought to open with a hinge-like motion to bind
to hemolymph lipophorins I and II. In addition to its function in lipid transport,
ALP-III has been found to be involved in immediate immunity (Wiesner et al.
1997), apoptosis (Sun et al. 1995), and pheromone secretion (Liu et al. 2000).
Since ALP-III can reversibly bind hydrophobic molecules and is expressed in the
male fire ant antenna, we plan to further evaluate this protein for a possible role
in pheromone reception.
Conclusions
In insect species with scarce or heterogeneous encapsulins, it may be possible
to purify candidate pheromones by affinity chromatography using proteins ex-
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Fig. 2. Pheromone-binding protein (PBP) attached to agarose beads. A. Insect
extract containing pheromone passed down a column of PBP-agarose. B.
Most of extract washes away, leaving pheromone bound to PBP on agarose beads.
pressed in bacteria from recombinant DNA sequences. In addition, other hydrophobic ligand binding proteins such as apolipophorin-III, which may be involved
in pheromone transport, could also be used for pheromone discovery.
Acknowledgment
Supported by a grant from the Texas Imported Fire Ant Research and Management
Project.
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