Article Morphology of the Novel Basimandibular Gland in the (, Formicidae)

Chu Wang 1,* , Michael Steenhuyse-Vandevelde 1, Chung-Chi Lin 2 and Johan Billen 1

1 Zoological Institute, University of Leuven, Naamsestraat 59, Box 2466, B-3000 Leuven, Belgium; [email protected] (M.S.-V.); [email protected] (J.B.) 2 Department of Biology, National Changhua University of Education, Changhua 50007, Taiwan; [email protected] * Correspondence: [email protected]

Simple Summary: form a diverse group of social insects that are characterized by an over- whelming variety of exocrine glands, that play a key function in the communication system and social organization of the colony. Our focus goes to the genus Strumigenys, that comprise small slow-moving ants that mainly prey on . We discovered a novel gland inside the mandibles of all 22 investigated , using light and electron microscopy. As the gland occurs close to the base of the mandibles, we name it ‘basimandibular gland’ according to the putative description given to this mandible region in a publication by the eminent British ant taxonomist Barry Bolton in 1999. The gland exists in both workers and queens and appeared most developed in the queens of Strumigenys mutica. These queens in addition to the basimandibular gland also have a cluster of gland cells near the tip of their mandibles. The queens of this species enter colonies of other Strumigenys species and parasitize on them. We expect that the peculiar development of these glands inside the mandibles of these S. mutica queens plays a role in this parasitic lifestyle, and hope that future   research can shed more light on the biology of these ants.

Citation: Wang, C.; Steenhuyse- Abstract: In 1999, Barry Bolton postulated the presence of a basimandibular gland in the mandibles Vandevelde, M.; Lin, C.-C.; Billen, J. in all species of the ant genus Strumigenys, solely based on scanning microscopy observations. We Morphology of the Novel now confirm the presence of this putative gland in the proximal outer part of the mandibles of Basimandibular Gland in the Ant 22 investigated species by histological and ultrastructural examination, including 10 short- and Genus Strumigenys (Hymenoptera, 12 long-mandibulate species. All species have a basimandibular gland, that is formed by 15–25 µm Formicidae). Insects 2021, 12, 50. https://doi.org/10.3390/insects thick epithelial cells and belongs to class-1 following the standard classification of exocrine 12010050 glands. We consider it a novel gland because of its peculiar bowl-shape and special arrangement of the microvilli that are confined to large vacuolar spaces instead of reaching the cuticle. The gland Received: 21 December 2020 is most pronounced in S. mutica, particularly in the queen. In addition to this gland, we also found Accepted: 4 January 2021 scattered class-3 intramandibular gland cells in the mandibles. Queens of S. mutica are peculiar in Published: 10 January 2021 having a cluster of these cells in the distal tip of their mandibles. As this species is a social parasite, further research is required to determine whether the development of these mandibular glands is Publisher’s Note: MDPI stays neu- related to its parasitic lifestyle. tral with regard to jurisdictional clai- ms in published maps and institutio- Keywords: exocrine glands; histology; ultrastructure; ants nal affiliations.

Copyright: © 2021 by the authors. Li- 1. Introduction censee MDPI, Basel, Switzerland. Social insects are well documented for their extraordinary variety of exocrine glands, This article is an open access article with ants having the most developed exocrine system currently comprising a total of distributed under the terms and con- 89 glands [1]. The glands are positioned all over the body, including the antennae and ditions of the Creative Commons At- legs [2,3]. Additionally, the mouthparts contain glandular tissue, with the mandibles espe- tribution (CC BY) license (https:// cially housing several exocrine glands. The most common and universal is the mandibular creativecommons.org/licenses/by/ gland that was first reported by Meinert back in 1860 [4]. The duct of this gland opens at 4.0/).

Insects 2021, 12, 50. https://doi.org/10.3390/insects12010050 https://www.mdpi.com/journal/insects Insects 2021, 12, 50 2 of 10

the upper surface at the base of each mandible, although the main secretory part occurs inside the head capsule. Besides this ‘extramandibular’ gland, a variety of glands have also been described inside the mandibles. These belong either to the epithelial class-1 glands or the bicellular class-3 glands according to the standard classification by Noirot and Quennedey [5]. The myrmicine ant genus Strumigenys comprises around 830 species of small and slow-moving ants in both the tropics and subtropics [6,7]. All Strumigenys species are predators equipped with specialized prey-seizing mandibles and belong to two major groups: the short- and long-mandibulate species [8]. Although Ward [9] recently moved Strumigenys to the Attini, for a long time it represented the largest genus among the tribe Dacetonini [10]. A distinctive characteristic in this genus according to Bolton [10] is the presence of five exocrine glands, that represent exocrine structures exclusively restricted to Strumigenys. Of these five glands, only the apicofemoral and apicotibial glands have yet been histologically confirmed [3,11]. The basimandibular gland, the ventral scape gland and the mesopleural gland, however, remain undocumented. Using scanning microscopy, Bolton [10] observed a pale patch or streak structure at the ventral or lateroventral part near the base of the mandible. Without any histological studies, he concluded that this region corresponds with an exocrine structure and named it ‘basimandibular gland’. Through surveying several Strumigenys species for the presence of exocrine glands in the mandibles of workers and queens, we aim to verify the existence of this presumed basimandibular gland. Our study particularly focuses on S. mutica, as this species is a social parasite of other Strumigenys species such as S. formosensis or S. solifontis.

2. Materials and Methods Workers and queens (and one male) belonging to 22 Strumigenys species were collected from their natural nests, representing both short- and long-mandibulate species (Table1 ). The anterior parts of the heads were cut off under a Leica MZ 12.5 microscope (Leica, Wetzlar, Germany) and fixed in 2% cold glutaraldehyde in a 50 mM Na-cacodylate buffer at pH 7.3 for 12 h, followed by postfixation in 2% osmium tetroxide in the same buffer for one hour. After dehydration in a graded acetone series, the tissues were embedded in araldite. Serial 1 µm semithin sections were made using a diamond knife with a Leica EM UC6 ultramicrotome (Leica, Wetzlar, Germany). Sections for light microscopy were stained with methylene blue and thionin and observed under an Olympus BX-51 microscope (Olympus, Tokyo, Japan). Double-stained 70 nm thin sections were studied under a Zeiss EM900 electron microscope (Zeiss, Oberkochen, Germany). Samples for the scanning microscopy study were put on aluminium stubs with double-adhesive tape, coated with palladium and viewed under a JEOL JSM-6360 scanning microscope (JEOL Ltd., Tokyo, Japan). Insects 2021, 12, x 3 of 10

Table 1. Survey of ants studied with their collection locality and indication of number of workers (W), alate queens (AQ) and males (M) examined. All 22 species were studied histologically with serial semithin sections. Species indicated with S Insects 2021 12 were, also, 50 examined with SEM, species indicated with T were also examined with TEM. 3 of 10

Species Collection Locality W AQ M S. benten T Yuchi Township, Nantou County, Taiwan 3 0 1 Table 1. Survey of ants studied with their collection locality and indication of number of workers (W), alate queens (AQ) S. canina Kobe, Japan 2 0 0 and males (M) examined. All 22 species were studied histologically with serial semithin sections. Species indicated with S S. elegantula T Yuchi Township, Nantou County, Taiwan 4 0 0 were also examined with SEM, species indicated with T were also examined with TEM. S. emmae S Baihe District, Tainan City, Taiwan 2 0 0 S. hexameraSpecies CollectionTakamatsu, Locality Japan W AQ 5 M0 0 S. kichijo S.T benten T YuchiLugu Township, Township, Nantou Nantou County, County, Taiwan Taiwan 3 0 4 0 1 0 S. canina S. leptothrix S,T Ren’ai Township,Kobe, Japan Nantou County, Taiwan 2 0 4 0 0 0 S. elegantula T Yuchi Township, Nantou County, Taiwan 4 0 0 S. membraniferaS. emmae S Ren’aiBaihe District,Township, Tainan Nantou City, Taiwan County, Taiwan 2 0 1 0 0 0 short-mandibulate S. mutica S.S,Thexamera Yuchi Township,Takamatsu, Nantou Japan County, Taiwan 5 0 8 5 0 0 S. kichijo T Lugu Township, Nantou County, Taiwan 4 0 0 S. sauteriS. leptothrix S,T Ren’aiLugu Township, Township, Nantou Nantou County, County, Taiwan Taiwan 4 0 2 2 0 0 S. chuchihensisS. membranifera Ren’aiJianshi Township, Township, Nantou Xinchu County, TaiwanCounty, Taiwan 1 0 0 1 0 0 short-mandibulate S,T S. formosensisS. muticaS,T YuchiRen’ai Township, Township, Nantou Nantou County, TaiwanCounty, Taiwan 8 5 6 0 0 0 S. sauteri Lugu Township, Nantou County, Taiwan 2 2 0 S. hispida Yuchi Township, Nantou County, Taiwan 3 0 0 S. chuchihensis Jianshi Township, Xinchu County, Taiwan 0 1 0 S. koningsbergeriS. formosensis S,T Ren’aiBogor Township, Botanical Nantou County,Garden, Taiwan Indonesia 6 0 1 0 0 0 S. lacunosaS. T hispida YuchiLugu Township, Township, Nantou Nantou County, County, Taiwan Taiwan 3 0 2 1 0 0 S. koningsbergeri Bogor Botanical Garden, Indonesia 1 0 0 S. liukueiensisS. lacunosa T LuguJiji Township, Township, Nantou Nantou County, County, Taiwan Taiwan 2 1 1 2 0 0 S. minutulaS. liukueiensisT HengchunJiji Township, Township, Nantou County, Pingtung Taiwan County, Taiwan 1 2 2 0 0 0 T S. nanzanensisS. minutula HengchunLanyu Township, Township, Pingtung Taitung County, County, Taiwan Taiwan 2 0 1 0 0 0 S. nanzanensis Lanyu Township, Taitung County, Taiwan 1 0 0 S. orchidensis Lanyu Township, Taitung County, Taiwan 1 0 0

long-mandibulate long-mandibulate S. orchidensis Lanyu Township, Taitung County, Taiwan 1 0 0 long-mandibulate S. perplexaS. perplexa ClydeClyde Mountain, Mountain, NSW, Australia NSW, Australia 2 0 2 0 0 0 S SS. rogeri Jiji Township, Nantou County, Taiwan 0 2 0 S. rogeri S. solifontis LuguJiji Township, Township, Nantou Nantou County, County, Taiwan Taiwan 6 0 0 2 0 0 S. solifontis Lugu Township, Nantou County, Taiwan 6 0 0

3.3. ResultsResults VentralVentral viewview examinationexamination withwith scanningscanning microscopymicroscopy confirmedconfirmed thethe existenceexistence ofof aa smoothsmooth proximalproximal portionportion ofof thethe ventralventral mandibularmandibular surfacesurface (Figure(Figure1 A,B).1A,B). The The cuticular cuticular surfacesurface inin thisthis areaarea isis devoiddevoid ofof hairshairs oror anyany sculpturationsculpturation asas isis foundfound elsewhereelsewhere onon thethe mandible,mandible, and therefore appears appears uniformly uniformly smooth. smooth. Observation Observation at higher at higher magnification, magnifica- tion,however, however, reveals reveals that thatthe smooth the smooth area area displa displaysys numerous numerous scattered scattered round round to ovoid to ovoid de- 2 depressionspressions of of 50–120 50–120 nm nm with with a adensity density around around 70 70 per per 100 100 µmµm2 (Figure(Figure 1C).1C).

FigureFigure 1.1. Ventral view scanning micr micrographsographs of of mandible mandible (Md). (Md). (A (A) S.) S. formosensis formosensis worker.worker. (B, (CB), CS.) S.leptothrix leptothrix worker.worker. Note Note smooth smooth surface surface of of proximal proximal part part of ofmandible, mandible, framed framed area area in in(B ()B appears) appears enlarged in (C). Lb: labium, Lp: labial palp, Mx: maxilla. enlarged in (C). Lb: labium, Lp: labial palp, Mx: maxilla.

SemithinSemithin sectionssections throughthrough thethe mandiblesmandibles ofof allall 2222 investigatedinvestigated speciesspecies (Table(Table1 )1) show show thethe presencepresence inin bothboth workersworkers andand queensqueens ofof anan epithelialepithelial glandgland whichwhich isis locatedlocated atat thethe proximal ventrolateral side of each mandible (Figures2 and3), which corresponds with the region of the smooth external cuticle. The cuticle overlaying the gland is approx. half as thick as the cuticle in the non-glandular region, which makes the gland look like a bowl-like extension into the cuticle (Figures2 and3). Many large vesicles are apically found in the glandular cells. The epithelium is formed by cylindrical cells with a thickness of 15–25 µm. Insects 2021, 12, x 4 of 10

proximal ventrolateral side of each mandible (Figures 2 and 3), which corresponds with

Insects 2021, 12, 50 the region of the smooth external cuticle. The cuticle overlaying the gland is approx.4 of half 10 as thick as the cuticle in the non-glandular region, which makes the gland look like a bowl- like extension into the cuticle (Figures 2 and 3). Many large vesicles are apically found in the glandular cells. The epithelium is formed by cylindrical cells with a thickness of 15–25 Theµm. extent The extent of the of glandular the glandular epithelium epithelium along along the lengththe length of the of the mandible mandible across across species species is similaris similar for for the the species species that that belong belong to the to short-mandibulatethe short-mandibulate group group (61.9 (61.9± 12.3 ± 12.3µm, µm,n = 14n )= and14) and these these belonging belonging to the to long-mandibulatethe long-mandibulate group group (62.8 (62.8± 12.4, ± 12.4, n = n 12). = 12). In S. In mutica S. mutica, of, whichof which we we had had both both castes castes available, available, the the thickness thickness of of the the epithelium epithelium is is 23.11 23.11± ±2.06 2.06µ µmm ((nn == 55)) in queens, whereas whereas in in workers workers it it measures measures 15.61 15.61 ± 2.41± 2.41 µmµ (nm = (n 8). = In 8). the In single the single male male(S. benten (S. benten) that) we that had we hadavailable, available, a similar a similar glandular glandular epithelium epithelium was was also also found found under under the theventral ventral proximal proximal part part of the of themandible mandible (Figure (Figure 2K).2K).

FigureFigure 2.2.Semithin Semithinsections sectionsthrough through head head and and mandibles mandibles of of various variousStrumigenys Strumigenysspecies. species. (A (A) Frontal) sectionFrontal throughsection through head of headS. mutica of S. queen,mutica queen, showing showing basimandibular basimandibular gland gland (BMG) (BMG) located located at outer at outer lateral side of mandibles, br: brain. (B,C) Cross sections through proximal part of left mandi- lateral side of mandibles, br: brain. (B,C) Cross sections through proximal part of left mandible ble of S. mutica queen and worker, showing both BMG and intramandibular gland (IMG). Arrow of S. mutica queen and worker, showing both BMG and intramandibular gland (IMG). Arrow in B indicates duct of class-3 gland crossing cuticle. (D–G,I) Cross sections through the base of left mandible of several Strumigenys species. (H,J) Longitudinal sections through mandible of S. kichijo

and S. elegantula workers, arrowheads indicate the large passage of sensillar cells through cuticle, SC: sensillar cells. (K) Cross section through mandible of S. benten male. Insects 2021, 12, x 5 of 10

in B indicates duct of class-3 gland crossing cuticle. (D–G,I) Cross sections through the base of left mandible of several Strumigenys species. (H,J) Longitudinal sections through mandible of S. kichijo and S. elegantula workers, arrowheads indicate the large passage of sensillar cells through cuticle, SC: sensillar cells. (K) Cross section through mandible of S. benten male.

Apart from the class-1 basimandibular gland, we also observe scattered class-3 intra- mandibular gland cells with a diameter of around 10 µm in all species, of which the ducts may open through the upper or lower mandibular surface as small pores. This is also the case in S. mutica, in which the queens display the additional characteristic of having a compact cluster of approx. 10 such gland cells at the upper mandibular tip (Figure 3). The ducts of this gland transverse the cuticle to open dorsally near the inner margin of the mandibular tip (Figure 3A, 3C, 3D), whereas in workers, the ducts open more laterally at Insects 2021, 12, 50 the outer side of the mandible (Figure 3B). The mandibles also contain several sensillar5 of 10 cells, that can be recognized by their large passage through the cuticle of 2–5 µm (Figure 2H, 2J).

FigureFigure 3.3. SemithinSemithin sections sections of of the the social social parasite parasite species species S. S.mutica mutica. (A.(,BA) ,CrossB) Cross section section through through mandiblemandible tip tip of of queen queen (A (A) and) and worker worker (B (),B showing), showing IMG IMG cells, cells, arrows arrows indicate indicate ducts ducts opening opening through through cuticle. EA: end apparatus. (C) Longitudinal section through head of queen, showing cuticle. EA: end apparatus. (C) Longitudinal section through head of queen, showing IMG dorsally IMG dorsally and BMG ventrally, arrow indicates duct, ab: base. (D) Cross section and BMG ventrally, arrow indicates duct, ab: antenna base. (D) Cross section through proximal part through proximal part of queen mandible, showing IMG dorsally and BMG ventrally, arrows indi- ofcate queen ducts. mandible, showing IMG dorsally and BMG ventrally, arrows indicate ducts. Apart from the class-1 basimandibular gland, we also observe scattered class-3 in- Ultrastructural examination of the glandular epithelium shows columnar cells with tramandibular gland cells with a diameter of around 10 µm in all species, of which the a basally located round nucleus with a diameter of 3–4 µm (Figure 4A,B). The horizontally ducts may open through the upper or lower mandibular surface as small pores. This is also layered cuticle overlaying the gland displays capricious transverse slits and spaces, that the case in S. mutica, in which the queens display the additional characteristic of having end up at the surface as minute pores around 100 nm (Figure 4A inset), and that corre- a compact cluster of approx. 10 such gland cells at the upper mandibular tip (Figure3). spond with the depressions observed with scanning microscopy (see Figure 1C). The ap- The ducts of this gland transverse the cuticle to open dorsally near the inner margin of the ical portion of the epithelium contains numerous large spaces that are filled with secretion mandibular tip (Figure3A,C,D), whereas in workers, the ducts open more laterally at the outer(Figure side 4A,E,F). of the In mandible their most (Figure apical3B). part, The these mandibles spaces alsocontain contain microvilli. several Most sensillar microvilli, cells, thathowever, can be do recognized not reach by the their cuti largecle, but passage remain through within the the cuticle large ofspace, 2–5 µ whichm (Figure means2H,J). the cuticleUltrastructural is lined underneath examination with ofa thin the glandularlayer of apical epithelium cytoplasm shows (Figure columnar 4C–E). cells The with cyto- a basallyplasm contains located round smooth nucleus endopl withasmic a diameter reticulum of 3–4and µseveralm (Figure mitochondria4A,B). The horizontally(Figure 4G). layeredWedged cuticle in between overlaying the glandular the gland displayscells are capriciousone or two transverse nerve fibres slits with and a spaces, diameter that of end 1.5 up at the surface as minute pores around 100 nm (Figure4A inset), and that correspond with the depressions observed with scanning microscopy (see Figure1C). The apical portion of the epithelium contains numerous large spaces that are filled with secretion (Figure4A,E,F ). In their most apical part, these spaces contain microvilli. Most microvilli, however, do not reach the cuticle, but remain within the large space, which means the cuticle is lined underneath with a thin layer of apical cytoplasm (Figure4C–E). The cytoplasm contains smooth endoplasmic reticulum and several mitochondria (Figure4G). Wedged in between the glandular cells are one or two nerve fibres with a diameter of 1.5 µm that are loaded with small dark-staining neurosecretory vesicles of 50–80 nm (Figure4H , see also Figure4B,F ). We did not find any muscle fibres associated with the basimandibular gland. Insects 2021, 12, x 6 of 10

Insects 2021, 12, 50 µm that are loaded with small dark-staining neurosecretory vesicles of 50–80 nm (Figure6 of 10 4H, see also Figure 4B,F). We did not find any muscle fibres associated with the basiman- dibular gland.

FigureFigure 4.4. Electron micrographs of of basimandibular basimandibular gland. gland. (A (A) General) General view view of ofgland gland epithelium epithelium in inS. S.leptothrix leptothrix workerworker with with basally basally located located nuclei nuclei (N) (N) and and large large vacuolar vacuolar spaces spaces (vs); (vs); inset inset shows shows detaildetail ofof apicalapical depressionsdepressions onon cuticularcuticular surface (arrowheads). ( B) Gland epithelium epithelium in in S.S. lacunosa lacunosa worker. Note nerve fibre penetrating between gland cells (NF). (C–E) Details of apical cytoplasm worker. Note nerve fibre penetrating between gland cells (NF). (C–E) Details of apical cytoplasm and microvilli (mv) in workers of S. minutula (C), S. elegantula (D) and S. leptothrix (E), showing and microvilli (mv) in workers of S. minutula (C), S. elegantula (D) and S. leptothrix (E), showing subcuticular cytoplasmic layer (arrowheads). (F) Gland epithelium in S. mutica worker showing subcuticularlarge vacuolar cytoplasmic spaces, micr layerovilli (arrowheads). and nerve fibre. (F) (G Gland) Central epithelium cytoplasm in S. in muticaS. minutulaworker worker showing with largemitochondria vacuolar spaces,(M) and microvilli smooth endoplasmic and nerve fibre. reticulum (G) Central (SER). cytoplasm(H) Detail inof S.nerve minutula fibre workercontaining with mitochondriadark staining (M)neurosecretory and smooth vesicles endoplasmic in S. mutica reticulum worker. (SER). ct: cuticle. (H) Detail of nerve fibre containing dark staining neurosecretory vesicles in S. mutica worker. ct: cuticle. The scattered intramandibular gland cells are characterized by their end apparatus, mitochondriaThe scattered and intramandibularGolgi apparatus gland(Figure cells 5A are,B). characterizedTheir accompanyi by theirng ducts end apparatus, cells open mitochondriathrough the cuticle and Golgi as narrow apparatus canals (Figure with 5aA,B). diameter Their of accompanying 0.25–0.5 µm (Figure ducts cells5C,D). open The throughnarrow gland the cuticle ducts as can narrow be easily canals distinguished with a diameter from the of 0.25–0.5much broaderµm (Figure sensillar5C,D). passages The narrowthrough gland the cuticle ducts (Figure can be easily 5D). distinguished from the much broader sensillar passages through the cuticle (Figure5D).

Insects 2021, 12, 50 7 of 10 Insects 2021, 12, x 7 of 10

FigureFigure 5.5. ElectronElectronmicrographs micrographs of of intramandibular intramandibular gland. gland. (A (A,B),B)General General view view of of gland gland cell cell in in worker ofworkerS. mutica of S.( Amutica) and (S.A) formosensis and S. formosensis(B), showing (B), showing end apparatus end apparatus (EA), mitochondria (EA), mitochondria (M) and (M) Golgi and Golgi apparatus (GA). (C) Duct cell (arrowhead) penetrating ventral mandibular cuticle (ct) in S. apparatus (GA). (C) Duct cell (arrowhead) penetrating ventral mandibular cuticle (ct) in S. mutica mutica worker (BMG: basimandibular gland). (D) Narrow gland ducts (arrowheads) and broader worker (BMG: basimandibular gland). (D) Narrow gland ducts (arrowheads) and broader sensillar sensillar processes (sp) adjacent to cuticle in S. mutica worker. ct: cuticle, N: nucleus. processes (sp) adjacent to cuticle in S. mutica worker. ct: cuticle, N: nucleus.

4.4. DiscussionDiscussion TheThe possibilitypossibility toto makemake histologicalhistological sectionssections throughthrough hardhard insectinsect cuticlecuticle usingusing epoxyepoxy resinsresins asas embedding embedding medium medium instead instead of of the the traditional traditional paraffin paraffin embedding embedding has has resulted resulted in thein the discovery discovery during during the recentthe recent decades decades of many of many glandular glandular structures struct insideures inside the mandibles: the man- thedibles: first the gland first described gland described inside the inside mandibles the mandibles of many ant of speciesmany ant in differentspecies in castes different was simplycastes was termed simply ‘intramandibular termed ‘intramandibular gland’. It is formedgland’. byIt is class-3 formed secretory by class-3 cells secretory underneath cells theunderneath outer margin the outer of the margin mandibles of the that mandibles open mainly that open at the mainly upper mandibularat the upper surfacemandibular [12]. InsurfaceOecophylla [12]. In longinoda Oecophylla[13 ]longinoda and Aneuretus [13] and simoni Aneuretus[14], thissimoni gland [14], has this ducts gland opening has ducts at bothopening the upperat both and the lowerupper surface. and lower A peculiar surface. modificationA peculiar modification of the class-3 of intramandibular the class-3 intra- glandmandibular is found gland in the is rarefound agroecomyrmecine in the rare agroecomyrmecineTatuidris tatusia , in which tatusia all duct, in openingswhich all assembleduct openings and open assemble through and a open sieve through plate at thea sieve proximal plate at ventral the proximal side near ventral the inner side edge near ofthe the inner mandibles edge of [ 15the]. InmandiblesAtta sexdens [15]. rubropilosa In Atta sexdens, both class-1rubropilosa and, class-3both class-1 intramandibular and class-3 glandsintramandibular have been glands reported have [16 ].been In the reported pest ant [16].Brachyponera In the pest sennaarensis ant Brachyponera, a large sennaarensisclass-1 in-, tramandibulara large class-1 glandintramandibular appears underneath gland appears the distinctive underneath mandibular the distinctive pit that mandibular is a diagnostic pit characterthat is a diagnostic for this genus. character The thick for this glandular genus. The epithelium thick glandular extends alongepithelium both the extends dorsal along and ventralboth the part dorsal of the and mandibles ventral part [17]. of Interestingly, the mandibles an epithelial[17]. Interestingly, intramandibular an epithelial gland intra- has beenmandibular described gland in Strumigenys has been described(formerly Pyramicain Strumigenys) membranifera (formerly[18], Pyramica however,) thismembranifera gland is located[18], however, at the inner this gland distal is tip located of the mandiblesat the inner which distal istip very of the different mandibles from which the position is very mentioneddifferent from by Boltonthe position [10]. In mentionedProtanilla wallaceiby Boltonworkers [10]. In and Protanilla queens, wallacei the epithelial workers intra- and mandibularqueens, the glandepithelial with intramandibular a proximal ventral gland position with appears a proximal similar ventral to the position basimandibular appears glandsimilar we to describethe basimandibular here [19], although gland we it doesdescribe not havehere the[19], bowl-shape although it anddoes also not lacks have thethe peculiarbowl-shape microvillar and also association lacks the pe withculiar vacuolar microvillar spaces association it has in Strumigenys with vacuolar. A conspicuousspaces it has epithelialin Strumigenys gland. A also conspicuous occurs ventrally epithelial in the gland mandibles also occurs of ventrally in japonicum the mandiblesworkers of butProceratium is totally japonicum absent in workers the related butDiscothyrea is totally absent sauteri in(Billen, the related unpubl. Discothyrea obs.). This sauteri indicates (Billen, theunpubl. distribution obs.). This of this indicates gland amongthe distribution the Formicidae of this gland is unpredictable among the and Formicidae phylogenetically is unpre- unstable.dictable and Our phylogenetically findings in all 22unstable. examined Our species findings of inStrumigenys all 22 examined, however, species do of confirm Strumi- thegenys existence, however, of the do basimandibularconfirm the existence gland of as the a novel basimandibular exocrine structure gland as and a novel a diagnostic exocrine characterstructure forand this a diagnostic genus [10 character]. Although for this ant genus males [10]. have Although much shorter ant males mandibles have much than workers and queens, the presence of a similar glandular epithelium in the single male

Insects 2021, 12, 50 8 of 10

we had available illustrates the basimandibular gland represents a common feature in Strumigenys ants. Class-1 intramandibular glands are also found in stingless bees [20,21] and in neotropical Polistinae wasps [22]. The presence of smooth endoplasmic reticulum is indicative of the elaboration of a non-proteinaceous and hence possibly pheromonal function. Secretion reaches the mandibular surface after passing through tortuous intracuticular spaces, that connect to minute superficial slits. Such slits have also been described for other class-1 glands in ants such as the apicofemoral and apicotibial glands in Strumigenys [11] and several other epithelial glands in the legs of various species [3] as well as for the mandibular pit gland in Brachyponera sennaarensis [17]. A peculiar ultrastructural feature that has not been found so far in any of the many exocrine glands in social insects is the presence of microvilli that are associated with large vacuolar spaces without reaching the apical cuticle. The functional significance of this arrangement remains unknown. It is also striking to find one or two nerve fibres that are wedged in between the gland cells. Not much is known about the neural regulation mechanisms of secretion release in social insects, although the presence of the nerve fibres loaded with neurosecretory vesicles is an indication that neural activation is most probable. The appearance and size of these nerve fibres is in agreement with that of the axonal projections that have been found in the salivary glands of ticks [23]. In these glands, however, the axons provide the innervation of myoepithelial cells that upon contraction cause secretion release. The basimandibular gland, however, is not associated with any muscle fibres, which makes it more difficult to understand how release of secretion is achieved. The function of the various intramandibular glands unfortunately remains undoc- umented. A study about the chemical composition of the intramandibular glands in Neoponera villosa suggests the compounds play a role of worker activity, and in the queen they may be related with a caste profile [24]. In Oecophylla longinoda, a peculiar behavior occurs when workers discover a new territory, a sugary food source, a prey or an alien ant, at such encounters they rub their mandibles onto the substrate as a marking signal to recruit their nestmates [13]. Special attention needs to be given to Strumigenys mutica, as this short-mandibulate species is an obligate social parasite in the nest of some long-mandibulate species such as S. formosensis or S. solifontis in Taiwan (C.-C. Lin, pers. comm.) or S. lewisi in Japan (F. Ito, pers. comm.). The young mated S. mutica queen enters a colony of one of these long-mandibulate species, where she eliminates the resident queen and takes over the reproductive command. Once the S. mutica queen has produced her own offspring, a mixed colony results with short-mandibulate mutica workers aside long-mandibulate workers of the host species (Figure6). How the mutica queen manages to enter the host colony, how she kills the resident queen and how she makes herself accepted by the host workers, is totally unknown so far. Our finding of a prominent class-3 gland that opens at the tip of the mandibles of the mutica queen makes it tempting to speculate that this gland offers the parasite queen the weaponry to attack the resident queen, as the mandibles form an obvious tool for attacking an opponent. This hypothesis finds support in our finding of this gland only in the queens of S. mutica, whereas the eventual class-3 cells in S. mutica workers and in other Strumigenys species are less obvious and moreover occur in a more proximal position inside the mandibles. Another possible support is that also in the slave-making social parasite , the usurper queen is equipped with obvious class-3 intramandibular gland cells near the inner tip of the sabre-shaped mandibles, although their function could not yet be elucidated [25]. Various glands and chemical strategies are used in this [25,26] as well as in other parasitic ants and this may also be the case in Strumigenys. Insects 2021, 12, 50 9 of 10 Insects 2021, 12, x 9 of 10

FigureFigure 6.6. View of mixed lab colony colony with with S.S. mutica mutica queenqueen (with (with enlarged enlarged thorax, thorax, blue blue arrowhead) arrowhead) surroundedsurrounded byby workersworkers ofofS. S. muticamutica(yellow (yellow arrowheads) arrowheads) and andS. S. solifontis solifontis(green (green arrowheads). arrowheads).

5.5. ConclusionsConclusions WeWe confirmconfirm thethe putative existence of of the the basimandibular basimandibular gland gland in in the the genus genus Strumi-Stru- migenysgenys byby histological histological and and ultrastructural ultrastructural examination. examination. The The gland gland is isconsidered considered to to be be a anovel novel exocrine exocrine structure structure in inants. ants. Although Although it appears it appears somehow somehow similar similar to the to ‘intraman- the ‘intra- mandibulardibular epithelial epithelial gland’ gland’ that thathas been has been described described in Protanilla in Protanilla wallacei wallacei [19] [and19] andthat thatmay mayalso occur also occur in some in some other otherant species, ant species, the no thevel novelbasimandibular basimandibular gland can gland be candistinguished be distin- guishedby its anatomical by its anatomical bowl-shape bowl-shape and peculiar and arrangement peculiar arrangement of microvilli of that microvilli are confined that are to confinedlarge vesicular to large spaces vesicular instead spaces of directly instead reac ofhing directly the cuticle. reaching In theaddition cuticle. to the In additionbasiman- todibular the basimandibular gland, we suggest gland, a potential we suggest function a potential during function colony duringfoundation colony of a foundation prominent ofdistal a prominent class-3 intramandibular distal class-3 intramandibular gland in the queens gland of inthe the social queens parasite of the S. socialmutica parasite. S. mutica. Author Contributions: Conceptualization, C.W. and J.B.; validation, formal analysis and investi- Authorgation, Contributions:C.W., M.S.-V., C.-C.L.Conceptualization, and J.B.; resources, C.W. and C.-C.L. J.B.; and validation, J.B.; writing, formal C.W. analysis and andJ.B.; investiga-visualiza- tion,tion, C.W.,C.W., M.S.-V., M.S.-V. C.-C.L.and J.B.; and supervision, J.B.; resources, J.B.; fund C.-C.L.ing andacquisition, J.B.; writing, C.W. C.W. All authors and J.B.; have visualization, read and C.W.,agreed M.S.-V. to the andpublished J.B.; supervision, version of J.B.;the manuscript. funding acquisition, C.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by China Scholarship Council, grant number 201906300043. Funding: This research was funded by China Scholarship Council, grant number 201906300043. Institutional Review Board Statement: Ethical review and approval were waived for this study, as Institutionalants are not considered Review Board as laboratory Statement: anEthicalimals according review and to approvalEU Directive were 2010/63. waived for this study, as ants are not considered as laboratory according to EU Directive 2010/63. Data Availability Statement: All microscopy data are available in the Zoological Institute, LSSE DataLab, AvailabilityUniversity of Statement: Leuven, Belgium.All microscopy data are available in the Zoological Institute, LSSE Lab, University of Leuven, Belgium. Acknowledgments: We are grateful to An Vandoren for her help in section preparation and Alex Acknowledgments:Vrijdaghs for scanningWe microscopy. are grateful We to An thank Vandoren three anonymous for her help reviewers in section for preparation their constructive and Alex Vrijdaghscomments for that scanning improved microscopy. the manuscript. We thank three anonymous reviewers for their constructive comments that improved the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the Conflictsdesign of ofthe Interest: study; inThe the authors collection, declare analyses, no conflict or interpretation of interest. The of fundersdata; in hadthe nowriting rolein of the the design man- ofuscript, the study; or in in the the decision collection, to publish analyses, the or results. interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References References 1. Billen, J. Diversidad y morfología de las glándulas exocrinas en las hormigas. In Hormigas de Colombia; Fernández, F., Guerrero, 1. Billen,R.J., Delsinne, J. Diversidad T., Eds.; y morfolog Universidadía de las Nacional glándulas de exocrinasColombia en Press: las hormigas. Bogota, Colombia, In Hormigas 2019; de Colombia pp. 165–174.; Ferná ndez, F., Guerrero, R.J., 2. Delsinne,Romani, R.; T., Eds.;Grasso, Universidad D.A.; Mori, Nacional A.; Isidoro, de Colombia N.; Le Moli, Press: F. Antennal Bogota, Colombia, glands of 2019;the slave-making pp. 165–174. ant Polyergus rufescens and its 2. Romani,slave species R.; Grasso, D.A.; cunicularia Mori,A.; (Hymenoptera, Isidoro, N.; Le Formicidae). Moli, F. Antennal Can. J. glandsZool. 2006 of, the 84,slave-making 490–494, doi:10.1139/z05-187. ant Polyergus rufescens and its slave species Formica cunicularia (Hymenoptera, Formicidae). Can. J. Zool. 2006, 84, 490–494. [CrossRef]

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