B.L. Cannell

DNA-Based Faecal Dietary Analysis: A Comparison of qPCR and High Throughput Sequencing Approaches

The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.

Individual Diving Strategies in the Little Penguin

Abstract Data on the diving activity of the Little Penguin (Eudyptula minor) as recorded by data loggers are few. We monitored the foraging parameters of free-ranging Little Penguins, breeding at Penguin Island, Western Australia, using a small, hydrodynamically shaped time-depth recorder. Data were obtained for one, two and three consecutive foraging trips of three, one and two birds, respectively. These data showed that individual Little Penguins presented distinct diving depths that are consistent from one trip to the next. Four birds exploited shallow depths, around 1-5 m, but two other birds always chose to dive deeper, to around 8-10 m. No trends could be established between the adult mass or the chick mass and the preferred diving depths. The causes and consequences of these individual diving strategies are discussed.

The pathology and pathogenicity of a novel Haemoproteus spp. infection in wild Little Penguins (Eudyptula minor)

One hundred and thirty four Little Penguin (Eudyptula minor) carcases found since 2004 in south west Australia were necropsied. The livers and spleens from ten of the penguins exhibited varying degrees of multifocal, randomly scattered areas of necrosis and varying numbers of parasites were associated with these areas. Hepatomegaly and splenomegaly were noted in many of these ten cases. Necrosis and parasites were also observed in the cardiac muscle of four of the cases and in the lung tissue in one of the penguins. Using PCR, the parasites were positively identified in four of the cases as Haemoproteus spp. and morphologically identical tissue stage parasites associated with histopathological changes were observed in all ten dead penguins. This is the first study to demonstrate both the in situ presence of the Haemoproteus parasite in any member of the Sphensicidae family and mortality due to its presence. We postulate the involvement of anomalous environmental conditions in a potential increase in local vectors.

Poorer breeding by little penguins near Perth, Western Australia is correlated with above average sea surface temperatures and a stronger Leeuwin Current

Using 20 years of data (1986 to 2008), we examined relationships between oceanographic variables (Fremantle sea level (FSL) – a proxy for the strength of the Leeuwin Current – and sea surface temperature (SST)) and five measures of little penguin, Eudyptula minor, breeding performance near Perth, Western Australia: namely (1) the laying date, (2) the number of chicks produced per pair, (3) the proportion of eggs that hatched, (4) the overall breeding success, defined as the proportion of total eggs laid that resulted in successful fledglings and (5) chick mass at fledging. The next three years of data (2009 to 2011) were used to test the performance of our statistical predictive models. FSL provided more accurate predictions of timing of laying, whereas SST provided more accurate predictions of breeding success. A later end to laying was associated with a high FSL during the summer (December to February) before breeding. Higher SSTs in the pre-breeding period from April to May corresponded to reduced breeding success, with lower fledgling success, fewer chicks per pair and generally a lower mean mass of chicks at fledging. The models predict that future oceanographic warming is expected to reduce the breeding success of this colony of little penguins.

Temperature inside nest boxes of little penguins

Abstract In order to assess the potential impact that artificial nest boxes may have on the occupation rate or physical condition of adults and chicks of little penguins (Eudyptula minor), we recorded temperature continuously for 37 days simultaneously inside 7 nest boxes and in surrounding bush. Temperature inside the boxes was always higher than that in the bush, the difference being greatest around noon. Solar radiation caused temperature inside the boxes to increase. Temperature differences between box interiors and exteriors were smaller on windy and dry days. To prevent hyperthermic conditions, we suggest improvements in the ventilation of nest boxes.

Effects of artificial social stimuli on the reproductive schedule and hormone levels of yellow-eyed penguins (Megadyptes antipodes)

The effects of social stimuli on avian reproductive behaviors such as breeding schedules and courtship behaviors are well known due to numerous field studies. However, studies that have simultaneously examined the effects of social stimuli on reproductive behavior and the mediating endocrine mechanisms have been largely restricted to captive populations, which may not be representative of free-living populations. This study, conducted over two breeding seasons, aimed to simultaneously measure the effects of experimentally increasing auditory stimuli on the breeding schedule and endocrinology (levels of total androgen, estradiol, progesterone and prolactin) on free-living yellow-eyed penguins (Megadyptes antipodes). The yellow-eyed penguin is the least colonial of all penguins, nesting far apart from each other under dense vegetation, and, therefore, is presumed to experience much lower levels of social stimuli than other penguins. Egg laying was significantly more synchronous and tended to be earlier when birds were exposed to playbacks of the calls of conspecifics in 1 year of the study. We also found that levels of total androgen and estradiol of males in 1 year, and prolactin in another year, were proportionally higher among treated birds compared control birds that received no artificial auditory stimuli. These results show that even among supposedly solitary nesters, social stimuli could still play a role in influencing reproductive behavior and physiology. For the first time in free-living seabirds, we have demonstrated that behavioral responses to increased social stimuli are associated with hormonal changes.

Micromelia in Little Penguins (Eudyptula minor)

ABSTRACT Two cases of unilateral micromelia are described in wild juvenile little penguins (Eudyptula minor). The first bird was found at San Remo, Phillip Island, Victoria, Australia. The birds left flipper was reduced in length, and radiography demonstrated severe shortening and dysplasia of the humerus. The second bird was found having difficulty swimming off the coast near Fremantle, Western Australia. Clinical examination revealed that the bird was underweight (0.55 kg) but otherwise in good condition with subadult plumage and had probably only recently fledged from nearby Garden or Penguin Islands. The left wing was markedly reduced in size and had limited range of elbow and carpal joint movement but sensation and motor control were normal. Radiography demonstrated a severe reduction in size and distortion of the humerus and shortening and fusion of the ulna, radius, metacarpals, and phalanges. The furcula was asymmetrical because the left clavicle was slightly thinner and more radiolucent than the right and was also slightly deviated to the right side. Necropsy results of histopathologic examination revealed no other significant external abnormalities other than a mild focal heterophilic ulcerative proventriculitis from a mild burden of Contracaecum eudyptulae. Concentrations of heavy metals in kidney, liver, and brain were: cadmium, <6 mg/kg; mercury, <0.05 mg/kg; lead, <2 mg/kg; and selenium, 1.30 mg/kg. Possible causes of unilateral micromelia in these penguins are discussed.

Anthropogenic trauma is the most prevalent cause of mortality in Little Penguins (Eudyptula minor) in Perth, Western Australia

Abstract Penguins both forage and travel in the marine environment and so spend a much greater proportion of their lives in this environment than other seabirds. This maximises their exposure to an increasing range of threats compared with other seabirds. From late 2003 to 2012, necropsies were performed on 159 Little Penguins found in the Perth region, Western Australia. Given the close proximity of the colonies to increasingly populated urban areas, the aims of this study were to: (1) determine the causes of mortality; (2) determine the proportion of deaths attributable to anthropogenic causes; (3) use this information to help guide management strategies; and (4) identify potential threats to coastal seabirds in general. In most cases, cause of mortality could be assigned to one of 11 categories. Trauma, most likely from watercraft, was the main cause of mortality. The next most common cause, starvation, was more likely to occur in spring and summer. Management strategies for colonies of Little Penguins near high levels of watercraft activity should take into account the risk of injury or death from watercraft strikes.

Augmenting mark-recapture with beach counts to estimate the abundance of little penguins on Penguin Island, Western Australia

Context Penguin Island supports the largest colony of little penguins in Western Australia. It is subjected to a suite of anthropogenic threats because of its proximity to an increasing urban population. For effective management of the colony, it is necessary to not only have knowledge of the size of the colony, but also the population trend of the colony. Aims To demonstrate a new cost-effective method of estimating the island-wide population of penguins on Penguin Island. Methods We estimated the island-wide population by combining mark-recapture sampling over 2 years on part of the island and beach counts of penguins arriving at night around the entire island. We estimated the abundance using closed population models, allowing for sex and time effects in capture probabilities. We had four capture occasions in 2008 only, and so considered heterogeneity of capture probabilities (Mh), using the Chao heterogeneity moment estimator. The proportion of all penguins counted that arrived at the four mark-recapture sites was then used to inflate the population estimate for the whole island. Key results In all, 62% of all penguins counted used the four mark-recapture sites. In 2007, there were an estimated 2369±198 penguins, and 1543±182 in 2008. When capture heterogeneity was allowed for in 2008, this estimate increased to 2069±1172. Conclusions Fewer eggs were laid and all measures of breeding performance were lower in 2008 than in 2007. Hence, the lower population estimate is most likely to represent fewer birds attempting to breed. However, further work on population estimates is required to determine whether capture heterogeneity occurs in both good and poor breeding years. Capture rates were affected by the presence of a full moon and high tides. Implications The estimate of the population can be used as part of the basis of a long-term monitoring program needed for effective management of the penguin colony. However, such studies must be coincident with the monitoring of a suite of reproductive and foraging parameters if short-term impacts of threats are to be recognised and well managed.

WITHDRAWN: Corrigendum to "The pathology and pathogenicity of a novel Haemoproteus spp. infection in wild Little Penguins (Eudyptula minor)" [Vet. Parasitol. 197 (1-2) (2013) 74-84].

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.vetpar.2014.07.021. The duplicate article has therefore been withdrawn.