Mandibular Gland Extracts Research Articles

Aggregation Role of Mandibular Gland Secretions in the Giant Tropical Ant Paraponera clavata (Formicidae: Paraponerinae)

Social insects exhibit sophisticated behavioral repertoires to maintain communication and regulate colony activities. To elucidate the behavioral mechanisms of social insects is essential a thorough understanding of morphophysiological characteristics, such as the functionality of exocrine gland secretions. In this study, it was analyzed whether the mandibular gland secretions influence the aggregation behavior of workers of the ant Paraponera clavata. Associated behavioral acts were also studied. Bioassays indicated that mandibular gland extract triggers aggregation behaviors in P. clavata workers. The observed behavioral acts include mandible opening, raising of the antennae, antennal contact with treated filter paper, selfgrooming, cleaning of the abdominal tip, and allogrooming. These observations demonstrate the potential multifunctionality of compounds secreted by mandibular glands within the behavioral repertoire of P. clavata.

Melipona beecheii (Hymenoptera, Apidae) foragers deposit a chemical mark on food to attract conspecifics

Stingless bees have a sophisticated system of chemical communication that helps conspecifics find food sources. In this study, we investigated whether Melipona beecheii foragers deposit a chemical mark on food to recruit conspecifics. Our results showed that foragers preferred to visit the feeders visited previously by conspecifics over clean feeders. We also found that foragers preferred visiting feeders baited with labial gland extracts over those baited with mandibular extracts or hexane. Labial gland extracts elicited higher forager antennal responses compared with those evoked by the mandibular gland extracts or hexane. Labial gland extracts and extracts from feeders visited by foragers contain a mixture of unsaturated hydrocarbons, followed by straight chain hydrocarbons and small quantities of esters. The main component is a mixture of alkene isomers C27:1.

Response of the neonate larvae of Cactoblastis cactorum to synthetic cactoblastins, a newly identified class of pheromonally-active chemicals found in the caterpillar’s mandibular glands

The pre-excavation activity of the neonate larvae of Cactoblastis cactorum (Lepidoptera: Pyralidae) is typically confined to an arena that encircles the base of their egg stick. If the caterpillars are unable to penetrate the host plant within the arena, they strike off en masse in search of a more favorable site, marking their pathway with secretions from their mandibular glands. One component of this secretion, 4-hydroxy-2-oleoylcyclohexane-1,3-dione (an acylcyclohexanedione, ACHD), has been previously shown to function as a trail pheromone. In this paper, we report the characterization, synthesis, and bioassays of a family of 3-acyl fatty acid methyl esters (which we named cactoblastins), a chemically distinct group of mandibular gland compounds that also elicit trail following behavior. The relative effectiveness in eliciting trail following of the ACHD and six, chemically distinct, synthetic cactoblastins was determined. The most bioactive of the cactoblastins was also compared to whole mandibular gland extract to assess its ability to disrupt the aggregative behavior of the neonates and to serve as a biorational alternative to pesticides for managing invasive populations of the caterpillar.

Larval Pheromone Disrupts Pre-Excavation Aggregation of Cactoblastis cactorum (Lepidoptera: Pyralidae) Neonates Precipitating Colony Collapse

Newly eclosed larvae of Cactoblastis cactorum Berg (Lepidoptera: Pyralidae) contain their activity to an arena formed at the base of their eggstick, marked with a mandibular gland pheromone. Laboratory and field studies were undertaken to determine if these pre-excavation aggregations, essential to their successful penetration of the host plant, could be disrupted with mandibular gland extract causing the incipient colonies to perish. Cladodes or whole plants were sprayed with the pheromone, obtained by extracting macerated caterpillars in hexane, hexane only, or left unsprayed, and the survivorship of caterpillars that eclosed from eggsticks attached to the cladodes recorded at a later date. In 4 separate experiments, the average survivorship of C. cactorum larvae from cohorts on cladodes sprayed with the extract (15%) differed markedly from survivorship of caterpillar cohorts on cladodes treated with the solvent only (84%) or left untreated (80%). This differential mortality was attributed to the elicitation of the independent dispersal of the caterpillars by the mandibular gland pheromone and their failure to reaggregate in numbers sufficient to mount a successful attack on the host plant. The potential for managing pest populations of caterpillars employing this target-specific alternative to conventional pesticides is discussed.

(S)-2-Heptanol, the alarm pheromone of the stingless bee Melipona solani (Hymenoptera, Meliponini)

Timely defence response is critical in any highly social bee species. Chemical signalling is closely linked to this behaviour, and several pheromones have been discovered. In this work, we identified the pheromone of the stingless bee Melipona solani and evaluated the electrophysiological and behavioural response of the bee to the identified compound. We determined that the mandibular glands serve as the reservoir of the alarm pheromone. Because enantiomeric recognition has been scarcely examined, we performed bioassays and electroantennographic (EAG) studies to determine the role of chirality in pheromone recognition. We found that (S)-2-heptanol was the active compound involved in the alarm response of this species. Although we did not find (R)-2-heptanol in the mandibular gland extracts, bees responded to it in a similar way to the (S)-isomer and to a racemic mixture. The behavioural response of M. solani was flight, different from other Melipona species behaviour. We discuss these findings in an evolutionary and ecological framework.

Associative learning of non-nestmate odor marks between colonies of the stingless bee Scaptotrigona mexicana Guérin (Apidae, Meliponini) during foraging

Stingless bees use chemical signals to communicate nestmates the location of rich food sources. Such information may be intercepted by conspecifics from other colonies. In this study, we investigated if chemical information from non-nestmates can be used to orient foragers of the stingless bee Scaptotrigona mexicana to food sources. In the first experiment, foragers were exposed to feeders that were differentially odor-marked by nestmates and non-nestmates, and their preferences for both types of feeders were recorded. In a second experiment, we marked different feeders with mandibular or labial gland extracts of nestmates and non-nestmates. Results from the first experiment indicate that foragers were able to associate odor marks from non-nestmates with rich food sources. In the second experiment, we observed that foragers did not differentiate between the gland extracts of nestmates and those from non-nestmates. We discuss these findings within a behavioral and ecological framework.

Collectively Facilitated Behavior of the Neonate Caterpillars of Cactoblastis cactorum (Lepidoptera: Pyralidae).

The behavioral biology of the first instar larva of Cactoblastis cactorum was studied from the time of eclosion until the colony penetrated and initiated excavation of the host plant. Hatching from an egg stick was asynchronous, requiring 20 h for the entire cohort to eclose at 50%–70% RH and significantly longer at a lower range of RHs. On eclosion, neonates aggregated in an arena at the base of their egg stick and did not attempt to excavate the cladode until an average of 25 caterpillars had collected, approximately 15 h after the onset of egg hatch. Typically only a single entrance hole was formed, limiting the active process of excavating to one or a few individuals at-a-time until the host was fully penetrated and enlarged internally. Olfactometer tests showed that the neonates are strongly attracted to volatile chemicals released when caterpillars chewed into the cladode, accounting for the strong fidelity of the whole cohort to the initial site of penetration. In one instance, the caterpillars were observed to deal with an explosive release of mucilage by imbibing the liquid until the flooded zone was drained and the caterpillars could reenter the plant through the original entrance hole. Once inside the cladode, marked individuals adopted a regular cycle of defecating at the surface at a mean interval of approximately 10 min when followed for 35 successive cycles. Blanket spraying cladodes with a mandibular gland extract prior to hatching led to the independent dispersal of neonates and a failure to form an arena. When the cladode was impenetrable at the site of eclosion, the active cohort of unfed neonates set off together in search of a new site, marking and following a persistent trail that allowed late-to-eclose caterpillars to join their departed siblings. The adaptive significance of these observations is discussed in the context of the life history of the caterpillar.

An unusual recruitment strategy in a mass-recruiting stingless bee, Partamona orizabaensis.

Foragers of several stingless bee species deposit attractive scent marks on solid substrates to precisely recruit nestmates to food. Interestingly, Partamona workers quickly recruit large numbers of nest mates to resources, likely even without the deposition of attractive scent marks. However, systematic studies of the recruitment system of these bees are lacking. We now studied the recruitment behavior of P. orizabaensis. Our findings show that foragers of this species can recruit large numbers of nestmates to food sources at a particular location. The precise nestmate recruitment does not rely on attractive scent marks deposited on substrates. We never observed any scent marking behavior and feeders baited with labial or mandibular gland extracts were not attractive for the bees. Chemical analyses showed that the foragers' labial gland secretions exclusively contain long chain hydrocarbons, which render their role in recruitment communication unlikely. Whether mandibular gland secretions, which contain esters and alcohols that are known as attractive pheromones in other bee species, are used to guide recruits toward food during flight, remains elusive. We conclude that Partamona's quick recruitment system that does not rely on conspicuous scent marks has evolved as a strategy against competition with sympatrically occurring and more aggressive bee species.

Male meliponine bees (Scaptotrigona aff. depilis) produce alarm pheromones to which workers respond with fight and males with flight.

In highly social hymenopteran societies, males mainly serve reproductive purposes. Semiochemicals enable the different hymenopteran sexes and castes to communicate with each other and to coordinate important functions within colonies. I hereby show that sexual dimorphism in meliponine bees incorporates the alarm and defence communication system. I chemically analysed the mandibular glands of Scaptotrigona aff. depilis using GCMS methods and conducted behavioural experiments in both males and female workers using cephalic and mandibular gland extracts. In addition, behaviour studies with male cephalic extracts were also conducted in Scaptotrigona bipunctata and Partamona cupira. Males and female worker bees showed differences in the content of the mandibular glands, which are responsible for alarm communication in meliponines. Males never attacked but usually fled when confronted with the mandibular gland extract content of other conspecific males or females. Interestingly, however, meliponine males were still able to raise alarm and to induce substantial amounts of aggression at nest entrances, which is different from the much better known and studied honey bees. Potential reasons are briefly discussed.

Defensive repertoire of the stingless bee Melipona flavolineata Friese (Hymenoptera: Apidae)

Despite the fact that Meliponini bee lost their sting apparatus (stingless bees), they did not lose their ability to defend themselves. A variety of defensive strategies is described for the group, such as bitting and resin deposition. Defensive behaviours are usually mediated by chemical communication such as alarm pheromones. This study describe the defensive strategies of the stingless bee Melipona flavolineata Friese 1900 towards known pheromone sources, the head secretions of the robber bee Lestrimelitta limao and the mandibular gland extract of conspecifics M. flavolineata workers. The pheromones provoked different defensive reactions. The head secretions of the robber bee repelled returning foragers, elicited the enclosing of the nest entrance tube with batumen balls and agglomeration of workers outside of the box. In contrast, the mandibular gland extract elicited aggression towards the pheromone site, transport of resin and generalised agitated flights. The results confirm the mandibular gland as a source of alarm pheromone for this specie and also the chemical triggering of defensive response for the known cleptoparasite L. limao.

Trail Marking by Larvae of the Cactus Moth,Cactoblastis cactorum

The cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), spends most of its larval life feeding within the cladodes of Opuntia cactuses, but the gregarious caterpillars begin their life outside the plant, and in the later instars make intermittent excursions over plant surfaces to access new cladodes and to thermoregulate. The study reported here showed that when the caterpillars move en masse, they mark and follow trails that serve to keep the cohort together. Artificial trails prepared from hexane extracts of the caterpillar's paired mandibular glands were readily followed by the caterpillars. The glands are remarkably large, and their fluid contents, which constitute approximately 1% of the total wet mass of a caterpillar, are secreted onto the substrate as they move. Although the caterpillars also lay down copious quantities of silk, the material in itself neither elicits trail following nor is it a requisite component of pathways that elicit trail following. Previous analyses of the mandibular glands of other species of pyralid caterpillars showed that they contain a series of structurally distinct 2-acyl-1,3 cyclohexane diones. Chemical analysis indicates that the glands of C. cactorum contain structurally similar compounds, and bioassays indicate that trail following occurs in response to these chemicals. While the mandibular glands' fluids have been shown to act as semiochemicals, effecting both interspecific and intraspecific behavior in other species of pyralids, the present study is the first to report their use as a trail pheromone.

Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.)

Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour's glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour's gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour's gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.

Effect of gland extracts on digging and residing preferences of red imported fire ant workers (Hymenoptera: Formicidae)

There is evidence that ant-derived chemical stimuli are involved in regulating the digging behavior in Solenopsis invicta Buren. However, the source gland(s) and chemistry of such stimuli have never been revealed. In this study, extracts of mandibular, Dufour's, postpharyngeal, and poison glands were evaluated for their effect on ant digging and residing preferences of S. invicta workers from three colonies. In the intracolonial bioassays, workers showed significant digging preferences to mandibular gland extracts in 2 of 3 colonies and significant residing preferences in 1 of 3 colonies; significant digging preferences to Dufour's gland extracts in 1 of 3 colonies and significant residing preferences in 2 of 3 colonies. No digging and residing preferences were found for postpharyngeal and poison gland extracts. In intercolonial bioassays, significant digging and residing preferences were found for mandibular gland extracts in 3 of 6 colony combinations. Significant digging preferences to Dufour's gland extracts were found in 4 of 6 colony combinations and significant residing preferences in all 6 colony combinations. For postpharyngeal gland extracts, significant digging preferences were found only in 1 of 6 colonial combinations and no significant residing preferences were found. For poison gland extracts, no significant digging preferences were found; significant residing preferences were found in 1 of 6 colony combinations. However, a significant residing deterrence (negative residing preference index) was found for 2 of 6 colony combinations. Statistical analyses using data pooled from all colonies showed that mandibular and Dufour's gland extracts caused significant digging and residing preferences in both intracolonial and intercolonial bioassays but not postpharyngeal and poison gland extracts. By analyzing the data pooled from the same three colonies used for gland extract bioassays, it was found that, in no cases, workers showed significant digging and residing preferences to 2-ethyl-3,6-dimethylpyrazine, an alarm pheromone component from mandibular gland.

Mandibular gland secretions of meliponine worker bees: further evidence for their role in interspecific and intraspecific defence and aggression and against their role in food source signalling

Like ants and termites some species of stingless bees (Meliponini), which are very important pollinators in the tropics, use pheromone trails to communicate the location of a food source. We present data on the communicative role of mandibular gland secretions of Meliponini that resolve a recent controversy about their importance in the laying of such trails. Volatile constituents of the mandibular glands have been erroneously thought both to elicit aggressive/defensive behaviour and to signal food source location. We studied Trigona spinipes and Scaptotrigona aff. depilis ('postica'), two sympatric species to which this hypothesis was applied. Using extracts of carefully dissected glands instead of crude cephalic extracts we analysed the substances contained in the mandibular glands of worker bees. Major components of the extracts were 2-heptanol (both species), nonanal (T. spinipes), benzaldehyde and 2-tridecanone (S. aff. depilis). The effect of mandibular gland extracts and of individual components thereof on the behaviour of worker bees near their nest and at highly profitable food sources was consistent. Independent of the amount of mandibular gland extract applied, the bees overwhelmingly reacted with defensive behaviour and were never attracted to feeders scented with mandibular gland extract or any of the synthetic chemicals tested. Both bee species are capable of using mandibular gland secretions for intra- and interspecific communication of defence and aggression and share 2-heptanol as a major pheromone compound. While confirming the role of the mandibular glands in nest defence, our experiments provide strong evidence against their role in food source signalling.

Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens.

Polyethism and nestmate recognition in the alarm reaction of Atta leaf‐cutting ants

Abstract Ethological studies are conducted under laboratory conditions using workers of Atta sexdens sexdens and Atta opaciceps collected from field colonies to investigate the mechanisms involved in the alarm response and intra‐specific recognition in leaf‐cutting ants. Hexane extracts from the heads of gardeners and generalist workers elicit higher levels of alarm response in foragers from the same colony than do mandibular gland extracts from foragers and soldiers, indicating that gardeners, generalists and foragers are primarily responsible for the production of alarm pheromone. Foragers subjected to extracts from non‐nestmates exhibit significantly greater alarm responses than are induced by similar extracts derived from nestmate workers, suggesting that the alarm pheromone may have a role in nestmate recognition.

Effects of insemination quantity on honey bee queen physiology.

Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.

Hexyl Decanoate, the First Trail Pheromone Compound Identified in a Stingless Bee, Trigona recursa

Foragers of many species of stingless bees guide their nestmates to food sources by means of scent trails deposited on solid substrates between the food and the nest. The corresponding trail pheromones are generally believed to be produced in the mandibular glands, although definitive experimental proof has never been provided. We tested the trail following behavior of recruits of Trigona recursa in field experiments with artificial scent trails branching off from natural scent trails of this stingless bee. First-time recruits (newcomers) did not follow these trails when they were laid with pure solvent or mandibular gland extract. However, they did follow trails made with labial gland extract. Chemical analyses of labial gland secretions revealed that hexyl decanoate was the dominant component (72.4 +/- 1.9% of all volatiles). Newcomers were significantly attracted to artificial trails made with synthetic hexyl decanoate, demonstrating its key function in eliciting scent-following behavior. According to our experiments with T. recursa, the trail pheromone is produced in the labial glands and not in the mandibular glands. Hexyl decanoate is the first component of a trail pheromone identified and proved to be behaviorally active in stingless bees.

Mandibular Gland Secretion of Melipona beecheii: Chemistry and Behavior

The mandibular gland secretion of Melipona beecheii contains a rich mixture of terpenoid and oxygenated compounds and unsaturated and saturated hydrocarbons. However, it differs markedly from the 11 other Brazilian species examined in previous studies, both by the absence of 2-heptanol and the presence of rose oxides. The most abundant compound was geranyl hexanoate, whereas the most volatile compounds were cis- and trans-rose oxide and geraniol. The complete blend and five individual components found in the gland secretion were tested by electroantennography (EAG) and behavioral assays. The complete mandibular gland extract and geraniol elicited the strongest EAG responses, whereas these and farnesyl acetate induced the strongest attack response from workers. The role of the rose oxides remains to be elucidated, as they do not appear to play a major role as an alarm pheromone of this species.

Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant Myrmecia gulosa.

In many species of social insects, the cuticular hydrocarbons of adults vary with both colony identity and individual physiology (oogenesis). Such variations have been shown in some ants and social wasps to function in nestmate recognition, but as yet there is no demonstration of their use by workers to recognize egg layers. We report that in the ant Myrmecia gulosa, workers can discriminate queens and fertile workers from infertile individuals based on distinctive blends of long-chained hydrocarbons present both on the cuticle and in the postpharyngeal gland. The purified hydrocarbon fraction of cuticular extracts from queens elicited high interest in workers, unlike the nonhydrocarbon fraction. However, both fractions were necessary to trigger a response of maximal intensity. In contrast, extracts of mandibular and Dufour glands from queens or infertile workers were not treated differentially by workers. We suggest that cuticular hydrocarbons function as pheromones allowing for recognition of the queen as well as egg-laying workers.