Zoologica Scripta, Vol. 17, No. 3, pp. 293-295,1988 0300-3256188 $3.00 + .OO Printed in Great Britain Pergamon Press plc 01988 The Norwegian Academy of Science and Letters The crenellate lining of the Dufour gland in the genus Aenictus: a new character for interpreting the phylogeny of Old World army ants (Hymenoptera, Formicidae, Dorylinae)

JOHAN P. J. BILLEN and WILLIAM H. GOTWALD JR

Accepted 7 July 1987

Billen, J. P. J. & Gotwald, W. H. Jr. 1988. The crenellate lining of the Dufour gland in the genus Aenictus: a new character for interpreting the phylogeny of Old World army ants (Hymenoptera, Formicidae, Dorylinae) .-Zool. Scr. 17: 293-295.

The Dufour gland epithelium in Aenictus has a crenellate appearance, a condition previously found only in the primarily African genus Dorylus. This character, taken alone, strongly suggests that these two genera share a common ancestry, as is presently reflected in their placement in the subfamily Dorylinae, and that an independent, convergent origin for them, as has been proposed in the triphyletic hypothesis for army ant evolution, is incorrect.

Johan P. J. Billen, Limburgs Universitair Centrum, Department SBM, B-3610 Diepenbeek, Belgium. Address for correspondence: Zoological Institute, University of Leuven, Naamsestraat 59, B-3000 Leuven, Belgium. William H. Gotwald Jr, Utica College of Syracuse University, Department of Biology, Utica, NY 13502. U.S.A.

Introduction Malaysia (Borneo), in June 1985 by one of us (WHG). Their posterior abdominal halves were fixed for 2-4 h in 2% glutaraldehyde in 50 mM sodium cacodylate buffer, pH 7.3, containing 150 mM saccharose. Army ants are characterized by their distinct behaviour of Tissues were kept in buffer solution until arrival in Belgium, where they nomadism and group predation (Wilson 1958) and by were post-fixed for 1 h in 2% osmium tetroxide in the same buffer. After their pantropical distribution. Two major groups can be dehydration in an acetone series, tissues were embedded in Araldite. Double stained thin sections were examined with a Philips EM 400 clearly distinguished on the basis of zoogeographical, electron microscope. morphological and ethological characteristics: the Ecitoninae in the New World and the Dorylinae in the Old World tropics (Gotwald 1982; Snelling 1979). The Results Dorylinae comprise two tribes, with the Dorylini (single genus Dorylus Fabricius, 1793) mainly confined to Africa The morphology of the Dufour gland is similar in the and the Aenictini (single genus Aenictus Shuckard, 1840) workers of all three Aenictus species investigated. The best represented in tropical Asia. There are considerable gland is a small, pyriform sac that opens in the sting base differences, however, between the members of both through a slit-like duct, ventral to the opening of the tribes, especially in their external morphology. Based on poison gland duct. As in other ants, the duct of each of these differences and on the geological interpretation of these sting glands has its own, independent muscular their actual geographical distribution the hypothesis of a supply. In the Dufour gland muscle fibres insert onto both triphyletic origin for the army ants has been advanced the dorsal and ventral duct wall. The poison gland duct, (Gotwald 1979). In this regard, both doryline groups have on the other hand, only shows muscular insertion onto its been believed to have arisen independently from each ventral side, while the dorsal wall displays a much other and independently from the Neotropical thickened, rigid cuticular lining (Fig. 1). The ultrastruc- Ecitoninae. tural organization in the muscular attachment region Our present paper is based on the recent discovery that shows the occurrence within the duct cells of bundles of the Dufour gland morphology represents a valuable microtubules that form the structural link between the character to distinguish between the Ecitoninae and the adhering myofilaments and the overlying cuticle, which African Dorylini (Billen 1985). This stimulated us to has a thickness of approx. 1 pm (Fig. 2). investigate the Dufour gland in Aenictus and to compare The secretory part of the Dufour gland is comprised of its morphology with that of Dorylus. a glandular epithelium that also lines the central reservoir space (Figs. 1, 3). The epithelial thickness is fairly con- stant, though it may vary from 4 to 10 pm between Material and methods individuals. Rounded or slightly ovoid nuclei occupy a basal position in the cells and have a diameter of between Worker individuals of Aenictus dentatus Forel, 1911 (Utica College COIL no. SAC-002), A. laeviceps (Fr. Smith, 1858) (SAC-009) and Aenictus 3 and 5 pm. The cytoplasm is characterized by a moderate sp. (SAC-013) were collected at Bako National Park, Sarawak, East network of smooth endoplasmic reticulum, relatively 293 Zoologica Scripta 17 294 J. P. J. Billen & W. H. Gotwald Jr

Figs. I-5.-1-3. Aenictus dentatus.-I. Longitudinal section showing the Dufour gland (Dg)and the poison gland duct (Pd).DdDufour gland duct; MFmuscle fibres.-2. Detail of the ventral wall of the Dufour gland duct, with intracellular bundles of microtubules (MT)and adhering muscle fibres (MF).ct cuticle.-3. Dufour gland epithelium. Note crenellate apical border and intercellular junctions mostly coinciding with intercrenellar tops (arrows). MF muscle fibres; M mitochondria; N nucleus.4. Crenellate Dufour gland epithelium in Doryfus nigricans.-5. Dufour gland epithelium of uniform thickness in Eciton burcheffi.Figures 4 and 5 from Billen (1986).

numerous mitochondria in the basal region and some- junction nearly always coinciding with an intercrenellar times some small electron-dense inclusions. A few elec- top (Figs. 1,3). As a result, the majority of the cells show tron-lucid vacuoles with a diameter of nearly 1 pm are an apical depression that corresponds with the narrow randomly dispersed (Fig. 3). crenel between adjacent tops. The epithelium has a crenellate apical border. The The apical cell membrane displays a simple topography lateral cell membranes are very much folded in the upper and closely adjoins the overlying cuticle, which has a cell half, with the most apical part of this intercellular uniform thickness between 0.20 and 0.25 pm (45 times Zoologica Scripta 17 Dufour gland morphology in Aenictus army ants 295 thinner than in the duct region). The basal plasmalemma between the glands in Aenictus and Dorylus suggests a shows only a very few invaginations. The gland is sur- common origin for these two genera. This evidence sup- rounded by a thin basement membrane (around 50 nm ports the current placement of both genera in the subfam- thick) and a few rather thin muscle fibres (Fig. 3). ily Dorylinae (see Snelling 1979; Gotwald 1982), implying The Dufour gland epithelium in Dorylini and a common ancestry for all of the Old World army ants, a Ecitoninae is shown for comparison (Billen 1986). Species group that furthermore arose independently of the New of Dorylus (and the subgenus Anomma)have a crenellate World army ants (subfamily Ecitoninae). epithelium, with a very well defined sequence of crenel The crenellate condition of the epithelial lining in both tops and depressions (Fig. 4). In Eciton, and later con- Old World genera does not support the triphyletic firmed in Labidus and Neivamyrmex, the entirely differ- hypothesis for the army ants proposed by Gotwald (1979) ent epithelium has a very uniform thickness and is charac- and supported by recent research on gastral exocrine terized by a basal accumulation of lateral cell membrane glands by Jensen (1986). Although the ancestral relation- foldings (Fig. 5). ships of the Old World army ants remain unclear, the results of this study imply a monophyletic origin.

Discussion Acknowledgements

AS in other ants, the Dufour gland in Aenictus is formed We are grateful to EIS Plaum for technical assistance in sample prep- by a reservoir sac, lined with a secretory epithelium, and aration for electron microscopy. JPJB thanks the Belgian National Fund for Scientific Research for a senior research assistantship. The research a slit-like duct. The latter contains an extensive muscular contributions of WHG were supported by National Science Foundation apparatus that allows the Dufour gland to discharge its (U.S.A.) grant BSR-8403385. secretion independently from the poison gland (Billen 1982, 1986). The cytoplasmic organization of the glandu- lar cells corresponds with that of the Dufour gland in References other ant species and is in accordance with the production Billen, J. 1982. The Dufour gland closing apparatus in Formica san- Of low weight lipophilic substances k guinea Latreille (Hymenoptera, Formicidae).-Zoomorphology 99: Quennedey 1974; Billen 1986). No information is avail- 235-244. able on the chemical composition of the Dufour gland in Billen, J. 1985. Comparative ultrastructure of the poison and Dufour glands in Old and New World army ants (Hymenoptera, For- Aenictus nor on its function. It is probably involved in micidae).-Actes Coil. lnsectes Soc. 2: 17-26. secretion of hydrocarbons with an eventual pheromonal Billen, J. 1986. Comparative morphology and ultrastructure of the function, as is common among the Formicidae (Blum & Dufour gland in ants (Hymenoptera, Formicidae).-Ent. gen. 11: 165-181. Hermann 1978). Blum, M. S. & Hermann, H. R. 1978. Venoms and venom apparatuses The most obvious character of the gland in Aenictus is of the Formicidae: Myrmeciinae, Ponerinae, Dorylinae, Pseudomyr- the crenellate aspect of its epithelial lining and the coinci- mecinae, Myrmicinae, and Formicinae. In Arthropod venoms (ed. S. Bettini): 801-869. Springer Verlag, Berlin. dence of intercellular junctions with the intercrenellar Jessen, K. 1987. Gastral exocrine glands in ants-functional and sys- tops. This particular condition is all the more remarkable, tematical aspects. In Chemistry and biology of social insects (eds J. because it is most similar to that of the Dufour gland in the Eder & H. Rembold): 445446. J. Peperny Verlag, Miinchen. Gotwald, W. H. 1979. Phylogenetic implications of army ant zoogeog- other Old World army ant genus Dorylus. No other ant raphy (Hymenoptera: Formicidae).-Ann. ent. Soc. Am. 72: 462- species investigated thus far shows a comparable epi- 467. thelial type, as was revealed by ultrastructural examin- Gotwald, W. H. 1982. Army ants. In Social insects 4 (ed. H. R. Hermann): 157-254. Academic Press, New York. ation of 60 species representing the 8 major subfamilies Noirot, C. & Quennedey, A. 1974. Fine structure of insect epidermal among the Formicidae (Billen 1986). glands.-A. Rev. Enf. 19: 61-80. Given the Dufour gland’s apparent systematic impor- Snelling, R. R. 1982. Systematics of social Hymenoptera. In Social insects2 (ed. H. R.Hermann): 369453. Academic Press, New York. tance in the ants in general (Billen 1986), and the army Wilson, E. 0. 1958. The beginnings of nomadic and group-predatory ants in particular (Billen 1985), the striking similarity behavior in the ponerine ants.-Evolution 12: 24-31.

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