Non-invasive sampling techniques : applications for mapping the generic variation of powerful owls

The Powerful Owl (Ninox strenua) is endemic to Australia, being resident in the three eastern mainland states and the Australian Capital Territory. It is classified nationally as of conservation significance and vulnerable in the state of Victoria. The elusive nature of this owl, along with its dispersed distribution, low population density and difficulty in identifying individual birds, limit the collection of ecological data. Molecular methods can be used to obtain crucial ecological information, essential for Powerful Owl conservation.

Non-invasive sampling is a relatively new method used for obtaining genetic material from free-ranging animals. This type of sampling however, is generally overlooked as a potential DNA source. Shed hair and feathers, faeces, urine, skins and eggshells are all potential sources of DNA. Non-invasive sampling regimes may be the only alternative for the genetic analysis of endangered and/or elusive species that are difficult to sample otherwise.

Powerful Owls moult annually. Shed feathers therefore, can be collected from under roost trees and used for genetic analysis. Feathers collected provide DNA that is unique to the individual and can provide additional ecological knowledge of the species.

In this study we collected shed Powerful Owl feathers during 2003 and 2004. In order to obtain samples from across the owl's large distribution, public awareness about the project via the way of flyers, mail-outs, media sources (radio, newspapers and magazines), email lists and public seminars was initiated. Overall, the collection strategy was very successful with over 500 Powerful Owl feather samples being collected.

Genetic information obtained from the analysis of DNA from feathers can enable a more rigorous assessment of population viability of the Powerful Owl. Specifically designed molecular markers will facilitate unequivocal identification of individual birds ("DNA fingerprinting"). Through the application of molecular techniques we can collect ecological information about the Powerful Owl such as, genetic divergence, population structure, dispersal patterns, migration and inbreeding. These questions can not be addressed via traditional data collection and will contribute significantly to the successful conservation of the Powerful Owl and potentially other raptor species.

Sexing accuracy and indicators of maturation status in captive Murray cod Maccullochella peelii peelii using non-invasive ultrasonic imagery

Macroscopic- and histological-based assessments of gonad condition were compared with ultrasound images to determine the feasibility of this technology as a non-invasive diagnostic tool for identifying sex and assessing maturation status of Murray cod. Four age-classes (1⁺, 2⁺, 3⁺ and 6⁺ years), were sub-sampled at monthly intervals throughout their annual reproductive cycle and scanned with a 5 MHz linear transducer. An interpretation of sex was made from the resulting images and maximum cross-sectional gonad diameter and area were recorded. Fish were subsequently dissected to confirm gender, and the weights and maturation status of gonads determined and then compared with their respective image profile. Ovaries of females were usually a distinctive feature in ultrasound images, being particularly obvious in older and/or more developed fish. In contrast, the identification of male testis was more problematic. Nonetheless, identifying sex from ultrasound images was consistently achieved by recording the presence/absence of a female ovary (96% total sexing accuracy). Maximum cross-sectional ovary diameter and area were highly correlated with gonad weight (r² = 0.90 and 0.89, respectively) suggesting that indices of maturation status, comparable to the gonadosomatic index (GSI), can be obtained non-destructively from ultrasound scans of females. A less distinct relationship occurred between these dimensions and weight of testes (r² = 0.41). Significant increases (P < 0.05) in mean gonad index (GI, calculated from gonad diameter) occurred for most gonad development stages. However, differences in mean GI between maturation stages were confounded by phenotypic variability, indicating that GI may be limited to population level studies. Nevertheless, ultrasound images of ovaries at each development stage were visually distinctive and enabled qualitative evaluations of maturity, thereby complementing quantitative GI assessments. Repeated serial-monitoring of the same population using ultrasound appears to have great potential for tracking maturation-induced changes in broodfish.

Performances of three minimally invasive cardiac output monitoring systems

We evaluated cardiac output (CO) using three new methods – the auto-calibrated FloTrac–Vigileo (CO_ed), the non-calibrated Modelflow (CO_mf ) pulse contour method and the ultra-sound HemoSonic system (CO_hs) – with thermodilution (CO_td) as the reference. In 13 postoperative cardiac surgical patients, 104 paired CO values were assessed before, during and after four interventions: (i) an increase of tidal volume by 50%; (ii) a 10 cm H₂O increase in positive end-expiratory pressure; (iii) passive leg raising and (iv) head up position. With the pooled data the difference (bias (2SD)) between CO_ed and CO_td, CO_mf and CO_td and CO_hs and CO_td was 0.33 (0.90), 0.30 (0.69) and −0.41 (1.11) l.min⁻¹, respectively. Thus, Modelflow had the lowest mean squared error, suggesting that it had the best performance. CO_ed significantly overestimates changes in cardiac output while CO_mf and CO_hs values are not significantly different from those of CO_td. Directional changes in cardiac output by thermodilution were detected with a high score by all three methods.

Relationship of nutritional and metabolic factors to non-invasive indices of macrovascular disease in diabetes

Factors which may account for the high frequency of macrovascular disease in diabetics are age, sex, cigarette smoking, hypertension, obesity, lack of exercise, diet, hyperglycaemia, hyperinsulinaeroia, hypercholesterolaemia, hypertriglyceridaemia, low HDL-cholesterol concentration, elevated free fatty acid concentration and enhanced platelet aggregation. Twenty seven (13 men and 14 women) non-insulin-dependent diabetics and thirty eight age, height and weight matched healthy subjects (10 men and 28 women) were studied. None of the subjects were smokers, or hypertensive. No subject had any clinical evidence of peripheral arterial disease, coronary heart disease or cerebrovascular disease. All had apparently normal peripheral pulses and normal ankle/arm blood pressure indices. Methods for determining arterial compliance in the segment between the left subclavian artery and each common femoral artery, and proximal resistance at the common femoral artery and posterior tibial artery, have been reviewed and developed. An appropriate food intake methodology for deriving food indices from food records was developed. Biochemical determinants have been made of glucose tolerance, glycosylated haemoglobin, serum total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, plasma free fatty acid and insulin. A significant decrease in the arterial compliance, and a significant increase in the arterial proximal resistance at the common femoral artery and posterior tibial artery in non-insulin-dependent diabetics, compared with their healthy controls, have been found. Significant negative correlation between arterial compliance and proximal resistance and, a significant positive correlation between the arterial proximal resistance of common femoral artery and posterior tibial artery were found. Differences between control (healthy subjects) and non-insulin-dependent diabetic groups indicate that preclinical peripheral arterial disease can be recognised even in mild diabetics by non-invasive measurement of arterial compliance or proximal resistance. There were significant and negative correlations between arterial compliance and each of blood glucose, blood glycosylated haemoglobin (HbAlC), plasma free fatty acid and plasma insulin concentration. There were significant and positive correlations between arterial proximal resistance of common femoral artery and posterior tibial artery and each of blood glucose, glycosylated haemoglobin and plasma free fatty acid concentration. Multivariate analysis to examine each of the biochemical factors Including blood glucose, blood glycosylated haemoglobin (HbAlC), plasma free fatty acid, plasma Insulin and lipids, showed that the factor which most influenced the arterial compliance and the proximal resistance of posterior tibial artery was the glucose level in the fasting state or the glucose response after a glucose load. In addition, the factors which most influenced proximal resistance of the common femoral artery were free fatty acid -level in the fasting state or glucose response after a glucose load. The factors which most influenced arterial compliance were glucose level in men, and the insulin level in the fasting state or the plasma free fatty acid response after a glucose load in women. These findings indicate that blood glucose, plasma free fatty acid and plasma insulin are risk factors for changes in arterial wall characteristic at a stage when no clinical evidence of macrovascular disease is apparent. Arterial compliance was decreased and the proximal resistance of posterior tibial artery was increased in those with a low intake of protective foods compared with those with a high intake whether healthy subjects or non-insulin-dependent diabetics. Arterial compliance was decreased in non-fish eaters compared with the fish eaters whether healthy subjects or non-insulin-dependent diabetics. Proximal resistance of the posterior tibia! artery in non-fish eaters was increased compared with fish eaters in healthy subjects. Overall, food variety, a protective food score consumption and fish consumption emerge as importance determinants of arterial wall characteristics at a stage when no clinical evidence of macrovascular disease is apparent.

Positive and negative effects of phoretic mites on the reproductive output of an invasive bark beetle

When multiple species coexist upon a single host, their combined effect on the host can be unpredictable. We explored the effect of phoretic mites on the reproductive output of the five-spined bark beetle, Ips grandicollis. Using correlative approaches and experimental manipulation of mite numbers we examined how mite load affected the number, size and condition of bark beetle offspring produced. We found that mites have both negative and positive consequences on different aspects of bark beetle reproduction. Females from which mites were removed were more fecund and produced larger offspring than females with mites, implying a cost of mite loads. However, when mites were present on females, those bearing the highest mite loads produced offspring that were larger and in better condition, indicating a beneficial effect of mites. These data suggest that phoretic interactions between mites and bark beetles differ over the course of the host’s lifespan, with either the mites interacting in different ways with different life stages of the host (parasitic on adult, mutualistic with larvae), and/or the beetles being host to different mite assemblages over their lifetime.

Predator control promotes invasive dominated ecological states

Invasive species are regarded as one of the top five drivers of the global extinction crisis. In response, extreme measures have been applied in an attempt to control or eradicate invasives, with little success overall. We tested the idea that state shifts to invasive dominance are symptomatic of losses in ecosystem resilience, due to the suppression of apex predators. This concept was investigated in Australia where the high rate of mammalian extinctions is largely attributed to the destructive influence of invasive species. Intensive pest control is widely applied across the continent, simultaneously eliminating Australia’s apex predator, the dingo (Canis lupus dingo). We show that predator management accounts for shifts between two main ecosystem states. Lethal control fractures dingo social structure and leads to bottom-up driven increases in invasive mesopredators and herbivores. Where control is relaxed, dingoes re-establish top–down regulation of ecosystems, allowing for the recovery of biodiversity and productivity.

Invasive plants – Do they devastate or diversify rural livelihoods? Rural farmers' perception of three invasive plants in Nepal

In this paper, we examine how rural people in the buffer zone of Chitwan National Park in Nepal perceive the effects of accidently transported invasive plant species, such as Mikania micrantha, Lantana camara and Chromolaena odorata, on their livelihoods. We found that their perception of the impact of each species on their livelihood varies with factors such as the duration of the presence of invasive plants in the landscape, and household characteristics. Results of a household survey indicate that farm households close to the forests have responded to the invasive species both as a victim and a beneficiary. Farm households are likely to adapt to the invaded environment as they have a history of interacting with invasive plants and can commoditise them through appropriate intervention. Additionally, the findings indicate that rural people are willing to invest in the control and management of invasive plants if appropriate technical assistance is available. Without assistance, they consider mitigating the infestation an unattainable mission and consider acceptance of the invasive species as a part of the rural ecosystem an inevitable outcome.

How many of Australia's ground-nesting birds are likely to be at risk from the invasive Cane Toad (Rhinella marina)?

Cane Toads (Rhinella marina; hereafter 'toads') are large, toxic American anurans that were introduced to Australia in 1935. Research on their ecological impact has focussed on the lethal ingestion of toxic toads by native frog-eating predators. Less attention has been paid to the potential impacts of Cane Toads as predators, although these large anurans sometimes eat vertebrates, such as nestling birds and bird eggs. We review published and unpublished data on interactions between Cane Toads and Australian ground-nesting birds, and collate distributional and breeding information to identify the avian taxa potentially at risk of having eggs or chicks eaten by Cane Toads. Cane Toads are currently sympatric with 80 ground-nesting bird species in Australia, and five additional species of bird occur within the predicted future range of the toad. Although many species of bird are potentially at risk, available data suggest there is minimal impact of Cane Toads on ground-nesting species. Future research could usefully address both direct and indirect impacts of the invasion by Cane Toads, ideally with detailed field observations of these impacts on nesting success and of changes in bird breeding success as a function of invasion by toads.

Development of novel microsatellite markers for the invasive Northern Pacific seastar, Asterias amurensis

The Northern Pacific seastar, Asterias amurensis, is a benthic marine predator, which has recently established several invasive populations in Australian waters. To investigate population structure, diversity and patterns of connectivity, we isolated and characterised 27 microsatellite loci and tested their polymorphism based on 46 individuals from two invasive populations. The mean allelic richness was 4.33; observed heterozygosity was 0.42, while the percentage of polymorphic loci was 92.6%. The polymorphic markers will prove useful in the assessment of population genetic parameters, in both invasive and native A. amurensis populations.

Non-invasive collection and examination of human corneal epithelial cells

Purpose. To report the development of a new apparatus for non-invasive collection of human corneal epithelial cells.

Methods. Previous methods of non-invasive, irrigative corneal cell collection resulted in low cell yields limiting potential analysis. A new ocular surface cell collection apparatus (OSCCA) was designed to collect more epithelial cells from direct irrigation of the corneal surface to allow for clinical comparisons. Forty-five samples were obtained (unilateral or bilateral over seven visits) from five human participants. Cell yield, size, phenotype, and corneal staining (prior and post eye wash) were examined.

Results. On average 364 ± 230 epithelial cells were collected from the cornea per eye. Epithelial cell sizes ranged from 8.21 to 51.69 μm in diameter, and 67.30 to 2098.85 μm2 area. The proportion of corneal specific cells collected per sample was 75 ± 14% as determined by positive K3 expression with AE5. On average, 77 ± 0.2% of epithelial cells harvested were nucleated, the remainder were non-nucleated ghost cells. Corneal staining was reduced in the OSCCA-washed vs. contralateral non-washed eyes (p = 0.02).

Conclusions. The OSCCA allows collection of human corneal epithelial cells with significantly higher yields, and greater specificity than previously reported. Reduced corneal staining observed post eye-wash demonstrated the safety of the technique, and its ability to remove cells directly from the corneal surface. The OSCCA could provide an objective non-invasive method of investigating pathological changes, effects of topical therapeutics, and impact of contact lenses and care-solutions of the cells of the ocular surface.

The power of myth : the (non) impact of invasive cane toads (Bufo marinus) on domestic chickens (Gallus gallus)

Myths about invasive species are widespread in the general community, even when contrary to scientific evidence. Public revulsion against invasive cane toads (Bufo marinus) in Australia has encouraged the belief that toads pose a significant risk to domestic poultry, by poisoning fowls that eat toads or that drink water contaminated by toads. Although discredited by scientists in 1938, within 2 years of the toads’ introduction to Australia, the myth continues to flourish. We conducted experimental trials to evaluate the vulnerability of chickens to toad-contaminated water, and to toad ingestion. No ill effects were seen, with one chicken consuming 45 small toads without falling ill. Thus, available evidence suggests that cane toads do not imperil domestic poultry.

Responses of Australian wading birds to a novel toxic prey type, the invasive cane toad Rhinella marina

The impact of invasive predators on native prey has attracted considerable scientific attention, whereas the reverse situation (invasive species being eaten by native predators) has been less frequently studied. Such interactions might affect invasion success; an invader that is readily consumed by native species may be less likely to flourish in its new range than one that is ignored by those taxa. Invasive cane toads (Rhinella marina) in Australia have fatally poisoned many native predators (e.g., marsupials, crocodiles, lizards) that attempt to ingest the toxic anurans, but birds are more resistant to toad toxins. We quantified prey preferences of four species of wading birds (Nankeen night heron, purple swamphen, pied heron, little egret) in the wild, by offering cane toads and alternative native prey items (total of 279 trays offered, 14 different combinations of prey types). All bird species tested preferred the native prey, avoiding both tadpole and metamorph cane toads. Avoidance of toads was strong enough to reduce foraging on native prey presented in combination with the toads, suggesting that the presence of cane toads could affect predator foraging tactics, and reduce the intensity of predation on native prey species found in association with toads.