Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini)

The digestive physiology and biochemistry of larvae of the leaf-cutting ant Acromyrmex subterraneus were investigated here. The activity of digestive enzymes was evaluated in the labial glands, midgut epithelium (soluble and particulate fractions), and in the lumen contents, separated into endo and ectoperitrophic regions. Enzymes with high levels of activity were partially characterised using chromatography and electrophoresis techniques. Microscope observations were carried out and the anatomy of the larval digestive tract was described here for the first time. Larvae fed with pH indicator solutions showed the anterior portion of the midgut to be acidic and the posterior portion neutral to alkaline, indicating that the pH of the different regions of the midgut could optimise certain enzyme activities, whilst inhibiting others. The flow rate of the intestinal contents was also evaluated in larvae fed with a dye solution. The slow flow rate is probably due to closure of the rear end of the larval midgut. No compartmentalisation of digestive enzymes acting on oligosaccharides and disaccharides in the ectoperitrophic space and on polysaccharides in the endoperitrophic space was observed here, which could also be related to the closure of the midgut. The digestive physiology of these larvae is therefore similar to ancestral Holometabola, a paradox when considering the highly evolved nature of these insects. The larval midgut demonstrated a large diversity of enzyme activities with high levels of alpha-amylase, alpha-mannosidase, chitinase, alpha-glucosidase, beta-glucosidase and proteinase. High levels of chitinase and amylase activities were detected in the labial glands of larvae. The enzyme profile reflected the necessity of the larvae to degrade the fungal substrate, their sole diet, and a probable source of some of the digestive enzymes detected here. When compared to adults, the larvae had a greater diversity and higher levels of enzyme activity, highlighting their importance as the "digestive caste" of the colony.     

Oral trophallaxis in adult leaf-cutting ants Acromyrmex subterraneus subterraneus (Hymenoptera, Formicidae)

Adult leaf-cutting ants of the subspecies Acromyrmex subterraneus subterraneus were fed with an Evans Blue dye solution, which allowed the investigation of subsequent exchange of liquids between ants by oral trophallaxis. Trophallactic behavior was filmed and the antennation patterns of donor and recipient ants were described. The ants’ crop capacity was measured following ad libitum feeding on dye solution. Ants previously fed on the dye solution (donors) were placed individually with unfed ants of the same caste (recipients) and the amount of dye solution passed from the donor to the recipient by oral trophallaxis was measured after one hour. The volume received was 0.26 ± 0.15μL (mean ± SD) and the residual volume of dye in the crop of the donors was 0.49 ± 0.23μL. There were 38 trophallactic events recorded for 50 pairs of ants. Trophallaxis was observed from 2.3 min to 21.5 min after initial exposure, with a mean latency of 8.4 ± 5.6 min. The mean duration of a trophallatic event was 2.3 ± 1.3 min. As far as we know, this is the first time that trophallaxis in leaf-cutting ants has been described and quantified. Received 2 December 2005; revised 6 April 2006; accepted 10 April 2006.     

Queen adoption in colonies of the leaf-cutting ant Acromyrmex subterraneus molestans (Hymenoptera: Formicidae)

Bioassays were conducted in both laboratory and the field to determine if monogynous colonies of Acromyrmex subterraneus molestans (Myrmicinae, Attini) adopt queens from other colonies of the same subspecies. The results suggest that the adoption of fertilized queens is a possible mechanism to explain the occurrence of colonies with multiple queens in this subspecies. Only minor workers were able to discriminate queens from other colonies and were aggressive toward them. Therefore, queen recognition differs among subcastes.     

Worker laying in leafcutter ant Acromyrmex subterraneus brunneus （Formicidae, Attini）

We studied the process of offspring production in queenless colonies of Acromyrmex subterraneus brunneus, and particularly evaluated the ovary development of workers as a function of their age. For this, subcolonies were set up and evaluated at different periods of isolation from the queen （2, 4 and 6 months）, besides individually labeled age groups. The subcolonies were assessed according to offspring production and ovaries containing oocytes or not. The evaluations showed worker oviposition and development of males originating from worker-laid eggs. At 2 months＇ absence of the queen, eggs and larvae were found, with eggs in a higher proportion than larvae. After 4 months, the proportion of eggs had reduced while larvae had increased, and a pupa was found in one subcolony. At 6 months, besides a higher share of larvae, one pupa and one adult male were found. Dissection of workers revealed ovaries containing oocytes during the periods of evaluation. Only a group of medium-sized and large workers, 23.3%, 20.9% and 37.5% of the population from each period assessed in queenless subcolonies respectively, presented developed oocytes in the ovary. The same was observed in colonies with a queen, with 17.6%, 19.6% and 7.8% of the group of dissected workers from each time period, respectively. With respect to worker age, we observed by dissection of the ovary, that the greatest percentage of individuals with ovarioles containing oocytes occurred at 45 days （6 weeks） up to 90 days （12 weeks）. These results probably are associated with the workers reproduction and the laying of trophic and reproductive eggs in colonies with and without a queen; these eggs have distinct functions in each situation.     

Imidacloprid Inhibits Behavioral Defences of the Leaf-Cutting Ant Acromyrmex subterraneus subterraneus (Hymenoptera:Formicidae)

Leaf-cutting ants have evolved a range of defensive strategies which complicate the use of entomopathogens for their control. One of these behavioral strategies is self-grooming, which increases when ants detect the presence of fungal conidia on their integuments. We have previously shown that insecticides, when used at ultra-low concentrations, can be synergists of entomopathogenic fungi. It is possible that certain insecticides could modify ant behavior in a way that increases the chances of a fungal infection taking hold. The current study investigated the effects of the neonicotinoid insecticide Imidacloprid (IMI) and the entomopathogenic fungus Beauveria bassiana on self-grooming and locomotion behavior of Acromrymex subterraneus subterraneus workers when ants were exposed to the agents separately or together. Initially 10, 20 and 40 ng/insect IMI was topically administered to worker ants. Four hours after administration, the ants were placed into an experimental arena and self-grooming and locomotion behaviors were recorded for 10 min. Separate groups of ants were exposed to filter paper discs impregnated with conidial suspensions and 4 h later their behavior was monitored for 10 min. Subsequently, ants were treated with 10 ng/insect IMI, immediately exposed to fungal suspensions and 4 h later their behavior was monitored. The 10 ng/insect IMI treatment resulted in higher locomotor activity and lower self-grooming when compared to vehicle, 20 ng and 40 ng IMI treatments. Exposure to fungal conidia produced low locomotion and high self-grooming behaviors. The 10 ng IMI + fungal conidia treatment resulted in lower self-grooming and higher locomotion behavior. These alterations caused by a low concentration of IMI could increase ant susceptibility to infection by entomopathogenic fungi and are thus of interest for future tests in the development of integrated pest management strategies.     

Male induction and function of workers' excitability during swarming in leaf-cutting ants (Atta and Acromyrmex) (Hymenoptera,Formicidae)

Worker excitation in species of Atta and Acromyrmex prior to and during the swarming of reproductives is initiated by secretions from the male mandibular glands. During this phase, workers are highly aggressive, attacking all foreign objects. Field data suggest that this behavior is not only advantageous in protecting sexuals as they swarm, but also to prime workers to execute queens trying to initiate colonies near mature colonies following nuptial flights.     

Age polyethism in the leaf-cutting ant Acromyrmex subterraneus brunneus Forel, 1911 (Hym., Formicidae)

Abstract  This study describes and quantifies the behavioural acts of two laboratory colonies of Acromyrmex subterraneus brunneus by investigating worker age polyethism. Twenty-nine behavioural acts were recorded during the 19-week observation period. Young individuals performed tasks inside the nest related to brood care and care for the fungus garden, whereas older individuals performed activities outside the nest such as foraging and activities in the waste chamber. The average longevity (±SD) was 108.21 ± 3.30, 109.15 ± 1.92 and 122.71 ± 1.55 days for large, medium and small workers, respectively. The small-sized workers presented a higher probability of reaching older age than large- and medium-sized workers. This study describes task switching according to age polyethism and the relationship of physical and temporal subcastes.     

Acromyrmex ameliae sp. n. （Hymenoptera： Formicidae）： A new social parasite of leaf-cutting ants in Brazil

The fungus-growing ants （Tribe Attini） are a New World group of〉 200 species, all obligate symbionts with a fungus they use for food. Four attine taxa are known to be social parasites of other attines. Acromyrmex （ Pseudoatta） argentina argentina and Acromyrmex （Pseudoatta） argentina platensis （parasites of Acromyrmex lundi）, and Acromyrmex sp. （a parasite of Acromyrmex rugosus） produce no worker caste. In contrast, the recently discovered Acromyrmex insinuator （a parasite of Acromyrmex echinatior） does produce workers. Here, we describe a new species, Acromyrmex ameliae, a social parasite of Acromyrmex subterraneus subterraneus and Acromyrmex subterraneus brunneus in Minas Gerais, Brasil. Like A. insinuator, it produces workers and appears to be closely related to its hosts. Similar social parasites may be fairly common in the fungus-growing ants, but overlooked due to the close resemblance between parasite and host workers.     

Foraging and leaf-cutting of the desert gardening ant Acromyrmex versicolor versicolor (Pergande) (Hymenoptera: Formicidae)

Summary Four colonies of the desert leaf-cutter ant Acromyrmex versicolor versicolor (Pergande) located 30 miles N.E. of Tempe, Arizona were observed over a 7 month period. The ants utilized trails in foraging, a characteristic of higher attines, as well as foraging singly, a typical pattern among the more primitive gardeners. The ants cut and collected both dry and green vegetation with dry grasses comprising the bulk of the forage. The ants increased their cutting of green vegetation after significant rainfall but collected dry grasses almost exclusively during dry periods. Detailed macro-motion picture analysis of leaf-cutting revealed that the desert gardener utilized a unique technique for cutting compound desert leaves.     

Chemical detection of the proteins and lipids in the fat body cells from workers of Attini ants (Hymenoptera: Formicidae)

The ultrastructural cytochemistry study and the chemical analysis carried out to examine the fat body of workers of the basal Attini Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata revealed very electrondense protein granules of varied sizes and shapes distributed throughout the cytoplasm of parietal and perivisceral trophocytes and oenocytes. Lipids were present in the cytoplasm of trophocytes as very electrondense granules. In M. parallelus, A. disciger, and A. laevigata, lipids were present as droplets. In parietal as well as perivisceral oenocytes, lipid granules were very electrondense. In A. laevigata, lipids were present as droplets. The chemical analysis using gas chromatography showed that the main lipid compounds present in trophocytes and oenocytes were saturated (myristic, palmitic, and stearic) and unsaturated fatty acids (oleic and linoleic). Also, in basal species, lipid compounds consisted of mainly saturated fatty acids, while in derived species saturated as well as unsaturated fatty acids were observed.     

Comparative reproductive biology of the social parasite Acromyrmex ameliae de Souza, Soares & Della Lucia and of its host Acromyrmex subterraneus subterraneus Forel (Hymenoptera: Formicidae)

Social parasites exhibit several characteristics that allow them to exploit their host species efficiently. The smaller size of parasite species is a trait commonly found in ants. In this work, we investigated several aspects of the reproductive biology of Acromyrmex ameliae De Souza, Soares & Della Lucia, a recently discovered parasite of Acromyrmex subterraneus subterraneus Forel. Sexuals of A. ameliae are substantially smaller than those from host species. Parasite queens laid significantly less worker eggs than host queens and inhibit sexual production of the host. The sex ratio of parasite species is highly female biased. Interestingly, we have observed parasite coupling on the laboratory, inside the nests and in the ground, opening the possibility to use controlled mating to study genetic approaches of parasitism in the ants.     

Ecological traits and evolutionary sequence of nest establishment in fungus-growing ants (Hymenoptera, Formicidae, Attini)

Neotropical ants in the tribe Attini share the obligatory behaviour of cultivating fungi as an essential food source. Of this complex and well-studied mutualism, little is known about the biological traits of the phylogenetically basal attines, which may offer important clues to the origins and early evolution of this intricate symbiosis. In this paper we focus on the founding of new nests, a key to understanding evolutionary changes in many social insects, which has received comparatively little attention in attine ants. We present a comparative survey of nest-founding behaviour in Attini, based on 441 foundress nests of 20 species in eight attine genera. In general, attine queens are semiclaustral and haplometrotic when founding nests. We show that attine foundresses of most species use an inert platform (discarded forewings, roots, or rocks) on which the incipient fungal garden is physically isolated during nest foundation, and they conduct semiclaustral foundation. This behaviour is not shared with Atta , which places the incipient garden directly on the soil floor of the initial nest chamber, and conducts claustral foundation. Nest-founding maps congruently onto the major clades in attine phylogeny, suggesting that the behavioural mechanisms used to isolate the incipient garden may have been key innovations in the early evolution of attine fungiculture. The evolutionary sequence of gardening in Attini suggests a transformation series from retention and use of detached wings (suspended from the chamber ceiling or placed over the chamber floor) to root-suspension, and finally use of bare soil. We also discuss transitions from semiclaustral to claustral founding, as well as from haplometrosis to pleometrosis, from ecological and evolutionary perspectives.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 39–48.     

The basicoxal gland, a new exocrine structure in poneromorph ants (Hymenoptera, Formicidae)

The tegumental epithelium of the outer dorsolateral region in the proximal part of the coxae in the mid‐ and hindlegs of both workers and queens of the ants Odontomachus rixosus and O. simillimus is differentiated into a conspicuous and hitherto unknown exocrine gland. The glandular cells display a clear microvillar differentiation of their apical cell membrane, and are lined with the tegumental cuticle, which in this part contains crack‐like channels perpendicular to its surface, that carry the glandular secretions to the outside. Apical microvilli support the transport of substances, and contain an extension of tubular smooth endoplasmic reticulum in their centre. The function of the gland may be that of providing lubricant substances to the articulation region of the generally heavily sclerotized ponerine ant species. The gland is also found in several other ponerine and amblyoponine species, but not in the ectatommine species studied. The foreleg coxae lack a basicoxal gland in all species examined, which may be explained by the more limited articulation between the thorax and the coxae in the forelegs compared to the mid‐ and hindlegs.     

Hierarchical establishment of information sources during foraging decision-making process involving Acromyrmex subterraneus (Forel, 1893) (Hymenoptera,Formicidae)

During foraging, worker ants are known for making use of many information sources to guide themselves in external environments, especially individual (memory) and social (trail pheromone) information. Both kinds of information act in a synergic way, keeping the foraging process efficient and organized. However, when social and individual information is conflicting face a trail bifurcation, it is necessary to establish a hierarchical order so prioritizing one of them. This study aims to verify which information (social or individual) is prioritized by Acromyrmex subterraneus workers when facing a bifurcation in a Y-trail system. Only one branch of the Y-trail leads to food resource and it had a section covered by filter paper where trail pheromone was deposited by workers. Pheromone deposition was here estimated by worker flow. After an individually marked forager (target-worker) made 1, 3 or 5 trips to the food resource, the filter paper was transferred to the branch which did not lead to the food. The time spent by target workers on branch selection and their right choice (branch with food) frequency were registered. Regardless of the target worker's previous trips to the resource, right choice frequency stood over 70%. In addition, the number of previous trips did not influence the time spent on decision making. However, the higher the flow of workers, the longer the time spent on decision making. By simulating a situation with conflicting information, it was possible to verify that a hierarchical order is established by A. subterraneus, which prioritized individual information (memory).     

The role of leaf-cutting ant workers (Hymenoptera: Formicidae) in fungus garden maintenance

Abstract.

• 1 We studied the role of leaf-cutting ant workers (Atta sexdens (L.) in fungus garden maintenance, by temporarily excluding workers from the garden. This increased its subsequent attractiveness, as expressed by an increase in the numbers of workers licking it.
• 2 The length of free mycelia on areas of the garden from which workers were excluded increased but was reduced again when workers were returned. Workers therefore removed hyphae from the garden surface.
• 3 The maximum‘isolation effect’was obtained by preventing ant access for 2–3 days, after which the effect declined. Removing staphylae from portions of garden kept ant-free for 4 and 6 days restored the effect, as the ants were not distracted by harvesting staphylae. Portions of garden kept ant-free for longer than this were no more attractive than non-isolated control garden.
• 4 Workers were highly efficient in detecting and removing contaminants from their fungus garden. Samples of garden could be isolated from workers for up to 12 days before major growth of contaminants occurred, and this contrasted with the maximum of 6 days for the isolation effect on licking. The isolation effect was therefore not a response to contaminant growths on the garden.
• 5 Workers on the garden surface may remove hyphae for nutritional reasons, or to‘prune’their fungus and stimulate its growth. In either case, the result is a regulation of fungal growth.

     

Labial gland disease in the genusFormica (Formicidae,Hymenoptera)

Summary Labial gland disease causes swelling of the labial glands in the pupal stage inFormica species. The resulting adults have enlarged thoraces and are called secretergates (Wasmann's pseudogynes). They infect the younger larvae, probably during feeding. The agent of the disease is unknown. It soon becomes non-infectious outside the labial glands. Diseased females were not seen to feed larvae. Yet their offspring contained secretergates. The discrepancy remains unexplained.     

Functional morphology of the metapleural gland in workers of the ant Crematogaster inflata (Hymenoptera,Formicidae)

Abstract. Workers of Crematogaster inflata possess the largest metapleural glands (relative to body size) known among ants, with reservoirs extending anteriorly up to the junction between the pro‐ and the mesothorax, and with over 1400 secretory cells on both sides together. This large secretory capacity is related to the gland's defensive function, which, in members of this species, is directed against larger arthropod and vertebrate enemies, and apparently not against microorganisms, in contrast to other ants, where the gland produces antibiotics. The gland is not equipped with any direct musculature. Secretion release is probably caused by contraction of the oblique longitudinal thorax muscles or by passive expulsion caused by external pressure.     

The influence of the scout upon the decision-making process of recruited workers in three Acromyrmex species (Formicidae: Attini)

We examined how the information communicated by a scout worker influences the plant species transported by recruited workers in Acromyrmex balzani, Acromyrmex rugosus, and Acromyrmex crassispinus, three species of leaf-cutting ant that have different substrate and habitat preferences. We verified that certain plant species were more likely to be transported than others and that recruitment occurs. We found that recruited workers were more likely to transport non-preferred plant species when they were recruited to these by scout workers. The results suggest that the scout worker can communicate information about plant identity to recruited workers, but that recruited workers integrate the information communicated with their own experience during the decision-making process.     

A phylogenetic analysis of the fungus-growing ants (Hymenoptera: Formicidae: Attini) based on morphological characters of the larvae

A phylogenetic hypothesis of the fungus-growing ants (subfamily Myrmicinae, tribe Attini) is proposed, based on a cladistic analysis utilizing forty-four morphological characters (109 states) of the prepupal worker larva. The fifty-one attine species analysed include representatives of eleven of the twelve currently recognized attine genera, excluding only the monotypic workerless parasite Pseudoatta ; the non-attines include two outgroups (species of the basal myrmicine genera Myrmica and Pogonomyrmex ), two myrmicine species presumed to be distantly related to the attines, and twelve species representing taxa that have been proposed by prior workers as possible sister groups of the Attini. There is strong character support for the monophyly of the Attini and for a sister-group relationship of the Attini and the Neotropical Blepharidatta brasiliensis. The Attini are divided into two distinct lineages, an 'apterostigmoid' clade (containing Apterostigma and Mycocepurus) and an 'attoid' clade (containing all other attine genera except Myrmicocrypta). The attine genus Myrmicocrypta appears to be paraphyletic with respect to these two groups; the species M.buenzlii in particular retains many attine plesiomorphies.
These results indicate that the fungus-growing behaviour had a single evolutionary origin in the ants. They also indicate that mycelium cultivation is plesiomorphic and that yeast cultivation is derived within the Attini, overturning the long-standing assumption that the yeast-growing Cyphomyrmex species are the most primitive Attini. Behavioural and ecological investigations into the origin and evolution of the fungus-growing behaviour might more profitably focus on species in the attine genus Myrmicocrypta , as well as those in the closely related non-attine genera Blepharidatta and Wasmannia.