TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

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

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

Quantifying impacts of an environmental intervention using environmental DNA.

Environmental laws around the world require some version of an environmental-impact assessment surrounding construction projects and other discrete instances of human development. Information requirements for these assessments vary by jurisdiction, but nearly all require an analysis of the biological elements of ecosystems. Amplicon-sequencing-also called metabarcoding-of environmental DNA (eDNA) has made it possible to sample and amplify the genetic material of many species present in those environments, providing a tractable, powerful, and increasingly common way of doing environmental-impact analysis for development projects. Here, we analyze an 18-month time series of water samples taken before, during, and after two culvert removals in a salmonid-bearing freshwater stream. We also sampled multiple control streams to develop a robust background expectation against which to evaluate the impact of this discrete environmental intervention in the treatment stream. We generate calibrated, quantitative metabarcoding data from amplifying the 12s MiFish mtDNA locus and complementary species-specific quantitative PCR data to yield multispecies estimates of absolute eDNA concentrations across time, creeks, and sampling stations. We then use a linear mixed effects model to reveal patterns of eDNA concentrations over time, and to estimate the effects of the culvert removal on salmonids in the treatment creek. We focus our analysis on four common salmonid species: cutthroat trout (Oncorhynchus clarkii), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), and sockeye salmon (Oncorhynchus nerka). We find that one culvert in the treatment creek seemed to have no impact while the second culvert had a large impact on fish passage. The construction itself seemed to have only transient effects on salmonid species during the two construction events. In the context of billions of dollars of court-mandated road culvert replacements taking place in Washington State, USA, our results suggest that culvert replacement can be conducted with only minimal impact of construction to key species of management concern. Furthermore, eDNA methods can be an effective and efficient approach for monitoring hundreds of culverts to prioritize culverts that are required to be replaced. More broadly, we demonstrate a rigorous, quantitative method for environmental-impact reporting using eDNA that is widely applicable in environments worldwide.

Sponge organic matter recycling: Reduced detritus production under extreme environmental conditions.

Sponges are a key component of coral reef ecosystems and play an important role in carbon and nutrient cycles. Many sponges are known to consume dissolved organic carbon and transform this into detritus, which moves through detrital food chains and eventually to higher trophic levels via what is known as the sponge loop. Despite the importance of this loop, little is known about how these cycles will be impacted by future environmental conditions. During two years (2018 and 2020), we measured the organic carbon, nutrient recycling, and photosynthetic activity of the massive HMA, photosymbiotic sponge Rhabdastrella globostellata at the natural laboratory of Bouraké in New Caledonia, where the physical and chemical composition of seawater regularly change according to the tide. We found that while sponges experienced acidification and low dissolved oxygen at low tide in both sampling years, a change in organic carbon recycling whereby sponges stopped producing detritus (i.e., the sponge loop) was only found when sponges also experienced higher temperature in 2020. Our findings provide new insights into how important trophic pathways may be affected by changing ocean conditions.

Phototrophic sponge productivity may not be enhanced in a high CO2 world.

Sponges are major components of benthic communities across the world and have been identified as potential "winners" on coral reefs in the face of global climate change as result of their tolerance to ocean warming and acidification (OA). Previous studies have also hypothesised that photosymbiont-containing sponges might have higher productivity under future OA conditions as a result of photosymbionts having increased access to CO2 and subsequently greater carbon production. Here we test this hypothesis for a widespread and abundant photosymbiont-containing sponge species Lamellodysidea herbacea at a CO2 seep in Papua New Guinea simulating OA conditions. We found seep sponges had relatively higher cyanobacterial abundance, chlorophyll concentrations and symbiont photosynthetic efficiency than non-seep sponges, and a three-fold higher sponge abundance at the seep site. However, while gross oxygen production was the same for seep and non-seep sponges, seep sponge dark respiration rates were higher and instantaneous photosynthesis: respiration (P:R) ratios were lower. We show that while photosymbiont containing sponges may not have increased productivity under OA, they are able to show flexibility in their relationships with microbes and offset increased metabolic costs associated with climate change associated stress.

Bioeroding sponge species from the Wakatobi region of southeast Sulawesi, Indonesia.

Sponges that excavate and inhabit calcareous substrate, predominantly of the Clionaidae, are widely distributed in marine habitats, but are particularly diverse and abundant on coral reefs. Unfortunately, their cryptic habit and difficult taxonomy mean respective taxa are poorly understood, and therefore they are rarely included in reef surveys. This is particularly true of the Southeast Asian Indo-Pacific, where a diverse faunistic record is contrasted with a very limited understanding of eco-physiological requirements of these sponges. In light of this, in situ surveys of the most common bioeroding sponges in the Wakatobi region of Southeast Sulawesi, Indonesia were conducted, followed by morphological and molecular analysis. The seven most common species in the Wakatobi included five species with a wide Indo-Pacific distribution and two new species. Four species, Cliona orientalis, Cliona aff. schmidtii, Spheciospongia cf. vagabunda trincomaliensis and Cliothosa hancocki have been previously reported from Indonesian waters, while Zyzzya criceta is a new record for Indonesia. Two brown sponges belonging to the Cliona viridis species complex, Cliona wakatobiensis sp. nov. and Cliona cribripora sp. nov., are here described as new to science. They were morphologically distinct from other species in the sample region and could not conclusively be allocated to any other known clionaid species. A maximum likelihood analysis of ITS1 rDNA revealed them to be phylogenetically closer to other Indo-Pacific species such as Cliona orientalis and Cliona thomasi than to other species within this taxon complex.

Interocean patterns in shallow water sponge assemblage structure and function.

Sponges are a major component of benthic ecosystems across the world and fulfil a number of important functional roles. However, despite their importance, there have been few attempts to compare sponge assemblage structure and ecological functions across large spatial scales. In this review, we examine commonalities and differences between shallow water (<100 m) sponges at bioregional (15 bioregions) and macroregional (tropical, Mediterranean, temperate, and polar) scales, to provide a more comprehensive understanding of sponge ecology. Patterns of sponge abundance (based on density and area occupied) were highly variable, with an average benthic cover between ~1 and 30%. Sponges were generally found to occupy more space (percentage cover) in the Mediterranean and polar macroregions, compared to temperate and tropical macroregions, although sponge densities (sponges m-2 ) were highest in temperate bioregions. Mean species richness standardised by sampling area was similar across all bioregions, except for a few locations that supported very high small-scale biodiversity concentrations. Encrusting growth forms were generally the dominant sponge morphology, with the exception of the Tropical West Atlantic, where upright forms dominated. Annelids and Arthropods were the most commonly reported macrofauna associated with sponges across bioregions. With respect to reproduction, there were no patterns in gametic development (hermaphroditism versus gonochorism), although temperate, tropical, and polar macroregions had an increasingly higher percentage of viviparous species, respectively, with viviparity being the sole gamete development mechanism reported for polar sponges to date. Seasonal reproductive timing was the most common in all bioregions, but continuous timing was more common in the Mediterranean and tropical bioregions compared to polar and temperate bioregions. We found little variation across bioregions in larval size, and the dominant larval type across the globe was parenchymella. No pattens among bioregions were found in the limited information available for standardised respiration and pumping rates. Many organisms were found to predate sponges, with the abundance of sponge predators being higher in tropical systems. While there is some evidence to support a higher overall proportion of phototrophic species in the Tropical Austalian bioregion compared to the Western Atlantic, both also have large numbers of heterotrophic species. Sponges are important spatial competitors across all bioregions, most commonly being reported to interact with anthozoans and algae. Even though the available information was limited for many bioregions, our analyses demonstrate some differences in sponge traits and functions among bioregions, and among macroregions. However, we also identified similarities in sponge assemblage structure and function at global scales, likely reflecting a combination of regional- and local-scale biological and physical processes affecting sponge assemblages, along with common ancestry. Finally, we used our analyses to highlight geographic bias in past sponge research, and identify gaps in our understanding of sponge ecology globally. By so doing, we identified key areas for future research on sponge ecology. We hope that our study will help sponge researchers to consider bioregion-specific features of sponge assemblages and key sponge-mediated ecological processes from a global perspective.

Seasonally Driven Sexual and Asexual Reproduction in Temperate Tethya Species.

Marine organisms that rely on environmental cues for reproduction are likely to experience shifts in reproductive phenology and output due to global climate change. To assess the role that the environment may play in the reproductive timing for temperate sponges, this study examined sexual and asexual reproduction in New Zealand sponge species (Tethya bergquistae and the Tethya burtoni complex) and correlated reproductive output with temperature, chlorophyll-a concentration, and rainfall. Histological analyses of sponges collected monthly (from February 2015 to February 2017) revealed that these sponges are oviparous and gonochoristic and that they sexually reproduce annually during the austral summer. Both monthly collections and in situ monitoring revealed that Tethya spp. asexually bud continuously, but with greater intensity in the austral spring and summer. Temperature was positively associated with both sexual reproduction and budding, with seasonal cues appearing important. Future shifts in the environment that alter such cues are expected to affect population dynamics of these sponges.

Interlaboratory study to validate a STR profiling method for intraspecies identification of mouse cell lines.

The Consortium for Mouse Cell Line Authentication was formed to validate Short Tandem Repeat (STR) markers for intraspecies identification of mouse cell lines. The STR profiling method is a multiplex polymerase chain reaction (PCR) assay comprised of primers targeting 19 mouse STR markers and two human STR markers (for interspecies contamination screening). The goals of the Consortium were to perform an interlaboratory study to-(1) validate the mouse STR markers to uniquely identify mouse cell lines (intraspecies identification), (2) to provide a public database of mouse cell lines with the National Institute of Standards and Technology (NIST)-validated mouse STR profiles, and (3) to publish the results of the interlaboratory study. The interlaboratory study was an international effort that consisted of 12 participating laboratories representing institutions from academia, industry, biological resource centers, and government. The study was based on 50 of the most commonly used mouse cell lines obtained from the American Type Culture Collection (ATCC). Of the 50 mouse cell lines, 18 had unique STR profiles that were 100% concordant (match) among all Consortium laboratory members, and the remaining 32 cell lines had discordance that was resolved readily and led to improvement of the assay. The discordance was due to low signal and interpretation issues involving artifacts and genotyping errors. Although the total number of discordant STR profiles was relatively high in this study, the percent of labs agreeing on allele calls among the discordant samples was above 92%. The STR profiles, including electropherogram images, for NIST-validated mouse cell lines will be published on the NCBI BioSample Database (https://www.ncbi.nlm.nih.gov/biosample/). Overall, the interlaboratory study showed that the multiplex PCR method using 18 of the 19 mouse STR markers is capable of discriminating at the intraspecies level between mouse cell lines. Further studies are ongoing to refine the assay including (1) development of an allelic ladder for improving the accuracy of allele calling and (2) integration of stutter filters to identify true stutter.

Hidden diversity in the genus Tethya: comparing molecular and morphological techniques for species identification.

Correctly determining species' identity is critical for estimating biodiversity and effectively managing marine populations, but is difficult for species that have few morphological traits or are highly plastic. Sponges are considered a taxonomically difficult group because they lack multiple consistent diagnostic features, which coupled with their common phenotypic plasticity, makes the presence of species complexes likely, but difficult to detect. Here, we investigated the evolutionary relationship of Tethya spp. in central New Zealand using both molecular and morphological techniques to highlight the potential for cryptic speciation in sponges. Phylogenetic reconstructions based on two mitochondrial markers (rnl, COI-ext) and one nuclear marker (18S) revealed three genetic clades, with one clade representing Tethya bergquistae and two clades belonging to what was a priori thought to be a single species, Tethya burtoni. Eleven microsatellite markers were also used to further resolve the T. burtoni group, revealing a division consistent with the 18S and rnl data. Morphological analysis based on spicule characteristics allowed T. bergquistae to be distinguished from T. burtoni, but revealed no apparent differences between the T. burtoni clades. Here, we highlight hidden genetic diversity within T. burtoni, likely representing a group consisting of incipient species that have undergone speciation but have yet to express clear morphological differences. Our study supports the notion that cryptic speciation in sponges may go undetected and diversity underestimated when using only morphology-based taxonomy, which has broad scale implications for conservation and management of marine systems.

Assessment of the stability of DNA in specimens collected under conditions for drug testing-A pilot study.

For forensic biological sample collections, the specimen donor is linked solidly to his or her specimen through a chain of custody (CoC) sometimes referenced as a chain of evidence. Rarely, a donor may deny that a urine or oral fluid (OF) specimen is his or her specimen even with a patent CoC. The goal of this pilot study was to determine the potential effects of short-term storage on the quality and quantity of DNA in both types of specimen under conditions that may be encountered with employment-related drug testing specimens. Fresh urine and freshly collected oral fluid all produced complete STR profiles. For the "pad" type OF collectors, acceptable DNA was extractable both from the buffer/preservative and the pad. Although fresh urine and OF produced complete STR profiles, partial profiles were obtained after storage for most samples. An exception was the DNA in the Quantisal OF collector, from which a complete profile was obtained for both freshly collected OF and stored OF.

Sediment impacts on marine sponges.

Changes in sediment input to marine systems can influence benthic environments in many ways. Sponges are important components of benthic ecosystems world-wide and as sessile suspension feeders are likely to be impacted by changes in sediment levels. Despite this, little is known about how sponges respond to changes in settled and suspended sediment. Here we review the known impacts of sedimentation on sponges and their adaptive capabilities, whilst highlighting gaps in our understanding of sediment impacts on sponges. Although the literature clearly shows that sponges are influenced by sediment in a variety of ways, most studies confer that sponges are able to tolerate, and in some cases thrive, in sedimented environments. Critical gaps exist in our understanding of the physiological responses of sponges to sediment, adaptive mechanisms, tolerance limits, and the particularly the effect of sediment on early life history stages.

Utility of the Y-STR typing systems Y-PLEX 6 and Y-PLEX 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States.

The Y-PLEX 6 and Y-PLEX 5 systems enable analysis for 11 Y-STR loci. We present here the utility of these systems in forensic casework. A total of 188 samples, including 127 evidence samples, were analyzed using either or both of the systems. The evidence sample types included fingernail scrapings, sperm or seminal fluid, epithelial cells, blood and other tissues. The Y-STR typing systems provided useful probative results in difficult cases. A reference database for Caucasian (n = 517), African American (n = 535), and Hispanic (n = 245) population groups within the United States was generated for estimating the haplotype frequency in forensic casework. Among the individuals profiled, 311 Caucasians, 412 African Americans, and 194 Hispanics provided unique profiles in their respective population datasets. This is the first report describing the haplotype database for the set of 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Linkage analysis reveals that the frequencies from forensically important autosomal loci can be multiplied with the Y-STR haplotype frequency. The results from Y-PLEX systems have been accepted in courts in the United States.

Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit.

PURPOSE: To determine the effects of immunization against lysostaphin on the bactericidal action of lysostaphin in ocular tissue and the possible induction of allergic reactions. METHODS: Rabbits were immunized against lysostaphin by subcutaneous, intranasal, or topical routes. Anti-lysostaphin antibody titers were determined by ELISA and by neutralization of lysostaphin. Methicillin-resistant Staphylococcus aureus was intrastromally or intravitreously injected into rabbit eyes. Eyes were treated either topically with drops of lysostaphin (0.3%) or with a single intravitreous injection (0.1 mL) of lysostaphin (0.1%). At the time of death, corneas or vitreous humors were cultured to determine the number of colony forming units (CFU). RESULTS: Rabbits in keratitis experiments that were immunized subcutaneously, intranasally, or topically had serum antibody titers of 10,240, 187, and 1,867, respectively, and neutralization titers of 8 or less. In both normal and immunized rabbits with keratitis, lysostaphin significantly reduced the log CFU to less than 1 log, whereas the untreated eyes contained more than 10(6) CFU/cornea (P < or = 0.0001). Rabbits that were subcutaneously or topically immunized for endophthalmitis experiments had serum antibody titers of 1636 or 137, respectively, and neutralization titers of 2 or less. A single intravitreous injection of lysostaphin (0.1%) sterilized all eyes of immunized and nonimmune rabbits with endophthalmitis. No adverse effects were observed with the administration of lysostaphin to either normal or immunized rabbit eyes. CONCLUSIONS: Lysostaphin treatment of immunized rabbits was effective in treating S. aureus-infected eyes, despite the presence of anti-lysostaphin antibody. No adverse reactions were produced by administration of lysostaphin to immunized rabbits.