Proteomic diversification of spermatostyles among six species of whirligig beetles.

Seminal fluid protein composition is complex and commonly assumed to be rapidly divergent due to functional interactions with both sperm and the female reproductive tract (FRT), both of which evolve rapidly. In addition to sperm, seminal fluid may contain structures, such as mating plugs and spermatophores. Here, we investigate the evolutionary diversification of a lesser-known ejaculate structure: the spermatostyle, which has independently arisen in several families of beetles and true bugs. We characterized the spermatostyle proteome, in addition to spermatostyle and FRT morphology, in six species of whirligig beetles (family Gyrinidae). Spermatostyles were enriched for proteolytic enzymes, and assays confirmed they possess proteolytic activity. Sperm-leucylaminopeptidases (S-LAPs) were particularly abundant, and their localization to spermatostyles was confirmed by immunohistochemistry. Although there was evidence for functional conservation of spermatostyle proteomes across species, phylogenetic regressions suggest evolutionary covariation between protein composition and the morphology of both spermatostyles and FRTs. We postulate that S-LAPs (and other proteases) have evolved a novel structural role in spermatostyles and discuss spermatostyles as adaptations for delivering male-derived materials to females.

The ultrastructure of the spermatheca of Mordellistena brevicauda (Coleoptera, Tenebrionoidea) and the associated bacterial cells.

The ultrastructural study on the female reproductive system of the beetle M. brevicauda (Mordellidae) confirmed the positive correlation between the length of the sperm and the size of the female seminal receptacle (Spermatheca). The spermatheca of the species is characterized by an apical bulb-like structure where the spermathecal duct forms numerous folds filled with sperm. At this level many bacterial cells are present intermingled with the duct folds. Some are organized in large structures, such as bacteriomes, while other are single bacteriocytes. The latter are often found near the basal lamina of duct epithelium. In addition, some bacteria are visible in the cytoplasm of the duct epithelial cells. Interestingly, bacterial cells have never been observed in the duct lumen. The possible function of the bacterial cells is discussed.

The sperm ultrastructure of Spermophagus kuesteri Schilsky, 1905 (Chrysomelidae, Bruchinae) and considerations on the relationships of Cucujiformia superfamilies.

The sperm ultrastructure of the bean-weevil Spermophagus kuesteri (Bruchinae) was studied to verify the congruence of the new position of the subfamily within Chrysomelidae. The results indicated a positive answer to the question supporting a close relationship between Chrysomelidae and Curculionidae, a finding confirmed also by molecular data. Moreover, the sperm morphology of Divales cinctus, a member of Melyridae (Cleroidea) allowed to confirm the different sperm organization between members of this superfamily and Phytophaga (Chrysomeloidea + Curculionoidea). While studying the spermiogenesis of S. kuesteri, some sperm cysts showed aberrant cells provided with two flagella in the same plasma membrane. These aberrant sperm could be the result, during early spermiogenesis, of irregular processes involving the canal rings between spermatids.

The sperm structure of Clinidium canaliculatum (Costa): A contribution to the systematic position of Rhysodidae (Coleoptera: Carabidae).

The systematic position and the phylogenetic relationship of Rhysodidae members is still debated, with some authors considering the group as a separate family of Adephaga, while for others they could be a subfamily of Carabidae. The group have morphological traits quite different from Carabidae and an aberrant behaviour compared to ground beetles being not predaceous. The sperm ultrastructure of C. canaliculatum was studied comparatively with other species of beetles, Carabidae in particular. The results indicate that the sperm structure of this species is similar to that of the Carabinae species. As in these species, C. canaliculatum has sperm conjugates with an apical conical cap protecting the heads and the initial region of flagella. This sperm appearance is also shared by another species of Rhysodidae, Omoglymmius hamatus. The material of the apical cap consists of an electron-dense material with a peculiar outer net configuration. Many species of Carabidae, however, can present a different type of sperm conjugation, the spermatostyle: a long rod-like structure where the individual sperms have only the most apical part inserted in the cortical area and the flagella are completely free. C. canaliculatum sperm are endowed with a mono-layered acrosome, a nucleus of variable shape along its length, a flagellum consisting of a typical axoneme 9 + 9+2, provided with 16 protofilaments in the tubular wall of accessory tubules, two asymmetric mitochondrial derivatives with the left one larger than the opposite one, and the right accessory body elongated and larger than the opposite one. These sperm characteristics, which are shared also by another member of the group, suggest the demotion of the family Rhysodidae to the subfamily Rhysodinae within Carabidae, a result also supported by recent molecular data.

Ultrastructure of the Spermiogenesis in Halyomorpha halys (Hemiptera: Pentatomidae): X-Irradiation and New Insights on the Centriolar Region Organization.

Halyomorpha halys (Heteroptera: Pentatomidae) is an insect pest native to Asia that has spread over the last two decades to most of the North America, parts of South America, Europe and North Africa. Its impact is significant as it can feed on more than 300 host plants, rendering affected fruits and vegetable crops unsellable or of lower quality. Various chemical and biological methods have been used to control this pest, with varying degrees of success. The sterile insect technique (SIT) is a pest control method involving the sterilization of insects via ionizing radiation and their subsequent mass release into the field. In the present contribution, the spermiogenesis of H. halys was studied from an ultrastructural point of view in both irradiated and non-irradiated adult males. In both cases, we observed ultrastructural characteristics typical of hemipteran sperm cells: bridges connecting the mitochondrial derivatives and the axonemal microtubules, the absence of accessory bodies, and the presence of two or three crystalline inclusions within the mitochondrial derivatives, an acrosome composed of tightly packed tubules, and an atypical, plaque-shaped microtubular organizing center (MTOC) in the centriolar region. Moreover, in the same region, we seldom observed the presence of two centrioles in the spermatids, one of which disappeared at a later stage of maturation. This feature is a novelty for insect spermiogenesis. The cysts of irradiated adults were not all uniformly affected by the radiation. However, irradiated cysts sometimes exhibited a general disorganization of sperm arrangement, incomplete divisions of sperm cells resulting in multiple copies of the same organelle within the same cell, failure to reabsorb the cytoplasm, and the lack of axonemes. Finally, rod-shaped viruses or virus-like particles were observed in vasa deferentia independently of irradiation.

Fine structure of the female genital system of diving beetle Stictonectes optatus (Seidlitz, 1887) (Dytiscidae-Hydroporinae) and evidence of mating plug formation.

The general organization of the female genital system of the diving beetle Stictonectes optatus was studied, clarifying the complex structure of the spermatheca and spermathecal gland. The two structures adhere closely to each other, sharing a small area of their cuticular epithelium. A long duct connects the bursa copulatrix to the spermatheca, where the sperm are stored. The sperm reach the common oviduct, where egg fertilization occurs, via a fertilization duct. The spermathecal gland cells have extracellular cisterns where secretions are stored. Thin ducts composed of duct-forming cells transport these secretions to the apical gland region and into the spermathecal lumen. Soon after mating, the bursa copulatrix is almost completely occupied by a plug secreted by the male accessory glands. The secretions of the bursa epithelium seem to contribute to plug formation. Later this plug becomes large and spherical, obstructing the bursa copulatrix.

The Structure of the Female Genital System of the Diving Beetle Scarodytes halensis (Fabricius, 1787) (Hydroporinae, Dytiscidae), and the Organization of the Spermatheca and the Spermathecal Gland Complex.

The fine structure of the female reproductive organs of the diving beetle Scarodytes halensis has been described, with particular attention to the complex organization of the spermatheca and the spermathecal gland. These organs are fused in a single structure whose epithelium is involved in a quite different activity. The secretory cells of the spermathecal gland have a large extracellular cistern with secretions; duct-forming cells, by their efferent duct, transport the secretions up to the apical cell region where they are discharged into the gland lumen. On the contrary, the spermatheca, filled with sperm, has a quite simple epithelium, apparently not involved in secretory activity. The ultrastructure of the spermatheca is almost identical to that described in a closely related species Stictonectes optatus. Sc. halensis has a long spermathecal duct connecting the bursa copulatrix to the spermatheca-spermathecal gland complex. This duct has a thick outer layer of muscle cells. Through muscle contractions, sperm can be pushed forwarding up to the complex of the two organs. A short fertilization duct allows sperm to reach the common oviduct where eggs will be fertilized. The different organization of the genital systems of Sc. halensis and S. optatus might be related to a different reproductive strategy of the two species.

The sperm structure of the diving beetles Stictonectes optatus (Seidlitz, 1887) and Scarodytes halensis (Fabricius, 1787) (Dytiscidae, Hydroporinae) with evidence of a spermatostyle in the sperm conjugation.

The structure of the male genital organs and spermiogenesis of two diving beetles, Stictonectes optatus and Scarodytes halensis were studied for the first time. S. optatus shows unifollicular testes consisting of a long tubule apically forming a globular structure. The deferent duct epithelia show a secretory activity involved in the spermatostyle organization. They are connected with two very large accessory glands. Sc. halensis has a more common structure of the male genital apparatus with unifollicular cylindrical testes and very long deferent ducts. Sc. halensis accessory glands are smaller than those of S. optatus. The sperm structure in both species is characterized by a small acrosome, a flattened nucleus with a lateral extension containing a centriole from which a long flagellum originates. Both species exhibit sperm conjugation with long sperm bundles showing nuclei orderly arranged in sperm-heads stacks and free flagella. In addition, S. optatus has a thick layer of secretion surrounds these sperm-head stacks. Such a secretion is considered a spermatostyle. This finding represents the first record about the presence of this structure among Dytiscidae. In the flagellum, a typical axoneme with a 9 + 9 + 2 microtubular complex, and two mitochondrial derivatives are present in both species. Those of S. optatus have a peculiar shape with the apical side, in cross-section, displaying pointed corners. Two small accessory bodies are located between the axoneme and the two mitochondrial derivatives.

Sperm structure of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Hydroporinae, Dytiscidae) and considerations on extracellular material surrounding sperm bundles.

The sperm cells of the diving beetle Deronectes moestus incospectus are characterized by sperm conjugation leading to the formation of sperm bundles of 64 units each. These bundles are formed at the end of spermatocyte cell divisions occurring in the testes and can be detected in the anterior region of the deferent ducts (first type of sperm conjugation). Fusions of some sperm bundles can occur at the end of the deferent ducts. The sperm bundles show sperm-head stacks (sperm rouleaux) and are surrounded by a cup of extracellular material secreted by the epithelial cells of the deferent ducts. This material extends posteriorly around the sperm bundle to cover the nuclei and the initial region of the sperm flagella. The cup extracellular material consists of fine tubules, and is no longer visible in sperm bundles at the posterior end of the deferent ducts. The sperm cells of D. moestus incospectus have an axoneme with a 9 + 9 + 2 pattern and unusual mitochondrial derivatives having a matrix showing dense dots and a small crystallized domain. Two thin elongated accessory bodies are located between the mitochondrial derivatives and the axoneme. The extracellular material can have different morphologies in the various families of Adephaga, but all are produced by the epithelium of the deferent ducts. Thus it is reasonable to assume that it has the same function in the different groups.

Morphology of male and female reproductive systems in the ground beetle Apotomus and the peculiar sperm ultrastructure of A. rufus (P. Rossi, 1790) (Coleoptera, Carabidae).

Relatively few studies have focused on evolutionary losses of sexually selected male traits. We use light and electron microscopy to study the male and female reproductive anatomy of Apotomus ground beetles (Coleoptera, Carabidae), a lineage that we reconstruct as likely having lost sperm conjugation, a putative sexually selected trait. We pay particular attention to the structure of the testes and spermatheca. Both of these organs share a strikingly similar shape-consisting of long blind canals arranged into several concentric overlapping rings measuring approximately 18 mm and 19.5 mm in total length, respectively. The similarity of these structures suggests a positive evolutionary correlation between female and male genital organs. Males are characterized by unifollicular testes with numerous germ cysts, which contain 64 sperm cells each, and we record a novel occurrence of sperm cyst "looping", a spermatogenic innovation previously only known from some fruit fly and Tenebrionid beetle sperm. The sperm are very long (about 2.7 mm) and include an extraordinarily long helicoidal acrosome, a short nucleus, and a long flagellum. These findings confirm the structural peculiarity of sperm, testis, and female reproductive tract (FRT) of Apotomus species relative to other ground beetles, which could possibly be the result of shifts in sexual selection.

Ultrastructure of the female reproductive organs of the diving beetle Deronectes moestus incospectus (Leprieur, 1876) (Dytiscidae, Hydroporinae).

We describe the ultrastructure of the female reproductive organs of Deronectes moestus (Dytiscidae Hydroporinae). The long spermathecal duct has a simple epithelium lined internally by a thin cuticle and externally by a thick layer of muscle cells. The wide duct lumen contains electron-dense material, among which remnants of extracellular material are visible. This material consists of tubular structures assembled around sperm bundles previously described in the male deferent ducts. The so-called gland, disposed along the spermathecal duct, is a structure with epithelial cells lined by an irregular cuticle bearing a rich system of microvilli. Many mitochondria are visible in the apical cytoplasm of the epithelial cells, and a few spheroidal bodies are close to the basal nuclei. Since the epithelial ultrastructure of the gland suggests it is involved in fluid uptake from the lumen rather than secretory activity, the term gland, coined by other authors to describe this organ, is inappropriate. The spermatheca is a large structure with a complex epithelium showing secretory and duct-forming cells. The lumen of this organ contains sperm with the distinctive ultrastructural features of those described in the male deferent ducts, namely having a mitochondrial matrix with a small crystallized area and electron-dense dots. Because to its overall organization, the spermatheca of D. moestus can be considered a more integrated organ than those in previously studied hydroporine species.

The sperm ultrastructure of members of basal Tenebrionoidea (Coleoptera).

The sperm ultrastructure of some beetles of Tenebrionoidea was studied with particular attention to those of the Ripiphoridae, Mordellidae, and Meloidae. These three groups are often thought to form a clade, which is the sister group of the remaining Tenebrionoidea. The testes of the two former families have thinner but longer spermatic cysts containing fewer and longer sperm. Within each cyst all sperm cells have the same orientation, but cross sections showed that the orientation of the axonemes alternate between adjacent cysts, possibly due to the cysts bending on themselves. In both families the sperm has a bilayered acrosome and the flagellum, which shows mitochondrial derivatives starting laterally to the nuclear base, has a typical 9 + 9+2 axoneme with accessory tubules provided with 16 protofilaments in their wall, and well-structured triangular shaped accessory bodies. In Mordellistena sp (Mordellidae) sperm, both mitochondrial derivatives and accessory bodies are somewhat asymmetrical. Moreover, the flagellum shows a very thin and long tail end provided with only accessory tubules. Meloidae species have testes with thicker sperm cysts containing numerous shorter sperm. Within the individual cysts the sperm flagella exhibit an alternating orientation of their axonemes as consequence of a peculiar spermatogenetic process. The flagellar structure is similar to that of the above-mentioned species, but the accessory bodies are not well defined and constituted by fuzzy material. In Mylabris hieracii (Meloidae) sperm, the acrosome is flat with a conspicuous perforatorium and its nucleus has a peculiar quadrangular section. Berberomeloe majalis sperm has a large acrosome with an unusual pentagonal perforatorium. The centriolar structure of Mylabris variabilis shows a complex of dense radial links connecting the microtubular structures to the plasma membrane. These results suggest that Ripiphoridae have a closer relationship with Mordellidae than with Meloidae. These findings are in agreement with results obtained with molecular data.

New Findings on the Sperm Structure of Tenebrionoidea (Insecta, Coleoptera).

The sperm ultrastructure of a few representative species of Tenebrionoidea was studied. Two species belong to the Mordellidae (Mordellistena brevicauda and Hoshihananomia sp.), one species to Oedemeridae (Oedemera nobilis), and one species to Tenebrionidae (Accanthopus velikensis). It is confirmed that Mordellidae are characterized by the lowest number of spermatozoa per cyst (up to 64), a number shared with Ripiphoridae. In contrast, in the two other families, up to 512 spermatozoa per cyst are observed, the same number present, for example, in Tenebrionidae. Also, as in the other more derived families of tenebrionoids studied so far, during spermatogenesis in O. nobilis and A. velikensis, sperm nuclei are regularly distributed in two sets at opposite poles of the cysts. On the contrary, the Mordellidae species do not exhibit this peculiar process. However, during spermiogenesis, the bundles of sperm bend to form a loop in their median region, quite evident in the Hoshihananomia sp., characterized by long sperm. This process, which also occurs in Ripiphoridae, probably enables individuals to produce long sperm without an increase in testicular volume. The sperm looping could be a consequence of the asynchronous growth between cyst size and sperm length. The sperm ultrastructure of the Mordellidae species reveals that they can be differentiated from other Tenebrionoidea based on the shape and size of some sperm components, such as the accessory bodies and the mitochondrial derivatives. They also show an uncommon stiff and immotile posterior flagellar region provided with only accessory tubules. These results contribute to a better knowledge of the phylogenetic relationship of the basal families of the large group of Tenebrionoidea.

The ultrastructure of sperm and female sperm storage organs in the water strider Gerris lacustris L. (Heteroptera) and a possible example of genital coevolution.

The fine structural organization of the male and the female inner reproductive apparatuses of the water-strider Gerris lacustris was studied. The sperm of the species shows a long helicoidal acrosome provided with longitudinal tubules, and a short nucleus. The flagellum is characterized by crescent mitochondrial derivatives and a 9 + 9 + 2 axoneme, as occurs in all Heteroptera. The female reproductive apparatus is characterized by an extremely long spermathecal duct, filled with sperm, which plays the role of the main sperm storage organ. The duct has a thin epithelium surrounded by a complex of secretory and duct-forming cells. The spermathecal duct flows into the gynatrial sac. This region, together with the fertilization chamber, exhibits a simple epithelium with deep apical plasma membrane invaginations, and it does not show conspicuous secretions. The basal cell region shows plasma membrane infoldings forming thin cytoplasmic bands hosting mitochondria and large intercellular spaces. This organization is typical of epithelia active in fluid reabsorption. Two lateral large gynatrial glands open into the gynatrial sac. Such glands also exhibit secretory and duct forming cells. The same structure of these glands is also present along the proximal region of the fecundation canal. The duct forming cells of these regions have very wide ducts with peculiar cuticular finger-like structures at their opening into the gland duct lumen. The results of the present study suggest the occurrence of a coevolution between the sperm and the spermathecal duct lengths.

Coevolution between female seminal receptacle and sperm morphology in the semiaquatic measurer bug Hydrometra stagnorum L. (Heteroptera, Hydrometridae).

The coevolution between sperm length and size of the female sperm-storage organs is described for the first time within Heteroptera. The long sperm of the measurer bug Hydrometra stagnorum is characterized by the unusually long acrosome with its anterior region helically arranged, and by a very short nucleus. The sperm flagellum has a 9 + 9+2 conventional axoneme and crystallized mitochondrial derivatives. The female spermatheca consists of an extraordinarily long spermathecal duct ending with an apical spermathecal bulb into which flows also the secretions of a relatively short spermathecal gland. Both spermathecal duct and gland have a thin epithelium lined by a cuticle, beneath which a complex of secretory and duct forming cells are present. The secretions of these two structures flow into the apical spermathecal bulb. A thick layer of muscle fibers surrounds the epithelium. These results confirm the opinion that the dimensions of the female reproductive sperm-storage organs are able to drive the sperm morphology.

The sperm ultrastructure of Pytho depressus (Linnaeus, 1767) (Coleoptera, Pythidae).

The sperm ultrastructure of Pytho depressus (Pythidae) is described in this study. The sperm are short cells, about 85-90 µm long, with an acrosome consisting of three layers, a cylindrical nucleus, which at its base has the initial region of two mitochondrial derivatives. The flagellum has two well-developed triangular accessory bodies, and a 9 + 9+2 axonemal pattern with accessory tubules provided with 16 protofilaments in their wall. The structure and shape of the accessory bodies are diagnostic characters within the superfamily. The sperm morphology of P. depressus can be easily distinguished from those of Ripiphoridae, Meloidae and Tenebrionidae. The P. depressus sperm are organized in cysts as in other species of the group but the sperm are not well aligned and show an antiparallel orientation, a feature also observed in other tenebrionids. The phylogenetic implications of the observed sperm features are discussed in the context of comparative sperm ultrastructure of other insect species.

A Stresipteran parasite extends the lifespan of workers in a social wasp.

In social wasps, female lifespan depends on caste and colony tasks: workers usually live a few weeks while queens as long as 1 year. Polistes dominula paper wasps infected by the strepsipteran parasite Xenos vesparum avoid all colony tasks, cluster on vegetation where parasite dispersal and mating occur, hibernate and infect the next generation of wasp larvae. Here, we compared the survival rate of infected and uninfected wasp workers. Workers' survival was significantly affected by parasite sex: two-third of workers parasitized by a X. vesparum female survived and overwintered like future queens did, while all workers infected by a X. vesparum male died during the summer, like uninfected workers that we used as controls. We measured a set of host and parasite traits possibly associated with the observed lifespan extension. Infected overwintering workers had larger fat bodies than infected workers that died in the summer, but they had similar body size and ovary development. Furthermore, we recorded a positive correlation between parasite and host body sizes. We hypothesize that the manipulation of worker's longevity operated by X. vesparum enhances parasite's fitness: if workers infected by a female overwinter, they can spread infective parasite larvae in the spring like parasitized gynes do, thus contributing to parasite transmission.

Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera).

In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, gamma-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible.

Centriole symmetry: a big tale from small organisms.

Centrioles are microtubule-based cylindrical organelles with a 9-fold symmetry. They are essential for axoneme formation in cilia and flagella and for centrosome organization. In the basal hexapods Acerentomon microrhinus, we discovered unusually large centrioles composed of 14 doublet microtubules that serve as templates for cilia and flagella and organize mitotic and meiotic spindles. These observations challenge the long-standing view that centriole symmetry is highly conserved among eukaryotes. Strikingly, daughter centrioles contain a transient cartwheel that is lost after maturation. The length of radial spokes is like that found in 9-fold cartwheels, whereas the diameter of the hub varies according to the dimensions of the centriole cylinder. This suggests that the hub may dictate the master plan for centriole geometry. Finally, the finding that 14-doublet centrioles arise from 9-doublet mothers points to an alternative model for centriole assembly.

Sperm ultrastructure of three species of ladybirds (Coleoptera, Coccinellidae).

The sperm of three coccinellid species belonging to the subfamilies Chilocorinae, Coccinellinae and Epilachninae were studied under light and transmission electron microscopy. The basic sperm structure of these ladybirds is common to that of the other previously studied species, especially the acrosome in front of the basal body and not the nucleus, with this latter running parallel with the flagellar components. In the Chilocorinae Platynaspis luteorubra (Platynaspidini) the sperm are of the type 1, as in Scymnini and Coccinellini, since they exhibited a cylindrical basal body with 9 + 0 and then 9 + 2, microtubules continuing further in an initial flagellar portion with the only axoneme devoid of accessory structures. The sperm exhibits a thin nucleus and mitochondrial derivatives. Such uniformity of sperm ultrastructure could be indicative of the occurrence of a close relationship between Platynaspidini and Scymnini as also proposed in the previous studies. Conversely, they differ markedly from the sperm of type 3 observed in the Chilocorini Exochomus quadripustulatus. In the Coccinellini Propylea quatuordecimpunctata the sperm are also of the type 1, but they can be easily differentiated from those of the other Coccinellinae studied so far, because of their very short acrosome without the posterior extension, the relatively thicker mitochondrial derivatives and the cylindrical nucleus. Epilachna clandestina sperm resemble those of E. quadripustulatus but differ from them, because they exhibit an elliptical nucleus which is anteriorly very thin, and the asymmetrical mitochondrial derivatives in the anterior extremity, with the greater one starting at the same level of the basal body, rather than at the nucleus level, as it occurs in E. quadripustulatus. Because of the differences observed in the sperm ultrastructure we propose a new sperm type (Type 4) for E. clandestina. This study on ladybird spermatozoon ultrastructure clearly indicates that the current classifications of coccinellids do not reflect the natural history of this well-known insect family.