Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

A Polymerase Chain Reaction Method for Identification of Five Major Meloidogyne Species

A polymerase chain reaction (PCR) method for discriminating Meloidogyne incognita, M. arenaria, M. javanica, M. hapla, and M. chitwoodi was developed. Single juveniles were ruptured in a drop of water and added directly to a PCR reaction mixture in a microcentrifuge tube. Primer annealing sites were located in the 3'' portion of the mitochondrial gene coding for cytochrome oxidase subunit II and in the 16S rRNA gene. Following PCR amplification, fragments of three sizes were detected. The M. incognita and M. javanica reactions produced a 1.7-kb fragment; the M. arenaria reaction, a 1.1-kb fragment; and the M. hapla and M. chitwoodi reactions resulted in a 0.52-kb fragment. Digestion of the amplified product with restriction endonucleases allowed discrimination among species with identically sized amplification products. Dra I digestions of the 0.52-kb amplification product produced a characteristic three-banded pattern in M. chitwoodi, versus a two-banded pattern in M. hapla. Hinf I digestion of the 1.7-kb fragment produced a two-banded pattern in M. javanica, versus a three-banded pattern in M. incognita. Amplification and digestion of DNA from juveniles from single isolates of M. marylandi, M. naasi, and M. nataliei indicated that the diagnostic application of this primer set may extend to less frequently encountered Meloidogyne species.     

Nesting biology of three Megachile (Hymenoptera: Megachilidae) species from Eastern Amazonia,Brazil

Megachile Latreille is a conspicuous genus of solitary bees distributed worldwide. However, the biology of tropical species is still little known. We present data on biology of Megachile brasiliensis Data Torre, Megachile sejuncta Cockerell and Megachile stilbonotaspis Moure found in two remnants of eastern Amazonian forest in northeastern Brazil. The study was conducted using the trap-nest methodology in two different areas during four periods. We collected a total of 24 nests of M. brasiliensis, 26 of M. sejuncta and 28 of M. stilbonotaspis. The differential abundance of collected nests may reflect the population size in each sampled place. The nesting activity was concentrated mainly between July and January and species presented a multivoltine pattern, except for M. sejuncta, which was partly univoltine. Assessed pollen use showed a predominant use of Attalea sp. (Arecaceae) and, for M. stilbonotaspis, Tylesia sp. and Lepidaploa sp. (Asteraceae). Babassu is a very common palm in the studied areas and the studied species seem to have a strong link with it. We also reported change of pollen use by M. sejuncta, probably due to competition with M. brasiliensis, which may have influenced the biased sex ratio observed in M. sejuncta toward males. Parasites reported here were also recorded for other Megachile species, such as Coelioxys, Brachymeria, Meloidae and Pyralidae species. Mites were observed in association with M. stilbonotaspis. The data presented here set up a background that encourages new studies on the ecology of these three Amazonian species, providing tools for proper biodiversity management and conservation.     

Re-establishment of Carabus (Cathoplius) aliai Escalera, 1944 as a separate valid species (Coleoptera,Carabidae)

Carabus (Cathoplius) aliai was described as a separate species by Escalera in 1944 but since the 1950–60s it has been considered as a subspecies of Carabus (Cathoplius) stenocephalus Lucas, 1866. This downgrading was adopted after examining only a few specimens, due to their rarity in collections. In recent years, an important population of this taxon was rediscovered in the Tan-Tan area in southern Morocco. By combining field observations with laboratory breeding experiments including hybridization trials, and through the morphological examination of a representative number of individuals, it is confirmed that Carabus aliai is indeed a valid species. Despite close geographic distribution, the morphological and biological characteristics of Carabus aliai and Carabus stenocephalus ifniensis Zarco, 1941, its northern substitutive taxon, are very different. Carabus aliai adults are characterized by a smaller size, a slender silhouette, a more brilliant aspect, a narrower pronotum, a coarser elytral sculpture, longer legs, and a wider and a little more curved apex of the median lobe of the aedeagus. Carabus aliai larvae are also characterized by a much smaller size and the Carabus aliai pupa has a narrower thoracic area and a different chaetotaxy compared to that of Carabus stenocephalus ifniensis. Contrary to this, Carabus aliai has a life cycle belonging to the annual univoltine winter semelparous type. Moreover, the duration of its development cycle is shorter. Carabus aliai is a sabulicolous steppe-wandering species with an intensive running activity, while Carabus stenocephalus ifniensis is a more sedentary taxon. Crossbreeding experiments showed a marked reproductive isolation between Carabus aliai and Carabus stenocephalus ifniensis. When F1 hybrids were crossed with one another, a very high mortality rate during embryonic, larval and pupal development was evident and no vital F2 neo-adults were obtained. Morphological and biological differences, together with the reproductive failure in Carabus aliai × Carabus stenocephalus ifniensis hybrids, clearly indicate that Carabus aliai is a separate Cathoplius species that is distributed in an area south of the Anti-Atlas chain, from Plage Blanche (Guelmim) to Lemsid and Bou Kra (south of Laâyoune). Carabus aliai is therefore both a Saharan desert endemic and an Atlantic resident. Moreover, it is the southernmost Carabus species of the western Palaearctic region.     

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

The het-c heterokaryon incompatibility gene in Aspergillus niger

Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent.     

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.     

One-third rules with equality: Second-order evolutionary stability conditions in finite populations

The one-third law of evolutionary dynamics [Nowak et al. 2004. Emergence of cooperation and evolutionary stability in finite populations. Nature 428, 246-650] describes a robustness criterion for evolution in a finite population: If at an A-frequency of 1/3, the fitness of an A player is greater (smaller) than the fitness of a B player, then a single A mutant that appears in a population of otherwise all B has a fixation probability greater (smaller) than the neutral threshold 1/N, the inverse population size. We examine the case where at an A-frequency of 1/3, the fitness of an A player is exactly equal to the fitness of a B player. We find that in this case the relative magnitude of the cross payoffs matters: If the payoff of A against B is larger (smaller) than the payoff of B against A, then a single A mutant has a fixation probability larger (smaller) than 1/N. If the cross payoffs coincide, we are in the special case of a partnership game, where the deviation cost from an inefficient equilibrium is exactly balanced by the potential gain of switching to the payoff dominant equilibrium. We show that in this case the fixation probability of A is lower than 1/N. Finally, we illustrate our findings by a language game with differentiated costs of signals.     

Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group

Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker.     

Immature Stages and Hosts of Two Plesiomorphic,Antillean Genera of Membracidae (Hemiptera) and a?new species of Antillotolania from Puerto Rico

The nymphs of Antillotolania Ramos and Deiroderes Ramos are described for the first time, along with the first host record for the genus Antillotolania, represented by Antillotolania myricae, sp. n. Nymphal features of both genera, such as a ventrally fused, cylindrical tergum IX (anal tube), the presence of abdominal lamellae, and heads with foliaceous ventrolateral lobes confirm their placement in Membracidae and are consistent with phylogenetic analyses placing them in Stegaspidinae but in conflict with a cladistic analysis showing a closer relationship to Nicomiinae. Head processes and emarginate forewing pads in the last instars of both genera support an earlier estimate, based on nuclear genes, that the two genera form a monophyletic group in Stegaspidinae. Distinguishing features of the four species of Antillotolania are tabulated.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Tylenchulus graminis n. sp. and T. palustris n. sp. (Tylenchulidae), from Native Flora of Florida,with Notes on T. semipenetrans and T. furcus

Tylenchulus graminis n. sp. and T. palustris n. sp. are described and illustrated from broomsedge (Andropogon virginicus L.) and pop ash (Fraxinus caroliniana Mill.), respectively. T. graminis resembles T. furcus in having a distinct anus, but T. graminis second-stage juveniles (J2) do not have a bifid tail. T. semipenetrans does not have a perceptible anus. The mature female of T. graminis has a mucronate pointed terminus while T. semipenetrans has a smooth and round terminus. T. graminis males have wider stylet knobs and basal bulb and a longer tail than T. semipenetrans males. T. graminis J2 have a longer posterior body portion (without large fat globules) than T. semipenetrans J2. T. palustris resembles T. semipenetrans in having an undetectable anus but differs by the short and conoid mature female postvulval section. The male of T. palustris has larger stylet knobs and basal bulb than those of T. semipenetrans and a bluntly rounded tail terminus, which is tapered in T. semipenetrans. T. palustris differs from T. furcus and T. graminis in having an undetectable anus, by the conoid postvulval section of mature females, by the shorter and rounded tail of males, and the shorter J2 posterior body section without large fat globules. T. graminis and T. palustris are parasites of indigenous flora of Florida.     

Canis accitanus nov. sp., a new small dog (Canidae, Carnivora, Mammalia) from the Fonelas P-1 Plio-Pleistocene site (Guadix basin, Granada, Spain)

This paper reports a new species of dog (Canis accitanus nov. sp.) from the Fonelas P-1 site (dated close to the Plio-Pleistocene boundary) in Granada, Spain. This new taxon shows cranial features more similar to coyote-like dogs (C. lepophagus, C. priscolatrans, C. arnensis or C. latrans) than to wolf-like dogs (C. etruscus, C. mosbachensis or C. lupus), such as a long and narrow muzzle, a little-developed sagittal crest and frontal bones raised only a little above the rostrum. However, it also shows a series of autapomorphic characteristics in its upper dentition, essentially in the first upper molar, which reflects a trophic adaptation towards a more abrasive diet than that eaten by other species of its genus. This new dog is the smallest representative of the genus Canis ever recorded for the European Pliocene or Pleistocene.     

Botryosphaeriaceae from tuart (Eucalyptus gomphocephala) woodland, including descriptions of four new species

Eucalyptus gomphocephala (tuart) is a tree native to the southwest coast of Western Australia, where, in some areas, there is a significant decline in the health of tuart. Botryosphaeriaceous taxa have been isolated as endophytes and canker pathogens from numerous hosts in many parts of the world and have been implicated in the decline of E. gomphocephala. In the present study, endophytic fungi were isolated from a wide variety of native woody plant species (Acacia cochlearis, A. rostellifera, Allocasuarina fraseriana, Agonis flexuosa, Banksia grandis, E. gomphocephala, E. marginata and Santalum acuminatum), at two locations in native E. gomphocephala woodland; a site in decline at Yalgorup National Park and a healthy site at Woodman Point Regional Park. Of the 226 isolates obtained, 154 were botryosphaeriaceous taxa, 80 % of which were found to be Neofusicoccum australe, isolated from all hosts at both collection sites. Four new species are described, Dothiorella moneti, Dothiorella santali, Neofusicoccum pennatisporum, and a species belonging to a genus only recently included in the Botryosphaeriaceae, Aplosporella yalgorensis. The other species isolated were Botryosphaeria dothidea on the new hosts A. rostellifera, A. cochlearis and E. marginata and Dichomera eucalypti, on the new host E. marginata. None of the new species formed lesions on excised stems of their host species, E. gomphocephala, or a common plantation species, E. globulus. However, Neofusicoccum australe formed lesions on excised stems of E. globulus and E. gomphocephala.     

Molecular-based estimate of species number,phylogenetic relationships and divergence times for the genus Stenotaenia (Chilopoda,Geophilomorpha) in the Italian region

Stenotaenia is one of the largest and most widespread genera of geophilid centipedes in the Western Palearctic, with a very uniform morphology and about fifteen species provisionally recognized. For a better understanding of Stenotaenia species-level taxonomy, we have explored the possibility of using molecular data. As a preliminary assay, we sampled twelve populations, mainly from the Italian region, and analyzed partial sequences of the two genes COI and 28S. We employed a DNA-barcoding approach, complemented by a phylogenetic analysis coupled with divergence time estimation. Assuming a barcoding gap of 10–16% K2P pairwise distances, we found evidence for the presence of at least six Stenotaenia species in the Italian region, which started diverging about 50 million years ago, only partially matching with previously recognized species. We found that small-sized oligopodous species belong to a single clade that originated about 33 million years ago, and obtained some preliminary evidence of the related genus Tuoba being nested within Stenotaenia.