Ultramorphology and histology of the larval salivary gland of Pachycondyla villosa (Fabricius) (Hymenoptera: Formicidae, Ponerinae)

Utramorfologia e histologia da glândula salivar larval de Pachycondyla villosa (Fabricius) (Hymenoptera: Formicidae, Ponerinae)

SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY

Ultramorphology and histology of the larval salivary gland of Pachycondyla villosa (Fabricius) (Hymenoptera: Formicidae, Ponerinae)

Utramorfologia e histologia da glândula salivar larval de Pachycondyla villosa (Fabricius) (Hymenoptera: Formicidae, Ponerinae)

Fernando J. Zara^I; Flávio H. Caetano^II

^IFaculdade de Ciências Matemáticas, da Natureza e Técnologia da Informação, Universidade Metodista de Piracicaba - UNIMEP (Campus Taquaral), Rod. do Açúcar, km 156, 13400-911, Piracicaba, SP

^IIDepto. Biologia, Instituto de Biociências, Universidade Estadual Paulista - UNESP, C. postal 199, 13506-900 Rio Claro, SP

ABSTRACT

The salivary gland possesses a single anterior duct with a columnar epithelium, two lateral ducts with simple cubical epithelium, which becomes columnar in the proximal portion, and two thin-walled reservoirs, from which two secretory regions arise. The dorsal and ventral branches of the secretory regions are connected via transverse commissures and ends in a loop. The secretory region is uniform and consists on a simple cubical epithelium. This paper also presents a review on the morphology of the salivary glands in insects, mainly in Hymenoptera-Aculeata.

Key words: Insect, ant, SEM, morphology

RESUMO

A glândula salivar apresenta-se com um duto anterior único, formado por um epitélio colunar, dois dutos laterais curtos, os quais apresentam-se com epitélio cúbico simples e que na sua porção mais proximal torna-se colunar. Posterior a estes, encontram-se os dois reservatórios, os quais possuem o epitélio bastante delgado e é neste reservatório que a região secretora da glândula se abre. Os ramos dorsal e ventral da região secretora da glândula conectam-se por meio de comissuras transversais, sendo que, posteriormente, a região secretora termina em forma de "alça". A região secretora é uniforme, não apresenta tipos celulares distintos e é formada por um epitélio cúbico simples. Neste trabalho é apresentada, também, a revisão sobre a morfologia da glândula salivar larval em insetos, principalmente com relação aos Hymenoptera-Aculeata.

Palavras-chave: Insecta, formiga, MEV, morfologia

The ant Pachycondyla villosa (Fabricius) is restricted to the Neotropical areas, and occurs from central Mexico to Northern Argentina (Kempf 1972). The ants usually construct their nests on trees and in some bromeliads of the species Aechmea bromeliifolia.

P. villosa presents four larval instars and has no exclusive components which could act as pheromone, detected by gas chromatography (Zara & Caetano 2001, Zara et al. 2002). Previous papers on the morphology of ant larval salivary glands indicate that some species possess reservoir and one or two secretory tubes on each side of the body. (Wheeler 1910, Athias-Henriot 1947, Gama 1985).

According to Petralia & Vinson (1980) and Petralia & Haut (1986), ant larval salivary glands may be involved in silk production and/or digestion. Digestive enzyme activity has been detected in the larval salivary glands of the ant, Acromyrmex octospinosus (Reich) (Febvay & Kermarrec 1981). In Solenopsis invicta (Buren), the proteolitical activity was detected and this activity can indicate digestive enzymes production (Petralia et al. 1980). Apart from this digestive function, Ponerinae ants as well as members of other subfamilies, cease feeding at the end of the last instar and begin to produce silk for their cocoon (Wheeler & Wheeler 1976).

Several papers have been published on the histology of Hymenoptera larvae salivary glands mostly concerning bees (Nelson 1924, Cruz-Landim & Mello 1981) and wasps (Edwards 1980) but only a few dealing with ants (Valentini 1951, Lappano 1958, Ofer 1970). None of those authors, however, worked with any member of the Ponerinae. In those papers a great variation on the Hymenoptera salivary glands morphology was described. However, these variations were not compared one to another (or among each other). In this paper, the ultramorphology and histology of salivary glands at the last larval instar of the P. villosa is described and a review on the morphology of the salivary glands of insects, mainly in Hymenoptera-Aculeata, is presented.

Materials and Methods

The Insects and Instars. One bromeliad of the genus Aechmea, where the ants had nested, was collected in the gardens of the UNESP (Univesidade Estadual de São Paulo), campus of Rio Claro County, (22°24'36''S; 47°33'36''W, 612 m average altitude). The larvae were separated and anaesthetized by thermal shock (3 to 5 min. at 4°C) for head capsule measurement. The last larval instar was determined according to Zara (1995) and Zara & Caetano (2001), which is based on the head capsule size.

Ultramorphology. For the scanning electron microscopy, salivary glands were removed and fixed in 4% paraformaldehyde and 4% glutaraldehyde, for 24h, dehydrated in an ascending ethanol series (70% to 100%), subjected to two15-min. baths each with 100% acetone and then critically pointed dried (Balzers CPD 030) using 100% acetone. After dehydration the material was placed on aluminium supports using double-sided sticky-tape and sputter coated with gold (Balzers SD 050 sputtering). The salivary glands were examined in Jeol P15 SEM.

Histology.P. villosa larvae were fixed directly in 4% paraformaldehyde in a 0.1M phosphate buffer (pH 7.2). The material was then dehydrated in 70%, 80%, 90% and 95% ethanol solutions for 20 min. and transferred to a resin solution (JB4 - Polaron Instruments/Bio Rad) for 72h at 4°C. Finally the material was transferred to resin-filled moulds containing a catalyst. Sections were stained with hematoxiline/eosine, according to routine procedure, or stained using the mercuric-bromophenol blue reaction to detect protein (Lison 1960, Gabe 1976).

Results

Ultramorphology. The salivary gland of the last instar of P. villosa consists of a single anterior duct, beginning at the labium, followed by two short lateral ducts, leading into two flattened reservoirs lateral to the digestive system. Reservoirs are followed by two secretory regions with a very convoluted appearance (Figs. 1 and 2). The region of the anterior duct presents a smoother surface in comparison with the lateral ducts, as well as a flatter appearance (Fig. 3). The reservoir region has a distinct morphology: flatter and wider in comparison with the preceding region with a lobular surface (Fig. 4). The differences between the two types of surfaces are apparent in the transition region between the reservoir and lateral ducts: the reservoir consisting of large trapezoidal cells, whist the ducts consist of smaller pentagonal cells (Fig. 4, arrows). When the reservoir is swollen with secretion, its cells resemble those of the lateral ducts, albeit being larger and pentagonal (Figs. 2 and ⁵).

Each secretory region presents two secretory tubes (dorsal and ventral), distally linked, forming a loop, each previously entering its respective reservoir (Fig. 2). The secretory region has a smooth surface and includes three transverse commissures connecting the dorsal and ventral tubes (Figs. 2 and ⁶). When the gland was split at several points, fibrous material was found in the lumen (Figs. 7).

Many tracheal tubes were present on the epithelium of the secretory region, reservoirs and anterior duct (Figs. 3, ⁵ and ⁶).

Histology. The reservoirs are located in the mid to fore region of the body, while the secretory region is found in the mid to rear part (Fig. 8). The anterior duct under the sub-oesophageal ganglion has a columnar epithelium, the cells of which have large elliptic nuclei and the basal region cells show various vacuoles (Figs. 9 and 10). The lumen is lined by a thin cuticle and gives a positive reaction to the mercuric-bromophenol blue test (Fig. 10). The distal portion of the lateral ducts has simple cubical epithelium, with large rounded nuclei. The cells also contain vacuoles in the basal region and have a very distinct brush border; no cuticle was found (Fig.13). The lateral ducts run along side the oesophagus, only withdrawing from it at the level where the anterior duct is formed, near the sub-oesophageal ganglion (Figs. 11 and 13). In the proximal portion of the lateral duct a mercuric-bromophenol blue positive secretion is present (Figs. 10 to 12), and the duct shows the same characteristics as in the anterior duct, including the presence of cuticle (Fig.12). The reservoirs have a very thin epithelium (Fig.14), with flattened cells, nuclei and cytoplasm containing several vacuoles (Fig. 15). In the lumen there is also evidence of mercuric-bromophenol blue positive material (Fig. 15) The secretory region has a simple cubical epithelium, with rounded or oval nuclei. The cytoplasm contains some vacuole and fibrous material can be observed in the lumen (Fig. 16), which also gives a positive reaction in the mercuric-bromophenol blue test. No histological differences were observed, either between the distal and proximal portions of the secretory region, or between these and the transverse commissures or the dorsal and ventral branches of the secretory region.

Discussion

Scanning electron microscopy allowed a more accurate observation of larval salivary gland morphology, in comparison with that obtained by the photo-stereomicroscopic system. The larval salivary glands of P. villosa have been found to differ from those described for Diptera larvae (Phillips & Swift 1965, Harrod & Kastritsis 1972, Riparbelli et al. 1993, Riparbelli et al. 1994), Lepidoptera (Cruz-Landim 1973, Wiley & Lai-Fook 1974, Akai 1983, Sorour et al. 1990, Victoriano 1998) and for those of other Hymenoptera - Aculeata, such as bees of the Apis genera (Nelson 1924, Cruz-Landim & Mello 1969), Melipona (Silva de Moraes 1972, Silva de Moraes & Cruz-Landim 1979) and Scaptotrigona (Cruz-Landim & Mello 1981) in that they possess a reservoir and have two secretory tubes, ending in a "blunt bottom", at each side of the body, therefore not ending in a "loop". Compared to the wasps Vespula germanica L. (Edwards 1980), Pseudopolybia vespiceps (de Saussure), Mischocyttarus atramentarius Zikan, Polistes versicolor Oliver and Occipitalia traili (Cameron.) (Caetano, unpublished data), the gland differs in that it has a reservoir, short lateral ducts, joints and by not ending in a blunt bottom. In the ant larvae, this gland differs by having two secretory tubes at each side of the body instead of a single one, as in Plagiolepis crosi (Sant.), Camponotus compressus (Fabr.), (Athia-Henriot 1947) Tapinoma sirothi Krausse, Pheidole pallidula (Nyl.) (Valentini 1951) and ending in "blunt bottom "as described for Camponotus rufipes Fabr. (Gama 1985), C. pennsylvanicus (Degeer.), Formica pallidefulva Smith. (Petrallia & Haut 1986), Camponotus silvaticus (Oliver) Monomorium ajier Bernard, and many other species described by Valentini (1951), Messor rufo-testaceus (Forster) and M. aegyptiacus (Em.) (Athias-Henriot 1947), Eciton burchelli Westw. (Lappano 1958), Neivamyrmex nigriscens (Cresson) (Wang & Happ 1974) and Formica pratensis males Retz (Emmert 1968). Morphological data of larval salivary gland for different subfamilies of ants are presented in Table 1.

Apart from these differences, P. villosa salivary glands have transverse commissures in the secretory region, not seen in any of the aforementioned species. Larvae of Bombus spp and B. attratus Franklin do have many secretory tubes on either side of the body, with short transverse commissures so that they anastomose across the middle, but in these species, they do not end in a "loop" (Flower & Kenchington 1967, Mello & Vidal 1979).

The comparative morphology of salivary glands for Hymenoptera - Aculeata is summarized in the Table 2. When compared to Hymenoptera - Symphyta salivary glands, the differences become more pronounced. In these insects, the salivary glands present isolated or bunched secretory units, linked to the reservoir epithelium by a secretory channel. The tubular reservoir has a single ramification at each side of the body in Neurotoma and Cladius. However, in Celphus cinctus Norton the tubular reservoirs are compound by two tubes, one shorter than the other (Kenchington 1972). The lumen of the P. villosa salivary glands secretory region presents fibrous material scattered along its entire length. In this respect it differs from the silkworm Bombyx mori L. (Akai 1983, Akai et al.1987) in which the silk protein is found in the lumen in spherical bodies, and only assumes a fibber shape when passing through the spinneret. The histology of anterior duct epithelium in P. villosa resembles that described for other species of ants, consisting of a columnar epithelium lined by cuticle (Valentini 1951, Lappano 1958, Emmert 1968). The denomination given to this salivary gland region varies widely, being described as a common duct (Lappano 1958, Emmert 1968), "canal impair" (Valentini 1951), "tube impair" (Athias-Henriot 1947) and final excretory duct (Gama 1985). The lateral ducts, also called "labial ducts" (Lappano 1958; Wang & Happ 1974), "canal d'écoulement lateral" (Athias-Henriot 1947) and "paired duct" (Emmert 1968, Petralia & Vinson 1980), possess a varied morphology, with the distal portion consisting of rounded nuclei cubical cells and the apex presenting a brush border without the presence of cuticle. The proximal region of the lateral ducts shows cells resembling those in the single anterior duct, the presence of cuticle also being observed. Within the larvae, the salivary glands are found near the oesophagus, the joining of the lateral ducts occurring between the sub-oesophageal ganglion and the oesophagus. This arrangement differs from that described for S. invicta (Petralia & Vinson 1980), which occurs between the sub-oesophageal ganglion and the protothoraxic ganglion, and for C. rufipes (Gama 1985), occurring at the last protothoraxic ganglion.

The pair of reservoirs strongly resemble the description of C. pennsylvanicus (Petralia & Haut 1986), C. rufipes (Gama 1985), S. invicta (Petralia & Vinson 1980, Petralia et al. 1980) Pheidole pallidula (Valentini 1951), Paedaulgus termitoletes (Forel) (Wheeler 1910), in which the epithelium is thin, consisting of flat cells with oval nuclei, containing many heterochromatine blocks. The reservoirs are quite ample and Wheeler (1918) speculates that they could function as food stores, to be provided to adults. This function, however, cannot be assigned to P. villosa reservoirs, needing experimental confirmation for Ponerinae (Hölldobler 1985, Hölldobler & Wilson 1990). The secretory region presents a simple cubical epithelium, its diameter consisting of four to five cells. The basal regions of the cells are very vacuolated, resembling that observed in C. rufipes, and this could indicate a capture of substances from the hemolymph to form glandular secretions (Terra & Bianchi 1975, Gama 1985). This type of epithelium seems to be the most common in Hymenoptera larval salivary glands, as observed in Melipona quadrifasciata (Lep.) (Mello & Vidal 1971), Scaptotrigona postica Latreille (Cruz-Landim & Mello 1981), E. burchelli (Lappano 1958) N. nigriscens (Wang & Happ 1974), C. rufipes (Gama 1985), S. invicta (Petralia & Vinson 1980), C. pennsylvanicus (Petralia & Haut 1986).

The presence of fibrous secretion can be observed in the secretory region, thus secretion related to the silk produced for building the cocoon, and the occurrence of this kind of secretion is also reported for C. rufipes (Gama 1985), Polirhachis simplex (Forel) (Ofer 1970). Besides the silk, the salivary system can also produce digestive enzymes, as suggested for E. burchelli (Lappano 1958) or for the Myrmicinae Acromyrmex octospinosus (Febvay & Kermarrec 1981) and S. invicta (Petralia et al. 1980). The production of digestive enzymes in this region of the gland may also be occurring in P. villosa, where the presence of small vesicles (microapocrine secretion) in the lumen was observed in larvae at the beginning of the last instar by transmission electron microscopy (Zara, unpublish data). Only a single cell type was identified in the secretory region, which agrees with the observations on Apis (Nelson 1924), M. quadrifasciata (Mello & Vidal 1971, Silva de Moraes & Cruz-Landim 1979), S. postica (Cruz-Landim & Mello 1981) and S. invicta (Petralia et al. 1980). The presence of protein identified by the mercuric-bromophenol blue test was observed in all regions of the gland, and a similar reaction was observed in S. invicta (Petralia et al. 1980). In summary, the salivary glands of the last larval instar of P. villosa differs from that observed in the majority of Hymenoptera currently studied, mainly because of their loop shape, three transverse commissures, and presence of mercuric-bromophenol blue positive fibrous material, probably silk, in the lumen.

Acknowledgments

This research work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) proc. 98/00576-8.

Literature Cited

Received 10/01/02

Accepted 25/09/02

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Publication Dates

• Publication in this collection
  03 June 2003
• Date of issue
  Jan 2003

History

• Accepted
  25 Sept 2002
• Received
  10 Jan 2002