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.     

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.     

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.     

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.     

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.     

Restricted specificity of a hyperglycaemic factor from the corpus cardiacum of the stick insect Carausius morosus

Extracts of the corpora cardiaca of the stick insect Carausius morosus elevate the haemolymph carbohydrate concentration in adult and 6th-instar larvae which are ligated behind the first pair of legs, but not in non-ligated (intact) insects. The increase in haemolymph sugars is due to trehalose elevation, is time dependent (with a maximal effect about 90–120 min after injection), and is dose dependent (needing 0.005 gland equivalents for a significant effect and a tenfold higher dose for a maximal response). The hyperglycaemic factor is localised entirely in the corpora cardiaca and appears to be specific to stick insects; corpora cardiaca extracts of two lepidopteran species (Acherontia atropos and Aglais urticae) and of Locusta migratoria have no effect, whereas corpora cardiaca extracts of the stick insects Cuniculina impigra and Sipyloidea sipylus have similar activity to those from C. morosus. This specificity is also shown when S. sipylus is used as the recipient. Synthetic adipokinetic hormone and red pigment concentrating hormone possess no hyperglycaemic activity in the stick-insect system. Two peaks of hyperglycaemic activity were obtained after column chromatography of corpora cardiaca extract of C. morosus on Sephadex G-25 and Sephadex LH-20. The factor seems to act via activation of fat-body glycogen phosphorylase, which, although 60% active in the control insects, is significantly increased to approx. 85% upon corpora cardiaca injection. However, the activation is demonstrated in ligated and intact insects. No significant decrease in the glycogen level of the fat body is observed after corpora cardiaca injection.     

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.     

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.     

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.     

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.     

On the head morphology of Phyllium and the phylogenetic relationships of Phasmatodea (Insecta)

Friedemann K., Wipfler B., Bradler S. and Beutel R.G. 2011 . On the head morphology of Phyllium and the phylogenetic relationships of Phasmatodea (Insecta). —Acta Zoologica (Stockholm) 00 : 1–16. External and internal head structures of Phyllium siccifolium are described in detail. The findings are compared with conditions found in other phasmatodeans and members of other neopteran lineages. The compiled 125 characters were analysed cladistically. A clade Eukinolabia (Phasmatodea + Embioptera) was confirmed. Synapomorphies of these two taxa are the shift of the origin of M. tentorioparaglossalis to the hind margin of the prementum, the presence of M. tentorioscapalis medialis, and antennal muscles that originate exclusively on the anterior tentorial arms. Within Eukinolabia, the position of Timema remains somewhat ambiguous because of missing anatomical data. However, it was confirmed as sister group of Euphasmatodea in a monophyletic Phasmatodea. Apomorphic groundplan features of Euphasmatodea are salivary ducts with separate external openings, apically rounded glossae, the presence of the galealobulus, and the reduction of the antennifer. The monophyly of Neophasmatidae was confirmed. Autapomorphies are the loss of M. frontobuccalis posterior, the anteriorly or dorsally directed maxillary palps, and the reduction of the mandibular incisivi. The analysis of characters of the head yielded three new autapomorphies of Phylliinae, the presence of a protuberance on the attachment site of the dorsal tentorial arms, dorsoventrally flattened maxillary‐ and labial palps, and possibly the narrow and U‐shaped field of trichomes on the apical part of the galea.     

A walking stick,Clonistria dominicana n. sp. (Phasmatodea: Diapheromeridae) in Dominican amber

A walking stick, Clonistria dominicana n. sp. (Phasmatodea: Diapheromeridae: Diapheromerinae: Diapheromerini) is described from Dominican amber. The new species can be distinguished from extant members of the genus by the long hind leg tarsomere II (over six times the length of the foreleg tarsomere II), the short metanotum, which is only half the length of the median segment and the prominent median carina. Two phasmatid eggs in separate pieces of Dominican amber are also presented. One of these has characteristics of the genus Clonistria Stål, 1875, which supports the generic placement of C. dominicana. The second egg resembles those of the genus Malacomorpha Rehn, 1906 (Pseudophasmatidae: Pseudophasmatinae). Both of these genera occur in Hispaniola today.     

A new genus and new species of Philippine stick insects (Insecta: Phasmatodea) and phylogenetic considerations

Based on characters of both sexes, a new genus and species of the basal euphasmatodean lineage Aschiphasmatidae is described and figured from the Philippines. Dallaiphasma eximius gen. et sp. n. displays interesting features for the group, including: a cone-shaped vertex, which is notably raised above the pronotum; the tibial area apicalis represented by a depressed membranous posterior lateral region, and a strongly sclerotized central apical region; the euplantulae consisting of smooth-type attachment pads; the pretarsal claws pectination reduced to minute denticulations; and the well-differentiated boundary between the metanotum and the first abdominal tergum. The phylogenetic information content of the new findings is discussed. Furthermore, as a result of this study, the Aschiphasmatidae are newly recorded from Mindoro island, and now include five genera and six species of the Philippines.     

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.     

Interpopulation differences in egg sculpturing of the stick insect,Clonopsis gallica (Charp.) (Phasmatodea: Bacillidae)

Eggs of 6 Clonopsis gallica (Phasmatodea: Bacillidae) populations have been investigated by means of scanning electron microscopy (SEM) to detect intraspecific differences. Two main chorionic patterns have been observed: type 1 is common to 5 populations, while type 2 is found in the Motril (South Spain) sample. Type-2 sculpturing differs from type-1 in the absence of prominences on the operculum; and hemispheric, isolated mamelons replace the smooth and diffuse prominences of the common pattern on the egg capsule. This suggests that a considerable degree of difference exists in the South Spanish C. gallica. In the 5 type-1 populations, only minor and partially overlapping differences in the opercular pattern have been detected, so that the existence of subspecific taxa is very unlikely among them. Therefore, the Calabrian subspecies C. gallica affinis does not appear to be tenable.This is the 1st report on egg interpopulation differences in the Phasmatodea. These findings suggest further field search, along with karyological and gene-enzyme system analysis, within the C. gallica complex.     

Structural heterozygosity and aneuploidy in the parthenogenetic stick insect Carausius morosus Br. (Phasmatodea: Phasmatidae)

The female chromosome complement of the thelytokous stick insect Carausius morosus Br. consists of three metacentric sex chromosomes, four metacentric and 57 acrocentric autosomes. The rare impaternate males have two sex chromosomes. The spermatogenesis is highly aberrant which is evident from the various numbers of univalents, homomorphic and unequal bivalents, and multivalents during first metaphase, and from abnormal segregation patterns during first and second anaphase. The abnormalities are due to aneuploidy and structural heterozygosity. The heterozygosity is maintained by the endomeiotic chromosome duplication in females. Translocations resulting from chiasmata in unequal associations are not formed during female meiosis. It has been discussed that the heterozygosity in males, and consequently in females, is caused by either chromosomal mutations, as indicated by at least ten interchanges and three inversions, or hybridization, indicated by allotriploidy.     

Investigating hybridization in the parthenogenetic New Zealand stick insect Acanthoxyla (Phasmatodea) using single-copy nuclear loci

The New Zealand stick insect genus Acanthoxyla Uvarov is extremely unusual among higher taxa of animals in that all known species are obligate parthenogens. We have used a combination of the mitochondrial DNA genes cytochrome oxidase subunits I and II, 28S nuclear ribosomal RNA, and the two single-copy nuclear genes elongation factor 1alpha and phosphoglucose isomerase to test hypotheses on the role of hybridization in the evolution of this genus. Alleles at the single-copy nuclear loci in three sampled species of Acanthoxyla were resolved by cloning the PCR products. Analysis of multilocus genotypes shows that most sampled individuals of Acanthoxyla possess three alleles at the single-copy nuclear loci, which we have interpreted to indicate triploidy. Because most of the alleles from Acanthoxyla form a monophyletic group, including sets of alleles possessed by the putative triploids, we have inferred that the extant parthenogenetic lineages formed via hybridization between species of Acanthoxyla, at least one of which must have been sexual. More recently, there have been multiple introgression events from the related species Clitarchus hookeri White, although C. hookeri does not appear to be involved with the origin of parthenogenesis in Acanthoxyla. Our study demonstrates the utility of cloning alleles from multiple single-copy nuclear genes for resolving the origins of parthenogenetic lineages.     

Phylogeographic history of the New Zealand stick insect Niveaphasma annulata (Phasmatodea) estimated from mitochondrial and nuclear loci

We have assessed the utility of a single-copy nuclear locus and mitochondrial DNA (mtDNA) in a phylogeographic study of the New Zealand stick insect Niveaphasma annulata (Hutton). We amplified sequences from the mitochondrial cytochrome oxidase subunit I (COI) gene and the single-copy nuclear gene elongation factor-1α (EF1α) from 97 individuals. Allelic phase at the EF1α locus was determined using Denaturing Gradient Gel Electrophoresis. Phylogenetic analyses showed broad congruence between the geographic distribution of three major COI clades and EF1α alleles, which suggested that the phylogenetic patterns reflect population history rather than lineage sorting. However, the geographic boundaries of these clades were not always in exact agreement between the two loci. Our data indicate that Niveaphasma annulata was most likely separated into a number of refugia during Pleistocene glacial advances. Subsequent to glacial retreat these refugial populations have expanded and now form a number of zones of secondary contact. We contrast these patterns with those observed from other New Zealand taxa. Our study offers compelling evidence for the use of nuclear genes alongside mtDNA for future phylogeographic studies.