MICROSCOPY RESEARCH AND TECHNIQUE 74:763–771 (2011) Morphology and Histochemistry of the Intramandibular Glands in Attini and Ponerini (Hymenoptera, Formicidae) Species 1 2 LUIZA CARLA BARBOSA MARTINS AND JOSE´ EDUARDO SERRA˜ O 1Departamento de Biologia Animal, Universidade Federal de Vic¸osa, Vic¸osa-36570-000, Minas Gerais, Brasil 2Departamento de Biologia Geral, Universidade Federal de Vic¸osa, Vic¸osa-36570-000, Minas Gerais, Brasil KEY WORDS ants; histology; exocrine gland; mandibles; phylogeny ABSTRACT An understanding of the internal morphology of the ant mandible is important in explaining the relationship between the exocrine system and the behavioral and phylogenetic char- acteristics of different subfamilies of Formicidae. In this study, we investigated the occurrence of intramandibular glands in ants from the Ponerini (Ponerinae) and Attini (Myrmicinae). These ants possess glands from classes I and III, and secretory epithelial cells with a reservoir. The intraman- dibular glands show a distinct histology in the studied species, varying in their location, degree of development, and chemical content. Using this information, it is possible to hypothesize that the glands from different tribes produce different substances, which may indicate a variety of functions, depending on the chemical nature of the cellular constituents. A cladistic analysis using the charac- ters of the intramandibular glands separated both tribes, suggesting that structural differences in the intramandibular glands may contribute to future phylogenetic studies of the Formicidae. Microsc. Res. Tech. 74:763–771, 2011. VC 2010 Wiley-Liss, Inc. INTRODUCTION epidermis in the pupa (Cruz-Landim and Abdalla, A common characteristic of social insects is the va- 2002). They were described for the first time in Atta riety of exocrine glands on the various parts of their sexdens rubropilosa ants by Toledo (1967). bodies. The high number and diversity of these Study of the morphology of the intramandibular glands relate to the many important functions that glands in ants can help us to understand the relation- secretions have in the lives of social insects (Billen, ship between the exocrine gland system and the behav- 2008; Billen and Morgan, 1998; Ho¨lldobler and ioral and phylogenetic characteristics of the different Wilson, 1990; Noirot and Quennedey, 1991). At this ant species. This study investigated the occurrence of time, a total of 105 different exocrine glands have intramandibular glands in ants from the tribes Poner- been recognized in the various groups of social ini (Ponerinae) and Attini (Myrmicinae). insects (Billen, 2008). MATERIALS AND METHODS Noirot and Quennedey (1991) classify the epidermal Ants glands of insects into three classes. Class I comprises epidermal cells that take on a secretory function, and Worker castes of the species of Formicidae listed in release compounds to the exterior of the body by diffu- Table 1 were obtained by field collection in the States sion through the cuticle. The class II glands comprise of Minas Gerais and Bahia (Brazil), and transferred to cells which also release secretions to the body surface Zamboni’s fixative solution (Stefanini et al., 1967). through the cuticle, but, in this case, since the cells are Voucher specimens were deposited in the Entomologi- not in contact with the cuticle, the secretion is first cal Regional Museum from Federal University of passed through an epidermal cell. In the class III Vic¸osa (UFVB). glands, the secretory cells usually originate from the epidermis. The secretory cells are usually spherical Histology and Histochemistry and are linked by a canal cell to a pore in the cuticle, The mandibles of three specimens each species anno- where the secretion is released. tated in the Table 1 were removed from the fixed speci- In the Hymenoptera, there are usually two types of mens, dehydrated in a graded ethanol series and em- mandibular glands: (i) the ectomandibular or mandibu- bedded in historesin (Leica). The mandibles were sec- lar glands and (ii) the mesomandibular or intraman- tioned longitudinally in 3-lm slices and stained with dibular glands (Cruz-Landim and Abdalla, 2002). The ectomandibular glands are the best known and stud- *Correspondence to: Jose´ Eduardo Serra˜o, Departamento de Biologia Geral, Universidade Federal de Vic¸osa, Avenida Peter Henry Rolfs, s/n, CEP. 36570- ied, and form the basis our knowledge of the ‘‘mandibu- 000-Vic¸osa, Minas Gerais, Brasil. E-mail: [email protected] lar glands’’ in general, in contrast to the less well Received 29 June 2010; accepted in revised form 20 September 2010 understood intramandibular glands (Cruz-Landim and Contract grant sponsors: SECTI/FAPESB (PNX0011/2009), CNPq and FAPE- Abdalla, 2002). MIG. DOI 10.1002/jemt.20956 The intramandibular glands are classified as tegu- Published online 17 November 2010 in Wiley Online Library (wileyonlinelibrary. mental glands and differentiated with the mandible com). VC 2010 WILEY-LISS, INC. 764 L.C.B. MARTINS AND J.E. SERRA˜ O TABLE 1. List of taxa analyzed and locality Wheeler, 1981) using Camponotus rufipes (Fabricius, Species Collect site 1775) (Formicinae) as the out-group. The characters present in the out-group were consid- Myrmicinae ered as plesiomorphic and coded as (0), and the apo- Attini Acromyrmex subterraneus Paraopeba, Minas Gerais morphic characters as (1) and (2) in the case of non- brunneus (Forel, 1911) ordered multi-state characters. The cladistic analysis Acromyrmex niger (Fr. Smith, 1858) Vic¸osa, Minas Gerais was conducted using the PAUP computer program, ver- Acromyrmex subterraneus Teixeiras, Minas Gerais molestans (Santschi, 1925) sion 4.0b10 (Swofford, 1998) with a heuristic search Atta bisphaerica (Forel, 1908) Teixeiras, Minas Gerais and TBR algorithm. The results were analyzed using Atta laevigata (Fr. Smith, 1858) Teixeiras, Minas Gerais the TreeView program, version 16.6. Atta sexdens rubropilosa (Forel, 1908) Teixeiras, Minas Gerais The evaluated characters of the intramandibular Ponerinae Ponerini glands (Fig. 1) were: Hypoponera sp1. Vic¸osa, Minas Gerais Leptogenys arcuata (Roger, 1861) Ilhe´us, Bahia 1. Type of epithelium: flattened (0), cuboidal (1), and Leptogenys sp1. Ilhe´us, Bahia columnar (2). Odontomachus haematodus Itajuı´pe, Bahia 2. Nucleus size of the epithelial cells: large when nu- (Linnaeus, 1758) > Pachycondyla sp1. Ibicuı´, Bahia cleus/cytoplasm ratio 0.20 (0) and small when nu- Pachycondyla crassinoda Porto Seguro, Bahia cleus/cytoplasm ratio 0.20 (1). (Latreille, 1802) 3. Type III gland: without a cytoplasm vacuole (0), Pachycondyla harpax Itajuı´pe, Bahia highly vacuolated (1), and weakly vacuolated (2). (Fabricius, 1804) Pachycondyla impressa Itajuı´pe, Bahia 4. Size of the nucleus of the type III gland cell: small Roger, 1861 when nucleus/cytoplasm ratio 0.14 (0), large when Pachycondyla stigma Boa Nova, Bahia nucleus/cytoplasm ratio >0.14 (1). (Fabricius, 1804) 5. Glandular reservoir: absent (0) and present (1). Pachycondyla veranae Guaratinga, Bahia (Forel, 1922) 6. Other types of cells: absent (0) and present (1). Pachycondyla villosa Itajuı´pe, Bahia 7. Cytoplasm granules: absent (0) and present (1). (Fabricius, 1804) Formicinae Camponotus rufipes Itajuı´pe, Bahia (Fabricius, 1775) RESULTS The intramandibular glands in the ants studied can hematoxyline and eosin. Some slices of mandible were be divided into three categories: (i) class I glands, char- also tested for histochemistry as follows: Mercury-bro- acterized by cuboidal or columnar epidermal cells; (ii) mophenol blue for protein staining; PAS (Periodic acid- class III unicellular glands, isolated cells in the inter- Schiff) for polysaccharide and glyco-conjugate; and nal cavity of the mandible characterized by the pres- Nile blue for lipid identification, according to Pearse ence of canaliculi that open to pores on the surface of (1985). the mandible; and (iii) secretory epithelial cells with a reservoir, formed by hypertrophy of the epidermal cells Morphometry in specific areas of the mandible and containing a wide Morphometric data on gland cells were obtained reservoir (Fig. 1). Characteristically, the pores of the from 10 sections/ant with aid of the software Image- class III glands occur on the superior surface of the Pro Plus version 4.5 (Media cybernetics). The total mandible (Figs. 2–4). However, the occurrence and area of the cell and the nucleus were obtained and used structure of these three types of gland, varies in the to determine the nucleus/cytoplasm ratio with the for- different ant species, as described below. mula: NCR 5 N/C 2 N, were N is the area of the nu- In the Attini, all of the Acromyrmex and Atta species cleus and C the area of the cell. studied the intermandibular epidermis has flattened cells with epithelial glands with a reservoir (Figs. 5 Scanning Electronic Microscope and 6) and unicellular glands of class III. In Acromyrmex subterraneus brunneus and Acromyr- Ant mandibles were removed, dehydrated in a mex niger, the flattened epithelium of the intramandib- graded ethanol series, transferred to hexamethyldisila- ular epidermis has cells with a smaller nucleus zane (HMDS) for 5 min and air dried. They were then compared with those of A. subterraneus molestans.In glued into aluminum supports, covered with gold (20 all of the Attini species, some regions of the intraman- nm) and observed in a scanning electron microscope, dibular epidermis contain cells that have become LEO VP1430, in the Microscopy and Microanalysis Nu- hypertrophied, forming