Venom alkaloids of monogyne and polygyne forms of the red imported fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>, in Taiwan

Both monogyne and polygyne colonies of Solenopsis invicta now occupy Taiwan. Although venom alkaloids of these ants have been described and synthesized, we here report on a quantitative analysis of the two social forms for the first time. The alkaloids were studied by gas chromatography coupled to mass spectrometry (GC-MS), and six major venom alkaloids were detectable in both types of workers. Both C₁₃:C_13:1 and C₁₅:C_15:1 ratios in alkaloid venom of monogyne workers were statistically higher than that of polygyne workers, but the sum of proportions of unsaturated alkaloids of polygyne workers was significantly higher than that of monogyne workers, regardless of growth temperature, sampling seasons or geographic location. Results of this study demonstrate that the difference in the proportions of unsaturated alkaloids and the ratios of C₁₃:C_13:1 and C₁₅:C_15:1 alkaloids might be a good indicator for differentiating monogyne and polygyne forms of S. invicta. Received 20 February 2008; revised 4 July 2008; accepted 5 August 2008.     

Evidence for mating plugs in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Summary Male inhibition of female re-mating is com-mon in many insects. Mating plugs, used by males to con-trol female re-mating, have been postulated in several ant species. Recent studies of bumblebees have described re-mating inhibition by male accessory gland secretions. Fire ants Solenopsis invicta possess accessory glands containing the same four fatty acids as do the bumblebees. Furthermore it appears that some of these acids are transferred to the female at mating. Thus, it is possible that single mating of fire ant females may be enforced by male mating plugs.     

Evolutionary history of <Emphasis Type="Italic">Wolbachia</Emphasis> infections in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background  

Wolbachia are endosymbiotic bacteria that commonly infect numerous arthropods. Despite their broad taxonomic distribution, the transmission patterns of these bacteria within and among host species are not well understood. We sequenced a portion of the wsp gene from the Wolbachia genome infecting 138 individuals from eleven geographically distributed native populations of the fire ant Solenopsis invicta. We then compared these wsp sequence data to patterns of mitochondrial DNA (mtDNA) variation of both infected and uninfected host individuals to infer the transmission patterns of Wolbachia in S. invicta.     

Thermoregulatory brood transport in the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Nest structure in ants is often designed to optimize the colony’s ability to thermoregulate, and this specialization is most highly developed in mound-building ant species. Solenopsis invicta invest a large amount of energy in building mounds and transporting their brood up and down in their nests as a means of thermoregulation. Because few ant species build true mounds, we wanted to determine the effectiveness of these mounds in harvesting solar heat as well as to distinguish what factors (temperature vs. circadian rhythm) govern where fire ants place their brood in the mound and when they place it. We measured temperature patterns in the mound over several days at different depths and under different conditions (under direct sunlight or shade), and then conducted a series of field experiments to manipulate the orientation and time of heating. On cool mornings in spring or fall, surface temperatures of the mound rise at the fastest rate on the side receiving the most direct sunlight (usually the south side). This heating causes a temperature gradient through different depths in the mound, and shows little difference from outside ground temperature at a depth greater than ~40 cm inside the nest. In the morning, fire ants move their brood up into the mound on the side most directly heated, and when temperatures exceed optimal (~32°C) they move their brood down the temperature gradient to lower depths in the nest. In addition to this, mound temperature does not only increase due to direct sunlight, but temperature also increases higher than ground temperatures when the mound is in the shade due to its low specific heat. Experiments in which sunlight was mirrored to the normally shaded side of the mound, or when mounds were heated at night, revealed that S. invicta primarily track temperature patterns and do not rely on behavioral habits or circadian rhythms for the thermoregulatory transport of their brood. When mounds were shaded, S. invicta brood was evenly distributed directly under the surface of the mound rather than aggregating towards a specific side. The fire ant mound is important for thermoregulation because, compared to moundless subterranean nests, it absorbs heat more rapidly both in direct sunlight and shady conditions. Temperature tracking within the nest is key to understanding thermoregulatory placement of fire ant brood, as well as insight into the production of sexual brood and reproduction. Received 9 August 2007; revised 31 January 2008; accepted 7 February 2008.     

Genetic and morphological variation over space and time in the invasive fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Social insects are among the most successful and damaging of invasive taxa. We studied spatial and temporal variation in two traits, colony genetic structure and worker mass, associated with social insect success in the introduced fire ant Solenopsis invicta. Our aim was to determine if changes in social structure occurred over time and if variation in worker size was related to worker genotype. We sampled 1139 workers from five multiple-queen S. invicta nests on six dates over a one-year period. The genotypes of workers were determined at ten microsatellite loci and at the selected locus general protein-9 (Gp-9). We found little evidence for genetic differentiation of workers sampled from distinct nests or from different dates at the microsatellite loci. However, worker Gp-9 genotype frequencies varied among nests and over time. In addition, worker mass was affected by nest-of-origin, sampling date, ploidy level, and Gp-9 genotype. Our results suggest that large numbers of queens contribute to the production of workers in introduced S. invicta nests throughout the year. Colony boundaries are semi-permeable, although the among-nest variation in Gp-9 genotype frequencies and worker mass does suggest that boundaries are present. In addition, selection operating on Gp-9 genotype depends on nest environment. Finally, worker mass is affected by both endogenous and exogenous factors in S. invicta. Overall, our data suggests that the key traits of colony social structure and worker size reflect the effects of variable selection in invasive social insects.     

Population genetic structure of the red imported fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>, in Taiwan

We generated and analyzed microsatellite genotypic data and mtDNA sequence data from the fire ant Solenopsis invicta collected from two separate infested areas (Taoyuan and Chiayi) in Taiwan to infer the population and colony structure of these recently established populations. These genetic analyses revealed the following patterns: 1) Relatedness among worker nestmates was significantly greater than zero for both social forms from both populations; 2) No significant isolation by distance was found among nests within each social form from either population; 3) Significant mtDNA but no nuclear differentiation occurs between sympatric social forms in Taoyuan; 4) Molecular signatures of genetic bottlenecks associated with recent introductions are evident in both populations; and 5) The two sampled populations, Taoyuan and Chiayi, are highly genetically differentiated at both the nuclear or mtDNA genomes and most likely derive from two separate introductions into Taiwan. While results from these analyses generally were consistent with predictions based on the known biology of these ants and similar studies of S. invicta in the U.S.A. and South America, some patterns likely reflect the recent introduction and human-mediated inadvertent transport of ants in Taiwan. This is the first study to investigate the population and colony structure of fire ants in Taiwan and results from our study represent an important contribution to the ongoing efforts aimed at eradicating this invasive pest in Taiwan. Received 18 June 2007; revised 11 October 2007; accepted 17 October 2007.     

Unexpected patterns of segregation distortion at a selfish supergene in the fire ant <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Background

The Sb supergene in the fire ant Solenopsis invicta determines the form of colony social organization, with colonies whose inhabitants bear the element containing multiple reproductive queens and colonies lacking it containing only a single queen. Several features of this supergene — including suppressed recombination, presence of deleterious mutations, association with a large centromere, and “green-beard” behavior — suggest that it may be a selfish genetic element that engages in transmission ratio distortion (TRD), defined as significant departures in progeny allele frequencies from Mendelian inheritance ratios. We tested this possibility by surveying segregation ratios in embryo progenies of 101 queens of the “polygyne” social form (3512 embryos) using three supergene-linked markers and twelve markers outside the supergene.

Results

Significant departures from Mendelian ratios were observed at the supergene loci in 3–5 times more progenies than expected in the absence of TRD and than found, on average, among non-supergene loci. Also, supergene loci displayed the greatest mean deviations from Mendelian ratios among all study loci, although these typically were modest. A surprising feature of the observed inter-progeny variation in TRD was that significant deviations involved not only excesses of supergene alleles but also similarly frequent excesses of the alternate alleles on the homologous chromosome. As expected given the common occurrence of such “drive reversal” in this system, alleles associated with the supergene gain no consistent transmission advantage over their alternate alleles at the population level. Finally, we observed low levels of recombination and incomplete gametic disequilibrium across the supergene, including between adjacent markers within a single inversion.

Conclusions

Our data confirm the prediction that the Sb supergene is a selfish genetic element capable of biasing its own transmission during reproduction, yet counterselection for suppressor loci evidently has produced an evolutionary stalemate in TRD between the variant homologous haplotypes on the “social chromosome”. Evidence implicates prezygotic segregation distortion as responsible for the TRD we document, with “true” meiotic drive the most likely mechanism. Low levels of recombination and incomplete gametic disequilibrium across the supergene suggest that selection does not preserve a single uniform supergene haplotype responsible for inducing polygyny.

     

Ecological dominance of the red imported fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>, in its native range

Despite the widespread impacts invasive species can have in introduced populations, little is known about competitive mechanisms and dominance hierarchies between invaders and similar taxa in their native range. This study examines interactions between the red imported fire ant, Solenopsis invicta, and other above-ground foraging ants in two habitats in northeastern Argentina. A combination of pitfall traps and baits was used to characterize the ant communities, their dominance relationships, and to evaluate the effect of phorid flies on the interactions. Twenty-eight ant species coexisted with S. invicta in a gallery forest gap, whereas only ten coexisted with S. invicta in a xerophytic forest grassland. S. invicta was the most numerically dominant species in the richest and complex habitat (gallery forest); however it performed better as discoverer and dominator in the simpler habitat. S. invicta was active during day and night. In spite of its poor capacity to discover resources, S. invicta showed the highest ecological dominance and the second-best behavioral dominance after Camponotus blandus. S. invicta won 78% of the interactions with other ants, mostly against its most frequent competitor, Pheidole cf. obscurithorax, dominating baits via mass recruitment and chemical aggression. P. cf. obscurithorax was the best food discoverer. S. invicta won 80% of the scarce interactions with Linepithema humile. Crematogaster quadriformis was one of the fastest foragers and the only ant that won an equal number of contests against S. invicta. The low presence of phorid flies affected the foraging rate of S. invicta, but not the outcome of interspecific interactions. This study revealed that the red imported fire ant ecologically dominated other terrestrial ants in its native range; however, other species were able to be numerically dominant or co-dominant in its presence.     

PBAN/pyrokinin peptides in the central nervous system of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species. This research was supported in part by a US-Israel Binational Science Foundation Grant (no. 2003367).     

Red imported fire ants (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>) and seasonality influence community refuge use

Refuges are fundamental to animal ecology as refuge availability affects many levels of biological organization—from the behavior and physiology of individuals to the interspecific dynamics of a community. Although frequently studied in the context of predator–prey interactions, refuges may also mediate interspecific competition between native and invasive taxa given the role of refuges as a valuable resource. Because interspecific interactions (e.g., competition and predation) can be modulated by temporal and biotic (e.g., trophic level) factors, we used a manipulative approach to investigate community-wide refuge-use patterns in the context of two important ecological factors: invasive species and seasonality. We surveyed refuge (artificial cover object) use of ants and vertebrates in a forest community for 2 years, and we systematically suppressed an established invasive species (red imported fire ant, Solenopsis invicta) to examine its impact on community refuge use. Native Camponotus ants appeared to co-exist and share refuges with S. invicta, but we found evidence for a negative effect of S. invicta on vertebrate refuge use that was also influenced by season. Vertebrates were more abundant under refuges undergoing suppression of S. invicta, and they were less abundant under refuges during the fall (the season characterized by the highest occupancy of refuges by S. invicta). Thus, researchers must continue to examine the entire community and to incorporate the effects of season when assessing the impact of invasive species (e.g., at our site, a survey conducted only in the summer or only on native ants would have indicated a negligible effect of S. invicta on community refuge use).     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Impact of worker longevity and other endogenous factors on colony size in the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Central to the survival and reproduction of social insect queens is the size of colonies at maturity. The influence of exogenous factors such as predation, food abundance, and seasonal changes in temperature on colony size are well studied. Less well studied are endogenous life-history factors such as a queen’s fertility and lifespan, duration of worker development from egg to adult and worker lifespan. Endogenous factors regulating the rate of colony growth and colony size were simulated using the fire ant, Solenopsis invicta. Assuming ideal environmental conditions of no predation, abundant food and uniform temperature, the simulation showed unequivocally that colony size is determined by two endogenous factors: a queen’s egg-laying rate (R _q) and worker longevity (L _w) (Colony size = R _q × L _w). Thus, we are left with an unanswered question: if worker longevity contributes directly to colony size, why is the lifespan of workers so short—in most cases, a small fraction of a queen’s lifespan?     

Evaluation of the toxicity of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis> extract

In this study, the methanol extract of Arthrospira (Spirulina) platensis was examined for acute and subchronic toxicities. The extract did not produce any sign of toxicity within 7 days after feeding it at a single high dose of 6 g kg⁻¹ body weight to female and male Swiss mice. For the subchronic toxicity test, the extract at doses of 6, 12, and 24 mg kg⁻¹ body weight was orally administered to six male and six female Wistar rats daily for 12 weeks. Throughout the study period, we did not observe any abnormalities on behavior, food and water intakes, and health status among the treated animals. The hematology and clinical chemistry parameters of treated groups did not significantly differ from those of the controls in both sexes. Postmortem examination of the test groups also showed no abnormalities in both gross and histological findings. These results thus suggest that the methanol extract of A. platensis did not cause acute or subchronic toxicity in our experimental animals.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

<Emphasis Type="Italic">Sex combs reduced</Emphasis> (<Emphasis Type="Italic">Scr</Emphasis>) regulatory region of <Emphasis Type="Italic">Drosophila</Emphasis> revisited

The Hox gene Sex combs reduced (Scr) is responsible for the differentiation of the labial and prothoracic segments in Drosophila. Scr is expressed in several specific tissues throughout embryonic development, following a complex path that must be coordinated by an equally complex regulatory region. Although some cis-regulatory modules (CRMs) have been identified in the Scr regulatory region (~75 kb), there has been no detailed and systematic study of the distinct regulatory elements present within this region. In this study, the Scr regulatory region was revisited with the aim of filling this gap. We focused on the identification of Initiator elements (IEs) that bind segmentation factors, Polycomb response elements (PREs) that are recognized by the Polycomb and Trithorax complexes, as well as insulators and tethering elements. To this end, we summarized all currently available information, mainly obtained from high throughput ChIP data projects. In addition, a bioinformatic analysis based on the evolutionary conservation of regulatory sequences using the software MOTEVO was performed to identify IE and PRE candidates in the Scr region. The results obtained by this combined strategy are largely consistent with the CRMs previously identified in the Scr region and help to: (i) delimit them more accurately, (ii) subdivide two of them into different independent elements, (iii) identify a new CRM, (iv) identify the composition of their binding sites and (v) better define some of their characteristics. These positive results indicate that an approach that integrates functional and bioinformatic data might be useful to characterize other regulatory regions.     

Identification of <Emphasis Type="Italic">S</Emphasis> haplotypes in <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis of <Emphasis Type="Italic">SP11</Emphasis> alleles

A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.