An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

The Impact of Videofluoroscopic Pulse Rate on Duration and Kinematic Measures in Infants and Adults with Feeding and Swallowing Disorders.

This investigation assessed the impact of temporal resolution during a videofluoroscopic evaluation of swallowing (VFSS) on measures of duration and kinematics. Thirty adult and ten infant swallow studies, all acquired at 30 frames and 30 pulses per second, were obtained from a New Mexico hospital. All swallow studies were altered to simulate 15 and 5 pulses per second. Duration measures included pharyngeal response time, duration of upper esophageal sphincter (UES) opening, velopharyngeal closure duration and total swallow duration. Kinematic measures were assessed in adults only and included peak hyoid position and extent of UES opening during the swallow. Analysis of outcome measures was performed and compared across the three temporal resolutions (30, 15, and 5 pulses per second). For data points where normative values are available, we evaluated the impact of temporal resolution on clinical determination (i.e., did a change in pulse rate alter the clinical classification). Kinematic and duration measures were altered with changes in pulse rate and these changes increased as temporal resolution decreased. For outcome measures where normative values are available, accuracy of clinical determination decreased with decreased pulse rate. Temporal resolution impacts duration and kinematic measures. However, the direction of these changes is unpredictable, indicating sensitivity and specificity are both affected. Without a predictable impact, the use of lower pulse rates may alter clinical impressions and treatment recommendations yielding inappropriate treatment goals and treatment duration.

Measuring the cost-effectiveness of a home-visiting intervention to promote early child development among rural families linked to the Rwandan social protection system.

Early childhood development (ECD) programmes are heralded as a way to improve children's health and educational outcomes. However, few studies in developing countries calculate the effectiveness of quality early childhood interventions. This study estimates the cost and cost-effectiveness of the Sugira Muryango (SM) trial, a home-visiting intervention to improve ECD outcomes through positive parent-child relationships. Cost-effectiveness analysis of ECD interventions is challenging given their potential to have multiple benefits. We propose a cost-effectiveness method using a single outcome, in this case the improvement in cognitive development per home-visit session, as an indication of efficiency comparable across similar interventions. The trial intervention cost US$456 per family. This cost will likely fall below US$200 if the intervention is scaled through government systems. The cost-effectiveness analysis suggests that while SM generated a relatively small impact on markers of early development, it did so efficiently. The observed improvements in cognitive development per home-visit are similar to other home-visiting interventions of longer duration. SM by focusing on the family had benefits beyond ECD, including reductions in violence against children and intermate partner violence, further analysis is needed to include these returns in the economic evaluation.

Developing model systems for dinoflagellates in the post-genomic era.

Dinoflagellates are a diverse group of eukaryotic microbes that are ubiquitous in aquatic environments. Largely photosynthetic, they encompass symbiotic, parasitic, and free-living lineages with a broad spectrum of trophism. Many free-living taxa can produce bioactive secondary metabolites such as biotoxins, some of which cause harmful algal blooms. In contrast, most symbiotic species are crucial for sustaining coral reef health. The year 2023 marked a decade since the first genome data of dinoflagellates became available. The growing genome-scale resources for these taxa are highlighting their remarkable evolutionary and genomic complexities. Here, we discuss the prospect of developing dinoflagellate models using the criteria of accessibility, tractability, resources, research support, and promise. Moving forward in the post-genomic era, we argue for the development of fit-to-purpose models that tailor to specific biological contexts, and that a one-size-fits-all model is inadequate for encapsulating the complex biology, ecology, and evolutionary history of dinoflagellates.

Genomic copy number variability at the genus, species and population levels impacts in situ ecological analyses of dinoflagellates and harmful algal blooms.

The application of meta-barcoding, qPCR, and metagenomics to aquatic eukaryotic microbial communities requires knowledge of genomic copy number variability (CNV). CNV may be particularly relevant to functional genes, impacting dosage and expression, yet little is known of the scale and role of CNV in microbial eukaryotes. Here, we quantify CNV of rRNA and a gene involved in Paralytic Shellfish Toxin (PST) synthesis (sxtA4), in 51 strains of 4 Alexandrium (Dinophyceae) species. Genomes varied up to threefold within species and ~7-fold amongst species, with the largest (A. pacificum, 130 ± 1.3 pg cell-1 /~127 Gbp) in the largest size category of any eukaryote. Genomic copy numbers (GCN) of rRNA varied by 6 orders of magnitude amongst Alexandrium (102- 108 copies cell-1) and were significantly related to genome size. Within the population CNV of rRNA was 2 orders of magnitude (105 - 107 cell-1) in 15 isolates from one population, demonstrating that quantitative data based on rRNA genes needs considerable caution in interpretation, even if validated against locally isolated strains. Despite up to 30 years in laboratory culture, rRNA CNV and genome size variability were not correlated with time in culture. Cell volume was only weakly associated with rRNA GCN (20-22% variance explained across dinoflagellates, 4% in Gonyaulacales). GCN of sxtA4 varied from 0-102 copies cell-1, was significantly related to PSTs (ng cell-1), displaying a gene dosage effect modulating PST production. Our data indicate that in dinoflagellates, a major marine eukaryotic group, low-copy functional genes are more reliable and informative targets for quantification of ecological processes than unstable rRNA genes.

Response of planktonic microbial assemblages to disturbance in an urban sub-tropical estuary.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.

Family Violence Reduction Within a Parenting Intervention in Rwanda: A Mixed-Methods Study.

BACKGROUND AND OBJECTIVES: A mixed-methods study of mechanisms of change through which a home-visiting-based early childhood development intervention, Sugira Muryango ("strong family"), reduced violent discipline and intimate partner violence in Rwanda. METHODS: The cluster-randomized trial of Sugira Muryango enrolled socioeconomically vulnerable families with children aged 6 to 36 months in rural Rwanda. We interviewed 18 female caregivers early in the intervention, and 21 female caregivers and 11 male intimate partners were interviewed after the intervention. Coded interviews identify risk factors for violence and mechanisms of intervention-related change in violence. Quantitative analyses included 931 caregivers (52.6% female) who lived with an intimate partner to examine risk factors for violence, intervention effects, and mechanisms of violence reduction. RESULTS: The qualitative data identified daily hardships and alcohol problems as risk factors for violent discipline and intimate partner violence. Through Sugira Muryango, caregivers learned that strong relationships between partners and engagement of male caregivers in child care has positive impacts on children's development. Techniques taught by community lay workers improved communication, promoted positive parent-child interactions, and reduced intimate partner violence and violent discipline. Quantitative analyses also found that daily hardships predict violent discipline and intimate partner violence. Sugira Muryango reduced violent discipline, increased father engagement, and increased female caregiving warmth. Moreover, pre- to postintervention change in caregiving warmth was associated with reduced use of violent discipline among female caregivers and marginally associated with reduced female victimization. CONCLUSIONS: Violence reduction can be integrated into early child development programs to reduce violent discipline and intimate partner violence.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae).

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL-1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL-1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

Morphology and molecular phylogeny of the benthic dinoflagellates (Dinophyceae, Peridiniales) Amphidiniopsis crumena n. sp. and Amphidiniopsis nileribanjensis n. sp.

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4' 3a 7″ ?C 4?S 6″' 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI ('crumena group').

Geographical distribution, molecular and toxin diversity of the dinoflagellate species Gambierdiscus honu in the Pacific region.

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns.

Correction: McLennan et al. Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species. Microorganisms 2021, 9, 510.

The authors wish to make the following corrections to this paper [...].

Temperature variability interacts with mean temperature to influence the predictability of microbial phenotypes.

Despite their relatively high thermal optima (Topt ), tropical taxa may be particularly vulnerable to a rising baseline and increased temperature variation because they live in relatively stable temperatures closer to their Topt . We examined how microbial eukaryotes with differing thermal histories responded to temperature fluctuations of different amplitudes (0 control, ±2, ±4°C) around mean temperatures below or above their Topt . Cosmopolitan dinoflagellates were selected based on their distinct thermal traits and included two species of the same genus (tropical and temperate Coolia spp.), and two strains of the same species maintained at different temperatures for >500 generations (tropical Amphidinium massartii control temperature and high temperature, CT and HT, respectively). There was a universal decline in population growth rate under temperature fluctuations, but strains with narrower thermal niche breadth (temperate Coolia and HT) showed ~10% greater reduction in growth. At suboptimal mean temperatures, cells in the cool phase of the fluctuation stopped dividing, fixed less carbon (C) and had enlarged cell volumes that scaled positively with elemental C, N, and P and C:Chlorophyll-a. However, at a supra-optimal mean temperature, fixed C was directed away from cell division and novel trait combinations developed, leading to greater phenotypic diversity. At the molecular level, heat-shock proteins, and chaperones, in addition to transcripts involving genome rearrangements, were upregulated in CT and HT during the warm phase of the supra-optimal fluctuation (30 ± 4°C), a stress response indicating protection. In contrast, the tropical Coolia species upregulated major energy pathways in the warm phase of its supra-optimal fluctuation (25 ± 4°C), indicating a broadscale shift in metabolism. Our results demonstrate divergent effects between taxa and that temporal variability in environmental conditions interacts with changes in the thermal mean to mediate microbial responses to global change, with implications for biogeochemical cycling.

Mapping the development of a Dinophysis bloom in a shellfish aquaculture area using a novel molecular qPCR assay.

Diarrhetic shellfish toxins produced by certain species of the marine dinoflagellate Dinophysis can accumulate in shellfish in high concentrations, representing a significant food safety issue worldwide. This risk is routinely managed by monitoring programs in shellfish producing areas, however the methods used to detect these harmful marine microbes are not usually automated nor conducted onsite, and are often expensive and require specialized expertise. Here we designed a quantitative real-time polymerase chain reaction (qPCR) assay based on the ITS-5.8S ribosomal region of Dinophysis spp. and evaluated its specificity, efficiency, and sensitivity to detect species belonging to this genus. We designed and tested twenty sets of primers pairs using three species of Dinophysis - D. caudata, D. fortii and D. acuminata. We optimized a qPCR assay using the primer pair that sufficiently amplified each of the target species (Dacu_11F/Dacu_11R), and tested this assay for cross-reactivity with other dinoflagellates and diatoms in the laboratory (11 species) and in silico 8 species (15 strains) of Dinophysis, 3 species of Ornithocercus and 2 species of Phalacroma. The qPCR assay returned efficiencies of 92.4% for D. caudata, 91.3% for D fortii, and 91.5% for D. acuminata, while showing no cross-reactivity with other phytoplankton taxa. Finally, we applied this assay to a D. acuminata bloom which occurred in an oyster producing estuary in south eastern Australia, and compared cell numbers inferred by qPCR to those determined by microscopy counts (max abund. ∼6.3 × 103 and 5.3 × 103 cells L-1 respectively). Novel molecular tools such as qPCR have the potential to be used on-farm, be automated, and provide an early warning for the management of harmful algal blooms.

Impacts of harmful algal blooms on marine aquaculture in a low-carbon future.

The IPCC Special Report on Global Warming of 1.5 °C highlights the potential for dietary shifts to reduce greenhouse gas emissions from livestock. Reductions in the consumption of terrestrial animal protein require increases in the consumption of other food categories, to maintain food security, balanced dietary patterns, and protein intake. Aquaculture has long been suggested as one way to meet future food security needs, and marine and estuarine aquaculture in particular is associated with comparatively low greenhouse gas emissions. However, marine and freshwater aquaculture is affected by factors including harmful algal blooms (HABs), which have been increasingly documented around the world, correlated to increases in worldwide aquaculture. In this study, we applied a global multi-region input-output model to capture the direct effects as well as the indirect and induced effects HABs might pose to a global dietary transition from terrestrial livestock to increased seafood consumption from marine and estuarine aquaculture sources. We found that marine and estuarine aquaculture has a substantial potential to replace meat consumption from terrestrial livestock sources, as increases in CO2 emissions from aquaculture were more than offset by reductions in emissions from mainly cattle grazing and associated land clearing. HABs were found to have a minor monetary impact, but the impact on protein supply was found to be potentially sizeable. For example, in a future setting where 40% of terrestrial protein sources were replaced by aquaculture, a HAB-caused global loss of 5% would set in motion numerous supply-chain cascades, affecting industries auxiliary to aquaculture, indirectly and ultimately reducing protein intake by 10-20%. Such reductions have the potential for pushing parts of Sub-Saharan populations into protein-energy malnutrition. Nevertheless, there remains a significant potential for a dietary transition to increased aquaculture seafood to contribute to reductions in GHG.

Using qPCR and high-resolution sensor data to model a multi-species Pseudo-nitzschia (Bacillariophyceae) bloom in southeastern Australia.

Harmful algal blooms, including those caused by the toxic diatom Pseudo-nitzschia, can have significant impacts on human health, ecosystem functioning and ultimately food security. In the current study we characterized a bloom of species of Pseudo-nitzschia that occurred in a south-eastern Australian oyster-growing estuary in 2019. Using light microscopy, combined with molecular (ITS/5.8S and LSU D1-D3 rDNA regions) and toxicological evidence, we observed the bloom to consist of multiple species of Pseudo-nitzschia including P. cf. cuspidata, P. hasleana, P. fraudulenta and P. multiseries, with P. cf. cuspidata being the only species that produced domoic acid (3.1 pg DA per cell). As several species of Pseudo-nitzschia co-occurred, only one of which produced DA, we developed a rapid, sensitive and efficient quantitative real-time polymerase chain reaction (qPCR) assay to detect only species belonging to the P. pseudodelicatissima complex Clade I, to which P. cf. cuspidata belongs, and this indicated that P. cuspidata or closely related strains may have dominated the Pseudo-nitzschia community at this time. Finally, using high resolution water temperature and salinity sensor data, we modeled the relationship between light microscopy determined abundance of P. delicatissima group and environmental variables (temperature, salinity, rainfall) at two sites within the estuary. A total of eight General Linear Models (GLMs) explaining between 9 and 54% of the deviance suggested that the temperature (increasing) and/or salinity (decreasing) data were generally more predictive of high cell concentrations than the rainfall data at both sites, and that overall, cell concentrations were more predictive at the more oceanic site than the more upstream site, using this method. We conclude that the combination of rapid molecular methods such as qPCR and real-time sensor data modeling, can provide a more rapid and effective early warning of harmful algal blooms of species of Pseudo-nitzschia, resulting in more beneficial regulatory and management outcomes.

A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis.

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish-Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography-tandem Mass Spectrometry) and LC-MS (Liquid Chromatography-Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0-150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60-262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25-100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

Assessing the Use of Molecular Barcoding and qPCR for Investigating the Ecology of Prorocentrum minimum (Dinophyceae), a Harmful Algal Species.

Prorocentrum minimum is a species of marine dinoflagellate that occurs worldwide and can be responsible for harmful algal blooms (HABs). Some studies have reported it to produce tetrodotoxin; however, results have been inconsistent. qPCR and molecular barcoding (amplicon sequencing) using high-throughput sequencing have been increasingly applied to quantify HAB species for ecological analyses and monitoring. Here, we isolated a strain of P. minimum from eastern Australian waters, where it commonly occurs, and developed and validated a qPCR assay for this species based on a region of ITS rRNA in relation to abundance estimates from the cultured strain as determined using light microscopy. We used this tool to quantify and examine ecological drivers of P. minimum in Botany Bay, an estuary in southeast Australia, for over ~14 months in 2016-2017. We compared abundance estimates using qPCR with those obtained using molecular barcoding based on an 18S rRNA amplicon. There was a significant correlation between the abundance estimates from amplicon sequencing and qPCR, but the estimates from light microscopy were not significantly correlated, likely due to the counting method applied. Using amplicon sequencing, ~600 unique actual sequence variants (ASVs) were found, much larger than the known phytoplankton diversity from this region. P. minimum abundance in Botany Bay was found to be significantly associated with lower salinities and higher dissolved CO2 levels.

Effect of a home-visiting parenting program to promote early childhood development and prevent violence: a cluster-randomized trial in Rwanda.

INTRODUCTION: Families living in extreme poverty require interventions to support early-childhood development (ECD) due to broad risks. This longitudinal cluster randomised trial examines the effectiveness of Sugira Muryango (SM), a home-visiting intervention linked to Rwanda's social protection system to promote ECD and reduce violence compared with usual care (UC). METHODS: Families with children aged 6-36 months were recruited in 284 geographical clusters across three districts. Cluster-level randomisation (allocated 1:1 SM:UC) was used to prevent diffusion. SM was hypothesised to improve child development, reduce violence and increase father engagement. Developmental outcomes were assessed using the Ages and Stages Questionnaire (ASQ-3) and the Malawi Development Assessment Tool (MDAT) and anthropometric assessments of growth. Violence was assessed using questions from UNICEF Multiple Indicators Cluster Survey (MICS) and Rwanda Demographic and Health Surveys (DHS). Father engagement was assessed using the Home Observation for Measurement of the Environment. Blinded enumerators conducted interviews and developmental assessments. RESULTS: A total of 541 SM families and 508 UC families were enrolled and included in the analyses. Study attrition (2.0% children; 9.6% caregivers) was addressed by hot deck imputation. Children in SM families improved more on gross motor (d=0.162, 95% CI 0.065 to 0.260), communication (d=0.081, 95% CI 0.005 to 0.156), problem solving (d=0.101, 95% CI 0.002 to 0.179) and personal-social development (d=0.096, 95% CI -0.015 to 0.177) on the ASQ-3. SM families showed increased father engagement (OR=1.592, 95% CI 1.069 to 2.368), decreased harsh discipline (incidence rate ratio, IRR=0.741, 95% CI 0.657 to 0.835) and intimate partner violence (IRR=0.616, 95% CI:0.458 to 0.828). There were no intervention-related improvements on MDAT or child growth. CONCLUSION: Social protection programmes provide a means to deliver ECD intervention. TRIAL REGISTRATION NUMBER: NCT02510313.

Revealing RNA virus diversity and evolution in unicellular algae transcriptomes.

Remarkably little is known about the diversity and evolution of RNA viruses in unicellular eukaryotes. We screened a total of 570 transcriptomes from the Marine Microbial Eukaryote Transcriptome Sequencing Project that encompasses a wide diversity of microbial eukaryotes, including most major photosynthetic lineages (i.e. the microalgae). From this, we identified thirty new and divergent RNA virus species, occupying a range of phylogenetic positions within the overall diversity of RNA viruses. Approximately one-third of the newly described viruses comprised single-stranded positive-sense RNA viruses from the order Lenarviricota associated with fungi, plants, and protists, while another third were related to the order Ghabrivirales, including members of the protist and fungi-associated Totiviridae. Other viral species showed sequence similarity to positive-sense RNA viruses from the algae-associated Marnaviridae, the double-stranded RNA (ds-RNA) Partitiviridae, as well as tentative evidence for one negative-sense RNA virus related to the Qinviridae. Importantly, we were able to identify divergent RNA viruses from distant host taxa, revealing the ancestry of these viral families and greatly extending our knowledge of the RNA viromes of microalgal cultures. Both the limited number of viruses detected per sample and the low sequence identity to known RNA viruses imply that additional microalgal viruses exist that could not be detected at the current sequencing depth or were too divergent to be identified using sequence similarity. Together, these results highlight the need for further investigation of algal-associated RNA viruses as well as the development of new tools to identify RNA viruses that exhibit very high levels of sequence divergence.

Intergenerational impacts of trauma and hardship through parenting.

BACKGROUND: Millions of people worldwide experience severe trauma in their lifetime. Trauma has immediate and long-term effects on emotional wellbeing. Moreover, the experiences of one generation may influence subsequent generations via social and biological pathways. Poor mental health and emotion dysregulation associated with trauma may affect parenting behaviours, which may have long-lasting effects on children's development. METHODS: We use longitudinal data from a unique sample of 732 caregivers of children aged 6-36 months living in extremely poor rural households in Rwanda to examine associations of caregiver lifetime trauma, recent daily hardships, mental health, and emotion dysregulation with parenting behaviours reflecting parental acceptance and rejection of their offspring. RESULTS: Cumulative trauma exposure (ß = .234, p < .001) and recent daily hardships (ß = .323, p < .001) are associated with higher levels of internalising symptoms. Trauma (ß = .257, p < .001) and daily hardships (ß = .323, p < 0.001) are also associated with post-traumatic stress disorder (PTSD) symptoms. Internalising symptoms predict more rejection (ß = .177, p = .001), but show no association with acceptance. Caregiver PTSD symptoms predict more rejection (ß = .277, p < .001) and less acceptance (ß = -.190, p = .003). Both internalising symptoms (ß = .557, p < .001) and PTSD symptoms (ß = .606, p < .001) are strongly associated with poor emotion regulation. Indirect effects suggest that caregiver trauma and hardships affect parenting indirectly via elevated caregiver internalising symptoms and PTSD and that some of these effects are accounted for by emotion dysregulation. CONCLUSIONS: Caregiver internalising and PTSD symptoms are important mechanisms through which caregiver trauma and hardship affect parenting behaviours. Emotion dysregulation is a shared mechanism linking caregivers' mental health problems with parenting behaviours that reflect acceptance and rejection of the child. Emotion regulation is indicated as a key target for prevention of adverse effects of caregiver trauma on mental health and child wellbeing.