A morphological and genetic description of pentastomid infective nymphs belonging to the family Sebekidae Sambon, 1922 in fish in Australian waters

Infective nymphal stages of the family Sebekidae Sambon, 1922 are reported from four species of fish in Australian waters for the first time. Infected fish were collected from locations in Western Australia, the Northern Territory and north Queensland. The infective nymphs of Alofia merki Giglioli in Sambon, 1922 and Sebekia purdieae Riley, Spratt et Winch, 1990 are reported and described for the first time. The remaining specimens were identified as belonging to the genus Sebekia Sambon, 1922 based on the combination of buccal cadre shape, shape and size of hooks, and overall body size, but could not be attributed to any of the other species of Sebekia already reported due to missing required morphological features. DNA sequences of members of the family Sebekidae are presented for the first time. The lack of knowledge on the pentastome fauna of wild crocodiles, and any potential intermediate hosts, in northern Australia, is also outlined.

Chromosome complements in the spermatogenesis of two penaeid prawns, Penaeus merguiensis and P. esculentus.

Although the fisheries for, and mariculture of, penaeid prawns are of major commercial importance, there has been relatively little research undertaken on the chromosome number, structure and composition in the Penaeidae. One reason for this is due to the relatively small size and large number of chromosomes, which makes production of histological material difficult. In this paper, we report a simple and effective technique for determining chromosome complements during spermatogenesis in two species of penaeid prawns, Penaeus merguiensis and P. esculentus in Australia. The first estimates of the number of chromosomes in these species are given.

Pig effluent-P application can increase risk of P transport: two case studies

Land application of piggery effluent (containing urine, faeces, water, and wasted feed) is under close scrutiny as a potential source of water resource contamination with phosphorus (P). This paper investigates two case studies of the impact of long-term piggery effluent-P application to soil. A Natrustalf (Sodosol) at P1 has received a net load of 3700 kg effluent P/ha over 19 years. The Haplustalf (Dermosol) selected (P2) has received a net load of 310 000 kg P/ha over 30 years. Total, bicarbonate extractable, and soluble P forms were determined throughout the soil profiles for paired (irrigated and unirrigated) sites at P1 and P2, as well as P sorption and desorption characteristics. Surface bicarbonate (PB, 0 - 0.05 m depth) and dilute CaCl2 extractable molybdate-reactive P (PC) have been significantly elevated by effluent irrigation (P1: PB unirrigated 23±1, irrigated 290±6; PC unirrigated 0.03±0.00, irrigated 23.9±0.2. P2: PB unirrigated 72±48, irrigated 3950±1960; PC unirrigated 0.7±0.0, irrigated 443±287 mg P/kg; mean±s.d.). Phosphorus enrichment to 1.5 m, detected as PB, was observed at P2. Elevated concentrations of CaCl2 extractable organic P forms (POC; estimated by non-molybdate reactive P in centrifuged supernatants) were observed from the soil surface of P1 to a depth of 0.4 m. Despite the extent of effluent application at both of these sites, only P1 displayed evidence of significant accumulation of POC. The increase in surface soil total P (0 - 0.05 m depth) due to effluent irrigation was much greater than laboratory P sorption (>25 times for P1; >57 times for P2) for a comparable range of final solution concentrations (desorption extracts ranged from 1-5 mg P/L for P1 and 50-80 mg P/L for P2). Precipitation of sparingly soluble P phases was evidenced in the soils of the P2 effluent application area.

An overview of land application of pig effluent-P using soil P chemistry and mass balance calculations.

Attention is directed at land application of piggery effluent (containing urine, faeces, water, and wasted feed) as a potential source of water resource contamination with phosphorus (P). This paper summarises P-related properties of soil from 0-0.05 m depth at 11 piggery effluent application sites, in order to explore the impact that effluent application has had on the potential for run-off transport of P. The sites investigated were situated on Alfisol, Mollisol, Vertisol, and Spodosol soils in areas that received effluent for 1.5-30 years (estimated effluent-P applications of 100-310000 kg P/ha in total). Total (PT), bicarbonate extractable (PB), and soluble P forms were determined for the soil (0-0.05 m) at paired effluent and no-effluent sites, as well as texture, oxalate-extractable Fe and Al, organic carbon, and pH. All forms of soil P at 0-0.05 m depth increased with effluent application (PB at effluent sites was 1.7-15 times that at no-effluent sites) at 10 of the 11 sites. Increases in PB were strongly related to net P applications (regression analysis of log values for 7 sites with complete data sets: 82.6 % of variance accounted for, p <0.01). Effluent irrigation tended to increase the proportion of soil PT in dilute CaCl2-extractable forms (PTC: effluent average 2.0 %; no-effluent average 0.6%). The proportion of PTC in non-molybdate reactive forms (centrifuged supernatant) decreased (no-effluent average, 46.4 %; effluent average, 13.7 %). Anaerobic lagoon effluent did not reliably acidify soil, since no consistent relationship was observed for pH with effluent application. Soil organic carbon was increased in most of the effluent areas relative to the no-effluent areas. The four effluent areas where organic carbon was reduced had undergone intensive cultivation and cropping. Current effluent management at many of the piggeries failed to maximise the potential for waste P recapture. Ten of the case-study effluent application areas have received effluent-P in excess of crop uptake. While this may not represent a significant risk of leaching where sorption retains P, it has increased the risk of transport of P by run-off. Where such sites are close to surface water, run-off P loads should be managed.

First records of the papaya strain of Papaya ringspot virus (PRSV-P) in French Polynesia and the Cook Islands

The papaya strain of Papaya ringspot virus (PRSV-P), the cause of papaya ringspot disease, was confirmed in French Polynesia and the Cook Islands by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). In French Polynesia, the virus has probably been on the islands of Tahiti and Moorea for several years, but appears not to have spread to eight other islands. In contrast, PRSV-P has only recently appeared in the Cook Islands and is now the subject of an eradication campaign.

Root-lesion nematodes (Pratylenchus thornei and P. neglectus): A review of recent progress in managing a significant pest of grain crops in northern Australia

Two species of root-lesion nematode (predominantly Pratylenchus thornei but also P. neglectus) are widespread pathogens of wheat and other crops in Australia's northern grain belt, a subtropical region with deep, fertile clay soils and a summer-dominant rainfall pattern. Losses in grain yield from P. thornei can be as high as 70% for intolerant wheat cultivars. This review focuses on research which has led to the development of effective integrated management programs for these nematodes. It highlights the importance of correct identification in managing Pratylenchus species, reviews the plant breeding work done in developing tolerant and resistant cultivars, outlines the methods used to screen for tolerance and resistance, and discusses how planned crop sequencing with tolerant and partially resistant wheat cultivars, together with crops such as sorghum, sunflower, millets and canaryseed, can be used to reduce nematode populations and limit crop damage. The declining levels of soil organic matter in cropped soils are also discussed with reference to their effect on soil health and biological suppression of root-lesion nematodes.

Identification of quantitative trait loci for resistance to two species of root-lesion nematode (Pratylenchus thornei and P. neglectus) in wheat

Pratylenchus thornei and P. neglectus are two species of root-lesion nematode that cause substantial yield losses in wheat. No commercially available wheat variety has resistance to both species. A doubled-haploid population developed from a cross between the synthetic hexaploid wheat line CPI133872 and the bread wheat Janz was used to locate and tag quantitative trait loci (QTLs) associated with resistance to both P. thornei and P. neglectus. Wheat plants were inoculated with both species of nematode in independent replicated glasshouse trials repeated over 2 years. Known locations of wheat microsatellite markers were used to construct a framework map. After an initial single-marker analysis to detect marker-trait linkages, chromosome regions associated with putative QTLs were targetted with microsatellite markers to increase map density in the chromosome regions of interest. In total, 148 wheat microsatellite markers and 21 amplified fragment length polymorphism markers were mapped. The codominant microsatellite marker Xbarc183 on the distal end of chromosome 6DS was allelic for resistance to both P. thornei and P. neglectus. The QTL were designated QRlnt.lrc-6D.1 and QRlnn.lrc-6D.1, for the 2 traits, respectively. The allele inherited from CPI133872 explained 22.0-24.2% of the phenotypic variation for P. thornei resistance, and the allele inherited from Janz accounted for 11.3-14.0% of the phenotypic variation for P. neglectus resistance. Composite interval mapping identified markers that flank a second major QTL on chromosome 6DL (QRlnt.lrc-6D.2) that explained 8.3-13.4% of the phenotypic variation for P. thornei resistance. An additional major QTL associated with P. neglectus resistance was detected on chromosome 4DS (QRlnn.lrc-4D.1) and explained a further 10.3-15.4% of the phenotypic variation. The identification and tagging of nematode resistance genes with molecular markers will allow appropriate allele combinations to be selected, which will aid the successful breeding of wheat with dual nematode resistance.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Ontogenetic depth partitioning by juvenile freshwater sawfish (Pristis microdon: Pristidae) in a riverine environment

The freshwater sawfish (Pristis microdon) is a critically endangered elasmobranch. Ontogenetic changes in the habitat use of juvenile P. microdon were studied using acoustic tracking in the Fitzroy River, Western Australia. Habitat partitioning was significant between 0+ (2007 year class) and larger 1+ (2006 year class) P. microdon. Smaller 0+ fish generally occupied shallower water (<0.6 m) compared with 1+ individuals, which mainly occurred in depths >0.6 m. Significant differences in hourly depth use were also revealed. The depth that 1+ P. microdon occupied was significantly influenced by lunar phase with these animals utilising a shallower and narrower depth range during the full moon compared with the new moon. This was not observed in 0+ individuals. Habitat partitioning was likely to be related to predator avoidance, foraging behaviours, and temperature and/or light regimes. The occurrence of 1+ P. microdon in deeper water may also result from a need for greater depths in which to manoeuvre. The present study demonstrates the utility of acoustic telemetry in monitoring P. microdon in a riverine environment. These results demonstrate the need to consider the habitat requirements of different P. microdon cohorts in the strategic planning of natural resources and will aid in the development of management strategies for this species.

Rapid Global Expansion of the Fungal Disease Chytridiomycosis into Declining and Healthy Amphibian Populations.

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.

Resistance to root-lesion nematodes (Pratylenchus thornei and P-neglectus) in synthetic hexaploid wheats and their durum and Aegilops tauschii parents.

Root-lesion nematodes (Pratylenchus thornei Sher and Allen and P. neglectus (Rensch) Filipijev and Schuurmans Stekhoven) cause substantial yield loss to wheat crops in the northern grain region of Australia. Resistance to P. thornei for use in wheat breeding programs was sought among synthetic hexaploid wheats (2n= 6x = 42, AABBDD) produced through hybridisations of Triticum turgidum L. subsp. durum (Desf.) Husn (2n= 4x = 28, AABB) with Aegilops tauschii Coss. (2n= 2x = 14, DD). Resistance was determined for the synthetic hexaploid wheats and their durum and Ae. tauschii parents from the numbers of nematodes in the roots of plants grown for 16 weeks in pots of pasteurised soil inoculated with P. thornei. Fifty-nine (32%) of 186 accessions of synthetic hexaploid wheats had lower numbers of nematodes than Gatcher Selection 50a (GS50a), a partially resistant bread wheat. Greater frequencies of partial resistance were present in the durum parents (72% of 39 lines having lower nematode numbers than GS50a) and in the Ae. tauschii parents (55% of 53 lines). The 59 synthetic hexaploids were re-tested in a second experiment along with their parents. In a third experiment, 11 resistant synthetic hexaploid wheats and their F-1 hybrids with Janz, a susceptible bread wheat, were tested and the F(1)s were found to give nematode counts intermediate between the respective two parents. Synthetic hexaploid wheats with higher levels of resistance resulted from hybridisations where both the durum and Ae. tauschii parents were partially resistant, rather than where only one parent was partially resistant. These results suggest that resistance to P. thornei in synthetic hexaploid wheats is polygenic, with resistances located both in the D genome from Ae. tauschii and in the A and/or B genomes from durum. Five synthetic hexaploid wheats were selected for further study on the basis of (1) a high level of resistance to P. thornei of the synthetic hexaploid wheats and of both their durum and Ae. tauschii parents, (2) being representative of both Australian and CIMMYT (International Maize and Wheat Improvement Centre) durums, and (3) being representative of the morphological subspecies and varieties of Ae. tauschii. These 5 synthetic hexaploid wheats were also shown to be resistant to P. neglectus, whereas GS50a and 2 P. thornei-resistant derivatives were quite susceptible. Results of P. thornei resistance of F(1)s and F(2)s from a half diallel of these 5 synthetic hexaploid wheats, GS50a, and Janz from another study indicate polygenic additive resistance and better general combining ability for the synthetic hexaploid wheats than for GS50a. Published molecular marker studies on a doubled haploid population between the synthetic hexaploid wheat with best general combining ability (CPI133872) and Janz have shown quantitative trait loci for resistance located in all 3 genomes. Synthetic hexaploid wheats offer a convenient way of introgressing new resistances to P. thornei and P. neglectus from both durum and Ae. tauschii into commercial bread wheats.

Cellular stages of root formation, root system quality and survival of Pinus elliottii var. elliottii x P. caribaea var. hondurensis cuttings in different temperature environments.

Time to first root in cuttings varies under different environmental conditions and understanding these differences is critical for optimizing propagation of commercial forestry species. Temperature environment (15, 25, 30 or 35 +/- A 2A degrees C) had no effect on the cellular stages in root formation of the Slash x Caribbean Pine hybrid over 16 weeks as determined by histology. Initially callus cells formed in the cortex, then tracheids developed and formed primordia leading to external roots. However, speed of development followed a growth curve with the fastest development occurring at 25A degrees C and slowest at 15A degrees C with rooting percentages at week 12 of 80 and 0% respectively. Cutting survival was good in the three cooler temperature regimes (> 80%) but reduced to 59% at 35A degrees C. Root formation appeared to be dependant on the initiation of tracheids because all un-rooted cuttings had callus tissue but no tracheids, irrespective of temperature treatment and clone.

Cellular stages of root formation, root system quality and survival of Pinuselliottii var. elliottii x P. caribaea var. hondurensis cuttings in different temperature environments.

Time to first root in cuttings varies under different environmental conditions and understanding these differences is critical for optimizing propagation of commercial forestry species. Temperature environment (15, 25, 30 or 352C) had no effect on the cellular stages in root formation of the Slash * Caribbean Pine hybrid over 16 weeks as determined by histology. Initially callus cells formed in the cortex, then tracheids developed and formed primordia leading to external roots. However, speed of development followed a growth curve with the fastest development occurring at 25C and slowest at 15C with rooting percentages at week 12 of 80 and 0% respectively. Cutting survival was good in the three cooler temperature regimes (>80%) but reduced to 59% at 35C. Root formation appeared to be dependant on the initiation of tracheids because all un-rooted cuttings had callus tissue but no tracheids, irrespective of temperature treatment and clone.

Specific detection of the Old World screwworm fly, Chrysomya bezziana, in bulk fly trap catches using real-time PCR.

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis-causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time-consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular-based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species-specific real-time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species-specific primers and an OWS-specific Taqman MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non-target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real-time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.

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.