Salt tolerance in mungbean is associated with controlling Na and Cl transport across roots, regulating Na and Cl accumulation in chloroplasts and maintaining high K in root and leaf mesophyll cells.

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na+ and Cl- in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells.

The spectrum of Ih ice using terahertz time-domain spectroscopy.

Here, we report the frequency-dependent spectrum of ice Ih in the range of 0.2-2 THz. We confirm the presence of a feature that blue-shifts from around 1.55-1.65 THz with a decreasing temperature from 260 to 160 K. There is also a change in the trend of the refractive index of ice corresponding to a dispersion, which is also around 1.6 THz. The features are reproduced in data acquired with three commercial terahertz time-domain spectrometers. Computer-simulated spectra assign the feature to lattice translations perpendicular to the 110 and 1̄10 planes of the ice Ih crystal. The feature's existence should be recognized in the terahertz measurements of frozen aqueous solution samples to avoid false interpretations.

Differences in foliar phosphorus fractions, rather than in cell-specific phosphorus allocation, underlie contrasting photosynthetic phosphorus use efficiency among chickpea genotypes.

Although significant intraspecific variation in photosynthetic phosphorus (P) use efficiency (PPUE) has been shown in numerous species, we still know little about the biochemical basis for differences in PPUE among genotypes within a species. Here, we grew two high PPUE and two low PPUE chickpea (Cicer arietinum) genotypes with low P supply in a glasshouse to compare their photosynthesis-related traits, total foliar P concentration ([P]) and chemical P fractions (i.e. inorganic P (Pi), metabolite P, lipid P, nucleic acid P, and residual P). Foliar cell-specific nutrient concentrations including P were characterized using elemental X-ray microanalysis. Genotypes with high PPUE showed lower total foliar [P] without slower photosynthetic rates. No consistent differences in cellular [P] between the epidermis and mesophyll cells occurred across the four genotypes. In contrast, high PPUE was associated with lower allocation to Pi and metabolite P, with PPUE being negatively correlated with the percentage of these two fractions. Furthermore, a lower allocation to Pi and metabolite P was correlated with a greater allocation to nucleic acid P, but not to lipid P. Collectively, our results suggest that a different allocation to foliar P fractions, rather than preferential P allocation to specific leaf tissues, underlies the contrasting PPUE among chickpea genotypes.

Comparison of in vitro growth characteristics of Cryptosporidium hominis (IdA15G1) and Cryptosporidium parvum (Iowa-IIaA17G2R1 and IIaA18G3R1).

Cryptosporidium is a major cause of diarrhoeal disease and mortality in young children in resource-poor countries, for which no vaccines or adequate therapeutic options are available. Infection in humans is primarily caused by two species: C. hominis and C. parvum. Despite C. hominis being the dominant species infecting humans in most countries, very little is known about its growth characteristics and life cycle in vitro, given that the majority of our knowledge of the in vitro development of Cryptosporidium has been based on C. parvum. In the present study, the growth and development of two C. parvum isolates (subtypes Iowa-IIaA17G2R1 and IIaA18G3R1) and one C. hominis isolate (subtype IdA15G1) in HCT-8 cells were examined and compared at 24 h and 48 h using morphological data acquired with scanning electron microscopy. Our data indicated no significant differences in the proportion of meronts or merozoites between species or subtypes at either time-point. Sexual development was observed at the 48-h time-point across both species through observations of both microgamonts and macrogamonts, with a higher frequency of macrogamont observations in C. hominis (IdA15G1) cultures at 48-h post-infection compared to both C. parvum subtypes. This corresponded to differences in the proportion of trophozoites observed at the same time point. No differences in proportion of microgamonts were observed between the three subtypes, which were rarely observed across all cultures. In summary, our data indicate that asexual development of C. hominis is similar to that of C. parvum, while sexual development is accelerated in C. hominis. This study provides new insights into differences in the in vitro growth characteristics of C. hominis when compared to C. parvum, which will facilitate our understanding of the sexual development of both species.

Subcellular Partitioning of Arsenic Trioxide Revealed by Label-Free Imaging.

Subcellular partitioning of therapeutic agents is highly relevant to their interactions with target molecules and drug efficacy, but studying subcellular partitioning is an enormous challenge. Here, we describe the application of nanoscale secondary ion mass spectrometry (NanoSIMS) analysis to define the subcellular pharmacokinetics of a cytotoxic chemotherapy drug, arsenic trioxide (ATO). We reasoned that defining the partitioning of ATO would yield valuable insights into the mechanisms underlying ATO efficacy. NanoSIMS imaging made it possible to define the intracellular fate of ATO in a label-free manner─and with high resolution and high sensitivity. Our studies of ATO-treated cells revealed that arsenic accumulates in the nucleolus. After prolonged ATO exposure, ∼40 nm arsenic- and sulfur-rich protein aggregates appeared in the cell nucleolus, nucleus, and membrane-free compartments in the cytoplasm, and our studies suggested that the partitioning of nanoscale aggregates could be relevant to cell survival. All-trans retinoic acid increased intracellular ATO levels and accelerated the nanoscale aggregate formation in the nucleolus. This study yielded fresh insights into the subcellular pharmacokinetics of an important cancer therapeutic agent and the potential impact of drug partitioning and pharmacokinetics on drug activity.

Salt tolerance in relation to elemental concentrations in leaf cell vacuoles and chloroplasts of a C4 monocotyledonous halophyte.

Halophytes accumulate and sequester high concentrations of salt in vacuoles while maintaining lower levels of salt in the cytoplasm. The current data on cellular and subcellular partitioning of salt in halophytes are, however, limited to only a few dicotyledonous C3 species. Using cryo-scanning electron microscopy X-ray microanalysis, we assessed the concentrations of Na, Cl, K, Ca, Mg, P and S in various cell types within the leaf-blades of a monocotyledonous C4 halophyte, Rhodes grass (Chloris gayana). We also linked, for the first time, elemental concentrations in chloroplasts of mesophyll and bundle sheath cells to their ultrastructure and photosynthetic performance of plants grown in nonsaline and saline (200 mM NaCl) conditions. Na and Cl accumulated to the highest levels in xylem parenchyma and epidermal cells, but were maintained at lower concentrations in photosynthetically active mesophyll and bundle sheath cells. Concentrations of Na and Cl in chloroplasts of mesophyll and bundle sheath cells were lower than in their respective vacuoles. No ultrastructural changes were observed in either mesophyll or bundle sheath chloroplasts, and photosynthetic activity was maintained in saline conditions. Salinity tolerance in Rhodes grass is related to specific cellular Na and Cl distributions in leaf tissues, and the ability to regulate Na and Cl concentrations in chloroplasts.

Recently photoassimilated carbon and fungus-delivered nitrogen are spatially correlated in the ectomycorrhizal tissue of Fagus sylvatica.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15 N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13 CO2 atmosphere. We analysed the short-term distribution of 13 C and 15 N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13 C to root parts that received more 15 N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13 C and 15 N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Role of roots in adaptation of soil-indifferent Proteaceae to calcareous soils in south-western Australia.

Very few of the >650 Proteaceae species in south-western Australia cope with the high calcium (Ca) levels in young, calcareous soils (soil indifferent); most are Ca sensitive and occur on nutrient-impoverished, acidic soils (calcifuge). We assessed possible control points for Ca transport across roots of two soil-indifferent (Hakea prostrata and Banksia prionotes) and two calcifuge (H. incrassata and B. menziesii) Proteaceae. Using quantitative X-ray microanalysis, we investigated cell-specific elemental Ca concentrations at two positions behind the apex in relation to development of apoplastic barriers in roots of plants grown in nutrient solution with low or high Ca supply. In H. prostrata, Ca accumulated in outer cortical cells at 20 mm behind the apex, but [Ca] was low in other cell types. In H. incrassata, [Ca] was low in all cells. Accumulation of Ca in roots of H. prostrata corresponded to development of apoplastic barriers in the endodermis. We found similar [Ca] profiles in roots and similar [Ca] in leaves of two contrasting Banksia species. Soil-indifferent Hakea and Banksia species show different strategies to inhabit calcareous soils: H. prostrata intercepts Ca in roots, reducing transport to shoots, whereas B. prionotes allocates Ca to specific leaf cells.

The barrier to radial oxygen loss impedes the apoplastic entry of iron into the roots of Urochloa humidicola.

Lack of O2 and high concentrations of iron (Fe) and manganese (Mn) commonly occur in waterlogged soils. The development of a barrier to impede radial O2 loss (ROL) is a key trait improving internal O2 transport and waterlogging tolerance in plants. We evaluated the ability of the barrier to ROL to impede the entry of excess Fe into the roots of the waterlogging-tolerant grass Urochloa humidicola. Plants were grown in aerated or stagnant deoxygenated nutrient solution with 5 µM or 900 µM Fe. Quantitative X-ray microanalysis was used to determine cell-specific Fe concentrations at two positions behind the root apex in relation to ROL and the formation of apoplastic barriers. At a mature zone of the root, Fe was 'excluded' at the exodermis where a suberized lamella was evident, a feature also associated with a strong barrier to ROL. In contrast, the potassium (K) concentration was similar in all root cells, indicating that K uptake was not affected by apoplastic barriers. The hypothesis that the formation of a tight barrier to ROL impedes the apoplastic entry of toxic concentrations of Fe into the mature zones of roots was supported by the significantly higher accumulation of Fe on the outer side of the exodermis.

Rod-plate interactions in sepiolite-LAPONITE® gels: microstructure, surface chemistry and rheology.

Pure and composite clay gels of sepiolite and LAPONITE® at 4 wt% solids were characterised microstructurally and rheologically. They all displayed time-dependent behaviour, i.e. yield stress increased with aged time. The microstructures captured by cryo-SEM on the samples prepared by high pressure and rapid freezing showed the presence of bonding between LAPONITE® sheets and sepiolite fibres. These bonds are likely to be formed between the positive sheet edge and the negative fibre silica face as suggested by the upright orientation adopted by most LAPONITE® sheets. The sheets formed isolated open networks at a low content and a continuous open network at a high content. The fibres of the pure sepiolite gel formed a relatively strong network. However, the composite networks formed at a low LAPONITE® content between 5 and 25% must be discontinuous as the yield stress is zero. At a higher LAPONITE® content, the composite structure displayed increasing strength. The time-dependent ageing behaviour is due to the electric double layer (EDL, face-face) repulsive force governing the development of the 3D network structure. The Leong model described this behaviour well. All the clay gels displayed a maximum yield stress at a pH of 9 to 10. Pyrophosphate additive reduced the maximum yield stress and shifted the yield stress-pH curve to a lower pH. The Herschel-Bulkley model described the flow behaviour of all the gels well. The variation of the model yield stress with gel composition is similar to that obtained by the vane yield stress technique.

Phosphorus toxicity, not deficiency, explains the calcifuge habit of phosphorus-efficient Proteaceae.

The calcifuge habit of plants is commonly explained in terms of high soil pH and its effects on nutrient availability, particularly that of phosphorus (P). However, most Proteaceae that occur on nutrient-impoverished soils in south-western Australia are calcifuge, despite their ability to produce cluster-roots, which effectively mobilize soil P and micronutrients. We hypothesize that the mechanism explaining the calcifuge habit in Proteaceae is their sensitivity to P and calcium (Ca), and that soil-indifferent species are less sensitive to the interaction of these nutrients. In this study, we analyzed growth, gas-exchange rate, and chlorophyll fluorescence of two soil-indifferent and four calcifuge Hakea and Banksia (Proteaceae) species from south-western Australia, across a range of P and Ca concentrations in hydroponic solution. We observed Ca-enhanced P toxicity in all analyzed species, but to different extents depending on distribution type and genus. Increasing P supply enhanced plant growth, leaf biomass, and photosynthetic rates of soil-indifferent species in a pattern largely independent of Ca supply. In contrast, positive physiological responses to increasing [P] in calcifuges were either absent or limited to low Ca supply, indicating that calcifuges were far more sensitive to Ca-enhanced P toxicity. In calcifuge Hakeas, we attributed this to higher leaf [P], and in calcifuge Banksias to lower leaf zinc concentration. These differences help to explain these species' contrasting sensitivity to Ca-enhanced P toxicity and account for the exclusion of most Proteaceae from calcareous habitats. We surmise that Ca-enhanced P toxicity is a major factor explaining the calcifuge habit of Proteaceae, and, possibly, other P-sensitive plants.

Edaphic niche characterization of four Proteaceae reveals unique calcicole physiology linked to hyper-endemism of Grevillea thelemanniana.

Endemism and rarity have long intrigued scientists. We focused on a rare endemic and critically-endangered species in a global biodiversity hotspot, Grevillea thelemanniana (Proteaceae). We carried out plant and soil analyses of four Proteaceae, including G. thelemanniana, and combined these with glasshouse studies. The analyses related to hydrology and plant water relations as well as soil nutrient concentrations and plant nutrition, with an emphasis on sodium (Na) and calcium (Ca). The local hydrology and matching plant traits related to water relations partially accounted for the distribution of the four Proteaceae. What determined the rarity of G. thelemanniana, however, was its accumulation of Ca. Despite much higher total Ca concentrations in the leaves of the rare G. thelemanniana than in the common Proteaceae, very few Ca crystals were detected in epidermal or mesophyll cells. Instead of crystals, G. thelemanniana epidermal cell vacuoles contained exceptionally high concentrations of noncrystalline Ca. Calcium ameliorated the negative effects of Na on the very salt-sensitive G. thelemanniana. Most importantly, G. thelemanniana required high concentrations of Ca to balance a massively accumulated feeding-deterrent carboxylate, trans-aconitate. This is the first example of a calcicole species accumulating and using Ca to balance accumulation of an antimetabolite.

Angiosperm symbioses with non-mycorrhizal fungal partners enhance N acquisition from ancient organic matter in a warming maritime Antarctic.

In contrast to the situation in plants inhabiting most of the world's ecosystems, mycorrhizal fungi are usually absent from roots of the only two native vascular plant species of maritime Antarctica, Deschampsia antarctica and Colobanthus quitensis. Instead, a range of ascomycete fungi, termed dark septate endophytes (DSEs), frequently colonise the roots of these plant species. We demonstrate that colonisation of Antarctic vascular plants by DSEs facilitates not only the acquisition of organic nitrogen as early protein breakdown products, but also as non-proteinaceous d-amino acids and their short peptides, accumulated in slowly-decomposing organic matter, such as moss peat. Our findings suggest that, in a warming maritime Antarctic, this symbiosis has a key role in accelerating the replacement of formerly dominant moss communities by vascular plants, and in increasing the rate at which ancient carbon stores laid down as moss peat over centuries or millennia are returned to the atmosphere as CO2 .

Calcium-enhanced phosphorus toxicity in calcifuge and soil-indifferent Proteaceae along the Jurien Bay chronosequence.

Many Proteaceae are highly phosphorus (P)-sensitive and occur exclusively on old nutrient-impoverished acidic soils (calcifuge), whilst a few also occur on young calcareous soils (soil-indifferent) that are higher in available calcium (Ca) and P. Calcium increases the severity of P-toxicity symptoms, but its underlying mechanisms are unknown. We propose that Ca-enhanced P toxicity explains the calcifuge habit of most Proteaceae. Four calcifuge and four soil-indifferent Proteaceae from South-Western Australia were grown in hydroponics, at a range of P and Ca concentrations. Calcium increased the severity of P-toxicity symptoms in all species. Calcifuge Proteaceae were more sensitive to Ca-enhanced P toxicity than soil-indifferent ones. Calcifuges shared these traits: low leaf zinc concentration ([Zn]), low Zn allocation to leaves, low leaf [Zn]:[P], low root : shoot ratio, and high seed P content, compared with soil-indifferent species. This is the first demonstration of Ca-enhanced P toxicity across multiple species. Calcium-enhanced P toxicity provides an explanation for the calcifuge habit of most Proteaceae and is critical for the management of this iconic Australian family. This study represents a major advance towards an understanding of the physiological mechanisms of P toxicity and its role in the distribution of Proteaceae.

Globular structures in roots accumulate phosphorus to extremely high concentrations following phosphorus addition.

Crops with improved uptake of fertilizer phosphorus (P) would reduce P losses and confer environmental benefits. We examined how P-sufficient 6-week-old soil-grown Trifolium subterraneum plants, and 2-week-old seedlings in solution culture, accumulated P in roots after inorganic P (Pi) addition. In contrast to our expectation that vacuoles would accumulate excess P, after 7 days, X-ray microanalysis showed that vacuolar [P] remained low (<12 mmol kg-1 ). However, in the plants after P addition, some cortex cells contained globular structures extraordinarily rich in P (often >3,000 mmol kg-1 ), potassium, magnesium, and sodium. Similar structures were evident in seedlings, both before and after P addition, with their [P] increasing threefold after P addition. Nuclear magnetic resonance (NMR) spectroscopy showed seedling roots accumulated Pi following P addition, and transmission electron microscopy (TEM) revealed large plastids. For seedlings, we demonstrated that roots differentially expressed genes after P addition using RNAseq mapped to the T. subterraneum reference genome assembly and transcriptome profiles. Among the most up-regulated genes after 4 hr was TSub_g9430.t1, which is similar to plastid envelope Pi transporters (PHT4;1, PHT4;4): expression of vacuolar Pi-transporter homologs did not change. We suggest that subcellular P accumulation in globular structures, which may include plastids, aids cytosolic Pi homeostasis under high-P availability.

Calcium modulates leaf cell-specific phosphorus allocation in Proteaceae from south-western Australia.

Over 650 Proteaceae occur in south-western Australia, contributing to the region's exceptionally high biodiversity. Most Proteaceae occur exclusively on severely nutrient-impoverished, acidic soils (calcifuge), whilst only few also occur on young, calcareous soils (soil-indifferent), higher in calcium (Ca) and phosphorus (P). The calcifuge habit of Proteaceae is explained by Ca-enhanced P toxicity, putatively linked to the leaf cell-specific allocation of Ca and P. Separation of these elements is essential to avoid the deleterious precipitation of Ca-phosphate. We used quantitative X-ray microanalysis to determine leaf cell-specific nutrient concentrations of two calcifuge and two soil-indifferent Proteaceae grown in hydroponics at a range of Ca and P concentrations. Calcium enhanced the preferential allocation of P to palisade mesophyll (PM) cells under high P conditions, without a significant change in whole leaf [P]. Calcifuges showed a greater PM [P] compared with soil-indifferent species, corresponding to their greater sensitivity. This study advances our mechanistic understanding of Ca-enhanced P toxicity, supporting the proposed model, and demonstrating its role in the calcifuge distribution of Proteaceae. This furthers our understanding of nutrient interactions at the cellular level and highlights its importance to plant functioning.

Salinity tolerance in chickpea is associated with the ability to 'exclude' Na from leaf mesophyll cells.

Salinity tolerance is associated with Na 'exclusion' from, or 'tissue tolerance' in, leaves. We investigated whether two contrasting chickpea genotypes, salt-tolerant Genesis836 and salt-sensitive Rupali, differ in leaf tissue tolerance to NaCl. We used X-ray microanalysis to evaluate cellular Na, Cl, and K concentrations in various cell types within leaflets and also in secretory trichomes of the two chickpea genotypes in relation to photosynthesis in control and saline conditions. TEM was used to assess the effects of salinity on the ultrastructure of chloroplasts. Genesis836 maintained net photosynthetic rates (A) for the 21 d of salinity treatment (60 mM NaCl), whereas A in Rupali substantially decreased after 11 d. Leaflet tissue [Na] was low in Genesis836 but had increased markedly in Rupali. In Genesis836, Na was accumulated in epidermal cells but was low in mesophyll cells, whereas in Rupali cellular [Na] was high in both cell types. The excessive accumulation of Na in mesophyll cells of Rupali corresponded to structural damage to the chloroplasts. Maintenance of photosynthesis and thus salinity tolerance in Genesis836 was associated with an ability to 'exclude' Na from leaflets and in particular from the photosynthetically active mesophyll cells, and to compartmentalize Na in epidermal cells.

DMSP biosynthesis by an animal and its role in coral thermal stress response.

Globally, reef-building corals are the most prolific producers of dimethylsulphoniopropionate (DMSP), a central molecule in the marine sulphur cycle and precursor of the climate-active gas dimethylsulphide. At present, DMSP production by corals is attributed entirely to their algal endosymbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, we show that coral juveniles produce DMSP in the absence of algal symbionts. DMSP levels increased up to 54% over time in newly settled coral juveniles lacking algal endosymbionts, and further increases, up to 76%, were recorded when juveniles were subjected to thermal stress. We uncovered coral orthologues of two algal genes recently identified in DMSP biosynthesis, strongly indicating that corals possess the enzymatic machinery necessary for DMSP production. Our results overturn the paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight the double jeopardy represented by worldwide declining coral cover, as the potential to alleviate thermal stress through coral-produced DMSP declines correspondingly.

Zinc and cadmium mapping by NanoSIMS within the root apex after short-term exposure to metal contamination.

Zinc as a micronutrient and cadmium as a nonessential toxic element share similar pathways for entering plant tissues and thus may be antagonistic. In nutrient solution culture, 17-day-old radish (Raphanus sativus L) plants were exposed to short-term (24 h) equimolar metal contamination (2.2 µM of each 70Zn and total Cd) to investigate the in situ Zn/Cd distribution in the apical root tissues using high-resolution secondary ion mass spectrometry (NanoSIMS) imaging. Inductively-coupled plasma mass spectrometry analysis of bulk root tissue confirmed large root uptake of both metal elements. After 24-h exposure the total root concentration (in µg/g DW) of 70Zn was 180 ±â€¯24 (mean±SE) and of total Cd 352 ±â€¯11. NanoSIMS mapping was performed on the cross sections of the radish root apex as a crucial component in root growth and uptake of water and nutrients from soil. Elemental maps of 70Zn and 114Cd isotopes revealed greater enrichment of both metals in the outer epidermal root layer than in cortical tissues and especially stele, confirming the epidermal root cells as preferential sites of metal uptake, and indicating relatively slow and less-intensive metal transport into other parts (edible hypocotyl, shoot) of metal-sensitive radish. NanoSIMS has been confirmed as a powerful tool for spatial detection and visualisation of some ultra-trace metal isotopes (e.g. 70Zn) in the fast-growing root tips. However, precise (sub)cellular mapping of diffusible metallic ions (Cd, Zn) remains a technically-challenging task in plant specimens given an unavoidable compromise between optimising methodology for structural preservation vs. authentic in vivo ion localisation.

Crown-of-Thorns Sea Star Acanthaster cf. solaris Has Tissue-Characteristic Microbiomes with Potential Roles in Health and Reproduction.

Outbreaks of coral-eating crown-of-thorns sea stars (CoTS; Acanthaster species complex) cause substantial coral loss; hence, there is considerable interest in developing prevention and control strategies. We characterized the microbiome of captive CoTS and assessed whether dysbiosis was evident in sea stars during a disease event. Most tissue types had a distinct microbiome. The exception was female gonads, in which the microbiomes were highly variable among individuals. Male gonads were dominated (>97% of reads) by a single Mollicutes-related operational taxonomic unit (OTU). Detailed phylogenetic and microscopy analysis demonstrated the presence of a novel Spiroplasma-related bacterium in the spermatogenic layer. Body wall samples had high relative abundance (43 to 64% of reads) of spirochetes, likely corresponding to subcuticular symbionts reported from many echinoderms. Tube feet were characterized by Hyphomonadaceae (24 to 55% of reads). Pyloric cecal microbiomes had high alpha diversity, comprising many taxa commonly found in gastrointestinal systems. The order Oceanospirillales (genera Endozoicomonas and Kistimonas) was detected in all tissues. A microbiome shift occurred in diseased individuals although differences between tissue types were retained. The relative abundance of spirochetes was significantly reduced in diseased individuals. Kistimonas was present in all diseased individuals and significantly associated with diseased tube feet, but its role in disease causation is unknown. While Arcobacter was significantly associated with diseased tissues and Vibrionaceae increased in diversity, no single OTU was detected in all diseased individuals, suggesting opportunistic proliferation of these taxa in this case. This study shows that CoTS have tissue-characteristic bacterial communities and identifies taxa that could play a role in reproduction and host health.IMPORTANCE Coral-eating crown-of-thorns sea stars (CoTS; Acanthaster species complex) are native to the Indo-Pacific, but during periodic population outbreaks they can reach extreme densities (>1,000 starfish per hectare) and function as a pest species. On the Great Barrier Reef, Australia, CoTS have long been considered one of the major contributors to coral loss. There has been significant investment in a targeted control program using lethal injection, and there is interest in developing additional and complementary technologies that can increase culling efficiencies. The biology of CoTS has been studied extensively, but little is known about their associated microbiome. This cultivation-independent analysis of the CoTS microbiome provides a baseline for future analyses targeting the functional role of symbionts, the identification of pathogens, or the development of reproduction manipulators.