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Coevolution between female seminal receptacle and sperm morphology in the semiaquatic measurer bug Hydrometra stagnorum L. (Heteroptera, Hydrometridae)
Arthropod Structure & Development 60 (2021) 101001
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Coevolution between female seminal receptacle and sperm morphology in the semiaquatic measurer bug Hydrometra stagnorum L. (Heteroptera, Hydrometridae) Romano Dallai*, Pietro Paolo Fanciulli, David Mercati, Pietro Lupetti Department of Life Sciences, University of Siena, Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2020 Accepted 7 October 2020 Available online xxx
The coevolution between sperm length and size of the female sperm-storage organs is described for the first time within Heteroptera. The long sperm of the measurer bug Hydrometra stagnorum is characterized by the unusually long acrosome with its anterior region helically arranged, and by a very short nucleus. The sperm flagellum has a 9 þ 9þ2 conventional axoneme and crystallized mitochondrial derivatives. The female spermatheca consists of an extraordinarily long spermathecal duct ending with an apical spermathecal bulb into which flows also the secretions of a relatively short spermathecal gland. Both spermathecal duct and gland have a thin epithelium lined by a cuticle, beneath which a complex of secretory and duct forming cells are present. The secretions of these two structures flow into the apical spermathecal bulb. A thick layer of muscle fibers surrounds the epithelium. These results confirm the opinion that the dimensions of the female reproductive sperm-storage organs are able to drive the sperm morphology. © 2020 Elsevier Ltd. All rights reserved.
Keywords: Semiaquatic bug Coevolution Electron microscopy Insect sperm morphology
1. Introduction It is well known that the fertilization success in different organisms depends on interplay between sperm length and the length of the female primary sperm storage organ, the seminal receptacle (Miller and Pitnick, 2002). This relationship was initially observed in Drosophila, namely throughout comparative studies on Drosophila pseudoobscura and Drosophila bifurca (Pitnick et al., 1999). It was hypothesized that the presence of long seminal receptacles drives the production of longer sperm, as these are capable to better displace and resist displacement by competitor sperm (Pattarini et al., 2006). Similar results were recently obtained on the obsolete groundelice Zorotypus impolitus and Zorotypus caudelli (Dallai et al., 2014) as well as on some Onthophagus beetles (Hunt et al., 2002; Werner and Simmons, 2011), suggesting that the evolution of sperm elongation in these species may be regarded as a possible consequence of the observed modifications in the female genital tract. Also studies on diving beetles (Dytiscidae) have
* Corresponding author. E-mail addresses: [email protected] (R. Dallai), [email protected] (P.P. Fanciulli), [email protected] (D. Mercati), [email protected] (P. Lupetti). https://doi.org/10.1016/j.asd.2020.101001 1467-8039/© 2020 Elsevier Ltd. All rights reserved.
revealed that sperm morphology within these groups was correlated with the dimension of the female reproductive tract and, in particular, evolved in response to changes in the organization of the female sperm-storage organs (Higginson et al., 2012a). Moreover, several evidences suggested that female reproductive morphology may evolve first and can act as a driver for the subsequent evolution of the sperm structures (complementary evolution) (Pitnick et al., 1995; Miller and Pitnick, 2002; Higginson et al., 2012b). These studies have, in fact, led to the hypothesis that post-copulatory sexual selection determined by female reproductive structures may be able to bias paternity by determining the best sperm for fertilization. Here we describe a novel example of coevolution between the male and female reproductive structures in the measurer bug Hydrometra stagnorum L, which is one of the most common species of the semiaquatic species of the infraorder Gerromorpha (Heteroptera) alongside Nepomorpha and Leptodomorpha (Andersen, 1982; Wheeler et al., 1993; Xie et al., 2008), all characterized by a long and peculiar sperm, possibly evolved as consequence of a very long female reproductive tract. These groups have an old origin, dating back to late Permian to Early Triassic (269-246 Ma) (Wang et al., 2016). H. stagnorum is a member of the Hydrometridae family. The infraorder Gerromorpha also comprises: Gerridae,
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1.5 mm long, and further with a thicker elliptical region 1.5 mm long and 0.15 mm wide, possibly the seminal vesicle. The most proximal tract of deferent duct is connected with that of the opposite side. At the end of the reproductive time, testes show a reduced dimension with the most proximal region becoming narrowed (Fig. 1A and B).
Veliidae, Hebridae, Macroveliidae and Paraphrynoveliida. A close relationship between Hydrometridae and Macrovelidae is well supported by phylogenetic analyses (Andersen and Weir, 2004; Damgaard, 2008). Hydrometridae is a monophyletic family supported by the prolonged head capsule, tuberculated posterior pair of cephalic trichobotria and apically modified antennae (Andersen and Weir, 2004).
3.2. Sperm structure 2. Material and methods The sperm of the measurer H. stagnorum are long cells (1520 mm) that are arranged in helical fashion within the testes (Fig. 1C and D). They are characterised by an extraordinarily long cylindrical acrosome (367 mm), a very short nucleus (8 mm) and a long cylindrical flagellum (1145 mm) (Fig. 1E). The acrosome, 0.5 mm in diameter, in its anterior region (about 200 mm long) shows a helical array (Fig. 1F); it contains a bundle of longitudinal tubules, 16 nm in diameter each, which fills the whole acrosome lumen except for a small eccentric area. These tubules are separated by dense material and have a twisted configuration (see also Dallai and Afzelius, 1980). The nucleus, 0.68 mm in diameter, contains compact chromatin material (Fig. 1G and H). The flagellum, 0.62 mm in diameter, comprises an axoneme 9 þ 9þ2 microtubules flanked by two long crescent-shaped mitochondrial derivatives, that embrace axonemal structure (Fig. 1H and I). Two filamentous bridges connect the mitochondrial derivatives to the flattened cisterns adherent to doublets 1e2 and 4e5 of the axoneme, as it occurs in Heteroptera in general (Dallai and Afzelius, 1980; Mercati et al., 2009). Two small dense spots are present above the acrosome. Three crystalline inclusions are evident in the mitochondrial matrix (Fig. 1I).
Males and females of the measurer bug H. stagnorum (L.) were collected in the neighbourhood of Siena and Grosseto (Italy, Tuscany). 2.1. Light and epifluorescence microscopic preparations The male and female genital systems of H. stagnorum were dissected in 0.1 M phosphate buffer to which 3% sucrose was added (PB1). A drop of sperm was taken from dissected deferent ducts of males, were spread on a histological slide and a drop of 1 mg/ml of the DNA specific dye Hoechst 33.258 in 0.1 M PB1 was added, and the sample was covered with a coverslip. The spermathecal complex, dissected out in PB1 from adult fertilized females was placed on a glass. As above described, a drop of Hoechst was added and a coverslip applied and gently pressed. Fluorescence observation of the labelled samples were carried out with a Leica DMRB light microscope equipped with UV light source, fluorescein and UV filters and a Zeiss AxioCam digital camera with dedicated imaging software. Confocal laser scanning microscopy on free sperm was carried out on a Zeiss LSM700.
3.3. Female genital apparatus and spermathecal complex 2.2. Electron microscopic preparations The genital system of H. stagnorum young female consists of an ovary with six long ovarioles (1.5 mm long) on each side, which flow in large calices. When the ovaries are immature, only three ovarioles are visible (Fig. 2AeC). The lateral oviducts, 600 mm long, fuse in a common oviduct, about 1.0 mm long, at the posterior end of which a large structure, the spermatheca, 500 mm long, is present. It is a flattened complex structure tightly surrounded by a layer of fat body (Fig. 2AeC). When dissected, such structure reveals to consist of an extraordinary long and thin convoluted cylindrical duct ending in an apical spherical bulb (Fig. 2D). The duct is about 6500 mm long and at its end, where it opens into the vagina, it is only 6 mm in diameter, while at half-length it is about 16e17 mm in diameter; where the duct flows into the apical bulb it is only 9.5e10 mm in diameter (Fig. 3). At this level it runs parallel to the spermathecal gland, an almost cylindrical duct, 450 mm long and about 12.5 mm in diameter, narrowing down to 10 mm at its free extremity. When the spermathecal and the glandular ducts flow into the apical bulb, the diameter of this region widens up to 33 mm. The apical spermathecal bulb is about 103 mm in diameter (Fig. 3). A cross section of the spermathecal duct shows a thin epithelium lined by a 0.8e0.85 mm thick cuticle, with a very thin epicuticle layer (Fig. 4A). In some regions, the epithelium thickens up to 3.0 mm with cells containing elliptical nuclei (4.3 mm 2.0 mm). Several scattered large secretory cells provided with a large cistern are visible beneath the epithelium (Fig. 4BeD). When observed at low magnification, long tracts of spermathecal duct devoid of such secretory cells alternate with others, where complexes of secretory cells are visible (Fig. 4AeD). These cells are rich in mitochondria, rough endoplasmic reticulum and show scattered Golgi bodies. They have a large nucleus (8.7 mm 6.3 mm) with a conspicuous nucleolus. The large extracellular cistern is bordered by long
Male reproductive apparatuses of the species, after dissection, were fixed overnight at 4 C in 3% glutaraldehyde in PB1. After rinsing in PB1, the material was post-fixed in 2% OsO4 in PB1 for 2 h. After rinsing, the material was dehydrated and embedding in EponAraldite mixture. Part of material was fixed according to Dallai and Afzelius (1990) using 1% tannic acid, omitting post-fixation with osmium and using block staining with 1% aqueous uranyl acetate. Female reproductive apparatuses of the species were dissected out in 0.1 M phosphate buffer to which 5% sucrose was added (PB2). The last tract of the common oviduct containing the spermathecal complex was removed and fixed overnight in 3% glutaraldehyde in PB2. After rinsing, the material was post-fixed in OsO4 in PB2 for 2 h, rinsed again, dehydrated and embedded in the mixture EponAraldite resin. For both male and female reproductive system of the species, semi-thin and ultrathin sections, obtained with a Reichert Ultracut ultramicrotome. Semi-thin sections were routinely stained with 0.1% toluidine blue and observed with a Leica DMRB interference light microscope equipped with a Zeiss AxioCam digital camera. Ultrathin sections were routinely stained with uranyl acetate and lead citrate, and observed in a Philips CM 10 transmission electron microscope operating at an electron voltage of 80 kV. 3. Results 3.1. Male genital system of H. stagnorum The mature male genital apparatus of H. stagnorum consists of a pair of elongated conical testes, each about 2.0 mm long and 0.2 mm wide, that basally continue with a very thin region, about 2
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Fig. 1. A-B- Light microscope views of mature male genital apparatus at the reproduction time (A) and at the end of this stage (B). Note the different aspect of the seminal vesicles (sv). T, testes. C- Fluorescent light microscopy of testes with large sperm bundles (sp) in helical fashion. D- Light microscope view of testes showing sperm bundles (sp) in helical fashion. E- The whole sperm under the fluorescent light microscope. The strong fluorescence of the acrosome (A) is due to the intrinsic FAD presence at the acrosome (see Miyata et al., 2011). The Hoechst staining marks the short nucleus (N). F, flagellum. F- Detail of the acrosome (A) to show the helical array of the anterior region. G- Detail of the short nucleus (N) between the acrosome (A) and the flagellum (F). H- Cross section of a sperm bundle within the spermathecal duct showing the acrosome (A) with tubules embedded in a dense material and a flagellar axoneme (ax) with two mitochondrial derivatives (md) embracing the axoneme. I- Higher magnification of a cross sectioned axoneme (ax) after tannic acid preparation. Note the protofilaments in the tubule walls and the bridges connecting the axoneme to the mitochondria (arrows). Note also the three regions of crystallization in the mitochondrial matrix. 3
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Arthropod Structure & Development 60 (2021) 101001
Fig. 2. A- Genital apparatus of a young female with immature ovaries (o), lateral oviducts (lov) and spermatheca (spt). B- Genital apparatus of a mature female with two eggs (egg) ready to cross the oviducts (ov). spt, spermatheca. C- Female genital apparatus at the end of reproductive time showing ovaries (o) with immature eggs. spt, spermatheca. D- Light interference microscope showing the apical spermathecal bulb (sptb), the long spermathecal duct (sptd) and the short spermathecal gland (sptg).
connecting duct fuses with that of the spermathecal duct and its lumen opens into the lumen of the duct (Fig. 5D and E). For long tracts, beneath the epithelial cells, the insertions of muscle cells are visible. The epithelial cells are transformed into typical structures adapted for the attachment of muscles to the thick cuticle lining the duct. They are filled with longitudinal bundles of microtubules anchoring on apical short cylindrical cuticular invaginations to form hemidesmosomes, while at the cell base they form desmosomes with the sarcolemma (Fig. 5C). Beneath the basal lamina
microvilli (Fig. 4BeD and Fig 5C). In the cistern lumen, small dense vesicles are visible between microvilli; in a few cases, the cistern appears filled with electron-dense secretion (Fig. 4C and D). This secretion is set up into a typical end apparatus consisting of a cuticular duct of 0.37 mm in diameter, surrounded by a 0.47 mm thick layer of loose filaments (Figs. 4D and 5A). This connecting duct is formed by a duct forming cell (canal cell), intermingled with epithelial cells, showing a thin layer of cytoplasm and a slender nucleus. Close to the spermathecal duct, the cuticular layer of the 4
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Fig. 3. Confocal light microscope of a spermatheca with the spermathecal apical bulb (sptb), the strong fluorescence of the spermathecal duct (sptd) due to the intrinsic fluorescence of the sperm acrosomes filling the duct lumen and the short spermathecal gland (sptg).
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Fig. 4. A- Longitudinal section of the spermathecal duct filled with many sperm (sp) cross sectioned at different levels. A, acrosomes, F, flagella. Note the thin epithelium (Ep) lined by a thick cuticle (ct). BeC-D-Cross sections of the spermathecal duct (sptd) showing large secretory cells (sec) beneath the thin epithelium (Ep). Note the expanded cisterns (ci) with long microvilli (mv) bordering their lumen. In C, the cistern (ci) contains a complex of dense granules and thin filaments. In D, the cytoplasm shows cisterns of rough endoplasmic reticulum (rer), numerous mitochondria (mt), vesicles and dense bodies (d). The axial region of the cistern shows the end apparatus of the duct forming cell (end). A large nucleus (N1) of the secretory cell is visible at the periphery of the cell, while an elliptical nucleus (N2) of the epithelial cell is beneath the cuticle.
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Fig. 5. A- Cross section trough a cistern of a secretory cell (sec) with the end apparatus (end) of a duct forming cell. This cell is beneath the epithelium (Ep) intermingled with secretory cells. ct, cuticle. B- Detail of the secretory cell with long microvilli (mv) bordering the lumen of the cistern (ci). Note the thick cuticle (ct) lining the epithelial cells (Ep). CSome epithelial cells are often adapted to allow the insertion of the muscle fibres (ms) in the cell complex surrounding the spermathecal duct. Apical hemidesmosomes (hd) and basal desmosomes (d) are formed and serve as an anchorage of microtubule bundles (mi). D-E-Cross sections through the spermathecal duct showing a thin epithelial cell (Ep) with elongated nucleus (N) and lined by thick cuticle (ct). The region corresponds to the point where the canal (ca) of the duct forming cell (dfc) leads into the spermathecal duct lumen (sptl). Numerous sperm (sp) are visible in the lumen. In E, the cistern (ci) of a secretory cell (sec) is also present.
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conspicuous muscle structures with myofibril filaments in the typical array of striated muscles are visible (Fig. 5C). Many sperm sectioned at different levels are visible in the spermathecal duct lumen. Towards the posterior region close to the duct entrance into the vagina, the duct epithelium becomes thin and it shows a narrowed lumen due to a thicker cuticle, up to 6.0e7.5 mm high (Fig. 6A). In the lumen a dense secretion obscures the presence of a few sperm (Fig. 6A). The region close to the entrance of the spermathecal duct into the spermathecal apical bulb is also characterized by a thin epithelium lined by a very thick cuticle (about 6.5e7.0 mm high) surrounding a narrow lumen filled with many sperm (Fig. 6B). The apical sperm receptacle has a simple epithelium, about 23e27 mm high, lined by a 2.5e3.0 mm high cuticle, without
secretory cells (Fig. 6D). The cytoplasm does not show many secretory structures, while beneath the cuticle numerous mitochondria are visible (Fig. 6D). In the lumen, bundles of sperm are intermingled with large amount of dense secretions. The spermathecal gland has a fine structure similar to that of the spermathecal duct with secretory and duct forming cells (Fig. 7). The secretory cells are regularly distributed along the gland length and discharge their secretions into the gland lumen via a thin efferent duct produced by the canal cell. At the level of the entrance of the two structures, i.e., the spermathecal duct and the spermathecal gland, into the apical sperm receptacle, a muscle complex is observable (Fig. 6C). When observed under the fluorescence microscope the sperm are visible along the spermathecal duct with the acrosomes directed towards the duct opening into the vagina (Fig. 3).
Fig. 6. A- Cross section of the spermathecal duct close to its entrance into the vagina. Note the thicker cuticle (ct) lining the epithelium (Ep) and the narrowed duct lumen (L) filled with dense material obscuring the presence of some sperm. B- Cross section of the region close to the entrance of the spermathecal duct into the spermathecal apical bulb. Note the thicker cuticle (ct) and the lumen filled with sperm (sp). C- Cross section of the muscle complex (ms) of the region close to the entrance of the spermathecal duct into the apical bulb. D- Cross section trough the spermathecal apical bulb. The apical epithelial region is rich in mitochondria (mt), while inclusions are scarse. N, nuclei; ct, cuticle.
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Fig. 7. Cross section trough a secretory cell (sec) of the spermathecal gland. In the cytoplasm several mitochondria (mt), cisterns of rough endoplasmic reticulum (rer) and Golgi bodies (G) are present. Close to the cistern, several dense bodies (d) are visible. Thin epithelial cells (Ep) are line by a thick cuticle (ct). N, nucleus.
fertilization success depends on the interaction between male and female reproductive organs (Pitnick et al., 2009). Example of a positive coevolution between female sperm-storage organs and their ducts with sperm length have been described, other than in fruit flies and particularly D. bifurca (Pitnick et al., 2009), in groundlice (Zoraptera) (Dallai et al., 2012, 2014), featherwing beetles (Dybas and Dybas, 1981), stalk eyed flies (Presgraves et al., 1999; Kotrba and Heß, 2013), dung flies (Minder et al., 2005), moths (Morrow and Gage, 2000), diving and scarab beetles (García-
4. Discussion An extensive literature pointed out that post-copulatory sexual selection is a major evolutionary driver in several insect species, responsible for modifications in the morphology of sperm and in the female reproductive tract, specifically of the spermatheca shape and size (Pitnick et al., 2009; Liebherr and Kipling, 1998; Higginson et al., 2012b). These variations are the consequence of a differential selection pressure (Pitnick et al., 2009), since the overall 9
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lez and Simmons, 2007; Higginson et al., 2012a) and also Gonza birds (Briskie and Montgomerie, 1992; Briskie et al., 1997). All these examples reinforce the “sexually selected sperm hypothesis”, according to which females may be able to enhance the probability of fertilizing their eggs with sperm selected from males producing more efficient sperm (Sivinski, 1984; Curtsinger, 1991; Keller and Reeve, 1995). H. stagnorum is a polyandric species accepting mating with multiple males. During laboratory observations, at least two males of H. stagnorum were directly observed to mate with a single female. Moreover, at least in three adult immature females we found that the apical spermathecal bulbs were filled with sperm, thus suggesting that males, under laboratory conditions, can transfer their sperm to females before egg maturation. The evidence that the females of this species exhibit a long spermathecal duct with apical spermathecal bulbs suggests that the female reproductive tract was able to drive the evolution of a particular sperm morphology. Sperm of Hydrometra, in fact, besides being unusually long, has also an unusually long acrosome. This latter is characterized by a helical array in its anterior region possibly acting as an aid to forward sperm progression within a narrow and convolute spermathecal duct. As explained by Lüpold et al. (2016) “longer sperm are superior at displacing, and resisting displacement by, shorter competitor sperm within the seminal receptacle, and longer seminal receptacles drive sperm-length evolution by enhancing this competitive advantage” (Miller and Pitnick, 2002, 2003; Pattarini et al., 2006; Lüpold et al., 2012). H. stagnorum sperm structure is similar to that of the recently described sperm of the water strider Aquarius remigis, (Miyata et al., 2011), which has a 2.5 mm long acrosome and a nucleus only 5 mm long, and to that of Gerris remigis, showing an extraordinarily long sperm (5 mm), with an acrosome 2.5 mm long and a nucleus 5 mm long (Tandler and Moriber, 1966). Among the species of the related infraorder group Nepomorpha, the back-swimmer Martarega bentoi is also provided with a long acrosome 300 mm long and a nucleus 19 mm long (Novais et al., 2017) and also Notonecta glauca has an acrosome 1 mm long and a nucleus 16 mm long (Tandler and Moriber, 1966; Afzelius et al., 1976; Lee et al., 1995; Jamieson et al., 1999). It is not yet clear, which is the functional significance of these extremely long acrosomes. And the hypothesis that this depends on the life of the species in water, is not plausible. Apart from these two infraorders, a long acrosome has been described in a relatively limited number of other insect sperm, as in the apterygotans Machilis distincta (Dallai, 1972) and Petrobius brevistylis (Wingstrand, 1973), the ground-louse Z. impolitus (Dallai et al., 2014), several ladybirds (Dallai et al., 2018) and more recently mez and Maddison, 2020; in the ground beetle Apotomus rufus (Go Dallai et al. MS in preparation). Among the aforementioned examples, however, only Z. impolitus was shown to have a giant sperm (3 mm long) correlated with large spermathecal structures (1.4 mm long) (Dallai et al., 2014b). A coevolution between giant sperm and a female sperm storage organ was indeed suggested for Z. impolitus as a result of post-copulatory sexual selection. In this species, however, the male performs external sperm transfer (Dallai et al., 2014), thus suggesting the occurrence of a different type of evolutionary force determining fertilization success. The presence of long acrosomes and short nuclei in H. stagnorum and the related species of Gerromorpha and Nepomorpha, is not a common finding and it is worth discussing. As to the functional significance of a long acrosome, it can be excluded that it serves to improve sperm mobility which, in turn, is supported by the activity of the flagellar axoneme (Werner and Simmons, 2008; Dallai, 2014). A long acrosome, anteriorly positioned, would, on the contrary, impair movement as the structure
has to be pushed forward by the posterior flagellar motor. It is also questionable that such an acrosome could contribute to the physiological process of fertilization as it occurs in other organisms, i.e. enabling sperm to penetrate the micropyle by acrosome reaction and membrane fusion. In Drosophila melanogaster it is not yet clear how the sperm entrance into the egg is realized. The acrosome, rather than undergoing exocytosis, remains intact after its entry through the micropyle (Perotti, 1975; Wilson et al., 2006). Moreover, in the water strider A. remigis, which, as above mentioned, shares with H. stagnorum a long acrosome and a short nucleus, it is unlikely that the long acrosomal matrix contributes to any physiological process within the fertilized egg, because it remains structurally intact in the fertilized egg, even after early gastrulation events (Miyata et al., 2011). Similarly, in D. melanogaster sperm, the mitochondrial derivatives in particular, and possibly also the acrosome, persist through the gastrula stages during the different developmental stages (Karr, 1996; Snook and Karr, 1998; Pitnick and Karr, 1998; Karr et al., 2009). Furthermore, it is known that in many wasps, as well as in Drosophila, eggs are activated by mechanical stimulation, when they are squeezed through the oviduct or ovipositor before egg laying (Doane, 1960; Callaini et al., 1999; Horner and Wolfner, 2008), and in some stick insects, eggs are activated by exposure to air (Went, 1982). In several insect species, the acrosome is missing altogether (Dallai, 2014). All these considerations strongly suggest that the acrosome in H. stagnorum, as in the other species characterized by unusually long acrosomes, could play a different role not directly connected with fertilization. The fine structure of the spermathecal duct and of its apical spermathecal bulb in H. stagnorum, as well as that of the spermathecal gland, resembles that of many insect ectodermal glands (Noirot and Quennedey, 1991; Quennedey, 1998) with large secretory and duct forming cells. What is peculiar in these structures is the association, along the duct, of several secretory cells with conspicuous cisterns filled with secretions whose possible role is to increase the viscosity of the duct content where the long sperm have to be displaced. The lumen of the apical spermathecal bulb is filled with the secretions either of the spermathecal duct or of the spermathecal gland intermingled with sperm. Whether these two secretions have a different composition cannot be established at present, but it is possible to speculate that the secretion of the spermathecal gland may play a role in the activation of the sperm mobility. Additional considerations can be made regarding the function of the spermathecal glands. The sperm of H. stagnorum, after mating, are transferred to the female storage organ by contractions of the muscles surrounding the long spermathecal duct. When removed from this district, the sperm are immotile. The secretions produced by secretory cells present in the spermathecal gland epithelium and discharged into the apical spherical structure of the spermatheca may hence have a role in the maintenance of the proper environment for stored sperm and/or to trigger sperm motility, when sperm have to migrate from the spermatheca to fertilize eggs (Chapman, 2013; Pascini and Martins, 2017). A consideration worth of being briefly discussed, in this context, is the description of the position of the sperm within the deferent and spermathecal ducts. These observations take advantage of the intrinsic fluorescence of the acrosome (Miyata et al., 2011). In the deferent ducts, the sperm are distributed along the length of the ducts in conspicuous bundles, often arranged in a helicoidal fashion. In the spermathecal bulb, the sperm of a fertilized female are mainly assembled with their acrosomes directed towards the spermathecal duct opening into the vagina. Considering the presence of few mature eggs close to the common oviduct, it is likely that these sperm are ready to exit from the spermathecal duct and to fertilize eggs. 10
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It would be interesting to know whether other heteropteran species, notably Gerris, which shares with Aquarius and Hydrometra a comparable sperm structure, and Notonecta, similarly characterized by a very long sperm (Afzelius et al., 1976), display a similar coevolution between sperm and spermatheca as observed in Hydrometra. This will be matter for studies in progress.
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Nature 533, 535e538. Mercati, D., Giusti, F., Dallai, R., 2009. A novel membrane specialization in the sperm tail of bug insects (Heteroptera). J. Morphol. 270, 825e833. Miller, G.T., Pitnick, S., 2002. Sperm-female coevolution in Drosophila. Science 298, 1230e1233. Miller, G.T., Pitnick, S., 2003. Functional significance of seminal receptacle length in Drosophila melanogaster. J. Evol. Biol. 16, 114e126. Minder, A.M., Hosken, D.J., Ward, P.I., 2005. Co-evolution of male and female reproductive characters across the Scathophagidae (Diptera). J. Evol. Biol. 18, 60e69. Miyata, H., Noda, N., Fairbairn, D.J., Oldenbourg, R., Cardullo, R.A., 2011. Assembly of the fluorescent acrosomal matrix and its fate in fertilization in the water strider, Aquarius remigis. J. Cell. Physiol. 226, 999e1006. Morrow, E.H., Gage, J.G., 2000. The evolution of sperm length in months. Proc. Roy. Soc. Lond. B. 267, 307e313. Noirot, C., Quennedey, A., 1991. Glands, gland cells, glandular units: some comments on terminology and classification. Ann. Soc. Entomol. Fr. 27, 123e128. Novais, A.M., Dias, G., Lino-Neto, J., 2017. Testicular, spermatogenesis and sperm morphology in Martarega bentoi (Heteroptera: notonectidae). Arthropod Struct. Dev. 46, 635e643. Pascini, T.V., Martins, G.F., 2017. The insect spermatheca: an overview. Zoology 121, 56e71. Pattarini, J.M., Starmer, W.T., Bjork, A., Pitnick, S., 2006. Mechanisms underlying the sperm quality advantage in Drosophila melanogaster. Evolution 60, 2064e2080. Perotti, M.E., 1975. Ultrastructural aspects of fertilization in Drosophila. In: Afzelius, B.A. (Ed.), The Functional Anatomy of the Spermatozoon. Pergamon Press, NewYork, pp. 57e68. Pitnick, S., Karr, T.L., 1998. Paternal products and byeproducts in Drosophila development. Proc. Roy. Soc. Lond. B. 265, 821e826. Pitnick, S., Spicer, G.S., Markow, T.A., 1995. How long is a giant sperm? Nature 375, 109. Pitnick, S., Markow, T.A., Spicer, G.S., 1999. Evolution of multiple kinds of female sperm-storage organs in Drosophila. Evolution 53, 1804e1822. Pitnick, S., Wolfner, M.F., Suarez, S.S., 2009. Ejaculate-female and sperm-female interactions. In: Birkhead, T.R., Hosken, D.J., Pitnick, S. (Eds.), Sperm Biology: an Evolutionary Perspective. Academic Press, Cambridge, MA, pp. 247e304. Presgraves, D.C., Baker, H.R., Gerald, S., Wilkinson, G.S., 1999. Coevolution of sperm and female reproductive tract morphology in stalk-eyed flies. Proc. Roy. Soc. Lond. B. 266, 1041e1047. Quennedey, A., 1998. Insect epidermal gland cells: ultrastructure and morphogenesis. In: Microscopic Anatomy of Invertebrates, vol. 11a. Insecta, Wiley-Liss, Inc., pp. 177e207 Sivinski, J., 1984. Sperm competition. In: Smith, R.L. (Ed.), Sperm Competition and the Evolution of Animal Mating Systems. Academic Press, New York, pp. 88e115. Snook, R.R., Karr, T.L., 1998. Only long sperm are fertilization competent in six sperm-heteromorphic Drosophila species. Curr. Biol. 8, 291e294. Tandler, B., Moriber, L.G., 1966. Microtubular structures associated with the acrosome during spermiogenesis in the water-strider, Gerris remigis (Say). J. Ultrastruct. Res. 14, 391e404. dei, D., Ban ar, P., Xie, Q., Stys, Wang, Y., Cui, Y., Re P., Damgaard, J., Chen, P., Yi, W., Wang, Y., Dang, K., Li, C., Bu, W., 2016. Phylogenetic divergences of the true bugs (Insecta: Hemiptera: Heteroptera), with emphasis on the aquatic lineages: the last piece of the aquatic insect jigsaw originated in the Late Permian/Early Triassic. Cladistics 32, 390e405. Went, D.F., 1982. Egg activation and parthenogenetic reproduction in insects. Biol. Rev. 57, 319e344.
Authors credit Romano Dallai: Conceptualization, Investigation, Writing Original Draft, Writing - Review & Editing, Supervision; Pietro Paolo Fanciulli: Conceptualization, Writing - Original Draft, Writing - Review & Editing; David Mercati: Investigation, Writing - Review & Editing, Visualization; Pietro Lupetti: Investigation, Writing Review & Editing. Acknowledgement Thanks are due to Dr. Fabio Cianferoni for the determination of the specimens used in the work. References Afzelius, B.A., Baccetti, B., Dallai, R., 1976. The giant spermatozoon of Notonecta. J. Submicr. Cytol. 8, 149e161. Andersen, N.M., 1982. The semiaquatic bugs (Hemiptera, Gerromorpha). Phylogeny, adaptations, biogeography and classification. In: Entomograph, vol. 3. Scandinavian Science Press, Klampenborg, p. 455. Andersen, N.M., Weir, T.A., 2004. Australian water bugs. Their biology and identification (Hemiptera-Heteroptera, Gerromorpha & Nepomorpha). Entomonograph 14, 1e344. Apollo Books, Stenstrup. Briskie, J.V., Montgomerie, R., 1992. Sperm size and sperm competition in birds. Proc. R. Soc. Lond. B Biol. Sci. 247, 89e95. Briskie, J.V., Montgomerie, R., Birkhead, T.R., 1997. The evolution of sperm size in birds. Evolution 51, 937e945. Callaini, G., Riparbelli, M.G., Dallai, R., 1999. Centrosome inheritance in insects: fertilization and parthenogenesis. Biol. Cell. 91, 355e366. Chapman, R.F., 2013. The Insects: Structure and Function, fifth ed. Cambridge University Press, Cambridge. Curtsinger, J.W., 1991. Sperm competition and the evolution of multiple mating. Am. Nat. 138, 93e102. Dallai, R., 1972. The arthropod spermatozoon. XVII. Machilis distincta janetsch (Insecta Thysanura). Monit. Zool. Ital. 6, 37e61. Dallai, R., 2014. Overview on spermatogenesis and sperm structure of Hexapoda. Arthropod Struct. Dev. 43, 257e290. Dallai, R., Afzelius, B.A., 1980. Characteristics of the sperm structure in Heteroptera (Hemiptera, insect). J. Morphol. 164, 301e309. Dallai, R., Afzelius, B.A., 1990. Microtubular diversity in insect spermatozoa. Results obtained with a new fixative. J. Struct. Biol. 103, 164e179. Dallai, R., Mercati, D., Gottardo, M., Machida, R., Mashimo, Y., Beutel, R.G., 2012. The fine structure of the female reproductive system of Zorotypus caudelli Karny (Zoraptera). Arthropod Struct. Dev. 41, 51e63. Dallai, R., Gottardo, M., Mercati, D., Machida, R., Mashimo, Y., Matsumura, Y., Beutel, R.G., 2014. Giant spermatozoa and a huge spermatheca: a case of coevolution of male and female reproductive organs in the ground louse Zorotypus impolitus (Insecta, Zoraptera). Arthropod Struct. Dev. 43, 135e151. Dallai, R., Lino-Neto, J., Dias, G., Nere, P.H.A., Mercati, D., Lupetti, P., 2018. Fine structure of the ladybird spermatozoa (Insecta, Coleoptera, Coccinellidae). Arthropod Struct. Dev. 47, 286e298. Damgaard, J., 2008. Phylogeny of the semiaquatic bugs (Hemiptera-Heteroptera, Gerromorpha). Insect Systemat. Evol. 39, 431e460. Doane, W.W., 1960. Completion of meiosis in uninseminated eggs of Drosophila melanogaster Science, vol. 132, pp. 677e678. Dybas, L.K., Dybas, H.S., 1981. Coadaptation and taxonomic differentiation of sperm and spermathecae in feather wing beetles. Evolution 35, 168e174. lez, F., Simmons, L.W., 2007. Paternal indirect genetic effects on García-Gonza offspring viability and the benefits of polyandry. Curr. Biol. 17, 32e36. mez, R.A., Maddison, D.R., 2020. Novelty and emergent patterns in sperm: Go morphological diversity and evolution of spermatozoa and sperm conjugation in ground beetles (Coleoptera: carabidae). J. Morphol. 281, 862e892. Higginson, D.M., Miller, K.B., Segraves, K.A., Pitnick, S., 2012a. Convergence, recurrence and diversification of complex sperm traits in diving beetles (Dytiscidae). Evolution 66, 1650e1661. Higginson, D.M., Miller, K.B., Segraves, K.A., Pitnick, S., 2012b. Female reproductive tract form drives the evolution of complex sperm morphology. Proc. Natl. Acad. Sci. U.S.A. 109, 4338e4543. Horner, V.L., Wolfner, M.F., 2008. Transitioning from egg to embryo: triggers and mechanisms of egg activation. Dev. Dynam. 237, 527e544. 11
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Arthropod Structure & Development 60 (2021) 101001 Wilson, K.L., Fitch, K.R., Bafus, B.T., Wakimoto, B.T., 2006. Sperm plasma membrane breakdown during Drosophila fertilization requires Sneaky, an acrosomal membrane protein Development, 133, pp. 4871e4879. Wingstrand, K.G., 1973. The spermatozoa of the thysanuran insects Petrobius brevistylis Carp. and Lepisma saccharina L. Acta Zool. Stockholm 54, 31e52. Xie, Q., Tian, Y., Zheng, L., Bu, W., 2008. 18S rRNA hyper-elongation and the phylogeny of Euhemiptera (Insecta: Hemiptera). Mol. Phylogenet. Evol. 47, 463e471.
Werner, M., Simmons, L.W., 2008. Insect sperm motility. Biol. Rev. 83, 191e208. Werner, M., Simmons, L.W., 2011. Ultrastructure of spermatozoa of Onthophagus taurus (Coleoptera, Scarabaeidae) exhibits heritable variation. Naturwissenschaften 98, 213e223. Wheeler, W.C., Schuh, R.T., Bang, R., 1993. Cladistic relationships among higher groups of Heteroptera: congruence between morphological and molecular data sets. Entomol. Scand. 24, 121e137.
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Coevolution between female seminal receptacle and sperm morphology in the semiaquatic measurer bug Hydrometra stagnorum L. (Heteroptera, Hydrometridae) Romano Dallai*, Pietro Paolo Fanciulli, David Mercati, Pietro Lupetti Department of Life Sciences, University of Siena, Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2020 Accepted 7 October 2020 Available online xxx
The coevolution between sperm length and size of the female sperm-storage organs is described for the first time within Heteroptera. The long sperm of the measurer bug Hydrometra stagnorum is characterized by the unusually long acrosome with its anterior region helically arranged, and by a very short nucleus. The sperm flagellum has a 9 þ 9þ2 conventional axoneme and crystallized mitochondrial derivatives. The female spermatheca consists of an extraordinarily long spermathecal duct ending with an apical spermathecal bulb into which flows also the secretions of a relatively short spermathecal gland. Both spermathecal duct and gland have a thin epithelium lined by a cuticle, beneath which a complex of secretory and duct forming cells are present. The secretions of these two structures flow into the apical spermathecal bulb. A thick layer of muscle fibers surrounds the epithelium. These results confirm the opinion that the dimensions of the female reproductive sperm-storage organs are able to drive the sperm morphology. © 2020 Elsevier Ltd. All rights reserved.
Keywords: Semiaquatic bug Coevolution Electron microscopy Insect sperm morphology
1. Introduction It is well known that the fertilization success in different organisms depends on interplay between sperm length and the length of the female primary sperm storage organ, the seminal receptacle (Miller and Pitnick, 2002). This relationship was initially observed in Drosophila, namely throughout comparative studies on Drosophila pseudoobscura and Drosophila bifurca (Pitnick et al., 1999). It was hypothesized that the presence of long seminal receptacles drives the production of longer sperm, as these are capable to better displace and resist displacement by competitor sperm (Pattarini et al., 2006). Similar results were recently obtained on the obsolete groundelice Zorotypus impolitus and Zorotypus caudelli (Dallai et al., 2014) as well as on some Onthophagus beetles (Hunt et al., 2002; Werner and Simmons, 2011), suggesting that the evolution of sperm elongation in these species may be regarded as a possible consequence of the observed modifications in the female genital tract. Also studies on diving beetles (Dytiscidae) have
* Corresponding author. E-mail addresses: [email protected] (R. Dallai), [email protected] (P.P. Fanciulli), [email protected] (D. Mercati), [email protected] (P. Lupetti). https://doi.org/10.1016/j.asd.2020.101001 1467-8039/© 2020 Elsevier Ltd. All rights reserved.
revealed that sperm morphology within these groups was correlated with the dimension of the female reproductive tract and, in particular, evolved in response to changes in the organization of the female sperm-storage organs (Higginson et al., 2012a). Moreover, several evidences suggested that female reproductive morphology may evolve first and can act as a driver for the subsequent evolution of the sperm structures (complementary evolution) (Pitnick et al., 1995; Miller and Pitnick, 2002; Higginson et al., 2012b). These studies have, in fact, led to the hypothesis that post-copulatory sexual selection determined by female reproductive structures may be able to bias paternity by determining the best sperm for fertilization. Here we describe a novel example of coevolution between the male and female reproductive structures in the measurer bug Hydrometra stagnorum L, which is one of the most common species of the semiaquatic species of the infraorder Gerromorpha (Heteroptera) alongside Nepomorpha and Leptodomorpha (Andersen, 1982; Wheeler et al., 1993; Xie et al., 2008), all characterized by a long and peculiar sperm, possibly evolved as consequence of a very long female reproductive tract. These groups have an old origin, dating back to late Permian to Early Triassic (269-246 Ma) (Wang et al., 2016). H. stagnorum is a member of the Hydrometridae family. The infraorder Gerromorpha also comprises: Gerridae,
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1.5 mm long, and further with a thicker elliptical region 1.5 mm long and 0.15 mm wide, possibly the seminal vesicle. The most proximal tract of deferent duct is connected with that of the opposite side. At the end of the reproductive time, testes show a reduced dimension with the most proximal region becoming narrowed (Fig. 1A and B).
Veliidae, Hebridae, Macroveliidae and Paraphrynoveliida. A close relationship between Hydrometridae and Macrovelidae is well supported by phylogenetic analyses (Andersen and Weir, 2004; Damgaard, 2008). Hydrometridae is a monophyletic family supported by the prolonged head capsule, tuberculated posterior pair of cephalic trichobotria and apically modified antennae (Andersen and Weir, 2004).
3.2. Sperm structure 2. Material and methods The sperm of the measurer H. stagnorum are long cells (1520 mm) that are arranged in helical fashion within the testes (Fig. 1C and D). They are characterised by an extraordinarily long cylindrical acrosome (367 mm), a very short nucleus (8 mm) and a long cylindrical flagellum (1145 mm) (Fig. 1E). The acrosome, 0.5 mm in diameter, in its anterior region (about 200 mm long) shows a helical array (Fig. 1F); it contains a bundle of longitudinal tubules, 16 nm in diameter each, which fills the whole acrosome lumen except for a small eccentric area. These tubules are separated by dense material and have a twisted configuration (see also Dallai and Afzelius, 1980). The nucleus, 0.68 mm in diameter, contains compact chromatin material (Fig. 1G and H). The flagellum, 0.62 mm in diameter, comprises an axoneme 9 þ 9þ2 microtubules flanked by two long crescent-shaped mitochondrial derivatives, that embrace axonemal structure (Fig. 1H and I). Two filamentous bridges connect the mitochondrial derivatives to the flattened cisterns adherent to doublets 1e2 and 4e5 of the axoneme, as it occurs in Heteroptera in general (Dallai and Afzelius, 1980; Mercati et al., 2009). Two small dense spots are present above the acrosome. Three crystalline inclusions are evident in the mitochondrial matrix (Fig. 1I).
Males and females of the measurer bug H. stagnorum (L.) were collected in the neighbourhood of Siena and Grosseto (Italy, Tuscany). 2.1. Light and epifluorescence microscopic preparations The male and female genital systems of H. stagnorum were dissected in 0.1 M phosphate buffer to which 3% sucrose was added (PB1). A drop of sperm was taken from dissected deferent ducts of males, were spread on a histological slide and a drop of 1 mg/ml of the DNA specific dye Hoechst 33.258 in 0.1 M PB1 was added, and the sample was covered with a coverslip. The spermathecal complex, dissected out in PB1 from adult fertilized females was placed on a glass. As above described, a drop of Hoechst was added and a coverslip applied and gently pressed. Fluorescence observation of the labelled samples were carried out with a Leica DMRB light microscope equipped with UV light source, fluorescein and UV filters and a Zeiss AxioCam digital camera with dedicated imaging software. Confocal laser scanning microscopy on free sperm was carried out on a Zeiss LSM700.
3.3. Female genital apparatus and spermathecal complex 2.2. Electron microscopic preparations The genital system of H. stagnorum young female consists of an ovary with six long ovarioles (1.5 mm long) on each side, which flow in large calices. When the ovaries are immature, only three ovarioles are visible (Fig. 2AeC). The lateral oviducts, 600 mm long, fuse in a common oviduct, about 1.0 mm long, at the posterior end of which a large structure, the spermatheca, 500 mm long, is present. It is a flattened complex structure tightly surrounded by a layer of fat body (Fig. 2AeC). When dissected, such structure reveals to consist of an extraordinary long and thin convoluted cylindrical duct ending in an apical spherical bulb (Fig. 2D). The duct is about 6500 mm long and at its end, where it opens into the vagina, it is only 6 mm in diameter, while at half-length it is about 16e17 mm in diameter; where the duct flows into the apical bulb it is only 9.5e10 mm in diameter (Fig. 3). At this level it runs parallel to the spermathecal gland, an almost cylindrical duct, 450 mm long and about 12.5 mm in diameter, narrowing down to 10 mm at its free extremity. When the spermathecal and the glandular ducts flow into the apical bulb, the diameter of this region widens up to 33 mm. The apical spermathecal bulb is about 103 mm in diameter (Fig. 3). A cross section of the spermathecal duct shows a thin epithelium lined by a 0.8e0.85 mm thick cuticle, with a very thin epicuticle layer (Fig. 4A). In some regions, the epithelium thickens up to 3.0 mm with cells containing elliptical nuclei (4.3 mm 2.0 mm). Several scattered large secretory cells provided with a large cistern are visible beneath the epithelium (Fig. 4BeD). When observed at low magnification, long tracts of spermathecal duct devoid of such secretory cells alternate with others, where complexes of secretory cells are visible (Fig. 4AeD). These cells are rich in mitochondria, rough endoplasmic reticulum and show scattered Golgi bodies. They have a large nucleus (8.7 mm 6.3 mm) with a conspicuous nucleolus. The large extracellular cistern is bordered by long
Male reproductive apparatuses of the species, after dissection, were fixed overnight at 4 C in 3% glutaraldehyde in PB1. After rinsing in PB1, the material was post-fixed in 2% OsO4 in PB1 for 2 h. After rinsing, the material was dehydrated and embedding in EponAraldite mixture. Part of material was fixed according to Dallai and Afzelius (1990) using 1% tannic acid, omitting post-fixation with osmium and using block staining with 1% aqueous uranyl acetate. Female reproductive apparatuses of the species were dissected out in 0.1 M phosphate buffer to which 5% sucrose was added (PB2). The last tract of the common oviduct containing the spermathecal complex was removed and fixed overnight in 3% glutaraldehyde in PB2. After rinsing, the material was post-fixed in OsO4 in PB2 for 2 h, rinsed again, dehydrated and embedded in the mixture EponAraldite resin. For both male and female reproductive system of the species, semi-thin and ultrathin sections, obtained with a Reichert Ultracut ultramicrotome. Semi-thin sections were routinely stained with 0.1% toluidine blue and observed with a Leica DMRB interference light microscope equipped with a Zeiss AxioCam digital camera. Ultrathin sections were routinely stained with uranyl acetate and lead citrate, and observed in a Philips CM 10 transmission electron microscope operating at an electron voltage of 80 kV. 3. Results 3.1. Male genital system of H. stagnorum The mature male genital apparatus of H. stagnorum consists of a pair of elongated conical testes, each about 2.0 mm long and 0.2 mm wide, that basally continue with a very thin region, about 2
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Fig. 1. A-B- Light microscope views of mature male genital apparatus at the reproduction time (A) and at the end of this stage (B). Note the different aspect of the seminal vesicles (sv). T, testes. C- Fluorescent light microscopy of testes with large sperm bundles (sp) in helical fashion. D- Light microscope view of testes showing sperm bundles (sp) in helical fashion. E- The whole sperm under the fluorescent light microscope. The strong fluorescence of the acrosome (A) is due to the intrinsic FAD presence at the acrosome (see Miyata et al., 2011). The Hoechst staining marks the short nucleus (N). F, flagellum. F- Detail of the acrosome (A) to show the helical array of the anterior region. G- Detail of the short nucleus (N) between the acrosome (A) and the flagellum (F). H- Cross section of a sperm bundle within the spermathecal duct showing the acrosome (A) with tubules embedded in a dense material and a flagellar axoneme (ax) with two mitochondrial derivatives (md) embracing the axoneme. I- Higher magnification of a cross sectioned axoneme (ax) after tannic acid preparation. Note the protofilaments in the tubule walls and the bridges connecting the axoneme to the mitochondria (arrows). Note also the three regions of crystallization in the mitochondrial matrix. 3
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Fig. 2. A- Genital apparatus of a young female with immature ovaries (o), lateral oviducts (lov) and spermatheca (spt). B- Genital apparatus of a mature female with two eggs (egg) ready to cross the oviducts (ov). spt, spermatheca. C- Female genital apparatus at the end of reproductive time showing ovaries (o) with immature eggs. spt, spermatheca. D- Light interference microscope showing the apical spermathecal bulb (sptb), the long spermathecal duct (sptd) and the short spermathecal gland (sptg).
connecting duct fuses with that of the spermathecal duct and its lumen opens into the lumen of the duct (Fig. 5D and E). For long tracts, beneath the epithelial cells, the insertions of muscle cells are visible. The epithelial cells are transformed into typical structures adapted for the attachment of muscles to the thick cuticle lining the duct. They are filled with longitudinal bundles of microtubules anchoring on apical short cylindrical cuticular invaginations to form hemidesmosomes, while at the cell base they form desmosomes with the sarcolemma (Fig. 5C). Beneath the basal lamina
microvilli (Fig. 4BeD and Fig 5C). In the cistern lumen, small dense vesicles are visible between microvilli; in a few cases, the cistern appears filled with electron-dense secretion (Fig. 4C and D). This secretion is set up into a typical end apparatus consisting of a cuticular duct of 0.37 mm in diameter, surrounded by a 0.47 mm thick layer of loose filaments (Figs. 4D and 5A). This connecting duct is formed by a duct forming cell (canal cell), intermingled with epithelial cells, showing a thin layer of cytoplasm and a slender nucleus. Close to the spermathecal duct, the cuticular layer of the 4
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Fig. 3. Confocal light microscope of a spermatheca with the spermathecal apical bulb (sptb), the strong fluorescence of the spermathecal duct (sptd) due to the intrinsic fluorescence of the sperm acrosomes filling the duct lumen and the short spermathecal gland (sptg).
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Fig. 4. A- Longitudinal section of the spermathecal duct filled with many sperm (sp) cross sectioned at different levels. A, acrosomes, F, flagella. Note the thin epithelium (Ep) lined by a thick cuticle (ct). BeC-D-Cross sections of the spermathecal duct (sptd) showing large secretory cells (sec) beneath the thin epithelium (Ep). Note the expanded cisterns (ci) with long microvilli (mv) bordering their lumen. In C, the cistern (ci) contains a complex of dense granules and thin filaments. In D, the cytoplasm shows cisterns of rough endoplasmic reticulum (rer), numerous mitochondria (mt), vesicles and dense bodies (d). The axial region of the cistern shows the end apparatus of the duct forming cell (end). A large nucleus (N1) of the secretory cell is visible at the periphery of the cell, while an elliptical nucleus (N2) of the epithelial cell is beneath the cuticle.
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Fig. 5. A- Cross section trough a cistern of a secretory cell (sec) with the end apparatus (end) of a duct forming cell. This cell is beneath the epithelium (Ep) intermingled with secretory cells. ct, cuticle. B- Detail of the secretory cell with long microvilli (mv) bordering the lumen of the cistern (ci). Note the thick cuticle (ct) lining the epithelial cells (Ep). CSome epithelial cells are often adapted to allow the insertion of the muscle fibres (ms) in the cell complex surrounding the spermathecal duct. Apical hemidesmosomes (hd) and basal desmosomes (d) are formed and serve as an anchorage of microtubule bundles (mi). D-E-Cross sections through the spermathecal duct showing a thin epithelial cell (Ep) with elongated nucleus (N) and lined by thick cuticle (ct). The region corresponds to the point where the canal (ca) of the duct forming cell (dfc) leads into the spermathecal duct lumen (sptl). Numerous sperm (sp) are visible in the lumen. In E, the cistern (ci) of a secretory cell (sec) is also present.
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conspicuous muscle structures with myofibril filaments in the typical array of striated muscles are visible (Fig. 5C). Many sperm sectioned at different levels are visible in the spermathecal duct lumen. Towards the posterior region close to the duct entrance into the vagina, the duct epithelium becomes thin and it shows a narrowed lumen due to a thicker cuticle, up to 6.0e7.5 mm high (Fig. 6A). In the lumen a dense secretion obscures the presence of a few sperm (Fig. 6A). The region close to the entrance of the spermathecal duct into the spermathecal apical bulb is also characterized by a thin epithelium lined by a very thick cuticle (about 6.5e7.0 mm high) surrounding a narrow lumen filled with many sperm (Fig. 6B). The apical sperm receptacle has a simple epithelium, about 23e27 mm high, lined by a 2.5e3.0 mm high cuticle, without
secretory cells (Fig. 6D). The cytoplasm does not show many secretory structures, while beneath the cuticle numerous mitochondria are visible (Fig. 6D). In the lumen, bundles of sperm are intermingled with large amount of dense secretions. The spermathecal gland has a fine structure similar to that of the spermathecal duct with secretory and duct forming cells (Fig. 7). The secretory cells are regularly distributed along the gland length and discharge their secretions into the gland lumen via a thin efferent duct produced by the canal cell. At the level of the entrance of the two structures, i.e., the spermathecal duct and the spermathecal gland, into the apical sperm receptacle, a muscle complex is observable (Fig. 6C). When observed under the fluorescence microscope the sperm are visible along the spermathecal duct with the acrosomes directed towards the duct opening into the vagina (Fig. 3).
Fig. 6. A- Cross section of the spermathecal duct close to its entrance into the vagina. Note the thicker cuticle (ct) lining the epithelium (Ep) and the narrowed duct lumen (L) filled with dense material obscuring the presence of some sperm. B- Cross section of the region close to the entrance of the spermathecal duct into the spermathecal apical bulb. Note the thicker cuticle (ct) and the lumen filled with sperm (sp). C- Cross section of the muscle complex (ms) of the region close to the entrance of the spermathecal duct into the apical bulb. D- Cross section trough the spermathecal apical bulb. The apical epithelial region is rich in mitochondria (mt), while inclusions are scarse. N, nuclei; ct, cuticle.
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Fig. 7. Cross section trough a secretory cell (sec) of the spermathecal gland. In the cytoplasm several mitochondria (mt), cisterns of rough endoplasmic reticulum (rer) and Golgi bodies (G) are present. Close to the cistern, several dense bodies (d) are visible. Thin epithelial cells (Ep) are line by a thick cuticle (ct). N, nucleus.
fertilization success depends on the interaction between male and female reproductive organs (Pitnick et al., 2009). Example of a positive coevolution between female sperm-storage organs and their ducts with sperm length have been described, other than in fruit flies and particularly D. bifurca (Pitnick et al., 2009), in groundlice (Zoraptera) (Dallai et al., 2012, 2014), featherwing beetles (Dybas and Dybas, 1981), stalk eyed flies (Presgraves et al., 1999; Kotrba and Heß, 2013), dung flies (Minder et al., 2005), moths (Morrow and Gage, 2000), diving and scarab beetles (García-
4. Discussion An extensive literature pointed out that post-copulatory sexual selection is a major evolutionary driver in several insect species, responsible for modifications in the morphology of sperm and in the female reproductive tract, specifically of the spermatheca shape and size (Pitnick et al., 2009; Liebherr and Kipling, 1998; Higginson et al., 2012b). These variations are the consequence of a differential selection pressure (Pitnick et al., 2009), since the overall 9
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lez and Simmons, 2007; Higginson et al., 2012a) and also Gonza birds (Briskie and Montgomerie, 1992; Briskie et al., 1997). All these examples reinforce the “sexually selected sperm hypothesis”, according to which females may be able to enhance the probability of fertilizing their eggs with sperm selected from males producing more efficient sperm (Sivinski, 1984; Curtsinger, 1991; Keller and Reeve, 1995). H. stagnorum is a polyandric species accepting mating with multiple males. During laboratory observations, at least two males of H. stagnorum were directly observed to mate with a single female. Moreover, at least in three adult immature females we found that the apical spermathecal bulbs were filled with sperm, thus suggesting that males, under laboratory conditions, can transfer their sperm to females before egg maturation. The evidence that the females of this species exhibit a long spermathecal duct with apical spermathecal bulbs suggests that the female reproductive tract was able to drive the evolution of a particular sperm morphology. Sperm of Hydrometra, in fact, besides being unusually long, has also an unusually long acrosome. This latter is characterized by a helical array in its anterior region possibly acting as an aid to forward sperm progression within a narrow and convolute spermathecal duct. As explained by Lüpold et al. (2016) “longer sperm are superior at displacing, and resisting displacement by, shorter competitor sperm within the seminal receptacle, and longer seminal receptacles drive sperm-length evolution by enhancing this competitive advantage” (Miller and Pitnick, 2002, 2003; Pattarini et al., 2006; Lüpold et al., 2012). H. stagnorum sperm structure is similar to that of the recently described sperm of the water strider Aquarius remigis, (Miyata et al., 2011), which has a 2.5 mm long acrosome and a nucleus only 5 mm long, and to that of Gerris remigis, showing an extraordinarily long sperm (5 mm), with an acrosome 2.5 mm long and a nucleus 5 mm long (Tandler and Moriber, 1966). Among the species of the related infraorder group Nepomorpha, the back-swimmer Martarega bentoi is also provided with a long acrosome 300 mm long and a nucleus 19 mm long (Novais et al., 2017) and also Notonecta glauca has an acrosome 1 mm long and a nucleus 16 mm long (Tandler and Moriber, 1966; Afzelius et al., 1976; Lee et al., 1995; Jamieson et al., 1999). It is not yet clear, which is the functional significance of these extremely long acrosomes. And the hypothesis that this depends on the life of the species in water, is not plausible. Apart from these two infraorders, a long acrosome has been described in a relatively limited number of other insect sperm, as in the apterygotans Machilis distincta (Dallai, 1972) and Petrobius brevistylis (Wingstrand, 1973), the ground-louse Z. impolitus (Dallai et al., 2014), several ladybirds (Dallai et al., 2018) and more recently mez and Maddison, 2020; in the ground beetle Apotomus rufus (Go Dallai et al. MS in preparation). Among the aforementioned examples, however, only Z. impolitus was shown to have a giant sperm (3 mm long) correlated with large spermathecal structures (1.4 mm long) (Dallai et al., 2014b). A coevolution between giant sperm and a female sperm storage organ was indeed suggested for Z. impolitus as a result of post-copulatory sexual selection. In this species, however, the male performs external sperm transfer (Dallai et al., 2014), thus suggesting the occurrence of a different type of evolutionary force determining fertilization success. The presence of long acrosomes and short nuclei in H. stagnorum and the related species of Gerromorpha and Nepomorpha, is not a common finding and it is worth discussing. As to the functional significance of a long acrosome, it can be excluded that it serves to improve sperm mobility which, in turn, is supported by the activity of the flagellar axoneme (Werner and Simmons, 2008; Dallai, 2014). A long acrosome, anteriorly positioned, would, on the contrary, impair movement as the structure
has to be pushed forward by the posterior flagellar motor. It is also questionable that such an acrosome could contribute to the physiological process of fertilization as it occurs in other organisms, i.e. enabling sperm to penetrate the micropyle by acrosome reaction and membrane fusion. In Drosophila melanogaster it is not yet clear how the sperm entrance into the egg is realized. The acrosome, rather than undergoing exocytosis, remains intact after its entry through the micropyle (Perotti, 1975; Wilson et al., 2006). Moreover, in the water strider A. remigis, which, as above mentioned, shares with H. stagnorum a long acrosome and a short nucleus, it is unlikely that the long acrosomal matrix contributes to any physiological process within the fertilized egg, because it remains structurally intact in the fertilized egg, even after early gastrulation events (Miyata et al., 2011). Similarly, in D. melanogaster sperm, the mitochondrial derivatives in particular, and possibly also the acrosome, persist through the gastrula stages during the different developmental stages (Karr, 1996; Snook and Karr, 1998; Pitnick and Karr, 1998; Karr et al., 2009). Furthermore, it is known that in many wasps, as well as in Drosophila, eggs are activated by mechanical stimulation, when they are squeezed through the oviduct or ovipositor before egg laying (Doane, 1960; Callaini et al., 1999; Horner and Wolfner, 2008), and in some stick insects, eggs are activated by exposure to air (Went, 1982). In several insect species, the acrosome is missing altogether (Dallai, 2014). All these considerations strongly suggest that the acrosome in H. stagnorum, as in the other species characterized by unusually long acrosomes, could play a different role not directly connected with fertilization. The fine structure of the spermathecal duct and of its apical spermathecal bulb in H. stagnorum, as well as that of the spermathecal gland, resembles that of many insect ectodermal glands (Noirot and Quennedey, 1991; Quennedey, 1998) with large secretory and duct forming cells. What is peculiar in these structures is the association, along the duct, of several secretory cells with conspicuous cisterns filled with secretions whose possible role is to increase the viscosity of the duct content where the long sperm have to be displaced. The lumen of the apical spermathecal bulb is filled with the secretions either of the spermathecal duct or of the spermathecal gland intermingled with sperm. Whether these two secretions have a different composition cannot be established at present, but it is possible to speculate that the secretion of the spermathecal gland may play a role in the activation of the sperm mobility. Additional considerations can be made regarding the function of the spermathecal glands. The sperm of H. stagnorum, after mating, are transferred to the female storage organ by contractions of the muscles surrounding the long spermathecal duct. When removed from this district, the sperm are immotile. The secretions produced by secretory cells present in the spermathecal gland epithelium and discharged into the apical spherical structure of the spermatheca may hence have a role in the maintenance of the proper environment for stored sperm and/or to trigger sperm motility, when sperm have to migrate from the spermatheca to fertilize eggs (Chapman, 2013; Pascini and Martins, 2017). A consideration worth of being briefly discussed, in this context, is the description of the position of the sperm within the deferent and spermathecal ducts. These observations take advantage of the intrinsic fluorescence of the acrosome (Miyata et al., 2011). In the deferent ducts, the sperm are distributed along the length of the ducts in conspicuous bundles, often arranged in a helicoidal fashion. In the spermathecal bulb, the sperm of a fertilized female are mainly assembled with their acrosomes directed towards the spermathecal duct opening into the vagina. Considering the presence of few mature eggs close to the common oviduct, it is likely that these sperm are ready to exit from the spermathecal duct and to fertilize eggs. 10
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It would be interesting to know whether other heteropteran species, notably Gerris, which shares with Aquarius and Hydrometra a comparable sperm structure, and Notonecta, similarly characterized by a very long sperm (Afzelius et al., 1976), display a similar coevolution between sperm and spermatheca as observed in Hydrometra. This will be matter for studies in progress.
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