Kr-h1 maintains distinct caste-specific neurotranscriptomes in response to socially regulated hormones.

Behavioral plasticity is key to animal survival. Harpegnathos saltator ants can switch between worker and queen-like status (gamergate) depending on the outcome of social conflicts, providing an opportunity to study how distinct behavioral states are achieved in adult brains. Using social and molecular manipulations in live ants and ant neuronal cultures, we show that ecdysone and juvenile hormone drive molecular and functional differences in the brains of workers and gamergates and direct the transcriptional repressor Kr-h1 to different target genes. Depletion of Kr-h1 in the brain caused de-repression of "socially inappropriate" genes: gamergate genes were upregulated in workers, whereas worker genes were upregulated in gamergates. At the phenotypic level, loss of Kr-h1 resulted in the emergence of worker-specific behaviors in gamergates and gamergate-specific traits in workers. We conclude that Kr-h1 is a transcription factor that maintains distinct brain states established in response to socially regulated hormones.

Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

The Scent of Ant Brood: Caste Differences in Surface Hydrocarbons of Formica exsecta Pupae.

Chemical communication is common across all organisms. Insects in particular use predominantly chemical stimuli in assessing their environment and recognizing their social counterparts. One of the chemical stimuli used for recognition in social insects, such as ants, is the suite of long-chain, cuticular hydrocarbons. In addition to providing waterproofing, these surface hydrocarbons serve as a signature mixture, which ants can perceive, and use to distinguish between strangers and colony mates, and to determine caste, sex, and reproductive status of another individual. They can be both environmentally and endogenously acquired. The surface chemistry of adult workers has been studied extensively in ants, yet the pupal stage has rarely been considered. Here we characterized the surface chemistry of pupae of Formica exsecta, and examine differences among sexes, castes (reproductive vs. worker), and types of sample (developing individual vs. cocoon envelope). We found quantitative and qualitative differences among both castes and types of sample, but male and female reproductives did not differ in their surface chemistry. We also found that the pupal surface chemistry was more complex than that of adult workers in this species. These results improve our understanding of the information on which ants base recognition, and highlights the diversity of surface chemistry in social insects across developmental stages.

Behavioral flexibility in Wasmannia auropunctata (Hymenoptera: Formicidae).

Worker division of labor is a defining trait in social insects. Many species are characterized by having behavioral flexibility where workers perform non-typical tasks for their age depending on the colony's needs. Worker division of labor and behavioral flexibility were examined in the little fire ant Wasmannia auropunctata (Roger, 1863), for which age-related division of labor has been found. Young workers perform nursing duties which include tending of brood and queens, and colony defense, while older workers forage. When nurses were experimentally removed from the colony, foragers were observed carrying out nursing and colony defense duties, yet when foragers were removed nurses did not forage precociously. We also administered juvenile hormone analog, methoprene, to workers. When methoprene was applied, foragers increased their nursing and defense activities while nurses became mainly idle. The behavioral flexibility of foragers of the little fire ant may be evidence of an expansion of worker's repertoires as they age; older workers can perform tasks they have already done in their life while young individuals are not capable of performing tasks ahead of time. This may be an important adaptation associated with the success of this ant as an invasive species.

Different lethal treatments induce changes in piperidine (1,1'-(1,2-ethanediyl)bis-) in the epidermal compounds of red imported fire ants and affect corpse-removal behavior.

Corpse-removal behavior of the red imported fire ant (RIFA) and the effects of lethal substances on RIFA signal communication were investigated in this study. The RIFA corpses, obtained through freezing, ether, 0.25 mg/L thiamethoxam, and starvation to death treatments, and naturally dead red fire ants were subjected to gas chromatography-mass spectrometry to identify the cuticular hydrocarbon profiles that had an effect on the corpse-removal behavior. The results showed that lethal toxic substances altered the epidermal compounds of RIFA and affected their corpse-removal behavior. Lethal toxic substances increased the number of worker touches with corpses and identification time of corpses. In addition, the content of piperidine (1,1'-(1,2-ethanediyl)bis-) on the surface of the corpse was different following the various treatments. Contamination with toxic substances resulted in the increased secretion of piperidine and led to increased identification time of corpses, number of touch with corpses, and total time for removal of corpses. Piperidine content was higher under conditions of natural death (4.67 ± 0.55%) and with thiamethoxam (10.43 ± 0.78%), freezing (0.83 ± 0.25%), and ether treatment (12.50 ± 0.70%) than under starvation treatment (0). The higher content of piperidine led to a longer number of touches with corpses and identification time. Piperidine compounds may be an element in warning information, which could affect the occurrence of different corpse-removal behaviors.

Identification of a novel plant-derived attractant for Acromyrmex lobicornis leaf-cutting ants.

Leaf-cutting ants are considered major pests of agriculture and forestry in the Neotropics. Attractive toxic baits are the prevailing method for managing them. Thus, there is great interest in identifying attractants to incorporate into these baits. Moreover, leaf-cutting ants can avoid toxic baits by associating the attractant with the toxin. We evaluated attractiveness of heptyl butyrate, a volatile compound found in fresh apples and plums. We conducted field experiments with ten colonies of Acromyrmex lobicornis. First, we evaluated the behaviour of ants exposed to heptyl butyrate at 1% by surrounding resources. Then, we compared the attractiveness of heptyl butyrate and orange pulp, the most commonly used attractant. Finally, we evaluated whether heptyl butyrate increases the attractiveness of a carbohydrate resource at varying doses. Heptyl butyrate at 1% attracted 92% more ants than the control and that it was as attractive as orange pulp. Heptyl butyrate paired with sucrose at concentrations of 0.001 and 0.1% was more attractive than sucrose alone, but greater concentrations did not increase sucrose's attractiveness. Therefore, heptyl butyrate could be added to toxic baits to manage A. lobicornis as it is as attractive as the most commonly used attractant and can be applied directly to the pellets.

Risk element accumulation in Coleoptera and Hymenoptera (Formicidae) living in an extremely contaminated area-a preliminary study.

The risk element accumulation ability of two groups of epigeic species, insects from families Coleoptera and Hymenoptera (namely Formicidae), was determined and related to soil risk element content and bioaccessibility. The study was conducted in the district of Príbram, Czech Republic, which was characterised by extremely high aged pollution in the soils, including risk elements, especially As, Pb, Zn and Cd, due to the former mining and smelting activity. Four sampling sites differing in their pseudo-total risk element contents were selected and composite samples of individuals representing either Coleoptera or Formicidae were sampled at the individual sampling points. The results indicate the ability of Coleoptera and Formicidae organisms to accumulate risk elements, especially at the location with extremely high soil risk element content. In soil containing up to 841 mg As kg-1, 84.6 mg Cd kg-1, 4250 mg Pb kg-1 and 8542 mg Zn kg-1, contents in insect bodies reached 239 mg As kg-1 As, 24.2 mg Cd kg-1, 70.4 mg Pb kg-1 and 335 mg Zn kg-1 in beetles and up to 20.9 mg As kg-1, 29.9 mg Cd kg-1, 111 mg Pb kg-1 and 657 mg Zn kg-1 in ants. Therefore, bioaccumulation factors (BAFs) varied between 0.02 and 0.55. Increasing Cd content in Coleoptera bodies with increasing soil pseudo-total element content was observed only among the investigated elements. However, the results indicate increasing BAF values with decreasing soil element levels, especially for Cd, Pb and Zn, indicating limited uptake of elements by the organisms living in contact with extremely contaminated soil.

Imidacloprid alters ant sociobehavioral traits at environmentally relevant concentrations.

Much research has focused on the effects of neonicotinoids on honey bees, however, relatively few studies have investigated their effects on ants, a taxonomically-related eusocial insect of high ecological importance. This study quantified how dietary exposures to environmentally-relevant levels of a neonicotinoid insecticide (imidacloprid) affected foraging and nest building of the western harvester ant over 14 days. Using a replicated design, statistically-significant reductions in ant foraging success (50% or greater) occurred at concentrations as low as 50 ppb compared to controls. Both the number of ants entering the maze and the percentage of foraging ants able to locate food were impacted by imidacloprid exposure. Ants exposed to 50 ppb also took three times longer than controls to find food in a test maze. This concentration is among the lowest levels of imidacloprid reported to affect ants and is well within the range of concentrations found in pollen and nectar of imidacloprid-treated plants. Ant foraging success was also impaired at comparable levels as those reported for the honey bee. Although more refinement and research are needed, results from this study suggest that the western harvester ant may be useful for screening the effects of neurotoxic chemicals on their navigation and foraging, two behaviors which are critical to maintaining colony health of ants and the ecological services they provide.

Phthalides as promising insecticides against Tuta absoluta (Lepidoptera: Gelechiidae).

In this study, the insecticide potential of eight phthalides derived from furan-2(5H)-one was evaluated against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) larvae. The potency of the most active phthalides and the susceptibility of six different T. absoluta populations to these compounds were determined. The toxicity of these molecules to two non-target species (Solenopsis saevissima Smith and Tetragonisca angustula Latreille) was also evaluated. Two phthalides (3 and 4) presented insecticide potential against T. absoluta. Phthalide 4 was as toxic as piperine (positive control) and both phthalides exhibited rapid action (LT50 < 2 hours). The variation in the susceptibility of T. absoluta populations to the phthalides 3 and 4 was low. Neither phthalide presented physiological selectivity for non-target species. Therefore, the phthalides 3 and 4 are promising molecules, or at least, a starting point for a chemical optimization program leading to formulations for the management of the tomato leafminer. The application of such products should be conducted according to the principles of ecological selectivity.

Tapping the biotechnological potential of insect microbial symbionts: new insecticidal porphyrins.

BACKGROUND: The demand for sustainable agricultural practices and the limited progress toward newer and safer chemicals for use in pest control maintain the impetus for research and identification of new natural molecules. Natural molecules are preferable to synthetic organic molecules because they are biodegradable, have low toxicity, are often selective and can be applied at low concentrations. Microbes are one source of natural insecticides, and microbial insect symbionts have attracted attention as a source of new bioactive molecules because these microbes are exposed to various selection pressures in their association with insects. Analytical techniques must be used to isolate and characterize new compounds, and sensitive analytical tools such as mass spectrometry and high-resolution chromatography are required to identify the least-abundant molecules. RESULTS: We used classical fermentation techniques combined with tandem mass spectrometry to prospect for insecticidal substances produced by the ant symbiont Streptomyces caniferus. Crude extracts from this bacterium showed low biological activity (less than 10% mortality) against the larval stage of the fall armyworm Spodoptera frugiperda. Because of the complexity of the crude extract, we used fractionation-guided bioassays to investigate if the low toxicity was related to the relative abundance of the active molecule, leading to the isolation of porphyrins as active molecules. Porphyrins are a class of photoactive molecules with a broad range of bioactivity, including insecticidal. The active fraction, containing a mixture of porphyrins, induced up to 100% larval mortality (LD50 = 37.7 µg.cm-2). Tandem mass-spectrometry analyses provided structural information for two new porphyrin structures. Data on the availability of porphyrins in 67 other crude extracts of ant ectosymbionts were also obtained with ion-monitoring experiments. CONCLUSIONS: Insect-associated bacterial symbionts are a rich source of bioactive compounds. Exploring microbial diversity through mass-spectrometry analyses is a useful approach for isolating and identifying new compounds. Our results showed high insecticidal activity of porphyrin compounds. Applications of different experiments in mass spectrometry allowed the characterization of two new porphyrins.

Alarm Pheromone Composition and Behavioral Activity in Fungus-Growing Ants.

Chemical communication is a dominant method of communication throughout the animal kingdom and can be especially important in group-living animals in which communicating threats, either from predation or other dangers, can have large impacts on group survival. Social insects, in particular, have evolved a number of pheromonal compounds specifically to signal alarm. There is predicted to be little selection for interspecific variation in alarm cues because individuals may benefit from recognizing interspecific as well as conspecific cues and, consequently, alarm cues are not normally thought to be used for species or nestmate recognition. Here, we examine the composition of the alarm pheromones of seven species of fungus-growing ants (Attini), including both basal and derived species and examine the behavioral responses to alarm pheromone of Acromyrmex leaf-cutting ants, the sister genus to the highly studied Atta leaf-cutting ants. We find surprisingly high interspecific variation in alarm pheromone composition across the attine phylogeny. Interestingly, the active component of the alarm pheromone was different between the two leaf-cutting ant genera. Furthermore, in contrast to previous studies on Atta, we found no differences among morphological castes in their responses to alarm pheromone in Acromyrmex but we did find differences in responses among putative age classes. The results suggest that the evolution of alarm communication and signaling within social insect clades can be unexpectedly complex and that further work is warranted to understand whether the evolution of different alarm pheromone compounds is adaptive.

Synthesis and Antimicrobial Evaluation of Fire Ant Venom Alkaloid Based 2-Methyl-6-alkyl-Δ1,6-piperideines.

The first synthesis of 2-methyl-6-pentadecyl-Δ1,6-piperideine (1), a major alkaloid of the piperideine chemotype in fire ant venoms, and its analogues, 2-methyl-6-tetradecyl-Δ1,6-piperideine (2) and 2-methyl-6-hexadecyl-Δ1,6-piperideine (3), was achieved by a facile synthetic method starting with glutaric acid (4) and urea (5). Compound 1 showed in vitro antifungal activity against Cryptococcus neoformans and Candida albicans with IC50 values of 6.6 and 12.4 µg/mL, respectively, and antibacterial activity against vancomycin-resistant Enterococcus faecium with an IC50 value of 19.4 µg/mL, while compounds 2 and 3 were less active against these pathogens. All three compounds strongly inhibited the parasites Leishmania donovani promastigotes and Trypanosoma brucei with IC50 values in the range of 5.0-6.7 and 2.7-4.0 µg/mL, respectively.

Essential Oils of Hyptis pectinata Chemotypes: Isolation, Binary Mixtures and Acute Toxicity on Leaf-Cutting Ants.

Leaf-cutting ants are pests of great economic importance due to the damage they cause to agricultural and forest crops. The use of organosynthetic insecticides is the main form of control of these insects. In order to develop safer technology, the objective of this work was to evaluate the formicidal activity of the essential oils of two Hyptis pectinata genotypes (chemotypes) and their major compounds on the leaf-cutting ants Acromyrmex balzani Emery and Atta sexdens rubropilosa Forel. Bioassays of exposure pathways (contact and fumigation) and binary mixtures of the major compounds were performed. The major compounds identified in the essential oils of H. pectinata were ß-caryophyllene, caryophyllene oxide and calamusenone. The essential oils of H. pectinata were toxic to the ants in both exposure pathways. Essential oils were more toxic than their major compounds alone. The chemotype calamusenone was more toxic to A. balzani in both exposure pathways. A. sexdens rubropilosa was more susceptible to the essential oil of the chemotype ß-caryophyllene in both exposure pathways. In general, the binary mixtures of the major compounds resulted in additive effect of toxicity. The essential oils of H. pectinata is a raw material of great potential for the development of new insecticides.

Essential Oil of Aristolochia trilobata: Synthesis, Routes of Exposure, Acute Toxicity, Binary Mixtures and Behavioral Effects on Leaf-Cutting Ants.

Plants of the genus Aristolochia have been frequently reported as important medicinal plants. Despite their high bioactive potential, to date, there are no reports of their effects on leaf-cutting ants. Therefore, the present study aimed to evaluate the insecticidal activity of the essential oil of Aristolochia trilobata and its major components on Atta sexdens and Acromyrmex balzani, two species of leaf-cutting ants. The bioassays were performed regarding routes of exposure, acute toxicity, binary mixtures of the major components and behavioral effects. Twenty-five components were identified in the essential oil of A. trilobata using a gas chromatographic system equipped with a mass spectrometer and a flame ionization detector. The components found in higher proportions were sulcatyl acetate, limonene, p-cymene and linalool. The essential oil of A. trilobata and its individual major components were efficient against A. balzani and A. sexdens workers when applied by fumigation. These components showed fast and efficient insecticidal activity on ants. The components acted synergistically and additively on A. balzani and A. sexdens, respectively, and caused a strong repellency/irritability in the ants. Thus, our results demonstrate the great potential of the essential oil of A. trilobata and its major components for the development of new insecticides.

Toxicity of new pyrethroid in pest insects Asciamonuste and Diaphania hyalinata, predator Solenopsis saevissima and stingless bee Tetragonisca angustula.

There is increasing demand for new products for vegetable pest management. Thus, the aim of this study was to assess the toxicity of pyrethroids with acid moiety modifications to measure the insecticidal activity of these compounds on the lepidopteran vegetable pests Diaphania hyalinata (L.) (Lepidoptera: Pyralidae) and Asciamonuste (Latreille) (Lepidoptera: Pieridae) and evaluate their selectivity for the predatory ant Solenopsis saevissima (F. Smith) (Hymenoptera: Formicidae) and pollinator Tetragonisca angustula (Latreille) (Hymenoptera: Apidae: Meliponinae). Racemic mixtures of five new pyrethroids (30 µg molecule mg-1 insect body weight) resulted in high (100%) and rapid (stable LD50 after 12 h) mortality in D. hyalinata and A. monuste. In A. monuste, the trans-pyrethroid [12] isomer showed similar toxicity to permethrin. For D. hyalinata, the trans-pyrethroid [9] isomer and cis-pyrethroid [10] isomer were as toxic as permethrin. Due to their low selectivity, these new pyrethroids should be applied on the basis of ecological selectivity principles to minimize impacts on nontarget organisms S. saevissima and T. angustula.

Suppression of savanna ants alters invertebrate composition and influences key ecosystem processes.

In almost every ecosystem, ants (Hymenoptera: Formicidae) are the dominant terrestrial invertebrate group. Their functional value was highlighted by Wilson (1987) who famously declared that invertebrates are the "little things that run the world." However, while it is generally accepted that ants fulfil important functions, few studies have tested these assumptions and demonstrated what happens in their absence. We report on a novel large-scale field experiment in undisturbed savanna habitat where we examined how ants influence the abundance of other invertebrate taxa in the system, and affect the key processes of decomposition and herbivory. Our experiment demonstrated that ants suppressed the abundance and activity of beetles, millipedes, and termites, and also influenced decomposition rates and levels of herbivory. Our study is the first to show that top-down control of termites by ants can have important ecosystem consequences. Further studies are needed to elucidate the effects ant communities have on other aspects of the ecosystem (e.g., soils, nutrient cycling, the microbial community) and how their relative importance for ecosystem function varies among ecosystem types (e.g., savanna vs. forest).

Juvenile hormone downregulates vitellogenin production in Ectatomma tuberculatum (Hymenoptera: Formicidae) sterile workers.

In the ant Ectatomma tuberculatum (Olivier 1792), workers have active ovaries and lay trophic eggs that are eaten by the queen and larvae. Vitellogenins are the main proteins found in the eggs of insects and are the source of nutrients for the embryo in the fertilized eggs and for adults in the trophic eggs. In social insects, vitellogenin titres vary between castes and affect reproductive social status, nursing, foraging, longevity, somatic maintenance, and immunity. In most insects, vitellogenin synthesis is mainly regulated by juvenile hormone. However, in non-reproductive worker ants, this relationship is poorly characterized. This study determined the effects of juvenile hormone on vitellogenin synthesis in non-reproductive E. tuberculatum workers. Juvenile hormone was topically applied onto workers, and the effect on vitellogenin synthesis in the fat body and vitellogenin titres in the haemolymph were analysed by ELISA and qPCR. Juvenile hormone downregulated protein synthesis and reduced vitellogenin titres in the haemolymph, suggesting that in workers of E. tuberculatum, juvenile hormone loses its gonadotrophic function.

Learning through the waste: olfactory cues from the colony refuse influence plant preferences in foraging leaf-cutting ants.

Leaf-cutting ants learn to avoid plants initially harvested if they prove to be harmful for their symbiotic fungus once incorporated into the nest. At this point, waste particles removed from the fungus garden are likely to contain cues originating from both the unsuitable plant and the damaged fungus. We investigated whether leaf-cutting ant foragers learn to avoid unsuitable plants solely through the colony waste. We fed subcolonies of Acromymex ambiguus privet leaves treated with a fungicide undetectable to the ants, then collected the produced waste, and placed it into the fungus chamber of naive subcolonies. In individual choice tests, naive foragers preferred privet leaves before waste was put into the fungus chamber, but avoided them afterwards. Evidence on the influence of olfactory cues from the waste on decision making by foragers was obtained by scenting and transferring waste particles from subcolonies that had been fed either fungicide-treated or untreated leaves. In choice experiments, foragers from subcolonies given scented waste originating from fungicide-treated leaves collected fewer sugared paper discs with that scent compared with foragers from subcolonies given scented waste from untreated leaves. The results indicate that foragers learn to avoid plants unsuitable for the fungus by associating plant odours and cues from the damaged fungus that are present in waste particles. It is argued that waste particles may contribute to spread information about noxious plants for the fungus within the colony.

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.

Synthesis of the Enantiomers of Tedanalactam and the First Total Synthesis and Configurational Assignment of (+)-Piplaroxide.

Highlighting the recently established methodology for the direct synthesis of glycidic amides from tertiary allyl amines, the synthesis of the enantiomers of tedanalactam were completed in two steps from the corresponding chiral dihydropiperidine. Additionally, the (+)- and (-)-enantiomers of piplaroxide were obtained from their respective tedanalactam precursor, and the absolute configuration of the naturally occurring (+)-piplaroxide was determined. The present approach represents not only the shortest synthesis of (-)-tedanalactam but also the first total synthesis of (+)-piplaroxide, a repellent against the leafcutter ant Atta cephalotes.