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Myzus persicae is a major pest of many crops including canola and Brassica vegetables, partly because it vectors plant viruses. Previously it has been reported that double-stranded RNA delivered to aphids by injection, artificial diet or transgenic plants has knocked down target genes and caused phenotypic effects. While these studies suggest that RNA interference (RNAi) might be used to suppress aphid populations, none have shown effects sufficient for field control. The current study analyses the efficacy of dsRNA directed against previously reported gene-targets on Green peach aphid (Myzus persicae) strains. No silencing effect was observed when dsRNA was delivered in artificial diet with or without transfection reagents. dsRNA produced in planta also failed to induce significant RNAi in M. persicae. Transcriptome analyses of the midgut suggested other potential targets including the Ferritin heavy chain transcripts, but they also could not be knocked down with dsRNA. Here we show that dsRNA is rapidly degraded by midgut secretions of Myzus persicae. Analysis of the transcriptome of the M. persicae midgut revealed that an ortholog of RNases from other insects was abundant.

Insecticide resistance is a paradigm of microevolution, and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster Here we use a naïve population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome-wide association studies (GWAS) of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase ( Strn-Mlck ), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliprole's mode of action. GWAS also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homolog of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naïve to this new insecticide chemistry and it foreshadows a selective response among natural populations to these chemicals., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Imidacloprid, the world's most used insecticide, has caused considerable controversy due to harmful effects on non-pest species and increasing evidence showing that insecticides have become the primary selective force in many insect species. The genetic response to insecticides is heterogeneous across populations and environments, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, have revealed a mixed genetic architecture to resistance involving major genes (Paramyosin and Nicotinic-Acetylcholine Receptor Alpha 3) and polygenes with a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicated enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations in which chromosomal inversions In(2 L)t, In(3 R)Mo and In(3 R)Payne harbour susceptibility alleles. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.

Over the last 40 years, many types of population genetic markers have been used to assess the population structure of the pest moth species Helicoverpa armigera. While this species is highly vagile, there is evidence of inter-continental population structure. Here, we examine Z-chromosome molecular markers within and between Chinese and Australian populations. Using 1352 polymorphic sites from 40 Z-linked loci, we compared two Chinese populations of moths separated by 700 km and found virtually no population structure (n = 41 and n = 54, with <1% of variation discriminating between populations). The levels of nucleotide diversity within these populations were consistent with previous estimates from introns in Z-linked genes of Australian samples (π = 0.028 vs. 0.03). Furthermore, all loci surveyed in these Chinese populations showed a skew toward rare variants, with ten loci having a significant Tajima's D statistic, suggesting that this species could have undergone a population expansion. Eight of the 40 loci had been examined in a previous study of Australian moths, of which six revealed very little inter-continental population structure. However, the two markers associated with the Cyp303a1 locus that has previously been proposed to be a target of a selective sweep, exhibited allele structuring between countries. Using a separate dataset of 19 Australian and four Chinese moths, we scanned the molecular variation distributed across the entire Z-chromosome and found distinct blocks of differentiation that include the region containing Cyp303a1. We recommend some of these loci join those associated with insecticide resistance to form a set of genes best suited to analyzing population structure in this global pest.

Insecticide resistance is considered a classic model of microevolution, where a strong selective agent is applied to a large natural population, resulting in a change in frequency of alleles that confer resistance. While many insecticide resistance variants have been characterized at the gene level, they are typically single genes of large effect identified in highly resistant pest species. In contrast, multiple variants have been implicated in DDT resistance in Drosophila melanogaster ; however, only the Cyp6g1 locus has previously been shown to be relevant to field populations. Here we use genome-wide association studies (GWAS) to identify DDT-associated polygenes and use selective sweep analyses to assess their adaptive significance. We identify and verify two candidate DDT resistance loci. A largely uncharacterized gene, CG10737 , has a function in muscles that ameliorates the effects of DDT, while a putative detoxifying P450, Cyp6w1 , shows compelling evidence of positive selection., (Copyright © 2017 by the Genetics Society of America.)

Background: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests., Results: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes., Conclusions: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Insecticide resistance evolves extremely rapidly, providing an illuminating model for the study of adaptation. With climate change reshaping species distribution, pest and disease vector control needs rethinking to include the effects of environmental variation and insect stress physiology. Here, we assessed how both long-term adaptation of populations to temperature and immediate temperature variation affect the genetic architecture of DDT insecticide response in Drosophila melanogaster. Mortality assays and behavioural assays based on continuous activity monitoring were used to assess the interaction between DDT and temperature on three field-derived populations from climate extremes (Raleigh for warm temperate, Tasmania for cold oceanic and Queensland for hot tropical). The Raleigh population showed the highest mortality to DDT, whereas the Queensland population, epicentre for derived alleles of the resistance gene Cyp6g1, showed the lowest. Interaction between insecticide and temperature strongly affected mortality, particularly for the Tasmanian population. Activity profiles analysed using self-organizing maps show that the insecticide promoted an early response, whereas elevated temperature promoted a later response. These distinctive early or later activity phases revealed similar responses to temperature and DDT dose alone but with more or less genetic variance depending on the population. This change in genetic variance among populations suggests that selection particularly depleted genetic variance for DDT response in the Queensland population. Finally, despite similar (co)variation between traits in benign conditions, the genetic responses across population differed under stressful conditions. This showed how stress-responsive genetic variation only reveals itself in specific conditions and thereby escapes potential trade-offs in benign environments., (© 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.)

Motivation: RNA interference (RNAi) technology is being developed as a weapon for pest insect control. To maximize the specificity that such an approach affords we have developed a bioinformatic web tool that searches the ever-growing arthropod transcriptome databases so that pest-specific RNAi sequences can be identified. This will help technology developers finesse the design of RNAi sequences and suggests which non-target species should be assessed in the risk assessment process., Availability and Implementation: http://rnai.specifly.org, Contact: crobin@unimelb.edu.au., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of 12 Drosophila species by examining the congruence of gene trees and species trees. Although the number of P450 genes varies from 74 to 94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes-with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster, we observe gene copy number polymorphism in ten P450 genes including multiple cases of interparalog chimeras. Nonallelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically, we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. Although we observe such interparalog exchange in our within-species data sets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala(301) to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala(301) to Ser resistance mutation and Met(360) to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser(301) homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser(301) change into an Ala(301) background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

A suite of polymorphic microsatellite markers and the complete mitochondrial genome sequence was developed by next generation sequencing (NGS) for the critically endangered orange-bellied parrot, Neophema chrysogaster. A total of 14 polymorphic loci were identified and characterized using DNA extractions representing 40 individuals from Melaleuca, Tasmania, sampled in 2002. We observed moderate genetic variation across most loci (mean number of alleles per locus = 2.79; mean expected heterozygosity = 0.53) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. De novo and reference-based genome assemblies performed using MIRA were used to assemble the N. chrysogaster mitochondrial genome sequence with mean coverage of 116-fold (range 89 to 142-fold). The mitochondrial genome consists of 18,034 base pairs, and a typical metazoan mitochondrial gene content consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a single large non-coding region (control region). The arrangement of mitochondrial genes is also typical of Avian taxa. The annotation of the mitochondrial genome and the characterization of 14 microsatellite markers provide a valuable resource for future genetic monitoring of wild and captive N. chrysogaster populations. As found previously, NGS provides a rapid, low cost and reliable method for polymorphic nuclear genetic marker development and determining complete mitochondrial genome sequences when only a fraction of a genome is sequenced.

Genetic polymorphisms underlying adaptive shifts in thermal responses are poorly known even though studies are providing a detailed understanding of these responses at the cellular and physiological levels. The Frost gene of Drosophila melanogaster is a prime candidate for thermal adaptation; it is up-regulated under cold stress and knockdown of this gene influences cold resistance. Here we describe an amino-acid INDEL polymorphism in proline repeat number in the structural component of this gene. The two main repeats, accounting for more than 90% of alleles in eastern Australia, show a strong clinal pattern; the 6P allele was at a high frequency in tropical locations, and the 10P allele was common in temperate populations. However, the frequency of these alleles was not associated with three different assays of cold resistance. Adult transcription level of Frost was also unrelated to cold resistance as measured through post chill coma mobility. The functional significance of the proline repeat polymorphism therefore remains unclear despite its clinal pattern. The data also demonstrate the feasibility of using Roche/454 sequencing for establishing clinal patterns., (© 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.)

Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa., (© 2012 Blackwell Publishing Ltd.)

Background: The Australian sheep blowfly Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) is a destructive pest of the sheep, a model organism for insecticide resistance research, and a valuable tool for medical and forensic professionals. However, genomic information on L. cuprina is still sparse., Results: We report here the construction of an embryonic and 2 larval cDNA libraries for L. cuprina. A total of 29,816 expressed sequence tags (ESTs) were obtained and assembled into 7,464 unique clusters. The sequence collection captures a great diversity of genes, including those related to insecticide resistance (e.g., 12 cytochrome P450s, 2 glutathione S transferases, and 6 esterases). Compared to Drosophila melanogaster, codon preference is different in 13 of the 18 amino acids encoded by redundant codons, reflecting the lower overall GC content in L. cuprina. In addition, we demonstrated that the ESTs could be converted into informative gene markers by capitalizing on the known gene structures in the model organism D. melanogaster. We successfully assigned 41 genes to their respective chromosomes in L. cuprina. The relative locations of these loci revealed high but incomplete chromosomal synteny between L. cuprina and D. melanogaster., Conclusions: Our results represent the first major transcriptomic undertaking in L. cuprina. These new genetic resources could be useful for the blowfly and insect research community.

Latitudinal body size clines in animals conforming to Bergmann's rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for approximately 16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession., Competing Interests: The authors have declared that no competing interests exist.

Serine repeat antigens (SERAs) are a family of secreted "cysteine-like" proteases of Plasmodium parasites. Several SERAs possess an atypical active-site serine residue in place of the canonical cysteine. The human malaria parasite Plasmodium falciparum possesses six "serine-type" (SERA1 to SERA5 and SERA9) and three "cysteine-type" (SERA6 to SERA8) SERAs. Here, we investigate the importance of the serine-type SERAs to blood-stage parasite development and examine the extent of functional redundancy among this group. We attempted to knock out the four P. falciparum serine-type SERA genes that have not been disrupted previously. SERA1, SERA4, and SERA9 knockout lines were generated, while only SERA5, the most strongly expressed member of the SERA family, remained refractory to genetic deletion. Interestingly, we discovered that while SERA4-null parasites completed the blood-stage cycle normally, they exhibited a twofold increase in the level of SERA5 mRNA. The inability to disrupt SERA5 and the apparent compensatory increase in SERA5 expression in response to the deletion of SERA4 provides evidence for an important blood-stage function for the serine-type SERAs and supports the notion of functional redundancy among this group. Such redundancy is consistent with our phylogenetic analysis, which reveals a monophyletic grouping of the serine-type SERAs across the genus Plasmodium and a predominance of postspeciation expansion. While SERA5 is to some extent further validated as a target for vaccine and drug development, our data suggest that the expression level of other serine-type SERAs is the only barrier to escape from anti-SERA5-specific interventions.

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.

This study is motivated by two data sets which employ a custom Plasmodium falciparum version of the Affymetrix GeneChip, containing only perfect match (PM) oligonucleotides. A PM-only chip cannot be analysed using the standard Affymetrix-supplied software. We compared the performance of three match-only algorithms on these data: the Match Only Integral Distribution (MOID) algorithm, Robust Multichip Analysis (RMA), and the Model Based Expression Index (MBEI). We validated the differential expression of several genes using quantitative reverse transcriptase-PCR. We also performed a comparison using two publicly available 'benchmarking' data sets: the Latin Square spike-in data set generated by Affymetrix, and the Gene Logic dilution series. Since we know what the true fold changes are in these special data sets, they are helpful for assessment of expression algorithms.

The malaria parasite Plasmodium falciparum undergoes antigenic variation to evade host immune responses through switching expression of variant surface proteins encoded by the var gene family. We demonstrate that both a subtelomeric transgene and var genes are subject to reversible gene silencing. Var gene silencing involves the SIR complex as gene disruption of PfSIR2 results in activation of this gene family. We also demonstrate that perinuclear gene activation involves chromatin alterations and repositioning into a location that may be permissive for transcription. Together, this implies that locus repositioning and heterochromatic silencing play important roles in the epigenetic regulation of virulence genes in P. falciparum.

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.

To establish infection in the host, malaria parasites export remodeling and virulence proteins into the erythrocyte. These proteins can traverse a series of membranes, including the parasite membrane, the parasitophorous vacuole membrane, and the erythrocyte membrane. We show that a conserved pentameric sequence plays a central role in protein export into the host cell and predict the exported proteome in Plasmodium falciparum. We identified 400 putative erythrocyte-targeted proteins corresponding to approximately 8% of all predicted genes, with 225 virulence proteins and a further 160 proteins likely to be involved in remodeling of the host erythrocyte. The conservation of this signal across Plasmodium species has implications for the development of new antimalarials.

The apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify P. falciparum. In this paper, we present a novel series of P. falciparum transfection vectors based around the Gateway recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other P. falciparum proteins.

The primary pathophysiological events contributing to fatal malaria are the cerebral syndrome, anemia, and lactic acidosis. The molecular basis of each event has been unclear. In the present study, microarray analysis of murine transcriptional responses during the development of severe disease revealed temporal, organ-specific, and pathway-specific patterns. More than 400 genes in the brain and 600 genes in the spleen displayed transcriptional changes. Dominant patterns revealed strongly suppressed erythropoiesis, starting early during infection, and highly up-regulated transcription of genes that control host glycolysis, including lactate dehydrogenase. The latter presents a mechanism that may contribute to metabolic acidosis. No evidence for hypoxia-mediated regulation of these events was observed. Interferon-regulated gene transcripts dominated the inflammatory response to cytokines. These results demonstrate previously unknown transcriptional changes in the host that may underlie the development of malarial syndromes, such as anemia and metabolic dysregulation, and increase the utility of murine models in investigation of basic malarial pathogenesis.

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

The recognition and invasion of human erythrocytes by the most lethal malaria parasite Plasmodium falciparum is dependent on multiple ligand-receptor interactions. Members of the erythrocyte binding-like (ebl) family, including the erythrocyte binding antigen-175 (EBA-175), are responsible for high affinity binding to glycoproteins on the surface of the erythrocyte. Here we describe a paralogue of EBA-175 and show that this protein (EBA-181/JESEBL) binds in a sialic acid-dependent manner to erythrocytes. EBA-181 is expressed at the same time as EBA-175 and co-localizes with this protein in the microneme organelles of asexual stage parasites. The receptor binding specificity of EBA-181 to erythrocytes differs from other members of the ebl family and is trypsin-resistant and chymotrypsin-sensitive. Furthermore, using glycophorin B-deficient erythrocytes we show that binding of EBA-181 is not dependent on this sialoglycoprotein. The level of expression of EBA-181 differs among parasite lines, and the importance of this ligand for invasion appears to be strain-dependent as the EBA-181 gene can be disrupted in W2mef parasites, without affecting the invasion phenotype, but cannot be targeted in 3D7 parasites.

The Plasmodium falciparum serine repeat antigen (SERA) has shown considerable promise as a blood stage vaccine for the control of malaria. A related protein, SERPH, has also been described in P. falciparum. Whereas their biological role remains unknown, both proteins possess papain-like protease domains that may provide attractive targets for therapeutic intervention. Genomic sequencing has recently shown that SERA and SERPH are the fifth and sixth genes, respectively, in a cluster of eight SERA homologues present on chromosome 2. In this paper, the expression and functional relevance of these eight genes and of a ninth SERA homologue found on chromosome 9 were examined in blood stage parasites. Using reverse transcriptase-PCR and microarray approaches, we demonstrate that whereas mRNA to all nine SERA genes is synthesized late in the erythrocytic cycle, it is those genes in the central region of the chromosome 2 cluster that are substantially up-regulated at this time. Using antibodies specific to each SERA, it was apparent that SERA4 to -6, and possibly also SERA9, are synthesized in blood stage parasites. The reactivity of antibodies from malaria-immune individuals with the SERA recombinant proteins suggested that SERA2 and SERA3 are also expressed at least in some parasite populations. To examine whether SERA genes are essential to blood stage growth, each of the eight chromosome 2 SERA genes was targeted for disruption. Whereas genes at the periphery of the cluster were mostly dispensable (SERA2 and -3 and SERA7 and -8), those in the central region (SERA4 to -6) could not be disrupted. The inability to disrupt SERA4, -5, and -6 is consistent with their apparent dominant expression and implies an important role for these genes in maintenance of the erythrocytic cycle.

Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class.

Analysis of nucleic acid polymorphism in the flagellin genes of Campylobacter jejuni was used to investigate genetic diversity among Campylobacter spp. in a commercial broiler flock. Three hundred single colonies of C. jejuni were isolated from fecal samples collected weekly for 3 weeks immediately before slaughter. Both the flaA and flaB genes were amplified by PCR, and the PCR product was digested with the restriction enzyme AluI. The fragments generated were then analyzed by agarose gel electrophoresis. Among the 300 recovered isolates, five different restriction fragment length polymorphism profiles were observed. Three of these profiles were dominant during the course of the study, and the other two profiles were detected at low frequency. Analysis of genetic variation in C. jejuni over the course of an experimental infection lasting 7 weeks indicated that there was no obvious drift in the flagellin gene type. These findings demonstrate that a range of bacterial genotypes can constitute the bacterial population within a commercial poultry flock, with the most likely sources of these types being multiple environmental exposure and/or genetic drift within the population. This degree of diversity must be considered in epidemiological analyses which utilize genetic typing methods that investigate Campylobacter contamination of any food source, including poultry, to ensure that the total gene pool for C. jejuni is evaluated.

The molecular cloning and sequence analysis of four structurally variant linked genes (omp1A,B,C,D) that encode the major outer membrane protein of Dichelobacter nodosus strain VCS1001 are described. The isolation of rearranged copies of omp1A and omp1B, and the identification in the 5' regions of all four genes of short cross-over-site sequences that were similar to the Din family of cross-over-site sequences, suggested that site-specific DNA inversion was involved in omp1 rearrangement. Evidence for site-specific inversion of the 497 bp DNA fragment, which was located between the divergently orientated omp1A and omp1B genes, and which contained the promoter and 5' coding sequence of Omp1, was obtained by polymerase chain reaction-mediated amplification of inverted forms of these genes. However, to account for all of the omp1 gene copies cloned in this study, a more widespread inversion phenomenon must be involved in the rearrangement of these genes and a model for multiple site-specific DNA inversions at the omp1 locus is described. In this model the four structurally variant omp1 genes can be assembled from one of four structurally variant C-terminal coding regions and a conserved N-terminal coding region and can be expressed from a single promoter. It is postulated that this genetic capability endows D. nodosus with the ability to switch the antigenic specificity of one of its major surface proteins.

PCR-based diagnostic tests using oligonucleotides specific to 16S rRNA were designed for the specific detection of the turkey pathogens Mycoplasma meleagridis and M. iowae. This method of detection was shown to be rapid, species specific, and unaffected by strain variation or the presence of other organisms. Detection of M. meleagridis in clinical samples by PCR was achieved and later confirmed by culture and growth inhibition. Definitive identification by culture and growth inhibition required up to 3 weeks, whereas positive results from PCR testing were obtained within a day and negative samples were confirmed within 4 days.

Using 3H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as "alkaline" ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.

Ribonuclease activity at pH 7.1 ("alkaline" ribonuclease) was determined in homogenates of rat superior cervical ganglion up to 5 days after postganglionic nerve injury under optimal conditions of assay. Measurements were performed in the presence and absence of the sulfhydryl blocking agent, N-ethylmaleimide, to assess the proportion of "alkaline" ribonuclease apparently bound to endogenous inhibitor. Total ribonuclease activity per ganglion was stimulated 1.3 fold by 1 day after injury and remained elevated over the 5 day period. Free ribonuclease activity accounted for about 60% of the observed increase in total activity at day 1, but had returned to control level by day 3. At day 3 the entire 90% increase in total activity was attributable to ribonuclease bound to endogenous inhibitor (i.e. latent activity). These changes are occurring at times after nerve injury when marked alterations in RNA turnover have been observed, implicating "alkaline" ribonucleases in the control of RNA metabolism during nerve regeneration.