Divergent mating patterns and a unique mode of external sperm transfer in Zoraptera: an enigmatic group of pterygote insects.

A remarkable external sperm transfer is described for the first time in a species of a group of winged insects (Pterygota), the enigmatic Zoraptera. Mating and sperm transfer of two species of the order were examined in detail, documented, and compared with each other and with patterns described for other species belonging to the order. The behavior differs strikingly in Zorotypus impolitus and Zorotypus magnicaudelli. A copula is performed by males and females of the latter, as it is also the case in other zorapteran species and generally in pterygote insects. In striking contrast to this, males of Z. impolitus do not copulate but deposit small (100 µm in diameter) spermatophores externally on the abdomen of the female. Each spermatophore contains only one giant spermatozoon (3 mm long and 3 µm wide), a unique feature in the entire Hexapoda. External sperm transfer in Pterygota is a highly unusual case of evolutionary reversal. The very small relict group Zoraptera displays a uniform general morphology but exhibits very different reproductive structures and patterns of mating behavior. This may be an extreme form of a more general situation in insects, with a specific form of selection resulting in an accelerated rate of evolution in the reproductive system.

Domestication of olive fly through a multi-regional host shift to cultivated olives: comparative dating using complete mitochondrial genomes.

The evolutionary history of the olive fly, Bactrocera oleae, was reconstructed in a phylogenetic and coalescent framework using full mitochondrial genome data from 21 individuals covering the entire worldwide distribution of the species. Special attention was given to reconstructing the timing of the processes under study. The early subdivision of the olive fly reflects the Quaternary differentiation between Olea europea subsp. europea in the Mediterranean area and the two lineages of Olea europea subsp. cuspidata in Africa and Asia, pointing to an early and close association between the olive fly and its host. The geographic structure and timing of olive fly differentiation in the Mediterranean indicates a clear connection with the post-glacial recolonization of wild olives in the area, and is irreconcilable with the early historical process of domestication and spread of the cultivated olive from its Levantine origin. Therefore, we suggest an early co-history of the olive fly with its wild host during the Quaternary and post-glacial periods and a multi-regional shift of olive flies to cultivated olives as these cultivars gradually replaced wild olives in historical times.

Sperm ultrastructure and spermatogenesis in the Japanese beetle Popillia japonica (Coleoptera, Scarabaeidae).

Popillia japonica is an invasive scarab beetle native to Japan that in 1916 invaded New Jersey in USA. From that moment onwards, the insect has spread invading several US states, Canada, the Azores, Italy and, recently, Switzerland. It is a severe agricultural pest included in the EU priority pest list being able to feed on more than 300 plant species and having an important biotic potential. The general morphology of the reproductive apparatus shows paired testes, each of them having six testicular lobes grouped in threes. From the ventral part of each testicular lobe, each containing about 20 follicles, an efferent vessel originates that fuses with the other efferent vessels to form the deferent duct. A pair of long tubular accessory glands is present. The deferent ducts and accessory glands fuse together into an ejaculatory duct before entering the aedeagus. The sperm is a typical pterygote sperm, 110 µm long, composed of a head and a tail. In the head a three-layered acrosome of about 6 µm in length and a nucleus of about 18 µm long are present. During sperm maturation two C-shaped structures appear in the cytoplasm from the opposite sides of the nucleus that then disappear in late spermatids. In the tail a typical 9 + 9 + 2 flagellar axoneme and two mitochondrial derivatives are present. Moreover, in the head-tail transition region the centriolar adjunct forms a sheath from which three elongated accessory bodies originate. Two of these accessory bodies are placed alongside the axoneme, whilst the third one is placed beneath the mitochondrial derivatives. Mature sperm are grouped in cysts containing about 256 sperm cells. A morphological comparison with related species is provided.

Acetylcholinesterase genes in the basal Hexapod Orchesella villosa.

Acetylcholinesterase (AChE) is a key enzyme of the cholinergic nerve system. Of the two forms found in insects, the predominant one is active in the synapses and is the target of organophosphate and carbamate insecticides, while the role of the second is currently unknown. Two acetylcholinesterase cDNAs from the basal hexapod Orchesella villosa have been characterized and compared with others reported form insects. One form conforms well to the typical structure, while the other is characterized by an unusual 3' region. No amino acid mutation could be directly associated with known resistance mutations in other insect species or to a clear signal of selection in the distribution of alleles, although the action of some population process is suggested.

The spermatozoon of arthropoda. XXX. The multiflagellate spermatozoon in the termite Mastotermes darwiniensis.

In this paper the spermatozoon of the termite Mastotermes darwiniensis is described. It is the first example of a multiflagellate sperm cell in animals. The sperm consists of a conical head and 100 flagella. Other remarkable features of this sperm cell are the absence of an acrosome, the presence of centrioles containing doublet microtubules instead of triplets, and the presence of axonemes devoid of central tubules and with doublets bearing only one arm. The flagella are feebly motile.

The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm.

Two unconventional sperm models (all motile) have been studied. The first one has only the outer arm on the doublets (the gall midge, Diplolaboncus); the second one, has only a well-developed inner arm (the eel, Anguilla). Both are devoid of central tubules and radial spokes. The gall midge sperm yields a single electrophoretic band migrating similarly to the sea urchin dynein band A; a major high-molecular-weight band is obtained from eel sperm which co-migrates with the sea urchin dynein band B. The present picture is consistent with the localization of dynein in the axoneme--namely, of an A-like band in the outer arm, and of the B band in the inner arm. Moreover, the D band is present only in the eel, where gamma-links are present. ATPase activity was localized histochemically and found to be associated with both inner and outer arms, as well as with the gamma-links.

Protein of insect sperm mitochondrial crystals. Crystallomitin.

Mitochondrial derivatives of insect sperm usually contain a crystalline protein that shows a 45-nm main period, made up of 20-nm subperiods, determined by the coiling of filament bundles. Filaments are 2 nm thick and have a globular appearance. The crystals contain two main polypeptides, 52,000 and 55,000 daltons. These polypeptides are closely related, contain a high percentage of proline, and are insoluble in sodium dodecyl sulfate due to disulfide cross links. We suggest for this class of protein the name crystallomitin.

Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin.

Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man. In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating cytotoxin A). Cytotoxic activity is associated with a > 600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37- and 58-kD fragments. Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six- or seven-fold radial symmetry. Within each monomer, two domains can clearly be distinguished, suggesting that the 37- and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer. Analysis of preparations of the toxin that had undergone extensive cleavage into the 37- and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure. The data suggest a structural similarity with AB-type toxins.

The peculiar extra-acrosomal structure of the Collembola (Hexapoda) spermatozoa.

The springtail Collembola are characterized by having rolled spermatozoa, with a long cylindrical extracellular structure adhering to the acrosome. This structure is produced by the secretory activity of the testes epithelial cells at almost the end of spermiogenesis. At the beginning of its formation, it is a thin extension with a helical wall and a dense axial region. Later the cylindrical structure shows an inner organization which is different in the several species examined: species of Entomobryidae contain material with a paracrystalline structure, whilst some of Symphypleona contain ovoid structures. The outer envelope of the extracellular structure consists of two overlapped layers orthogonally arranged, clearly identified by cryo-preparations. Immunoblot analysis and lectin stainings have indicated that the cylindrical structure has a glycoproteic composition. As the structure is no longer visible after the sperm transfer into the female spermatheca, it is suggested that it could contain enzymes able to activate the sperm unwinding process and possibly allowing the reacquisition of sperm motility.

The morphology and ultrastructure of salivary glands of Zoraptera (Insecta).

The salivary glands of two species of Zoraptera, Zorotypus caudelli and Zorotypus hubbardi, were examined and documented mainly using transmission electron microscopy (TEM). The results obtained for males and females of the two species are compared and functional aspects related to ultrastructural features are discussed. The salivary glands are divided into two regions: the secretory cell region and the long efferent duct, the latter with its distal end opening in the salivarium below the hypopharyngeal base. The secretory region consists of a complex of secretory cells provided with microvillated cavities connected by short ectodermal ducts to large ones, which are connected with the long efferent duct. The secretory cell cytoplasm contains a large system of rough endoplasmic reticulum and Golgi apparatus producing numerous dense secretions. The cells of the efferent duct, characterized by reduced cytoplasm and the presence of long membrane infoldings associated with mitochondria, are possibly involved in fluid uptaking from the duct lumen.

Subtle effect of Xenos vesparum (Xenidae, Strepsiptera) on the reproductive apparatus of its male host: Parasite or parasitoid?

Parasitic castration is an adaptive strategy where parasites usurp the hosts' reproductive physiology to complete their life cycle. The alterations in the host traits vary in their magnitude, from subtle changes in the host morpho-physiology and behaviour to the production of complex aberrant phenotypes, which often depend on the host gender. The strepsipteran macroparasite Xenos vesparum induces dramatic behavioural and physiological changes in its female host, the paper wasp Polistes dominula, while its effect on the male phenotype is largely unknown. In this study we investigated how a single X. vesparum parasite influences the functional morphology of P. dominula male reproductive apparatus. We performed morphometry and ultrastructure characterization of corpora allata, testes, seminal vesicles and accessory glands in parasitized and unparasitized males, and also in young and old males to control for the effect of age on the natural deterioration of these organs. Our results show that age significantly affects the development of male reproductive apparatus. A low parasite load - one parasite per host is the common prevalence in the field - has only a marginal impact on the reproductive morphology of P. dominula males, affecting quantitatively but not qualitatively the protein content of male accessory glands. Thus, in male P. dominula wasps, X. vesparum appears to behave as a true "parasite", in clear opposition to the role of "parasitoid" that it takes in female hosts where castration causes the reproductive death.

The mitochondrial genomes of Campodea fragilis and Campodea lubbocki (Hexapoda: Diplura): High genetic divergence in a morphologically uniform taxon.

Complete mitochondrial genome sequences are presented from two dipluran hexapods (i.e., a group of "primarily wingless insects") of the genus Campodea and compared to those of other arthropods. Their gene order is the same as in most other hexapods and crustaceans. Structural changes have occurred in tRNA-C, tRNA-R, tRNA-S1 and tRNA-S2 as well as in both ribosomal RNAs. These mtDNAs have striking biases in nucleotide and amino acid composition. Although the two Campodea species are morphologically highly similar, their genetic divergence is larger than expected, suggesting a long evolutionary history, perhaps under stable ecological conditions.

The aberrant spermatogenesis of the Haplothrips simplex (Buffa) (Thysanoptera): ultrastructural study.

The aberrant spermatogenesis of the haploid insect Haplothrips simplex (Thysanoptera) is described. The process, which occurs in the pupal instars, is characterized by two mitotic divisions, the second of which gives rise to two different-sized spermatids: the larger spermatids have a nucleus with diffuse chromatin and proceed into spermiogenesis, while the small spermatids have pycnotic nuclei and degenerate. Both types of spermatids contain two centrioles parallely rather than orthogonally oriented. The occurrence of two centrioles supports a close relationship between Thysanoptera and Phthyraptera. Before the beginning of spermiogenesis, however, the functional spermatids show the unusual presence of a third parallel centriole which is formed by the duplication of one of the two pre-existing centrioles.

Fine structure of the secretory and sensory organs on the cephalon and the first pereionite of Trichoniscus alexandrae Caruso (Crustacea, Isopoda).

Trichoniscus alexandrae Caruso is a blind troglobiont isopod; males possess secretory and sensory organs on the cephalon and 1st pereionite consisting of cuticular pits hosting a tuft of setae and gland openings. Such organs are absent in females. Three types of cuticular structures have been observed: (a) lamellar setae, which likely play a role in protecting the gland openings and favouring the evaporation of secretions; (b) contact chemoreceptors, each provided with six bipolar sensory cells, a scolopale cell and enveloping cells; (c) a secretory cell complex, consisting of a long cylindrical slender duct-forming cell, with the function of transporting to the cuticular surface a secretion produced by two deeper secretory cells. The duct-forming cell is characterized by the presence of numerous microtubules in its cytoplasm, and is provided with a flattened duct. It is suggested that the secretion produced by the secretory cells could serve for sex-recognition.

Giant spermatozoa of Diasemopsis (Diopsidae, Diptera) - Structural, ultrastructural and functional aspects.

The spermatozoa of Diasemopsis comoroensis and Diasemopsis 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 evolution of sperm axoneme structure and the dynein heavy chain complement in cecidomid insects.

The 9 + 2 axoneme of cilia and flagella is specialized machinery aimed at the production of efficient, finely tuned motility, and it has been evolutionarily conserved from protists to mammals. However, the sperm cells of several insects express unconventional axonemes, which represent unique models for studying the structural-functional relationships underlying axonemal function and evolution. Cecidomids comprise a group of dipterans characterized by an overall tendency to deviate from the standard axonemal pattern. In particular, the subfamily Cecidomyiinae shows a series of progressive modifications of the sperm axoneme. We previously analyzed the unusual sperm axonemes of Asphondylia ruebsaameni (Asphondyliidi) and Monarthropalpus buxi (Cecidomyiidi), which are characterized by the absence of any structure related to the control of motility (that is, the central pair complex, radial spokes and inner dynein arms); however, these sperm are motile, and motility is driven by the outer dynein arms only. This simplification of the motility machinery is accompanied by a parallel reduction in the dynein isoform complement. Here, we complete our survey of the axonemal organization and the parallel evolution of sperm dynein complement in cecidomids with the characterization of both the sperm ultrastructure and the dynein genes in Dryomyia lichtensteini, a representative of Lasiopteridi, the cecidomid taxon with aberrant and immotile sperm cells. On the basis of the whole set of our data, we discuss the potential molecular mechanism(s) underlying the progressive modification of axoneme in cecidomids, leading first to a reduction of dynein genes and eventually to the complete loss of motility.

The fine structure of the rectal pads of Zorotypus caudelli Karny (Zoraptera, Insecta).

The rectal pads of a species of the controversial polyneopteran order Zoraptera were examined using histological sections and TEM micrographs. Six pads are present along the thin rectal epithelium. Each pad consists of a few large principal cells surrounded by flattened junctional cells, which extend also beneath the principal cells. The cells are lined by a thin apical cuticle. No basal cells and no cavity have been observed beneath the pad. Principal cells have a regular layer of apical microvilli and are joined by intercellular septate junctions, which are interrupted by short dilatations of the intercellular space. At these levels the two adjacent plasma membranes are joined by short zonulae adhaerentes. In the cytoplasm, a rich system of strict associations between lateral plasma membranes and mitochondria forms scalariform junctions. Rectal pads share ultrastructural features with similar excretory organs of several neopteran groups, in particular with Blattodea (roaches and termites) and Thysanoptera, and are involved in fluid reabsorption and ion regulation.

Assembly of the zygotic centrosome in the fertilized Drosophila egg.

Zygotic centrosome assembly in fertilized Drosophila eggs was analyzed with the aid of an antiserum Rb188, previously shown to be specific for CP190, a 190 kDa centrosome-associated protein (Whitfield et al. (1988) J. Cell Sci. 89, 467-480; Whitfield et al. (1995) J. Cell Sci. 108, 3377-3387). The CP190 protein was detected in two discrete spots, associated with the anterior and posterior ends of the elongating nucleus of Drosophila spermatids. As the spermatids matured, this labelling gradually disappeared and was no longer visible in sperm dissected from spermathecae and ventral receptacles. gamma-Tubulin was also found in association with the posterior end of the sperm nucleus during spermiogenesis, but was not detected in mature sperm. This suggests that CP190 and gamma-tubulin are not present in detectable quantities in fertilizing sperm. CP190 was not detected in association with the sperm nucleus of newly fertilized eggs removed from the uterus, whereas many CP190-positive particles were associated with microtubules of the sperm aster from anaphase I to anaphase II. These particles disappeared during early telophase II and only one pair of CP190-positive spots remained visible at the microtubule focus of the sperm aster. These spots were associated with one aster through telophase, and then moved away to form two smaller asters from which the first mitotic spindle was organized. Colchicine treatment suggested that at least some CP190 protein is an integral part of the centrosome rather than merely being transported along microtubules. Centrosomal localization of the CP190 antigen was prevented by incubation of the permeabilized zygote in 20 mM EDTA.

Mating of Xenos vesparum (Rossi) (Strepsiptera, Insecta) revisited.

The controversial mating of the strepsipteran Xenos vesparum was studied to investigate the possible sperm routes for fertilization. The female, which is a neotenic permanent endoparasite of Polistes wasps, extrudes only its anterior region, the "cephalothorax," from the host abdomen. This region has an opening where both mating and larval escape occur. Observations with scanning and transmission electron microscopy revealed spermatozoa not only in the hemocoel, but also in the "ventral canal" (an extragenital duct peculiar to strepsipteran females) and in the "genital ducts" (ectodermal invaginations connecting the ventral canal to the hemocoel) of recently mated females. Xenos vesparum spermatozoa can reach the oocytes either through the hemocoel as a result of a hypodermic insemination, or by moving along the extragenital ducts, which are later used by first instar larvae to escape. The hypothesis of hypodermic insemination is reconsidered in the light of behavioral and ultrastructural evidence.

Spermatozoa and spermiogenesis of Holocnemus pluchei (Scopoli, 1763) (Pholcidae, Araneae).

Until now, the knowledge on pholcid spermatozoa is based on two species, Pholcus phalangioides and, incompletely, Holocnemus pluchei. To complete this knowledge and to reveal more potential phylogenetic characters, we have investigated sperm ultrastructure and spermiogenesis of H. pluchei. We found that the sperm cells of this species are clearly different from those of P. phalangioides with respect to: (1) the lack of specialization in the cylindrical acrosomal vacuole; (2) a nuclear canal which is located in the periphery and not in the center of the nucleus; (3) a more prominent postcentriolar elongation of the nucleus; (4) the presence of "inner microtubules" in the implantation fossa in early and mid-spermatids; (5) the absence of a helical band of nuclear material; (6) the proximal centriole which is not prolonged; (7) the types of secretion in the seminal fluid (only two types in H. pluchei). Similarities in the spermatozoa of both species concern: (1) a large implantation fossa which contains large amounts of glycogen in mature spermatozoa; (2) absence of a centriolar adjunct; (3) an axonemal basis located in the posterior part of the implantation fossa; (4) the formation of the so-called cleistospermia in the vas deferens. Our results strongly support systematic relationships within Pholcidae placing these two species in different subgroups.