Evidence for a transcellular route for vitellogenin transport in the telotrophic ovary of Podisus nigrispinus (Hemiptera: Pentatomidae)

Vitellogenin is the main yolk precursor protein in insect oocytes. It is synthesized in the fat body and released into the hemolymph. To reach the oocyte surface, vitellogenin must cross a single layer of follicular epithelium cells. The transport of vitellogenin across the follicular epithelium has been suggested to occur through the enlarged intercellular spaces (patency) by a paracellular route or by endocytosis by follicular cells and release onto oocyte surface in a transcelluar route. In this study, we investigated whether vitellogenin transport in the meroistic telotrophic ovary of Podisus nigrispinus (Hemiptera) occurs via a paracellular or transcellular route. Light and transmission electron microscopies showed that short cell–cell contacts with well-developed occluding septate junctions were present in follicular cells with patency. Immunofluorescence microscopy revealed the presence of vitellogenin receptors in the plasma membrane and of vitellogenin in the cytoplasm of follicular cells. Data suggest that cell–cell contacts serve as a barrier to large vitellogenin molecules and that this protein is transported via a transcellular route of receptor-mediated endocytosis.

vitellogenin must be transported through the follicular epithelium 25 , which surrounds the oocyte 2,6 and regulates the flow of vitellogenin to the oocyte 5,7 .
A model proposed over fifty years ago to explain the vitellogenin transport through the follicular epithelium remains almost intact. Extracellular canals formed by enlargement of the intercellular spaces between the follicular cells (patency) facilitate diffusion of soluble proteins from the hemolymph until the oocyte surface via a paracellular route. Transmission electron microscopy and immunohistochemical studies in Hyalophora cecropia (Lepidoptera) [26][27][28] and Bemisia tabaci (Hemiptera) 29 , dynamics of the vital dyes trypan blue in Aedes aegypti (Diptera) 30 and Evan's blue in Rhodnius prolixus (Hemiptera) 31 suggest the occurrence of paracellular transport of proteins trough the ovarian follicular epithelium. Conversely, in social Hymenoptera with meroistic polytrophic ovaries, ultrastructural studies indicate patency in the follicular epithelium 25 , but immunocytochemistry of vitellogenin and VgR show that both are localized in the plasma membrane and cytoplasm of follicular cells, suggesting a transcellular route in which vitellogenin initially diffuses through intercellular spaces to the follicular cells surface, bind to VgR, following clathrin-mediated endocytosis and transport to another plasma membrane domain (transcytosis) closely associated with oocyte surface 32 . This recent finding stimulated us to verify whether in other insects with meroistic telotrophic ovaries, patency is already an evidence of paracellular route for the vitellogenin transport to the oocyte surface for further endocytosis.
Podisus nigrispinus (Hemiptera: Pentatomidae) is an important generalist predator used in biological control of agricultural insect pests 33 . The development 34 , histology, cytology [35][36][37] , predator-prey interaction 38 , and biochemical processes 39 of this predator have been studied. However, to the best of our knowledge, data on the morphology and reproductive physiology of P. nigrispinus females are scarce 4,40 and the route for vitellogenin transport from the hemolymph to the oocyte surface has yet to be determined.
The objective of this study was to investigate whether vitellogenin follows a paracellular or transcellular route through the follicular epithelium in P. nigrispinus and thus contribute to the comprehension of this insect's reproduction cycle.

Results
Podisus nigrispinus oocytes in the vitellarium were enveloped by a single layer of binucleate follicular cells (Fig. 1A). Previtellogenic oocytes were characterized by their small size and homogeneous cytoplasm, lined by columnar follicular cells with narrow intercellular spaces (Fig. 1A). In advanced vitellogenesis, the perioocytic space (between oocyte surface and follicular cells) increased in size, eggshell (chorion) synthesis occurred, and intercellular spaces of follicular cells narrowed (Fig. 1D).
The ultrastructure of follicular cells during vitellogenesis in Hemiptera was previously described 2 . Therefore, we used transmission electron microscopy to evaluate intercellular contacts between follicular cells in previtellogenic and vitellogenic follicles. In the former, follicular cells showed narrow intercellular spaces along almost all their extension ( Fig. 2A) as well as some septate junctions (Fig. 2B). In the vitellogenic follicle, there were enlarged intercellular spaces, which were interrupted by short contacts between adjacent follicular cells ( Fig. 3A). At these cell-cell contact points, the intercellular space was narrow and plasma membranes showed septate junctions along almost the entire length (Fig. 3B,C).
To investigate whether vitellogenin is transported to the perioocytic space via a paracellular or transcellular route, we analyzed previtellogenic and vitellogenic follicles using immunofluorescence microscopy for detection of VgR. Western blotting showed that the antibody used recognized VgR, confirming the presence of this protein in the plasma membrane of P. nigrispinus oocytic follicles (Fig. 4). Immunofluorescence microscopy revealed the presence of VgR (Fig. 5) on the surface of follicular cells in both previtellogenic (Fig. 5A) and vitellogenic follicles, with a high fluorescent signal in the latter (Fig. 5B). Since we found high amount of VgR in the vitellogenic follicle, these were evaluated for the uptake of the vitellogenin, showing presence of this protein into the follicular cells (Fig. 6).

Discussion
Our findings show that the follicular epithelium in vitellogenic follicles of P. nigrispinus is differentiated into columnar and juxtaposed cells at the anterior and posterior poles, whereas follicular cells in lateral regions are cubic with dilated intercellular spaces. A similar morphology was observed in R. prolixus (Hemiptera) at the onset of vitellogenesis, when cells of the follicular epithelium at the anterior and posterior poles are morphologically distinct from cells in the lateral portion 41 . The intercellular spaces between follicular cells of the epithelium lateral to the oocyte enlarge progressively with oocyte growth, whereas polar follicular cells continue to possess narrow intercellular spaces and might act as a barrier for the passage of yolk proteins 42,43 , but the physiological significance of this difference remains unknown. www.nature.com/scientificreports www.nature.com/scientificreports/ Enlargement of intercellular spaces in the follicular epithelium (patency) during vitellogenesis, as observed in the present study in P. nigrispinus, has been reported in H. cecropia 26,27 , R. prolixus 28,41 and B. tabaci 29 . Those studies show accumulation of hemolymph proteins in the intercellular spaces of follicular cells, which has been interpreted as an evidence that these proteins, including vitellogenin, are transported to the oocyte surface via a paracellular route. However, in bees, ants, and wasps, although patency occurs, vitellogenin is transported to the oocyte surface by receptor-mediated endocytosis, that is, via a transcellular route 25,32 .
Light and transmission electron microscopies showed that patency occurs in the follicular epithelium of P. nigrispinus vitellogenic follicles but also revealed that follicular cells display septate junctions in small cell-cell contact regions. Septate junctions play an important role in maintaining the mechanical and physiological integrity of the epithelium 41,43,44 . In addition, septate junctions are important permeability barriers that control the transit of molecules through invertebrate epithelia [45][46][47][48] . Some chemical species such as lanthanum ions (La 3+ ), which have high charge density and occur in a hydrated state, can cross septate junctions 43,49,50 ; however, the passage of vitellogenin, which is a large protein with a molecular mass of 200-700 kDa 14,51 , seems unlikely, as septate junctions in different epithelia of insects are effective barriers to some ions and molecules larger than 7 kDa 49,52-54 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Together, ultrastructural and immunofluorescence data show that intercellular spaces are blocked by septate junctions and that the plasma membrane of follicular cells contains VgR and vitellogenin, suggesting a transcellular route of vitellogenin transport to the perioocytic space, as has been demonstrated in Hymenoptera 32 . However, the question remains as to why patency occurs in follicular cells of insects during vitellogenesis. A possible explanation would be that the almost complete separation of adjacent follicular cells allows the exposure of a larger area of the plasma membrane to the hemolymph and thus exposes many VgR molecules for vitellogenin uptake, resulting in a high transport rate of reserve proteins to the oocyte.
The present study provides insight into the transcellular route of vitellogenin transport through the follicular epithelium in meroistic telotrophic ovaries, which seems to be a conserved route in insects, as it also occurs in insects with meroistic polytrophic ovaries. Further investigation is needed to elucidate the routes of vitellogenin transport in insects with panoistic ovaries. The ovaries were transferred to 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 12 h. After being washed in the same buffer, samples were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in a graded ethanol series (70, 80, 90, 95, and 98%), and embedded in LR White resin. Ultrathin sections, obtained using an ultramicrotome equipped with a glass knife, were stained with 2% aqueous uranyl acetate and lead citrate and analyzed using a transmission electron microscope (Zeiss LIBRA 120). www.nature.com/scientificreports www.nature.com/scientificreports/ Western blot analysis. Podisus nigrispinus females were dissected, and the ovaries were homogenized in 50 mM Tris-HCl buffer, pH 7.5, containing 10% protease inhibitor cocktail (P2714-1BTL, Sigma-Aldrich) using a cordless motor. Procedures were performed in triplicate. Then, samples were centrifuged at 10,000 × g for 15 min, and the supernatant containing soluble proteins was collected. The pellet was resuspended in Tris-HCl buffer, pH 7.5, 1% Triton X-100, and 10% protease inhibitor, homogenized, placed in an ultrasonic bath for extraction of membrane proteins, and centrifuged at 10,000 × g for 15 min. Supernatants containing soluble membrane proteins were submitted to SDS-PAGE 55 . The gel was incubated in transfer buffer [0.58% (w/v) Tris-HCl, 0.28% (v/v) glycine, 20% (v/v) methanol, and ultrapure water] and transferred to a nitrocellulose membrane by electroblotting at 190 A and 4 °C for 3 h (Mini Trans-Blot Cell ® , Bio-Rad). Subsequently, the nitrocellulose membrane was incubated in Tris-buffered saline (TBS) (25 mM Tris and 150 mM NaCl, pH 7.5) with 1% Tween 20 (TBST) and 5% skimmed milk powder for 16 h. The nitrocellulose membrane was rapidly washed with TBST, washed twice with TBST for 15 min, and incubated for 2 h with primary antibodies against vitellogenin receptor (anti-VgR) 32 (1:500). After washing with TBST, the nitrocellulose membrane was incubated with peroxidase-conjugated anti-mouse IgG secondary antibody (1:100) for 2 h. Then, samples were revealed with a diaminobenzidine solution [0.1% (w/v) DAB, 50 mM Tris-HCl, and H 2 O 2 ].
Immunofluorescence microscopy. After dissection, the ovarioles had the peritoneal sheath removed with tweezers and were immersed in Zamboni fixative [4% (w/v) paraformaldehyde and 0.4% (w/v) picric acid in