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.     

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.     

Ontogenesis of hemocytes in the stick insect Bacillus rossius (Rossi) (Phasmatodea,Bacillidae) studied with an anti-hemocyte monoclonal antibody

The monoclonal antibody BrH1, specific for hemocytes of the stick insect Bacillus rossius, was employed to study the appearance of hemocytes during embryogenesis. Laid eggs were collected for eight weeks, and cryosections were probed with the antibody. First positive cells were detected at the fifth week, and increased in number onwards. No peculiar differences were observed in the overall morphology between embryonic and non-embryonic hemocytes.     

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.     

Speciation through androgenesis in the stick insect genus Clonopsis (Insecta Phasmatodea)

In Morocco, Clonopsis stick insects showed tangled reproductive interactions actually resulting into a network of phylogenetic relationships known as ‘reticulate evolution’. Peculiar to parthenogenetic C. gallica and C. soumiae (54 and 72 chromosomes, respectively) – closely related to the bisexual C. felicitatis (2n = 36) – is the finding of numerically polyploid karyotypes with a diploid structure. Androgenesis appeared to be the most parsimonious explanation accounting for both the low mitochondrial differentiation and the quick onset of those polyploids with structurally diploid karyotypes, paired with neat nuclear differentiations. According to a proposed model, hybrid triploid females would segregate balanced haploid and diploid 2nd oocytes immediately producing all kinds of parthenogens and androgens. Owing to these peculiar reproductive issues, we felt useful searching for stronger evidence by deeply analysing the mitochondrial genome. This new analysis showed a neat separation of sexual Tetouan haplotypes from the parthenogenetic and androgenetic ones, which are grouped in two slightly overlapping groups by network analysis: Moroccan parthenogens and androgens vs European C. gallica. It could be also envisaged that C. gallica has multiple origins, being a complex of parthenogenetic strains originated through independent hybridizations. The straightforward mechanism originating both triploid and tetraploid parthenogens well fits with both their widely ascertained low mitochondrial differentiation and the geographical closeness of the most similar samples, independently from their specific karyotype. Combining the outcomes of the hybridization events and androgenesis, which completely substitutes hybrid genomes with those of a related paternal species, would conceivably realize the observed picture of species structure and distribution. Owing to the reinforced data set, it now appears much more sensible to support androgenesis as a quick pathway to originate polyploids with numerically and genetically sharply differing chromosome sets, while maintaining, at the same time, high mitochondrial similarity.     

An enigmatic new stick insect from the Philippine Islands (Insecta: Phasmatodea)

A new genus and species of stick insect is described and figured from Mount Halcon, on the Philippine island of Mindoro. Conlephasma enigma gen. et sp. n. is a stout, flightless, and apparently ground-dwelling species with vivid integumental colors. When disturbed, specimens spray a defensive secretion from the prothoracic exocrine glands. The systematic position of Conlephasma within Euphasmatodea is unclear. The elongated galealobulus and the trichome area located laterally in the galea, represent unusual apomorphic characters of the maxilla that could indicate affinities with Necrosciinae or Pseudophasmatinae. All tibiae exhibit the anareolate condition. Euplantulae are of two types: those of tarsomeres I–IV feature a nubby microstructure, whilst the one on the ventral side of the pretarsus is smooth. Males are characterized by the presence of a well-developed vomer on the tenth abdominal segment. A distinctive and apomorphic trait of female terminalia is represented by the elongated tenth abdominal tergum. Conlephasma can represent an interesting taxon for studies on the evolution of the stick and leaf insects.     

Chromosomal and NOR patterns in the polyclonal stick insect Bacillus atticus atticus (Insecta; Phasmatodea).

Bacillus atticus atticus is a complex of thelytokous parthenogens, related to the bisexual Bacillus grandii, that ranges from Sardinia to Near Eastern countries. Karyotypic and cytogenetic differentiation of the B. atticus atticus diploid unisexual "isolates" is really higher than expected. Its standard karyotype has 2n = 34 chromosomes, but several instances of repatterned or even aneuploid complements have been found. The number and location of silver-stained NORs are particularly intriguing, since in addition to homozygous NOR patterns, simple or double hemizygous strains are found spread over specific and wide regions. The odd patterns are not due to Ag-NOR staining technique artifacts, since the FISH method, using rDNA probes, apparently labels the same ribosomal clusters. Transpositions and translocations have been suggested to account for some NOR patterns, but hybridizations between different NOR-bearing races are also a possible cause. This chromosomal survey clearly contributes to a better understanding of B. atticus phylogeny.     

The germarium of panoistic ovarioles of Bacillus rossius (Insecta Phasmatodea): Structure and function during imaginal life

Summary

In adult females of Bacillus rossius (Insecta Phasmatodea) the germarium, localized at the ovariole tip just below the terminal filament and above the vitellarium, progressively reduces in size and eventually disappears at the end of the ovulatory period. The observations with light and electron microscopes show that in the end-chamber most germ cells are arrested in a post-pachytenic diffuse stage, which just precedes diplotenic oocyte growth. These observations also indicate that the reduction in size of the germarium of ovulating females should probably be ascribed to a progressive and extensive activation of the resting germ cells. The average number of ovulated eggs per ovariole (6.7±0.9) is consistent with this view. However, occasional findings of lepto-zygotenic germ cells in some preovulatory ovarioles of adult females do not completely rule out the persistence of scarce undifferentiated germ elements (oogonia) in the larval germarium at the onset of adult life. Furthermore, the reduction of the germarium in ovulating females and its subsequent disappearance in post-ovulating ones also includes the somatic cells, which are always present among the germ cells in previous stages. Since each early growing oocyte becomes surrounded by a thin monolayer of follicle cells, the diminution of end-chamber somatic cells supports the view that they actually represent prefollicular cells, which are progressively utilized from the onset of imaginal life onwards.     

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.     

Automictic parthenogenesis in the stick insectBacillus rossius Rossi (Cheleutoptera,phasmidae)

The process which restores the zygoid number of chromosomes in the thelytokously parthenogenetic stick insectBacillus rossius Rossi, is described. During the first 15 days after oviposition approximately 200 haploid (A+X=18 chromosomes) pre-blastoderm cells develop in the egg-cortex. During the second period of 15 days up to about 2000 blastoderm cells develop in which were found: haploid mitoses, C-mitoses (A+X=18 chromosomes), endomitoses, diploid mitoses (2A+2X=36 chromosomes) and pycnosis. The next 10 days the number of pycnotic nuclei decreases gradually and the blastoderm cells multiply by diploid mitosis only. Thereafter, a diploid germ band is formed either directly or after a diapause. It is concluded that C-mitosis in haploid cells restores the somatic number of chromosomes (automictic parthenogenesis), that a portion of the cells in C-mitosis degenerates and that endomitosis leads to the formation of some polyploid cells only. This development is discussed in relation to the occurrence of geographic parthenogenesis and of impaternate males.     

DNA-RNA bodies in midgut cells of the stick insect, Bacillus rossius.

In the anterior part of the midgut and in the Malpighian tubules of the stick insect Bacillus rossius, about 10% of the epithelial cells develop endonuclear bodies which appear as DNA-RNA masses; in these cells the usual nucleoli are no longer evident. The DNA-RNA bodies are first formed in third instar larvae, become numerous in the fourth instar and persist in adults. In all larval instars and adults a different kind of DNA-body has been noticed in the epithelial cells of the posterior midgut. The DNA-RNA bodies of the anterior midgut and of the Malpighian tubules have been interpreted as the result of somatic gene amplification, whereas the DNA masses of the posterior midgut are likely due to a virus infection.     

Endogenous cellulase enzymes in the stick insect (Phasmatodea) gut

High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of the walking stick (Phasmatodea) Eurycantha calcarata. The enzyme was isolated and analyzed via mass spectrometry. RT-PCR revealed two endoglucanase genes, EcEG1 and EcEG2. Mascot analysis of the purified enzyme confirms it to be the product of gene EcEG1. Homologous cDNAs were also isolated from a distantly related species, Entoria okinawaensis, suggesting a general distribution of cellulase genes in phasmids. Phasmid cellulases showed high homology to endogenously-produced glycoside hydrolase family 9 (GH9) endoglucanases from insects, especially to those of termites, cockroaches, and crickets. The purified E. calcarata enzyme showed clear antigency against an anti-serum for termite GH9 cellulase, which, together with the sequence homology, further suggests an endogenous origin of the enzyme. This discovery suggests a possible nutritive value for cellulose in the leaf-feeding phasmids, unlike in herbivorous Lepidoptera.     

Development of ovarian follicle cells of the stick insect,Carausius morosus br. (Phasmatodea), in relation to their function

The development of follicle cells encompassing the growing oocytes of the stick insect, Carausius morosus (Phasmatodea) has been investigated cytologically and cytophotometrically. The nuclei become 64-ploid through 3 telophasic restitution cycles and 2 endoreduplications. The genomes are evenly distributed over the nucleus, which is also supported by the regular distribution of the nucleoli, and, consequently, fulfill their functions more economically in the (up to 80 μm) thick epithelium. Previtellogenesis is sustained by follicle cells containing 2c-8c DNA, vitellogenesis by 16c cells, vitelline envelope formation by 32c cells, and water uptake, endochorion and exochorion formation by 64c cells.     

An early winged crown group stick insect from the Early Eocene amber of France (Insecta,Phasmatodea)

The new stick insect family Gallophasmatidae, based on Gallophasma longipalpis gen. et sp.n. , from the Earliest Eocene French amber has a pattern of tegmina venation typical of Archaeorthoptera, also present in at least some Mesozoic ‘Phasmatodea’. On the other hand, Gallophasma displays in its body anatomy some apomorphies of the extant Euphasmatodea, e.g. fusion of metatergum and abdominal tergum 1, correlated with the reduction of abdominal sternum 1 to lateral triangular sclerites. A unique autapomorphy of Gallophasma is the presence of annulated and apparently multi‐segmented or pseudo‐segmented cerci; all other Phasmatodea have one‐segmented cerci. The venation of the tegmina of Gallophasma differs from that of extant winged Phasmatodea in the plesiomorphic absence of a knob‐like dorsal eversion. This and other differences in the wing venation between extant and extinct Phasmatodea might have been caused by the loss of wings at some point in the evolutionary history of the order and their secondary gain in a subclade of the extant phasmids.     

Influence of aminergic and peptidergic substances on heart beat frequency in the stick insect Carausius morosus (Insecta,Phasmatodea)

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi‐exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo‐HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo‐HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo‐HrTH‐II, thereby rendering the HrTH peptide “not active.” In ligated stick insects, 6.7 × 10^?8 M Carmo‐HrTH‐II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose‐dependent manner; inhibition was achieved with 10^?4 M of octopamine.     

External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations

External morphological features of adult males are described in the stick insect Hermarchus leytensis from the Philippines, a species belonging to the little-known euphasmatodean lineage Stephanacridini. Mouthparts are characterized by some likely specialized features: (1) a dentate dorsal cutting edge on the mandibles; (2) distinct differences in size and shape between the galeae; (3) absence of an apical field of trichomes on the galeae; and (4) lacinial setae not protruding from the mesal margin, which features three bearing-like protuberances. The latter character state represents a very unusual condition, not known in other phasmatodeans or even in polyneopteran insects. A distinctive characteristic of attachment devices is that each euplantula is divided into two separated pads with a smooth microstructure, as it also occurs in some members of the clade Schizodecema. Male terminalia exhibit character states previously unknown in Stephanacridini, including (1) a symmetrical type of vomer and (2) claspers equipped with ∼70 very minute ventral teeth on each thorn pad. Potential implications for the systematic placement of H. leytensis are discussed. The results also underline the importance of microanatomical investigations as source of substantial characters for future analyses on phasmatodean systematics.     

An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60-180 Kd. Following in vivo exposure to [(35)S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [(35)S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [(3)H]-leucine for time intervals ranging from 20-240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.     

Postendocytic vitellin processing in ovarian follicles of the stick insect Carausius morosus (Br.)

Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A₁, A₂, and A₃) and 2 (B₁ and B₂) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV follicles, Vit A resolved into 4 distinct vitellin polypeptides (A₀, A₁, A₂ and A₃). Using a panel of monoclonal antibodies, polypeptide A₀ proved to be immunologically related to polypeptide A₂. In follicles about to begin choriongenesis, polypeptide A₃ was gradually replaced by a lower M_r polypeptide. Over the same time period, polypeptide B₁ changed in charge, but not in M_r. To confirm the existence of a polypeptide processing in C. morosus, ovarian follicles of different developmental stages were exposed in vivo to [³⁵S]-methionine from 6 to 72 h. Data showed that A₀ and B₁ were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B₂ and A₃ were also labelled to some extent. With progressively longer exposures, polypeptides A₁ and A₂ also became labelled. In vivo exposure to [³H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of ovarian follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.     

Developmental instability, hybridization and heterozygosity in stick insects of the genus Bacillus (Insecta; Phasmatodea) with different modes of reproduction

Several genetic factors are assumed to influence developmental instability (DI). One is the level of heterozygosity, with higher levels often being associated with decreased DI; another is genetic incompatibility in hybrids, which in several cases has been shown to increase DI. The genus Bacillus includes species which have both amphigonic heterozygous reproducing populations and homozygous parthenogenetic reproducing populations (B. rossius rossius and B. r. redtenbacheri). Furthermore, Bacillus includes hybrid parthenogenetic species, which have very high levels of almost fixed heterozygosities (B. atticus, B. whitei, B. lynceorum). We investigated the phenotypic variance (σ²p) and the impact of hybridization and level of heterozygosity on DI in females from these populations and species of Bacillus. DI was estimated as fluctuating asymmetry (FA) for three bilateral traits: the labial palpus, the maxillary palpus and the antenna. For the labial palpus and maxillary palpus we found, in general, a lower level of DI in the amphigonic females compared with parthenogenetic counterparts from the same species and with parthenogenetic females from the three hybrid species. A higher DI of the antenna was found in the hybrid species when compared with both parthenogenetic and amphigonic populations of the nonhybrid species, suggesting that the genes controlling antenna development are located on the sex chromosomes. The development of the investigated bilateral characters in the hybrid species seemed to be affected more by factors relating to genetic incompatibilities as a consequence of hybridization than by the stabilizing force of increased heterozygosity. Only few differences in σ²p were observed, supporting the possibility that the observed differences in DI are related mainly to internal genetic factors. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87 , 249–259.     

The mitochondrial genome of Bacillus stick insects (Phasmatodea) and the phylogeny of orthopteroid insects

The Order Phasmatodea (stick and leaf insects) includes many well-known species of cryptic phytophagous insects. In this work, we sequenced the almost complete mitochondrial genomes of two stick insect species of the genus Bacillus. Phasmatodea pertain to the Polyneoptera, and represent one of the major clades of heterometabolous insects. Orthopteroid insect lineages arose through rapid evolutionary radiation events, which likely blurred the phylogenetic reconstructions obtained so far; we therefore performed a phylogenetic analysis to resolve and date all major splits of orthopteroid phylogeny, including the relationships between Phasmatodea and other polyneopterans. We explored several molecular models, with special reference to data partitioning, to correctly detect any phylogenetic signal lying in rough data. Phylogenetic Informativeness analysis showed that the maximum resolving power on the orthopteroid mtDNA dataset is expected for the Upper Cretaceous, about 80millionyears ago (Mya), but at least 70% of the maximum informativeness is also expected for the 150-200 Mya timespan, which makes mtDNA a suitable marker to study orthopteroid splits. A complete chronological calibration has also been computed following a Penalized Likelihood method. In summary, our analysis confirmed the monophyly of Phasmatodea, Dictyoptera and Orthoptera, and retrieved Mantophasmatodea as sister group of Phasmatodea. The origin of orthopteroid insects was also estimated to be in the Middle Triassic, while the order Phasmatodea seems to appear in the Upper Jurassic. The obtained results evidenced that mtDNA is a suitable marker to unravel the ancient splits leading to the orthopteroid orders, given a proper methodological approach.