Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

Towards better management of Australia’s shark fishery: genetic analyses reveal unexpected ratios of cryptic blacktip species Carcharhinus tilstoni and C. limbatus

The common blacktip shark (Carcharhinus limbatus) and the Australian blacktip shark (C. tilstoni) are morphologically similar species that co-occur in subtropical and tropical Australia. In striking contrast to what has been previously reported, we demonstrate that the common blacktip shark is not rare in northern Australia but occurs in approximately equal frequencies with the Australian blacktip shark. Management of shark resources in northern Australia needs to take account of this new information. Species identification was performed using nucleotide sequences of the control, NADH dehydrogenase subunit 4 (ND4) and cytochrome oxidase I (COI) regions in the mitochondrial genome. The proportion of overall genetic variation (FST) between the two species was small (0.042, P < 0.01) based on allele frequencies at five microsatellite loci. We confirm that a third blacktip species (C. amblyrhynchoides, graceful shark) is closely related to C. tilstoni and C. limbatus and can be distinguished from them on the basis of mtDNA sequences from two gene regions. The Australian blacktip shark (C. tilstoni) was not encountered among 20 samples from central Indonesia that were later confirmed to be common blacktip and graceful sharks. Fisheries regulators urgently need new information on life history, population structure and morphological characters for species identification of blacktip shark species in Australia.

Soybean Stem Fly, Melanagromyza sojae (Diptera: Agromyzidae), in the New World: detection of high genetic diversity from soybean fields in Brazil

Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner), belongs to the family Agromyzidae and is highly polyphagous, attacking many plant species of the family Fabaceae, including soybean and other beans. SSF is regarded as one of the most important pests in soybean fields of Asia (e.g., China, India), North East Africa (e.g., Egypt), parts of Russia, and South East Asia. Despite reports of Agromyzidae flies infesting soybean fields in Rio Grande do Sul State (Brazil) in 1983 and 2009 and periodic interceptions of SSF since the 1940s by the USA quarantine authorities, SSF has not been officially reported to have successfully established in the North and South Americas. In South America, M. sojae was recently confirmed using morphology and its complete mitochondrial DNA (mtDNA) was characterized. In the present study, we surveyed the genetic diversity of M. sojae, collected directly from soybean host plants, using partial mtDNA cytochrome oxidase I (COI) gene, and provide evidence of multiple (>10) maternal lineages in SSF populations in South America, potentially representing multiple incursion events. However, a single incursion involving multiple-female founders could not be ruled out. We identified a haplotype that was common in the fields of two Brazilian states and the individuals collected from Australia in 2013. The implications of SSF incursions in southern Brazil are discussed in relation to the current soybean agricultural practices, highlighting an urgent need for better understanding of SSF population movements in the New World, which is necessary for developing effective management options for this significant soybean pest. © FUNPEC-RP.

Mitochondrial DNA diversity of Cleruchoides noackae (Hymenoptera: Mymaridae): a potential biological control agent for Thaumastocoris peregrinus (Hemiptera: Thaumastocoridae)

Carpintero and Dellap, (Hemiptera: Thaumastocoridae) is a native Australian sap-feeding insect that has become invasive and seriously damaging to commercially grown in the Southern Hemisphere. Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of . If implemented as a biological control agent, this factor will need to be considered in collecting and releasing .

A new species of gall midge (Diptera: Cecidomyiidae) feeding on ornamental Cordyline fruticosa(Asparagaceae) in Australia

Larvae of an undescribed gall midge were found feeding on leaves and stems within leaf sheaths and between leaf blades of potted plants of Cordyline fruticosa (Asparagaceae) in a production nursery in Queensland. The following varieties of the host plant were infested: Apple Blossom', Glauca', Kilauea', Negra', Pink Diamond, 'Purple Prince' and Willy's Gold'. The new species, Dasineura cordylineaeKolesik sp. nov., is described and its cytochrome oxidase unit I mitochondrial gene segment is sequenced. The new species is the first known gall midge feeding on a plant species of the genus Cordyline. Orange larvae induce oval shallow swellings on the leaf and stem tissue, which becomes necrotised during the later stage of larval feeding. Necrotic areas remain visible to the end of leaves' lives and decrease the market value of the plants. In the production nursery investigated, the lesions caused by the gall midge provided an entry for a fungal infection by Fusarium sp. inflicting further injury to plants. Larvae of the new species were preyed on by larvae of Gaurax sp. (Diptera: Chloropidae). This is the first worldwide record of Chloropidae preying on Cecidomyiidae.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Detecting quantitative trait loci affecting beef tenderness on bovine chromosome 7 near calpastatin and lysyl oxidase

From a study of 3 large half-sib families of cattle, we describe linkage between DNA polymorphisms on bovine chromosome 7 and meat tenderness. Quantitative trait loci (QTL) for Longissimus lumborum peak force (LLPF) and Semitendonosis adhesion (STADH) were located to this map of DNA markers, which includes the calpastatin ( CAST) and lysyl oxidase (LOX) genes. The LLPF QTL has a maximum lodscore of 4.9 and allele substitution of approximately 0.80 of a phenotypic standard deviation, and the peak is located over the CAST gene. The STADH QTL has a maximum lodscore of 3.5 and an allele substitution of approximately 0.37 of a phenotypic standard deviation, and the peak is located over the LOX gene. This suggests 2 separate likelihood peaks on the chromosome. Further analyses of meat tenderness measures in the Longissimus lumborum, LLPF and LL compression (LLC), in which outlier individuals or kill groups are removed, demonstrate large shifts in the location of LLPF QTL, as well as confirming that there are indeed 2 QTL on bovine chromosome 7. We found that both QTL are reflected in both LLPF and LLC measurements, suggesting that both these components of tenderness, myofibrillar and connective tissue, are detected by both measurements in this muscle.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA3 treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.

Correlation of plasma IGF-I concentrations and growth rate in aquacultured finfish: a tool for assessing the potential of new diets

A recently developed radioimmunoassay (RIA) for measuring insulin-like growth factor (IGF-I) in a variety of fish species was used to investigate the correlation between growth rate and circulating IGF-I concentrations of barramundi (Lates calcarifer), Atlantic salmon (Salmo salar) and Southern Bluefin tuna (Thunnus maccoyii). Plasma IGF-I concentration significantly increased with increasing ration size in barramundi and IGF-I concentration was positively correlated to growth rates obtained in Atlantic salmon (r2=0.67) and barramundi (r2=0.65) when fed a variety of diet formulations. IGF-I was also positively correlated to protein concentration (r2=0.59). This evidence suggested that measuring IGF-I concentration may provide a useful tool for monitoring fish growth rate and also as a method to rapidly assess different aquaculture diets. However, no such correlation was demonstrated in the tuna study probably due to seasonal cooling of sea surface temperature shortly before blood was sampled. Thus, some recommendations for the design and sampling strategy of nutritional trials where IGF-I concentrations are measured are discussed

Cytochrome P450 aromatase in grey mullet: cDNA and promoter isolation; brain, pituitary and ovarian expression during puberty.

In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515 bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485 bp and encoded 495 predicted amino acid residues. The expression level of muCyp19b significantly increased in the brain as puberty advanced; however, its expression level in the pituitary increased only slightly with pubertal development. In the ovary, the muCyp19a expression level markedly increased as puberty progressed. The promoter regions of the two genes were also isolated and their functionality evaluated in vitro using luciferase as the reporter gene. The muCyp19a promoter sequence (650 bp) contained a consensus TATA box and putative transcription factor binding sites, including two half EREs, an SF-1, an AhR/Arnt, a PR and two GATA-3s. The muCyp19b promoter sequence (2500 bp) showed consensus TATA and CCAAT boxes and putative transcription binding sites, namely: a PR, an ERE, a half ERE, a SP-1, two GATA-binding factor, one half GATA-1, two C/EBPs, a GRE, a NFkappaB, three STATs, a PPAR/RXR, an Ahr/Arnt and a CRE. Basal activity of serially deleted promoter constructs transiently transfected into COS-7, [alpha]T3 and TE671 cells demonstrated the enhancing and silencing roles of the putative transcription factor binding sites. Quinpirole, a dopamine agonist, significantly reduced the promoter activity of muCyp19b in TE671. The results suggest tissue-specific regulation of the muCyp19 genes and a putative alternative promoter for muCyp19b.

Pasteurella multocida Expresses Two Lipopolysaccharide Glycoforms Simultaneously, but Only a Single Form Is Required for Virulence: Identification of Two Acceptor-Specific Heptosyl I Transferases

Lipopolysaccharide (LPS) is a critical virulence determinant in Pasteurella multocida and a major antigen responsible for host protective immunity. In other mucosal pathogens, variation in LPS or lipooligosaccharide structure typically occurs in the outer core oligosaccharide regions due to phase variation. P. multocida elaborates a conserved oligosaccharide extension attached to two different, simultaneously expressed inner core structures, one containing a single phosphorylated 3-deoxy-D-manno-octulosonic acid (Kdo) residue and the other containing two Kdo residues. We demonstrate that two heptosyltransferases, HptA and HptB, add the first heptose molecule to the Kdo1 residue and that each exclusively recognizes different acceptor molecules. HptA is specific for the glycoform containing a single, phosphorylated Kdo residue (glycoform A), while HptB is specific for the glycoform containing two Kdo residues (glycoform B). In addition, KdkA was identified as a Kdo kinase, required for phosphorylation of the first Kdo molecule. Importantly, virulence data obtained from infected chickens showed that while wild-type P. multocida expresses both LPS glycoforms in vivo, bacterial mutants that produced only glycoform B were fully virulent, demonstrating for the first time that expression of a single LPS form is sufficient for P. multocida survival in vivo. We conclude that the ability of P. multocida to elaborate alternative inner core LPS structures is due to the simultaneous expression of two different heptosyltransferases that add the first heptose residue to the nascent LPS molecule and to the expression of both a bifunctional Kdo transferase and a Kdo kinase, which results in the initial assembly of two inner core structures.

Identification of small juvenile scombrids from northwest tropical Australia using mitochondrial DNA cytochrome b sequences

Small juveniles of the nine species of scombrids in Australian waters are morphologically similar to one another and, consequently, difficult to identify to species level. We show that the sequence of the mitochondrial DNA cytochrome b gene region is a powerful tool for identification of these young fish. Using this method, we identified 50 juvenile scombrids collected from Exmouth Bay, Western Australia. Six species of scombrids were apparent in this sample of fish: narrow-barred Spanish mackerel (Scomberomorus commerson), Indian mackerel (Rastrelliger kanagurta), frigate tuna (Auxis thazard), bullet tuna (Auxis rochei), leaping bonito (Cybiosarda elegans), and kawakawa (Euthynnus affinis). The presence of Indian mackerel, frigate tuna, leaping bonito, and kawakawa is the first indication that coastal waters may be an important spawning habitat for these species, although offshore spawning may also occur. The occurrence of small juvenile S. commerson was predicted from the known spawning patterns of that species, but other mackerel species (Scomberomorus munroi, Scomberomorus queenslandicus, Scomberomorus semifasiciatus) likely to be spawning during the sampling period were not detected among the 50 small juveniles analyzed here.

Phosphorus nutrition and tolerance of cotton to water stress I. Seed cotton yield and leaf morphology

Seed cotton yield and morphological changes in leaf growth were examined under drying soil with different phosphorus (P) concentrations in a tropical climate. Frequent soil drying is likely to induce a decrease in nutrients particularly P due to reduced diffusion and poor uptake, in addition to restrictions in available water, with strong interactive effects on plant growth and functioning. Increased soil P in field and in-ground soil core studies increased the seed cotton yield and related morphological growth parameters in a drying soil, with hot (daily maximum temperature >33°C) and dry conditions (relative humidity, 25% to 35%), particularly during peak boll formation and filling stage. The soil water content in the effective rooting zone (top 0.4 m) decreased to -1.5 MPa by day 5 of the soil drying cycle. However, the increased seed cotton yield for the high-P plants was closely related to increasing leaf area with increased P supply. Plant height, leaf fresh mass and leaf area per plant were positively related to the leaf P%, which increased with increasing P supply. Low P plants were lower in plant height, leaf area, and leaf tissue water in the drying soil. Individual leaf area and the water content of the fresh leaf (ratio of dry mass to fresh mass) were significantly dependent on leaf P%.

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50-60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome.

The limit to wheat water-use efficiency in eastern Australia. I. Gradients in the radiation environment and atmospheric demand

In the wheatbelt of eastern Australia, rainfall shifts from winter dominated in the south (South Australia, Victoria) to summer dominated in the north (northern New South Wales, southern Queensland). The seasonality of rainfall, together with frost risk, drives the choice of cultivar and sowing date, resulting in a flowering time between October in the south and August in the north. In eastern Australia, crops are therefore exposed to contrasting climatic conditions during the critical period around flowering, which may affect yield potential, and the efficiency in the use of water (WUE) and radiation (RUE). In this work we analysed empirical and simulated data, to identify key climatic drivers of potential water- and radiation-use efficiency, derive a simple climatic index of environmental potentiality, and provide an example of how a simple climatic index could be used to quantify the spatial and temporal variability in resource-use efficiency and potential yield in eastern Australia. Around anthesis, from Horsham to Emerald, median vapour pressure deficit (VPD) increased from 0.92 to 1.28 kPa, average temperature increased from 12.9 to 15.2°C, and the fraction of diffuse radiation (FDR) decreased from 0.61 to 0.41. These spatial gradients in climatic drivers accounted for significant gradients in modelled efficiencies: median transpiration WUE (WUEB/T) increased southwards at a rate of 2.6% per degree latitude and median RUE increased southwards at a rate of 1.1% per degree latitude. Modelled and empirical data confirmed previously established relationships between WUEB/T and VPD, and between RUE and photosynthetically active radiation (PAR) and FDR. Our analysis also revealed a non-causal inverse relationship between VPD and radiation-use efficiency, and a previously unnoticed causal positive relationship between FDR and water-use efficiency. Grain yield (range 1-7 t/ha) measured in field experiments across South Australia, New South Wales, and Queensland (n = 55) was unrelated to the photothermal quotient (Pq = PAR/T) around anthesis, but was significantly associated (r2 = 0.41, P < 0.0001) with newly developed climatic index: a normalised photothermal quotient (NPq = Pq . FDR/VPD). This highlights the importance of diffuse radiation and vapour pressure deficit as sources of variation in yield in eastern Australia. Specific experiments designed to uncouple VPD and FDR and more mechanistic crop models might be required to further disentangle the relationships between efficiencies and climate drivers.