Functional morphology of the Dufour gland in the queenless ant Dinoponera quadriceps

Colonies of the queenless ponerine ant Dinoponera quadriceps are characterized by a social hierarchy among the workers, in which the gamergate occupies the alpha rank. She may be challenged, however, by a beta worker in an attempt to take over reproductive control in the colony. The gamergate does not engage in a direct con�ict, however, but only smears secretion from her Dufour gland onto the beta worker. This secretion then stimulates the low-ranked workers to immobilize the beta worker and thus prevent it from overthrowing the gamergate. Our histological and ultrastructural examination of the Dufour gland in gamergates, one-week-old workers and low-ranked foragers clearly shows that the gamergate has the most developed gland containing an abundant smooth endoplasmic reticulum and an obvious microvillar differentiation of the apical cell membrane. These �ndings are in line with the already existing chemical and behavioural observations of the peculiar pretender punishment in this species.


Introduction
It is a common strategy in crime movies and real-life ma a organizations that the top level criminals do not eliminate their opponents directly by themselves, but rely upon the help of lower accomplices to carry out the dirty work.A comparable approach also exists in some permanently queenless ant species in which a hierarchy among the workers determines which individual(s) will be in charge of heading the colony and producing new offspring.In such species, workers have the potential to mate and after being inseminated become egg-layers that are referred to as gamergates (Peeters 1993).This phenomenon of mated workers is only known in about 100 species among the three ant subfamilies Amblyoponinae, Ectatomminae and Ponerinae (Monnin and Peeters 2008).A well-documented example of a gamergate species is the large Neotropical ponerine Dinoponera quadriceps, that has colonies with on average 80 adult workers, including a single gamergate that occupies the alpha rank in a dominance hierarchy of 3-5 high-ranking workers (Monnin andPeeters 1998, 1999;Monnin et al. 2002).These high-ranking workers, with the highest chance for the beta individual, have the potential to replace the gamergate when she dies.Instead of waiting for the gamergate to die naturally, however, a high-rank worker can increase her inclusive tness by overthrowing the gamergate and produce her own offspring.In case of such con ict between a gamergate and a challenging high-rank worker, short ghts between both ants occur.The gamergate may perform 'sting smearing' against the pretender worker, during which the gamergate smears Dufour gland secretion onto her opponent.This results in the pretender being immobilized by low-ranking workers, which can last for several days, after which the pretender loses her high rank (Monnin et al. 2002).In this way, similar to the crime movies, the gamergate relies on a cooperation with the subordinate workers to eliminate her eventual pretenders.Chemical analysis of the Dufour gland revealed that the hydrocarbon composition in gamergates is clearly different from that in the beta and low-rank workers.Gamergates contain more high molecular mass hydrocarbons than low-rank workers with beta workers having intermediate values.Bioassays in which workers were smeared with Dufour gland secretion moreover showed that the gland chemicals of the gamergate do act as a signal that elicits immobilization (Monnin et al. 2002).
Considering the clear effect of the gamergate's Dufour gland secretion on the pretender immobilization as evidenced both by chemical analysis and behavioural bioassays (Monnin et al. 2002), we aimed to examine the histological and ultrastructural appearance of this gland in gamergates and compare it with high-and low-ranked workers.

Material and methods
Two colonies of Dinoponera quadriceps were excavated near Sambaiba, in Bahia state, Brazil, in October 1994.The collected ants were kept in the laboratory in plaster nests with various chambers.All ants as well as the newly eclosed workers were individually marked with numbers glued onto the thorax in order to record individual behaviour at known age as well as social status.Dufour glands of such individuals from two different colonies (2 gamergates, 4 one-week-old workers, 4 foragers) were dissected and xed in cold 2% glutaraldehyde, buffered at pH 7.3 with 50 mM Na-cacodylate and 150 mM saccharose.
Post xation occurred in 2% osmium tetroxide in the same buffer.Dehydration took place in a graded acetone series, followed by embedding in Araldite and sectioning with a Leica EM UC6 ultramicrotome.
Semithin sections with a thickness of 1 µm were stained with methylene blue and thionin and viewed in an Olympus BX-51 microscope; double stained 70 nm thin sections were examined in a Zeiss EM900 electron microscope.

Results
The Dufour gland appears as a long tubular sac with a central lumen that opens through the sting bulb, ventral to the opening of the venom gland (Billen 1987).Its epithelial wall is formed by class-1 glandular cells (following the gland classi cation by Noirot and Quennedey 1974).Semithin histological sections show a clear difference in the appearance of the gland epithelium in gamergates compared with that of other workers: the gamergate gland displays a thick epithelial with tall columnar cells with a height of up to 80 µm (Fig. 1a) while the epithelium in 1-week-old young (Fig. 1b) and forager workers (Fig. 1c) is much more wrinkled and has a thickness of 25 and 15 µm, respectively.The gland is surrounded by muscle bres, that appear more conspicuous in foragers probably because of the wrinkled condition of the gland (Fig. 1a-c).
Ultrastructural examination illustrates the tall cylindrical cells with basally located round nuclei in gamergates (Fig. 1d) and the wrinkled appearance in the other workers (Fig. 1e,f).At higher magni cation, the gamergate gland is characterized by additional differences.One is the occurrence of a regular border of microvilli with a length of 1 µm that represent the modi ed apical cell membrane (Fig. 1g).The microvilli, however, are much more dispersed in 1-week-old workers (Fig. 1h) or are even absent in old foragers (Fig. 1i).The other difference is the presence of abundant smooth endoplasmic reticulum in gamergates (Fig. 1g), whereas this is much less prominent in the other workers (Fig. 1h,i).

Discussion
Information on the exocrine system of Dinoponera ants is available for morphological descriptions of the (post)pharyngeal gland (Schoeters and Billen 1997), pygidial gland (Billen et al. 1995) and venom gland (Schoeters and Billen 1995).Oldham et al. (1994) performed a chemical analysis of the mandibular gland of all 14 individuals of a colony of D. australis, taking into account their social role in the colony.Although the function of the mandibular gland secretion in Dinoponera is not known, it is interesting that this study revealed that the gamergate contained by far the smallest amount of secretion of all individuals, while the most active forager had the largest with 2,5-dimethyl-3-(3'-methylbutyl)pyrazine as the major component (Oldham et al. 1994).This nding is very different for the cuticular hydrocarbons in D. quadriceps, that are most abundant in the gamergate with the major component 9-hentriacontene acting as a signal for fecundity (Monnin et al. 1998;Peeters et al. 1999).Clear differences also exist in the Dufour gland composition of D. quadriceps, with the gamergate and beta worker having signi cantly more hydrocarbons than low-rank workers, but the gamergate having the highest proportion of highmolecular-mass compounds (Monnin et al. 2002).
Our histological and ultrastructural examination of the Dufour gland con rms the chemical and behavioural differences according to social status as the gamergate gland clearly represents the highest metabolic activity.The abundant presence of smooth endoplasmic reticulum in the gamergate is entirely in line with the production of hydrocarbons, while the well-developed apical microvilli indicate high transportation activity (Billen and Morgan 1998).These features are hardly present in the glands of foragers that belong to the group of low-ranking workers.One-week-old workers show an intermediate development of their Dufour gland, which can be explained by their young age that makes them hopeful workers in terms of reproduction, as they may have the potential to reach the beta rank and then challenge the gamergate.Having a reasonably well-developed Dufour gland therefore can be understood as their being in standby for an eventual increase of their social hierarchy.Our current data thus provides clear morphological support for the known chemical and behavioural role of the Dufour gland in the peculiar pretender punishment in D. quadriceps (Monnin et al. 2002).
The Dufour gland secretion plays a similar role in other ant species.In Aphaenogaster cockerelli it triggers worker aggression against replacement-queen pretenders (Smith et al 2012), and in incipient colonies of Cataglyphis piliscapa it possibly triggers aggression between young queens vying for colony ownership, i.e. when monogyny is restored consecutively after colony foundation (Monnin et al. 2018).Determining whether the Dufour gland of these species shows similar queen/worker differences as observed in D. quadriceps would be interesting, in particular because of the variability of function this gland plays across the ants, e.g. in foraging (Morgan 2009, Witte 2007), alarm (Lenz et al. 2013), appeasement (Billen et al. 2001;Grasso et al. 2005), defense (Billen et al 2009) or production of sex pheromones (Walter et al 1993).

Figure 1 Comparison
Figure 1