Infection by the castrating parasitic nematode Sphaerularia bombi changes gene expression in Bombus terrestris bumblebee queens.

Parasitism can result in dramatic changes in host phenotype, which are themselves underpinned by genes and their expression. Understanding how hosts respond at the molecular level to parasites can therefore reveal the molecular architecture of an altered host phenotype. The entomoparasitic nematode Sphaerularia bombi is a parasite of bumblebee (Bombus) hosts where it induces complex behavioural changes and host castration. To examine this interaction at the molecular level, we performed genome-wide transcriptional profiling using RNA-Sequencing (RNA-Seq) of S. bombi-infected Bombus terrestris queens at two critical time-points: during and just after overwintering diapause. We found that infection by S. bombi affects the transcription of genes underlying host biological processes associated with energy usage, translation, and circadian rhythm. We also found that the parasite affects the expression of immune genes, including members of the Toll signalling pathway providing evidence for a novel interaction between the parasite and the host immune response. Taken together, our results identify host biological processes and genes affected by an entomoparasitic nematode providing the first steps towards a molecular understanding of this ecologically important host-parasite interaction.

Functional insights into the infective larval stage of Anisakis simplex s.s., Anisakis pegreffii and their hybrids based on gene expression patterns.

BACKGROUND: Anisakis simplex sensu stricto and Anisakis pegreffii are sibling species of nematodes parasitic on marine mammals. Zoonotic human infection with third stage infective larvae causes anisakiasis, a debilitating and potentially fatal disease. These 2 species show evidence of hybridisation in geographical areas where they are sympatric. How the species and their hybrids differ is still poorly understood. RESULTS: Third stage larvae of Anisakis simplex s.s., Anisakis pegreffii and hybrids were sampled from Merluccius merluccius (Teleosti) hosts captured in waters of the FAO 27 geographical area. Specimens of each species and hybrids were distinguished with a diagnostic genetic marker (ITS). RNA was extracted from pools of 10 individuals of each taxon. Transcriptomes were generated using Illumina RNA-Seq, and assembled de novo. A joint assembly (here called merged transcriptome) of all 3 samples was also generated. The inferred transcript sets were functionally annotated and compared globally and also on subsets of secreted proteins and putative allergen families. While intermediary metabolism appeared to be typical for nematodes in the 3 evaluated taxa, their transcriptomes present strong levels of differential expression and enrichment, mainly of transcripts related to metabolic pathways and gene ontologies associated to energy metabolism and other pathways, with significant presence of excreted/secreted proteins, most of them allergens. The allergome of the 2 species and their hybrids has also been thoroughly studied; at least 74 different allergen families were identified in the transcriptomes. CONCLUSIONS: A. simplex s.s., A. pegreffi and their hybrids differ in gene expression patterns in the L3 stage. Strong parent-of-origin effects were observed: A. pegreffi alleles dominate in the expression patterns of hybrids albeit the latter, and A. pegreffii also display significant differences indicating that hybrids are intermediate biological entities among their parental species, and thus of outstanding interest in the study of speciation in nematodes. Analyses of differential expression based on genes coding for secreted proteins suggests that co-infections presents different repertoires of released protein to the host environment. Both species and their hybrids, share more allergen genes than previously thought and are likely to induce overlapping disease responses.

Genomics and transcriptomics across the diversity of the Nematoda.

The diversity of biology in nematodes is reflected in the diversity of their genomes. Parasitic species in particular have evolved mechanisms to invade and outwit their hosts, and these offer opportunities for the development of control measures. Genomic analyses can reveal the molecular underpinnings of phenotypes such as parasitism and thus, initiate and support research programmes that explore the manipulation of host and parasite physiologies to achieve favourable outcomes. Wide sampling across nematode diversity allows phylogenetically informed formulation of research hypotheses, identification of core features shared by all species or important evolutionary novelties present in isolated clades. Many nematode species have been investigated through the use of the expressed sequence tag approach, which samples from the transcribed genome. Gene catalogues generated in this way can be explored to reveal the patterns of expression associated with parasitism and candidates for testing as drug targets or vaccine components. Analysis environments, such as NEMBASE facilitate exploitation of these data. The development of new high-throughput DNA-sequencing technologies has facilitated transcriptomic and genomic approaches to parasite biology. Whole genome sequencing offers more complete catalogues of genes and assists a systems approach to phenotype dissection. These efforts are being coordinated through the 959 Nematode Genomes initiative.

Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises.

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well-acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)-based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

Caenorhabditis elegans is a nematode.

Caenorhabditis elegans is a rhabditid nematode. What relevance does this have for the interpretation of the complete genome sequence, and how will it affect the exploitation of the sequence for scientific and social ends? Nematodes are only distantly related to humans and other animal groups; will this limit the universality of the C. elegans story? Many nematodes are parasites; can knowledge of the C. elegans sequence aid in the prevention and treatment of disease?

Duplication and divergence: the evolution of nematode globins.

In common with many other groups, nematodes express globins with unknown functions. Nematode globin-like genes can be divided into class 1 globins, similar to vertebrate myoglobins, and a wide range of additional classes. Here we show that class 1 nematode globins possess a huge amount of diversity in gene sequence and structure. There is evidence for multiple events of gene duplication, intron insertion and loss between species, and for allelic variation effecting both synonymous and non-synonymous sites within species. We have also examined gene expression patterns in class I globins from a variety of species. The results show variation in the degree of gene expression, but the tissue specificity and temporal specificity of expression may be more conserved in the phylum. Because the structure-function relationships for the binding and transport of oxygen by globins are well understood, the consequences of genetic variation causing amino acid changes are explored. The gene family shows great promise for discovering unique insights into both structure-function relationships of globins and their physiologial roles.

Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility.

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

Alimentary fibropapilloma in cattle: a spontaneous tumor, nonpermissive for papillomavirus replication.

Fibropapillomatosis of the upper alimentary canal of cattle is described. The tumors, found in the esophagus, esophageal groove, and rumen, showed involvement of the subepithelial fibroblasts as well as of the squamous epithelial layer. Although the fibropapilloma cells harbored multiple episomal copies of the genome of bovine papillomavirus type 2 (BPV-2) easily detected by hybridization techniques, no mature virus could be isolated from these lesions or seen by electron microscopy, and no viral antigen could be detected by immunohistochemical methods. It would appear, therefore, that within the limitations of the techniques employed the alimentary canal epithelium and the underlying fibroblasts, while allowing BPV-2 DNA replication, are nonpermissive for the expression of the viral vegetative functions and that transformation of the epithelial cells, like transformation of fibroblasts, can take place in the absence of infectious viral progeny.

Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset.

Analysis of the Haemonchus contortus Expressed sequence tag (EST) dataset revealed that almost 10% of all ESTs (1719 ESTs) belong to a family of related genes. Close analysis of the ESTs suggests that these represent two genes (called here Hc-nim-1 and Hc-nim-2) with multiple alleles of each. These genes show significant similarity to two genes from Caenorhabditis elegans, F54D5.3 (Wormbase accession WBGene00010049, corresponding protein WP:CE28033) and F54D5.4 (WBGene00010050, WP:CE03409) of unknown function. Reverse transcriptase coupled-PCR showed that both genes are transcribed from the L4 stage onwards and are transcribed in both male and female adult worms. A partial bacterial recombinant of the Hc-NIM-1 protein was made and used to raise antiserum in rabbits which recognised a 19 kDa antigen in the water soluble protein fraction of adult worms. By immunohistochemistry, the Hc-NIM-1 protein was localised in the hypodermis of the pharyngeal region of adult worms but not posterior in the hypodermis surrounding the reproductive tract. To investigate the function of this novel protein family we conducted a RNA interference experiment for the homologuous proteins in C. elegans. No visible phenotype was detected after simultaneous RNAi treatment for both Ce-F54D5.3 and Ce-F54D5.4.

Sequence, expression and evolution of the globins of the parasitic nematode Nippostrongylus brasiliensis.

The globins of the nematode parasite Nippostrongylus brasiliensis have oxygen affinities 100-fold higher than the rodent host's haemoglobins. Two isoforms are found, one located in the cuticle, and the other in the body of the nematode. Both isoforms have been cloned and analysed for clues as to function and evolution. The body globin isoform is first expressed upon invasion of the mammalian host. The abundant cuticular globin is expressed only by adult nematodes in the gut, and differs significantly from the body globin. Both globins are found as trans-spliced mRNAs: the developmental pattern of expression of the mRNA parallels the protein expression. The pattern of the nematode globin genes is complex. Comparison with other nematode globin sequences suggests that N. brasiliensis is more closely related to Caenorhabditis elegans than to ascarid species. At least two gene duplication events are predicted: gene duplication preceded the radiation of the important vertebrate-parasitic strongylid nematode species. Both N. brasiliensis globins have a central intron the exact position of which suggests that it arose from an independent insertion event in the strongylid-rhabditid line. The globins have been expressed in Escherichia coli as functional holenzymes as a prelude to studies to elucidate the origin of their extraordinary oxygen affinity.

Specific serodiagnosis of visceral leishmaniasis using a Leishmania donovani antigen identified by expression cloning.

A lambda gt11 expression library was constructed using mRNA from the promastigote form of Leishmania donovani (African isolate MHOM/ET/67/HU3). The library was screened with serum obtained from a patient who contracted visceral leishmaniasis in the Sudan. Several cDNA clones which expressed beta-galactosidase/L. donovani antigen fusion proteins were isolated. One of these clones corresponded to a 60 kDa membrane-associated antigen. By a Western blot assay antibodies against the fusion protein were detected universally in the sera of visceral leishmaniasis patients. They were not detected in sera from patients with cutaneous leishmaniasis or other parasitic protozoan infections. The gene coding for this antigen was specific to the genus Leishmania as judged by DNA hybridisation, and its chromosomal location was conserved. A 20 kb mRNA was detected on Northern blots of promastigote RNA.

Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi.

Isolation and cloning of abundant reverse transcriptase-polymerase chain reaction (RT-PCR) products from the filarial nematode Brugia malayi using the conserved nematode spliced leader sequence and poly A as amplification targets has allowed us to identify abundant, stage specific transcripts from infective and post-infective larvae. The predicted protein products of the most prominent full-length transcripts from mosquito-derived L3 parasites are: (i) Bm-ALT-1, a homologue of a Dirofilaria immitis abundant larval protein: (ii) Bm-CPI-1, a cystatin-type cysteine protease inhibitor; (iii) Bm-ALT-3, a novel predicted 6 kDa glycine/tyrosine-rich protein; and (iv) Bm-TPH-1, a homologue of a mammalian translationally-controlled tumour protein. Some transcripts were not full-length but had mis-primed at A-rich stretches of coding sequence: the most abundant of these was Bm-col-3, a which encodes a collagen homologous to Bp-COL-1 of Brugia pahangi. Similar analysis of abundant spliced leader (SL)/oligo-dT products from fourth-stage larvae 9 days post-infection yielded two dominant transcripts: (i) Bm-cdd-1, which encodes a protein with homology to cytidine deaminase, differing at only one amino acid position from its homologue described in Brugia pahangi; and (ii) the same truncated form of Bm-col-3 found in L3 preparations. Expression of the major transcripts was assessed by PCR amplification of cDNA libraries derived from each stage of the life cycle. alt1, alt-3 and cpi-1 were all found to be specific to the L3 stage, while cdd-1 was found only in the L4 cDNA library. Expression of these larval-specific transcripts was not detected in either microfilarial or adult libraries.

Gene structure, activity and localization of a catalase from intracellular bacteria in Onchocerca volvulus.

Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotide and encoded amino acid sequences revealed that they derive from intracellular bacteria, rather than the O. volvulus nuclear genome. The endobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene encodes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression system. The purified 54 kDa protein has CAT activity over a broad pH range, with a specific activity of 103,000 +/- 3000 U mg(-1). The optical spectrum of the endobacterial CAT shows that it is a ferric haem-containing protein with a Soret band at 405 nm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rRNA gene was amplified from total genomic DNA by a polymerase chain reaction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacterium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species were immunohistochemically stained using polyclonal antibodies raised against the recombinant endobacterial CAT. CAT was detected in the endobacteria in the hypodermis of adult male and female O. volvulus, O. ochengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also detected in onchocercal oocytes and all embryonic stages including intrauterine microfilariae as well as skin microfilariae. O. volvulus thus harbours Wolbachia-like endosymbionts which are transovarially transmitted and show particular affinity for the hypodermal tissues of the lateral chords.

Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus with homologues in other filarial nematodes and Caenorhabditis elegans.

The full-length cDNA corresponding to an Onchocerca volvulus antigen, OvMBP/11, which had been selected as a serodiagnostic tool was isolated, sequenced, and the native antigen encoded by the cDNA characterised. The cDNA encodes a protein of 20.5 kDa (termed Ov 20) containing a putative signal peptide. Southern blot analysis indicates that there is only a single O. volvulus gene corresponding to Ov 20 but it has significant sequence similarity to genes corresponding to two 20.5-kDa predicted proteins of Caenorhabditis elegans. Homologues of the Ov 20 gene were detected at high stringency by Southern blot in the other Onchocerca species O. gibsoni, and O. gutturosa and at lower stringency in the related filarial nematodes Brugia malayi and Acanthocheilonema viteae. The Ov 20 native antigen has two molecular mass forms, 20 and 22 kDa, in all the life cycle stages studied. These isoforms have different levels of N-linked glycosylation on a peptide backbone of 17.5 kDa. Immunolocalisation and in situ hybridisation studies demonstrated that Ov 20 is transcribed and translated in the body wall of adult females and also in microfilariae, third and fourth stage larvae. Antigen was detected in the supernatant of in vitro cultured adult female nematodes. The B. malayi and A. viteae homologues are antigenically cross-reactive to Ov 20, share the same size peptide backbone but differ in their degree of glycosylation.

Genes expressed in Brugia malayi infective third stage larvae.

We have used a tag sequencing approach to survey genes expressed in the third stage infective larvae of the human filarial nematode parasite Brugia malayi. RNA was isolated from late vector-stage L3 larvae after days 9 or 10 of infection in mosquitos, and converted to cDNA by reverse transcriptase. Double-stranded cDNA was produced by either conventional methods (non-SL cDNA library) or by PCR using the nematode spliced leader (SLI) and oligo(dT) primers (SL cDNA library). Two clone libraries (one from SL and one from non-SL cDNAs) were constructed in lambda ZapII. A set of these full-length clones was selected and 596 inserts were sequenced from the 5' end. We have identified 364 B. malayi genes (the majority of which are new) that encode housekeeping proteins, structural proteins, proteins of immediate immunological or drug-discovery interest as well as a large class of novel sequences which may prove to have significant involvement in host invasion. Extensive, genome-wide approaches to the analysis of larval gene expression are now possible for B. malayi. We present several examples of this approach.

Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

Phylogeny of Wolbachia in filarial nematodes.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.

Genes and genomes of Necator americanus and related hookworms.

The human hookworms (Necator americanus and Ancylostoma duodenale) infect over one billion people. The phylogenetic relationships of the human hookworms suggest independent acquisition of the human host. The hookworms probably have a haploid chromosome number n = 6, and an XX-XO sex determination mechanism is likely to be used. Genetic and molecular research on hookworms is in its infancy, but several important genes and gene products have already been identified. Of note are cathepsin genes, a family of secreted proteins known as Ancylostoma activation-associated proteins and a family of anticoagulants. The inception of an expressed sequence tag program on the human hookworm, N. americanus, promises to yield many new genes with novel functions in the biology of these important parasites.

The small subunit ribosomal RNA sequence of Strongyloides stercoralis.

The published small subunit rRNA (ssrRNA) gene sequences for Strongyloides ratti and Strongyloides stercoralis are remarkably divergent, particularly in the 5' 400 bases of the approximately 1700 base pair (bp) sequences. This level of divergence between species nominally in the same genus was unprecedented. We have redetermined the ssrRNA sequence of S. stercoralis and find that the published sequence is a chimaera of parasite and fungal segments. The true sequence for S. stercoralis ssrRNA is very similar to that of S. ratti.