Phylogenetic revision of the flightless Australian genus Apterotheca Gebien (Coleoptera : Tenebrionidae : Coelometopinae)

Members of the flightless genus Apterotheca Gebien (Coleoptera : Tenebrionidae) are mostly restricted to the high elevation rainforests of the Wet Tropics World Heritage Area of north-eastern Australia. This region has been recognised as an 'epicentre of evolution for low vagility animals'. The genus Apterotheca is the most diverse low vagility insect taxon known in this region. Forty-four species are included here in a revision of the genus. Three of these species were previously included in Apterotheca (A. antaroides (Pascoe), A. besti (Blackburn) and A. punctipennis Carter), four were previously included in other genera (A. australis (Kulzer), comb. nov. and A. punctifrons (Gebien), comb. nov. in Apterophenus Gebien, A. costata (Buck), comb. nov. in Caxtonana Buck and A. pustulosa (Carter), comb. nov. in Austropeus Carter) and 37 are new. The monotypic genera Austropeus Carter, syn. nov. and Caxtonana Buck, syn. nov. are proposed as new synonyms of Apterotheca. A lectotype for A. punctipennis and A. besti are designated. A key to the species of Apterotheca and a phylogenetic analysis based on the morphological features of adults, as well as a discussion of character evolution, are also included. Data presented here represent the framework for future studies on the determinants of the patterns of diversity found in the Wet Tropics.

Wolbachia density and virulence attenuation after transfer into a novel host

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. metanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. metanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

The adult ventral nerve cord as a phylogenetic character in brachyceran Diptera

Insect ganglia are often composed of fused segmental units or neuromeres. We estimated the evolution of the ventral nerve cord (VNC) in higher Diptera by comparing the patterns of neuromere fusion among 33 families of the Brachycera. Variation within families is uncommon, and VNC architecture does not appear to be influenced by body shape. The outgroup pattern, seen in lower Diptera, is fusion of neuromeres belonging to thoracic segments 1 and 2 (T1 and T2), and fusion of neuromeres derived from T3 and abdominal segment 1 (A1). In the abdomen, neuromeres A7-10 are fused into the terminal abdominal ganglion (TAG). Increased neuromere fusion is a feature of the Brachycera. No brachyceran shows less fusion than the outgroups. We established six pattern elements; (1) fusion of T1 and T2, (2) fusion of T3 and A1, (3) fusion of the T1/T2 andT3/A1 ganglia, (4) increase in the number of neuromeres comprising the TAG, (5) anteriorward fusion of abdominal neuromeres, and (6) the complete fusion of thoracic and abdominal neuromeres into a synganglion. States 1 and 2 are present in the outgroup lower Diptera, and state 3 in the Xylophagomorpha, Stratiomyomorpha, Tabanomorpha and Cyclorrhapha. State 4 is a feature of all Eremoneura. State 5 is present in Cyclorrhapha only, and state 6, fusion into a synganglion, has evolved at least 4 times in the Eremoneura. Synapomorphies are provided for the Cyclorrhapha and Muscoidea, and a grouping of three basal brachyceran infraorders Xylophagomorpha, Stratiomyomorpha and Tabanomorpha. The patterns of fusion suggest that VNC architecture has evolved irreversibly, in accordance with Dollo's law.

Development and survival of the diamondback moth (Lepidoptera : Plutellidae) at constant and alternating temperatures

Survival and development time from egg to adult emergence of the diamondback moth, Plutella xylostella (L.), were determined at 19 constant and 14 alternating temperature regimes from 4 to 40degreesC. Plutella xylostella developed successfully front egg to adult emergence at constant temperatures from 8 to 32degreesC. At temperatures from 4 to 6degreesC or from 34 to 40degreesC, partial or complete development of individual stages or instars was possible, with third and fourth instars having the widest temperature limits. The insect developed successfully from egg to adult emergence under alternating regimes including temperatures as low as 4degreesC or as high as 38degreesC. The degree-day model, the logistic equation, and the Wang model were used to describe the relationships between temperature and development rate at both constant and alternating temperatures. The degree-day model described the relationships well from 10 to 30degreesC. The logistic equation and the Wang model fit the data well at temperatures 32degreesC. Under alternating regimes, all three models gave good simulations of development in the mid-temperature range, but only the logistic equation gave close simulations in the low temperature range, and none gave close or consistent simulations in the high temperature range. The distribution of development time was described satisfactorily by a Weibull function. These rate and time distribution functions provide tools for simulating population development of P. xylostella over a wide range of temperature conditions.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

Patterns of resistance to angular leaf spot, anthracnose and common bacterial blight in common bean germplasm

Diseases and insect pests are major causes of low yields of common bean (Phaseolus vulgaris L.) in Latin America and Africa. Anthracnose, angular leaf spot and common bacterial blight are widespread foliar diseases of common bean that also infect pods and seeds. One thousand and eighty-two accessions from a common bean core collection from the primary centres of origin were investigated for reaction to these three diseases. Angular leaf spot and common bacterial blight were evaluated in the field at Santander de Quilichao, Colombia, and anthracnose was evaluated in a screenhouse in Popayan, Colombia. By using the 15-group level from a hierarchical clustering procedure, it was found that 7 groups were formed with mainly Andean common bean accessions (Andean gene pool), 7 groups with mainly Middle American accessions (Middle American gene pool), while 1 group contained mixed accessions. Consistent with the theory of co-evolution, it was generally observed that accessions from the Andean gene pool were resistant to Middle American pathogen isolates causing anthracnoxe, while the Middle American accessions were resistant to pathogen isolates from the Andes. Different combinations of resistance patterns were found, and breeders can use this information to select a specific group of accessions on the basis of their need.

In vivo and in vitro toxicities of pithraj and neem against rice green leafhopper (Nephotettix cincticeps Uhler)

Seed extracts of Aphanamixis polystachya Wall et Parker (pithraj) and Azadirachta indica A. Juss (neem) were evaluated for their in vivo and in vitro toxicity to Nephotettix cincticeps Uhler (rice green leafhopper). Crude extracts from both plants showed toxicity to leafhopper. Among them, the methanol extract of pithraj (MCX) was most toxic and showed 95% mortality effects at 72 h after treatment (HAT), followed by neem (74%). When LD50's were compared, it was found that the neem extract possessed the highest toxicity (LD50 16.59 μg/insect) at 72 HAT. Both the pithraj (MCX) and neem extracts showed their enzyme inhibition effectiveness against rice green leafhopper. The highest inhibition rate (IR) was caused by neem (60%) at the concentration of 2.0 mg/ml, followed by MCX (47%). The lowest IR50 value (0.97 mg/ml) was observed in neem at 30 min.

Sustained high levels of foliar herbivory of the mangrove Rhizophora stylosa by a moth larva Doratifera stenosa (Limacodidae) in north-eastern Australia

The moth larva, Doratifera stenosa (Lepidoptera: Limacodidae), was observed feeding voraciously in great numbers on mature leaves of Rhizophora stylosa in mangroves at Port Curtis in Central Queensland, NE Australia. This behaviour was considered unusual since mangroves, and the Rhizophora species in particular, reportedly harbour few herbivores and have relatively low levels of herbivory, less than 10%. During a two year period (1996-1998), larvae were observed consuming around 30-40% of leaves in the canopy each year, and the mangroves appeared able to sustain these high levels of herbivory. The impact on trees was assessed in conjunction with a study of the herbivore, its behaviour and life history, in an attempt to explain the occurrence. Larvae were 1-2 cm in length, bright green and gregarious, with numerous small, stinging hairs along their upper bodies. Feeding was in small cohort groups of 5-70 individuals that broke up immediately prior to each moult after which they regrouped in much larger numbers of mixed cohorts to form single-file processions across branches, stems and prop roots. In this way, they moved to neighbouring trees with less affected foliage. One of the outstanding characteristics of this herbivore was its ability to desist from killing host trees although it appeared quite capable of doing so had it remained on individual trees. By moving from tree to tree, the herbivore was able to heavily crop Rhizophora foliage in an apparently sustainable manner. These findings demonstrate the role and importance of foliar herbivory in severely affected forests and how such instances best not be ignored or treated as curiosities in future assessments of herbivory and forest turnover in mangrove ecosystems.

The Three-dimensional Solution Structure of NaD1, a New Floral Defensin from Nicotiana alata and its Application to a Homology Model of the Crop Defense Protein alfAFP

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of NaD1, a novel antifungal and insecticidal protein isolated from the flowers of Nicotiana alata. NaD1 is a basic, cysteine-rich protein of 47 residues and is the first example of a plant defensin from flowers to be characterized structurally. Its three-dimensional structure consists of an a-helix and a triple-stranded anti-parallel beta-sheet that are stabilized by four intramolecular disulfide bonds. NaD1 features all the characteristics of the cysteine-stabilized up motif that has been described for a variety of proteins of differing functions ranging from antibacterial insect defensins and ion channel-perturbing scorpion toxins to an elicitor of the sweet taste response. The protein is biologically active against insect pests, which makes it a potential candidate for use in crop protection. NaD1 shares 31% sequence identity with alfAFP, an antifungal protein from alfalfa that confers resistance to a fungal pathogen in transgenic potatoes. The structure of NaD1 was used to obtain a homology model of alfAFP, since NaD1 has the highest level of sequence identity with alfAFP of any structurally characterized antifungal defensin. The structures of NaD1 and alfAFP were used in conjunction with structure - activity data for the radish defensin Rs-AFP2 to provide an insight into structure-function relationships. In particular, a putative effector site was identified in the structure of NaD1 and in the corresponding homology model of alfAFP. (C) 2002 Elsevier Science Ltd. All rights reserved.

Secretory pathway of trypanosomatid parasites

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Tryanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles it? the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory), transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is similar to3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terininal untranslated regions and, at its 3-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 front the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that similar to 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Structure of the gut contents of Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae) larvae fixed in situ in eggs of its host Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

Trichogramma australicum larvae develop most rapidly in younger eggs of its host, the pest lepidopteran Helicoverpa armigera . To establish how the developmental stage of the host affects the diet of T. australicum , larvae were fixed in situ in eggs of H. armigera of different ages and the structure of the egg contents and parasitoid gut contents examined histologically. Larvae feeding on newly laid host eggs contain primarily yolk particles in their gut, while larvae feeding on older hosts contain necrotic cells and yolk particles. The gut of T. australicum larvae does not contain organised tissue remnants, indicating that larvae feed primarily by sucking food into their pharynx and feed best on a mixture of particulate semisolids in a liquid matrix. Secretory structures of T. australicum larvae that could be involved in modifying the host environment were examined. The hindgut is modified to form an anal vesicle with a number of attributes suggesting that it may be a specialised secretory structure. The paired salivary glands open to the exterior via a common duct.