https://orcid.org/0000-0002-3017-3987
Figures
Abstract
Edessinae is one of the ten subfamilies of Pentatomidae, and it is further divided into seven genera. Among these, Edessa Fabricius, 1803 is the most diverse genus, boasting around 300 species recognized for their ecological and economic significance worldwide. The inclusion of various pentatomids in the Edessa genus has led to mistakes in its taxonomy due to common morphological features and misidentifications. An alternative to avoid mistakes is to use diverse datasets to characterize and classify insects, such as the male reproductive system and sperm morphology, for their variability and conserved traits within a clade. Thus, we described the morphology of the male reproductive system, spermatozoa, and spermiogenesis of Edessa rufomarginata (De Geer, 1773) using light microscopy. We discovered that their male reproductive system consists of a pair of elongated testes with four follicles each. The analysis revealed for the first time the presence of dimorphic spermatozoa in Edessinae. There are two distinct morphotypes: spermatozoa type I, produced by follicles 1, 2, and 3, with a total length of approximately 325 μm and a nucleus of 34 μm and spermatozoa type II, produced by follicle 4, measuring approximately 156 μm in total length and 73 μm in the nucleus, and showing an aberrant sperm morphology with different morphology from what has been described in Pentatomidae. The presence of sperm dimorphism in E. rufomarginata are not reported in any other Pentatomidae to date, and it may contribute to establishing taxonomic limits within the subfamily Edessinae.
Citation: Paulo MdS, Rezende PH, Costa DA, Teixeira ACP, Nascimento FWSd, Lino-Neto J, et al. (2024) First report of aberrant sperm in Edessinae and analysis of the male reproductive system of Edessa rufomarginata (De Geer, 1773) (Hemiptera: Pentatomidae). PLoS ONE 19(12): e0311254. https://doi.org/10.1371/journal.pone.0311254
Editor: Abeer El Wakil, Alexandria University, EGYPT
Received: May 8, 2024; Accepted: September 12, 2024; Published: December 12, 2024
Copyright: © 2024 Paulo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was funded by the Brazilian Funding Agencies: CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (process number 88887.903938/2023-00– MSP) e CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico (Process 140905/2020–0 – PHR and research grant number 306486/2019–9 - JLN, in the payment of PhD scholarships. The Postgraduate Program in Cellular and Structural Biology, at the Federal University of Viçosa, Minas Gerais – Brazil, funded the publication of the article by PlosOne. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Introduction
Edessinae is one of the ten subfamilies of Pentatomidae [1, 2] and is subdivided into seven genera, including the richest genus, Edessa Fabricius, 1803 [3, 4], with around 300 species described [5]. However, due to the lack of clear generic boundaries, it has been referred to as a “repository of species” [6] or a “garbage group” [5], where species of Edessinae with no clear taxonomy position are included in this genus. According to Fernandes and Doesburg [7], many pentatomids have been erroneously identified as belonging to Edessa due to their generalist characteristics and identification errors, incomplete descriptions of specimens, variations in species in different regions, and difficulties in obtaining the holotypes are the factors contributing to this “state of chaos” in their taxonomy.
Since Pendergrast’s [8] work on the heteropteran reproductive apparatus and classification, this type of data, especially on the male reproductive system, has been used to characterize and classify certain groups based on their specific shared traits. The reproductive system in Heteroptera consists of a pair of testes that are covered by a pigmented connective capsule, a pair of vasa deferentia that contain seminal vesicles, an ejaculatory bulb, a common ejaculatory duct, and paired accessory glands. The number of testicular follicles in each testis can range from one to nine [8–16].
Insect sperm have been used in several studies to characterize groups. Their morphology and ultrastructure can provide valuable insights into evolutionary relationships, reproductive biology, and taxonomic classification within insect species [17–19]. Spermatozoa show notable morphological variation, even at the species level, related to size, shape, number of components, and ultrastructural organization [17]. Thus, it can and has been used in the systematics of different groups of insects, including Pentatomidae [11, 20].
Based on morphological variations observed in Pentatomidae sperm, Bowen [20, 21] found that this group of stink bugs produces polymorphic sperm (two or more sperm morphotypes developed in the same male individual). Sperm polymorphism in Pentatomidae can be explained by the “harlequin” lobe theory [22, 23]. Schrader [22, 23] proposed that meiosis occurs differently in one of the testicular follicles, resulting in aberrant spermatids (with a different number of chromosomes) [22–24]. These variations lie in the presence of more than one sperm class varying in size and cellular ultrastructure [17].
In this study, we described the morphology of the male reproductive system and the morphology of the spermatozoa of Edessa rufomarginata (De Geer, 1773). The analysis of these data in Edessa can provide additional evidence for understanding their reproductive biology, evolution, and taxonomic classification since sperm in Pentatomidae have unique characteristics.
Materials and methods
Adult individuals, males, and females of E. rufomarginata were collected between April and September 2022 in cultivated Solanum spp. in the municipalities of Viçosa (-20.759684, -42.862992) and Diamantina (-18.223516, -43.612088), Minas Gerais state, Brazil.
Ten males and eight females were dissected in 0.1 M sodium phosphate buffer pH 7.2 (PBS). To examine the histology of the testes and accessory glands, they were fixed in 2.5% glutaraldehyde + sucrose 3% in PBS for 24 hours and Carnoy solution (Ethanol 95% + Glacial acetic acid = 3:1), respectively. Then, the samples were washed in the same buffer, post-fixed in 1% osmium tetroxide (the accessory glands did not go through this stage), dehydrated in an increasing series of ethanol (30%, 50%, 70%, 90%, and 100%) and included in Historesin® plastic resin (Leica Historesin, Heidelberg, Germany) and polymerized at 60°C for 12 hours. Semi-thin sections (1.0–1.5 μm for the testicles and 2.0–2.5 μm for the accessory glands) were obtained with glass knives on a Leica RM 2255 automatic microtome and mounted on histological slides. The sections of the testes were stained with a 1:5 filtered Giemsa solution in distilled water for 30 minutes. Sections of the accessory glands were subjected to acid hydrolysis with Periodic Acid for 25 minutes, washed in distilled water, and stained with Shiff’s Reactive (P.A.S.) for 1 hour.
For sperm analysis, the vasa deferentia from males and the spermatheca from females were transferred to histological slides with a drop of PBS, where it was smeared. After drying at room temperature, the samples were washed with distilled water, dried again at room temperature, and stained with Giemsa. Some samples were stained with DAPI solution (0.2 mg/mL) + mounting medium and covered with a coverslip to determine the size of the nuclei. All samples (sperm slides and histological sections) were photographed using a photomicroscope (Olympus, BX-60, Olympus Corporation, Tokyo, Japan) with an Olympus Q-Color3 digital camera attached. After completing these steps, we used thirty sperm of both morphotypes from each male for measurements using the software Sperm-Sizer-1.6.6 [25]. All materials used in the research remain stored at room temperature in the Cellular Ultrastructure Laboratory, at the Federal University of Viçosa, Minas Gerais, Brazil.
Results
Anatomy of the male reproductive system and spermatozoa
Males of E. rufomarginata (Fig 1A) have a pair of elongated testes subdivided into four follicles. Adjacent to the testes are the vasa deferentia, which converge into an ejaculatory bulb and, subsequently, into an ejaculatory duct. Following the ejaculatory duct is the aedeagus. It is interesting to note that E. rufomarginata does not have dilated seminal vesicles. In this species, the vasa deferentia are relatively thick (nearly 0.5 mm in diameter and around 4.5 mm in length) and serve as sperm reservoirs until copulation. Two types of accessory glands are also present. One type consists of small paired accessory glands, while the second is a singular, more prominent, and intrinsically coiled gland, presenting long ducts with secretory activity. The testes and vasa deferentia are surrounded by red-pigmented connective tissue capsules (Fig 1B).
Male individual of Edessa rufomarginata (A) and its reproductive system (B). The male reproductive system of E. rufomarginata is composed of a pair of elongated testes (t), followed by a pair of long vas deferens (vd). Note the red-pigmented peritoneal sheath covering both structures. Two types of accessory glands (ag1 and ag2) are also observed, in addition to the ejaculatory bulb (eb) in the central region between the accessory glands. In the distal portion is the ejaculatory duct (ed). Scale bars: A: 2 mm; B: 1,5 mm.
Spermatozoa extracted from the vas deferens exhibited dimorphism, meaning there are two distinct cell morphotypes easily distinguishable under light microscopy (Fig 2A–2D). In each testis, testicular follicles 1, 2, and 3 produce spermatozoa type I. These thin and elongated cells were the only ones found in the female’s spermatheca. They are approximately 325 μm long, with a nucleus length of 34 μm (Fig 2A and 2B). On the other hand, spermatozoa type II originates exclusively from testicular follicle 4. These spermatozoa are shorter, measuring approximately 156 μm in length and 73 μm in nucleus length (Fig 2C and 2D). These sperm seem flattened, with notable dilations in both the nucleus and the flagellum. The acrosome was on the anterior tip of the nucleus, showing lighter staining in the techniques used and exhibiting a globular region positioned lateral to the tip of the nucleus (Fig 2C–2E). Notably, the nucleus of this sperm demonstrates variations in its structure, with some nuclear regions exhibiting more condensed chromatin, resulting in more pronounced dilation compared to areas where the chromatin is less condensed (Fig 2E). Furthermore, the end of the nucleus shows high chromatin condensation, and it appears to be a dilation in the nucleus-flagellum transition zone (Fig 2E and 2F).
(A): Spermatozoa type I evidenced by Giemsa stain and (B): nucleus (n) stained with DAPI. (C): Spermatozoa type II evidenced by Giemsa stain. (D): detail of acrosome (ac) and (E): nucleus (n) stain with DAPI. Note the nuclear region with low chromatin condensation (dashed lines) in C and E. (F): nuclei-flagellum transition (arrow) showing the final region of the nucleus with highly condensed chromatin inserted into the flagellum (f). Scale bars: A: 50 μm; B and E: 10 μm; C: 25 μm; D and F: 5 μm.
Histology of the male reproductive system and spermatogenesis
Longitudinal sections of testicular follicles of E. rufomarginata provide information about the process of spermatogenesis in male adult individuals. Cysts in various stages of germ cell differentiation were observed (Fig 3), indicating an ongoing process of spermatogenesis into adulthood. Despite the similar elongated morphology of all four testicular follicles, follicle 4 appears shorter (Fig 3). However, even with these length differences, the spermatogonia undergoing initial differentiation are consistently located in the apical region of all follicles. In contrast, mature sperm are found in the basal part of the testes, situated close to the efferent ducts (Fig 3). This organization suggests spatial differentiation along the longitudinal axis of testicular follicles, where germ cells progress through various stages of development, from the apical to the basal region. The continuous process of spermatogenesis in adult E. rufomarginata could indicate constant sperm production.
The testes comprise 4 testicular follicles (1, 2, 3, and 4) and are covered externally by the peritoneal sheath (ps). This sheath also internally separates the 4 follicles. Spermatozoa are organized into cysts. In the apical region of the follicles are the initial cysts, in the growth zone (gz); in the medial portion, the sperm cells advance to the maturation zone (mz); and in the distal region, the final cysts are in the differentiation zone (dz). At the end of their development, the spermatozoa are released into the efferent ducts (ed). The latter extends to the vasa deferentia (vd). Scale bar: 175 μm.
Both types of spermatozoa (I and II) similarly change their structural organization during the development. Thus, throughout the process, these changes can be visible in three zones of the testicular follicles: growth zone, maturation zone, and differentiation zone (Fig 3). In the growth zone, mitochondria fuse to form nebenkerns and elongate around the axoneme, forming mitochondrial derivatives (Figs 4B–4D and 6A–6D). The nuclei of spermatogonia, at the beginning of spermatogenesis, have a rounded shape (Figs 4A–4D and 6A–6C) and, in the maturation zone, the nucleus is elongated (Figs 5A–5C and 6D). In the differentiation zone, the nuclei and flagella complete their elongation. Spermatozoa have different ways of organizing themselves within cysts during spermiogenesis. Spermatozoa type I are organized neatly in the register in the cysts (Fig 5D), whereas spermatozoa type II appear disorganized within the cyst. In the latter type, sperm are positioned at varying heights and directions (Fig 6E). Many cell fragments were observed at the end of spermiogenesis (Figs 5D and 6E).
(A): cysts with developing spermatids, a stage marked by the fusion of mitochondria (mi), forming nebenkerns (nk). Note the rounded nuclei (n) in the four images. In (B), (C), and (D), the nebenkerns (nk) fission takes place, originating the mitochondrial derivatives (md), now in pairs. Mitochondrial derivatives (md) become elongated as the flagellum elongates. cc: cyst cell nuclei; ac: pre-acrosomal vesicle. Scale bars: 20 μm.
Cysts with developing spermatids. In (A), (B), and (C), it is possible to observe the nuclei (n) lengthening and the acrosome (ac) positioning itself above the nucleus (n). In (D), the cyst is in the differentiation zone, where the nuclei (n) and flagella (f) elongate, and there is the presence of cellular fragments (cf) being eliminated. ps: peritoneal sheath. Scale bars: 20 μm.
(A), (B), (C), and (D): cysts in the maturation zone, where mitochondria (mi) fuse. Note the rounded nuclei (n) in A-C. In B and C, nebenkerns fission occurs, giving rise to two mitochondrial derivatives (md). In (D), the nuclei (n) are elongated, and the acrosome (ac) and mitochondrial derivatives (md) are visible. In E, during the development process spermatozoa are in the differentiation zone. It is possible to observe sperm nuclei spread throughout the cyst and many cell fragments (cf) being eliminated. cc: cystic cell nucleus; ps: peritoneal sheath. Scale bars: 20 μm.
Both accessory glands have high secretory activity, and different types of secretion can be observed. The pair of small glands (ag1) (Fig 1B) have mesodermal origin. These glands are formed by cubic epithelial cells with predominantly rounded nuclei and partially decondensed chromatin (Fig 7A–7C). Several secretion vesicles form in the cytoplasm and are directed towards the gland’s lumen. Using P.A.S. techniques, we identified that in these secretions predominate glycoproteins.
Details of the accessory gland of mesodermal origin. In (A), it is possible to observe many glycoprotein granules (sc) in the lumen (lm) of the gland. Epithelial cells do not present a specific organization, but the nuclei (n) are visible, with partially condensed chromatin and the basement membrane (bm). In (B), secretion (sp) production is visible in several epithelium cells and directed to the lumen (lm). The nuclei (n) present varied morphology and decondensed chromatin. In (C), several secretory vesicles (sc) are directed to the lumen (lm) of the gland. The nuclei (n) also vary morphologically, and their chromatin is decondensed. Scale bars: 20 μm.
The other type (ag2) is a single, and large accessory gland (Fig 1B), with high secretory activity (Fig 8A). A thin cuticle lining the glandular lumen (Fig 8B), indicating it has an ectodermal origin. The glandular tissue is formed by slightly elongated, cubic epithelial cells with rounded nuclei and chromatin varying in condensation (Fig 8A–8D). Furthermore, many secretory vesicles are observed in the cytoplasm of these cells (Fig 8C), and the lumen is filled with secretions (Fig 8A–8D). After being released, the secretions fuse, forming large dense particles (Fig 8A and 8C–8E). Meanwhile, in specific cells, it is possible to observe the entire cellular content being released in holocrine secretions, including the nucleus region (quite evident), into the lumen of the accessory gland (Fig 8B).
Details of the accessory gland of ectodermal origin. In (A) it is possible to observe the dense secretion (sc) in the lumen (lm) of the gland. Epithelial cells are elongated, with nuclei (n) close to the medial-basal region. Furthermore, nuclei (n) show variation in chromatin compaction. In (B) and (D), the secretion vesicles (sv) are visible in the cytosol of the cells, and their release (secretion: sc) into the lumen (lm) of the gland. After release, these compactors merge, forming larger mixtures. In both photos (B and D), the cuticle (ct) is evident, indicating the ectodermal origin of the gland. In (C), holocrine secretions (sc) are directed to the lumen (lm) of the gland. The nuclei (n) of the epithelial cells mix with the secretion (sc) in the lumen (lm) of the gland, demonstrating cellular disorganization at the time of secreting the products. (E): Detail of a secretory granule (sc) fused into the lumen (lm) of the gland. Scale bars: 20 μm.
Discussion
In this study, we report the presence of dimorphic spermatozoa in Edessinae for the first time, which means they have two morphotypes. The spermatozoa type I of E. rufomarginata is considered typical to fertilization, while type II is classified as aberrant. These aberrant sperm have unique morphological characteristics [22–24] and probably perform different reproduction functions or strategies during copulation distinct from fertilizing spermatozoa.
Production of more than one morphological type of spermatozoa has been reported in different species of Pentatomidae, specifically in Edessinae, Discocephalinae, and Pentatominae [11, 20–24, 26–29]. In Pentatomidae, the number of sperm primarily ranges from two (dimorphism) or three (polymorphism) types. Dimorphism predominantly occurs in this family in species with at least four testicular follicles, where the first three are typically responsible for producing fertilizing sperm, while the 4th generates aberrant sperm. Souza and Itoyama [28] numbered each follicle of Euschistus heros (Fabricius, 1798) according to its position in relation to the vas deferens. In this case of this study, follicle 1 of E. ruformaginata is the one closest to the exit of the vas deferens, and the 4th is the one farthest away. Interestingly, Edessa bifida (Say, 1832) accounted for 5 testicular follicles, with follicles 2 and 4 serving as the site for producing typical larger cells, according to cytogenetic data [20]. This finding catches our attention since both E. bifida and E. rufomarginata belong to the same genus, and so far, none of the studies have reported the occurrence of abnormal meiosis in follicle 2, only from follicle 4 onwards. Recently, Santos et al. [30] suggested the relocation of E. bifida to the genus Ascra, based on morphological characteristics mainly of the genitalia. However, data on the male reproductive system and spermatozoa of E. bifida (Ascra bifida) have not yet been published. Due to this discrepancy in information regarding such closely related species, we consider verifying the data in question essential. The difference in the number of follicles and the presence or absence of aberrant sperm in this species may support the genus change.
Sperm polymorphism has generally been documented in heteropterans possessing 5 to 8 testicular follicles. Typically, two follicles produce longer sperm (4 and 6), while a single follicle produces shorter sperm (5), with the remaining follicles producing intermediate-size gametes (1, 2, and 3, or even 7 and 8). Notably, sperm size does not correlate with lobe size; sometimes, the follicle that produces shorter sperm is the largest, while those that produce longer sperm are the smallest ([22–24, 26, 27]. The "harlequin" lobe, named for variation during normal meiosis, corresponds to the largest follicle in genus Loxa Amyot & Serville, 1843, the testicular follicle 5 [24, 31], which is responsible for the production of short sperm. In Arvelius albopunctatus (De Geer, 1773), follicles 3 and 5 are smaller and responsible for producing longer sperm, while follicle 4 is the largest and produces short sperm [27]. Similar changes occur during the development of E. rufomarginata sperm cells; however, even though meiosis produces aberrant sperm, such as Type II, they are produced in the smallest lobe, the testicular follicle 4, different from what was previously described.
The aberrant Type II sperm produced by E. rufomarginata may play different roles during reproduction. We observed that aberrant sperm had greater motility compared to typical sperm, this could indicate that these type II spermatozoa possibly help in the transfer of type I spermatozoa [32]. Interestingly, in the spermatheca of E. rufomarginata females, we exclusively observed the longer and typical sperm (type I). In this sense, short sperm are generally not used to fertilize eggs. However, according to Schrader [22] and Swallow & Wilkinson [24], shorter sperm (with a smaller number of chromosomes than the usual complement) do not leave the male reproductive system or, at least, were not found in females. Based on this information, perhaps aberrant Type II sperm can be rapidly degraded in the female reproductive system. Schrader [22, 23, 31] raises hypotheses about the persistence of the production of sperm not used in fertilization. He proposed that they could provide supplemental nutrients, particularly nucleoproteins, to the developing egg, fertilizing sperm or to be used for sperm before being transferred to the female [24]. Other hypotheses include the role of aberrant spermatozoa in sperm competition [32–35]. If aberrant sperm reached the female reproductive system, non-fertilizing sperm could be used to fill the spermatheca or copulatory bursa of females, preventing other males from depositing their sperm (see [35, 36]). It could also increase the chance of paternity of this first male or even provide nutrients to females [32, 35].
Regardless of the function that aberrant sperm may play in the male and female reproductive physiology, its morphological characteristics are undoubtedly important for phylogeny and taxonomy. It is notable that, although dimorphic spermatozoa differ mainly in total size or nucleus size, they most often present a very similar morphology when observed under light microscopy [11, 24, 37]. However, the aberrant type II sperm of E. rufomarginata possess a distinctive and easily identifiable morphology compared to type I. Type II sperm is slightly flattened and is significantly shorter than typical fertilizing sperm. Notably, the nuclei of aberrant sperm are longer than those of typical sperm, covering more than half of their total length. Its nuclear characteristics, such as changes in chromatin compaction and its insertion into elements of the flagellum, further highlight its differentiated morphology.
The anatomy of the male reproductive system of E. rufomarginata is similar to other Pentatomidae, following their general pattern of organization. The testes are elongated as in Chinavia ubica Rolston, 1983, Oebalus insularis Stål, 1872, Piezodorus guildinii (Westwood, 1837) [15], and Apodiphus amygdali (Germar, 1817) [13]. However, some pentatomids may have elongated ovoid testes, as in Nezara viridula Linnaeus, 1758 [38], or even kidney-shaped in Halys dentatus (Fabricius, 1775) [39]. Other variations are in the color of the peritoneal sheath, being yellowish, red, or even orange [8, 13–15, 28, 40]. E. rufomarginata has four testicular follicles, few when compared to other Pentatomidae, which usually have seven follicles [16, 22–24]. These data can be helpful in taxonomy since the characteristics mentioned above tend to be conserved within a species, genus, or even family [8, 16, 41].
E. rufomarginata has two accessory glands that produce different types of secretions. In accessory gland 1, the secretions of merocrine type are predominantly glycoproteins, while in the accessory gland 2 were not positives to glycoprotein test and the secretions seem to be merocrine and holocrine. According to Happ [42], holocrine secretions are less common in insects, and they are reported in Drosophila melanogaster (Meigen, 1830) [43] and Musca domestica Linnaeus, 1758 [44], for example. The merocrine and apocrine secretions are the most frequently observed in insects [42]. This secretion can help in the formation of the mating plug in females through the polymerization of smaller particles [42, 45], thus preventing sperm from subsequent copulations [46] from reaching the female’s copulatory bursa and/or spermatheca. Therefore, accessory glands are sources of many glycoproteins and lipids that can trigger changes in the physiology and behavior of males and females [35, 47–50]. Overall, it increases the chances of fertilization by the male, giving it advantages in sperm competition.
Based on these distinctive characteristics, it is reasonable to infer that if the morphology of spermatozoa commonly used in fertilization is already helpful for taxonomic purposes, a species that presents two types of spermatozoa further enhances the morphological characteristics in the analyses. Additionally, we highlight that the type II sperm of E. rufomarginata, which has such a peculiar and aberrant morphology, will have a higher value for taxonomic purposes than those dimorphic spermatozoa considered typical that vary, most often, in total and nuclear length. Notably, the observations raised here will significantly contribute to establishing taxonomic limits in a group as chaotic as Edessa, or at least for the subfamily Edessinae.
Final considerations
In summary, our investigation into the reproductive system of E. rufomarginata reveals the presence of dimorphic spermatozoa, one typical and the other with an aberrant morphotype. The aberrant spermatozoa may play diverse functions in reproduction other than fertilization. Furthermore, the male reproductive system presents unique anatomical characteristics with taxonomic importance. We also highlight that the fourth testicular follicle presents a distinct spermiogenesis and pattern of organization in the cysts, differing from the other follicles and being responsible for producing the aberrant sperm morphotype.
The observed sperm dimorphism aligns with existing theories and contributes to our knowledge of Pentatomidae’s reproductive strategies. Future works, including ultrastructural analysis, may further complement the data collection of this very diverse group. These findings highlight the importance of understanding the characteristics of the male reproductive system in insect biology.
Acknowledgments
We want to thank Dr. Maura Pinheiro (Universidade Federal de Viçosa) for help with laboratory techniques and Dr. José Antônio Marin Fernandes (Universidade Federal do Pará - UFPA) for confirming the identification of the specimens. We also thank the Programa de Pós-graduação em Biologia Celular e Estrutural (Universidade Federal de Viçosa–UFV).
References
- 1.
Schuh RT, Slater JA. True bugs of the World (Hemiptera: Heteroptera). Classification and Natural History. Cornell University Press. 1995.
- 2. Grazia J, Schuh RT, Wheeler WC. Phylogenetic relationships of family groups in Pentatomoidea based on morphology and DNA sequences (Insecta: Heteroptera). Cladistics. 2008; 24(6): 932–976. pmid:34892882
- 3. Silva EJE, Fernandes JAM, Grazia J. Caracterização do grupo Edessa rufomarginata e descrição de sete novas espécies (Heteroptera, Pentatomidae, Edessinae). Iheringia—Série Zoologia. 2006; 96(3): 345–362.
- 4.
Panizzi AR, Grazia J. True Bugs (Heteroptera) of the Neotropics. Entomology in Focus. Springer. 2015.
- 5. Fernandes JAM, Silva VJ. A new species group to Edessa, the E. ovina group, with description of a new species (Heteroptera: Pentatomidae: Edessinae) from Brazil. Zootaxa. 2021; 4958(1): 628–642. pmid:33903484
- 6. Santos BTS, Nascimento ATS, Fernandes JAM. Proposition of a new species group in Edessa Fabricius, 1803 (Hemiptera: Heteroptera: Pentatomidae: Edessinae). Zootaxa. 2014; 3774(5): 441–459. pmid:24871512
- 7. Fernandes JAM, Doesburg PH van. The E. cervus-group of Edessa Fabricius, 1803 (Heteroptera: Pentatomidae: Edessinae). Zoologische Mededeligen. 2000; 74(8): 151–165. https://www.researchgate.net/publication/228491813_The_E_cervus-group_of_Edessa_Fabricius_1803_Heteroptera_Pentatomidae_Edessinae
- 8. Pendergrast JG. The Male Reproductive Organs of Nezara viridula (L.) with a Preliminar Account of Their Development (Heteroptera; Pentatomidae). Auckland University College. Transactions of the Royal Society of New Zealand. 1956; 84(1): 139–146.
- 9. Gomes MO, Castanhole MMU, Souza HV, Murkami AS, Firmino TSS, Saran PS et al. Morphological aspects of the testes of 18 species of terrestrial of Heteroptera from Northwestern São Paulo (Brazil). Biota Neotropica. 2013; 13(3): 131–135.
- 10. Candan S, Yilmaz FS, Suludere Z, Erbey M. Morphology of spermathecae of some pentatomids (Hemiptera: Heteroptera: Pentatomidae) from Turkey. Zootaxa. 2015; 3937(3): 500–516. pmid:25947482
- 11. Araújo VA, Lino-Neto J, Ramalho FS, Zanuncio JC, Serrão JE. Ultrastructure and heteromorphism of spermatozoa in five species of bugs (Pentatomidae: Heteroptera). Elsevier. Micron. 2011; 42(6): 560–567. pmid:21376606
- 12. Özyurt N, Candan S, Suludere Z. The Morphology and Histology of The Female Reproductive System of Graphosoma lineatum (Heteroptera: Pentatomidae) based on light and scanning electron microscope studies. IJSR—International Journal of Scientific Research. 2013; 2(12): 42–46.
- 13. Özyurt N, Candan S, Suludere Z. The morphology and histology of the male reproductive system in Apodiphus amygdali (Germar, 1817) (Hemiptera: Heteroptera: Pentatomidae). Life: The Excitement of Biology. 2014; 2(1): 31–41.
- 14. Özyurt N, Candan S, Suludere Z. Ultrastructure of Male Reproductive System of Eurydema ventrale Kolenati 1846 (Heteroptera: Pentatomidae). Microscopy Research and Technique. 2015; 78(8): 643–653. pmid:26031393
- 15. Araújo VA, Bacca T, Dias LG. Anatomy of male and female reproductive organs of stink bugs pests (Pentatomidae: Heteroptera) from soybean and rice crops. Biota Neotropica. 2020; 20(4): e20201045.
- 16. Grozeva S, Stoianova D, Konstantinov F, Simov N, Kuznetsova VG. A synopsis of the numbers of testicular follicles and ovarioles in true bugs (Heteroptera, Hemiptera)–Sixty-five years of progress after J. Pendergrast’s Review. ZooKeys. 2022; 1136: 71–123. pmid:36762052
- 17.
Jamieson BGM, Dallai R, Afzelius BA. Insects: their spermatozoa and phylogeny. Science Publishers, Inc, Enfield, NH, U.S.A. 1999.
- 18. Dallai R, Gottardo M, Beutel RG. Structure and evolution of insect sperm: new interpretations in the age of phylogenomics. Annual Review of Entomology. 2016; 61: 1–23. pmid:26982436
- 19. Gottardo M, Dallai R, Mercati D, Hörnschemeyer T, Beutel RG. The evolution of insect sperm—an unusual character system in a megadiverse group. Journal of Zoological Systematics and Evolutionary Research. 2016; 54(4): 237–256.
- 20. Bowen RH. Studies on Insect Spermatogenesis. IV. The Phenomenon of Polymegaly in the Sperm Cells of the Family Pentatomidae. Proceedings of the American Academy of Arts and Sciences. 1922; 57(16): 391–422.
- 21. Bowen RH. Studies on Insect Spermatogenesis. I. The History of the Cytoplasmic Components of the Sperm in Hemiptera. Biological Bulletin. 1920; 39(6): 316–362.
- 22. Schrader F. Cytological and evolutionary implications of aberrant chromosome behavior in the harlequin lobe of some Pentatomidae (Heteroptera). Chromosoma. 1960a; 11(1): 103–128. pmid:14443523
- 23. Schrader F. Evolutionary aspects of aberrant meiosis in some Pentatominae (Heteroptera). Evolution—International Journal of Organic Evolution. 1960b; 14(4): 498–508.
- 24. Swallow JG, Wilkinson GS. The long and short of sperm polymorphisms in insects. Biological Reviews. 2002; 77(2): 153–182. pmid:12056745
- 25. McDiarmid CS, Li R, Kahrl AF, Rowe M, Griffith SC. Sperm sizer: A program to semi-automate the measurement of sperm length. Behavioral Ecology Sociobiology. 2021; 75(84): 1–8.
- 26. Schrader F, Leuchtenberger C. A cytochemical analysis of the functional interrelations of various cell structures in Arvelius albopuntatus (De Geer). Experimental Cell Research. 1950; 1(3): 421–452.
- 27. Schrader F, Leuchtenberger C. The cytology and chemical nature of some constituents of the developing sperm. Chromosoma. 1951; 4: 404–428. pmid:14936149
- 28. Souza HV, Itoyama MM. Comparative Study of Spermatogenesis and Nucleolar Behavior in Testicular Lobes of Euschistus heros (Heteroptera: Pentatomidae). Psyche: A Journal of Entomology. 2010; 2010(1): 1–10.
- 29. Cremonez PSG, Matsumoto JF, Perrier JD, Dunn TP, Pinheiro DO, Neves PMOJ. Morphological and Morphometric Parameters of the Reproductive Organs of Euschistus heros (Hemiptera: Pentatomidae) Treated with a Sublethal Juvenile Hormone Analog. Journal of Agricultural Science. 2023; 15(3): 10–28.
- 30. Santos BTS, Silva VJ. Fernandes JAM. Revision of Ascra with proposition of the bifida species group and description of two new species (Hemiptera: Pentatomidae: Edessinae). Zootaxa. 2015; 4034(3): 445–470. pmid:26624452
- 31. Schrader F. Regular occurrence of heteroploidy in a group of Pentatomidae (Hemiptera). Biological Bulletin. 1945; 88: 63–70.
- 32.
Pitnick S, Hosken DJ, Birkhead TR. Sperm morphological diversity. In Sperm Biology: an evolutionary perspective. Academic Press. 20098; 69–149. https://doi.org/10.1016/B978-0-12-372568-4.00003-3
- 33.
Eberhard WG. Sexual Selection and Animal Genitalia. Harvard University Press. Cambridge, Massachusetts and London, England; 1985.
- 34. Pitnick S, Mille GT, Schneider K. Markow TA. Ejaculate-female coevolution in Drosophila mojavensis. Proceedings. Biological Sciences. 2003; 270(1523): 1507–1512. pmid:12965017
- 35. Parker GA. Conceptual developments in sperm competition: a very brief synopsis. Philosophical Transactions of the Royal Society B. 2020; 375(1813): 1–10. pmid:33070727
- 36. Bressac C, Hauschteck-Jungen E. Drosophila subobscura females preferentially select long sperm for storage and use. Journal of Insect Physiology. 1996; 42(4): 323–328.
- 37. Pasini ME, Redi CA, Caviglia O, Perotti ME. Ultrastructural and cytochemical analysis of sperm dimorphism in Drosophila subobscura. Tissue & Cell. 1996; 28, 165–175. pmid:8650670
- 38. Esquivel JF. Stages of gonadal development of the southern green stink bug (Hemiptera: Pentatomidae): improved visualization. Annals of the Entomological Society of America. 2009; 102(2): 303–309.
- 39. Jyoti G, Santos N, Ashok D. Revamp studies on morpho-histology of the male reproductive system of Halys dentatus Fabricius (Hemiptera: Pentatomidae). Annual Research & Review in Biology. 2015; 63(3): 176–183.
- 40. Souza HV, Murakami AS, Moura J, Almeida EC, Marques IFG, Itoyama MM. Comparative analysis of the testes and spermatogenesis in species of the family Pentatomidae (Heteroptera). European Journal of Entomology. 2011; 108(3): 333–345.
- 41. Rezende PH, Dias G, Folly C, Lino-Neto J. Male reproductive system and sperm morphology of Eccritotarsini plant bugs (Heteroptera: Miridae). Zoomorphology, 2021; 140: 257–267.
- 42.
Happ GM. Structure and Development of Male Accessory Glands in Insects. In: King RC, Akai H, editors. Insect Ultrastructure. Springer, Boston, MA. 1984; 2: pp. 365–396. https://doi.org/10.1007/978-1-4613-2715-8_10
- 43. Perotti ME. Microtubules as components of Drosophila male paragonia secretion: An electron microscopic study, with enzymatic tests. Journal of Submicroscopic Cytology. 1971; 3(3): 255–282. https://eurekamag.com/research/016/381/016381353.php
- 44. Reimann JG. Ultrastructure of the ejaculatory duct region producing the male housefly accessory material. Journal of Insect Physiology. 1973; 19(1): 213–223. pmid:4688689
- 45. Bishop GH. Fertilization in the honey-bee. I. The male sexual organs: Their histological structure and physiological functioning. Journal of Experimental Zoology. 1920; 31(2): 225–265.
- 46.
Simmons L, Siva-Jothy MT. Sperm competition in insects: mechanisms and the potential for selection. In: Birkhead T, Moller A, editors. Sexual Selection and Sperm Competition. Academic Press. 1998; 341–434.
- 47. Leopold RA. The role of male accessory glands in insect reproduction. Annual Review of Entomology. 1976; 21: 199–221.
- 48. Baldini F, Gabrieli P, Rogers DW, Catteruccia F. Function and composition of male accessory gland secretions in Anopheles gambiae: a comparison with other insect vectors of infectious diseases. Pathogens and Global Health. 2012; 106(2): 82–93. pmid:22943543
- 49. Polat I, Mutlu DA, Suludere Z. Accessory glands of male reproductive system in Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera: Acrididae): A light and electron microscopic study. Microscopy Research and Technique. 2019; 83(3): 232–238. pmid:31769117
- 50. Wu LJ, Li F, Song Y, Zhang ZF, Fan YL, Liu TX. Proteome Analysis of Male Accessory Gland Secretions in the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae). Insects. 2023; 14 (2): 132. pmid:36835702