Theileria gilberti n. sp. (Apicomplexa: Theileriidae) in the Gilbert's Potoroo (Potorous gilbertii)

ABSTRACT. The morphology and genetic characterisation of a new species of piroplasm identified in the blood of the Gilbert's potoroo ( Potorous gilbertii ) from the Two Peoples Bay Nature Reserve near Albany, Western Australia, is described from blood and tissue samples from 16 Gilbert's potoroos. Microscopy of blood showed these parasites are highly pleomorphic with a mean length of 1.8 μm and mean width of 0.85 μm. Phylogenetic analysis of 18S rRNA sequence data identified the piroplasm as a new species of Theileria that is closely related to other Australian marsupial piroplasm species. Based on biological and molecular data, it is proposed that the parasite from Gilbert's potoroo be given the name Theileria gilberti n. sp.     

Further observations on the potoroo (Potorous tridactylus) in captivity

The maintenance and breeding performance of potoroos in captivity over a 7-year period is described. By removing offspring from the pouch and allowing 29 days for completion of embryonic development and birth to occur, pouch young of known ages can be obtained.     

Detecting a genetic bottleneck in Gilbert's Potoroo (Potorous gilbertii) (Marsupialia: Potoroidae), inferred from microsatellite and mitochondrial DNA sequence data

Gilbert's Potoroo isAustralia's most critically endangeredmarsupial, known from a single population inthe Two Peoples Bay National Park in WesternAustralia. We present results from a study ofgenetic variation in microsatellite andmitochondrial DNA. Mean heterozygosity at fivemicrosatellite loci was 49.3%, and the amountof mtDNA variation was extremely low ( =0.0004). There was evidence for a bottleneckin both sets of markers, and this wasconsistent with a demographic decline. Effective population size was estimated usingtwo different models of mutation formicrosatellites (N _e = 243 and 362). The results from this study highlight theconcern for the long-term survival of thisspecies.     

Changes in milk composition during lactation in the potoroo, Potorous tridactylus (Marsupialia: Potoroinae)

Milk samples from captive potoroos were analysed for composition during weeks 3-25 of the lactation period. During pouch residence, up to week 16, carbohydrate levels were high, ranging from 9 g 100 ml-1 at week 5 to 15 g 100 ml-1 at week 15; fat levels were consistently low, at around 2 g 100 ml-1; protein levels gradually increased from 5 g 100 ml-1 before week 10 to 12 g 100 ml-1 at week 16. Growth rates during this period increased exponentially, from 1 g week-1 at week 3 to 40 g week-1 at week 16. Thereafter, as the young left the pouch, marked changes were seen in carbohydrate and fat levels: by week 25, carbohydrate levels had fallen to 2 g 100 ml-1, and fat levels had risen to 26 g 100 ml-1. Protein levels increased moderately, reaching 15 g 100 ml-1 by week 25. Growth rates further increased during this period, to reach 60 g week-1 by week 25. Thus, trends in milk composition previously observed in Macropus species were observed also in the potoroo, suggesting a consistent pattern across the macropodid family. Carbohydrate levels in potoroo milk tend to be higher than in other macropodids, but total milk intake is as important as composition in determining growth rates.     

Effect of cadmium on the reproductive organs of the male potoroo Potorous tridactylus (macropodidae)

Low (2.28 mg/kg) or high (4.56 mg/kg) doses of cadmium chloride were administered, either intraperitoneally or subcutaneously, to adult male potoroos (P. tridactylus). After 7 days, the testes, caput epididymis and spermatic cords of the high-dose groups all displayed a degree of cellular damage; however, the damage was not as extensive as that which occurs in some eutherian species after only 24 h. This time differential might be attributed to structural differences between the testicular blood supply in eutherians and marsupials.     

Growth and role of the corpus luteum throughout delayed gestation in the potoroo, Potorous tridactylus

The growth cycle of the corpus luteum (CL) of the potoroo is similar to that of other macropodids. During delayed gestation, the post-partum CL remains quiescent until it is reactivated by removal of the sucking pouch young. The CL then undergoes a period of growth, rapid from Day 6 until Day 12, followed by a gradual decline from Day 21 to Day 27. Excision of the CL before Day 6 of pregnancy either inhibited embryonic development or failed to support it. Excision of the CL between 6 and 21 days after removal of pouch young did not prevent embryos developing to full term but interfered with parturition. Excision on Day 25 after removal of pouch young allowed birth but impaired lactation, neonates dying within 2 days. By Day 27, the CL appeared to be no longer essential for embryonic development, birth or neonate survival. It is suggested that the CL of the potoroo is required for a slightly greater proportion of pregnancy than in most larger kangaroos because the birth canal must be prepared before each parturition.     

Using a species distribution model to guide NSW surveys of the long‐footed potoroo (Potorous longipes)

Knowledge of threatened species’ distributions is essential for effective conservation decision‐making. Species distribution models (SDMs) are widely used to map species’ geographic ranges, identify new areas of suitable habitat and guide field surveys. In New South Wales (NSW), Australia, there are grave doubts about whether populations of the critically endangered long‐footed potoroo (Potorous longipes) remain extant, and identification of occupied sites is a high priority for its conservation. We used an SDM (Maxent) to identify regions in NSW that may have suitable habitat for the potoroo. The SDM was built with seven climate layers and had strong predictive performance (cross‐validated AUC = 0.94). We then combined this information on habitat suitability with vegetation and topography, to identify 58 survey sites across NSW. From April 2016 to May 2017, we undertook six field trips deploying six to eight cameras at each site for 52–63 days, resulting in 25 120 camera trap nights. A total of 215 759 images captured 43 native and feral animal species, but no long‐footed potoroos. Following the survey, newly available, independent presence and absence data were used to validate our model. A Kruskal–Wallis H test indicated that habitat suitability values were significantly higher at presence locations than absence locations (H = 58.66, d.f. = 1, P < 0.001). Finally, we refitted the Maxent model with the new data and identified additional regions that future surveys could explore. We conclude, however, that if the long‐footed potoroo remains extant in NSW, it is extremely rare.     

Establishment of ectomycorrhizae on the roots of two species of Eucalyptus from fungal spores contained in the faeces of the long-nosed potoroo (Potorous tridactylus)

Abstract The long-nosed potoroo (Potorous tridactylus), a medium-sized, ground-dwelling marsupial, inhabits the foothills and coastal forests of southeastern mainland Australia. Faecal analysis confirmed the mycophagous habit of P. tridactylus at a eucalypt forest site in East Gippsland, Victoria; in one month, the spores of 33 different fungi were identified. The majority of species (27) were hypogeal or subhypogeal gasteromycetes that form ectomycorrhizae with forest trees and shrubs, and included the widespread and common Mesophellia pachytrix. The possible role of mycophagy in establishment of eucalypt-fungal mycorrhizae was investigated in several ways. Fungal spores were viable after passage through the gut of P. tridactylus; application of spore-bearing faeces to seedlings of Eucalyptus sieberi and Eucalyptus globoidea grown in sterile soil produced ectomycorrhizae. Spores of M. pachythrix were among the most common spores in these faeces and some of the resulting ectomycorrhizae were of the type expected from M. pachythrix. In contrast, direct application of M. pachythrix spores from sporocarp tissue to seedlings produced no ectomycorrhizae. Ectomycorrhizae also were established on seedlings grown in unsterilized forest soil, indicating that soil-borne spores had been conditioned, either by passage through the gut of a mammal or some other means, for reaction with the roots of eucalypts. Addition of faeces improved root and shoot growth of seedlings, although we could not determine whether this was primarily due to the formation of ectomycorrhizae, the addition of nutrients in the faeces, or a combination of both. Potorous tridactylus may enhance the re-establishment of mycorrhizal colonies in logged and/or burned forest sites by dispersing in its faeces, the spores of mycorrhizal fungi from sporocarps consumed within the disturbed area, as well as spores from sporocarps eaten in adjacent undisturbed habitat. In performing this function, P. tridactylus may also aid in the recovery of regenerating silvertop ash-stringybark forests.     

Sperm cryopreservation of the critically endangered olive barb (Sarpunti) Puntiussarana (Hamilton, 1822)

The present study focused on development of a sperm cryopreservation protocol for the critically endangered olive barb Puntiussarana (Hamilton, 1822) collected from two stocks within Bangladesh and reared in the Fisheries Field Laboratory, Bangladesh Agricultural University (BAU). The sperm were collected in Alsever’s solution prepared at 296 mOsmol kg⁻¹. Sperm were activated with distilled water (24 mOsmol kg⁻¹) to characterize motility. Maximum motility (90%) was observed within 15 s after activation, and sperm remained motile for 35 s. Sperm activation was evaluated in different osmolalities and motility was completely inhibited when osmolality of the extender was ?287 mOsmol kg⁻¹. To evaluate cryoprotectant toxicity, sperm were equilibrated with 5%, 10% and 15% each of dimethyl sulfoxide (DMSO) and methanol. Sperm motility was noticeably reduced within 10 min, when sperm were equilibrated with 15% DMSO, indicating acute toxicity to spermatozoa and therefore this concentration was excluded in further trials. Sperm were cryopreserved using DMSO at concentrations of 5% and 10% and methanol at 5%, 10% and 15%. The one-step freezing protocol (from 5 °C to −80 °C at 10 °C/min) was carried out in a computer-controlled freezer (FREEZE CONTROL® CL-3300; Australia) and 0.25-ml straws containing spermatozoa were stored in liquid nitrogen for 7–15 days at −196 °C. The highest motility in thawed sperm 61 ± 8% (mean ± SD) was obtained with 10% DMSO. The fertilization and hatching rates were 70% and 37% for cryopreserved sperm, and 72% and 62% for fresh sperm. The protocol reported here can be useful for hatchery-scale production of olive barb. The use of cryopreserved sperm can facilitate hatchery operations, and can provide for long-term conservation of genetic resources to contribute in the recovery of critically endangered fish such as the olive barb.     

Anatomical specializations for nocturnality in a critically endangered parrot, the Kakapo (Strigops habroptilus)

The shift from a diurnal to nocturnal lifestyle in vertebrates is generally associated with either enhanced visual sensitivity or a decreased reliance on vision. Within birds, most studies have focused on differences in the visual system across all birds with respect to nocturnality-diurnality. The critically endangered Kakapo (Strigops habroptilus), a parrot endemic to New Zealand, is an example of a species that has evolved a nocturnal lifestyle in an otherwise diurnal lineage, but nothing is known about its' visual system. Here, we provide a detailed morphological analysis of the orbits, brain, eye, and retina of the Kakapo and comparisons with other birds. Morphometric analyses revealed that the Kakapo's orbits are significantly more convergent than other parrots, suggesting an increased binocular overlap in the visual field. The Kakapo exhibits an eye shape that is consistent with other nocturnal birds, including owls and nightjars, but is also within the range of the diurnal parrots. With respect to the brain, the Kakapo has a significantly smaller optic nerve and tectofugal visual pathway. Specifically, the optic tectum, nucleus rotundus and entopallium were significantly reduced in relative size compared to other parrots. There was no apparent reduction to the thalamofugal visual pathway. Finally, the retinal morphology of the Kakapo is similar to that of both diurnal and nocturnal birds, suggesting a retina that is specialised for a crepuscular niche. Overall, this suggests that the Kakapo has enhanced light sensitivity, poor visual acuity and a larger binocular field than other parrots. We conclude that the Kakapo possesses a visual system unlike that of either strictly nocturnal or diurnal birds and therefore does not adhere to the traditional view of the evolution of nocturnality in birds.     

Interspecific interactions of the critically endangered Forest Owlet (Athene blewitti)

The Forest Owlet (Athene blewitti) is critically endangered and at extremely high risk of extinction owing to its restricted distribution. An expedition was organized to determine the density of the Forest Owlet in the Melghat Tiger Reserve in February 2004 where they had been observed sporadically in the previous 5 years. We hoped to identify as many individuals as possible and to observe interspecific interactions in order to understand the social framework in which the species survives. A total of 43.4 km of jungle roads was checked; we confirmed the presence of three of the 13 previously reported individuals, and found 11 previously undetected owlets. Owlets were found in areas with several interconnected forest clearings which allowed the owlets to forage in them. In all cases where the Forest Owlet occurred, a village or agricultural fields of the indigenous people (Adivasis) was within a 0.5-km radius. It appears that Forest Owlets preferred to establish feeding territories in areas disturbed by anthropogenic activity such as clearing dead trees and undergrowth for fire, trampling undergrowth while searching for firewood, burning areas around the agricultural plots, or driving herds of cattle through the area. All of these activities appear to optimize the habitat for the sit-and-wait foraging Forest Owlet, facilitating detection and tracking of prey in open areas with sparse and short undergrowth, allowing a better all-round view due to a lower density of trees.     

Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus)

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current 'critically endangered' status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈ throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

Mitochondrial DNA differentiation in the critically endangered Berg River redfin (Pseudobarbus burgi)

The Berg River redfin (Pseudobarbus burgi) is a critically endangered endemic cyprinid from South Africa. We investigated mitochondrial DNA control region variation among specimens representative of five populations drawn from two adjacent river systems. Phylogenetic analyses, a minimum spanning network, and an analysis of molecular variance underscore the pronounced genetic separation of redfins originating from the geographically closely allied Verlorevlei and Berg Rivers, two populations that may have remained isolated since the Pleistocene. Despite a lack of geographic structuring within the Berg River, historic female gene flow among the upper and middle/lower parts of the river appears to be limited and the contemporary populations are probably isolated due to deterioration of the mainstream of the river. Our results suggest that the Berg and Verlorevlei populations should be managed as distinct conservation units. We encourage the use of sanctuaries, particularly by private landowners within both river systems, as this approach may contribute effectively to preserving genetic diversity within the species.