Seasonal activity and abundance of Orosius orientalis (Hemiptera : Cicadellidae) at agricultural sites in Southeastern Australia

Orosius orientalis is a leafhopper vector of several viruses and phytoplasmas affecting a broad range of agricultural crops. Sweep net, yellow pan trap and yellow sticky trap collection techniques were evaluated. Seasonal distribution of O. orientalis was surveyed over two successive growing seasons around the borders of commercially grown tobacco crops. Orosius orientalis seasonal activity as assessed using pan and sticky traps was characterised by a trimodal peak and relative abundance as assessed using sweep nets differed between field sites with peak activity occurring in spring and summer months. Yellow pan traps consistently trapped a higher number of O. orientalis than yellow sticky traps.

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.

Anti-metabolic effects of Galanthus nivalis agglutinin and wheat germ agglutinin on nymphal stages of the common brown leafhopper using a novel artificial diet system

The common brown leafhopper, Orosius orientalis (Matsumura) (Homoptera: Cicadellidae), previously described as Orosius argentatus (Evans), is an important vector of several viruses and phytoplasmas worldwide. In Australia, phytoplasmas vectored by O. orientalis cause a range of economically important diseases, including legume little leaf (Hutton & Grylls, 1956), tomato big bud (Osmelak, 1986), lucerne witches broom (Helson, 1951), potato purple top wilt (Harding & Teakle, 1985), and Australian lucerne yellows (Pilkington et al., 2004). Orosius orientalis also transmits Tobacco yellow dwarf virus (TYDV; genus Mastrevirus, family Geminiviridae) to beans, causing bean summer death disease (Ballantyne, 1968), and to tobacco, causing tobacco yellow dwarf disease (Hill, 1937, 1941). TYDV has only been recorded in Australia to date. Both diseases result in significant production and quality losses (Ballantyne, 1968; Thomas, 1979; Moran & Rodoni, 1999). Although direct damage caused by leafhopper feeding has been observed, it is relatively minor compared to the losses resulting from disease (P Tr E bicki, unpubl.).

Diversity of Cicadellidae in agricultural production areas in the Ovens Valley, Northeast Victoria, Australia

There is a paucity of data on the distribution of Cicadellidae (leafhoppers) in Australia. This study quantifies the relative abundance, seasonal activity and diversity of leafhoppers in the Ovens Valley region of north-east Victoria, Australia. Species diversity and abundance was assessed at four field sites in and around the field borders of commercially grown tobacco crops using three sampling techniques (pan trap, sticky trap and sweep net). Over 51 000 leafhopper samples were collected, with 57 species from 11 subfamilies and 19 tribes identified. Greater numbers and diversity of leafhoppers were collected in yellow pan traps. The predominant leafhopper collected was Orosius orientalis (Matsumura). Twenty-three leafhopper species were recorded for the first time in Victoria and eight economically important pest species were recorded. Seasonal activity of selected leafhopper species, covering two sampling seasons, is presented.

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.

Characterisation of the tritrophic interactions between tobacco yellow dwarf virus, its vector and host-plants

Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus) is an economically important pathogen causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco (Nicotiana tabacum L.), respectively. Prior to the commencement of this project, little was known about the epidemiology of TbYDV, its vector and host-plant range. As a result, disease control strategies have been restricted to regular poorly timed insecticide applications which are largely ineffective, environmentally hazardous and expensive. In an effort to address this problem, this PhD project was carried out in order to better understand the epidemiology of TbYDV, to identify its host-plant and vectors as well as to characterise the population dynamics and feeding physiology of the main insect vector and other possible vectors. The host-plants and possible leafhopper vectors of TbYDV were assessed over three consecutive growing seasons at seven field sites in the Ovens Valley, Northeastern Victoria, in commercial tobacco and bean growing properties. Leafhoppers and plants were collected and tested for the presence of TbYDV by PCR. Using sweep nets, twenty-three leafhopper species were identified at the seven sites with Orosius orientalis the predominant leafhopper. Of the 23 leafhopper species screened for TbYDV, only Orosius orientalis and Anzygina zealandica tested positive. Forty-two different plant species were also identified at the seven sites and tested. Of these, TbYDV was only detected in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. Using a quadrat survey, the temporal distribution and diversity of vegetation at four of the field sites was monitored in order to assess the presence of, and changes in, potential host-plants for the leafhopper vector(s) and the virus. These surveys showed that plant composition and the climatic conditions at each site were the major influences on vector numbers, virus presence and the subsequent occurrence of tobacco yellow dwarf and bean summer death diseases. Forty-two plant species were identified from all sites and it was found that sites with the lowest incidence of disease had the highest proportion of monocotyledonous plants that are non hosts for both vector and the virus. In contrast, the sites with the highest disease incidence had more host-plant species for both vector and virus, and experienced higher temperatures and less rainfall. It is likely that these climatic conditions forced the leafhopper to move into the irrigated commercial tobacco and bean crop resulting in disease. In an attempt to understand leafhopper species diversity and abundance, in and around the field borders of commercially grown tobacco crops, leafhoppers were collected from four field sites using three different sampling techniques, namely pan trap, sticky trap and sweep net. Over 51000 leafhopper samples were collected, which comprised 57 species from 11 subfamilies and 19 tribes. Twentythree leafhopper species were recorded for the first time in Victoria in addition to several economically important pest species of crops other than tobacco and bean. The highest number and greatest diversity of leafhoppers were collected in yellow pan traps follow by sticky trap and sweep nets. Orosius orientalis was found to be the most abundant leafhopper collected from all sites with greatest numbers of this leafhopper also caught using the yellow pan trap. Using the three sampling methods mentioned above, the seasonal distribution and population dynamics of O. orientalis was studied at four field sites over three successive growing seasons. The population dynamics of the leafhopper was characterised by trimodal peaks of activity, occurring in the spring and summer months. Although O. orientalis was present in large numbers early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peak in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and was also associated with warmer temperatures and lower rainfall. To understand the feeding requirements of Orosius orientalis and to enable screening of potential control agents, a chemically-defined artificial diet (designated PT-07) and feeding system was developed. This novel diet formulation allowed survival for O. orientalis for up to 46 days including complete development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), on leafhopper survival and development was assessed. Both GNA and WGA were shown to reduce leafhopper survival and development significantly when incorporated at a 0.1% (w/v) concentration. In contrast, CpTi at the same concentration did not exhibit significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis, TbYDV and the other pathogens it vectors. Finally, an electrical penetration graph (EPG) was used to study the feeding behaviour of O. orientalis to provide insights into TbYDV acquisition and transmission. Waveforms representing different feeding activity were acquired by EPG from adult O. orientalis feeding on two plant species, Phaseolus vulgaris and Nicotiana tabacum and a simple sucrose-based artificial diet. Five waveforms (designated O1-O5) were observed when O. orientalis fed on P. vulgaris, while only four (O1-O4) and three (O1-O3) waveforms were observed during feeding on N. tabacum and the artificial diet, respectively. The mean duration of each waveform and the waveform type differed markedly depending on the food source. This is the first detailed study on the tritrophic interactions between TbYDV, its leafhopper vector, O. orientalis, and host-plants. The results of this research have provided important fundamental information which can be used to develop more effective control strategies not only for O. orientalis, but also for TbYDV and other pathogens vectored by the leafhopper.

Assessment of an environmentally friendly, semi-pelagic fish trawl

Minimising catches of non-target animals in a trawl fishery reduces the impact on a marine community and may help to sustain the fishery resource in the long term. Hence the desirability for trawls that minimise impacts on non-target species while maintaining catches of target species. This study resulted from a need to further develop easily handled, semi-pelagic style trawls for Australia's Northern Fish Trawl Fishery. In November 1993 we compared catches from three differently rigged versions of a demersal wing trawl: one fished in a standard demersal configuration with its footrope on the sea bed, and two fished semi-pelagically, with their footropes raised to either 0.4-0.5 or 0.8-0.9 m above the sea bed. At two sites in the northeast Gulf of Carpentaria, each trawl type was used on the same combination of sites, grids within sites and times of day. Catches of the main target species (Lutjanus malabaricus and Lutjanus erythropterus) by the three trawl types were not significantly different. However, the mean catches of both these species and of other commercially important snappers, were highest in the semi-pelagic trawl raised 0.4-0.5 m above the sea bed. This increase could be due to a larger trawl spread or to the whole rig fishing higher in the water column. Of the 107 species of fishes analysed, 61 were caught in greater abundance in the demersal trawl. Seven species were caught more effectively in the semi-pelagic trawl with the footrope 0.4-0.5 m above the substrate; none was caught most effectively with the footrope set at 0.8-0.9 m. Epibenthic byproduct species (squid and Thenus orientalis), fish bycatch, sponges and other epibenthic invertebrates were also caught in lower numbers in the semi-pelagic trawls. The semi-pelagic trawls convincingly caught less (in both numbers and biomass) of the unwanted species which are normally discarded. Semi-pelagic fish trawls of the types tested would be suitable for Australia's Northern Fish Trawl Fishery and probably other demersal trawl fisheries that would benefit from the conservation of non-target epibenthic communities.

Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution

Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10−9 to P = 1.8 × 10−40) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10−3 to P = 1.2 × 10−13). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.

DNA sequences and austral taxa indicate generic synonymy of Paratrichocladius Santos-Abreu with Cricotopus Wulp (Diptera: Chironomidae)

In the century since the description of the orthoclad genus Paratrichocladius Santos-Abreu (Diptera: Chironomidae), separation in any life stage from the cosmopolitan, diverse Cricotopus Wulp has been problematic. Molecular analysis reveals the presence of two species in Australia that conform in morphology to Paratrichocladius and which form a well-supported clade including Paratrichocladius micans (Kieffer) from Africa and a distinct southern African larva. This clade clusters with taxa allied with Cricotopus albitibia (Walker), in turn nested within all other sampled Australian Cricotopus. Relevant nodes strongly support Cricotopus as nonmonophyletic without inclusion of Paratrichocladius. We synonymize Paratrichocladius with Cricotopus syn.n, treating Paratrichocladius as a subgenus. Cricotopus (Paratrichocladius) australiensis Cranston sp.n. is described for Trichocladius pluriserialis Freeman from Australia, which is not the same species under that name in New Zealand. Cricotopus (Paratrichocladius) bifenestrus Cranston sp.n. from Australia is described, also in all life stages. The many new combinations, listed in an Appendix, include three replacement names for new secondary homonyms, namely: Cricotopus (Paratrichocladius) sinobicinctus Cranston & Krosch nom.n. for Paratrichocladius bicinctus Fu, Sæther & Wang, Cricotopus draysoni Cranston & Krosch nom.n. for Cricotopus brevicornis Drayson, Krosch & Cranston, and Cricotopus (Paratrichocladius) sikhotealinus Makarchenko & Makarchenko nom.n. for Cricotopus orientalis Kieffer. We conclude with comments on wider issues in the taxonomy of Paratrichocladius, especially concerning New Zealand species.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Vα14Jα18 TCR α-chain specific for glycolipid Ags presented by CD1d. Murine Vα14Jα18+ account for 30–50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Vα24Vβ11+ NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3+ cells) and blood (0.02%). In contrast to those in blood, most hepatic Vα24+ NKT cells express the Vβ11 chain. They include CD4+, CD8+, and CD4−CD8− cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Vα24+ T cells are potent producers of IFN-γ and TNF-α, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, α-galactosylceramide. Vα24+Vβ11+ cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-γ in response to α-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.