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Arthropod Structure & Development 42 (2013) 197e208

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Arthropod Structure & Development

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The pygidial defense gland system of the Steninae (Coleoptera, Staphylinidae): Morphology, ultrastructure and evolution

Andreas Schierling*, Konrad Dettner

Institute of Animal Ecology II, University of Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany article info abstract

Article history: The pygidial defense glands of the Steninae consist of two big (r1) and two smaller (r2) secretion ﬁlled Received 4 February 2013 sac-like reservoirs with associated secretory tissues and basal eversible membrane structures. Accepted 4 March 2013 The secretion is made up of deterrent and antimicrobial alkaloids stored in r1 as well as terpenes in r2. The gland cells ﬁlling r1 form a band shaped secretory tissue (g1) in an invagination of the reservoir Keywords: membrane. The content of r2 is secreted by a tissue (g2) surrounding the efferent duct of r1 opposite to Staphylinidae r2. In both gland tissues the secretion is produced in type IIIt gland cells and accumulates in an extra- Steninae cellular cavity surrounded by numerous microvilli of the gland cell membrane. After exocytosis the Pygidial defense glands Ultrastructure secretion enters an epicuticular duct and is transported to the corresponding reservoir via a conducting Evolution canal enclosed in at least one canal cell. While the structure of g1 is very similar in all species of the Steninae, g2 is often reduced. This reduction of the system r2/g2 is accompanied by a decreasing amount of terpenes in the total secretion and could be of interest for phylogenetic studies in the subfamily of the Steninae. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction While the big gland system r1/g1 is dominant in every Stenus species, the smaller system r2/g2 is often reduced and difficult to Rove beetles of the genera Stenus Latreille and Dianous Gyllenhal localize. Probably for this reason it was reported only for Stenus exhibit the typical slim Staphylinid habitus with short elytra and comma LeConte and Stenus biguttatus Linnaeus (Schildknecht, 1970; a flexible but largely unprotected abdomen. This body shape allows Schildknecht et al., 1975, 1976; Whitman et al., 1990; Lusebrink, the beetles to colonize habitats with small interstices but signifi- 2007), but it is present in all Stenus species investigated and may cantly increases the danger of infestation by microorganisms and possibly serve as a character for phylogenetic studies of the Steninae. predation (Dettner, 1991, 1993). To reduce this threats most The reservoir r1 is filled with g1 synthesized piperidine, piper- Staphylinidae possess abdominal defense glands in which they ideine and pyridine alkaloids, r2 contains terpenes produced by synthesize and store antimicrobial and deterrent compounds g2 (Schildknecht, 1970; Schildknecht et al., 1975; Kohler, 1979; (Araujo, 1978; Dettner, 1987, 1991, 1993). The paired pygidial de- Lusebrink, 2007; Lusebrink et al., 2009; Müller et al., 2012). Nearly all fense glands of the Steninae are located lateral to the gut and dorsal secretion compounds show significant antibiotic and deterrent ac- to the gonads in the last three to four abdominal segments (Jenkins, tivity (Lusebrink et al., 2009; Schierling et al., 2013) and thus can serve 1957). They consist of two big sac-like reservoirs (r1) with a band as potent chemical defense compounds. When molested, the beetles shaped secretory tissue (g1) situated in an invagination of the r1 bend their abdomen toward the source of irritation, evert their glands membrane and a second smaller pair of reservoirs (r2) that open and moisten the aggressor with their secretion. In addition the beetles into the basal efferent duct of r1 (Fig. 1). Opposite to but separated use their pygidial gland secretion to coat their body surface and thus from r2, the associated secretory tissue (g2) surrounds the efferent avoiding infection by microorganisms (Betz, 1999). duct of r1. The caudal parts of r1 expand to larger cylindrical Moreover some species of the Steninae living on the banks of membrane structures, which can be everted lateral to the anus by water use the pygidial defense gland secretion for an exceptional increasing the haemolymph pressure. Retraction of the glands is form of locomotion called skimming. This phenomenon was first accomplished by retractor muscles (Jenkins, 1957). described for Steninae by Piffard (1901),aswellasforStenus cicindeloides Schaller and Stenus tarsalis Ljungh by Billard and Bruyant (1905). Supported on the water surface by their hydrophobic tarsi * Corresponding author. Tel.: þ49 921 552734; fax: þ49 921 552743. the beetles touch the surface with the tip of their abdomen and E-mail address: [email protected] (A. Schierling). release small amounts of secretion from the everted pygidial glands.

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Abbreviations mv microvilli of gc membrane nu nucleus bl basal lamina op opening of ed for release of se cac canal cell pf pore field cd epicuticular ducts pl plaques at the microvilli apices cc secretion-conducting canal r1 big reservoirs ccl lumen of cc r2 small reservoirs basal to r1 ec extracellular cavity rc secretion-receiving canal ed efferent duct of r1 rcl lumen of rc em eversible membrane parts rf reservoir wall filaments ep epicuticle/epicuticular material ri reservoir intima et epithelium cells rs ribosomes ev evaginations of the g2 rc rw reservoir wall fifilamentous layer/filament layer surrounding rc se secretion g1 secretory tissue filling r1 SEM scanning electron microscopy g2 secretory tissue filling r2 ser smooth endoplasmic reticulum gc gland cell te tergite (9th abdominal tergite) go golgi apparatus TEM transmission electron microscopy gu gut tz transition zone between cc and rc mc mitochondria ve vesicles mf myofibrils

Certain gland compounds quickly spread on the water and thereby This paper provides a detailed study of the morphology and fine rapidly propel the beetle ahead. By directed bending of the abdomen structure of the pygidial defense gland systems of S. comma and the beetle can determine the direction of its movement and thus S. biguttatus. Comparisons to other Stenus species are drawn in escape potential predators or return to the banks (Schildknecht, order to investigate the possible evolution of the gland systems and 1976). The spreading ability of the secretion is mostly attributed to their secretions. the alkaloid compounds that reveal high spreading pressures and velocities on the water surface (Schildknecht et al., 1975; Schierling 2. Material and methods et al., 2013; Lang et al., 2012). The morphology of the big gland system r1/g1 has already been 2.1. Collection and identification of the beetles investigated representatively for all Steninae in Dianous coerulescens by Jenkins (1957) with light microscopic techniques. How- All beetles of the genus Stenus were collected in the surround- ever, no electron microscopic studies were performed on the ings of Bayreuth, Germany; D. coerulescens was gathered from a ultrastructure of r1/g1 and nothing is known about the function small stream near Baiersbronn, Germany. The identification of the and fine structure of the smaller gland system part r2/g2. species was accomplished using the key of Lohse (1964) as modified by Lohse (1989) and Assing and Puthz (1998). Species investigated: Dianous coerulescens, Stenus bifoveolatus, Stenus biguttatus, Stenus bimaculatus, Stenus binotatus, Stenus comma, Stenus flavipalpis, Stenus flavipes, Stenus fulvicornis, Stenus juno, Stenus latifrons, Stenus picipes, Stenus providus, Stenus pubescens, Stenus similis, Stenus solutus.

2.2. Morphological and ultrastructural studies

For SEM studies of the pygidial defense gland system the beetles were killed by freezing for 30 min to 24 C. The glands were dissected and without any fixation macerated for 20e25 min in 10% KOH at 90 C. The macerated glands were rinsed in H2O and dehy- drated in acetone (30%, 50%, 70%, 90%, 2 100%, 10 min each). After that the specimens were critical point dried (transitional medium CO2, Balzers CPD 020) and coated with gold (Edwards S150A). SEM observations were performed with a Zeiss Leo 1530 FESEM. For TEM studies the beetles were killed with EtOAc and the dissected glands were fixed for at least one hour in cold 0.1 M cacodylate buffer (C2H7AsO2, pH 7.3, Sigma) containing 2.5% glutaraldehyde (C5H8O2, Serva). Thereafter, the samples were embedded in 2% agarose (Roth) to prevent them from mechanical Fig. 1. Pygidial defense gland system of Stenus comma and S. biguttatus. Left side: damage and again fixed in glutaraldehyde/cacodylate buffer over- natural position in the abdominal tip. Right side: schematic diagram. em e eversible night. Subsequently the samples were postfixed with 2% OsO for membrane parts, g1 e band shaped gland tissue associated to r1, g2 e gland tissue 4 associated to r2, gu e gut, r1 e big reservoir, r2 e small reservoir (modified according 2 h and stained with 2% uranyl acetate (C4H6O6U, Plano) for 90 min. to Whitman et al., 1990). After dehydration with EtOH (30%, 50%, 70%, 95%, 3 100%, 15 min Author's personal copy

A. Schierling, K. Dettner / Arthropod Structure & Development 42 (2013) 197e208 199 each) the specimens were transferred stepwise into propylenoxide studied. The following reports of the ﬁne structure of r1/g1 concern (C3H6O, Serva) and then embedded in epon (Serva). After poly- the two species S. comma and S. biguttatus, but the results apply merization the resin embedded samples were trimmed (Leica EM equally well to all other species we examined (except for structure Trim) and cut to 50 nm ultrathin sections using a Leica Ultracut dimensions). UCT microtome equipped with a diamond knife (MicroStar). The large reservoir/gland system r1/g1 consists of the trans- The sections were attached to polioform-coated copper grids lucent sac-like reservoir r1 of about 1.3e1.5 mm length and 250 mm and stained with saturated uranyl acetate and lead citrate width and the corresponding secretory tissue g1 (Fig. 1). The gland (Pb(NO3)2 þ Na3(C6H5O7) 1:1, Merck). TEM observations were tissue g1, approximately 1.2 mm long and 70 mm wide, is made up performed with a Zeiss EM 902 at 80 kV. of elongated gland cells situated in an invagination of the r1 wall and covered by several layers of unspecialized non-secretory cells. 2.3. Terminology and measurements of cell structures An overview of the morphology and ultrastructure of the big gland system r1/g1 is given in Fig. 2. The terminology of the gland structures follows that of Noirot According to Jenkins (1957), each secretory active cell of g1 and Quennedey (1974, 1991) and Quennedey (1998). As a result of bears an extracellular cavity, in which the products are secreted by macerating and drying procedures several structures are shrunk exocytosis and drained by an epicuticular duct (Fig. 3AeD). After and may therefore differ from their natural size in the SEM mi- maceration with KOH the cuticular parts of a duct remain as crographs. If not otherwise stated the measurements are based on tripartite cylindrical structures consisting of a basal secretion- light microscope or TEM analysis. conducting part about 15 mm in length and 0.7 mm in width, an elongated secretion-receiving structure approximately 9 mm long 3. Results and 0.3 mm wide connected by a short transition zone (SEM size measurements, Fig. 3A, B). The distal secretion-receiving structure 3.1. Structure of the big gland system r1/g1 is located in the middle of the extracellular cavity of the gland cell. It is lined with a porous, granular epicuticular material of about The gross morphology of the big gland system r1/g1 has already 50 nm thickness, surrounded by a 120e300 nm wide ﬁlamentous been described for D. coerulescens by Jenkins (1957). We concur mass that did not resist maceration. To achieve a high exocytosis with these general results and extend them to all Steninae we rate, the surface of the gland cell membrane surrounding the

Fig. 2. Schematic drawings of the big gland system r1/g1 of Stenus comma and S. biguttatus. A. Overview of cross section through r1/g1. The band shaped gland tissue consisting of the gland cells and protective epithelium cells is located in an invagination of the r1 wall (modified according to Jenkins, 1957). B. Detail of A. Gland cell with microvilli surrounded secretion-receiving duct and canal cell with conducting canal. C. Cross section through the secretion-receiving structure of a g1 cell. bl e basal lamina, cac e canal cell, cc e conducting canal, ec e extracellular cavity, ep e epicuticle, et e epithelium cells, fi e filamentous layer, gc e gland cell, go e golgi apparatus, mc e mitochondria, mv e microvilli, nu e nucleus, rc e receiving canal, rcl e lumen of the receiving canal, rw e reservoir wall, ve e secretion containing vesicles. Schematic illustrations not drawn scale. Author's personal copy

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Fig. 3. Structure of the gland system r1/g1 of S. comma (AeB, EeF) and S. biguttatus (CeD). A. SEM micrograph of macerated gland tissue g1. Both, the secretion-conducting ducts and the epicuticular lining of the reservoir resist treatment with KOH. The ducts are made up of a slim secretion-receiving structure (rc) and a thicker secretion-conducting part (cc). Author's personal copy

A. Schierling, K. Dettner / Arthropod Structure & Development 42 (2013) 197e208 201 extracellular cavity is increased by numerous microvilli (Fig. 3C, D). membrane for secreting the synthesized compounds via exocytosis Inside and around the microvilli the gland cells exhibit large, (Fig. 6AeD). Transverse sections reveal that the distal secretion- partially elongated mitochondria with cristae of variable length and receiving part is lined with an up to 60 nm wide porous epicu- frequency. In the electron dense cytoplasm of the gland cells ticle. The latter is surrounded by a network of filaments about 8 nm several vesicles and partial golgi systems are visible, containing in diameter building up an approximately 600 nm broad mesh-like electron lucent material (Fig. 3C, D). Each gland cell exhibits an filament layer (Fig. 6BeD). The fine filaments did not resist ovoid nucleus approximately 5 mm in diameter. Although expected maceration, so the whole end apparatus appears thinner in the SEM for a gland cell, an endoplasmic reticulum could not be observed. micrographs (Fig. 5) than in the TEM images (Fig. 6). The mostly The proximal secretion-conducting part of the epicuticular duct electron lucent microvilli bear an electron dense material in their is made up of a solid, 150e200 nm thick wall lined with a cuticulin apical region, where they reach the filament layer (Fig. 6BeD). layer of 10 nm (Fig. 3E). The canal is situated in an elongated canal Electron dense material also occurs in the cytoplasm of the whole cell that penetrates the gland cell and is only slightly greater in gland cell (Fig. 6C). The gland cells are often filled with smooth diameter than the canal itself. Being an extracellular structure, the endoplasmic reticulum (Fig. 6C). Sparse ovoid mitochondria with conducting canal is located in an extracellular cavity within the short cristae occur near the microvilli. Golgi systems, free ribo- surrounding cell (Fig. 3E). The canal cell is not secretory, which is somes and rough endoplasmic reticulum are seldom. Each gland apparent from missing microvilli and the impermeable, nonporous cell is coated by an 85 nm wide basal lamina (Fig. 6E) and equipped epicuticle of the canal. The gland cell and the canal cell are con- with an ovoid nucleus 5e6 mm in diameter. nected via septate junctions (Fig. 3E). The proximal secretion-conducting part of the epicuticular The wall of the secretion-storing reservoir r1 is lined by a 70e ducts transports the secreted compounds into the reservoir. The up 100 nm thick epicuticular intima (Fig. 3F). The stability and flexi- to 50 mm long (SEM size measurement) tubular ducts with a bility of the reservoir wall is due to a presumably single layer of diameter of 0.7 mm exhibit numerous evaginations all over their epithelium cells containing numerous right angular arranged surface (Figs. 5C, E, F, 7AeD). In S. biguttatus these evaginations are myofibrils. The epithelium cells are covered by a basal lamina of an ovoid form (Fig. 5C, E), whereas in S. comma they are more flat, (Fig. 3F). but still clearly visible (Fig. 5F). The secretion-conducting ducts of g2 are situated in at least one 3.2. Structure of the small gland system r2/g2 canal cell, which penetrates the gland cell and nearly comes up to the secretion-receiving part of the canal (Fig. 7A). As an extracel- In contrast to the gland system r1/g1 there are great interspe- lular structure, the conducting canal is surrounded by an extra- cific differences concerning morphology and ultrastructure of the cellular cavity (Fig. 7AeC). In contrast to the secretion-receiving smaller gland system r2/g2. In the following we first restrict our canals the conducting canals are enclosed by a 60 nm thick reports to r2/g2 of S. comma and S. biguttatus, because in these compact non-perforated epicuticle, lined with a cuticulin layer of species the situation is nearly identical and most complex. Subse- 5e8 nm width (Fig. 7C). The very slim canal cells are poor in inner quently the variations in the morphology of r2/g2 of further species structures and cell organelles, but with an ovoid nucleus. They are are described (see Section 3.4). Fig. 4 provides an overview of the surrounded by a basal Lamina (Fig. 7B). Proximally the canal cells morphology and ultrastructure of the small gland system r2/g2 of are bundled on a pore field where the canals open into the efferent S. comma and S. biguttatus. duct of r1 (Figs. 5B, 7D). The distal secretion-receiving and the The small gland system r2/g2 consists of a clear, secretion filled proximal secretion-conducting parts of the ducts are linked by a reservoir r2 (up to 330 mm long and 130 mm wide), which opens into short evagination free transition section with non perforated the efferent duct of the big reservoir r1 (Figs. 1 and 5A). The asso- epicuticle (Figs. 5D, 7A). In SEM and TEM micrographs the attach- ciated secretory tissue g2 encloses the efferent duct of r1 at the ment sites of the gland cell membranes are visible (Figs. 5D and 7A). opening of r2, with the main part being located opposite to r2 Gland cell and canal cell are interconnected via septate junctions. (Figs. 1 and 5A). g2 consists of numerous globular gland cells 16e The pore field on which the g2 canals open into the efferent duct 22 mm in diameter that make up a “cauliflower like” structure of r1 is situated exactly opposite to the reservoir r2 (Figs. 5A and visible with the light microscope. Each gland cell is equipped with 7E). In the living beetles the r1 efferent duct collapses and the pore an epicuticular duct that collects the secretion and conducts it into plate is pressed on the opening of r2 (Fig. 7E). the reservoir through at least one canal cell (Figs. 5e7). After The r2 wall consists of epithelium cells secreting a layer of fil- removal of the soft g2 tissue parts by maceration with KOH, the aments covered by a puckered epicuticular intima 8e13 nm in cuticular ducts become visible (Fig. 5). The canals measure up to width (Fig. 7F). The epithelium cells do not contain any muscle fi- 60 mm in total length (SEM size measurement) and can be divided brils as it was observed in the r1 wall. into a distal secretion-receiving, a proximal secretion-conducting, and a transition section. 3.3. Structure of the eversible membranes The about 12 mm long (SEM size measurement) and 0.9 mm wide distal secretion-receiving structure of the epicuticular ducts The eversible membrane parts represent the secretion-releasing (Fig. 5C, D, F) is situated in an extracellular cavity of the gland cell structures of the pygidial glands of the Steninae and do not vary in (Fig. 6AeC). As in the secretory cells of g1, the extracellular cavity of different species. The eversible membranes are situated at the base the g2 cells is filled with numerous microvilli of the inner gland cell of the efferent ducts of the reservoirs r1 and can be extruded

B. Detail of A. The secretion-receiving and conducting regions are connected by a transition zone (tz). C. TEM sections through a g1 gland cell. The secretion-receiving canal is situated in an extracellular cavity (ec) inside the gland cell (gc). The gland cell membrane sends microvilli (mv) into the extracellular cavity. Several mitochondria (mc) are located inside and around the microvilli. Close beside a golgi apparatus (go) is visible. D. Receiving canal surrounded by a porous epicuticle (arrow) and a filamentous layer (fi). Mito- chondria and vesicles (ve) accumulate around and inside the microvillus. Differences in microvilli morphology between C and D are due to individual cell age and secretion activity. E. TEM section through a canal cell (cac) penetrating a gland cell. The canal cell bears an extracellular cavity surrounding the conducting canal. Canal and gland cells are interconnected via septate junctions (insert). The wall of the secretion-conducting canal consists of epicuticular material (ep) lined with a thin cuticulin layer (arrowhead). F. Section through the reservoir wall made up of a reservoir intima (ri) and epithelium cells (et) containing myofibrils (mf). The epithelium cells are covered by a basal lamina (bl). Scale bars: A e 3 mm, B e 0.4 mm, C e 1 mm, D e 0.5 mm, E e 0.5 mm (insert 50 nm), F e 1 mm. Author's personal copy

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Fig. 4. Schematic drawings of the small gland system r2/g2 of Stenus comma and S. biguttatus. A. Overview of r2/g2 with secretion-conducting canals gathering on a pore field on the efferent duct membrane of r1 opposite to r2. B. Diagram of the secretion producing, receiving and conducting structures of g2. bl e basal lamina, cac e canal cell, cc e conducting canal, ec e extracellular cavity, ed e efferent duct of r1, ep e epicuticle, ev e evagination of the conducting canal, fi e filament layer, gc e gland cell, go e golgi apparatus, mc e mitochondria, mv e microvilli, nu e nucleus, pf e pore field, pl e plaques at the microvilli apices, rc e receiving canals, rs e ribosomes, ser e smooth endoplasmic reticulum. Schematic illustrations not drawn scale. laterally to the anus between the 9th tergite and sternite (Fig. 8). Their comparatively short conducting canals do not exhibit They are each made up of a tubular membrane, which is inverted at any evaginations and the distinct secretion-receiving structure the tip and thereby forms a cylindrical double walled structure observed in S. comma and S. biguttatus is replaced by a filamentary bearing a pore at the end for secretion release. While the inner receiving canal. Overall their organization is reminiscent of that of membrane represents an extension of the r1 efferent duct, the the g1 canals (Fig. 9C). This is also the case for S. flavipes. outer membrane of the eversible cylinder is continuous with a membrane separating the gut and pygidial gland apparatus from 4. Discussion the gonads and other abdominal organs (Fig. 8). For more details concerning the morphology of the eversible membrane parts refer 4.1. Morphology and ultrastructure of the pygidial defense gland to Jenkins (1957). apparatus

3.4. Intraspecific differences in the morphology of r2/g2 The pygidial defense gland apparatus of the Steninae consists of two reservoirs r1 and r2 with associated secretory tissues g1 and As described above there are no structural and functional g2, as well as an eversible membranous part for the secretion interspecific differences in the large gland system r1/g1 and the release. The gland system r1/g1 has already been well described by eversible membrane parts. However, r2/g2 is massively reduced Jenkins (1957) in D. coerulescens, which is representative for all and functionally altered in many species. The best developed and Steninae, but the ultrastructure and function of the whole pygidial most complex r2/g2 gland system can be found in S. comma and gland system is described in the current study for the first time. S. biguttatus. All other 16 species examined showed distinct re- The gland cells of both, g1 and g2 of S. comma and S. biguttatus ductions of either g2, r2 or both components. While r2 of S. comma can be classified as terminal class IIIt gland cells according to Noirot is up to 330 mm long, in S. bimaculatus, a species, which is actually and Quennedey (1974, 1991) and Quennedey (1998). The synthe- about 1 mm larger then S. comma, r2 only reaches a length of sized secretion is gathered in a receiving structure within an approximately 60 mm(Fig. 9A). Furthermore, in S. bimaculatus its extracellular cavity of the gland cell, drained by a secretion- conic form is lost and it becomes a small tubular appendix of the r1 conducting canal located in a canal cell, and transported to the efferent duct. An even more extreme form of reduction of r2 occurs reservoir (Pasteels, 1968; Noirot and Quennedey, 1974, 1991; in S. fulvicornis, S. juno and S. picipes. Species like D. coerulescens, Quennedey, 1998). Thus, the gland cells and the corresponding S. providus, S. solutus, S. pubescens, S. latifrons and others exhibit a canals, inclusive canal cells, together form functional glandular small reservoir r2 that keeps its conic form, but its size is also units (Noirot and Quennedey, 1991). While the secretion-receiving dramatically reduced. S. flavipes is the only species that possesses a canals of g1 and g2 gland cells both exhibit a porous, granular reservoir r2 with a reservoir size/body size ratio comparable to that epicuticle for reception of the secreted compounds, the conducting of S. comma and S. biguttatus. canals are lined with a continuous epicuticle and an additional In all species except S. comma and S. biguttatus, the epicuticular cuticulin layer. This is important to minimize the risk of self- ducts of g2 do not gather on a pore field, but open within a wide- intoxication with cytotoxical defense secretions (Noirot and spread area not opposite to r2 into the efferent duct of r1 (Fig. 9B). Quennedey, 1974, 1991; Quennedey, 1998). Author's personal copy

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Fig. 5. Structure of the small gland system r2/g2 of S. biguttatus (AeE) and S. comma (F). A. SEM overview of a macerated r2/g2 gland system. The secretory tissue g2 is located opposite to the small reservoir r2 at the efferent duct (ed) of r1. After removal of the soft secretory tissue by KOH treatment the epicuticular lining of the reservoirs and the cuticular ducts (cd) of g2 remain. B. Detail of A. The cuticular ducts gather on a pore ﬁeld (pf) exactly opposite to the opening of r2. C. SEM micrograph of the g2 secretion-receiving (rc) and conducting (cc) structures. The conducting canals are covered with ovoid evaginations (ev). D. Detail of C. The secretion-receiving and conducting regions are connected by an evagination free transition zone (tz). The visible ring structures (arrows) indicate cell membrane attachment sites. E. SEM detail of a secretion-conducting canal with evaginations. F. SEM micrograph of macerated g2 secretion-receiving and conducting structures of S. comma. Compared to S. biguttatus the evaginations are less distinct. Scale bars: A, B e 20 mm, C e 2 mm, D e 0.5 mm, E e 1 mm, F e 3 mm. Author's personal copy

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Fig. 6. TEM sections through the secretion-receiving end apparatus of g2 gland cells of S. comma. A. Secretion-receiving canal (rc) penetrating the extracellular cavity (ec) of the gland cell (gc). Numerous microvilli (mv) of the gland cell membrane extend into the extracellular cavity. B. Receiving canal surrounded by a porous epicuticle (arrow) that is followed by a filament layer (fi). C. Gland cell with end apparatus bearing a granular, electron dense material (asterisks) between possibly secretion containing vesicles (ve) and large amounts of smooth endoplasmic reticulum (ser). D. Detail of C. The filaments surrounding the porous epicuticle (ep) make up a mesh like layer. Microvilli reaching the filaments exhibit apical electron dense plaques (arrowheads). Secretion (se) is visible in the lumen of the secretion-receiving canal (rcl). E. Basal lamina (bl) externally surrounding a gland cell. Scale bars: A e 2 mm, B, C e 1 mm, D e 0.2 mm, E e 0.5 mm.

As non-proteinous secretions of low molecular weight, the did not reveal any significant differences, so it all might be the same defense compounds of the Steninae are thought to be synthesized material. in smooth endoplasmic reticulum and golgi systems. While the g2 In the g2 gland cells, the filament layer surrounding the gland cells are completely filled with smooth endoplasmic reticu- receiving canal probably does not represent accumulating secre- lum, these structures are lacking in g1 gland cells. Thus, the cellular tion. Although the fine filaments did not resist maceration by 10% origin of the alkaloids of r1/g1 remains unknown. KOH, they are nevertheless proposed to be of epicuticular origin, A sponge-like or filamentous layer surrounding the receiving which was lost due to the hard maceration process during prepa- canal of a gland cell, as it is found in both secretory tissues of the ration for SEM microscopy. Structures similar to these filaments Steninae, is usually interpreted as protein or epicuticular filaments could neither be observed in the lumen of the receiving nor in the (Noirot and Quennedey, 1991). Such structures are common and conducting canal, as it would be expected for secretion or secretion have been described for several insect gland cells (Forsyth, 1970; precursors. In contrast to the filamentous layer of the g1 secretion- Happ and Happ, 1973; Cazals and Juberthie-Jupeau, 1983; Araujo receiving apparatus, the single filaments of g2 cells are clearly and Pasteels, 1985; Biemont et al., 1990, 1992; Drilling et al., distinguishable as a built-up mesh-like structure. Furthermore 2010). Alternatively filaments are considered as secretion or there are plaques with high electron density visible at the apices of secretion-precursor material accumulating around the receiving many microvilli probably representing epicuticular material canal of a gland cell (Happ et al., 1966). In the case of g1 this second deposited by the gland cell (Locke and Huie, 1979). According to interpretation might be the case, because the filaments are visible Biemont et al. (1992) such cuticular filament structures could as a filamentous mass, not as distinct single filaments. Furthermore contain enzymes or compounds modificating the secretion by the comparison of the structure of the filamentous mass with the passing into the lumen of the receiving canal. In this manner content of the receiving canal and some vesicles in the gland cell nontoxic precursors could be synthesized in the gland cell, and not Author's personal copy

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Fig. 7. TEM sections through g2 secretion-conducting cells and r2 reservoir wall of S. biguttatus (AeD) and S. comma (E, F). A. The secretion-conducting canal cell (cac) containing the conducting canal (cc) with evaginations (ev) penetrates the gland cell (gc). The secretion-receiving (rc) and the conducting canals are linked by the evagination free transition zone (tz). In the gland cell the receiving canal with surrounding filament layer (fi) and extracellular cavity (ec) is visible. B. Section through a canal cell of g2 showing basal lamina (bl), conducting canal and evaginations. As well as the receiving canal the conducting canal is situated in an extracellular cavity. C. Detail of B. The conducting canal wall consists of a compact epicuticle (ep) and a thin cuticulin layer (arrow). Secretion (se) is visible in the lumen of the canal (ccl) and the evaginations. D. Secretion-conducting canals opening in the efferent duct (ed) of the big reservoir r1. All canals come together on a pore field (pf). E. Section through the efferent duct of r1 in the region of the opening of r2. Under resting conditions the r1 efferent duct collapses. F. Reservoir wall made up of reservoir intima (ri) followed by reservoir wall filaments (rf), epithelium cells (et) and basal lamina. In the lumen of the reservoir secretion (se) is visible. Scale bars: A, B e 0.5 mm, C e 0.2 mm, D, E e 5 mm, F e 1 mm. Author's personal copy

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those reported for E. longicollis (Eisner et al., 1964), but here its position is of fundamental significance for the functionality of r2/ g2. The pore plate is situated opposite to the opening of the reservoir r2, so the secreted compounds would have to pass the r1 efferent duct to enter the reservoir r2. The compounds found in r2 could not be detected in r1 (Schierling et al., 2013), so a mechanism must exist preventing the g2 synthesized compounds from entering r1. Because of the distinctive position of the pore plate exactly opposite the opening of r2, a transfer of the secretion from g2 to r2 could be facilitated by collapsing of the r1 efferent duct under resting conditions (no secretion release) that could result in the pore plate being pressed on the opening of r2. The wall of the reservoirs r1 and r2 is lined by a tight epicuticular intima that prevents the haemolymph and organs from being contaminated with toxic secretion. Furthermore the reservoir walls are reinforced by myofilaments (r1) or epicuticular filaments (r2). If the reservoir r1 is not maximally filled, the myofilaments in the epithelium cells are contracted, and the intima is puckered. A puckered intima was also observed in the r2 wall, albeit there are epicuticular filaments instead of muscle fibrils. However, in this Fig. 8. SEM micrograph of the tip of the abdomen of S. nitidiusculus with dissected fl eversible membrane parts (em) above the 9th tergite (te). The membranes are everted manner the reservoirs are exible structures that can be adjusted to to release the secretion through the opening (op) on their tip. Scale bar: 50 mm. the actual secretion level. The flat muscular epithelium surrounding r1 is not very thick, so it is probably not responsible for the rapid develop their full toxicity until being sieved through the extracel- gland eversion and secretion release, which is actually achieved by lular spongy receiving canal coating. increasing haemolymph pressure, as proposed by Jenkins (1957). In addition to the filament layer, the evaginations of the g2 Jenkins (1957) describes the whole pygidial defense gland conducting canals might act as reaction chambers in which apparatus of the Steninae as an invagination of the pleural mem- secreted compounds are gradually modified as it was described by brane. The duct structures of both secretory tissues g1 and g2, as Eisner et al. (1964) for the tenebrionid beetle Eleodes longicollis well as the corresponding reservoirs are lined with epicuticular ’ ’ LeConte. The chemical modification could result in an increasing material, which supports Jenkins proposal of the glands epidermal toxicity during passage through the canal. The conspicuous length origin (Noirot and Quennedey, 1974; Quennedey, 1998). of the g2 conducting canals would support this hypothesis. Such processes, however, would require enzymes or other compounds 4.2. Intraspecific differences and evolutionary aspects located in the evaginations, but there were no structures found that suggest secretory activity of the canal cells. Thus, secretion modi- The appearance of a second smaller reservoir r2 was first fying compounds or enzymes would have to be also secreted from described for S. comma by Schildknecht (1970). Later it was re- the g2 gland cells. ported for S. biguttatus and S. comma (Lusebrink, 2007), but it has The secretion-conducting canals of g2 all gather on a moderate never been described for other Steninae. Jenkins (1957) mentioned sklerotized pore plate on the efferent duct of r1, comparable to some structural changes at the basal parts of the gland tissue r1 in

Fig. 9. Reductions and variations of the small gland system r2/g2 of further Stenus species. SEM micrographs after maceration with KOH. A. Strongly reduced small reservoir r2 of S. bimaculatus with corresponding epicuticular ducts (cd) and efferent duct of r1 (ed). B. Epicuticular ducts opening on a widespread area into the efferent duct of r1ofS. bifoveolatus. The small reservoir r2 is situated cranially and not opposite to the canal openings. C. g2 secretion-receiving (rc) and conducting (cc) canals with connecting transition zone (tz) of S. juno. The canals do not signiﬁcantly differ in morphology and ﬁne structure from these of g1. The spherical structures visible on the canal surface represent remains of the macerated gland tissue. Scale bars: A, B e 10 mm, C e 0.5 mm. Author's personal copy

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D. coerulescens. He had probably found the tissue of g2, but was Germany) is thanked for numerous helpful hints concerning the unable to identify it as independent from g1. In our study we found Stenus phylogeny and J. Woodring (Animal Ecology II, University of r2 as well as the associated gland tissue g2 in every species Bayreuth) for correcting the English of our Manuscript. Support for examined, including D. coerulescens. However, there are great this research by a grant from the German Research Foundation species-dependent reductions in the morphology, ultrastructure (DFG: SE-595/14-1 and DE 258/12-1) is gratefully acknowledged. and function. As described above, the position of the pore plate exactly opposite to the reservoir r2 is important for the correct transfer of secretion References from g2 to r2 in S. comma and S. biguttatus. Only by this the pore plate Araujo, J., 1978. Anatomie compareé des systèmes glandulaires de défense chimique can be pressed on the opening of r2 while the r1 efferent duct is des Staphylinidae. Archives de Biologie 89, 217e250. collapsed. In the other species examined for this study the secretion- Araujo, J., Pasteels, J.M., 1985. Ultrastructure de la glande defensive de Drusilla conducting canals open into the efferent duct of r1 cranial to r2, so canaliculata (Fab.) (Coleoptera: Staphylinidae). Archives de Biologie 96, 81e99. the secretion transfer cannot work. As a consequence the reservoir r2 Assing, V., Puthz, V., 1998. 55. Gattung: Stenus Latreille. In: Lucht, W., Klausnitzer, B. is reduced, as it can be seen in various species. Furthermore, the (Eds.), Die Käfer Mitteleuropas, vol. 15. Gustav Fischer Verlag, Jena, pp. 130e131. epicuticular ducts of species with reduced r2/g2 are very similar in Betz, O., 1996. Function and evolution of the adhesive prey capture apparatus of e fi Stenus species (Coleoptera, Staphylinidae). Zoomorpholgy 116, 15 34. structure to these of g1, so it is dif cult to determine weather there Betz, O., 1998. Life forms and hunting behavior of some Central European Stenus are structurally modified g2 cells, or just tightly packed and round species (Coleoptera, Staphylinidae). Applied Soil Ecology 9, 69e74. shaped g1 cells opposite the small reservoir r2. Betz, O., 1999. A behavioral inventory of adult Stenus species (Coleoptera: Staphy- linidae). Journal of Natural History 33, 1691e1712. The small reservoirs r2 contain terpene compounds like among Biemont, J.C., Chauvin, G., Hamon, C., 1990. Morphology and ultrastructure of the others a-pinene, 1,8-cineol (eucalyptol) and 6-methyl-5-hepten-2- abdominal integumentary glands of Acanthoscelides obtectus (Say) (Coleop- on (Schildknecht, 1970; Schildknecht et al., 1975, 1976; Lusebrink, tera: Bruchidae). International Journal of Insect Morphology and Embryology a 19, 1e11. 2007). While -pinene and 1,8-cineol were found in many Stenus Biemont, J.C., Mahamadou, C., Pouzat, J., 1992. Localization and fine structure of the species, 6-methyl-5-hepten-2-on could only be identified in the r2/ female sex pheromone-producing glands in Bruchidius atrolineatus (Pic) g2 secretion of S. comma and S. biguttatus (Lusebrink, 2007; (Coleoptera: Bruchidae). International Journal of Insect Morphology and e Schierling et al., 2013), which show the best developed r2/g2 gland Embryology 21, 251 262. Billard, G., Bruyant, C.,1905. Sur un mode particulier de locomotion de certains Stenus. system within the whole genus. Furthermore, the amount of a- Comptes Rendus des Séances et mémoires de la Société de Biologie 59, 102e103. pinene and 1,8-cineol is maximized within these two species. In Cazals, M., Juberthie-Jupeau, L.J., 1983. Ultrastructure of a tubular sternal gland in other Steninae the terpenes occur, if at all, only in traces (Lusebrink, the males of Speonomus hydrophilus (Coleoptera: Bathyscinae). Canadian Jour- nal of Zoology 61, 673e681. 2007; Schierling et al., 2013). Because S. comma and S. biguttatus Dettner, K., 1987. Chemosystematics and evolution of beetle chemical defense. represent the only species exhibiting distinct evaginations of the Annual Reviews of Entomology 32, 17e48. secretion-conducting canals, there may be a correlation. Dettner, K., 1991. Chemische Abwehrmechanismen bei Kurzflüglern (Coleoptera: Staphylinidae). Jahrbücher der Naturwissenschaftlichen Vereinigung Wupper- S. comma and S. biguttatus both reveal lateral borders at their tal 44, 50e58. abdominal tergites. This character is classified as phylogenetically Dettner, K., 1993. Defensive secretions and exocrine glands in free-living Staphylinid primitive, so the two species are arranged at the base of the genus beetles e their bearing on phylogeny (Coleoptera: Staphylinidae). Biochemical Systematics and Ecology 21, 143e162. Stenus (Puthz, 2006, 2010). Because of their better adaption in Drilling, K., Dettner, K., Klass, K., 2010. Morphology of the pronotal compound nearly all areas, the alkaloid compounds of r1 are effective for de- glands in Tritoma bipustulata (Coleoptera: Erotylidae). Organisms Diversity and fense against predators or microorganisms and for locomotion via Evolution 10, 205e214. Eisner, T., McHenry, F., Salpeter, M.M., 1964. Defense mechanisms of arthropods XV. skimming (Schierling et al., 2013; Lang et al., 2012). Thus, most Morphology of the quinine-producing glands of a Tenebrionid beetle (Eleodes phylogenetically advanced species have nearly lost or at least longicollis Lec.). Journal of Morphology 115, 355e400. dramatically reduced their r2 compounds, as well as the structures Forsyth, D.J., 1970. The ultrastructure of the pygidial defence glands of the carabid e responsible for their synthesis and storing. Pterostichus madidus F. Journal of Morphology 131, 397 416. Happ, G.M., Happ, C.M., 1973. Fine structure of the pygidial glands of Bledius Dianous, the sister genus of Stenus with its single middle Euro- mandibularis (Coleoptera: Staphylinidae). Tissue and Cell 5, 215e231. pean species D. coerulescens has hitherto been classified as a Happ, G.M., Strandberg, J.D., Happ, C.M., 1966. The terpene-producing glands of a phylogenetically basal species of the Steninae due to the lack of an phasmid insect. Cell morphology and histochemistry. Journal of Morphology 119, 143e160. adhesive prey capture apparatus that probably represents an apo- Jenkins, M.F., 1957. The morphology and anatomy of the pygidial glands of Dianous morphy in Stenus (Puthz, 1981; Betz, 1996, 1998, 1999; Leschen and coerulescens Gyllenhal (Coleoptera: Staphylinidae). Proceedings of the Royal Newton, 2003). New molecular and chemotaxonomic analysis of Entomological Society of London 32, 159e169. Koerner, L., Laumann, M., Betz, O., Heethoff, M. Loss of the sticky harpoon e COI the phylogeny of the Steninae indicated that the genus Dianous sequences indicate paraphyly of Stenus with respect to Dianous (Staphylinidae, should be integrated into the genus Stenus as a species that might Steninae). Zoologischer Anzeiger, in press, http://dx.doi.org/10.1016/j.jcz.2012. have secondarily reduced the prey capture apparatus (Koerner 09.002. Kohler, P., 1979. Die absolute Konfiguration des Stenusins und die Aufklärung et al., in press; Schierling et al., 2013). This hypothesis is in accor- weiterer Inhaltsstoffe des Spreitungsschwimmers S. comma. PhD thesis, Uni- dance with the morphology of the small gland system r2/g2 of versity of Heidelberg, Germany. D. coerulescens that exists only in a strongly reduced form compa- Lang, C., Seifert, K., Dettner, K., 2012. Skimming behaviour and spreading potential of Stenus species and Dianous coerulescens (Coleoptera: Staphylinidae). Natur- rable to that of the phylogenetically advanced Stenus species. wissenschaften 99, 937e947. Leschen, R.A.B., Newton, A.F., 2003. Larval description, adult feeding behaviour, and Disclosure statement phylogenetic placement of Megalopinus (Coleoptera: Staphylinidae). Co- leopterists Bulletin 57, 469e493. Locke, M., Huie, P., 1979. Apolysis and the turnover of plasma membrane plaques The authors declare that there are no conflicts of interest. during cuticle formation in an insect. Tissue and Cell 11, 277e291. Lohse, G.A., 1964. 23. Familie: Staphylinidae. In: Die Käfer Mitteleuropas, vol. 4. Acknowledgments Goecke & Evers, Krefeld. Lohse, G.A., 1989. Ergänzungen und Berichtigungen zu Freude Harde Lohse “Die Käfer Mitteleuropas”. In: Lohse, G.A., Lucht, W. (Eds.), 1989. Die Käfer Mitte- We would like to thank R. Grotjahn (Cell Biology and Electron leuropas, vol. 12. Goecke & Evers, Krefeld, pp. 121e184. Microscopy, University of Bayreuth) for the assistance with the TEM Lusebrink, I., 2007. Stereoisomerie, Biosynthese und biologische Wirkung des Stenusins sowie weitere Inhaltsstoffe der Pygidialdrüsen der Kurz- and M. Heider as well as B. Förster (BIMF, University of Bayreuth) flüglergattung Stenus (Staphylinidae, Coleoptera). PhD thesis, University of for the help with the SEM analysis. V. Puthz (Castle Museum Schlitz, Bayreuth, Germany. Author's personal copy

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