The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

An extreme case of plant-insect co-diversification: figs and fig-pollinating wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale co-phylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on average, wasps had sequences from 77% of six genes (5.6kb), figs had sequences from 60% of five genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based co-phylogenetic analyses further support the co-diversification hypothesis. Biogeographic analyses indicate that the presentday distribution of fig and pollinator lineages is consistent with an Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term co-diversification.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

What limits insect fecundity? Body size- and temperature-dependent egg maturation and oviposition in a butterfly

1. Large female insects usually have high potential fecundity. Therefore selection should favour an increase in body size given that these females get opportunities to realize their potential advantage by maturing and laying more eggs. However, ectotherm physiology is strongly temperature-dependent, and activities are carried out sufficiently only within certain temperature ranges. Thus it remains unclear if the fecundity advantage of a large size is fully realized in natural environments, where thermal conditions are limiting. 2. Insect fecundity might be limited by temperature at two levels; first eggs need to mature, and then the female needs time for strategic ovipositing of the egg. Since a female cannot foresee the number of oviposition opportunities that she will encounter on a given day, the optimal rate of egg maturation will be governed by trade-offs associated with egg- and time-limited oviposition. As females of different sizes will have different amounts of body reserves, size-dependent allocation trade-offs between the mother’s condition and her egg production might be expected. 3. In the temperate butterfly Pararge aegeria , the time and temperature dependence of oviposition and egg maturation, and the interrelatedness of these two processes were investigated in a series of laboratory experiments, allowing a decoupling of the time budgets for the respective processes. 4. The results show that realized fecundity of this species can be limited by both the temperature dependence of egg maturation and oviposition under certain thermal regimes. Furthermore, rates of oviposition and egg maturation seemed to have regulatory effects upon each other. Early reproductive output was correlated with short life span, indicating a cost of reproduction. Finally, large females matured more eggs than small females when deprived of oviposition opportunities. Thus, the optimal allocation of resources to egg production seems dependent on female size. 5. This study highlights the complexity of processes underlying rates of egg maturation and oviposition in ectotherms under natural conditions. We further discuss the importance of temperature variation for egg- vs. time-limited fecundity and the consequences for the evolution of female body size in insects.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5 alpha and EQ1

Aims: To examine Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 for known pathogenic mechanisms. Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d postinfection. Conclusions: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. Significance and Impact of the Study: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

Fertilisers and insect herbivores: a meta-analysis

Despite the importance of a thorough understanding of the effect of synthetic fertiliser on insect population dynamics, existing literature is conflicting and an area of intense debate. Here, a categorical random-effects meta-analysis and a vote count meta-analysis are employed to examine the effects of nitrogen(N), phosphorus (P), potassium (K) and NPK fertiliser on insect population dynamics. In agreement with the general consensus, insects were found to respond positively, overall, to fertilisers. Sucking insects showed a much stronger response to fertilisers than chewing insects. The environment in which a study is conducted can have a marked effect on insect responses to fertiliser, with natural environments showing the potential for buffering effects of nitrogen fertilisers in particular. As well as highlighting the potential shortfall in the amount of research investigating particularly the effects of potassium and phosphorus, this study provides an invaluable flag post in the ongoing research investigating fertiliser effects on ecosystems.

Detecting insect pollinator declines on regional and global scales.

Recently there has been considerable concern about declines in bee communities in agricultural and natural habitats. The value of pollination to agriculture, provided primarily by bees, is >$200 billion/year worldwide, and in natural ecosystems it is thought to be even greater. However, no monitoring program exists to accurately detect declines in abundance of insect pollinators; thus, it is difficult to quantify the status of bee communities or estimate the extent of declines. We used data from 11 multiyear studies of bee communities to devise a program to monitor pollinators at regional, national, or international scales. In these studies, 7 different methods for sampling bees were used and bees were sampled on 3 different continents. We estimated that a monitoring program with 200–250 sampling locations each sampled twice over 5 years would provide sufficient power to detect small (2–5%) annual declines in the number of species and in total abundance and would cost U.S.$2,000,000. To detect declines as small as 1% annually over the same period would require >300 sampling locations. Given the role of pollinators in food security and ecosystem function, we recommend establishment of integrated regional and international monitoring programs to detect changes in pollinator communities.

Identifying key knowledge needs for evidence-based conservation of wild insect pollinators: a collaborative cross-sectoral exercise

In response to evidence of insect pollinator declines, organisations in many sectors, including the food and farming industry, are investing in pollinator conservation. They are keen to ensure that their efforts use the best available science. We convened a group of 32 ‘conservation practitioners’ with an active interest in pollinators and 16 insect pollinator scientists. The conservation practitioners include representatives from UK industry (including retail), environmental non-government organisations and nature conservation agencies. We collaboratively developed a long list of 246 knowledge needs relating to conservation of wild insect pollinators in the UK. We refined and selected the most important knowledge needs, through a three-stage process of voting and scoring, including discussions of each need at a workshop. We present the top 35 knowledge needs as scored by conservation practitioners or scientists. We find general agreement in priorities identified by these two groups. The priority knowledge needs will structure ongoing work to make science accessible to practitioners, and help to guide future science policy and funding. Understanding the economic benefits of crop pollination, basic pollinator ecology and impacts of pesticides on wild pollinators emerge strongly as priorities, as well as a need to monitor floral resources in the landscape.

The utility of aerial pan-trapping for assessing insect pollinators across vertical strata

Insect pollinators provide a critical ecosystem service by pollinating many wild flowers and crops. It is therefore essential to be able to effectively survey and monitor pollinator communities across a range of habitats, and in particular, sample the often stratified parts of the habitats where insects are found. To date, a wide array of sampling methods have been used to collect insect pollinators, but no single method has been used effectively to sample across habitat types and throughout the spatial structure of habitats. Here we present a method of ‘aerial pan-trapping’ that allows insect pollinators to be sampled across the vertical strata from the canopy of forests to agro-ecosystems. We surveyed and compared the species richness and abundance of a wide range of insect pollinators in agricultural, secondary regenerating forest and primary forest habitats in Ghana to evaluate the usefulness of this approach. In addition to confirming the efficacy of the method at heights of up to 30 metres and the effects of trap color on catch, we found greatest insect abundance in agricultural land and higher bee abundance and species richness in undisturbed forest compared to secondary forest.

Variation in genome organization of the plant pathogenic fungus Colletotrichum lindemuthianum

The genome structure of Colletotrichum lindemuthianum in a set of diverse isolates was investigated using a combination of physical and molecular approaches. Flow cytometric measurement of genome size revealed significant variation between strains, with the smallest genome representing 59% of the largest. Southern-blot profiles of a cloned fungal telomere revealed a total chromosome number varying from 9 to 12. Chromosome separations using pulsed-field gel electrophoresis (PFGE) showed that these chromosomes belong to two distinct size classes: a variable number of small (< 2.5 Mb) polymorphic chromosomes and a set of unresolved chromosomes larger than 7 Mb. Two dispersed repeat elements were shown to cluster on distinct polymorphic minichromosomes. Single-copy flanking sequences from these repeat-containing clones specifically marked distinct small chromosomes. These markers were absent in some strains, indicating that part of the observed variability in genome organization may be explained by the presence or absence, in a given strain, of dispensable genomic regions and/or chromosomes.

Persistent reduced ecosystem respiration after insect disturbance in high elevation forests

Amid a worldwide increase in tree mortality, mountain pine beetles (Dendroctonus ponderosae Hopkins) have led to the death of billions of trees from Mexico to Alaska since 2000. This is predicted to have important carbon, water and energy balance feedbacks on the Earth system. Counter to current projections, we show that on a decadal scale, tree mortality causes no increase in ecosystem respiration from scales of several square metres up to an 84 km2 valley. Rather, we found comparable declines in both gross primary productivity and respiration suggesting little change in net flux, with a transitory recovery of respiration 6–7 years after mortality associated with increased incorporation of leaf litter C into soil organic matter, followed by further decline in years 8–10. The mechanism of the impact of tree mortality caused by these biotic disturbances is consistent with reduced input rather than increased output of carbon.

Avoiding a bad apple: insect pollination enhances fruit quality and economic value

Insect pollination is important for food production globally and apples are one of the major fruit crops which are reliant on this ecosystem service. It is fundamentally important that the full range of benefits of insect pollination to crop production are understood, if the costs of interventions aiming to enhance pollination are to be compared against the costs of the interventions themselves. Most previous studies have simply assessed the benefits of pollination to crop yield and ignored quality benefits and how these translate through to economic values. In the present study we examine the influence of insect pollination services on farmgate output of two important UK apple varieties; Gala and Cox. Using field experiments, we quantify the influence of insect pollination on yield and importantly quality and whether either may be limited by sub-optimal insect pollination. Using an expanded bioeconomic model we value insect pollination to UK apple production and establish the potential for improvement through pollination service management. We show that insects are essential in the production of both varieties of apple in the UK and contribute a total of £36.7 million per annum, over £6 million more than the value calculated using more conventional dependence ratio methods. Insect pollination not only affects the quantity of production but can also have marked impacts on the quality of apples, influencing size, shape and effecting their classification for market. These effects are variety specific however. Due to the influence of pollination on both yield and quality in Gala, there is potential for insect pollination services to improve UK output by up to £5.7 million per annum. Our research shows that continued pollinator decline could have serious financial implications for the apple industry but there is considerable scope through management of wild pollinators or using managed pollinator augmentation, to improve the quality of production. Furthermore, we show that it is critically important to consider all production parameters including quality, varietal differences and management costs when valuing the pollination service of any crop so investment in pollinator management can be proportional to its contribution.

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long-standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes ‘gone rogue’ as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.

Reversing insect pollinator decline

Pollination by insects enables the reproduction of flowering plants and is critical to UK agriculture.1 Insect pollinators have declined globally, with implications for food security and wild habitats. This POSTnote summarises the causes for the recent trends, gaps in knowledge and possible strategies for reversing pollinator decline.