Protoplasma DOI 10.1007/s00709-015-0843-0

ORIGINAL ARTICLE

Changes in follicular cells architecture during vitellogenin transport in the ovary of social Hymenoptera Milton Ronnau 1,2 & Dihego Oliveira Azevedo 1,3 & Maria do Carmo Queiroz Fialho 4 & Wagner Gonzaga Gonçlaves 1 & José Cola Zanuncio 5 & José Eduardo Serrão 1

Received: 27 February 2015 / Accepted: 2 June 2015 # Springer-Verlag Wien 2015

Abstract Vitellogenins are the major yolk proteins, synthesized in the fat body, released into the hemolymph and captured by the developing oocytes, but the mechanisms by which these proteins cross the follicular cell layer are still poorly understood. This study describes the actin distribution in follicular cells during vitellogenin transport to the oocyte in social Hymenoptera represented by bees Apis mellifera and Melipona quadrifasciata, the wasp Mischocyttarus cassununga and the ant Pachycondyla curvinodis. In oocytic chambers of vitellogenic follicles, vitellogenin was found within the follicular cells, perivitelline space and oocyte, indicating a transcellular route from the hemolymph to the perivitelline space. The cortical actin cytoskeleton in follicular cells underwent reorganization during transport of vitellogenin across this epithelium suggesting that in the ovary of social hymenopterans, vitellogenin delivery to oocytes requires a dynamic cytoskeletal rearrangement of actin filaments in the follicular cells.

Handling Editor: Douglas Chandler * José Eduardo Serrão [email protected] 1

Department of General Biology, Federal University of Viçosa, 36570-000 Viçosa, MG, Brazil

2

Department of Biology, Federal University of Paraná, Palotina, PR, Brazil

3

Federal Insitute of Espírito Santo, Ibatiba, ES, Brazil

4

Department of Morphology, Federal University of Amazonas, Manaus, AM, Brazil

5

Department of Entomology, Federal University of Viçosa, Viçosa, MG, Brazil

Keywords Actin . Ants . Bees . Cytoskeleton . Reproduction . Wasp . Yolk

Introduction In eusocial insects, queens and workers differ morphologically and have a sophisticated division of labor. Their colonies are usually large with a complex system of communication and an elaborate nest structure (Bourke 1999), where workers have some labor division on basis of their age (Amdam and Omholt 2003; Fagundes et al. 2006). The female reproductive tract of insects consists of a pair of ovaries interconnected by a pair of lateral oviducts and an unpaired common oviduct (Snodgrass 1935; Chapman 2013). The ovary itself is divided into functional units called ovarioles (Snodgrass 1935; Chapman 2013). In Hymenoptera the ovarioles are of the meroistic polytrophic type, which have three regions: terminal filament, formed by connective tissue in the distal region, germarium where the germ line cells occurs, and vitellarium, a relatively large portion of the ovariole, which is characterized by the presence of growing oocytes with their nurse cells (Snodgrass 1935; Martins and Serrão 2004). In the vitellarium, both oocytes and nurse cells are lined by a single-layered follicular epithelium resulting in oocytic and in nurse chambers (Snodgrass 1935; Lisboa et al. 2005). In the vitellarium occurs the maturation of the oocyte by the transference of cytoplasmic material from the nurse cells to oocyte (previtellogenesis) followed by a period of rapid deposition of yolk (vitellogenesis) (Buning 1994; Souza et al. 2007). The vitellogenesis involves the large-scale production of vitellogenin in the fat body, its release into the hemolymph and uptake by oocytes, and its storage as yolk granules in the

M. Ronnau et al.

ooplasm (Engelmann 1979; Raikhel and Dhadialla 1992; Tufail and Takeda 2008). To reach the oocyte, vitellogenin must cross the follicular epithelium surrounding the oocyte. However, the mechanisms by which these proteins cross the follicular cell layer remain unknown. In some, insects are formed intercellular spaces between follicle cells (termed patency), suggesting a paracellular route for the transport of vitellogenin to the oocyte (Anderson and Telfer 1970; Huebner and Anderson 1972; Bast and Telfer 1976; Abu Hakima and Davey 1977; Telfer 1961; Davey et al. 1993). In fact, in the honey bee Apis mellifera tiny channels have been observed between the follicular cells, suggesting an effective transport mechanism for the vitellogenin to the perivitelline space, located between the oocyte and follicular cell layer (Engels 1973; Cruz-Landim et al. 2013). Nevertheless, it is uncertain whether vitellogenin is transported to the surface of oocytes via this intercellular route or by a transcellular route through the follicular cell cytoplasm. Indeed, vitellogenin has been found in granules in the cytoplasm of follicular cells (Fleig 1995). Seehuus et al. (2007) reported the accumulation of vitellogenin in the perivitelline space of A. mellifera, suggesting that transport can occur by both trans and paracellular routes. In both trans and paracellular routes, the cytoskeletal organization may be called upon to change the follicular cell architecture to allow the vitellogenin transport to the oocyte. Actin filaments are known to play an important functional role in the cytoskeleton of eukaryotic cells (Alberts et al. 2014) and likely important in changes the cell cortical architecture important to vitellogenin transport. In this study, we provide new evidence for vitellogenin transport by the transcellular route and we evaluate the reorganization of the actin cytoskeleton in follicle cells during this process. In addition, our study, for the first time, provides evidence of vitellogenin transport in social Hymenoptera such as, honeybee, stingless bee, ponerine ant, and polistine wasp, thus contributing to our understanding of the reproductive physiology of these insects.

Queens were cryo-anesthetized at 0 °C for 5 min and the ovaries were dissected in the presence of 125 mM NaCl plus protease inhibitor cocktail (Sigma-Aldrich).

Transmission electron microscopy The ovaries were dissected and transferred to 2.5 % glutaraldehyde in sodium cacodylate buffer 0.1 M pH 7.2 for 30 min. After, the ovarioles were sectioned in their previtellogenic (characterized by the presence of nurse chamber larger than the oocytic chamber) and vitellogenic (characterized by oocytic chamber larger than nurse chamber) portions. Then the samples were post-fixed in 1 % osmium tetroxide in the same buffer, dehydrated in a graded ethanol series (70, 80, 90, 95 %), and embedded in LR White resin.

Materials and methods Insects Females laying eggs of the wasp Mischocyttarus cassununga, queens of the honeybee A. mellifera, and of the stingless bee Melipona quadrifasciata were obtained in the apiary of the Federal University of Viçosa (UFV). Queens of the ant Pachycondyla curvinodis were obtained from colonies maintained at the Department of General Biology at UFV.

Fig. 1 Confocal micrographs of the oocytic chamber. a Vitellogenic follicle of Apis mellifera showing the presence of vitellogenin (green) into follicular cells (FC) and into oocyte (OO). b Vitellogenic follicle of Pachycondyla curvinodis showing the presence of vitellogenin (green) within the follicular cells (FC) and in the oocyte (OO). N—nucleus of follicular cells

Vitellogenin transport in hymenoptera

Ultrathin sections (60–90 nm thick) were stained with 1 % uranyl acetate and lead citrate and analyzed in an EM 109 transmission electron microscope Zeiss at Nucleus of Microscopy and Microanalysis (NMM) at UFV. Immunofluorescence After dissection, the peritoneal sheath of the ovarioles were removed with forceps and they were transferred to 4 % paraformaldehyde fixative solution in sodium phosphate buffer Fig. 2 Transmission electron micrographs of oocytic chamber. a Previtellogenic follicular epithelium in Apis mellifera showing narrowed lateral intercellular spaces (arrows) and basal regions with enlarged subepithelial space filled with granular material (arrowheads). b Previtellogenic follicular epithelium in Mischocyttarus cassununga showing apical region of cell-cell contact with narrow intercellular space (arrowhead) and an electrondense belt at the apex (arrow). c Detail of the apical belt showing an adherens junction. d Vitellogenic follicle of M. cassununga showing the general aspect of follicular cells with enlarged intercellular spaces in the cell apex and base containing granular material (asterisks) which is also present in the perivitelline space (PVS). BL—basal lamina, M— mitochondria, N—nucleus, Nu— nucleolus, OO—oocyte, PS— peritoneal sheath

0.1 M, pH 7.2 (PBS) for 3 h at 4 °C followed by washes in PBS containing Triton X-100 0.3 % (PBST). Then, the samples were pre-incubated in 2 % normal rabbit serum in PBST for 2 h at room temperature, followed by incubation for 3 days 4 °C in anti-vitellogenin primary antibody (Azevedo et al. 2011) 1:500 in PBST. Then the samples were transferred to secondary antibody anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) 1:500 in PBST for 60 h in the dark and incubated in RNAse 1:50 in PBST for 40 min. The samples were then transferred to propidium iodide (5 mg/mL) for

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5 min and washed in PBST for 2 h, mounted with 50 % sucrose and analyzed in a confocal laser fluorescence microscope Zeiss 510 Meta at NMM. Detection of actin After dissection, the ovaries were transferred to Zamboni fixative solution (Stefanini et al. 1967) for 30 min. They were then washed three times with PBST. Then, the ovaries were incubated in the dark with phalloidin conjugated with rhodamine 1:100 in PBST for 30 min, mounted with 50 % sucrose and analyzed in a confocal laser fluorescence microscope Zeiss 510 Meta.

filaments (Fig. 3c) that coincided with ultrastructural changes in these lateral faces during vitellogenin transport as documented in Fig. 2d. In addition, at the apical surface of these follicular cells, there occurred an increase in the amount of filamentous actin with the new cortical network exhibiting short projections toward the oocyte (Fig. 3c).

Discussion In all species analyzed, the oocytic chambers of vitellogenic follicles have vitellogenin in the follicular cell cytoplasm, perivitelline space, and ooplasm. Anderson and Telfer

Results In the four studied species of social Hymenoptera, the wall of the vitellogenic oocytic chamber was formed by a single layer of cubic follicular cells. Fluorescence immunocytochemistry combined with laser scanning confocal microscopy revealed an accumulation of vitellogenin within the cytoplasm of follicle cells and a lesser amount within the ooplasm of each species eggs (Fig. 1a, b). In all studied species of Hymenoptera, the follicular cells of the previtellogenic ovarian follicles showed a well-developed nucleus containing decondesed chromatin and a nucleolus (Fig. 2a). The cell cytoplasm was rich in mitochondria (M) and some myelin figures (MF) were present (Fig. 2a). The follicular cell-cell contact regions had a narrow intercellular space (Figs. 2a, b) with an apical electron-dense belt (Fig. 2b), which at high resolution was showed to be an adherens junction (Fig. 2c). During the vitellogenesis, the apical region of the follicular cells exhibited occasional myelin figures while both the apical and perinuclear regions were rich in mitochondria (Fig. 2d). The main difference found in the vitellogenic follicles, compared to previtellogenic ones, were enlarged intercellular spaces in the apical and basal regions of the lateral faces between follicle cells (asterisks, Fig. 2d) while the central regions of these faces remained in close apposition. The enlarged intercellular space was filled with granular material, which was also found in the perivitelline space (Fig. 2d). These morphological changes in follicular cells were accompanied by a reorganization of the actin cytoskeleton (Fig. 3). Laser-scanning confocal microscopy showed that in previtellogenic follicles a dense cortical actin filament layer was seen applied to all plasma membrane faces (e.g., apical and lateral) but that there were interruptions in this layer at the basal plasma membrane of some cells (asterisks, Fig. 3a, b). A similar distribution of cortical actin filaments was seen in the follicular cells of vitellogenic follicles with one important exception: the lateral regions demonstrated an absence of actin

Fig. 3 Confocal micrographs of oocytic chamber. a Actin filaments (red) in the follicular cells (FC) of previtellogenic follicle of Melipona quadrifasciata showing interruptions (asterisks) in the basal portion and homogeneous distribution in the lateral region (arrows). b Actin filaments in the follicular cells (FC) of previtellogenic follicle of Apis mellifera showing interruptions (asterisks) in the cell base and continuous distribution in the apical region (arrow). c Actin filaments in the follicular cells (FC) of vitellogenic follicle of Apis mellifera showing the disruption in the basal (asterisks) and lateral cell regions (arrows). Note a high occurrence of actin filaments at the cell apex with projections toward the oocyte (OO)

Vitellogenin transport in hymenoptera

(1970) state that the passage of vitellogenin by the follicle epithelium occurs through canals in the intercellular spaces of the follicular cells during vitellogenesis in Cecropia sp (Lepidoptera), suggesting a paracellular route. A paracellular route has been also suggested in Hyalophora cecropia (Lepidoptera) (Telfer 1961), Rhodnius prolixus (Hemiptera) (Davey 1981), Locusta migratoria (Orthoptera) (Lauverjat et al. 1984), and mosquitoes (Raikhel and Lea 1991). Interestingly in social Hymenoptera here studied, vitellogenin was found inside the follicular cells indicating a transcellular route for vitellogenin transport to the perivitelline space. In R. prolixus, it has been reported that the follicular cells may participate in the vitellogenin synthesis (Melo et al. 2000), but this seems not to be the case during the vitellogenesis in social Hymenoptera because the follicular cells have no features of secretory cells producing large amounts of protein such as well-developed rough endoplasmic reticulum and secretory granules (Alberts et al. 2014). The social Hymenoptera species studied here have a belt of adherens junction in the apical region of cell–cell contact of follicular cells in both pre- and vitellogenic follicles. Membrane junctions in the apical region are essential for controlling epithelial permeability (Huebner and Injeyan 1980; Serrão et al. 2004), supporting the hypothesis of the transcellular transport of vitellogenin by follicular cells to the oocyte. Although Cruz-Landim et al. (2013) describe that lanthanum ions cross the intercellular spaces of the follicular cells in A. mellifera, this molecule is too small compared with circulating vitellogenin in the hemolymph of insects (Wheeler et al. 1999; Tufail and Takeda 2008), which in bees and ants has a molecular weight between 300 and 500 kDa (Azevedo et al. 2011). Noteworthy is that our immunofluorescence analyses show vitellogenin in the follicular cell cytoplasm suggesting a possible transcellular together with the paracellular route. This intracellular vitellogenin localization will be studied in the future using high resolution electron microscopic immunolocalization. The different arrangements of actin filaments between the follicular cells of previtellogenic and vitellogenic follicles of social Hymenoptera suggest the rearrange of actin filaments in response to the vitellogenin transport. Firstly, during vitellogenic transport/patency, the oocyte undergoes changes in its volume allowing the opening of canals between the follicular cells to expose the hemolymph the surface of the oocyte (Huebner and Injeyan 1980). The formation of these intercellular canals is under juvenile hormone control, requiring a rearrangement of the cytoskeleton and an increase in cell volume after vitellogenin uptake with the basal intercellular region controlling the passage of vitellogenin to the oocyte (Davey 2007). In all species studied here, cortical actin filaments have interruptions at the basal surface and disorganization in the lateral region of the follicular cells in the oocytic

chamber of vitellogenic follicles, which may be due to the enlargement of the intercellular spaces. Although Patrício and Cruz-Landim (2006, 2011) have pointed out that F-actin play important roles in the ovary structure as well as in cell motility in the germarium of bees, studies on F-actin in the insect ovary are largely restricted to its organization in the nurse cells and oocyte during early vitellogenesis (the previtellogenic stage herein) (Gutzeit and Huebner 1986; Theurkauf et al. 1992; Patrício and CruzLandim 2006, 2011; Amaral and Machado-Santelli 2009). The current study has expanded our knowledge by showing that changes in follicular cell architecture during vitellogenesis proper are likely actin based and are important for improving the transport of vitellogenin from the hemolymph to the oocyte. Acknowledgments This research was supported by Brazilian research Agencies, FAPEMIG, CNPq, and CAPES. Authors are grateful to Nucleus of Microscopy and Microanalysis at Federal University of Viçosa for technical assistance and to anonymous reviewers for criticism and manuscript improvement. Conflict of interest The authors declare that they have no conflict of interest.

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