The evolving story of sulfoxaflor resistance in the green peach aphid, Myzus persicae (Sulzer).

BACKGROUND: The green peach aphid, Myzus persicae (Sulzer), is one of the most economically important crop pests worldwide. Insecticide resistance in this pest was first detected over 60 years ago, with resistance in M. persicae now spanning over 80 active ingredients. Sulfoxaflor is a relatively new insecticide that is primarily used to control sap-feeding insects. In 2018 resistance to sulfoxaflor was discovered in field populations of M. persicae in Australia. This study aimed to determine the current distribution and phenotypic levels of sulfoxaflor resistance in Australian clones of M. persicae and to investigate how these patterns relate to clonal type. RESULTS: For the first time, we show there is low-level resistance (8-26-fold) distributed across Australia, with resistance being detected in aphids collected from approximately 20% of all M. persicae collected and screened. Furthermore, this study shows sulfoxaflor resistance is found in two M. persicae haplotypes, providing evidence that there have been multiple independent evolutionary events which have given rise to sulfoxaflor resistance in this species. CONCLUSION: These findings have important implications for the chemical control of M. persicae in Australia, especially when considering the broader genetic background of these aphids which are known to harbour a number of other insecticide resistance mechanisms. We recommend continuous monitoring of sulfoxaflor resistance in field populations of M. persicae (in Australia and elsewhere) and further research into the underlying genetic mechanisms conferring resistance to sulfoxaflor in both clonal haplotypes. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Heterogeneous patterns of heterozygosity loss in isolated populations of the threatened eastern barred bandicoot (Perameles gunnii).

Identifying and analysing isolated populations is critical for conservation. Isolation can make populations vulnerable to local extinction due to increased genetic drift and inbreeding, both of which should leave imprints of decreased genome-wide heterozygosity. While decreases in heterozygosity among populations are frequently investigated, fewer studies have analysed how heterozygosity varies among individuals, including whether heterozygosity varies geographically along lines of discrete population structure or with continuous patterns analogous to isolation by distance. Here we explore geographical patterns of differentiation and individual heterozygosity in the threatened eastern barred bandicoot (Perameles gunnii) in Tasmania, Australia, using genomic data from 85 samples collected between 2008 and 2011. Our analyses identified two isolated demes undergoing significant genetic drift, and several areas of fine-scale differentiation across Tasmania. We observed discrete genetic structures across geographical barriers and continuous patterns of isolation by distance, with little evidence of recent or historical migration. Using a recently developed analytical pipeline for estimating autosomal heterozygosity, we found individual heterozygosities varied within demes by up to a factor of two, and demes with low-heterozygosity individuals also still contained those with high heterozygosity. Spatial interpolation of heterozygosity scores clarified these patterns and identified the isolated Tasman Peninsula as a location where low-heterozygosity individuals were more common than elsewhere. Our results provide novel insights into the relationship between isolation-driven genetic structure and local heterozygosity patterns. These may help improve translocation efforts, by identifying populations in need of assistance, and by providing an individualised metric for identifying source animals for translocation.

P450-mediated resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) reduces the efficacy of neonicotinoid seed treatments in Brassica napus.

BACKGROUND: The prophylactic use of seeds treated with neonicotinoid insecticides remains an important means of controlling aphid pests in canola (Brassica napus) crops in many countries. Yet, one of the most economically important aphid species worldwide, the peach potato aphid (Myzus persicae), has evolved mechanisms which confer resistance to neonicotinoids, including amplification of the cytochrome P450 gene, CYP6CY3. While CYP6CY3 amplification has been associated with low-level resistance to several neonicotinoids in laboratory acute toxicity bioassays, its impact on insecticide efficacy in the field remains unresolved. In this study, we investigated the impact of CYP6CY3 amplification on the ability of M. persicae to survive neonicotinoid exposure under laboratory and semi-field conditions. RESULTS: Three M. persicae clones, possessing different copy numbers of CYP6CY3, were shown to respond differently when exposed to the neonicotinoids, imidacloprid and thiamethoxam, in laboratory bioassays. Two clones, EastNaernup209 and Osborne171, displayed low levels of resistance (3-20-fold), which is consistent with previous studies. However, in a large-scale semi-field trial, both clones showed a surprising ability to survive and reproduce on B. napus seedlings grown from commercial rates of neonicotinoid-treated seed. In contrast, an insecticide-susceptible clone, of wild-type CYP6CY3 copy number, was unable to survive on seedlings treated in the same manner. CONCLUSION: Our findings suggest that amplification of CYP6CY3 in M. persicae clones substantially impairs the efficacy of neonicotinoid seed treatments when applied to B. napus. These findings highlight the potentially important real-world implications of resistances typically considered to be 'low level' as defined through laboratory bioassays. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Spirotetramat resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) and its association with the presence of the A2666V mutation.

BACKGROUND: Chemicals are widely used to protect field crops against aphid pests and aphid-borne viral diseases. One such species is Myzus persicae (Sulzer), a global pest that attacks a broad array of agricultural crops and transmits many economically damaging plant viruses. This species has evolved resistance to a large number of insecticide compounds as a result of widespread and repeated chemical use in many parts of the world. In this study, we investigated the evolution of resistance to a new plant protection product, spirotetramat, following reported chemical control failures. RESULTS: Our study provides clear phenotypic and genotypic evidence of spirotetramat resistance in populations of M. persicae from Australia. We show there is cross-resistance to other insecticides within the same chemical group, namely spiromesifen and spirodiclofen. We also demonstrate that resistance is associated with the previously reported mutation, A2226V in the target site of spirotetramat, acetyl-CoA carboxylase. Our genetic analysis found all resistant M. persicae populations belong to the same multi-locus clonal type and carry the A2226V mutation, which appears to be inherited as a dominant trait in this species. CONCLUSION: Our findings provide new insight into the resistance conferred by A2226V and have implications for the control of M. persicae in Australia and worldwide. A diagnostic assay developed in this study should serve as a valuable tool for future resistance monitoring and to support the implementation of pest management strategies. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Australian Bryobia mites (Trombidiformes: Tetranychidae) form a complex of cryptic taxa with unique climatic niches and insecticide responses.

BACKGROUND: Bryobia (Koch) mites belong to the economically important spider mite family, the Tetranychidae, with >130 species described worldwide. Due to taxonomic difficulties and most species being asexual, species identification relies heavily on genetic markers. Multiple putative Bryobia mite species have been identified attacking pastures and grain crops in Australia. In this study, we collected 79 field populations of Bryobia mites and combined these with 134 populations that were collected previously. We characterised taxonomic variation of mites using 28S rDNA amplicon-based DNA metabarcoding using next-generation sequencing approaches and direct Sanger sequencing. We then undertook species distribution modelling of the main genetic lineages and examined the chemical responses of multiple field populations. RESULTS: We identified 47 unique haplotypes across all mites sampled that grouped into four distinct genetic lineages. These lineages have different distributions, with three of the four putative lineages showing different climatic envelopes, as inferred from species distribution modelling. Bryobia mite populations also showed different responses to a widely used insecticide (the organophosphate, omethoate), but not to another chemical (the pyrethroid, bifenthrin) when examined using laboratory bioassays. CONCLUSION: Our findings indicate that cryptic diversity is likely to complicate the formulation of management strategies for Bryobia mites. Although focussed on Australia, this study demonstrates the challenges of studying Bryobia and highlights the importance of further research into this complex group of mites across the world. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Overexpression of UDP-glucuronosyltransferase and cytochrome P450 enzymes confers resistance to sulfoxaflor in field populations of the aphid, Myzus persicae.

The green peach aphid, Myzus persicae, is a highly damaging, globally distributed crop pest that has evolved multiple resistance to numerous insecticides. It is thus imperative that insecticides that are not strongly compromised by pre-existing resistance are carefully managed to maximise their effective life span. Sulfoxaflor is a sulfoximine insecticide that retains efficacy against M. persicae clones that exhibit resistance to older insecticides. In the current study we monitored the efficacy of sulfoxaflor against M. persicae populations collected in Western Australia, following reports of control failures in this region. We identified clones with low (4-23-fold across multiple independent bioassay experiments), but significant, levels of resistance to sulfoxaflor compared with a reference susceptible clone. Furthermore, we demonstrate that sulfoxaflor resistance can persist after many months of culturing in the laboratory in the absence of insecticide exposure. Resistance was not conferred by known mechanisms of resistance to neonicotinoid insecticides, that act on the same target-site as sulfoxaflor, i.e. the R81T mutation or overexpresssion of the P450 gene CYP6CY3. Rather, transcriptome profiling of multiple resistant and susceptible clones identified the P450 CYP380C40 and the UDP-glucuronosyltransferase UGT344P2 as highly overexpressed (21-76-fold and 6-33-fold respectively) in the resistant clones. Transgenic expression of these genes demonstrated that they confer, low, but significant, levels of resistance to sulfoxaflor in vivo. Taken together, our data reveal the presence of low-level resistance to sulfoxaflor in M. persicae populations in Australia and uncover two novel mechanisms conferring resistance to this compound. The findings and tools generated in this study provide a platform for the development of strategies that aim to slow, prevent or overcome the evolution of more potent resistance to sulfoxaflor.

A molecular method for biomonitoring of an exotic plant-pest: Leafmining for environmental DNA.

Understanding how invasive species respond to novel environments is limited by a lack of sensitivity and throughput in conventional biomonitoring methods. Arthropods in particular are often difficult to monitor due to their small size, rapid lifecycles, and/or visual similarities with co-occurring species. This is true for the agromyzid leafminer fly, Liriomyza sativae, a global pest of vegetable and nursery industries that has recently established in Australia. A robust method based on environmental DNA (eDNA) was developed exploiting traces of DNA left inside "empty" leaf mines, which are straightforward to collect and persist longer in the environment than the fly. This extends the window of possible diagnosis to at least 28 days after a leaf mine becomes empty. The test allowed for visually indistinguishable leafmining damage caused by L. sativae to be genetically differentiated from that of other flies. Field application resulted in the identification of new local plant hosts for L. sativae, including widely distributed weeds and common garden crops, which has important implications for the pest's ability to spread. Moreover, the test confirmed the presence of a previously unknown population of L. sativae on an island in the Torres Strait. The developed eDNA method is likely to become an important tool for L. sativae and other leafmining species of biosecurity significance, which, historically, have been difficult to detect, diagnose and monitor. More generally, eDNA is emerging as a highly sensitive and labour-efficient surveillance tool for difficult to survey species to improve outcomes for agricultural industries, global health, and the environment.

Strip spraying delays pyrethroid resistance in the redlegged earth mite, Halotydeus destructor: a novel refuge strategy.

BACKGROUND: Pesticide resistance has seen control options for the redlegged earth mite (RLEM), Halotydeus destructor, dwindle for Australian grain farmers. The recent discovery of high recessiveness for pyrethroid resistance in RLEM provided an opportunity to examine the feasibility of a refuge strategy to slow the evolution of resistance. Unlike lepidopterous pests in Bt crops, where refuge strategies are routinely practiced, RLEM is a slow-moving pest, which will impact the design of susceptible refuges. RESULTS: Firstly, we confirmed the pyrethroid resistant allele is recessive to the susceptible (wildtype) allele (in terms of resistance) across spatially separated Australian populations. Secondly, we demonstrated that a small, localized resistant mite population can revert to susceptibility at field relevant scales and conditions. Next, we used a simulation modelling approach to design a practical refuge strategy to maintain susceptibility to pyrethroids in populations with a low incidence of resistance. Certain configurations (e.g. a pesticide strip width of 50 m and refuge spacing of 10 m) maintained low levels of resistance across a 10-year time horizon, with lower mite abundance and minimal yield loss. A larger refuge proportion did not always delay resistance, and, under certain conditions, increased resistance frequency. CONCLUSION: Strip spraying to maintain refuges can be readily incorporated into RLEM management programs where sprayer widths in commercial cropping contexts are typically between 20-40 m. A refuge approach to RLEM management that uses strip spraying may enhance long term control options in the absence of new chemical registrations but will now require field validation. © 2021 Society of Chemical Industry.

Learnings from over a decade of increasing pesticide resistance in the redlegged earth mite, Halotydeus destructor (Tucker).

BACKGROUND: The redlegged earth mite, Halotydeus destructor (Tucker), is a destructive and economically important pest of winter grain crops and pastures in Australia. It is largely controlled by pesticides, but this mite has evolved resistance to pyrethroid and organophosphate chemicals. A national Resistance Management Strategy has been developed for pro-active management to delay further resistance evolution, though its success is reliant on a detailed understanding of the incidence, patterns of spread, current distribution and the nature of resistance in the field. Here, we report on a long-term resistance surveillance programme undertaken between 2006 and 2019 informed by resistance risk forecasting. RESULTS: By mapping the Australian distribution of resistance through time, we show that resistance is present across three Australian states and covers more than 3000 km. This current range includes a recently identified population exhibiting organophosphate resistance representing the most easterly location of resistance in H. destructor. Using field history information, we identify associations for the first time between crop management practices employed by farmers and the presence of pyrethroid resistance. Management strategies that could minimize the risk of further resistance include limiting local spread of resistance through farm hygiene practices, crop rotations and reducing pesticide usage. CONCLUSION: This study highlights the challenges of resistance in H. destructor but also indicates how quantitative resistance risk analysis can be developed to target field surveillance and delay further resistance. The management strategies highlighted in this study can help maintain the effectiveness of control options but will depend on farmer engagement and adoption. © 2021 Society of Chemical Industry.

Incursion pathways of the Asian tiger mosquito (Aedes albopictus) into Australia contrast sharply with those of the yellow fever mosquito (Aedes aegypti).

BACKGROUND: Understanding pest incursion pathways is critical for preventing new invasions and for stopping the transfer of alleles that reduce the efficacy of local control methods. The mosquitoes Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are both highly invasive disease vectors, and through a series of ongoing international incursions are continuing to colonize new regions and spread insecticide resistance alleles among established populations. This study uses high-resolution molecular markers and a set of 241 reference genotypes to trace incursion pathways of Ae. albopictus into mainland Australia, where no successful invasions have yet been observed. We contrast these results with incursion pathways of Ae. aegypti, investigated previously. RESULTS: Assignments successfully identified China, Japan, Singapore and Taiwan as source locations. Incursion pathways of Ae. albopictus were entirely different to those of Ae. aegypti, despite broad sympatry of these species throughout the Indo-Pacific region. Incursions of Ae. albopictus appeared to have come predominantly along marine routes from key trading locations, while Ae. aegypti was mostly linked to aerial routes from tourism hotspots. CONCLUSION: These results demonstrate how genomics can help decipher otherwise cryptic incursion pathways. The inclusion of reference genotypes from the Americas may help resolve some unsuccessful assignments. While many congeneric taxa will share common incursion pathways, this study highlights that this is not always the case, and incursion pathways of important taxa should be specifically investigated. Species differences in aerial and marine incursion rates may reflect the efficacy of ongoing control programmes such as aircraft disinsection. © 2020 Society of Chemical Industry.

Heterogeneous genetic invasions of three insecticide resistance mutations in Indo-Pacific populations of Aedes aegypti (L.).

Nations throughout the Indo-Pacific region use pyrethroid insecticides to control Aedes aegypti, the mosquito vector of dengue, often without knowledge of pyrethroid resistance status of the pest or origin of resistance. Two mutations (V1016G + F1534C) in the sodium channel gene (Vssc) of Ae. aegypti modify ion channel function and cause target-site resistance to pyrethroid insecticides, with a third mutation (S989P) having a potential additive effect. Of 27 possible genotypes involving these mutations, some allelic combinations are never seen whereas others predominate. Here, five allelic combinations common in Ae. aegypti from the Indo-Pacific region are described and their geographical distributions investigated using genome-wide SNP markers. We tested the hypothesis that resistance allele combinations evolved de novo in populations versus the alternative that dispersal of Ae. aegypti between populations facilitated genetic invasions of allele combinations. We used latent factor mixed-models to detect SNPs throughout the genome that showed structuring in line with resistance allele combinations and compared variation at SNPs within the Vssc gene with genome-wide variation. Mixed-models detected an array of SNPs linked to resistance allele combinations, all located within or in close proximity to the Vssc gene. Variation at SNPs within the Vssc gene was structured by resistance profile, whereas genome-wide SNPs were structured by population. These results demonstrate that alleles near to resistance mutations have been transferred between populations via linked selection. This indicates that genetic invasions have contributed to the widespread occurrence of Vssc allele combinations in Ae. aegypti in the Indo-Pacific region, pointing to undocumented mosquito invasions between countries.

Tracking genetic invasions: Genome-wide single nucleotide polymorphisms reveal the source of pyrethroid-resistant Aedes aegypti (yellow fever mosquito) incursions at international ports.

Biological invasions are increasing globally in number and extent despite efforts to restrict their spread. Knowledge of incursion pathways is necessary to prevent new invasions and to design effective biosecurity protocols at source and recipient locations. This study uses genome-wide single nucleotide polymorphisms (SNPs) to determine the origin of 115 incursive Aedes aegypti(yellow fever mosquito) detected at international ports in Australia and New Zealand. We also genotyped mosquitoes at three point mutations in the voltage-sensitive sodium channel (Vssc) gene: V1016G, F1534C and S989P. These mutations confer knockdown resistance to synthetic pyrethroid insecticides, widely used for controlling invertebrate pests. We first delineated reference populations using Ae. aegypti sampled from 15 locations in Asia, South America, Australia and the Pacific Islands. Incursives were assigned to these populations using discriminant analysis of principal components (DAPC) and an assignment test with a support vector machine predictive model. Bali, Indonesia, was the most common origin of Ae. aegypti detected in Australia, while Ae. aegypti detected in New Zealand originated from Pacific Islands such as Fiji. Most incursives had the same allelic genotype across the three Vsscgene point mutations, which confers strong resistance to synthetic pyrethroids, the only insecticide class used in current, widely implemented aircraft disinsection protocols endorsed by the World Health Organization (WHO). Additionally, all internationally assigned Ae. aegypti had Vssc point mutations linked to pyrethroid resistance that are not found in Australian populations. These findings demonstrate that protocols for preventing introductions of invertebrates must consider insecticide resistance, and highlight the usefulness of genomic data sets for managing global biosecurity objectives.

Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion.

Parthenogenetic reproduction is taxonomically widespread and occurs through various cytological mechanisms, which have different impact on the genetic variation of the offspring. Extatosoma tiaratum is a facultatively parthenogenetic Australian insect (Phasmatodea), in which females oviposit continuously throughout their adult lifespan irrespective of mating. Fertilized eggs produce sons and daughters through sexual reproduction and unfertilized eggs produce female offspring via parthenogenesis. Here, we developed novel microsatellite markers for E. tiaratum and characterized them by genotyping individuals from a natural population. We then used the microsatellite markers to infer the cytological mechanism of parthenogenesis in this species. We found evidence suggesting parthenogenesis in E. tiaratum occurs through automixis with terminal fusion, resulting in substantial loss of microsatellite heterozygosity in the offspring. Loss of microsatellite heterozygosity may be associated with loss of heterozygosity in fitness related loci. The mechanism of parthenogenetic reproduction can therefore affect fitness outcomes and needs to be considered when comparing costs and benefits of sex versus parthenogenesis.

Genetic rescue increases fitness and aids rapid recovery of an endangered marsupial population.

Genetic rescue has now been attempted in several threatened species, but the contribution of genetics per se to any increase in population health can be hard to identify. Rescue is expected to be particularly useful when individuals are introduced into small isolated populations with low levels of genetic variation. Here we consider such a situation by documenting genetic rescue in the mountain pygmy possum, Burramys parvus. Rapid population recovery occurred in the target population after the introduction of a small number of males from a large genetically diverged population. Initial hybrid fitness was more than two-fold higher than non-hybrids; hybrid animals had a larger body size, and female hybrids produced more pouch young and lived longer. Genetic rescue likely contributed to the largest population size ever being recorded at this site. These data point to genetic rescue as being a potentially useful option for the recovery of small threatened populations.

Discovery of metabolic resistance to neonicotinoids in green peach aphids (Myzus persicae) in Australia.

BACKGROUND: Myzus persicae is a serious pest that attacks a broad range of agricultural crops. This species has developed chemical resistance to many insecticides globally, and within Australia resistance to multiple chemical groups has been identified. Resistance to neonicotinoid insecticides has been discovered in several countries, but has not previously been confirmed in Australia. We use biomolecular assays and bioassays on field-collected populations to investigate neonicotinoid resistance in M. persicae within Australia. RESULTS: Several geographically and genetically distinct populations showed evidence for resistance in bioassays. Genetic markers identified that the mechanism of neonicotinoid resistance in Australia is metabolic resistance through the enhanced expression of a cytochrome P450 gene, CYP6CY3. CONCLUSION: M. persicae populations in parts of Australia are now resistant to four different insecticide chemical groups, raising concerns about the long-term management of this pest. While higher copy numbers of CYP6CY3 were seen in all resistant populations, the number of gene copies was not strongly correlated with the level of resistance as determined by LD50 values generated through bioassays. This finding sheds further light on the complexity of the P450 genes in regulating neonicotinoid resistance. © 2016 Society of Chemical Industry.

I Environmental DNA sampling is more sensitive than a traditional survey technique for detecting an aquatic invader.

Effective management of alien species requires detecting populations in the early stages of invasion. Environmental DNA (eDNA) sampling can detect aquatic species at relatively low densities, but few studies have directly compared detection probabilities of eDNA sampling with those of traditional sampling methods. We compare the ability of a traditional sampling technique (bottle trapping) and eDNA to detect a recently established invader, the smooth newt Lissotriton vulgaris vulgaris, at seven field sites in Melbourne, Australia. Over a four-month period, per-trap detection probabilities ranged from 0.01 to 0.26 among sites where L. v. vulgaris was detected, whereas per-sample eDNA estimates were much higher (0.29-1.0). Detection probabilities of both methods varied temporally (across days and months), but temporal variation appeared to be uncorrelated between methods. Only estimates of spatial variation were strongly correlated across the two sampling techniques. Environmental variables (water depth, rainfall, ambient temperature) were not clearly correlated with detection probabilities estimated via trapping, whereas eDNA detection probabilities were negatively correlated with water depth, possibly reflecting higher eDNA concentrations at lower water levels. Our findings demonstrate that eDNA sampling can be an order of magnitude more sensitive than traditional methods, and illustrate that traditional- and eDNA-based surveys can provide independent information on species distributions when occupancy surveys are conducted over short timescales.

High levels of resistance to carbamate and pyrethroid chemicals widespread in Australian Myzus persicae (Hemiptera: Aphididae) populations.

The green peach aphid, Myzus persicae (Sulzer), is a serious pest throughout the world, attacking a broad range of crop plants across numerous agricultural industries. This species has a high propensity to develop chemical resistance, and has the unenviable title of having resistance to more insecticides than any other insect species. An extensive survey of field populations was undertaken across Australia, and showed widespread and high levels of resistance to carbamates and synthetic pyrethroids in M. persicae. Moderate levels of resistance to organophosphates were also observed in many populations, while there is new evidence of resistance developing to neonicotinoids. Isofemale (clonal) lines of M. persicae were generated and subsequently tested across a range of insecticides; individual genetic clones were found to contain resistance to multiple chemical classes. Resistance genotyping of these aphids were consistent with published literature of known resistant mechanisms. The high and widespread levels of resistance identified within Australia are concerning. Resistance in M. persicae has spread quickly across Australia, and thus farmers are likely to have fewer chemical control options in the future. There is a need to develop resistance management strategies that rotate insecticides, spray insecticides only when economically necessary, and incorporate nonchemical control options.

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus.

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as 'endangered' under the Environment Protection and Biodiversity Conservation Act 1999, and 'vulnerable' under the International Union for Conservation of Nature's Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F ST = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.

Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica.

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO(3)) to generate shells or skeletons. Studies of potential effects of future levels of pCO(2) on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO(2) levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (Ω(Ar) = 0.71), the CaCO(3) polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.