Concentrations of calcium and 25-hydroxycholecalciferol (vitamin D3) in plasma of wild kakapo (Strigops habroptilus) living on two islands in New Zealand.

AIMS This preliminary study had the objectives of describing the concentrations of ionised calcium and 25-hydroxycholecalciferol (25(OH)D3) in the blood of wild kakapo (Strigops habroptilus) living on two islands in New Zealand, and to determine the effects of supplementary feeding on these blood parameters. METHODS Blood samples were obtained from 33 kakapo living on two offshore islands during routine health checks in 2015. Birds on Hauturu were sampled in May (n=5) and birds on Whenua Hou were sampled in July (n=15) and November (n=26). Of the birds sampled on Whenua Hou in November, 15 received supplementary food prior to sampling. Samples were analysed for pH, and concentrations of ionised calcium, total calcium, phosphorous, total protein, albumin, globulin, uric acid and 25(OH)D3. RESULTS Concentrations of ionised calcium did not differ between unsupplemented birds on the two islands, nor between supplemented (median 1.17 (95% CI=1.12-1.20) mmol/L) and unsupplemented (median 1.09 (95% CI=1.08-1.14) mmol/L) birds sampled in November on Whenua Hou (p>0.05), and were comparable with published normal ranges for other psittacines. Concentrations of 25(OH)D3 did not differ between unsupplemented birds on the two islands (p>0.05), but were higher in supplemented (median 8.00 (95% CI=4.76-8.45) nmol/L) than unsupplemented (median 0.00 (95% CI=-0.16-0.48) nmol/L) birds on Whenua Hou (p<0.001). All values were much lower than published ranges for healthy psittacines. There was no difference between male and female birds on Whenua Hou for any parameter measured (p>0.05). CONCLUSIONS AND CLINICAL RELEVANCE The calcium status of the kakapo in this study was comparable to other wild psittacines, however concentrations of 25(OH)D3 were much lower. The concentrations of 25(OH)D3 may be within the normal range for the species, however further data are required to confirm this. The significant increase in concentrations of 25(OH)D3 in supplementary fed birds suggests that this food was providing more of the nutrient than the wild diet at that time of year, although the effects of this are unknown. Further investigation is required into the calcium and vitamin D3 status of kakapo, across a wider range of locations, seasons and ages. This would help define normal ranges for these parameters, allow interpretation in clinically abnormal individuals, and guide the refinement of supplementary foods. This information would, therefore, assist the future conservation management of this critically endangered species.

Quantifying the impact of storage procedures for faecal bacteriotherapy in the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus).

The endemic New Zealand kakapo is classified as 'critically endangered' and, in an effort to prevent extinction and restore the kakapo population, intensive handling of rare kakapo chicks is often utilised to reduce mortality and improve health outcomes among juveniles. Due to concerns that hand-reared chicks may not receive a full bacterial complement in their gut in the absence of regurgitated food from their mother, conservation workers feed a suspension of frozen adult faeces to captive chicks. However, the efficacy of this practice is unknown, with no information about the viability of these bacteria, or whether certain bacterial taxa are selected for or against as a consequence of freezing. In this study we experimentally determined the effects of freezing and reanimation on bacterial cell viability and diversity, using a faecal sample obtained from a healthy adult kakapo. Freezing reduced the number of viable bacterial cells (estimated by colony-forming units, CFU) by 99.86%, although addition of a cryoprotectant prior to freezing resulted in recovery of bacterial cells equivalent to that of non-frozen controls. Bacterial taxonomic diversity was reduced by freezing, irrespective of the presence of a cryoprotectant. While this study did not address the efficacy of faecal supplementation per se, the obtained data do suggest that faecal bacteriotherapy using frozen faeces (with a cryoprotectant) from healthy adult birds warrants further consideration as a conservation strategy for intensively managed species.

Hidden impacts of conservation management on fertility of the critically endangered kakapo.

Background: Animal conservation often requires intensive management actions to improve reproductive output, yet any adverse effects of these may not be immediately apparent, particularly in threatened species with small populations and long lifespans. Hand-rearing is an example of a conservation management strategy which, while boosting populations, can cause long-term demographic and behavioural problems. It is used in the recovery of the critically endangered kakapo (Strigops habroptilus), a flightless parrot endemic to New Zealand, to improve the slow population growth that is due to infrequent breeding, low fertility and low hatching success. Methods: We applied Bayesian mixed models to examine whether hand-rearing and other factors were associated with clutch fertility in kakapo. We used projection predictive variable selection to compare the relative contributions to fertility from the parents' rearing environment, their age and previous copulation experience, the parental kinship, and the number of mates and copulations for each clutch. We also explored how the incidence of repeated copulations and multiple mates varied with kakapo density. Results: The rearing status of the clutch father and the number of mates and copulations of the clutch mother were the dominant factors in predicting fertility. Clutches were less likely to be fertile if the father was hand-reared compared to wild-reared, but there was no similar effect for mothers. Clutches produced by females copulating with different males were more likely to be fertile than those from repeated copulations with one male, which in turn had a higher probability of fertility than those from a single copulation. The likelihood of multiple copulations and mates increased with female:male adult sex ratio, perhaps as a result of mate guarding by females. Parental kinship, copulation experience and age all had negligible associations with clutch fertility. Conclusions: These results provide a rare assessment of factors affecting fertility in a wild threatened bird species, with implications for conservation management. The increased fertility due to multiple mates and copulations, combined with the evidence for mate guarding and previous results of kakapo sperm morphology, suggests that an evolutionary mechanism exists to optimise fertility through sperm competition in kakapo. The high frequency of clutches produced from single copulations in the contemporary population may therefore represent an unnatural state, perhaps due to too few females. This suggests that opportunity for sperm competition should be maximised by increasing population densities, optimising sex ratios, and using artificial insemination. The lower fertility of hand-reared males may result from behavioural defects due to lack of exposure to conspecifics at critical development stages, as seen in other taxa. This potential negative impact of hand-rearing must be balanced against the short-term benefits it provides.

The promise and challenges of characterizing genome-wide structural variants: A case study in a critically endangered parrot.

There is growing interest in the role of structural variants (SVs) as drivers of local adaptation and speciation. From a biodiversity genomics perspective, the characterization of genome-wide SVs provides an exciting opportunity to complement single nucleotide polymorphisms (SNPs). However, little is known about the impacts of SV discovery and genotyping strategies on the characterization of genome-wide SV diversity within and among populations. Here, we explore a near whole-species resequence data set, and long-read sequence data for a subset of highly represented individuals in the critically endangered kakapo (Strigops habroptilus). We demonstrate that even when using a highly contiguous reference genome, different discovery and genotyping strategies can significantly impact the type, size and location of SVs characterized genome-wide. Further, we found that the mean number of SVs in each of two kakapo lineages differed both within and across generations. These combined results suggest that genome-wide characterization of SVs remains challenging at the population-scale. We are optimistic that increased accessibility to long-read sequencing and advancements in bioinformatic approaches including multireference approaches like genome graphs will alleviate at least some of the challenges associated with resolving SV characteristics below the species level. In the meantime, we address caveats, highlight considerations, and provide recommendations for the characterization of genome-wide SVs in biodiversity genomic research.

Non-invasive real-time genomic monitoring of the critically endangered kakapo.

We used non-invasive real-time genomic approaches to monitor one of the last surviving populations of the critically endangered kakapo (Strigops habroptilus). We first established an environmental DNA metabarcoding protocol to identify the distribution of kakapo and other vertebrate species in a highly localized manner using soil samples. Harnessing real-time nanopore sequencing and the high-quality kakapo reference genome, we then extracted species-specific DNA from soil. We combined long read-based haplotype phasing with known individual genomic variation in the kakapo population to identify the presence of individuals, and confirmed these genomically informed predictions through detailed metadata on kakapo distributions. This study shows that individual identification is feasible through nanopore sequencing of environmental DNA, with important implications for future efforts in the application of genomics to the conservation of rare species, potentially expanding the application of real-time environmental DNA research from monitoring species distribution to inferring fitness parameters such as genomic diversity and inbreeding.

ENVIRONMENTAL FACTOR INVESTIGATION OF EXUDATIVE CLOACITIS IN KAKAPO (STRIGOPS HABROPTILUS) ON WHENUA HOU (CODFISH ISLAND), NEW ZEALAND.

Kakapo (Strigops habroptilus) are critically endangered nocturnal parrots endemic to New Zealand. Exudative cloacitis is a disease only affecting the breeding population of Kakapo on Whenua Hou (Codfish Island), for which a consistent primary pathogenic organism involved has not been identified. This epidemiological study was conducted to identify the environmental factors contributing to the initiation of this disease in Kakapo by 1) producing and describing a case series; 2) mapping the geographic distribution of exudative cloacitis cases; 3) investigating the chemical characteristics of Kakapo roosting sites; and 4) assessing the effects of climatic factors on the incidence of exudative cloacitis each year. Soil samples from the Kakapo roost sites and other areas of the Whenua Hou were examined for pH, ammonium, and moisture contents. From 2002 to 2017, 22 sporadic cases of exudative cloacitis have been diagnosed and the disease distribution on Whenua Hou overlaps the Kakapo distribution. A mixed group of adults and juveniles was affected and there was no evidence of spatial or temporal clustering of the disease. Current findings on the chemical characteristics of Kakapo roosting sites do not show any evidence that these factors are involved in the initiation of the exudative cloacitis. Nevertheless, the results suggest that the ammonium and moisture levels of the roosts are worthy of more detailed study in future cases. We were not able to demonstrate any significant influence of broad measures of climate on the incidence of exudative cloacitis on Whenua Hou. Prospective data collection would help for a complete epidemiological investigation of this disease in future cases.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

Preferred natural food of breeding Kakapo is a high value source of calcium and vitamin D.

The Kakapo, a large NZ native parrot, is under severe threat of extinction. Kakapo breed only in years when the local podocarps, including rimu (Dacrydium cupressinum), are fruiting heavily, and the fruit are the preferred food both in the diet of breeding females and for provisioning chicks. Attempts to provide a supplementary food during years of poor fruit supply have failed to encourage breeding. Nutrient analysis of rimu berries reveals high calcium content (8.4mg/g dry matter) which would be essential for both egg shell production and the growing skeleton of the chick. Vitamin D is also critical for these processes and for the maintenance of calcium homeostasis, but the source of vitamin D for these nocturnal, ground-dwelling vegetarians is unknown. OBJECTIVE: To examine the vitamin D status of adult Kakapo, and to investigate the possibility that rimu berries provide vitamin D as well as calcium, thus differentiating them from the supplementary foods provided to date. METHOD: Previously collected and frozen serum from 10 adult birds (6 females, 4 males) was assayed for 25(OH)D3 and D2. Two batches of previously frozen rimu berries were analysed for vitamin D3 and D2. RESULTS: Vitamin D status of the 10 adult birds was very low; mean 4.9nmol/l, range 1-14nmol/l 25(OH)D3. No 25(OH)D2 was detected in any of the birds. High levels of D2 and moderate levels of D3 were found in the rimu berries. CONCLUSION: Traditionally it has been considered that the D3 isoform of this endogenously produced secosteroid is produced only in animals. However, D3 has been reported in the leaves of plants of the Solanacae family (tomato, potato, capsicum). The avian vitamin D receptor (VDR) is thought to have a much greater affinity for the D3 form. Therefore if rimu fruit are able to provide breeding Kakapo with D3, and are a plentiful source of calcium, they could be the perfect food package for breeding and nesting birds. Of wider importance, this finding challenges conventional understanding that D3 production is exclusive to animals, and that there are no high concentration food sources of vitamin D.