Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity.

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

Comprehensive Detoxification Mechanism Assessment of Red Imported Fire Ant (Solenopsis invicta) against Indoxacarb.

The red imported fire ant (Solenopsis invicta) is one of the deadliest invasive ant species that threatens the world by disrupting biodiversity, important functions within a natural ecosystem, and community structure. They are responsible for huge economic losses in the infested countries every year. Synthetic insecticides, especially indoxacarb, have been broadly used to control S. invicta for many years. However, the biochemical response of S. invicta to indoxacarb remains largely undiscovered. Here, we used the sublethal doses of indoxacarb on the S. invicta collected from the eight different cities of Southern China. The alteration in the transcriptome profile of S. invicta following sublethal dosages of indoxacarb was characterized using high-throughput RNA-seq technology. We created 2 libraries, with 50.93 million and 47.44 million clean reads for indoxacarb treatment and control, respectively. A total of 2018 unigenes were regulated after insecticide treatment. Results indicated that a total of 158 differentially expressed genes (DEGs) were identified in the indoxacarb-treated group, of which 100 were significantly upregulated and 58 were downregulated, mostly belonging to the detoxification enzymes, such as AChE, CarE, and GSTs. Furthermore, results showed that most of these DEGs were found in several KEGG pathways, including steroid biosynthesis, other drug metabolizing enzymes, glycerolipid metabolism, chemical carcinogenesis, drug-metabolizing cytochrome P450, glutathione metabolism, glycerophospholipid metabolism, glycolysis/gluconeogenesis, and metabolism of xenobiotics. Together, these findings indicated that indoxacarb causes significant alteration in the transcriptome profile and signaling pathways of S. invicta, providing a foundation for further molecular inquiry.

Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies.

The symbiotic partnership between leaf-cutting ants and fungal cultivars processes plant biomass via ant fecal fluid mixed with chewed plant substrate before fungal degradation. Here we present a full proteome of the fecal fluid of Acromyrmex leaf-cutting ants, showing that most proteins function as biomass degrading enzymes and that ca. 85% are produced by the fungus and ingested, but not digested, by the ants. Hydrogen peroxide producing oxidoreductases were remarkably common in the proteome, inspiring us to test a scenario in which hydrogen peroxide reacts with iron to form reactive oxygen radicals after which oxidized iron is reduced by other fecal-fluid enzymes. Our biochemical assays confirmed that these so-called Fenton reactions do indeed take place in special substrate pellets, presumably to degrade plant cell wall polymers. This implies that the symbiotic partnership manages a combination of oxidative and enzymatic biomass degradation, an achievement that surpasses current human bioconversion technology.

Colonies of tropical leaf-cutting ants live in underground nests where a fungus grows that feeds them. The ants, in turn, provide the fungus with the freshly-cut leaf fragments it needs for nutrition. The relationship between the ants and the fungus, in which they live close together and help one another survive, is known as symbiosis. It is an ancient, extremely well integrated relationship, in which neither species can survive without the other. However, the details of how the ants and the fungus work together to break down the leaf fragments so they can be used for nutrition are not well understood. When the ants eat the fungus, they do not digest its enzymes (the proteins that accelerate chemical reactions in a cell). Instead, the fungal enzymes travel through the ants' gut and into their fecal liquid, which gets deposited on the fresh-cut leaves when the ants collect them. The ants then make temporary pellets out of the new leaf fragments before providing them to the fungus. To better understand how each species contributes to the breakdown of the leaf fragments, Schiøtt and Boomsma identified all the proteins present in the fecal fluid of the ants. Once they had a complete list of about 100 proteins, they determined which of them were produced by the fungus and which by the ant. Schiøtt and Boomsma observed that certain combinations of fungal and ant enzymes could trigger a Fenton reaction ­ a chemical reaction that efficiently begins the breakdown of the tough walls around plant cells. This reaction is so aggressive that it is rarely found in nature, but it could help explain the high efficiency of the fungus and the ants symbiotically processing leaf fragments. But could a Fenton reaction actually proceed in the ants' nest without hurting the ants or affecting the rest of the fungal garden? The evidence obtained suggested that the temporary pellets made by the ants serve to isolate the reaction, so the aggressive chemistry takes place away from the ants and detached from the fungal gardens. Schiøtt and Boomsma showed that the symbiotic relationship between the ants and the fungus has led to a sustainable and efficient way of breaking down plant materials to use them for nutrition. The Fenton reaction is economically important in many industries, including bioethanol production, the detergent industry, and food production. Emulating the methods used by leaf-cutting ants, which have been fine-tuned by millions of years of natural selection, may allow humans to develop more efficient technologies for breaking down organic compounds.

Interaction of fipronil and the red imported fire ant (Solenopsis invicta): Toxicity differences and detoxification responses.

Fipronil (FIP), a phenyl-pryazole pesticide, has been widely used for crop protection due to its broad insecticidal spectrum, especially for urban insect management. FIP also serves as the active ingredient of major baits used for the control of the red imported fire ant (RIFA; Solenopsis invicta). Although a vast majority of laboratory-based research has been performed using worker ants as a model, limited information is available regarding the toxicity of FIP in individuals from different castes and developmental stages. In this study, we investigated the interaction between FIP and this important pest, including FIP toxicity and transformation, RIFA enzyme activity and responses to FIP exposure. The topical and feeding toxicity of FIP in five adult castes, worker larvae and worker pupae were determined and compared. Topical toxicity assays showed that there were significant differences in FIP toxicity among adult workers (LD50 = 1.17 µg/g), larvae (LD50 = 1891.00 µg/g) and pupae (LD50 = 23981.00 µg/g). Although, no obvious differences in topical toxicity were observed among the adult castes, the differences in feeding toxicity were significant. For example, the LC50 value for the workers was 3.96-fold lower than that for soldiers at 24 h, and the LC50 value was slightly lower for male alates than for female alates at day 3 and day 4, respectively. The activities of detoxification enzymes in individuals of different castes and developmental stages were investigated with or without FIP treatment. Cytochrome P450 activity was approximately 24-fold higher in larvae than in workers, and adult workers exhibited 4-fold higher FIP-induced cytochrome P450 activity than individuals from other adult castes. In addition, in vitro experiments demonstrated that FIP was transformed into FIP-sulfone, and this process may be primarily mediated by RIFA P450(s).

Caste-Specific Expression of Na+/K+-ATPase in the Asian Weaver Ant, Oecophylla smaragdina (Fabricius, 1775).

Social insect colonies adopt different levels of survival strategies and exhibit well-defined reproductive division of labour. Oecophylla smaragdina (Fabricius, 1775) has physiological and behavioral adaptations that enable them to forage at extreme environmental conditions and are lethal to most other insects. Ion homeostasis is the key process in an organism's survival mechanism. Among ion pumps, the ATP-dependent sodium-potassium ion pump is essential for maintaining the Na+ and K+ ionic balance and is well known as the primary consumer of energy. Oecophylla smaragdina plays pivotal role as a model among social insects for understanding ion homeostasis at the organization level of the castes. We have evaluated the expression and activity of Na+/K+-ATPase among various castes of O. smaragdina (worker subcastes, queen and male). Real-time PCR and immunoblotting analyses revealed the differential expression of Na+/K+-ATPase in the castes. Significantly higher expression of Na+/K+-ATPase mRNA and protein were observed in the minor workers, queen, major workers and males respectively. These results suggest that in the weaver ant colony, the castes might have variously adapted and evolved with a well-developed ion transport mechanism which allows them to perform allocated tasks within the nest and could be a key to their adaptive benefits towards division of labour.

Developmental DNA methyltransferase expression in the fire ant Solenopsis invicta.

DNA methylation is accomplished in animals by 2 classes of enzymes known as DNA methyltransferases, DNMT3 and DNMT1, which perform de novo methylation and maintenance methylation, respectively. Several studies of hymenopteran eusocial insects suggest that DNA methylation is capable of influencing developmental plasticity. However, fundamental questions remain about the patterning of DNA methylation during the course of insect development. In this study, we performed quantitative real-time PCR (qPCR) on transcripts from the single-copy orthologs of DNMT1 and DNMT3 in the red imported fire ant, Solenopsis invicta. In particular, we assessed the expression of S. invicta Dnmt1 and Dnmt3 mRNA during 7 stages of worker development, among behaviorally distinct adults, and among male and female gonads. Dnmt3 was most highly expressed during embryonic development, whereas Dnmt1 was similarly expressed throughout the course of development. Moreover, Dnmt1 and Dnmt3 were highly expressed in testes and ovaries. Neither Dnmt was significantly differentially expressed among heads of behaviorally distinct adult castes. Our results support the hypothesis that extensive patterning of DNA methylation occurs during gametogenesis and embryogenesis in the insect order Hymenoptera.

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions.

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

An ant-plant mutualism through the lens of cGMP-dependent kinase genes.

In plant-animal mutualisms, how an animal forages often determines how much benefit its plant partner receives. In many animals, foraging behaviour changes in response to foraging gene expression or activation of the cGMP-dependent protein kinase (PKG) that foraging encodes. Here, we show that this highly conserved molecular mechanism affects the outcome of a plant-animal mutualism. We studied the two PKG genes of Allomerus octoarticulatus, an Amazonian ant that defends the ant-plant Cordia nodosa against herbivores. Some ant colonies are better 'bodyguards' than others. Working in the field in Peru, we found that colonies fed with a PKG activator recruited more workers to attack herbivores than control colonies. This resulted in less herbivore damage. PKG gene expression in ant workers correlated with whether an ant colony discovered an herbivore and how much damage herbivores inflicted on leaves in a complex way; natural variation in expression levels of the two genes had significant interaction effects on ant behaviour and herbivory. Our results suggest a molecular basis for ant protection of plants in this mutualism.

Genomic exaptation enables Lasius niger adaptation to urban environments.

BACKGROUND: The world is rapidly urbanizing, and only a subset of species are able to succeed in stressful city environments. Efficient genome-enabled stress response appears to be a likely prerequisite for urban adaptation. Despite the important role ants play in the ecosytem, only the genomes of ~13 have been sequenced so far. Here, we present the draft genome assembly of the black garden ant Lasius niger - the most successful urban inhabitant of all ants - and we compare it with the genomes of other ant species, including the closely related Camponotus floridanus. RESULTS: Sequences from 272 M Illumina reads were assembled into 41,406 contigs with total length of 245 MB, and N50 of 16,382 bp, similar to other ant genome assemblies enabling comparative genomic analysis. Remarkably, the predicted proteome of L. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, DNA repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. With respect to environmental stress, we note a proliferation of various detoxification genes, including glutatione-S-transferases and those in the cytochrome P450 families. Notably, the CYP9 family is highly expanded with 19 complete and 21 nearly complete members - over twice as many compared to other ants. This family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. Gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation. CONCLUSIONS: Our results suggest that the success of L. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the CYP9 cytochrome family due to coevolution with parasitic fungi, the diversification of DNA repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. Diversification of cytochromes and DNA repair systems along with reduced odorant communication are the basis of L. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources.

Queen pheromones modulate DNA methyltransferase activity in bee and ant workers.

DNA methylation is emerging as an important regulator of polyphenism in the social insects. Research has concentrated on differences in methylation between queens and workers, though we hypothesized that methylation is involved in mediating other flexible phenotypes, including pheromone-dependent changes in worker behaviour and physiology. Here, we find that exposure to queen pheromone affects the expression of two DNA methyltransferase genes in Apis mellifera honeybees and in two species of Lasius ants, but not in Bombus terrestris bumblebees. These results suggest that queen pheromones influence the worker methylome, pointing to a novel proximate mechanism for these key social signals.

Higher-Order Assembly of BRCC36-KIAA0157 Is Required for DUB Activity and Biological Function.

BRCC36 is a Zn(2+)-dependent deubiquitinating enzyme (DUB) that hydrolyzes lysine-63-linked ubiquitin chains as part of distinct macromolecular complexes that participate in either interferon signaling or DNA-damage recognition. The MPN(+) domain protein BRCC36 associates with pseudo DUB MPN(-) proteins KIAA0157 or Abraxas, which are essential for BRCC36 enzymatic activity. To understand the basis for BRCC36 regulation, we have solved the structure of an active BRCC36-KIAA0157 heterodimer and an inactive BRCC36 homodimer. Structural and functional characterizations show how BRCC36 is switched to an active conformation by contacts with KIAA0157. Higher-order association of BRCC36 and KIAA0157 into a dimer of heterodimers (super dimers) was required for DUB activity and interaction with targeting proteins SHMT2 and RAP80. These data provide an explanation of how an inactive pseudo DUB allosterically activates a cognate DUB partner and implicates super dimerization as a new regulatory mechanism underlying BRCC36 DUB activity, subcellular localization, and biological function.

Evolutionary history of the Azteca-like mariner transposons and their host ants.

Three different complete mariner elements were found in the genome of the ant Tapinoma nigerrimum. One (Tnigmar-Mr) was interrupted by a 900-bp insertion that corresponded to an incomplete member of a fourth mariner element, called Azteca. In this work, we isolate and characterize full-length Tnigmar-Az elements in T. nigerrimum. The purpose of this study is to clarify the evolutionary history of Azteca elements and their hosts as well as the possible existence of horizontal transfer processes. For this, Azteca-like elements were also retrieved from the available sequences of various ant genomes, representing four different ant subfamilies: Dolichoderinae, Formicinae, Myrmicinae, and Ponerinae. The tree topology resulting for the Azteca-like elements bore very little resemblance to that of their respective hosts. The pervasive presence of Azteca-like elements in all ant genomes, together with the observation that extant copies are usually younger than the genomes that host them, could be explained either by lineage sorting or by recent horizontal transfer of active elements. However, the finding of closer genetic relationships between elements than between the ants that host them is consistent with the latter scenario. This is clearly observed in Sinvmar-Az, Tnigmar-Az, Acepmar-Az, and Cflomar-Az elements, suggesting the existence of horizontal transfer processes. On the contrary, some elements displayed more divergence than did the hosts harboring them. This may reflect either further horizontal transfer events or random lineage sorting.

[The amplification of CYP9 genes as a preadaptation of the black garden ant Lasius niger to urban conditions].

Ants are one of the most ancient and successful groups of eusocial animals and they are spread all over the world. The nucleotide sequences of the genomes of eight ant species were determined by the year 2014. In these species, the mechanisms of ecological success, cast differentiation, and social communication were studied at genomic level. In ants, the genes of the cytochromes P450 involved in metabolism of xenobiotics and various endogenic substances are amplified. Although the substrates for several cytochrome P450 families have been identified, the functions of the ninth family, which is one of the most amplified, remain unknown. The black garden ant Lasius niger is one of the spices that have successfully adapted to urban conditions. To study the mechanisms of adaptation, we have read and annotated the nucleotide sequence of the L. niger genome; we have predicted the functions of the CYP9 genes using virtual screening. The obtained data allow us to suggest that cytochromes P450 are involved in the metabolism of various xenobiotics such as phytotoxins, mycotoxins, and insecticides. We assume that the functional divergence of the new CYP9 duplications was initially aimed at developing resistance to various mycotoxins, in particular to those produced by Fusarium fungi and, subsequently, to other xenobiotics.

Phylogeography of the sand dune ant Mycetophylax simplex along the Brazilian Atlantic Forest coast: remarkably low mtDNA diversity and shallow population structure.

BACKGROUND: During past glacial periods, many species of forest-dwelling animals experienced range contractions. In contrast, species living outside such moist habitats appear to have reacted to Quaternary changes in different ways. The Atlantic Forest represents an excellent opportunity to test phylogeographic hypotheses, because it has a wide range of vegetation types, including unforested habitats covered predominantly by herbaceous and shrubby plants, which are strongly influenced by the harsh environment with strong wind and high insolation. Here, we investigated the distribution of genetic diversity in the endemic sand dune ant Mycetophylax simplex across its known range along the Brazilian coast, with the aim of contributing to the understanding of alternative phylogeographic patterns. We used partial sequences of the mitochondrial gene cytochrome oxidase I and nuclear gene wingless from 108 specimens and 51 specimens, respectively, to assess the phylogeography and demographic history of this species. To achieve this we performed different methods of phylogenetic and standard population genetic analyses. RESULTS: The observed genetic diversity distribution and historical demographic profile suggests that the history of M. simplex does not match the scenario suggested for other Atlantic Forest species. Instead, it underwent demographic changes and range expansions during glacial periods. Our results show that M. simplex presents a shallow phylogeographic structure with isolation by distance among the studied populations, living in an almost panmictic population. Our coalescence approach indicates that the species maintained a stable population size until roughly 75,000 years ago, when it underwent a gradual demographic expansion that were coincident with the low sea-level during the Quaternary. Such demographic events were likely triggered by the expansion of the shorelines during the lowering of the sea level. CONCLUSIONS: Our data suggest that over evolutionary time M. simplex did not undergo dramatic range fragmentation, but rather it likely persisted in largely interconnected populations. Furthermore, we add an important framework about how both glacial and interglacial events could positively affect the distribution and diversification of species. The growing number of contrasting phylogeographic patterns within and among species and regions have shown that Quaternary events influenced the distribution of species in more ways than first supposed.

Ant sperm storage organs do not have phenoloxidase constitutive immune activity.

The prophenoloxidase system (proPO-AS) is a primordial constituent of insect innate immunity. Its broad action spectrum, rapid response time, and cytotoxic by-products induced by phenoloxidase (PO) production contribute to the effective clearing of invading pathogens. However, such immune reactions may not be optimal for insect organs that evolved to have mutualistic interactions with non-self-cells. Ant queens are long-lived, but only mate early in adult life and store the sperm in a specialized organ, the spermatheca. They never re-mate so their life-time reproductive success is ultimately sperm-limited, which maintains strong selection for high sperm viability before and after storage. The proPO-AS may therefore be inappropriate for the selective clearing of sexually transmitted infections, as it might also target sperm cells that cannot be replaced. We measured PO enzymatic activity in the sperm storage organs of three ant species before and after mating. Our data show that no PO is produced in the sperm storage organs, relative to other somatic tissues as controls, and that these negative results are not due to non-detection in small volumes as non-immune-relevant catalase activity in single spermatheca fluid samples of both virgin and mated queens was significant. The lack of PO activity in sperm storage organs across three different ant species may represent an evolutionarily conserved adaptation to life-long sperm storage by ant queens. We expect that PO activity will be similarly suppressed in queen spermathecae of other eusocial Hymenoptera (bees and wasps) and, more generally, of insect females that store sperm for long periods.

PO-CALC: a novel tool to correct common inconsistencies in the measurement of phenoloxidase activity.

A broad range of physiological and evolutionarily studies requires standard and robust methods to assess the strength and activity of an individual's immune defense. In insects, this goal is generally reached by spectrophotometrically measuring (pro-) phenoloxidase activity, an enzymatic and non-specific process activated after wounding and parasite infections. However, the literature surprisingly lacks a standard method to calculate these values from spectrophotometer data and thus to be able to compare results across studies. In this study, we demonstrated that nine methods commonly used to extract phenoloxidase activities (1) provide inconsistent results when tested on the same data sets, at least partly due to their specific sensitivity to the noise regularly present in enzymatic reaction curves. To circumvent this issue, we then (2) developed a novel, free and simple R-based program called PO-CALC and (3) demonstrated the robustness of its calculations for the different types of noises. Overall, we show that PO-CALC corrects overlooked though important inconsistencies in the measurement of phenoloxidase activities, and claim that its broad use would increase the significance and general validity of studies on invertebrate immunity.

Variable interaction specificity and symbiont performance in Panamanian Trachymyrmex and Sericomyrmex fungus-growing ants.

BACKGROUND: Cooperative benefits of mutualistic interactions are affected by genetic variation among the interacting partners, which may have consequences for interaction-specificities across guilds of sympatric species with similar mutualistic life histories. The gardens of fungus-growing (attine) ants produce carbohydrate active enzymes that degrade plant material collected by the ants and offer them food in exchange. The spectrum of these enzyme activities is an important symbiont service to the host but may vary among cultivar genotypes. The sympatric occurrence of several Trachymyrmex and Sericomyrmex higher attine ants in Gamboa, Panama provided the opportunity to do a quantitative study of species-level interaction-specificity. RESULTS: We genotyped the ants for Cytochrome Oxidase and their Leucoagaricus fungal cultivars for ITS rDNA. Combined with activity measurements for 12 carbohydrate active enzymes, these data allowed us to test whether garden enzyme activity was affected by fungal strain, farming ants or combinations of the two. We detected two cryptic ant species, raising ant species number from four to six, and we show that the 38 sampled colonies reared a total of seven fungal haplotypes that were different enough to represent separate Leucoagaricus species. The Sericomyrmex species and one of the Trachymyrmex species reared the same fungal cultivar in all sampled colonies, but the remaining four Trachymyrmex species largely shared the other cultivars. Fungal enzyme activity spectra were significantly affected by both cultivar species and farming ant species, and more so for certain ant-cultivar combinations than others. However, relative changes in activity of single enzymes only depended on cultivar genotype and not on the ant species farming a cultivar. CONCLUSIONS: Ant cultivar symbiont-specificity varied from almost full symbiont sharing to one-to-one specialization, suggesting that trade-offs between enzyme activity spectra and life-history traits such as desiccation tolerance, disease susceptibility and temperature sensitivity may apply in some combinations but not in others. We hypothesize that this may be related to ecological specialization in general, but this awaits further testing. Our finding of both cryptic ant species and extensive cultivar diversity underlines the importance of identifying all species-level variation before embarking on estimates of interaction specificity.

Esterase in imported fire ants, Solenopsis invicta and S. richteri (Hymenoptera: Formicidae): activity, kinetics and variation.

Solenopsis invicta and Solenopsis richteri are two closely related invasive ants native to South America. Despite their similarity in biology and behavior, S. invicta is a more successful invasive species. Toxic tolerance has been found to be important to the success of some invasive species. Esterases play a crucial role in toxic tolerance of insects. Hence, we hypothesized that the more invasive S. invicta would have a higher esterase activity than S. richteri. Esterase activities were measured for workers and male and female alates of both ant species using α-naphthyl acetate and ß-naphthyl acetate as substrates. Esterase activities in S. invicta were always significantly higher than those in S. richteri supporting our hypothesis. In S. invicta, male alates had the highest esterase activities followed by workers then female alates for both substrates. In S. richetri, for α-naphthyl acetate, male alates had the highest activity followed by female alates then workers, while for ß-naphthyl acetate, female alates had the highest activity followed by male alates then workers. For workers, S. richteri showed significantly higher levels of variation about the mean esterase activity than S. invicta. However, S. invicta showed significantly higher levels of variation in both female and male alates.

Selenophosphate synthetase in the male accessory glands of an insect without selenoproteins.

Selenoproteins (containing the 21st proteinogenic amino acid selenocysteine) play important roles throughout all domains of life. Surprisingly, a number of taxa have small selenoproteomes, and Hymenopteran insects appear to have fully lost selenoproteins. Nevertheless, their genomes contain genes for several proteins of the selenocysteine insertion machinery, including selenophosphate synthetase 1 (SELD/SPS1). At present, it is unknown whether this enzyme has a selenoprotein-independent function, and whether the gene is actually translated into a protein in Hymenoptera. Here, we report that SELD/SPS1 is present as a protein in the accessory glands of males of the ant Cardiocondyla obscurior. It appears to be more abundant in the glands of winged disperser males than in those of wingless, local fighter males. Mating increases the lifespan and fecundity of queens in C. obscurior, and mating with winged males has a stronger effect on queen fitness than mating with a wingless male. SELD/SPS 1 has been suggested to play an important role in oxidative stress defense, and might therefore be involved in the life-prolonging effect of mating.

Ant-fungus species combinations engineer physiological activity of fungus gardens.

Fungus-gardening insects are among the most complex organisms because of their extensive co-evolutionary histories with obligate fungal symbionts and other microbes. Some fungus-gardening insect lineages share fungal symbionts with other members of their lineage and thus exhibit diffuse co-evolutionary relationships, while others exhibit little or no symbiont sharing, resulting in host-fungus fidelity. The mechanisms that maintain this symbiont fidelity are currently unknown. Prior work suggested that derived leaf-cutting ants in the genus Atta interact synergistically with leaf-cutter fungi (Attamyces) by exhibiting higher fungal growth rates and enzymatic activities than when growing a fungus from the sister-clade to Attamyces (so-called 'Trachymyces'), grown primarily by the non-leaf cutting Trachymyrmex ants that form, correspondingly, the sister-clade to leaf-cutting ants. To elucidate the enzymatic bases of host-fungus specialization in leaf-cutting ants, we conducted a reciprocal fungus-switch experiment between the ant Atta texana and the ant Trachymyrmex arizonensis and report measured enzymatic activities of switched and sham-switched fungus gardens to digest starch, pectin, xylan, cellulose and casein. Gardens exhibited higher amylase and pectinase activities when A. texana ants cultivated Attamyces compared with Trachymyces fungi, consistent with enzymatic specialization. In contrast, gardens showed comparable amylase and pectinase activities when T. arizonensis cultivated either fungal species. Although gardens of leaf-cutting ants are not known to be significant metabolizers of cellulose, T. arizonensis were able to maintain gardens with significant cellulase activity when growing either fungal species. In contrast to carbohydrate metabolism, protease activity was significantly higher in Attamyces than in Trachymyces, regardless of the ant host. Activity of some enzymes employed by this symbiosis therefore arises from complex interactions between the ant host and the fungal symbiont.