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Sex differences in metabolic rates in field crickets and their dipteran parasitoids.

Sex differences in metabolic rate (MR) can result from dimorphism in the performance of energetically demanding activities. Male crickets (Teleogryllus oceanicus) engage in costly calling and aggressive activity not performed by females. Consistent with this difference, we found higher maximal MR, factorial scope, and fat content in males than females. T. oceanicus song is also costly because it attracts the parasitoid fly Ormia ochracea. Parasitized crickets had reduced maximal MR consistent with a metabolic cost to harboring larvae. This cost was greater for females, either because females invest more heavily into reproduction at the expense of metabolic capacity, or because males are under stronger selection to respond to infection. Little is known about O. ochracea outside of its auditory system and parasitic lifestyle. We observed greater resting MR in male flies, possibly reflecting a sex difference in the requirement for metabolic power output, because male flies perform potentially costly mating behavior not seen in females. We found a positive relationship between larval density within a cricket and pupal resting MR, suggesting that crickets in good condition are able to both harbor more larvae and produce larvae with higher resting MR. These results reveal a complex interplay between the metabolism of crickets and their fly parasitoids.

Analysis of Variance↗

The genome of Rickettsia prowazekii and some thoughts on the origin of mitochondria and hydrogenosomes.

The sequence of an alpha-proteobacterial genome, that of Rickettsia prowazekii, is a substantial advance in microbial and evolutionary biology. The genome of this obligately aerobic intracellular parasite is small and is apparently still undergoing reduction, reflecting gene losses attributable to its intracellular parasitic lifestyle. Evolutionary analyses of proteins encoded in the genome contain the strongest phylogenetic evidence to date for the view that mitochondria descend from alpha-proteobacteria. Although both Rickettsia and mitochondrial genomes are highly reduced, it appears that genome reduction in these lineages has occurred independently. Rickettsia's genome encodes an ATP-generating machinery that is strikingly similar to that of aerobic mitochondria. But it does not encode homologues for the ATP-producing pathways of anaerobic mitochondria or hydrogenosomes, leaving an important issue regarding the origin and nature of the ancestral mitochondrial symbiont unresolved.

Animals↗

Localisation, quantitation, and characterisation of neuropeptide F- and FMRFamide-immunoreactive peptides in turbellarians and a monogenean: a comparative study.

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a mere sedentary parasitic lifestyle.

Animals↗

Down the slippery slope: plastid genome evolution in Convolvulaceae.

Cuscuta (dodder) is the only parasitic genus found in Convolvulaceae (morning-glory family). We used long PCR approach to obtain large portions of plastid genome sequence from Cuscuta sandwichiana in order to determine the size, structure, gene content, and synteny in the plastid genome of this Cuscuta species belonging to the poorly investigated holoparasitic subgenus Grammica. These new sequences are compared with the tobacco chloroplast genome, and, where data are available, with corresponding regions from taxa in the other Cuscuta subgenera. When all known plastid genome structural rearrangements in parasitic and nonparasitic Convolvulaceae are considered in a molecular phylogenetic framework, three categories of rearrangements in Cuscuta are revealed: plesiomorphic, autapomorphic, and synapomorphic. Many of the changes in Cuscuta, previously attributed to its parasitic mode of life, are better explained either as plesiomorphic conditions within the family, i.e., conditions shared with the rest of the Convolvulaceae, or, in most cases, autapomorphies of particular Cuscuta taxa, not shared with the rest of the species in the genus. The synapomorphic rearrangements are most likely to correlate with the parasitic lifestyle, because they represent changes found in Cuscuta exclusively. However, it appears that most of the affected regions, belonging to all of these three categories, have probably no function (e.g., introns) or are of unknown function (a number of open reading frames, the function of which, if any, has yet to be discovered).

Base Sequence↗

Proteome composition in Plasmodium falciparum: higher usage of GC-rich nonsynonymous codons in highly expressed genes.

The parasite Plasmodium falciparum, responsible for the most deadly form of human malaria, is one of the extremely AT-rich genomes sequenced so far and known to possess many atypical characteristics. Using multivariate statistical approaches, the present study analyzes the amino acid usage pattern in 5038 annotated protein-coding sequences in P. falciparum clone 3D7. The amino acid composition of individual proteins, though dominated by the directional mutational pressure, exhibits wide variation across the proteome. The Asn content, expression level, mean molecular weight, hydropathy, and aromaticity are found to be the major sources of variation in amino acid usage. At all stages of development, frequencies of residues encoded by GC-rich codons such as Gly, Ala, Arg, and Pro increase significantly in the products of the highly expressed genes. Investigation of nucleotide substitution patterns in P. falciparum and other Plasmodium species reveals that the nonsynonymous sites of highly expressed genes are more conserved than those of the lowly expressed ones, though for synonymous sites, the reverse is true. The highly expressed genes are, therefore, expected to be closer to their putative ancestral state in amino acid composition, and a plausible reason for their sequences being GC-rich at nonsynonymous codon positions could be that their ancestral state was less AT-biased. Negative correlation of the expression level of proteins with respective molecular weights supports the notion that P. falciparum, in spite of its intracellular parasitic lifestyle, follows the principle of cost minimization.

Amino Acids↗

Identification and preliminary characterization of cuticular surface proteins of Haemonchus contortus.

Cuticular surface antigens of the XL3 and L4 stages of Haemonchus contortus have been studied by surface labeling and immunological techniques. Live worms were labeled with 125I and extracted with sodium dodecyl sulfate (SDS) followed by SDS + 2-mercaptoethanol. The SDS-soluble surface proteins of XL3s and L4s were found to consist of relatively few major species. The pattern of labeled polypeptides was distinctive for each developmental stage. These proteins are refractory to digestion by bacterial collagenase. Several of the proteins are glycosylated. Further extraction of labeled worms with SDS + 2-mercaptoethanol solubilized additional labeled proteins that appeared to be primarily collagens. Rabbit antisera prepared against native XL3 and L4-cuticles reacted strongly with the surfaces of live worms in immunofluorescence assays. In contrast, antisera prepared against SDS-extracted cuticles reacted weakly or not at all with live worms in similar experiments. Rabbit antisera prepared against adult cuticles failed to react with live XL3s or L4s. These studies suggest that the major surface antigens of XL3s and L4s are solubilized by SDS and that there are different antigens present on the cuticular surfaces of XL3s, L4s and adults. Stage-specificity in cuticular surface proteins may contribute to the successful parasitic lifestyle of this nematode.

Animals↗

Characterization of RP 333, a gene encoding CapD of Rickettsia prowazekii with UDP-glucose 4-epimerase activity.

The genetic mechanism of membrane assembly in Rickettsia prowazekii, the causative agent of epidemic typhus, is unknown. Recent progress in genomics of this small intracellular bacterium prompted us to explore membrane biogenesis as it pertains to identification and characterization of gene products that are of importance in polysaccharide biosynthesis. We chose to define the function of the R. prowazekii capD gene (RP 333) product designated as a putative epimerase. We detected a capD transcript in yolk sacs infected with R. prowazekii at ten days post-infection. The capD gene was PCR amplified and cloned into the entry vector pENTR/SD/D-TOPO and transferred to a low copy arabinose-inducible pBAD/Myc-HisA expression plasmid constructed in our laboratory. The corresponding recombinant protein (CapD) was expressed in PL2, a galE28 mutant of Escherichia coli, and has been shown to epimerize UDP-galactose to UDP-glucose without the addition of NAD+. CapD characterization is important for defense strategies targeted towards design of an R. prowazekii vaccine, generation of new antibiotics, and opening the way for research in membrane biogenesis with respect to the parasitic lifestyle of Rickettsia.

Base Sequence↗

Gene expression profiles associated with the transition to parasitism in Ancylostoma caninum larvae.

Ancylostoma caninum is a common canine parasite responsible for anemia and death in infected dogs. Gene expression profiling was used to investigate molecular differences between two different forms of the third larval stage (L3s): infective free-living larvae and in vitro serum-stimulated larvae that mimic the initial stages of parasitism of a host. We developed an A. caninum cDNA microarray consisting of 4191 EST clones, and used it to identify a set of 113 genes that are differentially regulated between infective and parasitic larval stages. Real-time RT-PCR was used to confirm the expression differences of a subset of the genes. Of the genes repressed upon serum stimulation, seven encode members of the 'Ancylostoma secreted protein' ASP family, while another transcript encoding a 24 kDa excretory protein with similarity to ASP was up-regulated in serum-stimulated L3s. This suggests that different members of a protein family that has important implications for the hookworm's parasitic lifestyle are regulated in a complementary manner in response to serum stimulation. Comparison of two strains of A. caninum from North Carolina and Maryland only identified a single gene, one of the members of the ASP family, that was differentially repressed upon serum stimulation.

Ancylostoma↗

Phylogeny of Arthropoda inferred from mitochondrial sequences: strategies for limiting the misleading effects of multiple changes in pattern and rates of substitution.

In this study, mitochondrial sequences were used to investigate the relationships among the major lineages of Arthropoda. The data matrix used for the analyses includes 84 taxa and 3918 nucleotides representing six mitochondrial protein-coding genes (atp6 and 8, cox1-3, and nad2). The analyses of nucleotide composition show that a reverse strand-bias, i.e., characterized by an excess of T relative to A nucleotides and of G relative to C nucleotides, was independently acquired in six different lineages of Arthropoda: (1) the honeybee mite (Varroa), (2) Opisthothelae spiders (Argiope, Habronattus, and Ornithoctonus), (3) scorpions (Euscorpius and Mesobuthus), (4) Hutchinsoniella (Cephalocarid), (5) Tigriopus (Copepod), and (6) whiteflies (Aleurodicus and Trialeurodes). Phylogenetic analyses confirm that these convergences in nucleotide composition can be particularly misleading for tree reconstruction, as unrelated taxa with reverse strand-bias tend to group together in MP, ML, and Bayesian analyses. However, the use of a specific model for minimizing effects of the bias, the "Neutral Transition Exclusion" (NTE) model, allows Bayesian analyses to rediscover most of the higher taxa of Arthropoda. Furthermore, the analyses of branch lengths suggest that three main factors explain accelerated rates of substitution: (1) genomic rearrangements, including duplication of the control region and gene translocation, (2) parasitic lifestyle, and (3) small body size. The comparisons of Bayesian Bootstrap percentages show that the support for many nodes increases when taxa with long branches are excluded from the analyses. It is therefore recommended to select taxa and genes of the mitochondrial genome for inferring phylogenetic relationships among arthropod lineages. The phylogenetic analyses support the existence of a major dichotomy within Arthropoda, separating Pancrustacea and Paradoxopoda. Basal relationships between Pancrustacean lineages are not robust, and the question of Hexapod monophyly or polyphyly cannot be answered with the available mitochondrial sequences. Within Paradoxopoda, Chelicerata and Myriapoda are each found to be monophyletic, and Endeis (Pycnogonida) is, surprisingly, associated with Acari.

Animals↗

Nutritional and respiratory pathways to parasitism exemplified in the Turbellaria.

Symbiosis is a dominant trait in the Platyhelminthes. The Neodermata (Aspidogastrea, Monogenea, Digenea, Udonellidea, Cestoda) are wholly parasitic and even the predominantly free-living Turbellaria have almost 200 species from 35 families living in permanent associations with other animals. In the simplest turbellarian symbioses, ectosymbiotes such as the Temnocephalida, some other Rhabdocoela and a few Tricladida live on the body surfaces or in the branchial chambers of their mainly arthropodan or chelonian hosts. They feed on the same types of prey as their free-living relatives but supplement their diet by opportunistic commensalism. Their digestive physiology and food reserves are the same as in free-living species. The entosymbiotic Umagillidae, Graffillidae, Pterastericolidae, Fecamplidae and Acholadidae live in internal body cavities or body wall derivatives of echinoderms, molluscs or arthropods and show increasing metabolic dependence on their hosts. Patterns of digestive physiology and food storage generally differ markedly from those of ectosymbiotic and free-living species. Some umagillids, in echinoids, feed as entozoic predators on co-symbiotic protozoa, supplemented by opportunistic ingestion of the hosts' ingesta, gut cells or coelomocytes. Others, in holothurians, feed mainly on gut cells, which also provide some digestive enzymes, and to a lesser extent on host ingesta and co-symbiotes. Graffillids, in molluscs, lack endogenous digestive enzymes and rely entirely on those taken in with host ingesta and gut tissues. Pterastericolids, in asteroids, similarly utilise gut tissues both as food and enzyme sources. The climax to metabolic dependence occurs in the Fecamplidae and Acholadidae. The former, in crustacean haemocoels and myzostomid tissues, lack conventional alimentary systems and absorb soluble nutrients through the epidermis. In the latter the only known species lives in the tube feet of its asteroid host, lacks a normal endodermal gut, but has a modified epidermis performing both digestive and absorptive functions. Most of these entosymbiotes show a shift from the lipid storage characteristic of free-living and ectosymbiotic species to the glycogen storage predominating in the Neodermata. In both groups this emphasis on carbohydrate metabolism is often independent of the PO2 of their environment. Both groups also show high fecundity and it is suggested that there is a direct relationship between this and glycogen storage. High fecundity, while clearly of adaptive value in entosymbiotes, is arguably primarily related to the assured food supply conferred by the entosymbiotic habit and thus can be viewed as a consequence of the latter rather than a prerequisite for it. Some entosymbiotic Turbellaria have evolved physiologically active haemoglobins, allowing them to abstract oxygen preferentially from host tissues; some have also evolved facultative glycolytic mechanisms comparable to those of the Cestoda. All these adaptations to ecto- and entosymbiotic life in the Turbellaria exemplify possible pathways to wholly parasitic lifestyles, with total metabolic dependence on the hosts, which may have been followed during the evolution of the Neodermata.

Animal Nutritional Physiological Phenomena↗

A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites.

Extracts of ectoparasitic mites of birds (Dermanyssus gallinae), sheep (Psoroptes ovis) and plants (Tetranychus urticae) and of free-living mites (Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar profiles and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only beta-galactosidase and beta-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25-33.5kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit.

Animals↗

Inferring protein function from genomic sequence: Giardia lamblia expresses a phosphatidylinositol kinase-related kinase similar to yeast and mammalian TOR.

Functional assays of genes have historically led to insights about the activities of a protein or protein cascade. However, the rapid expansion of genomic and proteomic information for a variety of diverse taxa is an alternative and powerful means of predicting function by comparing the enzymes and metabolic pathways used by different organisms. As part of the Giardia lamblia genome sequencing project, we routinely survey the complement of predicted proteins and compare those found in this putatively early diverging eukaryote with those of prokaryotes and more recently evolved eukaryotic lineages. Such comparisons reveal the minimal composition of conserved metabolic pathways, suggest which proteins may have been acquired by lateral transfer, and, by their absence, hint at functions lost in the transition from a free-living to a parasitic lifestyle. Here, we describe the use of bioinformatic approaches to investigate the complement and conservation of proteins in Giardia involved in the regulation of translation. We compare an FK506 binding protein homologue and phosphatidylinositol kinase-related kinase present in Giardia to those found in other eukaryotes for which complete genomic sequence data are available. Our investigation of the Giardia genome suggests that PIK-related kinases are of ancient origin and are highly conserved.

Amino Acid Sequence↗

Nucleic acid structure and intracellular immunity: some recent ideas from the world of RNAi.

Cells face a constant struggle against unwanted instructions that arrive in the form of viruses and transposons. At the core of this battle are two issues: how can cellular machinery recognize certain informational molecules as 'unwanted' and how can the cell use this recognition to effectively silence malicious genetic activity. While defenses against some specific parasites may be triggered by individual nucleic acid or protein sequences, such sequence-specific mechanisms have the limitation of allowing the parasite to evade following relatively minor evolutionary change. A more general set of defense mechanisms is based on recognition of structural features that are intrinsic aspects of one or more parasitic lifestyle. Recognition of extended regions of double-stranded RNA (dsRNA) provides cells with one such defensive modality. Essentially absent during 'normal' gene expression, long stretches of dsRNA within a cell serve as a dramatic warning that a segment of information may be replicating as RNA. In addition to exemplifying many of the mechanistic issues in genome defense, the cellular response to dsRNA provides several examples of the logic by which organisms attempt to focus their limited immunity resources on the most immediate and dangerous targets.

Biophysical Phenomena↗

Peptidoglycan structure of Salmonella typhimurium growing within cultured mammalian cells.

The cell wall structure of Salmonella typhimurium has been studied for the first time during transit from free-living to parasitic lifestyles. Peptidoglycan of S. typhimurium proliferating within human epithelial cells contains a high proportion of previously unidentified muropeptides (5-10-fold higher than in extracellular bacteria). Amino acid and mass-spectrometry analyses showed that these new components consist of dimeric cross-linked muropeptides lacking one of the two disaccharide (N-acetyl-glucosamine-beta-(1-->4)-N-acetyl-muramic acid) molecules. This unique structure suggests an active role for an N-acetyl-muramyl-L-alanine-amidase in remodelling the peptidoglycan of intracellular S. typhimurium. Additional alterations observed included: (i) the absence of glycine-containing muropeptides; (ii) the increase in the relative proportion of muropeptides cross-linked by L(meso)-diaminopimelyl-D(meso)-diaminopimelic acid (L-D) peptide bridges; and, (iii) the decrease in the global cross-linkage of the macromolecule. The structural alterations observed in the peptidoglycan of intracellular bacteria do not produce loss of the cell envelope. These results show that intracellular residence of S. typhimurium within epithelial cells is accompanied by significant changes in the bacterial cell wall. Remodelling of peptidoglycan structure may constitute another sophisticated strategy of this pathogen for adapting to and colonizing the intracellular niche of eukaryotic cells.

Carbohydrate Conformation↗

Trichinellosis: now and forevermore?

The re-emergence of the zoonotic importance of trichinellosis reminds us that this parasite's unique biological features serve it well in helping it to persist in the face of 150 years of efforts to eliminate it from the food chain. Because of Trichinella's re-emergence as an important public health risk, and the consequent stimulation of research, our knowledge of the parasite has grown, especially on the complexity of its biology and epidemiology. This overview will highlight the major features of this parasite's adaptive traits and the human factors that account for its re-emergence in many regions, along with the actions of the International Trichinellosis Commission to address these issues. The attractiveness of Trichinella as a research subject for study of the parasitic lifestyle will also be emphasized, including areas for future research.

Animals↗

Targeted gene disruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen.

Unidirectional transfer of nutrients from plant host to pathogen represents a most revealing aspect of the parasitic lifestyle of plant pathogens. Whereas much effort has been focused on sugars and amino acids, the identification of other significant metabolites is equally important for comprehensive characterization of metabolic interactions between plants and biotrophic fungal pathogens. Employing a strategy of targeted gene disruption, we generated a mutant strain (gpdhDelta) defective in glycerol-3-phosphate dehydrogenase in a hemibiotrophic plant pathogen, Colletotrichum gloeosporioides f.sp. malvae. The gpdhDelta strain had severe defects in carbon utilization as it could use neither glucose nor amino acids for sustained growth. Although the mutant mycelia were able to grow on potato dextrose agar medium, they displayed arrhythmicity in growth and failure to conidiate. The metabolic defect of gpdhDelta could be entirely ameliorated by glycerol in chemically defined minimal medium. Furthermore, glycerol was the one and only metabolite that could restore rhythmic growth and conidiation of gpdhDelta. Despite the profound defects in carbon source utilization, in planta the gpdhDelta strain exhibited normal pathogenicity, proceeded normally in its life cycle, and produced abundant conidia. Analysis of plant tissues at the peripheral zone of fungal infection sites revealed a time-dependent reduction in glycerol content. This study provides strong evidence for a role of glycerol as a significant transferred metabolite from plant to fungal pathogen.

Althaea↗

Nitric oxide and Mycobacterium leprae pathogenicity.

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.

Mycobacterium leprae↗

Cell division gene cluster in Spiroplasma kunkelii: functional characterization of ftsZ and the first report of ftsA in mollicutes.

Spiroplasma kunkelii is a helical, wall-less bacterium that causes corn stunt disease. In adaptation to its phloem-inhabiting parasitic lifestyle, the bacterium has undergone a reductive evolutionary process and, as a result, possesses a compact genome with a gene set approaching the minimal complement necessary for multiplication and pathogenesis. We cloned a much-reduced cell division gene cluster from S. kunkelii and functionally characterized the key division gene, ftsZ(sk). The 1236-bp open reading frame of ftsZ(sk) is capable of encoding a protein with a calculated molecular mass of 44.1 kDa. Protein sequence alignment revealed that FtsZ(sk) is remarkably similar to FtsZ proteins from other eubacteria, and possesses the conserved GTP-binding and hydrolyzing motifs. We demonstrated that overexpression of ftsZ(sk) in Escherichia coli causes transgression of the host cell division, resulting in a filamentous phenotype. We also report, for the first time, the presence of a ftsA gene in the cell division cluster of a mollicute species.

Amino Acid Sequence↗