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Spermiogenesis cells stained with lacto-acetic orcein ((a)–(h)) and silver impregnated ((i)–(t)). (a)–(d) Development of spermatids in lobes 1–3 and 5. (a) Round spermatids showing heteropyknotic material at the periphery and center of the nucleus (arrows) and a vesicle in the posterior region (arrowhead); (b) spermatid in elongation with heteropyknotic material at the periphery of the nucleus (arrow); (c) in a following stage, the heteropyknotic chromatin is visualized on only one side (arrow); (d) in a later stage, it becomes indistinguishable; (e)–(h) spermatids of lobes 4 and 6; (e) round spermatids showing a vesicle in the posterior region (arrowhead); (f) spermatid in elongation; (g) spermatid at the later stage of development with chromatin stained weakly and in the posterior region with a protuberance (arrow); (h) sperm showing a thin heteropyknotic chromatin on one side of the head (arrow); ((i)–(m)) round spermatid with several silver-positive bodies; ((j)–(l)) during the subsequent phases were observed three silver-positive bodies: one rod-shape and lighter close to region of tail formation ((j), smaller arrow), one round intensely stained (arrowhead), both in the posterior region of the spermatid and another smaller than others (bigger arrow) that was visualized in the middle region of the spermatid ((k), (l)); (m) spermatid in development with only one silver-positive body in the posterior region of the nucleus (arrow); (n) spermatozoon of lobes 1–3 with continuous silver-positive staining in the posterior region of the head (arrow) and in lobe 5 (o) a continuous positive silver impregnation (arrow) and several small bodies in all the nucleus (arrowhead). ((p)–(t)) Spermiogenesis of lobes 4 and 6; (p) spermatid in development showing strong silver-positive body in the posterior region of the head (arrow), several small silver-positive bodies in the middle region and a line-shaped silver staining from middle to anterior region (arrowhead); ((q)–(t)) developed spermatids showing four silver-positive behaviors: (q) continuous and linear (arrow); (r) large and amorphous; (s) linear and throughout the extent of the head and several round and small silver-positive bodies and (t) with the same previous characteristics, but the silver-positive bodies are larger and smaller in number. Bar= 10 μm
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In some testicular lobes of the Pentatomidae there may be occurrence of atypical spermatogenesis or polymegaly, leading to the production of nonfertile sperm. The comparative analysis of spermatogenesis and nucleolar behavior in testicular lobes of Euschistus heros showed cells with polymegaly in lobes 4 and 6. Generally, when these lobes are prese...
Citations
... In E. heros, it was possible to observe the presence of holocentric chromosomes and a diploid number 2n = 12A + XY, with the sex chromosomes showing a subtle difference in size, with the Y chromosome being the smallest, as previously observed in the works of Souza and Itoyama (2010), Bardella et al. (2013a), and Aguiar et al. (2017). In addition to the presence of holocentric chromosomes, other cytogenetic characteristics attributed to insects of the suborder Heteroptera are the presence of chiasmatic autosomal bivalents in contrast to the achiasmatic sex chromosomes and the occurrence of inverted meiosis in the sex chromosomes (Papeschi et al. 2003;Viera et al. 2009;Dionisio et al. 2020). ...
Transposable elements (TEs) are DNA sequences capable of moving within the genome. Their distribution is very dynamic among organisms, and despite advances, there are still gaps in the understanding of the diversity and evolution of TEs in many insect species. In the case of Euschistus heros, considered the main stink bug in the soybean crop in Brazil, little is known about the participation of these elements. Therefore, the objective of the current work was to identify the different groups of transposable elements present in the E. heros transcriptome, evidencing their chromosomal distribution. Through RNA-Seq and de novo assembly, 60,009 transcripts were obtained, which were annotated locally via Blastn against specific databases. Of the 367 transcripts identified as TEs, 202 belong to Class II, with emphasis on the TIR order. Among Class I elements or retrotransposons, most were characterized as LINE. Phylogenetic analyses were performed with the protein domains, evidencing differences between Tc1-mariner sequences, which may be related to possible horizontal transfer events. The transposable elements that stood out in the transcriptome were selected for fluorescent in situ hybridization. DNA transposon probes hAT, Helitron, and Tc1-mariner showed mostly scattered signals, with the presence of some blocks. Retrotransposon probes Copia, Gypsy, Jockey, and RTE showed a more pulverized hybridization pattern, with the presence of small interstitial and/or terminal blocks. Studies like this one, integrating functional genomics and molecular cytogenetic tools, are essential to expanding knowledge about transcriptionally active mobile elements, and their behavior in the chromosomes.
... It is known that the testes are externally covered by a CT capsule that projects inside the interior of the organ, separating each follicle. This tissue has been described in vertebrates as tunica albuginea, although it is referred to as external tunic as similar observed in insects, and its intern ramification are called internal tunics (Souza & Itoyama, 2010;Alevi, Oliveira, Rosa, & Oliveira, 2014). ...
The aim of this study was to determine the effect of the insecticide pyriproxyfen (Tiger™ 100 CE), juvenile hormone analogue, when applied in a sublethal LC 30 concentration (0.668 mL a. i. L-1), on the morphological and morphometric parameters of external and internal connective tissue (CT) of the Neotropical-brown stink bug Euschistus heros testicles. The insecticide was applied on nymphs from the 4th instar using a Potter tower with a working pressure of 82.73 kPa (12 lb pol-²) and 1 mL of the emulsion per replicate. A completely randomized experimental design was used, consisting of two treatments (control and pyriproxyfen-treated), five repetitions, and 10 adults of E. heros per experimental unit. The insects were maintained under controlled conditions until the emergence of adults. After 48 h of emergence of adults, the testicles were collected, fixed, and processed for morphological and morphometric analyses. A change was observed in the collagen fibers of the CT of treated insects when compared with those of controls. It was also observed that both types of CT (dense irregular and loose) over the internal tunic of the treated insect were thinner than those in control insects. The analysis showed that pyriproxyfen significantly reduced the external and internal CT width and the conformation of its fibers in all the observed regions when compared with the controls; this may affect the production of the three different types of sperm present in this species.
... The testes of E. heros are anatomically paired, connected to an ejaculatory common duct by vas deferens as described for other Pentatomidae insects (Araújo et al., 2011;Cremonez et al., 2017). The morphological differences between the testicular follicles of E. heros were previously observed (Souza & Itoyama, 2010;Aguiar et al., 2017), demonstrating that F1, F2 and F3 are regular sized follicles, while F4 and F6 are slightly larger and F5 is much thinner than the others. ...
Euschistus heros is an important pest in Brazilian agriculture growing importance in the general Neotropical realm. Its reproductive potential is the key factor for its characterization as a pest in major crops such as soybean and cotton. The aims of this study were to characterize morphometric parameters of testicles and testicular accessory cells-TACs nuclei of adults E. heros treated with chitin biosynthesis inhibitors (CBIs). The insecticides lufenuron (Match® 50 EC) and buprofezin (Applaud® 250 WP) were applied individually in 4th instar nymphs, remained in controlled conditionsuntil the emergence of adult males. The testicles were identified and removed 72 h after emergency, fixed, photographed for anatomic analysis, and processed for morphometric analyses of the TACs nuclei. It was possible to observe that lufenuron and buprofezin decreased the testicular area. Buprofezin decreased the mean nuclear area analyzed in the TACs, and nuclear hypertrophy can indicate an activity on support and nutrition of germ line cells, presenting a possible effect on protein synthesis. The intense reaction for Fast green in control compared to buprofezin treatment may indicate that total protein (histones and non-histones) has been altered. The tested insecticides, with special focus on buprofezin may affect the final stages of reproductive development of E. heros, with potential to be used in field to the control of this pest.
... Entomological Science (2017) 20, 279-287 observed in specimens collected in the State of São Paulo by Souza and Itoyama (2010a). The number of lobes varies from three to eight, with the prevalence of seven, among species of the family Pentatomidae (Souza & Itoyama 2010a). ...
... Entomological Science (2017) 20, 279-287 observed in specimens collected in the State of São Paulo by Souza and Itoyama (2010a). The number of lobes varies from three to eight, with the prevalence of seven, among species of the family Pentatomidae (Souza & Itoyama 2010a). ...
... In all the examined individuals, the fifth lobe was thinner than the others, and had spermatic cells larger in diameter than those of the first to third lobes but smaller than those of the fourth and sixth lobes, as reported by Souza and Itoyama (2010a). The presence of spermatozoa with different sizes and diameters was corroborated in E. heros: Cossolin (2015) described three types of sperms different in the morphology and ultrastructure. ...
Euschistus heros (Fabricius, 1798) is one of the most harmful insect pests damaging Brazilian soybean crops and has become a major problem due to its high population density and resistance to insecticides. Currently, there are no data on whether alterations of testicular morphology and chromosomal behavior are associated with the resistance mechanisms related to the action of insecticides. This study integrated analyses of the testicular morphology, meiocyte cell division, and chromosomal structure and behavior in the process of spermatogenesis in E. heros. We compared these features among wild-caught individuals, insecticide-susceptible and -resistant strains. The resistant strain was established through a selection experiment exposing the bugs to insecticides (thiamethoxam + lambda-cyhalothrin) for 15 generations. No differences were detected in the examined features among the three groups of experimental individuals: the testis comprised six lobes, with the fifth lobe thinner than the others; the karyotype was 2n = 14 (12 + XY), with no evident changes in chromosomal breakage, rearrangement or behavior in the meiosis; and abundant spermatozoa were observed in all testicular lobes. Thus, any effects of the long-term (15 generations) experimental selection by exposure to the insecticides were not detected on the male germinal tissue and chromosomes, suggesting the irrelevancy of the examined features to insecticide-resistance mechanisms in E. heros.
... They also have a variable number, for example, in the Coreidae it varies from three to seven (Souza et al., 2007a). According to the literature, the number of lobes in the family Lygaeidae is variable, with species with two, four, six and seven lobes, where seven is considered the ancestral number (Grozeva & Kuznetsova, 1992 (Souza & Itoyama, 2010) includes six species of the subfamily Asopinae, in which only one species (Euthyrhynchus floridanus) has six testicular lobes and the others seven. In the other subfamilies, the distribution of lobes differs, ranging from four to eight in the Discocephalinae, four to five in the Edessinae and three to seven in the Pentatominae. ...
... In the Discocephalinae, for example, the harlequin is usually lobe 5, numbered from the ejaculatory duct. In most cases, when lobes 4 and 6 show polymegaly, lobe 5 shows atypical meiosis, with the only exception being Euschistus heros (Pentatomidae: Carpocorini), which in addition to lacking a harlequin lobe, has a very narrow fifth lobe (Souza & Itoyama, 2010). The present study found that Proxys albopunctulatus (Pentatomidae: Carpocorini) belongs this select group, i.e., it lacks a harlequin lobe, and lobe 5 is flanked by lobes that produce differentiated cells and has a small diameter. ...
There are few studies on the morphology and meiosis in the testicles of Heteroptera, but are extremely important, especially for the family Pentatomidae, because in some species in this family meiosis in the testicular lobes results in the production of non-fertile spermatozoa. With the aim of improving the level of understanding of this phenomenon the morphology of the testes and spermatogenesis in 10 species of Pentatomidae were recorded and compared. All of them have testes covered by a pigmented peritoneal sheath. In some species the internal membrane or just inside the peritoneal sheath is pigmented. The pigmentation of both membranes varied. The pigmentation of both was either yellow, or the internal membrane was yellowish and the external one reddish. When the membrane is pigmented, the colour is red or yellow. The number of lobes varied from 3 to 7, with intermediate numbers of 4 and 6. The size and diameter of the lobes are similar in all the species studied, except Proxys albopunculatus, in which the diameter of the third and fifth is smaller than that of the other lobes. The behaviour of the cells during spermatogenesis was the same in all lobes of most species, except in P. albopunctulatus, in which the harlequin lobe is absent and the cells in lobes 4 and 6 exhibited characteristics different from those of cells in lobes 1-3 and 5. Chlorocoris complanatus and Loxa deducta (both Pentatominae) have a harlequin lobe (lobe 5). The chromosome complements recorded were: 2n = 12 (10A + XY) in Dichelops melacanthus and Edessa collaris, 2n = 14 (12A + XY) in C. complanatus, Edessa meditabunda, Ladeaschistus sp., Loxa deducta, P. albopunctulatus, Piezodorus guildinii and Thyanta perditor and 2n = 16 (14A + XY) in Edessa affinis. Thus, this study extends the knowledge of characteristics, such as the pigmentation of the peritoneal sheath, number of testicular lobes, the occurrence of meiotic cells in some testicular lobes, and the chromosome complements of the family Pentatomidae.
Taxonomic data on Coreidae have been fragmented over time and need to be revised. Likewise, data related to the development of germ cells and the features of the male reproductive system, including sperm, will contribute to understanding the biological mechanisms of reproduction and the systematics of its representatives. Aiming to provide these data, we describe the morphology of the male reproductive system and spermatozoa of Leptoglossus zonatus using light and transmission electron microscopies, respectively. Each of the two testes is surrounded by a bright red‐pigmented sheath and formed by seven follicles arranged side by side. The two vasa deferentia are filled with individualized sperm, especially in their final portion, which is dilated and curved. After dilation, the vasa deferentia receive the ducts of the accessory glands of mesodermal origin. The other unpaired accessory gland is of ectodermal origin and opens into the ejaculatory duct. Both glandular types are densely coiled and have lumens filled with secreted material. Testicular follicles contain cysts with germ cells at different stages of spermatogenesis, indicating continuous production of gametes throughout adult life. Mature sperm measure around 310 μm long, with a nucleus of 36 μm and a flagellum formed only by an axoneme of 9 + 9 + 2 microtubules and two symmetrical mitochondrial derivatives. Like the sperm of other Heteroptera, the acrosome has a single structure (without perforatorium), there are no accessory bodies in the flagella, and the mitochondrial derivatives are connected to the axonemes, supporting the synapomorphic condition of these characteristics for this suborder of bedbugs.
Research Highlights
The Leptoglossus zonatus sperm are slender and long, about 310 μm in length, and a nucleus 36 μm long.
Spermatogenesis occurs throughout adult life and equally in the seven testicular follicles.
The centriole adjunct in L. zonatus sperm does not give rise to accessory bodies.
The ectodermal gland produces a filamentous secretion, whereas in the ectodermal sac, the secretion is globular.
Representatives of the Coreidae, include species of economic importance that are pests in some crops, such as Machtima crucigera (Fabricius, 1775) pest of acerola crops. Morphological studies can contribute to the understanding of the evolutionary history of insects and to elucidate their reproductive strategies. This study describes the structure of the male reproductive tract of this insect. The males have two pectin-shaped testes, with seven follicles each, and many cysts at different developmental stages. In the testicular follicles, three spermatogenesis regions are recognized: the growth, maturation and differentiation zones. The reproductive tract also includes a pair of deferent ducts, each which enlarge into two seminal vesicle, accessory glands of mesodermal and ectodermal origins, an ejaculatory bulb, an ectodermal sac and an ejaculatory duct ending in the aedeagus. The testes and seminal vesicles are covered externally by an orange-pigmented peritoneal sheath. The morphological features of the male reproductive tract of M. crucigera and its similarities with other Heteroptera are discussed. These characters have potential to elucidate reproductive strategies of this agricultural pest and the evolutionary history of the group.
Insects of the suborder Heteroptera are known for their odor, for being pests, or for being disease carriers. To gain better insight into the cytogenetic characteristics of heteropterans, 18 species of terrestrial Heteroptera belonging to eight families were studied. The presence of heteropycnotic corpuscles during prophase I, terminal or interstitial chiasmas, telomeric associations between chromosomes, ring disposals of autosomes during metaphase, and late migrations of the sex chromosomes during anaphase were analyzed. These features showed identical patterns to other species of Heteroptera previously described in the literature. Another studied characteristic was chromosome complements. The male chromosome complements observed were 2n = 12 chromosomes [10A + XY, Galgupha sidae (Amyot & Serville) (Corimelaenidae) and Pachycoris torridus (Scopoli) (Scutelleridae)]; 2n = 13 [10A + 2m + X0, Harmostes serratus (Fabricius), Harmostes apicatus (Stål), Jadera haematoloma (Herrich-Schaeffer), Jadera sanguinolenta (Fabricius), Jadera sp. (Rhopalidae)], and Neomegalotomus parvus (Westwood) (Alydidae); 2n = 13 [12A + X0, Stenocoris furcifera (Westwood) (Alydidae); 2n = 14 [12A + XY, Dictyla monotropidia (Stål) (Tingidae)]; 2n = 19 [18A + X0, Acanonicus hahni (Stål) (Coreidae)]; 2n = 21 [18A + 2m + X0, Acanthocephala sp. (Dallas) (Coreidae)]; 2n = 27 [24A + 2m + X0, Anisoscelis foliacea marginella (Dallas) (Coreidae)]; 2n = 18 [16A + XY, Oncopeltus fasciatus (Dallas) (Lygaeidae)]; 2n = 17 [14A + X1X2Y, Oxycarenus hyalinipennis (Costa) (Lygaeidae)]; 2n = 16 [12A + 2m + XY, Pachybrachius bilobatus (Say) (Lygaeidae)]; 2n = 26 [24A + XY, Atopozelus opsinus (Elkins) (Reduviidae)]; and 2n = 27 [24A + X1X2Y, Doldina carinulata (Stål) (Reduviidae)]. The diversity of the cytogenetic characteristics of Heteroptera was reflected in the 18 studied species. Thus, this study extends the knowledge of these characteristics, such as the variations related to chromosome complements, sex chromosome systems, and meiotic behavior.
Different techniques have been used to evaluate post-meiotic nucleolar activity during spermatogenesis. However, the results have not been consistent, and transcriptional activity during spermiogenesis is still a highly debated issue. Through the use of cytogenetic tools, we evaluated whether the nucleolus is active during spermiogenesis in triatomines, more specifically, in Triatoma lenti. During spermatid differentiation, the nucleolus was observed using the cytogenetic technique of silver staining. Through the Feulgen reaction, we found that the silver ions had labeled the ribosomal DNA (rDNA). Using molecular cytogenetic techniques (DAPI and FISH), we were able to highlight the nucleolus and the Nucleolus Organizing Region (NOR) in spermatid images. Using the C-banding technique, we found rDNA and the NOR to be completely heterochromatic. Since this nuclear structure is present throughout the spermatid stage, we believe that the rDNA and NOR are inactivated by epigenetic factors, because we found a direct relationship between the nucleolus, rDNA, the NOR, and heterochromatin. Because heterochromatization of DNA is an important way to inactivate genetic material, we believe that the entire transcriptional activity required for cellular differentiation during spermiogenesis is mediated by the chromatoid body.
The Heteroptera are known for their odour, for being pests or for being disease
carriers. However, they are still not extensively studied, perhaps because they form a very large
group. Therefore, with the aim of enhancing the knowledge of the morphology of the testes of this
insect order, we collected and analysed 18 species of terrestrial Heteroptera from the northwestern
part of São Paulo. The analysis of these species revealed some differences between the
testicles of these species, including their shape (elongated, oval, round or pecten), the
morphology of the testicular lobes (elongated and paired side by side or united in a single
region), the colours of the peritoneal sheath that surrounds the lobes (red, orange,
yellow or translucent), and the number of testicular lobes (one, two, four, five, six or
seven). Because the aspects analysed were highly variable, our study suggests a need for
further analysis of Heteropteran testicular morphological differences.
