Horizontal Starch Gel Electrophoresis as a Tool for Studying GP-9

Most of the early research on Gp-9 was conducted using horizontal starch gel electrophoresis (HSGE) coupled with non-specific amido black staining as a method for surveying variation in the protein product of the gene, and the method continues to serve as a workhorse for efficiently conducting assays for a variety of uses in GP-9 research. In reference to this work, LI08 state that “… [GP-9] gel profiles were not documented in the literature” and “…native polyacrylamide gels (PAGE) … provides more consistent and reproducible profiles…”. Because much of what appears in the remainder of the current paper is based on results from HSGE, we offer the following comments with respect to the quality, reproducibility, and utility of the technique.

Details of gel and buffer compositions, running conditions, staining procedures, and absolute and relative protein migration distances for GP-9 using HSGE were provided a decade ago by DeHeer et al. [28]. Because HSGE separates proteins almost exclusively on the basis of net charge [29], [30], this technique is expected to distinguish only two classes of GP-9 proteins, those encoded by alleles of the b clade, which feature a charge-changing Glu151Lys replacement (Table 1), and those encoded by the remaining Gp-9 alleles, which lack this replacement [8]–[10]. This expectation has been confirmed using a large number of parallel protein/DNA assays in several fire ant species, with the relative HSGE mobilities of the protein products of Gp-9^b and the other alleles always as predicted from the inferred amino acid sequences [8], [10], [31] (e.g., Fig. 1). The fact that the derived, charge-variant Gp-9^b alleles are the only b-like alleles known to occur in the U.S.A. [8] (see also below) has led to the use of HSGE as a reliable, high-throughput tool for detecting polygyny in large sample sets from these invasive populations [e.g.], [32,33].

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Figure 1. Separation and visualization of GP-9 protein in S. invicta using HSGE coupled with amido black staining.

The GP-9 band of higher mobility is encoded by the B allele, whereas the band of lower mobility is encoded by the b allele. Material from which GP-9 was extracted and electrophoresed is as follows. Gel 1: lanes a–b, individual polygyne reproductive queen thoraces (both genotype Bb); lanes c–d, individual polygyne alate queen thoraces (both genotype bb); lanes e–f, individual polygyne alate queen thoraces (both genotype Bb); lanes g–h, individual polygyne alate queen thoraces (both genotype BB); lane i, individual triploid polygyne alate queen thorax (genotype BBb). Gel 2: lane a, individual polygyne alate queen head (genotype BB); lane b, individual polygyne alate queen head (genotype bb) ; lanes c–d, individual polygyne alate queen heads (both genotype Bb). Gel 3: lane a, individual adult worker head+thorax (genotype BB); lane b, individual adult worker head+thorax (genotype Bb); lane c, eggs (several hundred pooled) from polygyne nest (both B and b allelic proteins present); lane d, larvae (several hundred pooled 2nd instar) from polygyne nest (GP-9 not apparent). Gel 4: lane a, individual polygyne alate queen thorax (genotype Bb); lane b, monogyne adult male thoraces (50 pooled, only B allelic protein apparent). Gel 5: lane a, individual polygyne reproductive queen thorax (genotype Bb); lane b, individual polygyne reproductive queen hemolymph (genotype Bb); lane c, monogyne adult worker hemolymph (pooled from ten individuals, only B allelic protein apparent).

https://doi.org/10.1371/journal.pone.0007713.g001

We note that HSGE/GP-9 assays have been employed in several other contexts that, in aggregate, validate assay sensitivity and reproducibility. These assays were used: i) to predict the occurrence of b-like Gp-9 alleles that lack the charge-changing amino acid replacement characteristic of b alleles (the former class designated as b′ alleles; Table 1) in both S. invicta [26] and the related fire ant S. richteri [31], ii) to determine the ontogeny of expression of GP-9 in brood and adult females [5], as later confirmed using specific mRNA and polyclonal antibody assays [34, D. Gotzek et al. unpubl. data], iii) to confirm the presence of GP-9 at low levels in adult males [34] and in eggs [D. Gotzek et al. unpubl. data], as initially deduced using specific mRNA and antibody assays (see Fig. 1), and iv) to validate triploidy of S. invicta females, as confirmed in conjunction with microsatellite genotyping [35] (Fig. 1). Moreover, the equivalence of HSGE and SDS-PAGE coupled with amido black staining for separating and purifying GP-9 protein was demonstrated by Krieger and Ross [8; their Note 8 and Supplementary Online Material]. Although HSGE is conducted on pooled material (multiple individuals from single colonies) for some applications, the method is sufficiently sensitive to be used on individual adult queen heads and all but the smallest adult minor worker thoraces+heads [12], [36] (Fig. 1). Importantly, where we have judged HSGE to be inadequate to address particular questions, alternative molecular methods for assaying variation at the Gp-9 gene and its product have been employed; these include allele-specific PCR [9], [12], [37], [38], restriction fragment length polymorphism (RFLP) analysis [8], [31], direct sequencing of cDNA and genomic DNA [8]–[10], [38], microarray expression assays [20], and specific mRNA and polyclonal antibody assays [D. Gotzek et al. unpubl. data].

Expression of the Gp-9^b Allele in Polygyne S. invicta Colonies

A central claim of LI08 is that there exist “…two types of polygyne colonies, one that does not express GP-9^b (monogyne-like) and the other expressing both proteins, GP-9^B and GP-9^b.” This claim was based on the inability of the authors to detect the b allelic protein among workers (and apparently queens as well, see their Fig. 10) in three of the four ostensibly polygyne S. invicta colonies they studied. Such a finding hypothetically could be attributable either to a lack of workers in a polygyne colony bearing the b allele or to the inability of workers bearing the allele to express it (these possibilities were not distinguished nor discussed by LI08). To give a sense of the extraordinary nature of the claim that three-quarters of their polygyne study colonies lacked workers expressing GP-9^b, we summarize below the published evidence supporting an invariant link between the expression of polygyne social organization and both the presence and expression of b-like Gp-9 alleles in a fire ant colony's workers (and queens). We note that for S. invicta in the U.S.A., the association may be more narrowly defined as an invariant link between allele Gp-9^b (or its product) and polygyny, assuming that this is the only b-like allele that occurs in this newly colonized part of the range [8] (see also below).

Fifteen studies have documented the presence of b-like alleles in all polygyne colonies examined (Table 2); these studies surveyed a total of 1259 polygyne colonies of five different fire ant species (mostly S. invicta) sampled from three continents. Only eight of these studies explicitly tested for the presence of this class of Gp-9 alleles in the worker caste, but the remainder tested reproductive queens. All such queens possessed at least one b-like allele copy so, barring some unreported form of non-Mendelian inheritance of the gene [cf. 5], some proportion of workers in these study colonies can also be inferred to have possessed such alleles.

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Table 2. Studies documenting invariant presence of b-like Gp-9 alleles in inhabitants of polygyne fire ant colonies.

https://doi.org/10.1371/journal.pone.0007713.t002

Five studies have documented that the b allele not only is present but invariably is expressed in workers from polygyne S. invicta colonies in the introduced U.S.A. range (Table 3). This conclusion was based on the presence of the b allelic protein as assessed by HSGE in variable numbers of individual workers from all 225 colonies studied. Moreover, five studies have employed HSGE to show that, among thousands of reproductive queens sampled from many hundred polygyne colonies located across the U.S.A. range, each queen expressed the b allelic protein (Table 3).

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Table 3. Studies documenting expression of b allele of Gp-9 in adult female inhabitants of polygyne S. invicta colonies using HSGE.

https://doi.org/10.1371/journal.pone.0007713.t003

In order to directly examine the claim of LI08 that contradicts these previous findings, we conducted extensive sampling and testing of polygyne S. invicta colonies from College Station, Texas, a collection locality for one of the ostensibly polygyne samples in which workers reportedly failed to express the b allelic protein. Because the authors could not supply detailed collection locality information, we sampled 89 putative polygyne colonies from three sites dispersed around the city (see Table S1). Social organization of each colony initially was inferred in the field on the basis of nest distribution patterns, worker size distributions, and observation of multiple dealate queens (see Table 1 for terminology) [4], [39], [40]. However, because such information is not completely reliable, we definitively determined social organization for each colony as follows.

First, the mating status and reproductive development of up to four dealate queens per colony were determined; multiple mated queens with developed ovaries were found in 79 of the colonies. Next, for each of the remaining ten colonies from which only a single mated dealate queen was recovered, we determined genotype distributions for ten nestmate workers at six polymorphic allozyme loci. Worker genotype distributions in nine of these ten colonies were inconsistent with a simple-family (monogyne) colony structure in which all workers are the full-sister offspring of a single mother queen mated to a single (haploid) male [38], [41] (see Table 4); thus, we conclude that these were polygyne colonies from which we failed to recover more than a single reproductive queen during nest excavation. The final colony that we sampled (Col. 43) did contain workers whose genotype distributions implicated them as full sisters; moreover, the single mated dealate queen recovered possessed a multilocus genotype fully consistent with her being the mother of these workers (Table 4). Thus, the genotype composition of this colony is consistent with monogyne social organization. Finally, we assayed GP-9 of each sampled dealate queen from the 89 study colonies (n = 247). With the exception of the single queen from Col. 43, which exhibited only the single fast band characteristic of genotype BB, every other queen exhibited both bands, characteristic of genotype Bb. This is significant in that polygyne reproductive queens of S. invicta in the U.S.A. effectively always possess the Bb genotype, while monogyne queens bear the BB genotype [6]. In summary, we conclusively identified 88 of the College Station S. invicta colonies we sampled as polygyne, while one was determined to be monogyne.

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Table 4. Genotype distributions at six polymorphic allozyme loci for inhabitants of ten College Station S. invicta colonies from which only a single mated dealate queen was collected.

https://doi.org/10.1371/journal.pone.0007713.t004

We employed a sequential HSGE procedure to efficiently test for the presence and expression of the Gp-9^b allele in workers from our College Station study colonies (Fig. 2). In an initial screen, protein extracts pooled from 6–8 mature adult workers per colony were assayed for the presence of the b allelic protein. Given that as few as 10% of workers in polygyne colonies may bear a b allele [11,12, K. G. Ross unpubl. data], there is a chance that such a worker would not be included in a pool of so few workers sampled from any single colony (or that so few Bb workers were included that the b protein band would be comparatively faint). Thus, we conducted a secondary screen of another pooled 6–8 workers on any colonies for which the b allelic protein band was not visible and pronounced on the initial gel. Finally, for the remaining few colonies for which the b band still was not pronounced on the secondary gel, we screened individual extracts from an additional 8–10 workers per colony. The results of this sequential procedure clearly demonstrate that each of our 88 verified polygyne colonies contained workers bearing and expressing the Gp-9^b allele (Fig. 2). Moreover, the HSGE results described above demonstrate that every screened dealate queen from these colonies also bore and expressed the allele. Notably, there was no evidence for an expressed b allele among any of the workers from the single monogyne colony in our sample (Col. 43) screened singly (n = 24) or as pooled samples (n = 16) (Fig. 2), consistent with the invariant absence of such alleles previously reported in fire ant colonies of this social form. These results from our College Station S. invicta samples fully support previous findings that Gp-9^b (or some other b-like allele) invariably is present in and expressed by some proportion of workers (as well as all queens) in polygyne colonies, thus contradicting a central claim of LI08.

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Figure 2. Results of sequential HSGE procedure for testing presence and expression of Gp-9^b allele in adult S. invicta workers from College Station, Texas, U.S.A.

In the initial screen, each lane contained the pooled protein extracts of 6–8 adult worker nestmates (head+thorax); the 89 colonies were arranged sequentially across the lanes in the three initial screen gels (identification codes for groups of colonies appear above lanes). Asterisks in the initial screen gels indicate colonies for which additional pooled extracts of another 6–8 nestmate workers were analyzed in the secondary screen (colony identification codes appear above lanes in the secondary screen gel). Asterisks in the secondary screen gel indicate colonies that were subsequently screened using individual extracts from 8–10 workers per colony (colony identification codes appear above each group of lanes in the final screen gels). In the small final screen gel, “Q” denotes the lane containing extract from the presumed mother queen from Colony 43, the only monogyne colony in the sample, whereas “w” denotes lanes containing worker extracts (fourteen additional Col. 43 workers not depicted were individually screened; all possessed genotype BB). For all gels, groups of lanes containing test material are bracketed by standards (white arrows, standards with both B and b allelic proteins; black arrows, standards with B allelic protein only).

https://doi.org/10.1371/journal.pone.0007713.g002

The failure of LI08 to detect the b allele protein in three ostensibly polygyne samples (colonies), including one from the College Station area, can be attributed to any of several potential causes irrelevant to the proposed biological role of Gp-9 in inducing polygyny. An obviously important, but often poorly appreciated, requirement for any study of the fire ant social forms is that the social organization of each study colony be correctly identified. Polygyne and monogyne colonies can be difficult to differentiate using the field criteria of worker size and nest dispersion patterns, especially when colonies of the two types are interspersed. The presence of multiple dealate queens is not in itself sufficient to reliably identify any particular colony as polygyne, because monogyne colonies can naturally contain numerous virgin, non-reproductive dealate queens under some circumstances [4], [42]–[44], and traumatic dealation can occur in association with nest excavation. To our knowledge, only four definitive criteria exist for distinguishing polygyne from monogyne colonies of S. invicta in the introduced U.S.A. range: i) presence of multiple dealate queens that are mated and possess developed ovaries [45], [46], ii) presence of multiple matrilines represented among the workers [47] (diagnostic except in the rare case of recent queen turnover in a monogyne colony [32]), iii) presence of diploid males [48], and iv) presence of triploid females [35]. Evaluation of the latter three criteria requires the application of genetic methods, with the result that these criteria rarely have been used outside of our circle of collaborators [e.g.], [ 9], [32], [38], [49]–[51]; evaluation of all four criteria requires that colony fragments be returned to the laboratory for study.

The four colonies studied in LI08 were not evaluated with respect to any of these criteria by the authors; instead, they point to the collectors of the colonies as having determined the social organization “at the time of the field collections” [27]. For the two Texas colonies, neither Dr. K. M. Heinz, who supplied the material to the authors, nor Dr. S. B. Vinson, who supplied the colonies to Heinz, were able to provide us with information on the provenance or chain of custody of the samples, nor could they tell us on what basis these samples were determined to have originated from polygyne colonies. Moreover, the paper's authors were unable to make any of their samples available to us for confirmation of social form. Thus, the social organization of the three colonies from Texas and from California for which LI08 failed to detect the b allele protein in workers must be considered uncertain (but see below for the California colony).

Even assuming that all the study colonies of LI08 were polygyne, their sampling procedures for cDNA sequence analyses may not have been adequate to detect the b allele product in any particular study colony. The authors sequenced no more than 28 Gp-9 clones from mRNA extracts of workers from each of the four colonies. The probability of selecting at least one Gp-9^b clone for sequencing out of 28 depends on several factors, including the number of sampled ants from which the clones were obtained and the genotype proportions in the source colony. Although it is not possible to confidently follow their sampling scheme based on the information provided, no more than five workers from each colony (and possibly as few as one for each of the Texas colonies) served as sources for the mRNA in LI08. Simple calculations of joint binomial probabilities show that if a source colony contains 30% or fewer Bb workers, then there is a good chance (20–60%) of missing a Gp-9^b clone even with a sample of 28 clones distributed evenly across five workers (Fig. 3). If fewer workers are sampled, then the chances of selecting a Gp-9^b clone decline precipitously. The frequency of workers bearing Gp-9^b in polygyne colonies varies considerably in the wild, from around 80% to as low as 10% [11, K. G. Ross unpubl. data], and this lower proportion has been shown to be sufficient to elicit polygyne behavior in experimental laboratory colonies [11], [12]. (Virtually all b-bearing workers in polygyne colonies are Bb heterozygotes because of the apparent low viability of bb individuals [5], [31], [36].) We conclude from our probability calculations that the sampling effort of LI08 for their cDNA sequencing was not adequate to substantiate the existence of a previously unknown type of polygyne colony in which workers fail to express the GP-9^b protein.

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Figure 3. Binomial distribution probabilities of detecting Gp-9^b transcript in sample of 28 clones distributed across samples of 1–5 worker ants obtained from colonies with differing Gp-9 genotype proportions.

The proportions of Bb workers in the source colony represent the range that exists in the wild in polygyne S. invicta in the U.S.A.

https://doi.org/10.1371/journal.pone.0007713.g003

Surprisingly, the claim of LI08 that GP-9^b protein was not expressed in workers in their California study colony, based both on cDNA sequencing and PAGE, is directly contradicted by results of a subsequent experiment they conducted to identify a potential phosphorylation site in GP-9. A reported peptide fragment recovered from adult workers of the California colony in the latter experiment contains two amino acid residues that are fully diagnostic for the GP-9^b protein of S. invicta in the U.S.A. (Fig. 4). Clearly, Gp-9^b was expressed in workers of this colony.

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Figure 4. Amino acid sequences predicted for a peptide fragment of the B and b allelic variants of GP-9 from S. invicta in the U.S.A. and reported for the same fragment recovered from the California colony studied by LI08.

The LI08 fragment was obtained by N-terminal sequencing of Lys-C digested GP-9. Amino acid positions for the native protein are indicated, with positions of fragment residues diagnostic for the B and b proteins in the U.S.A. highlighted. All b′ proteins from S. invicta (known only from the native South American range) feature a combination of Val¹³⁶ and Ile¹³⁹ residues [8]–[10].

https://doi.org/10.1371/journal.pone.0007713.g004

From a technical standpoint, the California inconsistency raises doubts about the ability of the methods employed in LI08 to differentiate between the B and b variants encoded by Gp-9. Although the sampling issues discussed above must also factor into considerations of this inconsistency, there are causes for concern regarding the technical adequacy of the cDNA sequencing methods related to primer design. Specifically, the “gene specific” reverse primer RIFA-GP9br, presumably designed by LI08 with the aim of enriching amplification of the b transcript for subsequent sequencing, has the critical bases meant to confer allele binding specificity located near its 5′ end rather than within a few nucleotides of the 3′ end, so it is unlikely to have achieved the intended selective amplification [e.g., 52]. Compounding the difficulties in evaluating the sources of the California discrepancy, the authors did not indicate what nucleotide variation they used to distinguish between B and b alleles, and they did not deposit recovered nucleotide sequences in GenBank or another publicly available sequence depository. These issues are relevant in the following respect.

The charge-variant b-like alleles of Gp-9, including the b allele found in S. invicta in the U.S.A., represent only a portion of the diversity of b-like alleles associated with polygyny in S. invicta (Table 1) [8]–[10]. Given the few samples of introduced S. invicta for which Gp-9 has been surveyed at the DNA sequence level (16 females from four localities [8]), it is conceivable that b′ alleles, the class of b-like alleles lacking the charge-changing amino acid replacement of Gp-9^b, occur in some areas of the U.S.A. in association with polygyny. Because such b′ alleles encode products that are expected to be inseparable from the B allele product using charge-based electrophoretic methods, their presence in the California and Texas study colonies of LI08 presumably would be undetectable using native PAGE. Thus, the failure of LI08 to appreciate the full scope of variation at Gp-9 and design assays accordingly could explain their inability to detect relevant GP-9 variation using PAGE in three of four ostensibly polygyne colonies. Lending credence to this scenario, the inferred native source population for S. invicta in the U.S.A., determined to reside in northeastern Argentina [53], contains both types of b-like alleles [9], [37]. Mitigating against it, a comprehensive survey of GP-9 in polygyne colonies from six widely dispersed sites in the U.S.A. failed to uncover evidence for electrophoretically cryptic b′ alleles [32].

In order to learn if b′ alleles occur along with b alleles in S. invicta in the U.S.A., we undertook an expanded survey of Gp-9 variation, using as source material genomic DNA from 41 males obtained from both polygyne and monogyne colonies located in southeastern Mississippi, U.S.A. This area was chosen for sampling because of its proximity (<50 km) to Mobile, Alabama, the site of introduction of S. invicta to the U.S.A. [4], as well as the apparent genetic resemblance of populations in the area to the original founder population [32], [54]. Each of the 48 full-length Gp-9 sequences we recovered is identical to one of the three B haplotypes (B1, B2, B3) or the single b haplotype (b1) described previously from the U.S.A. by Krieger and Ross [8] (see Table 1). In view of the minimal nuclear genomic differentiation that exists across the U.S.A. range of S. invicta [32], we believe that effectively all Gp-9 variants in the introduced range have now been documented. Because none of these is a b′ allele, it is unlikely that the presence of these electrophoretically cryptic b-like alleles, which typically occur in colonies to the exclusion of b alleles [31], contributed to the failure of LI08 to detect the b protein product using PAGE in three of their four study colonies.

In summary, a central claim of LI08, that a second type of polygyne colony of S. invicta exists in which adult workers (and reproductive queens) do not express the GP-9^b protein, is not supported by our detailed review of previous studies, our re-examination of the methods and results of LI08, or our expanded sampling and HSGE analyses of a source population for one of the LI08 study colonies. We conclude that the claim is almost certainly based on artifactual data resulting from improper determination of the social form of the study colonies, inadequacy of sampling, and/or technical deficiencies in the methods used to assay for GP-9^b transcripts and protein.

GP-9 as a General Hemolymph Protein

The second central empirical claim of LI08, derived from their experiments with native PAGE, is that GP-9 protein is present in the hemolymph, and thus distributed throughout the body, of adult fire ant workers and queens. Gotzek and Ross [6] previously stated that “GP-9 protein routinely is extracted from whole-thorax or head homogenates of adult females, and it is abundant in thoracic hemolymph…, which suggests that it may circulate throughout the hemocoel”, a statement based largely on unpublished HSGE results such as shown in Figure 1. An important inference of this finding in the view of LI08 is that any primary role for GP-9 as a pheromonal chaperone or stimulator of neuronal activity within chemosensilla in the manner of the “gold standard” OBPs (Table 1) can be ruled out, because these confirmed chemosensory transducers are limited in their expression to chemosensilla (indeed, this is one of their defining features) [21], [55]. Nonetheless, LI08 demonstrate that GP-9 is present in the antennal club of fire ant workers [their Figure 8, see also 56], and other authors have suggested that OBPs with ubiquitous expression may have primary olfactory functions when they are expressed in olfactory organs [57; see also 58]–[61]. A somewhat stronger case that GP-9 does not function in a manner analogous to the “gold standard” OBPs can perhaps be made in light of the evidence presented by LI08 that it undergoes phosphorylation, an unknown form of post-translational modification among these proteins [60], [62, but see 63],[64]. Because comprehensive biochemical and expression data have been obtained for relatively few OBPs demonstrated to function specifically in chemosensory signal transduction [21], [55], [60], generalizations regarding their forms of post-translational modification and sites of expression should be viewed cautiously.

GP-9 and the Genetic Regulation of Fire Ant Social Organization

The hemolymph distribution of GP-9 as well as the purported lack of expression of a b-like allelic protein in some polygyne S. invicta colonies led LI08 to conclude that “…it is highly unlikely that GP-9s are involved in olfactory mediation of social organization of the red imported fire ant.” In order to evaluate this very general conclusion, it is helpful to distinguish two separate, but interrelated, levels of explanation for the genetic basis of regulation of fire ant social organization, the specific biochemical and physiological pathways in which GP-9 functions and the manner in which variation in these functions affects production and perception of the stimuli modulating worker behaviors toward queens.

Regarding the biochemical level, the idea that the products of Gp-9 may play primary chemosensory (olfactory or gustatory) roles similar to those proposed for the “gold standard” OBPs emerged from the convergence of two lines of evidence, the identification of GP-9 as a member of the OBP family [8] and the strong association of individual Gp-9 genotypes of queens and workers with aggressive behaviors and responses known to be mediated by semiochemicals and central to worker control of colony queen number [6], [7], [16]. Initial enthusiasm for the idea that Gp-9 plays a direct, exclusive role as a pheromone signal transducer has since given way to a more pluralistic outlook [6]. The more recent scenarios incorporate an increased recognition that chemosensory sensitivity and specificity is determined at multiple anatomical and physiological levels and sites [60], [65]-[67], with GP-9 potentially involved at any number of these. Among the possibilities considered by Gotzek and Ross [6] was that GP-9 may function “as a hemolymph carrier protein serving some primary function other than chemical communication, perhaps as a transporter of small hydrophobic endocrine factors”, a proposal echoed in the statement by LI08 that “…GP-9s are more likely involved in the transport of lipids or small ligands in the hemolymph…”. Indeed, the possibility that Gp-9 plays no role whatsoever in the regulation of fire ant social organization, serving only as a functionally irrelevant marker for other genes with such roles, was considered explicitly in some early papers [5], [26], although it has since been discounted on the following grounds: i) the sex specificity and time course of GP-9 expression in queens and workers parallel patterns of semiochemical release and behavioral responses involved in regulating queen number, ii) positive selection has driven the molecular evolution of Gp-9 (not linked genes) specifically in the context of the joint evolutionary origins of b-like alleles and polygyne social organization, and iii) the obligate association of b-like alleles with polygyny in S. invicta and related species has persisted in the face of recombination over evolutionary time scales [see 6], [9]. In light of these data, we feel that LI08's seeming conclusion that Gp-9 is unlikely to be involved in any way in olfactory mediation of fire ant social organization is premature.

With regard to the behavioral level of explanation of Gp-9 effects, a large obstacle to forming testable hypotheses is the lack of a detailed understanding of pheromonal communication systems of fire ants, this despite the fact that S. invicta is the best studied ant in this regard. Nonetheless, available general information on chemical communication in ants, as well as specific information on the relationship between Gp-9 and fire ant social organization, certainly must be accommodated into theoretical frameworks if they are to be relevant to guiding empirical work on the Gp-9 system (see Table 5). In overlooking or misappropriating much significant information, LI08 paint an unnecessarily confused, and in some respects irrelevant, picture of the pheromonal basis of regulation of queen number in fire ants. Queen primer pheromones of S. invicta, a focus of much attention in LI08, have been shown to attract workers to tend reproductive queens, prevent reproduction by alate queens in the nest, inhibit sexualization of female larvae, and, in monogyne colonies, mediate worker imprinting on the mother queen and execution of supernumerary reproductives [reviewed in 4]. Whether any of these diverse functions are mediated by the same releaser pheromone involved in the Gp-9 phenomenology, as LI08 seem to believe, is unknown but seems doubtful. For instance, the need to disable queen imprinting effects when studying Gp-9 effects [6], [7], as well as the lack of effect of queen reproductive status or ovarian development on queen acceptability once Gp-9 genotype is taken into account [7], [68] — in contrast to a strong effect of these factors on many primer pheromone responses [4], [69]–[72] — suggest strongly that at least some of the above functions are served by pheromonal systems unrelated to Gp-9.

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Table 5. Information on pheromonal communication in ants and on Gp-9 and the behavioral regulation of fire ant colony social organization.

https://doi.org/10.1371/journal.pone.0007713.t005

LI08 prominently cite as background to their work two studies that were conducted on such presumably unrelated systems and that did not attempt to account for Gp-9 effects [69], [73], thus making their relevance to the higher-level role of Gp-9 in regulating colony queen number questionable. LI08 then compound this problem by mischaracterizing the state of knowledge of the Gp-9 system. Specifically, they mistakenly attribute the social form of source and recipient colonies, rather than the Gp-9 genotypes of introduced queens and of recipient colony workers, as the major determinants of the outcomes of decisive behavioral assays (see Table 5). This oversight stems at least in part from their apparent reliance on an outdated secondary account of fire ant biology [74], information in which has been superseded by recent authoritative reviews of fire ant social biology [4], [6] as well as comprehensive behavioral studies aimed specifically at testing the relative roles of Gp-9 genotype and other factors in the regulation of colony queen number [7], [11], [12].

HSGE Analyses of S. invicta from College Station, Texas

We collected samples from 89 suspected polygyne S. invicta colonies at three sites in College Station, Texas (see Table S1), a locality from which one of the four study colonies of LI08 originated (their Texas A&M colony). The sampled colonies were the only ones suspected to be polygyne, based on initial field criteria [39], [40], out of several hundred colonies inspected at the three sites. The focal nests were excavated and samples of 20–100 workers were aspirated from each; dealate queens were collected opportunistically. All samples were immediately placed on liquid nitrogen in the field and then held in a −80°C freezer upon return to the laboratory. Social organization initially inferred for each colony in the field was definitively determined in the following way. The mating status and reproductive development of up to four dealate queens per colony were determined by dissecting the spermathecae and ovaries of 247 queens from the 89 colonies; queens were judged to be mated when the spermatheca was white and opaque, and they were judged to be reproductively active when the ovaries contained multiple fully developed eggs [e.g., 46]. For the subset of ten colonies in which only a single mated queen was recovered, we determined genotype distributions for ten randomly chosen nestmate workers at six polymorphic allozyme loci using HSGE coupled with specific histochemical staining [75]. Monogyne fire ant colonies display a simple family structure in which all workers are the full-sister offspring of a single mother queen mated to a single (haploid) male [38], [41]; therefore, genotype distributions inconsistent with such structure provide strong evidence of polygyny. For bi-allelic loci, such distributions feature three unique genotypes, two unique homozygous genotypes, or two unique genotypes that depart from a 1∶1 ratio (determined using binomial tests).

Expression of Gp-9 and variation in the protein products were studied in the College Station samples using the HSGE methods detailed in DeHeer et al. [28]. Soluble proteins were extracted from the head+thorax of individual workers or pools of workers by macerating the material in 50 mM tris-HCl buffer solution (15 µL/individual). GP-9 bands were stained and visualized as described in DeHeer et al. [28]. GP-9 standards derived from individuals of known Gp-9 genotype (BB and Bb) were included in multiple lanes of every gel.

With the following exceptions, these same procedures were used to generate the gel images shown in Figure 1. To detect GP-9 in the hemolymph, the fluid was directly expressed onto paper wicks soaked in tris-HCl buffer by applying gentle pressure to the anterior opening of decapitated thoraces; the wicks were then loaded into gels and subjected to electrophoresis. Protein extracts for pooled samples of eggs, larvae, and adult males were concentrated in a vacuum concentrator prior to loading into gels for electrophoresis.

Sequence Analyses of Gp-9 in S. invicta from Southeastern Mississippi

Nucleotide sequences of Gp-9 were obtained from the genomic DNA of 41 S. invicta males collected around the cities of Hurley and Pascagoula, Mississippi; sources of these samples were 15 polygyne and 26 monogyne colonies [see 32 for details]. Sequence data were generated as described in Krieger and Ross [8] and Gotzek et al. [9]. Two sequences were recovered from each of seven heterozygous diploid males, while only a single sequence was recovered from the remaining 34 haploid or homozygous diploid males. The resulting 48 sequences were visually checked and aligned using the program Lasergene v8.0 (DNAStar), and are deposited in GenBank under accession numbers GU086668–GU086715.

Binomial Probability Calculations

We evaluated the sampling sufficiency for the cDNA sequencing that LI08 employed to determine if workers present in a colony express Gp-9^b. We used binomial distributions to calculate the joint probabilities of detecting a b transcript given that 28 clones were obtained from 1–5 sampled workers from single source colonies containing different proportions of BB and Bb workers. We used genotype proportions in the source colonies that span the range observed in polygyne S. invicta colonies in the U.S.A. [11, K. G. Ross unpubl. data]. These colonies were assumed to lack workers with the bb genotype — because of their apparent low viability, such workers are rarely found [5], [36]. We further assumed that the 28 clones were distributed evenly across the sampled workers.