The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

International audience

Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation

Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.

UNDERSTANDING LATE SEASON FRUIT ROT PATHOSYSTEMS AND INSECT INTERACTIONS IN MID-ATLANTIC VINEYARDS

Fungal fruit rots and insect pests are among the most important problems negatively affecting the yield and quality of mid-Atlantic wine. In pathogenicity trials of fungi recovered from diseased Chardonnay and Vidal blanc grapes, Alternaria alternata, Pestalotiopsis telopeae, and Aspergillus japonicus were found to be unreported fruit rot pathogens in the region. Additionally, P. telopeae and A. japonicus had comparable virulence to the region’s common fruit rot pathogens. Furthermore, a timed-exclusion field study was implemented to evaluate vineyard insect-fruit rot relationships. It was found that clusters exposed to early-season insect communities that included Paralobesia viteana had a significantly greater incidence of sour rot than clusters protected from insects all season. These results were contrary to the current assumption that fall insects are the primary drivers of sour rot in the region. This research provides diagnostic tools and information to develop management-strategies against fungal and insect pests for mid-Atlantic grape growers.

Bacterial community associated to the pine wilt disease insect vectors Monochamus galloprovincialis and Monochamus alternatus

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialiswere collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

Agronomic performance of selected sweet potato cultivars under greenhouse and field infested with meloigogyne incognita and soilborne insect pests.

The root knot nematode (RKN), Meloidogyne incognita, is widespread worldwide and a major pathogen of several cultivated crops. The use of resistant genotypes is the most effective and environmentally sound way to manage RKN. In this study, we screened 16 selected sweet potato cultivars including Amanda, Bárbara, Beatriz, Beauregard, Brazlândia Branca, Brazlândia Rosada, Brazlândia Roxa, BRS Amélia, BRS Cuia, BRS Rubissol, Carolina Vitória, Duda, Júlia, Marcela, PA-26/2009, and Princesa obtained from Embrapa and Universidade Federal do Tocantins? germplasm bank. Studies were conducted under greenhouse and field conditions and the agronomic performance of the cultivars was evaluated in a nematode and soilborne insect-infested field. All 16 sweet potato cultivars tested were rated as resistant to this nematode both under greenhouse and field conditions with reproduction factors < 1. In the field infested with M. incognita, sweet potato cultivars Duda, BRS Amélia, Beauregard, Brazlândia Rosada, and Brazlândia Roxa stood out as superior cultivars, with average yield ranging from 26 to 47 tons per ha. Overall, most cultivars exhibited a fusiform to near fusiform root shape, a good characteristic for the market, and were moderately affected by insects (attack incidence 1 to 30%). As global demand for energy continues to rise, selecting new cultivars of sweet potatoes with increased resistance to nematode diseases and with high yield will be important for food security and biofuel production.

Influence of host plant structure and microclimate on the abundance and behaviour of Tephritid fly

Microclimate and host plant architecture significantly influence the abundance and behavior of insects. However, most research in this field has focused at the invertebrate assemblage level, with few studies at the single-species level. Using wild Solanum mauritianum plants, we evaluated the influence of plant structure (number of leaves and branches and height of plant) and microclimate (temperature, relative humidity, and light intensity) on the abundance and behavior of a single insect species, the monophagous tephritid fly Bactrocera cacuminata (Hering). Abundance and oviposition behavior were signficantly influenced by the host structure (density of foliage) and associated microclimate. Resting behavior of both sexes was influenced positively by foliage density, while temperature positively influenced the numbers of resting females. The number of ovipositing females was positively influenced by temperature and negatively by relative humidity. Feeding behavior was rare on the host plant, as was mating. The relatively low explanatory power of the measured variables suggests that, in addition to host plant architecture and associated microclimate, other cues (e.g., olfactory or visual) could affect visitation and use of the larval host plant by adult fruit flies. For 12 plants observed at dusk (the time of fly mating), mating pairs were observed on only one tree. Principal component analyses of the plant and microclimate factors associated with these plants revealed that the plant on which mating was observed had specific characteristics (intermediate light intensity, greater height, and greater quantity of fruit) that may have influenced its selection as a mating site.

Towards continuous surveillance of fruit flies using sensor networks and machine vision

In Australia, the Queensland fruit fly (B. tryoni), is the most destructive insect pest of horticulture, attacking nearly all fruit and vegetable crops. This project has researched and prototyped a system for monitoring fruit flies so that authorities can be alerted when a fly enters a crop in a more efficient manner than is currently used. This paper presents the idea of our sensor platform design as well as the fruit fly detection and recognition algorithm by using machine vision techniques. Our experiments showed that the designed trap and sensor platform is capable to capture quality fly images, the invasive flies can be successfully detected and the average precision of the Queensland fruit fly recognition is 80% from our experiment.

Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Vectors and alternative hosts of tobacco yellow dwarf virus in southeastern Australia

Factors that determine the epidemiology of Tobacco yellow dwarf virus (TbYDV), including alternative host plants and insect vector(s), were assessed over three consecutive growing seasons at four field sites in Northeastern Victoria in commercial tobacco growing properties. In addition, these factors were assessed for one growing season at three bean growing properties. Overall, 23 leafhopper species were identified at the 7 sites, with Orosius orientalis as the predominant leafhopper. Of the leafhoppers collected, only O. orientalis and Anzygina zealandica tested positive for TbYDV by polymerase chain reaction (PCR). The population dynamics of O. orientalis was assessed using sweep net sampling over three growing seasons and a trimodal distribution was observed. Despite large numbers of O. orientalis occurring early in the growing season (September–October), TbYDV was only detected in these leafhoppers between late November and end of January. The peaks in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and were associated with warmer temperatures and lower rainfall. Spatial and temporal distribution of vegetation at selected sites was determined using quadrat sampling. Of the 40 plant species identified, TbYDV was detected only in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. The proportion of host and non-host availability for leafhoppers was associated with climatic conditions.

Inter-and Intra-Site Genetic Diversity of Natural Field Populations of Rice Tungro Bacilliform Virus in the Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host /environmental shifts.

The complete mitochondrial genome of the gall-forming fly, Fergusonina taylori(Nelson and Yeates) (Diptera: Fergusoninidae)

The monogeneric family Fergusoninidae consists of gall-forming flies that, together with Fergusobia (Tylenchida: Neotylenchidae) nematodes, form the only known mutualistic association between insects and nematodes. In this study, the entire 16,000 bp mitochondrial genome of Fergusonina taylori Nelson and Yeates was sequenced. The circular genome contains one encoding region including 27 genes and one non-coding A þT-rich region. The arrangement of the proteincoding, ribosomal RNA (rRNA) and transfer RNA (tRNA) genes was the same as that found in the ancestral insect. Nucleotide composition is highly A þ T biased. All of the protein initiation codons are ATN, except for nad1 which begins with TTT. All 22 tRNA anticodons of F. taylori match those observed in Drosophila yakuba, and all form the typical cloverleaf structure except for tRNA-Ser (AGN) which lacks a dihydrouridine (DHU) arm. Secondary structural features of the rRNA genes of Fergusonina are similar to those proposed for other insects, with minor modifications. The mitochondrial genome of Fergusonina presented here may prove valuable for resolving the sister group to the Fergusoninidae, and expands the available mtDNA data sources for acalyptrates overall.

A mitochondrial genome phylogeny of termites (Blattodea: Termitoidae) : robust support for interfamilial relationships and molecular synapomorphies define major clades

Despite their ecological significance as decomposers and their evolutionary significance as the most speciose eusocial insect group outside the Hymenoptera, termite (Blattodea: Termitoidae or Isoptera) evolutionary relationships have yet to be well resolved. Previous morphological and molecular analyses strongly conflict at the family level and are marked by poor support for backbone nodes. A mitochondrial (mt) genome phylogeny of termites was produced to test relationships between the recognised termite families, improve nodal support and test the phylogenetic utility of rare genomic changes found in the termite mt genome. Complete mt genomes were sequenced for 7 of the 9 extant termite families with additional representatives of each of the two most speciose families Rhinotermitidae (3 of 7 subfamilies) and Termitidae (3 of 8 subfamilies). The mt genome of the well supported sister group of termites, the subsocial cockroach Cryptocercus, was also sequenced. A highly supported tree of termite relationships was produced by all analytical methods and data treatment approaches, however the relationship of the termites + Cryptocercus clade to other cockroach lineages was highly affected by the strong nucleotide compositional bias found in termites relative to other dictyopterans. The phylogeny supports previously proposed suprafamilial termite lineages, the Euisoptera and Neoisoptera, a later derived Kalotermitidae as sister group of the Neoisoptera and a monophyletic clade of dampwood (Stolotermitidae, Archotermopsidae) and harvester termites (Hodotermitidae). In contrast to previous termite phylogenetic studies, nodal supports were very high for family-level relationships within termites. Two rare genomic changes in the mt genome control region were found to be molecular synapomorphies for major clades. An elongated stem-loop structure defined the clade Polyphagidae + (Cryptocercus + termites), and a further series of compensatory base changes in this stem loop is synapomorphic for the Neoisoptera. The complicated repeat structures first identified in Reticulitermes, composed of short (A-type) and long (B-type repeats) defines the clade Heterotermitinae + Termitidae, while the secondary loss of A-type repeats is synapomorphic for the non-macrotermitine Termitidae.

EPG monitoring of the probing behaviour of the common brown leafhopper Orosius orientalis on artificial diet and selected host plants

The common brown leafhopper Orosius orientalis (Hemiptera: Cicadellidae) is a polyphagous vector of a range of economically important pathogens, including phytoplasmas and viruses, which infect a diverse range of crops. Studies on the plant penetration behaviour by O. orientalis were conducted using the electrical penetration graph (EPG) technique to assist in the characterisation of pathogen acquisition and transmission. EPG waveforms representing different probing activities were acquired from adult O. orientalis probing in planta, using two host species, tobacco Nicotiana tabacum and bean Phaseolus vulgaris, and in vitro using a simple sucrose-based artificial diet. Five waveforms (O1–O5) were evident when O. orientalis fed on bean, whereas only four waveforms (O1–O4) and three waveforms (O1–O3) were observed when the leafhopper fed on tobacco and on the artificial diet, respectively. Both the mean duration of each waveform and waveform type differed markedly depending on the food substrate. Waveform O4 was not observed on the artificial diet and occurred relatively rarely on tobacco plants when compared with bean plants. Waveform O5 was only observed with leafhoppers probing on beans. The attributes of the waveforms and comparative analyses with previously published Hemipteran data are presented and discussed, but further characterisation studies will be needed to confirm our suggestions.