Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera,Drosophilidae): Cytochemical,structural and ultrastructural characterization

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1–3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.     

Taxonomic boundaries,phylogenetic relationships and biogeography of the <Emphasis Type="Italic">Drosophila willistoni</Emphasis> subgroup (Diptera: Drosophilidae)

The Drosophila willistoni subgroup represents a complex with varying taxonomic levels. It encompasses D. willistoni and its five sibling species: D. equinoxialis, D. insularis, D. paulistorum, D. pavlovskiana and D. tropicalis. Of these, D. equinoxialis, D. tropicalis and D. willistoni present differentiation at subspecific level, whereas D. paulistorum represents a superspecies, formed by six semispecies. Despite this taxonomic and evolutionary complexity, many of these semi and subspecific taxa have not yet had their phylogenetic status tested in an explicitly molecular study. Aiming to contribute to the understanding of the evolution of this challenging group, we analyzed nucleotide sequences from two mitochondrial and four nuclear datasets, both individually and simultaneously, through different phylogenetic methods. High levels of incongruence were detected among partitions, especially concerning the mitochondrial sequences. As this incongruence was found to be statistically significant and robust to the use of different models and approaches, and basically restricted to mitochondrial loci, we suggest that it may stem mainly from hybridization-mediated asymmetrical introgression. Despite this, our nuclear data finally led to a phylogenetic hypothesis which further refines several aspects related to the willistoni subgroup phylogeny. In this respect, D. insularis, D. tropicalis, D. willistoni and D. equinoxialis successively branched off from the willistoni subgroup main stem, which recently subdivided to produce D. paulistorum and D. pavlovskiana. As regards the semispecies evolution, we found evidence of a recent diversification, which highly influenced the obtained results due to the associated small levels of genetic differentiation, further worsened by the possibly associated incompletely sorted ancestral polymorphisms and by the possibility of introgression. This study also raises the question of whether these semispecies are monophyletic at all. This reasoning is particularly interesting when one considers that similar levels of reproductive isolation could be attained through infection with different Wolbachia strains.     

Phylogeny of Drosophila and related genera inferred from the nucleotide sequence of the Cu,Zn Sod gene

The phylogeny and taxonomy of the drosophilids have been the subject of extensive investigations. Recently, Grimaldi (1990) has challenged some common conceptions, and several sets of molecular data have provided information not always compatible with other taxonomic knowledge or consistent with each other. We present the coding nucleotide sequence of the Cu,Zn superoxide dismutase gene (Sod) for 15 species, which include the medfly Ceratitis capitata (family Tephritidae), the genera Chymomyza and Zaprionus, and representatives of the subgenera Dorsilopha, Drosophila, Hirtodrosophila, Scaptodrosophila, and Sophophora. Phylogenetic analysis of the Sod sequences indicates that Scaptodrosophila and Chymomyza branched off the main lineage before the major Drosophila radiations. The presence of a second intron in Chymomyza and Scaptodrosophila (as well as in the medfly) confirms the early divergence of these two taxa. This second intron became deleted from the main lineage before the major Drosophila radiations. According to the Sod sequences, Sophophora (including the melanogaster, obscura, saltans, and willistoni species groups) is older than the subgenus Drosophila; a deep branch splits the willistoni and saltans groups from the melanogaster and obscura groups. The genus Zaprionus and the subgenera Dorsilopha and Hirtodrosophila appear as branches of a prolific “bush” that also embraces the numerous species of the subgenus Drosophila. The Sod results corroborate in many, but not all, respects Throckmorton's (King, R.C. (ed) Handbook of Genetics. Plenum Press, New York, pp. 421–469, 1975) phylogeny; are inconsistent in some important ways with Grimaldi's (Bull. Am. Museum Nat. Hist. 197:1–139, 1990) cladistic analysis; and also are inconsistent with some inferences based on mitochondrial DNA data. The Sod results manifest how, in addition to the information derived from nucleotide sequences, structural features (i.e., the deletion of an intron) can help resolve phylogenetic issues. Correspondence requests to: F. J. Ayala     

Sex-specific methylation in <Emphasis Type="Italic">Drosophila</Emphasis>: an investigation of the <Emphasis Type="Italic">Sophophora</Emphasis> subgenus

Epigenetic phenomena have been widely characterized in the genomes of vertebrates and DNA methylation is a key mechanism of epigenetic regulation. The DNA methylation systems of invertebrates and vertebrates show several notable differences. However, the evolutionary implications of those differences only recently began to be revealed. Our study investigated the recurrence of sex-specific methylation, as previously described for the species Drosophila willistoni, in other species of the Sophophora subgenus that present close evolutionary relationship. The MSRE and Southern blot techniques were used to analyze rDNA of some species of the willistoni, melanogaster, saltans and obscura groups of Drosophila and the results suggested that differential DNA methylation between sexes only occurs in Drosophila tropicalis and D. insularis, two sibling species of the willistoni subgroup. However, only using the MSRE technique we could detect sex-specific patterns of DNA methylation in all species of willistoni subgroup. These results indicate that DNA methylation may present important differences, even between closely related species, shedding new light on this Neotropical species complex.     

Comparative study of sperm ultrastructure of Donax hanleyanus and Donax gemmula (Bivalvia: Donacidae)

The ultrastructure of bivalve spermatozoa can be species‐specific and often provides important taxonomic traits for systematic reviews and phylogenetic reconstructions. Young individuals of the Donacidae species Donax hanleyanus are often identified as samples of Donax gemmula. Hence, the spermatozoa ultrastructure of both species was described in the present work, aiming to identify characters that could be useful for further taxonomic and phylogenetic analyses. D. hanleyanus and D. gemmula spermatozoa were different especially in relation to acrosomal characteristics and chromatin condensation. The spermatozoon produced by D. hanleyanus had a nucleus (exhibiting granular chromatin with a rope‐like appearance) capped by a long and conical acrosomal vesicle, which extended itself outward beyond the anterior nuclear fossa. Otherwise, the nucleus of the sperm cell of D. gemmula showed well‐compacted chromatin, and its acrosome, which was partially inserted into the anterior nuclear fossa, had a bubble‐like tip. In conclusion, the conspicuous ultra‐structural differences found between the spermatozoan morphologies were helpful for the discrimination of the species. In conclusion, our results suggest that analyses of sperm ultrastructure of the bivalves in the family Donacidae can be valuable to investigate their taxonomic relatedness. The present results also contribute to assess the monophyletic status of the family.     

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Background  

The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.     

Evidence for interspecific transfer of the transposable element mariner betweenDrosophila andZaprionus

Summary The transposable element mariner occurs widely in themelanogaster species group ofDrosophila. However, in drosophilids outside of themelanogaster species group, sequences showing strong DNA hybridization with mariner are found only in the genusZaprionus. the mariner sequence obtained fromZaprionus tuberculatus is 97% identical with that fromDrosophila mauritiana, a member of themelanogaster species subgroup, whereas a mariner sequence isolated fromDrosophila tsacasi is only 92% identical with that fromD. mauritiana. BecauseD. tsacasi is much more closely related toD. mauritiana than isZaprionus, the presence of mariner inZaprionus may result from horizontal transfer. In order to confirm lack of a close phylogenetic relationship between the genusZaprionus and themelanogaster species group, we compared the alcohol dehydrogenase (Adh) sequences among these species. The results show that the coding region of Adh is only 82% identical betweenZ. tuberculatus andD. mauritiana, as compared with 90% identical betweenD. tsacasi andD. mauritiana. Furthermore, the mariner gene phylogeny obtained by maximum likelihood and maximum parsimony analyses is discordant with the species phylogeny estimated by using the Adh genes. The only inconsistency in the mariner gene phylogeny is in the placement of theZaprionus mariner sequence, which clusters with mariner fromDrosophila teissieri andDrosophila yakuba in themelanogaster species subgroup. These results strongly suggest horizontal transfer.     

Ultrastructure of the spermatozoa of Psammotettix striatus (Linnaeus) and Exitianus nanus (Distant) (Hemiptera: Auchenorrhyncha: Cicadellidae: Deltocephalinae)

Previous studies of insect spermatozoa indicate that these specialized cells have undergone significant morphological evolution and exhibit traits useful for reconstructing phylogenetic relationships. Although leafhoppers (Cicadellidae) are among the largest and most economically important insect families, few comparative studies of their spermatozoa have been published. Here, the ultrastructure of mature spermatozoa of two leafhoppers Psammotettix striatus (Linnaeus) and Exitianus nanus (Distant), representing two different tribes of the largest leafhopper subfamily, Deltocephalinae, was examined by light and transmission electron microscopy. The shape and ultrastructure of spermatozoa of the two species are very similar to those of other Cicadellidae as well as other Auchenorrhyncha, comprising a conical acrosome invaginated to form a subacrosomal space, a filiform homogeneously condensed nucleus, a lamellate centriolar adjunct connecting the nucleus with the mid-piece/flagellum, a long flagellum with a 9 + 9 + 2 axoneme pattern and two symmetrical mitochondrial derivatives with an orderly array of peripheral cristae, and two drop-shaped accessory bodies. They may be distinguished by the size of the sperm, and the shape of the nucleus, accessory bodies, and paracrystalline region of mitochondrial derivatives. The fine morphology and ultrastructure of spermatozoon in P. striatus and E. nanus are illustrated, along with a brief discussion of the implications for classification and phylogenetic analyses of the subfamily.     

Presence of only eupyrene spermatozoa in adult males of the genus Micropterix hübner and its phylogenetic significance (Lepidoptera : Zeugloptera,Micropterigidae)

The morphology of the male genital organs and sperm ultrastructure were studied by light and transmission electron microscopy in adult males of two species of the genus Micropterix (Micropterigidae; Zeugloptera; Lepidoptera). The results are compared with findings from other primitive Lepidoptera and Trichoptera, and evaluated from a phylogenetic viewpoint. Both Micropterix species examined agree with regard to essential characters of the male genital organs. The paired testes are separate and show no internal compartmentalization. The male genital organs contain only mature nucleate (eupyrene) spermatozoa. Anucleate (apyrene) spermatozoa characteristically found in all other Lepidoptera were not observed. The eupyrene spermatozoa are filiform, measure 100 μm in length, and contain an elongated nucleus, 2 mitochondrial derivatives without paracrystalline materials, and a 9 + 2 axoneme without accessory tubules; the nucleus extends for almost the entire length of the spermatozoa. The absence of apyrene spermatozoa in Micropterix is in contrast to their presence in another species of the same family.     

Comparative ultrastructure of ant spermatozoa (Formicidae: Hymenoptera)

Mature spermatozoa from spermathecae of founding queens were obtained from 5 species of ants, representing the major subfamilies Myrmicinae (Acromyrmex versicolor, Crematogaster sp.) and Dolichoderinae (Tapinoma sessile, Conomyrma insana, Conomyrma wheeleri). The ultrastructure of ant spermatozoa has many features in common with that of higher insects and is similar to that of other Hymenoptera. Structural similarities to spermatozoa of other Hymenoptera include an acrosome containing an internal rod that extends into the nucleus, two elongate mitochondrial derivatives, a centriolar adjunct, and an axonemal arrangement of 9 + 9 + 2 that includes well-developed coarse, or accessory, tubules. Spermatozoa obtained from A. versicolor, a species that is known to store and utilize viable sperm from this supply for over 10 years, show greater development of the mitochondrial derivatives than do the other species. The most distinctive feature of ant spermatozoa in comparison to other Hymenoptera is the large size of the centriolar adjunct relative to the other organelles. The centriolar adjunct is located posterior to the nucleus, anterior to the mitochondrial derivatives, and opposite the axoneme.     

The sperm structure of <Emphasis Type="Italic">Galloisiana yuasai</Emphasis> (Insecta,Grylloblattodea) and implications for the phylogenetic position of Grylloblattodea

The sperm ultrastructure of the Grylloblattodea Galloisiana yuasai was described and the sperm characters were comparatively examined in several orthopteroid insect orders for inferring the phylogenetic placement of the Grylloblattodea. The spermatozoa of G. yuasai are joined in bundles (spermatodesms) containing 200 units. Major features of these spermatozoa include a monolayered acrosome, a 9+9+2 axoneme with 16-pfs accessory microtubules and expanded intertubular material, and an evident “centriole adjunct”. The diffused material observed between the axoneme and the mitochondrial derivatives is considered to be an extension of the three connecting bands observed in other orthopteroid taxa, similar to what happens in some orthopteran lineages. The presence of the connecting bands, even though modified in G. yuasai, suggests that the Grylloblattodea are to be placed in a clade with Mantophasmatodea, Mantodea and Orthoptera.     

Structure and ultrastructure of spermatozoa and spermiogenesis in three species of Lucilia Robineau‐desvoidy, 1830 (Diptera: Calliphoridae)

Morphology of male internal reproductive organs, spermatozoa, and spermiogenesis of the blow‐flies Lucilia cuprina, Lucilia eximia, and Lucilia peruviana is first described here, using light and transmission electron microscopy. Spermiogenesis follows the characteristics described for others insect species. The spermatozoa of L. cuprina are similar to those described for other Brachycera. However, in L. eximia and L. peruviana, some differences were found. In L. cuprina and L. eximia species, the spermatozoa are long and thin, measuring about 211 μm and 146 μm in length, of which the head region measures approximately 19 μm and 17 μm, respectively. A polymorphism was observed in L. cuprina and L. eximia spermatozoa. In all three species, the head includes a monolayered acrosome with electron‐lucent material. The shape of the nucleus, in cross sections, varies from circular to oval with completely condensed chromatin. Implantation of the axoneme was observed in the middle region of the nucleus, known as the “peg” region. In the next region, the beginning of two mitochondrial derivatives of similar diameter and different lengths in L. cuprina and only one in L. eximia and L. peruviana was observed. In the overlap region, the following structures were observed: nucleus, centriolar adjunct, mitochondrial derivatives, and axoneme. The axoneme is of a conventional insectan type with a 9 + 9 + 2 microtubular arrangement. The male internal reproductive tract consists of testis, deferent ducts, a strongly developed seminal vesicle, accessory glands, and ejaculatory duct. These features are consistent with the structural diversity of the dipteran reproductive tract and spermatozoa, comprising an essential tool for understanding the complex variations found in the Diptera. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Identification of the sibling species of the Drosophila willistoni subgroup through the electrophoretical mobility of acid phosphatase-1

The Drosophila willistoni group consists of 23 species of which six are sibling species and belong to the D. willistoni subgroup: D. willistoni, Drosophila equinoxialis, Drosophila tropicalis, Drosophila insularis, Drosophila pavlovskiana and Drosophila paulistorum. These sibling species are abundant in the Neotropical region and can hardly be differentiated by the usual taxonomic traits. Four of them (D. willistoni, D. equinoxialis, D. tropicalis and D. paulistorum) cover extensive geographic distribution areas overlapping in places while two of them are endemic (D. insularis and D. pavlovskiana). In this study, we presented a method for the identification of five sibling species of the D. willistoni subgroup based on the allozyme variation of acid phosphatase‐1 (Acph‐1) in acrylamide gel electrophoresis. Our work showed that Acph‐1 allozyme differences can be used for species‐diagnostic characterization. This method was shown to be a more efficient tool for species identification than others because it is both quicker and produces reliable results.     

Occurrence of biflagellate spermatozoa in Cetopsidae, Aspredinidae, and Nematogenyidae (Teleostei: Ostariophysi: Siluriformes)

In the present study spermiogenesis was investigated in Cetopsis coecutiens (Cetopsidae), and Bunocephalus amazonicus (Aspredinidae), while spermatozoa ultrastructure was investigated in C. coecutiens, B. amazonicus, and Nematogenys inermis (Nematogenyidae). Aspredinidae and Cetopsidae share a spermatogenesis of the semicystic type, and a particular type of spermiogenesis process not reported in any fish group. In the three species analyzed, spermatozoa are biflagellate with flagella having the classical axoneme formulae (9 + 2). The analysis of thirteen characters showed the presence of eight characters shared by Cetopsidae and Aspredinidae, and six characters shared by Cetopsidae and Nematogenyidae, which may suggest that these three families may be more related than actually hypothesized, comprising a very primitive siluriform lineage originated after Diplomystidae.     

Ultrastructure of spermatozoa of members of Calappidae,Aethridae and Menippidae and discussion of their phylogenetic placement

The families Aethridae and Calappidae were originally considered as part of the same family; however, their morphology and molecular biology separate them into two families. In this context, we describe the ultrastructure of spermatozoa of species of the Calappidae, Aethridae and Menippidae to elucidate the relationships among taxa. The vasa deferentia were submitted to routine protocols for transmission electron microscopy. Our results indicate that the morphology of the spermatozoa of Hepatus pudibundussupports its exclusion from the Superfamily Calappoidea due to the presence of the apical striated layer. The spermatozoa of Menippe nodifrons is very similar to H. pudibundus and corroborates the recent phylogenetic analysis using sequence data of nuclear genes. Moreover, our results evidence two morphological patterns of spermatozoa within Calappidae. Calappa ocellata and C. cinerea show spermatozoa with a wide acrosome vesicle, a thick operculum shaped as a shallow “W” and a large thickened ring. Calappa gallusand C. hepatica show spermatozoa with a longer acrosome vesicle, a pointed operculum and a slender thickened ring. Our ultrastructure results conform with previous molecular proposal and show that spermatozoa ultrastructure can be an effective tool to adjust phylogenetic relationship when used in association with molecular data.     

Polymorphism of spermatozoa in Largus rufipennis Laporte 1832 (Heteroptera: Pyrrhocoroidea: Largidae)

The production of polymorphic spermatozoa has been registered in various insect orders such as Diptera, Lepidoptera, and Hemiptera. In this work, morphology of two types of spermatozoa produced by Largus rufipennis was reported for the first time in the Largidae family. For this, techniques including optical and transmission electron microscopy were used. Spermatozoa measured, on the average, 260 and 200 μm, and both types possessed a nucleus measuring on the average 65 μm. No ultrastructural differences were observed between the two spermatozoa types from L. rufipennis. The head region is composed of an acrosome, a nucleus, and part of the centriolar adjunct. The centriolar adjunct is in parallel with the nucleus and followed by mitochondrial derivates. The flagellum consists of an axoneme (9 + 9 + 2 microtubules) and two mitochondrial derivatives; no other accessory bodies were observed. The mitochondrial derivatives are symmetric in size and diameter. A similar quantity of the two spermatozoa types was observed in the seminal vesicle (57% of the large type and 43% of the small type), while in the spermatheca of the female, the larger spermatozoa were preferentially stored (87%). These results permit discussions concerning of the species biology reproduction, most specifically sperm competition strategies.     

Spermatogenesis and the structure of the testes in Nemertodermatida

The ultrastructure of the testes in two representatives of the enigmatic taxon Nemertodermatida was studied using transmission electron microscopy. Nemertoderma westbladi has paired testes, which are delineated by lining cells. Within each testis, different follicles, each surrounded by a membrane-like structure, are found. Flagellophora apelti has genuinely follicular testes, consisting of several follicles, each containing a certain stage of spermatogenesis. As the gametes are not enclosed by a structure that can be called a true gonad, the structure of the testes differs from most bilaterian animals, but resembles the organization of gametogenic areas of ctenophores. Each stage of spermatogenesis in F. apelti is described, enabling the inference of the origin of the structures seen in mature spermatozoa. The overall structure of the mature spermatozoa is similar in all nemertodermatids and unique within the Metazoa: an elongated head containing the nucleus; a middle piece containing an axoneme, mitochondrial derivatives and in F. apelti granular derivatives; and a flagellar tail.     

Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae)

This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 μm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9?+?9?+?2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.