Differential Behavioral Responses of Solenopsis invicta (Hymenoptera: Formicidae) Workers Toward Nestmate and Non-Nestmate Corpses.

The removal of corpses (aka 'necrophoric behavior') is critical to sanitation in ant colonies. However, little is known about differences in the necrophoric responses of Solenopsis invicta workers towards corpses of nestmates and non-nestmates. We introduced corpses of S. invicta workers from either intracolony (i.e., nestmate) or intercolony (i.e., non-nestmate) origin at the entrance of artificial nests, and recorded workers' aggressive responses and necrophoric behaviors for analysis. Solenopsis invicta workers displayed distinct responses towards corpses of different origins. Specifically, resident workers were more likely to remove fresh non-nestmate corpses than nestmate corpses, but there was no difference regarding corpses that had been dead for 15 min or longer. Resident workers reacted more aggressively to, and removed more quickly, fresh non-nestmate corpses than corpses of their nestmates. On the other hand, there was no significant difference in the removal time between nestmate and non-nestmate corpses that had been dead for 15 min or longer. Resident workers always displayed stronger aggressiveness towards non-nestmate corpses than nestmate corpses, excepting to corpses that had been dead for 6 h, which elicited a response. No significant correlation between the removal times and aggressiveness levels were detected in any treatments. It remains to be tested whether this differential response is adaptive in how it influences colony fitness and competition.

Wound treatment and selective help in a termite-hunting ant.

Open wounds are a major health risk in animals, with species prone to injuries likely developing means to reduce these risks. We therefore analysed the behavioural response towards open wounds on the social and individual level in the termite group-hunting ant Megaponera analis During termite raids, some ants get injured by termite soldiers (biting off extremities), after the fight injured ants get carried back to the nest by nest-mates. We observed treatment of the injury by nest-mates inside the nest through intense allogrooming at the wound. Lack of treatment increased mortality from 10% to 80% within 24 h, most likely due to infections. Wound clotting occurred extraordinarily fast in untreated injured individuals, within 10 min. Furthermore, heavily injured ants (loss of five extremities) were not rescued or treated; this was regulated not by the helper but by the unresponsiveness of the injured ant. Interestingly, lightly injured ants behaved 'more injured' near nest-mates. We show organized social wound treatment in insects through a multifaceted help system focused on injured individuals. This was not only limited to selective rescuing of lightly injured individuals by carrying them back (thus reducing predation risk), but, moreover, included a differentiated treatment inside the nest.

Intra- and inter-specific variation in alarm pheromone produced by Solenopsis fire ants.

Some fire ants of the genus Solenopsis have become invasive species in the southern United States displacing native species by competition. Although the displacement pattern seems clear, the mechanisms underlying competitive advantage remain unclear. The ability of ant workers to produce relatively larger amount of alarm pheromone may correspond to relative greater fitness among sympatric fire ant species. Here we report on quantitative intra-specific (i.e. inter-caste) and inter-specific differences of alarm pheromone component, 2-ethyl-3,6-dimethylpyrazine (2E36DMP), for several fire ant species. The alarm pheromone component was extracted by soaking ants in hexane for 48 h and subsequently quantified by gas chromatography-mass spectrometry at single ion monitoring mode. Solenopsis invicta workers had more 2E36DMP than male or female alates by relative weight; individual workers, however, contained significantly less pyrazine. We thus believe that alarm pheromones may serve additional roles in alates. Workers of Solenopsis richteri, S. invicta, and hybrid (S. richteri × S. invicta) had significantly more 2E36DMP than a native fire ant species, Solenopsis geminata. The hybrid fire ant had significantly less 2E36DMP than the two parent species, S. richteri and S. invicta. It seems likely that higher alarm pheromone content may have favored invasion success of exotic fire ants over native species. We discuss the potential role of inter-specific variation in pyrazine content for the relationship between the observed shifts in the spatial distributions of the three exotic fire ant species in southern United States and the displacement of native fire ant species.

On the Molecular Basis of Division of Labor in Solenopsis invicta (Hymenoptera: Formicidae) Workers: RNA-seq Analysis.

The fire ant Solenopsis invicta Buren is an important invasive pest. Among S. invicta workers behavioral changes depend on age where younger ants are nurses and older ants foragers. To identify potential genes associated with this division of labor, we compared gene expression between foragers and nurses by high-throughput sequencing. In total, we identified 1,618 genes significantly differently expressed between nurses and foragers, of which 542 were upregulated in foragers and 1,076 were upregulated in nurses. Several pathways related to metabolism were significantly enriched, such as lipid storage and fatty acid biosynthesis, which might contribute to the division of labor in S. invicta. Several genes involved in DNA methylation, transcription, and olfactory responses as well as resistance to stress were differentially expressed between nurses and foragers workers. Finally, a comparison between previously published microarray data and our RNA-seq data in S. invicta shows 116 genes overlap, and the GO term myofibril assembly (GO: 0030239) were simultaneously significantly enriched. These results advance knowledge of potentially important genes and molecular pathways associated with worker division of labor in S. invicta. We hope our dataset will provide . candidate target genes to disrupt organization in S. invicta as a control strategy against this invasive pest.

Wound-dependent leg amputations to combat infections in an ant society.

Open wounds pose major infection and mortality risks in animals.1,2 To reduce these risks, many animal species apply antimicrobial compounds on their wounds.1,2,3,4 Ant societies use antimicrobial secretions from the metapleural gland to combat pathogens,5,6,7,8,9,10 but this gland has been lost over evolutionary time in several genera, including Camponotus.11 To understand how infected wounds are handled without the use of antimicrobial secretions from the metapleural gland, we conducted behavioral and microbiological experiments in Camponotus floridanus. When we experimentally injured a worker's leg at the femur, nestmates amputated the injured limb by biting the base (trochanter) of the leg until it was severed, thereby significantly increasing survival compared to ants that did not receive amputations. However, when the experimental injury was more distal (at the tibia), nestmates did not amputate the leg and instead directed more wound care to the injury site. Experimental amputations also failed to improve survival in ants with infected tibia injuries unless the leg was amputated immediately after pathogen exposure. Micro-CT scans revealed that the muscles likely responsible for leg hemolymph circulation are predominantly in the femur. Thus, it is likely that femur injuries, by attenuating hemolymph flow, provide sufficient time for workers to perform amputations before pathogen spread. Overall, this study provides the first example of the use of amputations to treat infected individuals in a non-human animal and demonstrates that ants can adapt their type of treatment depending on the location of wounds.

Hitting the Road: Haplotype Diversity of Fire Ants Nesting on Disturbed Atlantic Forest Habitats.

Ants of the genus Solenopsis are globally distributed, presenting high diversity and many generalist species. In South America, the dominant species is Solenopsis saevissima (Smith, 1855), commonly found nesting in grassy fields surrounding humanized areas. In spite of being so common, there has been no research evaluating the effect of human disturbances on the mitochondrial DNA (mtDNA) haplotype diversity in this species. In this context, we here characterized the mtDNA haplotype diversity in S. saevissima nests by highway roadsides, dust roads, and forest borders of Atlantic Forest, based on partial sequences of cytochrome c oxidase subunit I (COI). Based on the facts that the species is a rapid colonizer of disturbed habitats, we specifically probed how the genetic diversity of native S. saevissima is impacted by highways and roads infrastructure expanding around the rainforest. Species diagnosis was established both by morphological characters and obtained mtDNA COI sequences. Overall, the species exhibited high haplotypes and nucleotide diversity, particularly around forest borders; though all haplotypes seemed closely related across the different habitats. We identified seven mitochondrial haplotypes (H1 to H7), where haplotype H1 was exclusively found in highway roadside nests, and H7 on dust roads; the remaining haplotypes were recorded from all habitats. Haplotype H1 was geographically isolated to the south of the Atlantic Forest, supporting previous suggestions that it acts as a biogeographical barrier. The pattern is suggestive of a recent species expansion, probably resulting from extensive habitat fragmentation. Taken together, our data demonstrates fire ant haplotypes prevailing in some anthropized habitats, characterizing how a native species lining the remnants of the Brazilian Atlantic Forest might be a concern for environmental conservation.

From species descriptions to diversity patterns: the validation of taxonomic data as a keystone for ant diversity studies reproducibility and accuracy.

Research findings in natural sciences need to be comparable and reproducible to effectively improve our understanding of ecological and behavioural patterns. In this sense, knowledge frontiers in biodiversity studies are directly tied to taxonomic research, especially in species-rich tropical regions. Here we analysed the taxonomic information available in 470 studies on Brazilian ant diversity published in the last 50 years. We aimed to quantify the proportion of studies that provide enough data to validate taxonomic identification, explore the frequency of studies that properly acknowledge their taxonomic background, and investigate the primary resources for ant identification in Brazil. We found that most studies on Brazilian ant diversity (73.6%) explicitly stated the methods used to identify their specimens. However, the proportion of papers that provide complete data for the repository institutions and vouchered specimens is vanishingly small (5.8%). Additionally, only 40.0% of the studies consistently presented taxon authorities and years of description, rarely referencing taxonomic publications correctly. In turn, the number of specialists and institutions consulted for ant identification in Brazil has increased in the last years, along with the number of studies that explicitly provide their taxonomic procedures for ant identification. Our findings highlight a shift between generations regarding the recognition of taxonomy as fundamental science, deepening our understanding of biodiversity.

Cuticular Hydrocarbon Profiling by Fractionation and GC-MS in Socially Parasitic Ants.

Ants use cuticular hydrocarbon (CHC) as a semiochemical for recognizing their nestmates. For socially parasitic ants, deceiving the CHC is an important survival strategy. Profiling and quantifying CHC is a potent approach to understanding such nestmate discrimination behavior. Thus, a highly efficient, stable, and reproducible extraction method for CHC is essential for this purpose. This paper describes a method for socially parasitic ants to disguise the host species' CHC profile under laboratory conditions, as well as the extraction and measurement of CHC from ants (from a previous study). First, the artificial isotopic substance is applied to the host worker; then, the socially parasitic ant disguises the host-like CHC profile against the above host worker. Next, the CHC is extracted and fractionated from a socially parasitic ant using hexane and silica gel. After concentrating the fractionated product, this product is then used for measurement by gas chromatographymass spectrometry (GC-MS). The CHC extraction protocol described in this paper may be used for various ant species.

The Intranidal Myrmecophiles of the Maltese Islands with Notes on Messor Nests as Repositories of Biodiversity.

The intranidal myrmecophilous arthropod fauna of the Maltese Islands is reviewed. Thirty species from nine orders are found to be obligate myrmecophiles, of which four species are recorded from the Maltese archipelago for the first time: Phrurolithus sp. (Araneae: Phrurolithidae), Pogonolaelaps canestrinii (Berlese, 1904), Gymnolaelaps messor Joharchi, Halliday, Saboori & Kamali, 2011 and G. myrmecophilus (Berlese, 1892) (Mesostigmata: Laelapidae). Phrurolithus also represents the first record of the family Phrurolithidae in Malta. Notes on the biology and local distribution of each species are provided, including ant-myrmecophile associations, of which two appear to be previously unknown: the occurrence of Smynthurodes betae Westwood, 1849 (Hemiptera: Aphididae) in the nest of Plagiolepis pygmaea (Latreille, 1798) and Phrurolithus in the nest of Pheidole pallidula (Nylander, 1849). Fourteen additional species are found to be either only occasionally myrmecophilic, accidental ant-guests or potentially myrmecophilous, the latter remaining ambiguous due to a lack of knowledge of their biology. Of these, the family Caeculidae (Arachnida: Trombidiformes) represents a new record for the Maltese Islands, on the basis of Microcaeculus sp. occurring in a nest of Camponotus barbaricus Emery, 1905. Preliminary results indicate that Messor nests may be repositories of considerable myrmecophile diversity, with the most unique symbionts.

Corrieoponenouragues gen. nov., sp. nov., a new Ponerinae from French Guiana (Hymenoptera, Formicidae).

This study describes the worker and queen castes of the Neotropical ponerine Corrieoponenouragues gen. nov., sp. nov., an ant from the tropical rainforest in French Guiana. Worker morphology of the taxon is compared with those of other Ponerinae and the similarities between them are discussed, refining the definition of character states for some diagnostic characters at the generic level, providing an identification key to the Neotropical genera, and making some adjustments to the taxonomic framework within the subfamily. Descriptions, diagnosis, character discussion, identification key, and glossary are illustrated with more than 300 images and line drawings. Open science is supported by providing access to measurement data for specimens of the new genus, a matrix of character states for all ponerine taxa evaluated in this study, and specimen data for all examined material. The new or revived combinations presented here are Pachycondylaprocidua Emery, comb. rev., Neoponeracuriosa (Mackay and Mackay), comb. nov., Leptogenysbutteli (Forel), comb. nov., and Bothroponeraescherichi (Forel), comb. nov. In addition, Leptogenysbutteli is synonymized with Leptogenysmyops (Emery), syn. nov.

New Tools for Conservation Biological Control: Testing Ant-Attracting Artificial Nectaries to Employ Ants as Plant Defenders.

Knowledge of the role of ants in many agroecosystems is relatively scarce, and in temperate regions the possibility to exploit ants as biocontrol agents for crop protection is still largely unexplored. Drawing inspiration from mutualistic ant-plant relationships mediated by extrafloral nectaries (EFNs), we tested the use of artificial nectaries (ANs) in order to increase ant activity on pear trees and to evaluate the effects on the arthropods, plant health and fruit production. While EFNs secrete a complex solution mainly composed of sugars and amino acids, ANs were filled with water and sucrose only. The results suggest that ANs can be used as manipulative instruments to increase ant activity over long periods of time. High ant activity was significantly linked to lower incidence of the pathogen fungus Venturia pyrina (pear scab) on pear leaves, and of the presence of Cydia pomonella (codling moth) caterpillars on pear fruit production. These results further encourage exploring underrated possibilities in the development of new tools for conservation biological control (CBC).

Queen venom isosolenopsin A delivers rapid incapacitation of fire ant competitors.

Fire ant venom contains insecticidal alkaloids named 'solenopsins'. Whilst species-specific differences are reported, little attention has been given to caste-specific venom adaptations. The venom of fire ant queens has remained particularly poorly studied, though studies have shown it to be strikingly similar across different species, in being primarily composed of the alkaloid isosolenopsin A, regardless of the chemical configuration in workers. We predict that this is the evolutionary outcome of stabilising selection, implying that a shared mechanism is responsible for the conserved venom composition among fire ant queens. The present investigation tests whether venom plays a role in nest founding, when queens must succeed in isolation in the field against competitor species. Here, we report that fire ant queen venom and isosolenopsin A are faster to incapacitate alien ants than the venom of fire ant workers. Representative sympatric competitor ant species were selected and exposed on their heads to the venom of workers and queens of the invasive fire ant species Solenopsis invicta and S. geminata. Queen venom was found to incapacitate rival foragers quicker than worker venom. The effects were reproduced using synthetic solenopsins, establishing that solenopsin A analogues are particularly effective contact neurotoxins. Overall, the venom of S. invicta is more lethal than of S. geminata, regardless of the incapacitation speed. We believe these are fundamental aspects of the chemical ecology of the invasive ants which remain overlooked, and emphasise the need for further studies into the venom biology of founding queens.

Fire Ant Venom Alkaloids Inhibit Biofilm Formation.

Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from Solenopsis invicta) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium Pseudomonas fluorescens. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.

Electrophysiological and Alarm Responses of Solenopsis invicta Buren (Hymenoptera: Formicidae) to 2-Ethyl-3,5-dimethylpyrazine (Short Title: EAG and Behavioral Responses of Fire Ants to Pyrazine).

2-Ethyl-3,5-dimethylpyrazine is an isomer of 2-ethyl-3,6-dimethylpyrazine, the alarm pheromone component of the red imported fire ant, Solenopsis invicta Buren. The pyrazine was synthesized and its alarm activity was investigated under laboratory conditions. It elicited significant electroantennogram (EAG) activities, and released characteristic alarm behaviors in fire ant workers. The EAG and alarm responses were both dose-dependent. Two doses of the pyrazine, 1 and 100 ng, were further subjected to bait discovery bioassays. Fire ant workers excited by the pyrazine were attracted to food baits, and their numbers increased over time. Ants displayed very similar response patterns to both low and high doses of the pyrazine. The pyrazine impregnated onto filter paper disc attracted significantly more fire ant workers than the hexane control for all observation time intervals at the low dose, and in the first 15 min period at the high dose. The pyrazine loaded onto food bait directly tended to attract more fire ant workers than the hexane control. These results support the potential use of 2-ethyl-3,5-dimethylpyrazine to enhance bait attractiveness for the control of S. invicta in invaded regions.

Speedy milking of fresh venom from aculeate hymenopterans.

A straightforward method for extracting aculeate arthropod venoms by centrifugation is described, based on adapting a glass insert containing a piece of metal mesh or glass wool into a centrifuge tube. Venom apparatuses are centrifuged for 30 s intervals at ≈2000-6000 g, with samples being dislodged between cycles. Venom from fire ants, honeybees, and a social wasp were extracted within minutes. The method is suited for small-scale bioassays and allows for faithful descriptions of unmodified toxin cocktails.