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Morphology and ultrastructure of the spermatozoa of Lonchoptera lutea Panzer, 1809 (Diptera: Lonchopteridae)
Abstract
Lonchoptera lutea males produce giant spermatozoa that are more than 2000 μm long and 1.4 μm wide. Unlike the typical brachyceran spermatozoon, they have a highly asymmetrical cross-section with only a single, albeit very large, mitochondrial derivative and a pair of massive accessory bodies, one of which extends throughout the entire length of the sperm tail. The accessory bodies consist of an electron-dense matrix in which numerous peculiar electron-lucid substructures are embedded. In the mated female, the giant spermatozoa are found inside two tubular spermathecae which are also extremely long, measuring 4000 μm or more.
Among species of the spear-winged flies (Lonchopteridae) there is remarkable variation in sperm size, with some species producing giant spermatozoa. With a length of 7500 μm and a width of 1.3 μm the spermatozoon of Lonchoptera fallax ranks among the largest known to date. In the present study body size, testis size, sperm size, and spermatid number per bundle and per testis were examined across 11 Lonchoptera species. Results are discussed in terms of how these characters are related with each other and how their evolution affects the resource allocation amongst spermatozoa. Based on some discrete morphological characters and a molecular tree derived from DNA barcodes a phylogenetic hypothesis of the genus Lonchoptera is proposed. The occurrence of giant spermatozoa in Lonchopteridae is compared to convergent occurrences reported in other taxa.
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.
The objective of this study was to characterize, structurally and ultrastructurally, the spermatozoa of the screwworm flies
Cochliomyia hominivorax and Cochliomyia macellaria. To visualize the ultrastructure of microtubules and identify basic proteins, techniques such as the tannic acid fixation
and the cytochemical method of ethanolic phosphotungstic acid (EPTA) were used. These methods of fixation are important because
they reinforce the evidence of the protofilaments present in the microtubular wall and identify basic proteins, respectively.
With the tannic acid fixative it was possible to observe a significant number of microtubules in the cell cytoplasm during
spermiogenesis. Microtubules were observed in all regions of spermatids (head, ‘overlap’ zone and tail). The EPTA technique
highlighted the presence of basic proteins on the border of the nucleus and nuclear envelope in the two species analyzed,
and in the centriolar adjunct and on the border of mitochondrial derivatives in C. macellaria. The axoneme is of a conventional insect type with a 9 + 9 + 2 microtubular arrangement and the spermatozoa of C. hominivorax and C. macellaria are similar to those described for other Brachycera. The spermatozoa are long and thin in these two species, ∼190 µm in length,
of which the head region measures ∼26 µm in C. hominivorax and 29 µm in C. macellaria. A polymorphism was observed in C. hominivorax and C. macellaria. These features are consistent with the structural diversity of the dipteran spermatozoa, constituting an essential tool
for understanding the complex variations found in the Diptera order.
A cladistic analysis of the Empidoidea and basal lineages of the Cyclorrhapha, based on morphological characters, confirms the monophyly of both groups as well as that of the Eremoneura. The resulting final trees are used to revise the classification of the Empidoidea to include the following five families: Empididae, Hybotidae, Atelestidae (including Nemedininae n. subfam.), Brachystomatidae rev. stat. (comprising the subfamilies Brachystomatinae, Ceratomerinae and Trichopezinae), and Dolichopodidae s.lat. The family Microphoridae is not recognized, and the Microphorinae and Parathalassiinae are assigned to the Dolichopodidae s.lat. The Dolichopodidae s.str. includes 15 subfamilies that were previously recognized within the family. Within the Empidoidea we found support for Atelestidae as the sister group to the Hybotidae and for the monophyly of Parathalassiinae + Dolichopodidae s.str. The Empididae remains poorly defined and the genera Homalocnemis Philippi, Iteaphila Zetterstedt, Anthepiscopus Becker, and Oreogeton Schiner are classified as incertae sedis within the Empidoidea. In addition, the following higher taxa are proposed: Symballophthalmini n. tribe, Bicellariini n. tribe, Oedaleinae rev. stat., and Trichininae rev. stat., which are all assigned to the Hybotidae. The genus Sematopoda Collin is tentatively assigned to Trichopezinae, and Xanthodromia Saigusa is transferred from Hemerodromiinae to Brachystomatinae. All morphological characters are extensively discussed and illustrated, including details of the antennae, mouthparts, internal thoracic structures, wings, and male and female terminalia. In addition, a key to families and unplaced genus groups of the Empidoidea is provided. Feeding habits are also discussed in terms of the empidoid ground plan condition.
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.
The spermatozoa of Chrysomya megacephala are similar to those described for other Brachycera. In this species, the spermatozoa are long and thin, measuring about 590microm in length, of which the head region measures approximately 60microm. The head includes a monolayered acrosome with electron-lucid material, and the shape of the nucleus, in cross-sections, varies from circular to oval with completely condensed chromatin. The centriole was observed in the zone of flagellar implantation, below the "peg" region. In the region of overlap, the followings structures are observed: nucleus, centriolar adjunct, mitochondrial derivatives and axoneme. The two mitochondrial derivatives are of different lengths but similar diameter. The axoneme is of a conventional insectan type with a 9+9+2 microtubular arrangement, with accessory tubules flanked by the electron-dense intertubular material. The male internal reproductive tract consists of testis, vas deferens, seminal vesicle, accessory glands and ejaculatory duct.
Large-scale production of the black soldier fly [Hermetia illucens (L.) (Diptera: Stratiomyidae)] for use as aquaculture and poultry feed has developed into a global industry. Successful commercialization of the black soldier fly relies on optimizing the production of fecund adults. However, current mass-rearing protocols result in variable production of fertile eggs. To help lay a foundation for a better understanding of factors that may play a role in this variability, the morphology of the black soldier fly male reproductive tract and spermatozoa, associated spermatogenesis process, impact of age on the process, and the female spermatheca morphology were examined with various microscopic techniques (e.g., scanning electron microscope, transition electron microscope, and dissecting scope). The gross morphology of the male reproductive tract and female spermatheca appear to be similar to those found in other brachyceran flies. Male spermatozoa are long (~860 µm overall, ~8 µm head), apparently motile, and possess flagella with a typical 9 + 9 + 2 axoneme triplets. Germ cells go through incomplete mitotic divisions surrounded by somatic cyst cells in the testes. Spermatogenesis appears to be initiated during immature development (cryptocephalic pupa stage). From <24 h to 7 d post-emergence, male aging appeared to impact sperm production.
Sperm structure and ultrastructure of Hermetia illucens was determined by light microscopy and transmission electron microscopy. The main sperm components were similar as for other Dipteran subspecies, while the ultrastructure revealed distinguishing features in the zone of overlap and anterior flagellar region. Sperm varied in size indicating sperm polymorphism. The head region is lacking an acrosome. The zone of overlap consisted of uniquely organized centriolar adjunct material, partly forming electron dense areas to finally form an outer ring separating the mitochondrial derivatives from the 9 + 9 + 2 axoneme. Accessory bodies arising from the zone of overlap are flanked by smaller to large mitochondrial derivatives into the anterior flagellum. This study confirms sperm structure diversity between brachyceran subspecies and support its relationship with nematoceran subspecies.
The Drosophilidae family is formed by Brachycera Diptera distributed widely across different regions of the planet. It is composed of about 4,000 species, 304 of which are found in Brazil. The objective of this work was to characterize morphologically the structure of the male internal reproductive apparatus and the ultrastructure of the spermatozoon in four Neotropical (Drosophila cardini, D. mercatorum, D. nebulosa and D. sturtevanti) and two invasive (D. simulans and Zaprionus indianus) species of drosophilids. The structural aspect of the internal reproductive apparatus corresponds with that described for other drosophilids; however, there are differences in the size and coloration of the structures, such as the testes, in each species analyzed. The spermatozoon of these species was seen to be long and fine, presenting morphological variation. The ultrastructure of the spermatozoon revealed that the morphological pattern is similar to that found in the majority of insects. The head region presents a nucleus with condensed chromatin and the acrosome positioned laterally to the nucleus. In the tail region, the axoneme presents the 9 + 9 + 2 pattern commonly described for other species of Diptera. The species presented differences regarding the shape and size of the mitochondrial derivatives. Cytochemical analysis using EPTA also revealed differences in terms of the location of the basic proteins in the mitochondrial derivates. The results obtained contribute to expanding the database for the Drosophilidae family, providing information that may contribute to intra- and inter-specific identification and supplying phylogenetic analyses.
The spermatozoa of Diasemopsis comoroensis and D. meigenii differ from the conventional brachyceran type in several respects. Not only are they very long but they are also extraordinarily wide, especially at the very end of the tail. The latter is effected by two cellular components: oversized mitochondrial derivatives and a prominent central band, which is a peculiar structure not known from any other spermatozoa. Based on its position with respect to the other organelles and its origin during early spermiogenesis, the central band is interpreted as a derivative of the centriolar adjunct material. Like the axoneme, the mitochondrial derivatives and the central band extend through the entire length of the spermatozoon tail. The spermatozoon tail is helical and can be coiled up into a tight cone-shaped spiral with a peculiar corkscrew shaped end portion. The potential adaptive significance of these features and their coevolution with the morphology of the female's multi-chambered ventral receptacle are discussed, as are sperm competition and cryptic female choice.
The main characteristics of the sperm structure of Hexapoda are reported in the review. Data are dealing with the process of spermatogenesis, including the aberrant models giving rise to a reduced number of sperm cells. The sperm heteromorphism and the giant sperm exceeding the usual sperm size for length and width are considered. The characteristics of several components of a typical insect sperm are described: the plasma membrane and its glycocalyx, the nucleus, the centriole region and the centriole adjunct, the accessory bodies, the mitochondrial derivatives and the flagellar axoneme. Finally, a detailed description of the main sperm features of each hexapodan group is given with emphasis on the flagellar components considered to have great importance in phylogenetic considerations. This study may be also useful to those requiring an introduction to hexapod reproduction.
The purpose of this work is to present a critical resume of all research reports on the ultrastructure of mature uniramian sperm. The Uniramia comprise Onychophora, Myriapoda and Hexapoda. The book also reviews phylogenetic studies and describes the use of spermatozoal ultrastructure for taxonomy and phylogeny. The text is supplemented by illustrations, an author index and a subject index.
See also Facsimile paperback 2011 ISBN 978-0-521-27941-3
In this study we describe a new kind of sperm gigantism in the stalk-eyed fly, Diasemopsis comoroensis (Diptera, Diopsidae). The sperm cells have a length of up to 1.7mm and can be coiled into a compact 'slinky' spiral. Their ultrastructure involves a prominent electron dense central band, which runs the entire length of the sperm tail and in some regions constitutes its largest element in cross section. We propose that this organelle is either a giant centriole adjunct or a kind of accessory body derived from it, and that it takes part in coiling the sperm tail. To our knowledge, no comparable structure has been described before.
Musca domestica L., house fly, spermiogenesis is illustrated by transmission electron micrographs. The basic morphologic features of spermiogenesis which are presented include: elongation of the nucleus, and the development of the centriole adjunct, acrosome, and microtubules. A two-part centriole adjunct and a basal body (centriole derivative) that contains three central tubules are reported for the first time in developing insect sperm. It is suggested that the three tubules are related to the transitory microtubules in the sperm cytoplasm. Finally, it is postulated that at least three sperm types in insects exist with respect to the associations which the mitochondrial derivatives, the ciliary bundle (axial filament), and the centriole adjunct have with the base of the nucleus. These three forms can be categorized on the basis of their respective nuclei as truncate, concave and convex.
When viewed by scanning electron microscopy (SEM), the spermatozoon of the phorid dipteran Megaselia scalaris appears threadlike, lacking distinct head and tail areas. These areas can be observed, however, in appropriately stained material. Measurements of Feulgen-stained material reveal average lengths of the head, tail, and total cell of 18.7, 128.7, and 147.4 μm, respectively. When tested for sulfhydryl and disulfide groups, the head displays only disulfide groups. Transmission electron microscopy (TEM) reveals 12 different regions: three (1–3) in the head, four (9–12) in the tail, and five (4–8) in a short zone of overlap between the head and tail. Most of the cell lies in regions 9 and 11 of the tail and 3 of the head, accounting for, respectively, 37.3%, 45.7%, and 11.2% of the total length. A tubelike acrosome indents the anterior end of the nucleus. The tail originates asymmetrically in relation to the long axis of the cell as a peglike structure associated with the dorsolateral region of the nucleus. No centriole is visible, and the nucleus has a notched appearance in longitudinal sections. Two mitochondrial derivatives and an axoneme displaying a 9+9+2 microtubule configuration and ATPase activity extend throughout most of the tail. In regions 9 and 10, an asymmetrically arranged accessory body is also present. Features having possible taxonomic utility include the asymmetrically arranged accessory body, the size and shape of the acrosome, and the notched appearance of the nucleus. The present report is apparently the first to describe the spermatozoon of a cyclorrhaphous dipteran which is not a member of the Schizophora.
1Until now 9 Lonchoptera species have been recorded in the GDR: L. tristisMEIG., L. furcata (FALL.), L. lutea Pz., L. strobliDE MEIJ., L. meijereiCOLL., L.fallaxDE MEIJ., L. nigrociliataDUDA, L. scutellataSTEIN and L. nitidifronsSTROBL.2Large numbers of individuals and many localities exist of the following species: L. tristis, L. furcata and L. lutea.3L. tristis is most abundant in August, whereas L. furcata and L. lutea have been caught especially in September and October.4First fully developed oocytes of L. tristis in an small number of females occur as early as June but in > 90% only between August and October.5The widely distributed L. furcata was found only to be parthenogenetic over the whole area which has been investigated.6A key of the known species in the GDR completes the ecofaunistic records.
The spermatozoa of the 2 fruit fly species, Dacus oleae (Gmel.) and Drosophila melanogaster Meig. (Diptera), have been fixed in a glutaraldehyde-tannic acid mixture, followed by uranyl acetate postfixation, and the sperm tail studied by high resolution electron microscopy. Spermatozoa of both species were seen to possess a centriole containing 9 microtubular triplets. The centriolar A-, B- and C-subtubules have 13, 10 and 9 protofilaments, respectively. The outer subtubules (the C-subtubules) seem to be continuous with the accessory tubules of the main part of the tail. The accessory tubules and their intertubular material have a complex fine structure similar to that described in some other brachyceran flies. In the end piece of the sperm tail, the A-subtubules undergo degeneration, characterized by swelling and a loss of dynein arms and spokes. The tail region appears to be immotile, possibly to enable it better to stick to receptors on the egg micropyle.
The architecture of the tail axoneme of spermatozoa from 49 insect species representing 20 insect orders has been examined. Whereas the microtubular walls of the nine doublet tubules and the two central singlet tubules were found to consist of a fixed number of protofilaments, the walls of the accessory tubules may consist of 13, 15, 16, 17, or 19 microtubules and have diameters from 24 to 38 nm. Cytoplasmic microtubules in the spermatid cytoplasm, outside the axoneme, may have 13, 14, or 16 protofilaments. The number of protofilaments seems constant for each class of accessory tubule and, except in Diptera, seems fixed in each insect order. Pterygote insects have 16 protofilaments and this was found in all orders except Ephemeroptera and Psocoptera with 13, Phasmida with 17, and Trichoptera with 19 protofilaments. Within Diptera, 16 protofilaments were found within the family Mycetophilidae, which for this reason can be regarded as the most primitive, extant dipteran group; the majority of dipteran flies have 13 protofilaments. Two nematoceran families, Bibionidae and Culicidae, have axonemes of a 9 + 9 + “1” type and accessory tubules with 15 protofilaments. We feel that axonemal ultrastructure is important in studies of insect phylogeny. We conclude that spermatids can effect specification of different protofilament numbers with numerical exactitude for the different types of microtubules.
Fixed tissue is dehydrated with tertiary butyl alcohol overnight. The following day it is cleared in toluene, infiltrated and embedded in Araldite resin-hardener-accelerator mixture without dibutyl phthalate, and polymerized at 60° C. More rapid than previous techniques, this method gives blocks which do not fracture unduly on trimming and provides sections of soft tissues at 1 μ for phase contrast microscopy, as well as ultrathin sections which cut as easily with glass knives as sections of methacrylate. Araldite manufactured in the U.S.A. and in England are different. Satisfactory proportions for the American are: hardener DDSA, 3.5 ml; casting resin 6005, 5.0 ml; accelerator B, 0.12 ml. For the British product, these are: hardener 964 B, 5.0 ml; casting resin M, 5.0 ml; accelerator 964 C, 0.25 ml. The use of 2% agar for orienting small specimens in Araldite is feasible. Mallory's borax-methylene blue has been applied to the staining of Araldite sections as thin as 0.5 μ mounted on glass slides.
The "zipper line" of Drosophila melanogaster and of Drosophila species characterized by giant spermatozoa (D. hydei, D. kanekoi and D. bifurca) was studied by electron microscopy using conventional thin-sections, lectin labeling and freeze-fracture replicas. In cross sections the membrane specializations are located either at the level of the short cistern close to the large mitochondrial derivative where a small tuft of glycocalyx is visible or, in species characterized by long spermatozoa, along a cistern beneath the plasma membrane. In correspondence of such cistern, the plasma membrane exhibits a thick and extended glycocalyx. At this level, as well as at the short tuft of D. melanogaster, alpha-mannose residues were detected. The "zipper" of D. melanogaster consists of rows of intramembrane particles longitudinally disposed along the sperm tail and associated with the external face of the plasma membrane. On the protoplasmatic face a narrow ribbon of transversal grooves is visible. Freeze-fracture replicas have revealed, in the region characterized by extended glycocalyx, the presence of a large ribbon of intramembrane particles disposed in parallel transversal rows, associated with the protoplasmatic membrane face. On the complementary external face a ribbon of parallel transversal grooves was observed. It is suggested that membrane specializations are mechanical devices to protect spermatozoa from torsion and bending in the seminal vesicles and then in the female storage organ.
Die Lonchopteren des palaearktischen Gebietes
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Drosophila spermiogenesis: big things come from little packages
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Phylogeny of Diptera
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How long is a giant sperm?
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