Micron 127 (2019) 102757
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Short communication
Sperm morphology of Elasmus polistis Burks, 1971 (Hymenoptera: Chalcidoidea: Eulophidae) Pedro Nerea, Glenda Diasa, Helen P. Santosb, André De Souzac, José Lino-Netoa,
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a
Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900, Viçosa, Minas Gerais, Brazil Instituto Federal de Minas Gerais, 36415-000, Congonhas, Minas Gerais, Brazil c Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto da Universidade de São Paulo, 14040-901, Ribeirão Preto, São Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Insects Elasmidae Ultrastructure Electron microscopy Systematics
The sperm morphology of the parasitoid Elasmus polistis (Eulophidae) has been investigated with light and transmission electron microscopy. The sperm were filiform and spiraled, with 165.6 ( ± 4.6) μm in length, and showed a distinctive head, formed by a one-layered small acrosome and a nucleus, and a flagellar region. An extracellular sheath from which many long filaments radiated out covered the acrosome and part of the nucleus. The spiral nucleus, with 24.1 ( ± 1.3) μm in length, was filled with homogeneously compact chromatin. In the nucleus-flagellum transition, the centriole adjunct extended posteriorly from the nuclear base in a spiral around the basal body, which has two central microtubules, and axoneme for approximately 1.1 μm. The two mitochondrial derivatives began roughly at the same level and at the base of the centriole adjunct. In cross-section, they were symmetrical, with a slightly oval shape and a smaller diameter in comparison to the axoneme. The latter, also spiraled, consisted of 9 + 9 + 2 microtubules that was formed from the basal body situated just below and aligned with the nucleus. The E. polistis sperm showed the same basic structures and morphological characteristics as observed in other Chalcidoidea. However, it was possible to distinguish the sperm of this species from those of other Eulophidae by (i) the long length of the centriole adjunct on the flagellum, and (ii) the presence of two central microtubules within the basal body. The sperm characteristics suggest that Eulophidae is closely related to Trichogrammatidae and both families are more similar to Eurytomidae, Pteromalidae, and Torymidae than Agaonidae.
1. Introduction
secondary parasitoids (hyperparasitoids) of Braconidae and Ichneumonidae (Coote, 1997), the majority is a primary parasitoid of Lepidoptera, Coleoptera and, a few, of Vespidae (Hymenoptera), as Elasmus polistis that parasites wasp pupae of the genus Polistes (Dorfey and Kohler, 2011). Although there are some studies using molecular and traditional morphological data, there still remain many doubts regarding the systematic of Chalcidoidea. Some of many difficulties faced by taxonomists are due to (a) the morphological homoplasy abundance present in the group (Heraty et al., 2013) and (b) insufficient resolution upon applying a limited number of molecular markers (Munro et al., 2011). Throughout the evolutionary process, the morphological variations that spermatozoa accumulated provide a surprising richness in the number of components (see Gottardo et al., 2016), in addition to shapes and interactions between them. For this reason, the morphological diversity of the male germ cells has provided data sets that have been used to help in the understanding of the phylogenetic and taxonomic
Chalcidoidea is the second most specious superfamily among the Hymenoptera including about 22,500 described species (Heraty et al., 2011, 2013; Heraty, 2017; Huber, 2017; Noyes, 2018). They are small parasitoid wasps; generally between 1.0 and 2.0 mm, have a global broad distribution and are known as parasites of Coleoptera, Diptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera, Neuroptera, Odonata, Orthoptera, Psocoptera, Siphonaptera, Strepsiptera, Thysanoptera, among others. For this reason, these wasps have been widely used in biological control programs meant for controlling pest insects (Quicke, 1997; Heraty, 2017). Within Chalcidoidea, Eulophidae is one of the most specious families comprising 4472 described species, distributed in 297 genera. The genus Elasmus, currently the only member of the tribe Elasmini (Eulophidae: Eulophinae), has 258 described species, distributed in 36 countries (Noyes, 2018). Although some species of Elasmus are
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Corresponding author. E-mail address:
[email protected] (J. Lino-Neto).
https://doi.org/10.1016/j.micron.2019.102757 Received 2 May 2019; Received in revised form 8 August 2019; Accepted 20 September 2019 Available online 22 September 2019 0968-4328/ © 2019 Elsevier Ltd. All rights reserved.
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Subsequently, the slides were stained with Giemsa for 15 min, washed in running water, and left to dry at room temperature. Lastly, the analysis and photo documentation were carried out under an Olympus BX-60 photomicroscope.
relationships in various groups of animals, including insects (Jamieson et al., 1999; Pereira et al., 2008; Pitnick et al., 2009; Dias et al., 2013; Dallai, 2014; Barcellos et al., 2015; Dallai et al., 2016, 2018). Although there is an extensive list of described species of Chalcidoidea, the number of works on sperm morphology of these insects is still small (Wilkes and Lee, 1965; Lino-Neto et al., 1999, 2000; Lino-Neto and Dolder, 2001; Fiorillo et al., 2008; Brito et al., 2009; Silva, 2010; Gonçalves, 2012; Santos et al., 2013, 2016). In this context, an overview on morphology and structural components of the Chalcidoidea spermatozoa includes: (1) the fully spiral spermatozoa; (2) the presence of a filamentous extracellular sheath covering the acrosome and part of the nucleus; (3) very small acrosome, generally less than 1.0 μm; (4) the centriole adjunct, which begins laterally to the nuclear base and ends at the tips of the two mitochondrial derivatives; (5) symmetrical and very thin mitochondrial derivatives; and (6) at the end of the axoneme, the accessory tubules finishing before the others. Therefore, the present work aims to describe the structure and ultrastructure of E. polistis sperm and also discuss the new information in light of data already reported in the literature.
2.1.2. Nucleus measurement For measuring nucleus, the staining of some slides with 0.2 μg/ml DAPI in phosphate-buffered saline for 20 min, was followed by washing in running water and subsequent mounting with a 50% sucrose solution. Further, the nuclei were photographed using an epifluorescence microscope (Olympus, BX-60) equipped with BP360-370 nm filter. All the measurements of the images were carried out using Image-J free software (http://rsbweb.nih.gov/ij/) with the measurement of at least 10.0 sperm and 10.0 nuclei per individual. 2.2. Transmission Electron Microscopy (TEM) In order to image the samples using TEM, the dissection of seminal vesicles of five individuals in 0.1 M sodium cacodylate buffer (pH 7.2), was followed by fixing in 2,5% glutaraldehyde, 2.0% sucrose, and 5.0 mM CaCl2 solution in the same buffer for 2.0 h. Subsequently, the material was washed, post-fixed in a 1.0% osmium tetroxide aqueous solution for 2.0 h, dehydrated in an increasing series of acetone, and embedded in Epoxy resin (Epon 812). Further, ultrathin sections (˜ 60 nm) were made using an automatic ultramicrotome equipped with a diamond knife, collected on copper grids, and stained with 1.0% uranyl acetate in distilled water, followed by 2.0% lead citrate in distilled water. The analyses with the subsequent image capturing were carried out using transmission electron microscope Zeiss EM109 operating at 80 kV, in Núcleo de Microscopia e Microanálise from UFV.
2. Materials and methods Specimens of E. polistis (Fig. 1) were obtained from the host Polistes versicolor (Hymenoptera: Vespidae) collected in October 2015 in the urban area of Congonhas, Minas Gerais (MG), Brazil (20° 29′S, 43° 51′W), and kept in the General Biology Department of Universidade Federal de Viçosa (UFV), Viçosa, MG. Moreover, this is the first record of E. polistis occurrence in Minas Gerais, followed by the second appearance in Brazil, being the first in the Rio Grande do Sul (Dorfey and Kohler, 2011). The species was identified by Dr. Valmir Antonio Costa, from the Biological Institute of Campinas, São Paulo, who also prepared and deposited the voucher specimens in the “Oscar Monte” Entomophagous Insect Collection at that same institute.
3. Results The analysis revealed that E. polistis sperm were filiform and spiraled, with a total length of 165.6 ± 4.6 (157.1–174.7) μm (n = 30; Fig. 2A). Further, when stained with Giemsa the head and flagellum regions of the sperm were easily distinguished (see Fig. 2A and B), specifically the head, which was formed by the nucleus and a small acrosome (see Fig. 2A–G). The acrosome consisted of only a small vesicle (˜170 nm in length) apparently without any perforatorium (see Fig. 2D and E). Moreover, an extracellular sheath (˜760 nm in length and 40 nm in thickness) covered the small acrosome and anterior nuclear region, from which several long and fine filaments radiated out (Fig. 2D and E). The nucleus, also filiform and in spiral, measured about 24.1( ± 1.3) μm in length, and showed a gradual tapering from the median region to the tip, with a diameter ranging from 265 nm to 70 nm (see Fig. 2A–C). Additionally, a homogeneously compacted chromatin filled it completely (see Figs. 2E–G and 3 A–C), which was often evidenced as a circular shape in cross-sections (see Fig. 2G). But, in some sections, it showed irregular shape due to its twisting (see Fig. 2F). The nuclear posterior region was truncated with a slightly reduced diameter laterally, where the anterior projection of the halfmoon shaped centriole adjunct (˜ 50 nm) fits tightly (see Fig. 3A–C). In the nucleus-flagellum transition region, a basal body (or centriole) and a centriole adjunct was present. The basal body, from which the axoneme arises, measured about 0.20 μm in length (for equal diameter) and was closely associated as well as was in line with the truncated nuclear base. Further, a pair of microtubules inside the basal body was observed (see Fig. 3A and B). The centriole adjunct, that connected the nucleus to flagellar components, measuring 1.6 μm in total length, extended spirally around the nucleus for about 0.5 μm (see Fig. 3A–C) and1.0 μm around the basal body and axoneme (see Fig. 3D and E), ending just over the anterior tips of the mitochondrial derivatives (MD) (see Fig. 3B). The flagellum consisted of an axoneme and two mitochondrial derivatives (see Fig. 3F–H). These structures were also spiraled, with
2.1. Light microscopy (LM) 2.1.1. Sperm measurement For carrying out sperm measurement, initially, seminal vesicles of three individuals were dissected and the content was smeared on histological slides in the presence of 0.1 M sodium phosphate buffer (pH 7.2), followed by its fixation with 4.0% paraformaldehyde for 20 min.
Fig. 1. Female of Elasmus polistis Burks, 1971. Photo: Dr. Valmir Antonio Costa (Instituto Biológico, Campinas/SP). 2
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Fig. 2. Light (A–C) and transmission electron micrographs (D–G) of E. polistis sperm in transversal (E–G) and longitudinal (D) sections. (A, B) Spermatozoa stained with Giemsa showing the head (h) and flagellum (f) regions. (C) Nucleus (n) stained with DAPI. (D–G) Sections of the head region showing the nucleus (n), the small acrosome (a) and the filaments (sf) radiating from the extracellular sheath (s).
complete turns at regular intervals of approximately 1.55 μm (see Fig. 3F). Because of this arrangement, cross-sections of the axoneme did not show all the microtubules sectioned at right angles (see Fig. 3G and H). Indeed, the axoneme follows the usual pattern of 9 + 9 + 2 microtubules, with nine outer microtubules (accessory microtubules), nine peripheral doublets, and two central microtubules connected to the last by evident radial spokes (see Fig. 3H). Moreover, the two mitochondrial derivatives started at approximately 1.0 μm below the base of the
nucleus, but by being in contact with the end of the centriole adjunct (see Fig. 3B). In cross-section, the mitochondrial derivatives were symmetrical, slightly oval, with each one having an area of only 1/5 of that of the axoneme (see Fig. 3G and H). However, in longitudinal sections, they show parallel mitochondrial cristae at regular intervals of approximately 35 nm (see Fig. 3F). In the final flagellar region, one mitochondrial derivative finishes immediately before the other and both just before the axoneme (see Fig. 3I and J). In this last structure, 3
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Fig. 3. Transmission electron micrographs of E. polistis sperm in longitudinal (A, B, F) and transverse (C–E, G–L) sections. (A–E) The nucleus-flagellum transition region shows the nucleus (n), the basal body (bb) with two central microtubules (arrows), spiraled axoneme (ax), the half-moon shaped centriole adjunct (ca) partially enveloping the nucleus, the basal body and the beginning of the axoneme. Note that mitochondrial derivatives (md) start under the ca posterior extremity. (F) Flagellum segment showing the mitochondrial derivatives and axoneme in spiral. The arrows indicate mitochondrial cristae. (G) Spermatozoa cut at different levels showing the acrosome (a), extracellular sheath (s) from which several filaments radiate (sf), nucleus (n), axonema (ax) and mitochondrial derivatives (md). (H) Median region of the flagellum showing the mitochondrial derivatives (md) and axonema (ax) consisting of the peripheral doublets (arrowhead), peripheral pairs (large arrow), the central pair (small arrows) and radial spokes (*). (I–L) Final portion of the flagellum showing that one mitochondrial derivative (md) ends shortly before the other and both before the axoneme. In this, the accessory tubules are the first structures to finish, followed by the central pair, and, finally, the peripheral doublets.
the accessory microtubules were the first to finish, followed by the central pair and, finally, the peripheral doublets (see Fig. 3J–L).
sheath coating the acrosome and an anterior portion of the nucleus has been observed. This sheath, from which innumerable filaments radiate, can vary in thickness, length, and the way it associates with the nucleus. In Palmistichus elaeisis sperm (Eulophidae, Santos et al., 2013) it is thin and very short (˜0.3 μm), while in the agaonid Idarnes sp2, it is thick and very long (˜25 μm), covering the whole region of the head and, also, an anterior portion of the flagellum (Silva, 2010). On the other hand, no sheath was observed in the spermatozoa of Idarnes sp1 and Idarnes sp3 (Silva, 2010). Similarly, Wilkes and Lee (1965) did not report the presence of this structure in the Dahlbominus fuscipennis sperm (Eulophidae). In E. polistis sperm, as in others Eulophidae (Melittobia hawaiiensis and M. australica Brito et al., 2009; P. elaeisis, Santos et al., 2013), as well as Eurytomidae (Bephratelloides pomorum, Lino-Neto
4. Discussion The E. polistis sperm showed the basic morphological characteristics of those observed in most of the Chalcidoidea studied and described previously. However, some characteristics such as (1) the nucleus length and the total length of the sperm, (2) the centriole adjunct length over the axoneme, and (3) the presence of a microtubules central pair within the basal body differentiate sperm of this species from those of other Chalcidoidea. In the spermatozoa of Chalcidoidea, generally an extracellular 4
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0.5 μm. However, in the two Melittobia species (Brito et al., 2009) it does not extend posteriorly beyond the basal body, while in P. elaeisis and T. diatraeae (Santos et al., 2013) it overlaps the basal body for a short distance and in the species of this study it extends spirally around the axoneme for about 0.8 μm. The transition between nucleus and flagellum showed to be promising in the Chalcidoidea systematic, since it was possible to group the species belonging to the same families based on the similarities between the interactions of the components present in this region. The microtubules of the basal body were embedded in the dense centriole adjunct material that makes difficult to see the microtubular composition. However, the occurrence of two central microtubules, an unusual condition for this flagellar component (Avidor-Reiss, 2018), was easily observed. In insects, the occurrence of these microtubules inside the basal body was observed in Orthoptera (Melanoplus differentialis, Phillips, 1970), Diptera (Drosophila melanogaster, Dallai et al., 2008; Carvalho-Santos et al., 2012) and Coleoptera (Coccinellidae, Dallai et al., 2018). Carvalho-Santos et al. (2012) described that in D. melanogaster spermatogenesis a single microtubule is found inside most of the basal body, it extends into the axoneme where the second microtubule of the pair forms adjacent to it. It is possible that in E. polistis the emergence of two central microtubules occurred this way. However, Avidor-Reiss (2018) says that this condition may be due to the growth of the central pair of axonemal microtubules to the interior of the basal body, as occurs in many amniotes during spermatogenesis and remained in mature sperm. The specie of this studied was the first Hymenoptera that exhibited this feature, indicating that this may be a characteristic of the genus, or even of the species. Radial spokes are components of the axoneme and, together with the microtubules, are responsible for sperm motility. As the protein composition and structure of these components are not constant, it is possible that distinct organisms exhibit a varied morphology (Zhu et al., 2017). In the studied Chalcidoidea, these components are very evident, with the structure being similar in all species, suggesting that this is a conserved characteristic in the group. As in most Chalcidoidea, the E. polistis sperm axoneme is the last structure of the flagellum to end; first the accessory microtubules followed by the central pair. However, in Pegoscapus (Agaonidae, Fiorillo et al., 2008), the central pair finishes first. The axonema in T. diatraeae (Santos et al., 2013), when viewed in longitudinal section, has transverse striations along the entire length, something never before observed in any Chalcidoidea. The mitochondrial derivatives in this eulophid are oval, symmetrical, and with smaller diameter than the axonema, similar to those observed in other Eulophidae (Wilkes and Lee, 1965; Santos et al., 2013), Eurytomidae (Lino-Neto et al., 1999), Trichogrammatidae (LinoNeto et al., 2000; Lino-Neto and Dolder, 2001), Torymidae (Gonçalves, 2012), and Pteromalidae (Santos et al., 2016). However, the mitochondrial derivatives of M. hawaiiensis and M. australica (Brito et al., 2009) are asymmetrical, possibly representing a unique feature for this Eulophidae genus. In Pegoscapus (Fiorillo et al., 2008), mitochondrial derivatives differ from those mentioned above only because they are slightly asymmetrical. In Idarnes (Silva, 2010), they are larger in area and initially encircle the axoneme completely, with later acquiring triangular shape when observed in cross-section. Thus, this possibly indicates that the subfamilies to which the two genera of agaonids are included (Agaoninae and Sycophaginae, respectively) are distantly related to each other. With the exception of Idarnes, in which the mitochondrial derivatives begin at the nucleus base and above the centriole adjunct, in the other Chalcidoidea studied, the two mitochondrial derivatives begin approximately together and at the base of the centriole adjunct, and extend in a spiral form until near the end of the flagellum, where one derivative ends immediately before the other. Nevertheless, in T. diatraeae (Santos et al., 2013) being the only exception, the two mitochondrial derivatives end up in quite different levels. In addition to its function as an energetic source for the cell, the mitochondrial derivatives can reduce the flexibility of the flagellum
et al., 1999), Trichogrammatidae (Lino-Neto et al., 2000; Lino-Neto and Dolder, 2001), Agaonidae (Pegoscapus spp, Fiorillo et al., 2008; Idarnes spp, Silva, 2010) and Pteromalidae (Muscidifurax uniraptor, Santos et al., 2016), the extracellular sheath is closely attached to the nucleus. In contrast, in the agaonid Pegoscapus tonduzi (Fiorillo et al., 2008) and eulophid Trichospilus diatraeae (Santos et al., 2013) the sperm exhibited an electro-lucid space between the sheath and the nucleus. A filamentous extracellular sheath was also observed in the sperm of the parasitic wasps Braconidae (Newman and Quicke, 1998) and Ichneumonidae (Moreira et al., 2010), suggesting a close relationship between Chalcidoidea and Ichneumonoidea. On the other hand, there is no record of this structure in Aculeata (bees, ants, and wasps) and Symphyta sperm. The function of this structure remains unknown, possibly playing a mechanical and/or structural role, serving as a barrier that protects the nucleus and specially the acrosome. A very small acrosome was commonly observed in most Chalcidoidea sperm. An one-layer acrosome, formed only by the acrosomal vesicle, as in the Eulophidae studied here, was also observed in others species of this family (M. australica, M. hawaiiensis, Brito et al., 2009; T. diatraeae, Santos et al., 2013), as well as in Trichogrammatidae (Lino-Neto et al., 2000) and Agaonidae (Silva, 2010). The presence of a perforatorium, with the base inserted into a cavity at the anterior tip of the nucleus and covered by the acrosomal vesicle, was described in at least one species for each studied family of Chalcidoidea (Lino-Neto et al., 1999; Lino-Neto and Dolder, 2001; Silva, 2010; Gonçalves, 2012; Santos et al., 2013, 2016). In contrast, none of the structures that compose the acrosome were observed in the Pegoscapus spp (Agaonidae, Fiorillo et al., 2008). It is important to point out that, in Chalcidoidea, the presence (or absences) of the acrosomal components, mainly the perforatorium, should be considered with caution since they are very small structures (generally less than 0.2 μm). In addition, the acrosomal components may exhibit electrodensity similar to that of the nucleus, which may result in misinterpretation (Santos et al., 2016, Fig. 1B–E). In the Chalcidoidea the sperm nucleus are spiraled, and when observed under fluorescence microscopy (stained with DAPI) or scanning electron microscopy, they show two basic forms; (i) First, species in which sperm have robust and small nucleus that do not exceeds 20 μm in length, with a drop shape in the posterior region, and which taper sharply from 1/3 of their length towards the apex [see in Trichogramma pretiosum, T. atopovirilia (Lino-Neto et al., 2000), T. dendrolimi (LinoNeto and Dolder, 2001), M. hawaiiensis, M. australica (Brito et al., 2009), Idarnes sp1 (Pereira et al., 2008) and T. diatraeae (Santos et al., 2013)]. (ii) Second, species with spermatic nuclei longer than 24 μm with a needle-like shape and smooth tapering from the base to apex [see in D. fuscipennis (Wilkes and Lee, 1965), B. pomorum (Lino-Neto et al., 1999), Pegoscapus spp (Fiorillo et al., 2008), Idarnes sp2, Idarnes sp3 (Pereira et al., 2008), P. elaeisis (Santos et al., 2013) and this study]. Despite the differences, it is possible that in both cases the chromatin amounts are similar since the thicker basal area in the first may compensate for their reduced size. The centriole adjunct varies greatly in shape, size, and in the way it interacts with other sperm components in the nucleus-flagellum transition region. In Agaonidae, the centriole adjunct does not overlap the nuclear posterior end. Indeed, it was restricted to the basal body region in Pegoscapus (Fiorillo et al., 2008). However, in Idarnes (Silva, 2010), it begins after the mitochondrial derivatives and remains enveloped by them for at least 4.0 μm. In Eurytomidae (Lino-Neto et al., 1999), Torymidae (Gonçalves, 2012), and Pteromalidae (Santos et al., 2013) the centriole adjunct extends anteriorly, together and spiral with nucleus, for a long length (˜ 4–8 μm), but posteriorly (in the flagellar region) it extends for only 0.3–0.5 μm. In the three Trichogrammatidae species (Lino-Neto et al., 2000; Lino-Neto and Dolder, 2001), this structure does not measure more than 0.5 μm in length, extending anteriorly for about 0.3 μm and ending posteriorly just after the basal body. In Eulophidae, the centriole adjunct is very similar to that of the trichogrammatid sperm, not overlapping the nucleus for more than 5
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(see Dallai et al., 2018), which added to the spiral arrangement of the entire flagellum, may alters the flagellar beating pattern, influencing the sperm motion. The sperm flagellum of the Chalcidoidea, in general, does not exhibit accessory bodies, and when discernible, they are very slender, as in P. elaeisis (Santos et al., 2013), B. pomorum (Lino-Neto et al., 1999), and M. uniraptor (Santos et al., 2016). This characteristic differentiates the Chalcidoidea from most Hymenoptera (see in Jamieson et al., 1999; Zama et al., 2005; Araújo et al., 2009; Moreira et al., 2012). A center-flagellar material commonly occurs in the spermatozoa of Hymenoptera, especially in Symphyta (Newman and Quicke, 1999; Lino-Neto et al., 2008) and Aculeata (Zama et al., 2001; Mancini et al., 2009). In Chalcidoidea, including the species studied here, this material has not been observed except in Eurytomidae (B. pomorum, Lino-Neto et al., 1999) and Pteromalidae (M. uniraptor, Santos et al., 2016), an indication that the two families can be closely related.
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5. Conclusion These results show that the morphology of Chalcidoidea sperm indicate that Eulophidae is closely related to Trichogrammatidae, as observed by Heraty et al. (2013) and Peters et al. (2018) and both families are closer to Eurytomidae, Pteromalidae, and Torymidae than Agaonidae. In the last, the spermatozoa of Pegoscapus are more similar to the five families, with the similarity being closer to Idarnes, agreeing with the non-monophyletic condition of Agaonidae proposed by Rasplus et al. (1998) based on molecular data. Gauthier et al. (2000) demoted the Elasmidae to Elasmini tribe and placed this latter in Eulophinae (Eulophidae). This classification was confirmed by other works such as those Burks et al. (2011); Munro et al. (2011); Heraty et al. (2013) and Peters et al. (2018). Whence, in the present study, we can also observe a great morphological similarity between the E. polistis sperm and those of the other Eulophidae. Declaration of Competing Interest None. Acknowledgments We would like to thank to Dr. Valmir Antonio Costa (Biological Institute of Campinas, São Paulo) for the identification, preparation and deposition of the voucher specimens in the “Oscar Monte” Entomophagous Insect Collection, to the Nucleus of Electron Microscopy and Microanalysis of the UFV, and to GlobalEdico (www. globaledico.com) for reviewing English language. This research was funded by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). References Araújo, V.A., Moreira, J., Lino-Neto, J., 2009. Structure and ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse Fabricius1804 (Hymenoptera: Apoidea: Crabronidae). Micron 40, 719–723. Avidor-Reiss, T., 2018. Rapid evolution of sperm produces diverse centriole structures that reveal the most rudimentary structure needed for function. Cells 7, 67. Barcellos, M.S., Martins, L.C., Cossolin, J.F., Serrão, J.E., Delabie, J.H., Lino-Neto, J., 2015. Testes and spermatozoa as characters for distinguishing two ant species of the genus Neoponera (Hymenoptera: Formicidae). Fla. Entomol. 98, 1254–1256. Brito, P., Lino-Neto, J., Dolder, H., 2009. Sperm structure and ultrastructure of the Melittobia hawaiiensis, Perkins and M. australica, Girault (Chalcidoidea: Eulophidae). Tissue Cell 41, 113–117. Burks, R.A., Heraty, J.M., Gebiola, M., Hansson, C., 2011. Combined molecular and morphological phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with focus on the subfamily Entedoninae. Cladistics 27, 581–605. Carvalho-Santos, Z., Machado, P., Alvarez-Martins, I., Gouveia, S.M., Jana, S.C., Duarte, P., Amado, T., Branco, P., Freitas, M.C., Silva, S.T.N., Antony, C., Bandeiras, T.M.,
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