Validation of eDNA as a viable method of detection for dangerous cubozoan jellyfish

Stings from certain species of cubozoan jellyfish are dangerous to humans and their seasonal presence in tropical marine waters poses a significant risk to coastal communities. The detection of cubozoans is difficult due to high spatial and temporal variation in their occurrence and abundance. Environmental DNA (eDNA) has the potential to detect rare species and therefore offers potential to detect cubozoans, not only pelagic medusae, but presence of cryptic polyp life stages. The objective of this study was to validate the use of eDNA as a viable detection method for four cubozoan species (Chironex fleckeri, Copula sivickisi, Carybdea xaymacana, and Carukia barnesi). Species‐specific primers were developed for each of these four cubozoans and an eDNA approach validated utilizing both laboratory and field trials. Laboratory DNA degradation experiments demonstrated that C. sivickisi DNA degraded quickly but could still be detected in sea water for up to 9 days post‐jellyfish removal. Positive detection was found for C. fleckeri, C. xaymacana, and C. sivickisi medusae in the waters surrounding Magnetic Island, Queensland, in the Austral spring/summer (September‐January). Based on visual surveys, there was a poor relationship between concentration of eDNA and abundance of jellyfish. Positive eDNA amplification was also found from water sampled near the substratum when C. sivickisi medusae were out of season and absent. This suggests the eDNA analysis was likely detecting C. sivickisi polyps located within the substratum. Consequently, eDNA is an effective tool to detect both the medusae and likely polyps of cubozoans. This approach provides the means to reduce the risk of envenomation to swimmers and enhance our knowledge of cubozoan ecology

Microbiome diversity and composition varies across body areas in a freshwater turtle

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles

Testudines as sentinels for monitoring the dissemination of antibiotic resistance in marine environments: an integrative review

Dissemination of antibiotic resistance (AR) in marine environments is a global concern with a propensity to affect public health and many ecosystems worldwide. We evaluated the use of sea turtles as sentinel species for monitoring AR in marine environments. In this field, antibiotic resistant bacteria have been commonly identified by using standard culture and sensitivity tests, leading to an overrepresentation of specific, culturable bacterial classes in the available literature. AR was detected against all major antibiotic classes, but the highest cumulative global frequency of resistance in all represented geographical sites was against the beta-lactam class by a two-fold difference compared to all other antibiotics. Wastewater facilities and turtle rehabilitation centres were associated with higher incidences of multidrug-resistant bacteria (MDRB) accounting for an average of 58% and 49% of resistant isolates, respectively. Furthermore, a relatively similar prevalence of MDRB was seen in all studied locations. These data suggest that anthropogenically driven selection pressures for the development of AR in sea turtles and marine environments are relatively similar worldwide. There is a need, however, to establish direct demonstrable associations between AR in sea turtles in their respective marine environments with wastewater facilities and other anthropogenic activities worldwide

Methods for exploring the faecal microbiome of premature infants: a review

The premature infant gut microbiome plays an important part in infant health and development, and recognition of the implications of microbial dysbiosis in premature infants has prompted significant research into these issues. The approaches to designing investigations into microbial populations are many and varied, each with its own benefits and limitations. The technique used can influence results, contributing to heterogeneity across studies. This review aimed to describe the most common techniques used in researching the preterm infant microbiome, detailing their various limitations. The objective was to provide those entering the field with a broad understanding of available methodologies, so that the likely effects of their use can be factored into literature interpretation and future study design. We found that although many techniques are used for characterising the premature infant microbiome, 16S rRNA short amplicon sequencing is the most common. 16S rRNA short amplicon sequencing has several benefits, including high accuracy, discoverability and high throughput capacity. However, this technique has limitations. Each stage of the protocol offers opportunities for the injection of bias. Bias can contribute to variability between studies using 16S rRNA high throughout sequencing. Thus, we recommend that the interpretation of previous results and future study design be given careful consideration

The interplay of fungal and bacterial microbiomes on rainforest frogs following a disease outbreak

Emerging infectious diseases are a serious threat to wildlife populations, and there is growing evidence that host microbiomes play important roles in infection dynamics, possibly even mitigating diseases. Nevertheless, most research on this topic has focused only on bacterial microbiomes, while fungal microbiomes have been largely neglected. To help fill this gap in our knowledge, we examined both the bacterial and fungal microbiomes of four sympatric Australian frog species, which had different population-level responses to the emergence of chytridiomycosis, a widespread disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We sequenced 16,884 fungal amplicon sequence variants (ASVs) and 41,774 bacterial ASVs. Bacterial communities had higher richness and were less variable within frog species than were fungal communities. Nevertheless, both communities were correlated for both ASV richness and beta diversity (i.e., frogs with similar bacterial richness and community composition tended to also have similar fungal richness and community composition). This suggests that either one microbial community was having a large impact on the other or that they were both being driven by similar environmental factors. For both microbial taxa, we found little evidence of associations between Bd (prevalence or intensity) and either individuals' ASVs or beta diversity. However, there was mixed evidence of associations between richness (both bacterial and fungal) and Bd, with high richness potentially providing a protective effect. Surprisingly, the relative abundance of bacteria that have previously been shown to inhibit Bd was also positively associated with Bd infection intensity, suggesting that a high relative abundance of those bacteria provides poor protection against infection

The interplay of fungal and bacterial microbiomes on rainforest frogs following a disease outbreak

Emerging infectious diseases are a serious threat to wildlife populations, and there is growing evidence that host microbiomes play important roles in infection dynamics, possibly even mitigating diseases. Nevertheless, most research on this topic has focused only on bacterial microbiomes, while fungal microbiomes have been largely neglected. To help fill this gap in our knowledge, we examined both the bacterial and fungal microbiomes of four sympatric Australian frog species, which had different population-level responses to the emergence of chytridiomycosis, a widespread disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We sequenced 16,884 fungal amplicon sequence variants (ASVs) and 41,774 bacterial ASVs. Bacterial communities had higher richness and were less variable within frog species than were fungal communities. Nevertheless, both communities were correlated for both ASV richness and beta diversity (i.e., frogs with similar bacterial richness and community composition tended to also have similar fungal richness and community composition). This suggests that either one microbial community was having a large impact on the other or that they were both being driven by similar environmental factors. For both microbial taxa, we found little evidence of associations between Bd (prevalence or intensity) and either individuals' ASVs or beta diversity. However, there was mixed evidence of associations between richness (both bacterial and fungal) and Bd, with high richness potentially providing a protective effect. Surprisingly, the relative abundance of bacteria that have previously been shown to inhibit Bd was also positively associated with Bd infection intensity, suggesting that a high relative abundance of those bacteria provides poor protection against infection

Digital droplet PCR-based environmental DNA tool for monitoring Cryptocaryon irritans in a marine fish farm from Hong Kong

The adoption of new investigative strategies based on environmental DNA (eDNA) can be used to monitor parasites, associated bacterial microbiomes, and physical-chemical parameters in fish farms. In this study, we used the economically important and globally distributed fish ciliate parasite Cryptocaryon irritans as a model to understand the parasite abundance and potential drivers of its presence in marine fish farms. Environmental (rainfall) and physical-chemical (temperature, oxygen, salinity, pH) data collected from a marine fish farm in Hong Kong were analyzed together with the eDNA approach targeting C. irritans abundance based on digital droplet PCR and 16S metagenomics to determine associations and triggers between parasites and specific bacterial groups. Rainfall and temperature demonstrated positive associations with high abundance of C. irritans (eDNA) at the studied marine fish cage farm. However, rainfall was the only parameter tested that demonstrated a significant association with parasite eDNA, indicating that the raining season is a risky period for fish farmers in Hong Kong. Coraliomargarita was the bacterial genus with the most significant relationship with low abundance of C. irritans in water. Understanding the environmental triggers of ciliate parasites propagation and associated bacterial microbiome could elucidate new insights into environmental control, microbial management, and promote the reduction of chemical use in marine fish farms

microDecon: a highly accurate read‐subtraction tool for the post‐sequencing removal of contamination in metabarcoding studies

Contamination is a ubiquitous problem in microbiome research and can skew results, especially when small amounts of target DNA are available. Nevertheless, no clear solution has emerged for removing microbial contamination. To address this problem, we developed the R package microDecon (https://github.com/donaldtmcknight/microDecon), which uses the proportions of contaminant operational taxonomic units (OTUs) or amplicon sequence variants (ASVs) in blank samples to systematically identify and remove contaminant reads from metabarcoding data sets. We rigorously tested microDecon using a series of computer simulations and a sequencing experiment. We also compared it to the common practice of simply removing all contaminant OTUs/ASVs and other methods for removing contamination. Both the computer simulations and our sequencing data confirmed the utility of microDecon. In our largest simulation (100,000 samples), using microDecon improved the results in 98.1% of samples. Additionally, in the sequencing data and in simulations involving groups, it enabled accurate clustering of groups as well as the detection of previously obscured patterns. It also produced more accurate results than the existing methods for identifying and removing contamination. These results demonstrate that microDecon effectively removes contamination across a broad range of situations. It should, therefore, be widely applicable to microbiome studies, as well as to metabarcoding studies in general

The bacterial gut microbiome of probiotic-treated very-preterm infants: changes from admission to discharge

Background: Preterm birth is associated with the development of acute and chronic disease, potentially, through the disruption of normal gut microbiome development. Probiotics may correct for microbial imbalances and mitigate disease risk. Here, we used amplicon sequencing to characterise the gut microbiome of probiotic-treated premature infants. We aimed to identify and understand variation in bacterial gut flora from admission to discharge and in association with clinical variables. Methods: Infants born <32 weeks gestation and <1500 g, and who received probiotic treatment, were recruited in North Queensland Australia. Meconium and faecal samples were collected at admission and discharge. All samples underwent 16S rRNA short amplicon sequencing, and subsequently, a combination of univariate and multivariate analyses. Results: 71 admission and 63 discharge samples were collected. Univariate analyses showed significant changes in the gut flora from admission to discharge. Mixed-effects modelling showed significantly lower alpha diversity in infants diagnosed with either sepsis or retinopathy of prematurity (ROP) and those fed formula. In addition, chorioamnionitis, preeclampsia, sepsis, necrotising enterocolitis and ROP were also all associated with the differential abundance of several taxa. Conclusions: The lower microbial diversity seen in infants with diagnosed disorders or formula-fed, as well as differing abundances of several taxa across multiple variables, highlights the role of the microbiome in the development of health and disease. This study supports the need for promoting healthy microbiome development in preterm neonates

Exploring the long-term colonisation and persistence of probiotic-prophylaxis species on the gut microbiome of preterm infants: a pilot study

Preterm infants suffer from a higher incidence of acute diseases such as necrotising enterocolitis and sepsis. This risk can be mitigated through probiotic prophylaxis during admission. This reduction in risk is likely the result of acute modulation of the gut microbiome induced by probiotic species, which has been observed to occur up until discharge. We aimed to determine if this modulation, and the associated probiotic species, persisted beyond discharge. We conducted both a cross-sectional analysis (n= 18), at similar to 18 months of age, and a longitudinal analysis (n= 6), from admission to 18 months of the gut microbiome of preterm infants using both shotgun metagenomics and 16S rRNA profiling respectively. The 16S amplicon sequencing revealed that the microbial composition of the probiotic-supplemented infants changed dramatically over time, stabilising at discharge. However, species from the probiotic Infloran (R), as well as positive modulatory effects previously associated with supplementation, do not appear to persist beyond discharge and once prophylaxis has stopped. Conclusions: Although differences exist between supplemented and non-supplemented groups, the implications of these differences remain unclear. Additionally, despite a lack of long-term colonisation, the presence of probiotics during early neonatal life may still have modulatory effects on the microbiome assembly and immune system training