Characterization of microsatellite loci in the stick insects Bacillus rossius rossius,Bacillus rossius redtenbacheri and Bacillus whitei (Insecta: Phasmatodea)

Five microsatellite markers were obtained from a dinucleotide enriched genomic library of the stick insect Bacillus rossius rossius. The markers were tested in three species of Bacillus. All loci were polymorphic when tested across species. The number of alleles at each locus was low (maximum four alleles), but different allelic patters were observed among the species.     

Morphology of the terminalia of the stick insect Dajaca napolovi from Vietnam (Insecta: Phasmatodea)

Aschiphasmatinae is a small group of stick insects from the Oriental region whose genital morphology has been rarely described in detail. The subfamily is of particular interest, as phylogenetic studies have shown Aschiphasmatinae to be the sister group to the remaining Euphasmatodea. In this paper, the male and female terminalia are described for the first time in Dajaca napolovi Brock, a little known aschiphasmatine species from Vietnam. In the male, the transversally undivided abdominal sternum IX and gently incurved cerci with a conspicuous apical tooth represent apomorphies of Aschiphasmatinae. Thorn pads on the hind margin of abdominal tergum X consist of only a single row of 6–7 ventrally oriented teeth. The simple thorn pad structure of Aschiphasmatinae can represent an ancestral condition for Euphasmatodea. The vomer on venter X is well‐developed and features two unusually large basal apodemes and a strongly developed apical spine showing a specialized streaked surface micropattern. Female terminalia are characterized by an unkeeled abdominal sternum VIII covering the reduced primary ovipositor. Gonapophysis VIII does not extend beyond the tip of gonapophysis IX. An asymmetry concerning the size of the paired gonapophyses is reported for the first time in Phasmatodea.     

Allozymic characterization of Sardinian Bacillus rossius (Rossi) and B. atticus Brunner (Insecta Phasmatodea)

The allozymic characterization of newly discovered populations of B. atticus and B. rossius is given. Most Sardinian B. rossius demes belong to the subspecies B. r. rossius, Southeastern populations are B. r. redtenbacheri, while the central ones appear to be intermediate. Like all Italian B. atticus, also Sardinian demes belong to B. a. caprai. The presence of B. atticus alleles in syntopic B. rossius populations is explained through gene introgression due to past hybridization events between the two taxa.     

The follicle cell-oocyte interaction in ovarian follicles of the stick insect Bacillus rossius (Rossi): (Insecta: Phasmatodea)

Developing ovarian follicles of Bacillus rossius have been examined ultrastructurally in an attempt to understand how inception of vitel-logenesis is controlled. Early vitellogenic follicles are characterized by a thick cuboidal epithelium that is highly interlocked with the oocyte plasma membrane. Gap junctional contacts are present both at the follicle cell/oocyte interface and in between adjacent follicle cells. In addition, microvilli of follicle cells protrude deeply into the cortical ooplasm of these early vitellogenic oocytes. With the onset of vitellogenesis, wide intercellular spaces appear in the follicle cell epithelium and at the follicle cell/oocyte interface. Gap junctions become progressively reduced both on the follicle cell surface and on the oocyte plasma membrane. Microvilli from the two cell types no longer interlock. From a theoretical standpoint each of the two structural differentiations present at the follicle cell/oocyte interface—gap junctions and follicle cell microvilli—could potentially trigger inception of vitellogenesis. Gap junctions might permit the passage of a regulatory molecule, transferring from follicle cells to oocyte, which would control the assembly of coated pits on the oocyte plasma membrane. Alternatively cell interaction via microvilli might induce the appearance of coated pits, thus creating a membrane focus for vitellogenin receptors. Both possibilities are discussed in relation to current literature.     

Auto- and heterosynthetic processes in developing ovarian follicles of Bacillus rossius (Rossi) (Phasmatodea : Bacillidae)

The relative contribution to yolk deposition by heterosynthetic and autosynthetic processes has been examined in developing ovarian follicles of Bacillus rossius (Phasmatodea : Bacillidae). To accomplish this goal, early and late vitellogenic follicles have been examined ultrastructurally. In addition, follicle proteins, labelled both after in vivo and in vitro exposure to radioactive amino acids, have been characterized by gel electrophoresis on polyacrylamide gels. Finally, the intracellular distribution of radioactivity, due to incorporation of (³H)-leucine under both in vivo and in vitro conditions, has been examined in developing ovarian follicles by autoradiography and scintillation counting.Based on the evidence obtained in the present study, it is concluded that most, if not all, yolk deposited in developing ovarian follicles of B. rossius is obtained by exogenous processes, that is, taken up from the hemolymph by endocytosis. The endogenous contribution is limited to the formation of yolk-like vesicular bodies, whose major function has been interpreted in relation to the intracellular digestion and recycling of aged cell organelles.     

Centrosome dynamics and inheritance in related sexual and parthenogenetic Bacillus (Insecta Phasmatodea)

In animals, some general features of centrosome dynamics and inheritance have been widely recognized. The most acknowledged model assigns to sperm the contribution of a centriole to the fertilized egg, which in turn provides the pericentriolar materials, including gamma-tubulin, recruiting them from the cytoplasm: the main zygote microtubule organizing center (MTOC) is thus reconstituted to organize first the spermaster and then the full first embryonic spindle. Obviously the model cannot apply to parthenogenetic systems, which actually rely on egg components alone. In stick insects of the Bacillus genus, the spindle of both somatic and germ cells is clearly anastral, therefore we have been investigating their centrosome in sexual and parthenogenetic taxa by analyzing its component dynamics and transmission through the use of monoclonal beta- and gamma-tubulin antibodies and transmission electron microscopy (TEM). It has been shown that in sexually reproducing species the spermatozoon does not contribute the centriole, so that the egg wholly provides the MTOC and the ensuing anastral spindle of the embryo: MTs appear to derive from pronuclear chromatin surroundings and no asters are observed. The parthenogenetic embryo development is the same as the sexual one if syngamy is excepted. The parthenogenetic mechanism realized by these panoistic insects appears to differ from that observed in the meroistic hymenopteran and drosophilid species, where the embryo spindle derives from asters formed in the egg cortex. In stick insects, the lack of sperm contribution to embryonic centrosome appears to be a major trait accounting for the widespread occurrence of facultative and obligate parthenogenesis within the order.     

The spermatogenesis and sperm structure of Timema poppensis (Insecta: Phasmatodea)

The ultrastructure of spermatogenesis and spermatozoa was studied in Timema poppensis Vickery & Sandoval, 1999, a putative basal taxon of Phasmatodea. The apical portion of testis follicles consists of spermatogonial cells with polymorphic nuclei. Primary spermatocytes display very short primary cilia originating from the peripheral centrosomes. Early spermatids develop a conspicuous “nebenkern” consisting of fused mitochondria. They have a single peripheral centriole with microtubular triplets, which expresses a 3.6-μm-long cilium featuring a 9?+?2 axonemal pattern. In a later stage, the centriole and the ciliary shaft displace toward the inner part of the cytoplasm by an infolding of the plasma membrane. Mature spermatids exhibit a derived centriole with microtubule doublets devoid of dynein arms, which is equipped with a dense arc-like outer structure. Ciliary degeneration was not observed during spermiogenesis. Spermatozoa are short flagellate cells about 55–60?μm in length. They are characterized by a three-layered acrosomal complex. The distinctive bell-shaped morphology of the acrosome vesicle is likely an autapomorphic trait of Timema. The flagellum has a 9?+?9?+?2 axoneme, two accessory bodies, two flattened cisterns, and two elongated mitochondrial derivatives. Results support the hypothesis that Phasmatodea, comprising Timema?+?Euphasmatodea, form a monophyletic group. The presence of 17 protofilaments in the wall of accessory microtubules and the flattened configuration of the flagellum are potential apomorphic groundplan features of the order. Within Phasmatodea, a key evolutionary divergence was from the conventional insect spermiogenesis and sperm structure of Timema, to the unusual spermiogenetic process and peculiar sperm structure of Euphasmatodea. As a result, Timema retains more sperm character states found in the polyneopteran ground pattern, while Euphasmatodea have evolved outstanding sperm autapomorphies, like the loss of mitochondria and flattened cisterns, and the presence of strongly expanded accessory bodies.     

Egg envelopes of Baetis rhodani and Cloeon dipterum (Ephemeroptera, Baetidae): a comparative analysis between an oviparous and an ovoviviparous species

A comparative ultrastructural investigation on the eggshell (vitelline and chorionic envelopes) has been carried out in the nymphs of two mayfly species encompassed into the Baetidae, namely Baetis rhodani and Cloeon dipterum. During oocyte differentiation in the meroistic telotrophic ovarioles, gametes are connected to discrete nurse cells by trophic cords. In B. rhodani, each ovariole contains several eggs arranged in sequence, whereas in C. dipterum each contains a single egg. Follicle cells are competent for vitelline and chorionic envelope synthesis. Baetis rhodani is an oviparous species and the chorion is fairly thick, formed by an alveolate endochorion and a fibrillar exochorion delimited by a honey‐comb roof. Cloeon dipterum stands out among Ephemeroptera for being ovoviviparous. In B. rhodani, ovulation starts in the older nymphs with dark wing‐pads, whereas in C. dipterum choriogenesis ends in the imaginal stage. Here the chorion is very thin and laid eggs hatch almost immediately, allowing the larvulae to move out. The maturation of a single egg per ovariole is synchronized with the achievement of the adult stage. The acquisition of ovoviviparity has led to remarkable changes in the ovariole organization along with a simplification of the eggshell structure.     

The ovary ofCatajapyx aquilonaris (Insecta,Entognatha): ultrastructure of germarium and terminal filament

Summary Each of the two ovaries ofCatajapyx aquilonaris is composed of seven segmentally (metamerically) arranged ovarioles. The two lateral oviducts that join and bear ovarioles extend throughout the abdomen. In the ovariole three regions can be recognized: the terminal filament, the germarium and the vitellarium. The terminal filaments do not fuse with each other but attach separately (by means of muscle fibres) to the closest lobes of the fat body. Germ cells in the germarium are not joined by intercellular bridges and do not form clusters. Thus the ovarioles ofC. aquilonaris are interpreted as being primarily panoistic. The results obtained support the hypothesis that both dipluran subgroups (Campodeina and Japygina) do not form a monophyletic unit.     

The eggs of leaf insects (Insecta: Phasmida)

The morphology of seventeen samples of phylliine egg is described and figured, and an attempt is made to relate these egg forms to the alleged identity of the adult insects.     

Two new species of Phraortes from the Ryukyus,Japan (Phasmatodea: Phasmatidae)

Two new species of Phraortes are described from the Ryukyus of Japan. Phraortes kumensis sp. nov. is described from Kume‐jima Island, and P. yonaguniensis sp. nov. from Yonaguni‐jima Island. The morphology of the females, males and eggs of the new species is illustrated.     

The female postabdomen and internal genitalia of the basal moth genus Agathiphaga (Insecta: Lepidoptera: Agathiphagidae): morphology and phylogenetic implications

The female postabdomen of Agathiphaga vitiensis terminates in a telescope‐type extensible oviscapt with an apial ‘oviscapt probe’ composed of fused segments behind VIII. Exceptionally within the Lepidoptera two pairs of long ‘anterior apophyses’ arise from segment VIII, from the dorsum and venter. Agathiphaga has the most elaborate postabdominal musculature recorded from female Lepidoptera, comprising 24 muscle sets of which nine may be family autapomorphies. Apophysis musculature does not permit unambiguous homologizing of the single anterior apophysis in Lepidoptera–Glossata with either the dorsal or ventral pair in Agathiphaga, but is compatible with an interpretation of the glossatan anterior apophyses as a composite formation. Nine muscle sets shared with rhyacophilid caddisflies are ascribed to the amphiesmenopteran ground plan. The spermathecal duct represents an intermediate stage between the simple type present in Micropterigidae and the ‘two‐compartment type’ characteristic of almost all other Lepidoptera. The spermatheca has no lagena. The bursa copulatrix is small and simple. Accessory glands are very large, simple sacs. There are 40 + ovarioles per ovary. A terminal cloaca is extremely short. The numerous ovarioles potentially support a sister‐group relationship between Agathiphagidae and all other Lepidoptera, whereas the spermathecal duct histology supports the alternative conservative placement of the family as sister group of all nonmicropterigid Lepidoptera. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 905–920.     

Structure and function of the arolium of Mantophasmatodea (Insecta)

All species of the insect order Mantophasmatodea characteristically keep the 5th tarsomere and pretarsus (arolium plus two claws) turned upwards and off the substrate. The unusually large arolium was studied in two species of Mantophasmatodea using bright field light microscopy, reflection microscopy, fluorescence microscopy, TEM, SEM, and Cryo‐SEM. It contains an epithelial gland, numerous tracheoles, and nerves. The gland consists of enlarged epithelial cells with large nuclei, mitochondria, RER, golgi complexes, microtubules, and numerous secretion vesicles. Evidence for exocytosis of the vesicles into the gland reservoir between the epithelial gland and the thick cuticle could be observed. Cryo‐SEM revealed that the ventral side of the arolium and distal part of its dorsal side are covered with a liquid film. Fluid footprints of arolia of individuals walking on a glass plate also indicate the presence of secretory fluid on the arolium surface. Behavioral experiments using animals with ablated arolia showed that representatives of Mantophasmatodea do not need their arolia to detect and respond to vibratory communication signals nor to catch small to medium‐sized prey. Individuals with ablated arolia were not able to move upside down on a smooth glass plate. We conclude that Mantophasmatodea use their arolia for attachment when additional adhesion force is required (e.g. windy conditions, handling large prey, mating). They can bring their arolia in contact with the surface in a very fast reflex (18.0 ± 9.9 ms). The secretory fluid found on the surface is produced by the gland and transported to the outside, presumably through small pore channels, to enhance adhesion to the substrate. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea)

Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic. Hybridogenetic strains (B. rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B. whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division. New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions. On the other hand, in B. whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs. The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B. whitei from hemiclonal strains. In addition, B. whitei gains a closer resemblance to B. lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features. It is here suggested that B. lynceorum originated from the incorporation of an "atticus" genome into a B. whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids.     

Formation and structure of nutritive cords in telotrophic ovarioles of snake flies (Insecta: Raphidioptera)

Telotrophic ovariole of Raphidia spp. is composed of the anteriorly located terminal filament, tube-shaped tropharium, the vitellarium and the ovariole stalk. The tropharium consists of a central syncytial core surrounded by one cell thick layer of tapetum cells. Early previtellogenic oocytes differentiate at the base of tropharium. Both the oocytes and the tapetum cells are connected with the central syncytium by delicate intercellular bridges. At the onset of previtellogenic growth, the anterior parts of the oocytes become extended and form long cytoplasmic projections--nutritive cords. Each nutritive cord contains numerous microtubules that show no preferential orientation within the cord but diminishing anterior-posterior gradient of distribution. Irregular arrangement of microtubules indicates that this cytoskeletal scaffold does not play any role in directed transport within the ovariole but instead constitutes one of the elements of the structural framework of the nutritive cord. Besides microtubules, the stability of the nutritive cords in Raphidia ovarioles is maintained by the rim-shaped membrane foldings lined with microfilaments.     

Allozyme analysis and phyletic relationships of two new stick-insects from north-west Sicily: Bacillus grandii benazzii and B. rossius-grandii benazzii (Insecta Phasmatodea)

Two new Sicilian stick-insects have been discovered within the genus Bacillus. Evidence concerning the subspecific differentiation of Bacillus grandii benazzii and the hybrid constitution of B. rossius-grandii benazzii by means of allozyme analysis is given, and is consistent with morphological and karyological data on the differentiation of these two north-west Sicilian morphs from south-eastern B. g. grandii and B. whitei. B. g. benazzii is more polymorphic than B. g. grandii and B. rossius-g. benazzii embodies its genetic variability, thus differing sharply from the south-eastern parallel hybrid B. whitei ( = B. rossius × B. g. grandii). Reproductive mechanisms also appear to be different in the two interspecific hybrids, involving parthenogenesis in the latter and hybridogenesis in the former. Finally, the phyletic relationships among all Sicilian Bacillus taxa are summarized in a revised scheme, which also takes into account new evidence that B. atticus has contributed to hybrid constitution of the triploid B. lynceorum.