Direct and indirect impacts of synthetic biology on biodiversity conservation.

The world's biodiversity is in crisis. Synthetic biology has the potential to transform biodiversity conservation, both directly and indirectly, in ways that are negative and positive. However, applying these biotechnology tools to environmental questions is fraught with uncertainty and could harm cultures, rights, livelihoods, and nature. Decisions about whether or not to use synthetic biology for conservation should be understood alongside the reality of ongoing biodiversity loss. In 2022, the 196 Parties to the United Nations Convention on Biological Diversity are negotiating the post-2020 Global Biodiversity Framework that will guide action by governments and other stakeholders for the next decade to conserve the worlds' biodiversity. To date, synthetic biologists, conservationists, and policy makers have operated in isolation. At this critical time, this review brings these diverse perspectives together and emerges out of the need for a balanced and inclusive examination of the potential application of these technologies to biodiversity conservation.

The Pacific Biosciences de novo assembled genome dataset from a parthenogenetic New Zealand wild population of the longhorned tick, Haemaphysalis longicornis Neumann, 1901.

The longhorned tick, Haemaphysalis longicornis, feeds upon a wide range of bird and mammalian hosts. Mammalian hosts include cattle, deer, sheep, goats, humans, and horses. This tick is known to transmit a number of pathogens causing tick-borne diseases, and was the vector of a recent serious outbreak of oriental theileriosis in New Zealand. A New Zealand-USA consortium was established to sequence, assemble, and annotate the genome of this tick, using ticks obtained from New Zealand's North Island. In New Zealand, the tick is considered exclusively parthenogenetic and this trait was deemed useful for genome assembly. Very high molecular weight genomic DNA was sequenced on the Illumina HiSeq4000 and the long-read Pac Bio Sequel platforms. Twenty-eight SMRT cells produced a total of 21.3 million reads which were assembled with Canu on a reserved supercomputer node with access to 12 TB of RAM, running continuously for over 24 days. The final assembly dataset consisted of 34,211 contigs with an average contig length of 215,205 bp. The quality of the annotated genome was assessed by BUSCO analysis, an approach that provides quantitative measures for the quality of an assembled genome. Over 95% of the BUSCO gene set was found in the assembled genome. Only 48 of the 1066 BUSCO genes were missing and only 9 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.

mtDNA polymorphism and metabolic inhibition affect sperm performance in conplastic mice.

Whereas a broad link exists between nucleotide substitutions in the mitochondrial genome (mtDNA) and a range of metabolic pathologies, exploration of the effect of specific mtDNA genotypes is on-going. Mitochondrial DNA mutations are of particular relevance for reproductive traits, since they are expected to have profound effects on male specific processes as a result of the strict maternal inheritance of mtDNA. Sperm motility is crucially dependent on ATP in most systems studied. However, the importance of mitochondrial function in the production of the ATP necessary for sperm function remains uncertain. In this study, we test the effect of mtDNA polymorphisms upon mouse sperm performance and bioenergetics by using five conplastic inbred strains that share the same nuclear background while differing in their mitochondrial genomes. We found that, while genetic polymorphisms across distinct mtDNA haplotypes are associated with modification in sperm progressive velocity, this effect is not related to ATP production. Furthermore, there is no association between the number of mtDNA polymorphisms and either (a) the magnitude of sperm performance decrease, or (b) performance response to specific inhibition of the main sperm metabolic pathways. The observed variability between strains may be explained in terms of additive effects of single nucleotide substitutions on mtDNA coding sequences, which have been stabilized through genetic drift in the different laboratory strains. Alternatively, the decreased sperm performance might have arisen from the disruption of the nuclear DNA/mtDNA interactions that have coevolved during the radiation of Mus musculus subspecies.

Introduction of a male-harming mitochondrial haplotype via 'Trojan Females' achieves population suppression in fruit flies.

Pests are a global threat to biodiversity, ecosystem function, and human health. Pest control approaches are thus numerous, but their implementation costly, damaging to non-target species, and ineffective at low population densities. The Trojan Female Technique (TFT) is a prospective self-perpetuating control technique that is species-specific and predicted to be effective at low densities. The goal of the TFT is to harness naturally occurring mutations in the mitochondrial genome that impair male fertility while having no effect on females. Here, we provide proof-of-concept for the TFT, by showing that introduction of a male fertility-impairing mtDNA haplotype into replicated populations of Drosophila melanogaster causes numerical population suppression, with the magnitude of effect positively correlated with its frequency at trial inception. Further development of the TFT could lead to establishing a control strategy that overcomes limitations of conventional approaches, with broad applicability to invertebrate and vertebrate species, to control environmental and economic pests.

Spatial utilization predicts animal social contact networks are not scale-free.

While heterogeneity in social behaviour has been described in many human contexts it is often assumed to be less common in the animal kingdom even though scale-free networks are observed. This homogeneity raises the question of whether the patterns of behaviour necessary to account for scale-free social contact networks, where the degree distribution follows a power law, i.e. a few individuals are very highly connected but most have only a few connections, occur in animals, or whether other mechanisms are needed to produce realistic contact network architectures. We develop a space-utilization model for individual animal behaviour to predict the individuals' social contact network. Using basic properties of the χ2 distribution we present a simple analytical result that allows the model to give a range of predictions with minimal computational effort. The model results are tested on data collected in New Zealand for the social contact networks of the wild brushtail possum (Trichosurus vulpecula). Our model provides a better prediction of network architecture than other simple models, including a scale-free model.

Is Avian Malaria Playing a Role in Native Bird Declines in New Zealand? Testing Hypotheses along an Elevational Gradient.

The mosquito-borne disease avian malaria (Plasmodium spp.) has impacted both captive populations and wild individuals of native New Zealand bird species. However, whether or not it is a cause of concern to their wild populations is still unclear. In Hawaii, the disease has been a major factor in the population declines of some native forest bird species, often limiting their elevational distribution due to an inverse relationship between force of infection and elevation. While studies have investigated latitudinal patterns of infection in New Zealand, elevational patterns are unexplored. To address this, a survey was conducted in Nelson Lakes National Park, a site experiencing native bird declines in which disease has been suggested as playing a role, to investigate whether there is a similar inverse relationship in New Zealand. Results from blood samples (n = 436) collected over three seasons across a broad elevational range (650-1400 m) support there being such a relationship. In addition, an overall higher prevalence in non-native (14.1%) versus native birds (1.7%) may indicate differential impacts on these two groups, while particularly high prevalence in non-native Turdus spp. supports previous suggestions that they are key reservoir hosts for the disease. Overall, these findings add weight to the hypothesis that avian malaria is playing a role in ongoing declines of native New Zealand birds.

Optimising survey effort to monitor environmental variables: A case study using New Zealand kiwifruit orchards.

Environmental monitoring is increasingly used to assess spatial and temporal trends in agricultural sustainability, and test the effectiveness of farm management policies. However, detecting changes in environmental variables is often technically and logistically challenging. To demonstrate how survey effort for environmental monitoring can be optimised, we applied the new statistical power analysis R package simr to pilot survey data. Specifically, we identified the amount of survey effort required to have an 80% chance of detecting specified trends (-1 to -4% pa) in 13 environmental variables on New Zealand kiwifruit orchards within an 11-year period. The variables assessed were related to soil status, agricultural pests (birds), or ecosystem composition (birds). Analyses were conducted on average values (for each orchard and year combination) to provide a consistent scale for comparison among variables. Survey frequency varied from annual (11 surveys) to every 5 years (3 surveys). Survey size was set at either 30, 60, 150 or 300 orchards. In broad terms, we show the power to detect a specified range of trends over an 11-year period in this sector is much higher for 'soil status' than for 'agricultural pest' or 'ecosystem composition'. Changes in one subset of native bird species (nectar-feeders) requiring a particularly high level of relative survey effort to detect with confidence. Monitoring soil status can thus be smaller and less frequent than those which also want to detect changes in agricultural pests or ecosystem composition (with the latter requiring the most effort) but will depend on the magnitude of changes that is meaningful to detect. This assessment thus allows kiwifruit industry in New Zealand to optimise survey design to the desired information, and provides a template for other industries to do likewise. Power analyses are now more accessible through the provision of the simr package, so deploying and integrating them into design and decision-making should be routine to reduce the risk of inefficiencies and opportunity costs.

Mitonuclear interactions, mtDNA-mediated thermal plasticity, and implications for the Trojan Female Technique for pest control.

Pest species pose major challenges to global economies, ecosystems, and health. Unfortunately, most conventional approaches to pest control remain costly, and temporary in effect. As such, a heritable variant of the Sterile Insect Technique (SIT) was proposed, based on the introduction of mitochondrial DNA mutations into pest populations, which impair male fertility but have no effects on females. Evidence for this "Trojan Female Technique" (TFT) was recently provided, in the form of a mutation in the mitochondrial cytochrome b gene (mt:Cyt-b) of Drosophila melanogaster which reduces male fertility across diverse nuclear backgrounds. However, recent studies have shown that the magnitude of mitochondrial genetic effects on the phenotype can vary greatly across environments, with mtDNA polymorphisms commonly entwined in genotype-by-environment (G × E) interactions. Here we test whether the male-sterilizing effects previously associated with the mt:Cyt-b mutation are consistent across three thermal and three nuclear genomic contexts. The effects of this mutation were indeed moderated by the nuclear background and thermal environment, but crucially the fertility of males carrying the mutation was invariably reduced relative to controls. This mutation thus constitutes a promising candidate for the further development of the TFT.

Sex difference in the survival rate of wild brushtail possums (Trichosurus vulpecula) experimentally challenged with bovine tuberculosis.

The main wildlife reservoir of bovine tuberculosis (TB) in New Zealand is the introduced brushtail possum (Trichosurus vulpecula), with spillover of infection from possums to livestock being regarded as the largest barrier to eradicating TB from the country. Past studies have experimentally challenged possums with Mycobacterium bovis (the causative agent of TB) to quantify infection parameters. However, the challenge models used are invariably non-representative of natural infection due to their resulting in much faster rates, and different clinical patterns of disease progression. We monitored the survival of 16 wild free-living possums, fitted with VHF mortality collars and experimentally challenged with a new model, out to six months post-challenge. The aim was to assess whether the new model does indeed result in an ongoing pathogenesis trajectory that is more reflective of natural TB in possums. The mean survival period of challenged possums (~4.6months) did not differ from that estimated for wild free-living possums with naturally acquired TB. In addition, and unexpectedly, infected males survived on average for five weeks longer than females. This significant difference has not been previously observed in experimental trials with other TB/possum challenge models. If this is reflective of natural disease (as is the survival period produced by the percutaneous challenge model), TB infected males in the wild may be generating more secondary cases of infection than infected females. This insight has important implications for understanding the dynamics of, and managing, the disease in its New Zealand wildlife reservoir.

Discovery and complete genome sequence of a novel circovirus-like virus in the endangered rowi kiwi, Apteryx rowi.

Circoviruses are circular, non-enveloped, single-stranded DNA viruses around 2000 nucleotides (nt) in length and include the pathogenic species, Porcine circovirus 1 and Beak and feather disease virus, capable of causing significant morbidity and mortality. This group of viruses may be robust to degradation by external environments, and avian circoviruses are known to move between closely related hosts. Using a de novo metagenomic approach, followed by confirmatory PCR, we identify for the first time a circular Rep-encoding single-stranded (CRESS) DNA virus in New Zealand kiwi, Apteryx spp., derived from faecal matter of the rowi kiwi (A. rowi) showing signs of verminous dermatitis. The entire 2085 nt genome was cloned and sequenced and contains both capsid and replicase genes, as well as a conserved 9 nt motif. Phylogenetic analyses place it within Circoviridae, adjacent to other environmental CRESS-DNA viruses, and most closely related to badger circovirus-like virus (Meles meles circovirus-like virus). As the rowi is the most critically endangered kiwi, it is vital to understand the role of rowi kiwi circovirus-like virus as a possible pathogen and also any potential cross-species transmission.

The Trojan Female Technique for pest control: a candidate mitochondrial mutation confers low male fertility across diverse nuclear backgrounds in Drosophila melanogaster.

Pest species represent a major ongoing threat to global biodiversity. Effective management approaches are required that regulate pest numbers, while minimizing collateral damage to nontarget species. The Trojan Female Technique (TFT) was recently proposed as a prospective approach to biological pest control. The TFT draws on the evolutionary hypothesis that maternally inherited mitochondrial genomes are prone to the accumulation of male, but not female, harming mutations. These mutations could be harnessed to provide trans-generational fertility-based control of pest species. A candidate TFT mutation was recently described in the fruit fly, Drosophila melanogaster, which confers male-only sterility in the specific isogenic nuclear background in which it is maintained. However, applicability of the TFT relies on mitochondrial mutations whose male-sterilizing effects are general across nuclear genomic contexts. We test this assumption, expressing the candidate TFT-mutation bearing haplotype alongside a range of nuclear backgrounds and comparing its fertility in males, relative to that of control haplotypes. We document consistently lower fertility for males harbouring the TFT mutation, in both competitive and noncompetitive mating contexts, across all nuclear backgrounds screened. This indicates that TFT mutations conferring reduced male fertility can segregate within populations and could be harnessed to facilitate this novel form of pest control.

Discovery of novel virus sequences in an isolated and threatened bat species, the New Zealand lesser short-tailed bat (Mystacina tuberculata).

Bats harbour a diverse array of viruses, including significant human pathogens. Extensive metagenomic studies of material from bats, in particular guano, have revealed a large number of novel or divergent viral taxa that were previously unknown. New Zealand has only two extant indigenous terrestrial mammals, which are both bats, Mystacina tuberculata (the lesser short-tailed bat) and Chalinolobus tuberculatus (the long-tailed bat). Until the human introduction of exotic mammals, these species had been isolated from all other terrestrial mammals for over 1 million years (potentially over 16 million years for M. tuberculata). Four bat guano samples were collected from M. tuberculata roosts on the isolated offshore island of Whenua hou (Codfish Island) in New Zealand. Metagenomic analysis revealed that this species still hosts a plethora of divergent viruses. Whilst the majority of viruses detected were likely to be of dietary origin, some putative vertebrate virus sequences were identified. Papillomavirus, polyomavirus, calicivirus and hepevirus were found in the metagenomic data and subsequently confirmed using independent PCR assays and sequencing. The new hepevirus and calicivirus sequences may represent new genera within these viral families. Our findings may provide an insight into the origins of viral families, given their detection in an isolated host species.

Emerging infectious diseases of wildlife: a critical perspective.

We review the literature to distinguish reports of vertebrate wildlife disease emergence with sufficient evidence, enabling a robust assessment of emergence drivers. For potentially emerging agents that cannot be confirmed, sufficient data on prior absence (or a prior difference in disease dynamics) are frequently lacking. Improved surveillance, particularly for neglected host taxa, geographical regions and infectious agents, would enable more effective management should emergence occur. Exposure to domestic sources of infection and human-assisted exposure to wild sources were identified as the two main drivers of emergence across host taxa; the domestic source was primary for fish while the wild source was primary for other taxa. There was generally insufficient evidence for major roles of other hypothesized drivers of emergence.

Piroplasms of New Zealand seabirds.

Blood and ectoparasitic ticks were collected from migratory seabirds in New Zealand, including Australasian gannets (n = 13) from two sites and red-billed gulls (n = 9) and white-fronted terns (n = 2) from a third location. Blood smears were screened for parasite presence by microscopy, while DNA from blood samples was subjected to PCR for the presence of tick-transmitted protozoan haemoparasites belonging to the order Piroplasmida. Parasites were identified by comparing small subunit ribosomal RNA (18S rDNA) gene sequences to related sequences on GenBank. Analyses indicated that nine birds were infected with unknown variants of a Babesia poelea-like parasite (recorded as genotypes I and II), while four harboured a piroplasm that was genetically similar to Babesia kiwiensis. There was no parasite stratification by bird species; both the gannets and gulls were positive for all three parasites, while the terns were positive for the B. kiwiensis-like and the B. poelea-like (genotype I) parasites. The B. kiwiensis-like parasite found in the birds was also found in two species of ticks: Carios capensis and Ixodes eudyptidis. This represents the first report of Babesia-positive ticks parasitising seabirds in New Zealand. The lack of host specificity and evidence of wide ranging distributions of the three piroplasm genotypes suggests there is a high degree of haemoparasite transmission occurring naturally between New Zealand seabird populations and species.

New alphacoronavirus in Mystacina tuberculata bats, New Zealand.

Because of recent interest in bats as reservoirs of emerging diseases, we investigated the presence of viruses in Mystacina tuberculata bats in New Zealand. A novel alphacoronavirus sequence was detected in guano from roosts of M. tuberculata bats in pristine indigenous forest on a remote offshore island (Codfish Island).

Exploring the potential for Ross River virus emergence in New Zealand.

Ross River virus (RRV) is an exotic vector-borne disease considered highly likely to emerge as a future human health issue in New Zealand, with its range expansion from Australia being driven by exotic mosquito introduction and improving conditions for mosquito breeding. We investigated our ability to assess the potential for such emergence using deterministic modeling and making preliminary predictions based on currently available evidence. Although data on actual mosquito densities (as opposed to indices) were identified as a need for predictions to be made with greater confidence, this approach generated a contrasting prediction to current opinion. Only limited potential for RRV emergence in New Zealand was predicted, with outbreaks in the human population more likely of concern in urban areas (mainly should major exotic vectors of the virus establish). The mechanistic nature of the model also allowed the understanding that if such outbreaks do occur, they will most likely be driven by virus amplification in dense human populations (as opposed to the spillover infection from wildlife common in Australia). With implications for biosecurity and health care resource allocation, modeling approaches such as that employed here have much to offer both for disease emergence prediction and surveillance strategy design.

The Trojan female technique: a novel, effective and humane approach for pest population control.

Humankind's ongoing battle with pest species spans millennia. Pests cause or carry disease, damage or consume food crops and other resources, and drive global environmental change. Conventional approaches to pest management usually involve lethal control, but such approaches are costly, of varying efficiency and often have ethical issues. Thus, pest management via control of reproductive output is increasingly considered an optimal solution. One of the most successful such 'fertility control' strategies developed to date is the sterile male technique (SMT), in which large numbers of sterile males are released into a population each generation. However, this approach is time-consuming, labour-intensive and costly. We use mathematical models to test a new twist on the SMT, using maternally inherited mitochondrial (mtDNA) mutations that affect male, but not female reproductive fitness. 'Trojan females' carrying such mutations, and their female descendants, produce 'sterile-male'-equivalents under natural conditions over multiple generations. We find that the Trojan female technique (TFT) has the potential to be a novel humane approach for pest control. Single large releases and relatively few small repeat releases of Trojan females both provided effective and persistent control within relatively few generations. Although greatest efficacy was predicted for high-turnover species, the additive nature of multiple releases made the TFT applicable to the full range of life histories modelled. The extensive conservation of mtDNA among eukaryotes suggests this approach could have broad utility for pest control.

Predicted responses of invasive mammal communities to climate-related changes in mast frequency in forest ecosystems.

Predicting the dynamics and impacts of multiple invasive species can be complex because ecological relationships, which occur among several trophic levels, are often incompletely understood. Further, the complexity of these trophic relationships exacerbates our inability to predict climate change effects on invaded ecosystems. We explore the hypothesis that interactions between two global change drivers, invasive vertebrates and climate change, will potentially make matters worse for native biodiversity. In New Zealand beech (Nothofagus spp.) forests, a highly irruptive invasive mammal community is driven by multi-annual resource pulses of beech seed (masting). Because mast frequency is predicted to increase with climate change, we use this as a model system to explore the extent to which such effects may influence invasive vertebrate communities, and the implications of such interactions for native biodiversity and its management. We build on an established model of trophic interactions in the system, combining it with a logistic probability mast function, the parameters of which were altered to simulate either contemporary conditions or conditions of more or less frequent masting. The model predicts that increased mast frequency will lead to populations of a top predator (the stoat) and a mesopredator (the ship rat) becoming less irruptive and being maintained at appreciably higher average abundances in this forest type. In addition, the ability of both current and in-development management approaches to suppress invasive mammals is predicted to be compromised. Because invasive mammals are key drivers of native fauna extinction in New Zealand, with the additional loss of associated functions such as pollination and seed dispersal, these predictions imply potentially serious adverse impacts of climate change for the conservation of biodiversity and ecosystem function. Our study also highlights the importance of long-term monitoring data for assessing and managing future impacts of global change drivers.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination.

BACKGROUND: Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS: Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS: Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION: The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.