Contents lists available at ScienceDirect

Micron

journal homepage: www.elsevier.com/locate/micron

Morphology and ultrastructure of the infrabuccal pocket and its liningepithelium in workers of Ectomomyrmex javanus (Hymenoptera: Formicidae)

Chu Wanga, Johan Billenb, Xingran Pana, Hong Hea,⁎

a College of Forestry, Northwest A&F University, Yangling, 712100, PR Chinab Zoological Institute, KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium

A R T I C L E I N F O

Keywords:Ectomomyrmex javanusInfrabuccal pocketMorphologyUltrastructure

A B S T R A C T

This paper describes the morphology and ultrastructure of the infrabuccal pocket in workers of the carnivorousant Ectomomyrmex javanus, using SEM and TEM. The infrabuccal pocket is a flexible bag with a diameter of330 ± 30 μm. In its anterior part, the pocket wall reaches a thickness of 24.45 ± 3.45 μm, which is thicker thanthe wall lining the rest of the infrabuccal pocket, where it measures 6.87 ± 1.12 μm. The epidermal cells of thewall form a thickened epithelium with a thickness of 10.18 ± 2.50 μm. There are different kinds of hairs insidethe pocket, that help in filtering solid pellets. Literature data on the infrabuccal pocket are limited and thefunction of the thicker epithelium is not yet known. It may provide mechanical strength as the pocket undergoesdaily size changes because of the filling up and spitting out of pellets.

1. Introduction

Ants are among the best known social insects in terrestrial ecosys-tems (Hölldobler and Wilson, 1990). They can feed on both solid andliquid food, although solid particles cannot directly reach the midgutbecause of the complex structure of the proventriculus between thecrop and midgut (Wheeler and Bailey, 1920). To protect the proven-triculus and midgut from being blocked, the infrabuccal pocket acts as aprimary filtering structure that allows only liquids and small particles topass. This infrabuccal pocket is a ventral invagination of the lowerpharyngeal surface (Janet, 1895) with sizable shape that ensures thatlarger particles are gathered and compacted into a pellet. When thepocket is filled up, the pellets are expelled (Eisner and Happ, 1962;Hansen et al., 1999).

In addition, the infrabuccal pocket can prevent the invasion andspreading of microbial parasites in fungus-growing ants (Bailey, 1920;Little et al., 2003). In these Attini ants, the infrabuccal pocket cancollect both general debris and some detrimental pathogenic sporeswhen the workers groom their fungus garden. The pellets are laterexpelled in dump chambers inside or outside the nest.

Ants are characterized by their developed exocrine system withglands distributed in all body parts. Many new glands have been de-scribed recently (Billen, 1993; Billen and Morgan, 1998; Billen, 2009,2015). Eelen et al. (2004) were the first to report on the existence of aninfrabuccal gland in workers and queens of Monomorium pharaonis. The

gland has also been found in Protanilla wallacei (Billen et al., 2013) andMyrmoteras iriodum (Billen et al., 2015), but there is no informationwhether it also occurs in other species.

Ectomomyrmex javanus is a widespread soil-dwelling predatory antin North China and Southeast Asia (Xu, 1998). In this paper, we in-vestigated the morphology and ultrastructure of the infrabuccal pocketand its lining epithelium in workers of E. javanus, which is the first suchreport dealing with a predatory ant. This study forms part of a moregeneral study of the digestive characteristics of this and other Chineseant species (Zhang et al., 2018).

2. Material and methods

Ectomomyrmex javanus (formerly known as Pachycondyla javanaprior to the Ponerinae revision by Schmidt and Shattuck (2014)) is aground-dwelling predatory ant with workers foraging solitarily forprey. Foragers of E. javanus were collected in the campus of NorthwestA&F University, Yangling, Shaanxi province, China. Voucher specimenswere identified by Prof. Zhou Shanyi and deposited in the collection atGuangxi Normal University. The ants were put in a refrigerator for10min to reduce their activity, then they were dissected under a LeicaEZ4HD microscope.

https://doi.org/10.1016/j.micron.2018.09.001Received 6 July 2018; Received in revised form 31 August 2018; Accepted 2 September 2018

⁎ Corresponding author.E-mail addresses: mdjwangchu@126.com (C. Wang), johan.billen@kuleuven.be (J. Billen), xingran@nwafu.edu.cn (X. Pan), hehong@nwsuaf.edu.cn (H. He).

Micron 115 (2018) 50–53

Available online 05 September 20180968-4328/ © 2018 Elsevier Ltd. All rights reserved.

T

2.1. Light microscopy (LM)

The anterior part of the head of 10 workers was cut off in the regionof the compound eyes to allow proper penetration of the various che-micals during the subsequent steps of tissue processing for microscopy.The tissues were fixed in cold 2.5% glutaraldehyde in a Na-phosphatebuffer (100mM, pH 7.2) for 12 h and postfixed in cold 1% osmiumtetroxide for 2 h. After dehydration in a graded ethanol series, thesamples were impregnated in a mixture of ethanol and LR-white beforeembedding in pure LR-white (Zhou et al., 2018). Semithin sections of1 μm were made with a Leica RM2265 ultramicrotome and stained withammonium methylbenzene blue. Observations were made with anOlympus BX43 microscope.

2.2. Transmission electron microscopy (TEM)

For ultrastructural examination, thin sections of 70 nm were double-stained with lead citrate and uranyl acetate and viewed in a HitachiHT7700 transmission electron microscope.

2.3. Scanning electron microscopy (SEM)

The lateral side of the heads of 4 workers was longitudinally cut offwith a surgery knife to study the external appearance of the infrabuccalpocket. From 5 other workers the infrabuccal pockets were dissectedand opened to look at their internal appearance. These tissues werefixed in 2.5% glutaraldehyde, dehydrated in a graded ethanol series andtransferred to isoamyl acetate. The samples were critical-point driedusing an Emitech K850 instrument, mounted on stubs and gold-coatedusing a Hitachi E-1045 sputtering device. The samples were observedand photographed under a Hitachi S4800 scanning electron micro-scope.

3. Results

The infrabuccal pocket is a spherical sac-like extension of the ven-tral pharynx wall that occurs anteriorly in the head, underneath thepharynx (Fig. 1A). The lower dorsal lip of the prepharynx forms aventrally oriented flap that acts as a valve-like dorso-anterior lining ofthe infrabuccal pocket (Fig. 1B). At its ventro-anterior side, an upwardextension of the labium forms the other part of the infrabuccal pocket’sconnection to the prepharynx (Fig. 1B). We did not observe any musclefibres surrounding the pocket (Fig. 1A, B). The maximum diameter ofthe full pocket that we observed is 330 ± 30 μm (Fig. 1A, B), the size ofthe infrabuccal pocket being flexible according to the amount of itsaccumulated solid contents. Our dissections of workers in the morningalways showed them with an empty infrabuccal pocket (n=8 workers,100% empty), whereas the majority of workers dissected in the after-noon had full pockets (n=77 workers, 91% full).

The external surface of the infrabuccal pocket looks like an irregular

lattice (Fig. 2A, C). Regular hairs with hook-like curved terminal tipsand a length of 12.7 ± 2.5 μm are found in the lower lip of the pre-pharynx (Fig. 2B). The internal surface of the infrabuccal pocket isornamented with an imbricate pattern of polygonal scales of 5–10 μm.Each scale displays a number of uniform finger-like denticulate hairsthat are oriented toward the entrance of the pocket (Fig. 3A–C). Thenumber and length of the hairs decrease posteriorly. The finger-likehairs are long and bushy at the entrance of the pocket with a length of7.2 ± 2.5 μm and with a number up to 17 (Fig. 3A, D). More poster-iorly, the length of the hairs becomes less than 1 μm (Fig. 3C, E), whilein the most posterior part, the scales do not show any hairs anymore(Fig. 3A–C, E).

The pocket wall reaches a thickness of 24.45 ± 3.45 μm in itsanterior part, but elsewhere only measures 6.87 ± 1.12 μm. The wallconsists of an epidermal layer covered by three cuticular layers (anelectron-light endocuticle, an exocuticle of intermediate darkness andan electron-dense epicuticle). The epidermis is formed by a single layerof cuboidal cells lining the anterior part of the pocket with a thicknessof 10.18 ± 2.50 μm (Fig. 4A, C), whereas it measures only3.18 ± 0.97 μm in the posterior part of the pocket. The epidermal cellscontain a centrally located oval nucleus of approx. 2.5× 2 μm. Theapical cell membrane is differentiated into an irregular border withshort microvilli. The cytoplasm of the epidermal cells contains somemitochondria and glycogen. The intercellular membrane appears in-terdigitated in the apical part of the epithelium (Fig. 4C). The lumen ofthe infrabuccal pocket contains numerous fungus particles (Fig. 4A).The filtering hairs appear as a tapering continuation of the epicuticle(Fig. 4B).

4. Discussion

Janet (1895) was the first to study the morphology of the infra-buccal pocket with the suggestion that its main function was filteringthe solid food so that it could not enter into the pharynx and thuskeeping the digestive tract unblocked (Bailey, 1920). Eisner and Happ(1962) further verified the filter function through feeding experimentsby using different sized particles in Camponotus pennsylvanicus. Thepocket in addition is used to temporarily store large solid particleswhen liquid food is ingested. In a number of ant species, additionalfunctions have been reported for the infrabuccal pocket, such as a rolein food exchange during trophallaxis, especially in leaf-cutting ants(Bailey, 1920; Eisner and Happ, 1962). A particular function occurs innewly mated attine ant queens, in which the infrabuccal pocket servesas a receptacle for the storage of fungal spores that they will need whenfounding a new colony (Quinlan and Cherrett, 1978). As the labialgland opens anteriorly to the infrabuccal pocket, the ingested food hasalready been impregnated with saliva, so that it can be digested duringthe period it is stored in the pocket due to the enzymatic activity of thesaliva (Febvay and Kermarrec, 1981).

Our observations can be summarized in a number of interpretations

Fig. 1. Semithin longitudinal sectionthrough anterior part of head of E. javanusworker, A shows location of infrabuccalpocket (IBP), with detail in B.B, brain; CL, clypeus; Fh, filter hairs; TE,thickened epithelium of anterior infrabuccalpocket wall; Lb, labium; Li, lower lip pre-pharynx; Ph, pharynx; PPG, postpharyngealgland; ProPG, propharyngeal gland.

C. Wang et al. Micron 115 (2018) 50–53

51

on the functioning of the infrabuccal pocket in E. javanus workers: (1)The size and number of the finger-like denticulate hairs are highestanteriorly in the pocket and decrease towards the posterior part. Thisgradual arrangement allows an efficient filtering mechanism: following

the pathway of food particles inside the pocket as shown in Fig. 5 ofHansen et al. (1999), solid particles pile up in the posterior part of thepocket. The anteriorly pointing hairs are arranged in a direction op-posite to that of the ingested food particles, which accounts for an

Fig. 2. Scanning micrographs of infrabuccalpocket. A. General view of anterior portion oflongitudinally split head showing infrabuccalpocket (IBP), separated from the pharynx (Ph)by the lower lip of prepharynx (Li); B. Detail ofhairs of prepharynx lower lip (framed part inA); C. Folded view of infrabuccal pocketshowing its interior (IS) and exterior surface(ES).

Fig. 3. Scanning micrograps of inside lining of infrabuccal pocket. A–C. Variation of number and length of finger-like hairs on the scales from anterior toposterior; D. Filtering hairs at anterior part of pharynx; E. General view of internal surface of infrabuccal pocket (arrow pointing to the anterior) with pellet (P).

Fig. 4. Ultrastructure of infrabuccal pocketunder TEM. A. Electron micrograph of thick-ened epithelium (TE) in anterior part of infra-buccal pocket and lining cuticular layers (ed,endocuticle; ex, exocuticle; ep, epicuticle).Note presence of fungus (F) in pocket lumen; B.Detail of epicuticular filtering hairs (Fh); C.Cytoplasm of thickened epidermis.bm, basement membrane; FM, folded inter-cellular membrane; gl, glycogen; Mt, mi-tochondria; Mv, microvilli; N, nucleus.

C. Wang et al. Micron 115 (2018) 50–53

52

efficient filtering system. Also the higher number and longer size of thehairs anteriorly contribute in this regard. When the pocket is filled withpellets, they are expelled to make the pocket empty again. This emp-tying happens on a daily basis, as we found workers to have a fullpocket in the afternoon whereas it is always empty in the morning. Suchspitting out of stored pellets has also been documented by Febvay andKermarrec (1981) for Acromyrmex octospinosus. During the filling up ofthe pocket and the spitting out of pellets, the lower lip of the pre-pharynx and the upward extension of the labium act as a dorsal andventral valve, respectively, that controls the opening and closing of thepocket. The liquid portion of the food goes straight to the crop andmidgut through the pharynx and oesophagus without passing throughthe pocket. The absence of muscles surrounding the pocket indicatesthat the emptying of the pocket is achieved by changes in the hemo-lymph pressure in the head. (2) The differentiation of the thickenedepithelium in E. javanus workers only occurs at the anterior part of thepocket. In Monomorium pharaonis, only the epithelium lining the dorsalside of the pocket is thickened and forms a glandular epithelium (Eelenet al., 2004), while in Protanilla wallacei, the entire pocket wall isglandular, with an additional glandular thickening of the pharyngealgland wall (Billen et al., 2013). Our observation of a thicker anteriorepithelium in the infrabuccal pocket of E. javanus may suggest aglandular differentiation. The epithelium is not as conspicuouslythickened as it is in M. pharaonis and P. wallacei, however, and thusmakes a glandular differentiation rather questionable. The relativethickness of the epithelium with regard to the entire pocket wallmoreover is similar for the anterior and posterior portion of the pocket,and therefore does not support a glandular function. As the thickenedepidermal cells in addition do not contain granular endoplasmic re-ticulum, they most likely are not involved in any digestive function,which provides another argument for a non-glandular function. Thethickening may rather form a structural reinforcement of the anteriorportion of the pocket because E. javanus is a carnivorous ant andtherefore the intake of solid food and formation of solid pellets mayneed more specific structural adaptations for the formation and com-pression of pellets. The occurrence of interdigitated cell junctions canbe explained in this regard as it provides mechanical strength for thecompression of such solid pellets. (3) The surface of the lower lip of theprepharynx is also covered by fine filter hairs with a curved distal endthat act as a second filtering device to prevent the passage into thepharynx of any large solid particles that may already have passedthrough the filter of the infrabuccal pocket. (4) Our observations in-dicate the possible presence of fungus in the infrabuccal pocket. Itsappearance resembles the yeast Wickerhamiella sp. that has alreadybeen found in the infrabuccal pocket of Camponotus japonicus (Zhanget.al., 2018). Mankowski and Morrell (2004) described Debaryomycespolymorphus as the main yeast in the infrabuccal pocket of C. vicinus,while Hansen et al. (1999) found only one yeast in the infrabuccalpocket of C. modoc, with bacteria as the dominant microorganisms. Theexact role of these microorganisms in the infrabuccal pocket is not yetknown, but is most likely related with the digestion and processing ofthe food the ants have ingested.

Acknowledgments

We are grateful to Prof. Zhou Shanyi for the identification of the antspecies studied in this paper. This work was supported by the NationalNatural Science Foundation of China (Grant No. 31070342, 31570388),Forestry Industry Research Special Funds for Public Welfare Projects(Grant No. 201404302-4) and International Science and TechnologyCooperation Project of Northwest A&F University (2016, 2018).

References

Bailey, I.W., 1920. Some relations between ants and fungi. Ecology 1, 174–189.Billen, J., 1993. Morphology of the exocrine system in ants. Kipyatkov, V.E. (Ed.),

Proceedings of the Colloquia on Social Insects, 2nd Colloquium of the Russian-Speaking Section (IUSSI) 1–15.

Billen, J., 2009. Diversity and morphology of exocrine glands in ants. Proceedings of theXIX Symposium of Mirmecologia 1–6.

Billen, J., 2015. Functional morphology and diversity of exocrine glands in ants. Dias,R.K.S., Majer, J. (Eds.), 10th ANeT International Conference, 23–26 October 201513–20.

Billen, J., Morgan, E.D., 1998. Pheromone communication in social insects: sources andsecretions. In: Vander Meer, R.K., Breed, M.D., Espelie, K.E., Winston, M.L. (Eds.),Pheromone Communication in Social Insects: Ants, Wasps, Bees, and Termites.Westview Press, Boulder Colorado, pp. 3–33.

Billen, J., Bauweleers, E., Hashim, R., Ito, F., 2013. Survey of the exocrine system inProtanilla wallacei (Hymenoptera, Formicidae). Arthropod Struct. Dev. 42, 173–183.

Billen, J., Mandonx, T., Hashim, R., Ito, F., 2015. Exocrine glands of the ant Myrmoterasiriodum. Entomol. Sci. 18, 167–173.

Eelen, D., Børgesen, L.W., Billen, J., 2004. Morphology of a novel glandular epitheliumlining the infrabuccal cavity in the ant Monomorium pharaonis (Hymenoptera,Formicidae). Arthropod Struct. Dev. 33 471-451.

Eisner, T., Happ, G.M., 1962. The infrabuccal pocket of a formicine ant: a social filtrationdevice. Psyche 69, 107–116.

Febvay, G., Kermarrec, A., 1981. Morphologie et fonctionnement du filtre infrabuccalchez une attine Acromyrmex octospinosus (Reich) (Hymenoptera: Formicidae): Role dela poche infrabuccale. Int. J. Insect Morphol. Embryol. 10, 441–449.

Hansen, L.D., Spangenberg, W.J., Gaver, M.M., 1999. The infrabuccal chamber ofCamponotus modoc (Hymenoptera: Formicidae): ingestion, digestion, and survey ofbacteria. Robinson, W.H., Rettich, F., Rambo, G.W. (Eds.), Proceedings of the 3rdInternational Conference on Urban Pests 211–219.

Hölldobler, B., Wilson, E.O., 1990. The Ants. Harvard University Press, Cambridge, Mass.Janet, C., 1895. Études sur les fourmis, les guêpes et les abeilles. Note 9: Sur Vespa crabro

L. Histoire d’un nid depuis son origine. Mém. Soc. Zool. France 8, 1–140.Little, A.E., Murakami, T., Mueller, U.G., Currie, C.R., 2003. The infrabuccal pellet piles of

fungus-growing ants. Naturwissenschaften 90, 558–562.Mankowski, M.E., Morrell, J.J., 2004. Yeasts associated with the infrabuccal pocket and

colonies of the carpenter ant Camponotus vicinus. Mycologia 96, 226–231.Quinlan, R.J., Cherrett, J.M., 1978. Studies on the role of the infrabuccal pocket of the

leaf-cutting ant Acromyrmex octospinosus (Reich) (Hym., Formicidae). Insectes Soc.25, 237–245.

Schmidt, C.A., Shattuck, S.O., 2014. The higher classification of the ant subfamilyPonerinae (Hymenoptera: Formicidae), with a review of ponerine ecology and be-havior. Zootaxa 3817, 1–242.

Wheeler, W.M., Bailey, I.W., 1920. The feeding habits of pseudomyrmine and other ants.Trans. Am. Philos. Soc. 22, 235–279.

Xu, Z.H., 1998. Description of Pachycondyla ants in China. J. Southwest For. Coll. 18,209–220.

Zhang, K.X., Wei, C., Nan, X.N., Wang, Y.G., He, H., 2018. Composition and diversity ofmicrobes in the infrabuccal pocket of Camponotus japonicus (Hymenoptera:Formicidae). Acta Entomol. Sin. 61, 686–697.

Zhou, Y., Li, C.Y., Billen, J., He, H., 2018. Morphology and ultrastructure of Dufour’s andvenom glands in the ant Camponotus japonicus Mayr (Hymenoptera: Formicidae).Micron 104, 72–78.

C. Wang et al. Micron 115 (2018) 50–53

53