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Background and aims: Proteaceae are a prominent plant family in south-western Australia. Most Proteaceae are 'calcifuge', occurring exclusively on old phosphorus (P)-impoverished acidic soils, with a few 'soil-indifferent' species also found on young P-richer calcareous soils. Calcium (Ca)-enhanced P toxicity explains the calcifuge habit of Proteaceae. However, previous research has so far been focused exclusively on the roles of Ca and P in determining Proteaceae distribution, and consequently there is little knowledge on how other soil-based strategies influence this distribution. We aimed to study the effects of young calcareous soils on four soil-grown Proteaceae and assess differences between calcifuge and soil-indifferent Proteaceae to better understand their natural distribution. Methods: Two calcifuge and two soil-indifferent Proteaceae from south-western Australia were grown in six contrasting soils, including young calcareous, and old acidic soils. Results: When grown in calcareous soils all species showed root growth inhibition, micronutrient deficiency, Ca-enhanced P toxicity, and negative impacts on physiology. Calcifuge species were more sensitive to calcareous soils than soil-indifferent ones, although this varied between genera. Soil-indifferent species tended to produce more cluster roots, release more carboxylates per root mass, and allocate less Ca to their leaves, compared with calcifuges; they also had smaller seeds and were less sensitive to Ca-enhanced P toxicity. Conclusion: We surmise that a combination of these traits allows soil-indifferent species to tolerate calcareous soils. This study provides insight into how Proteaceae respond to young calcareous soils and how this influences their distribution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Jurien Bay dune chronosequence in south‐western Australia's biodiversity hotspot comprises sites differing in nutrient availability, with phosphorus (P) availability declining strongly with increasing soil age. We have explored the exceptionally high photosynthetic P‐use efficiency (PPUE) of Proteaceae in this region, triggering the question what the PPUE of co‐occurring species in other families might be along the Jurien Bay chronosequence.We explored how traits associated with PPUE, photosynthetic nitrogen (N)‐use efficiency (PNUE) and leaf respiration might converge along the chronosequence, and whether Proteaceae and non‐Proteaceae species differ in leaf traits associated with nutrient use.Seven to 10 species were sampled at three sites differing in nutrient availability (ranging from N‐ to P‐limited). Measurements of leaf light‐saturated photosynthesis and dark respiration were integrated with measurements of total N and P concentration in both mature and senesced leaves, and leaf mass per unit area (LMA).Contrary to what is known for other chronosequences, rates of photosynthesis and respiration did not decrease with increasing soil age and LMA along the Jurien Bay chronosequence. However, they increased when expressed per unit leaf P. Both N and P were used much more efficiently for photosynthesis on nutrient‐poor sites, in both Proteaceae and non‐Proteaceae species. Proteaceae had the fastest rate of photosynthesis per unit leaf P, followed by species that preferentially allocate P to mesophyll cells, rather than epidermal cells.Synthesis. Our results show that with declining soil P availability, photosynthetic P‐use efficiency of all investigated species from different families increased. Plants growing on the oldest, most nutrient‐impoverished soils exhibited similar rates of CO2 exchange as plants growing on more nutrient‐rich younger soils, and extraordinarily high photosynthetic P‐use efficiency. This indicates convergence in leaf traits related to photosynthetic nutrient use on severely P‐impoverished sites. [ABSTRACT FROM AUTHOR]

Background: Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). Results: Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. Conclusions: These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement. [ABSTRACT FROM AUTHOR]

Colonies of Coscinaraea marshae corals from Rottnest Island, Western Australia have survived for more than 11 months in various bleached states following a severe heating event in the austral summer of 2011. These colonies are situated in a high-latitude, mesophotic environment, which has made their long-term survival of particular interest as such environments typically suffer from minimal thermal pressures. We have investigated corals that remain unbleached, moderately bleached, or severely bleached to better understand potential survival mechanisms utilised in response to thermal stress. Specifically, Symbiodinium (algal symbiont) density and genotype, chlorophyll- a concentrations, and δC and δN levels were compared between colonies in the three bleaching categories. Severely bleached colonies housed significantly fewer Symbiodinium cells ( p < 0.05) and significantly reduced chlorophyll- a concentrations ( p < 0.05), compared with unbleached colonies. Novel Symbiodinium clade associations were observed for this coral in both severely and moderately bleached colonies, with clade C and a mixed clade population detected. In unbleached colonies, only clade B was observed. Levels of δN indicate that severely bleached colonies are utilising heterotrophic feeding mechanisms to aid survival whilst bleached. Collectively, these results suggest that these C. marshae colonies can survive with low symbiont and chlorophyll densities, in response to prolonged thermal stress and extended bleaching, and increase heterotrophic feeding levels sufficiently to meet energy demands, thus enabling some colonies to survive and recover over long time frames. This is significant as it suggests that corals in mesophotic and high-latitude environments may possess considerable plasticity and an ability to tolerate and adapt to large environmental fluctuations, thereby improving their chances of survival as climate change impacts coral ecosystems worldwide. [ABSTRACT FROM AUTHOR]

A growing number of indigenous trypanosomes have been reported to naturally infect a variety of Australian wildlife with some species of Trypanosoma implicated in the population decline of critically endangered marsupials. However, the mode of transmission of Australian trypanosomes is unknown since their vectors remain unidentified. Here we aimed to fill this current knowledge gap about the occurrence and identity of indigenous trypanosomes in Australian invertebrates by conducting molecular screening for the presence of Trypanosoma spp. in native ticks collected from south-west Australia. A total of 231 ticks (148 collected from vegetation and 83 retrieved directly from 76 marsupial hosts) were screened for Trypanosoma using a High-Resolution Melt (HRM) qPCR assay. An overall Trypanosoma qPCR positivity of 37% (46/125) and 34% (26/76) was detected in questing ticks and host-collected (i.e., feeding) ticks, respectively. Of these, sequencing revealed 28% (35/125) of questing and 28% (21/76) of feeding ticks were infected with one or more of the five species of trypanosome previously reported in this region (T. copemani, T. noyesi, T. vegrandis, T. gilletti, Trypanosoma sp. ANU2). This work has confirmed that Australian ticks are capable of harbouring several species of indigenous trypanosome and likely serve as their vectors. [ABSTRACT FROM AUTHOR]

Trypanosomes are global blood parasites that infect a wide range of vertebrate hosts. Several species of Trypanosoma cause disease in humans and domesticated animals, and the majority are transmitted between hosts by haematophagous invertebrate vectors. Ticks have long been speculated as vectors for Australian trypanosomes. Recent studies using advanced molecular techniques have refocused attention on these arthropods, and whilst they have renewed discussions about Trypanosoma species and their vectors, these reports have simultaneously led to premature conclusions concerning the role of ticks as vectors. Here the controversy surrounding ticks as trypanosome vectors is discussed. We highlight the unanswered questions concerning the role played by ticks in trypanosome transmission and suggest future approaches to resolving these key knowledge gaps. Trypanosomes (genus Trypanosoma) are blood-borne protozoan parasites of vertebrates that typically require a haematophagous invertebrate as a vector. Triatomine bugs and several biting flies are recognized as their main vectors. Ticks, as blood feeders, are predisposed to the ingestion of various parasites during feeding and have therefore long been proposed as vectors for Trypanosoma species. Trypanosomes are reported in Australian wildlife, and early indications suggest that some species may adversely affect the health of native hosts. The vectors responsible for transmitting Australian Trypanosoma species between vertebrate hosts are unknown. Ticks are suggested as vectors for several Australian trypanosomes based on microscopic and molecular detection of trypanosomes in ticks removed from wildlife. However, there are no experimental transmission studies to support this hypothesis to date. [ABSTRACT FROM AUTHOR]

Tabanids (syn. horse flies) are biting-flies of medical and veterinary significance because of their ability to transmit a range of pathogens including trypanosomes – some species of which carry a combined health and biosecurity risk. Invertebrate vectors responsible for transmitting species of Trypanosoma between Australian wildlife remains unknown, thus establishing the role of potential vector candidates such as tabanids is of utmost importance. The current study aimed to investigate the presence of indigenous trypanosomes in tabanids from an endemic area of south-west Australia. A total of 148 tabanids were collected, with morphological analysis revealing two subgenera: Scaptia (Pseudoscione) and S. (Scaptia) among collected flies. A parasitological survey using an HRM-qPCR and sequencing approach revealed a high (105/148; 71%) prevalence of trypanosomatid DNA within collected tabanids. Individual tissues - proboscis (labrum, labium and mandibles, hypopharynx), salivary glands, proventriculus, midgut, and hindgut and rectum - were also tested from a subset of 20 tabanids (n = 140 tissues), confirming the presence of Trypanosoma noyesi in 31% of screened tissues, accompanied by T. copemani (3%) and T. vegrandis/T.gilletti (5%). An unconfirmed trypanosomatid sp. was also detected (9%) within tissues. The difference between tissues infected with T. noyesi compared with tissues infected with other trypanosome species was statistically significant (p < 0.05), revealing T. noyesi as the more frequent species detected in the tabanids examined. Fluorescence in situ hybridisation (FISH) and scanning electron microscopy (SEM) confirmed intact parasites within salivary glands and the proboscis respectively, suggesting that both biological and mechanical modes of transmission could occur. This study reveals the presence of Australian Trypanosoma across tabanid tissues and confirms intact parasites within tabanid salivary glands and the proboscis for the first time. Further investigations are required to determine whether tabanids have the vectorial competence to transmit Australian trypanosomes between wildlife. • Molecular and microscopic evidence of Australian Trypanosoma within tabanid tissues • First observation of Trypanosoma parasites within salivary glands of wild-caught Australian tabanids • First visualisation of a trypanosome flagellate within the proboscis of a wild-caught Australian tabanid • Evidence to suggest tabanids as potential vectors for indigenous species of Australian Trypanosoma [ABSTRACT FROM AUTHOR]

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge., (© 2021. The Author(s).)

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10 bp sequence) and CSB-2 (8 bp sequence) present lower interspecies homology, while CSB-3 (12 bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257 bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© Crown copyright 2018.)

Plants allocate nutrients to specific leaf cell types, with commelinoid monocots preferentially allocating phosphorus (P) to the mesophyll and calcium (Ca) to the epidermis, whereas the opposite is thought to occur in eudicots. However, Proteaceae from severely P-impoverished habitats present the same P-allocation pattern as monocots. This raises the question of whether preferential P allocation to mesophyll cells is a phylogenetically conserved trait, exclusive to commelinoid monocots and a few Proteaceae, or a trait that has evolved multiple times to allow plants to cope with very low soil P availability. We analysed the P-allocation patterns of 16 species from 10 genera, eight families and six orders within three major clades of eudicots across different P-impoverished environments in Australia and Brazil, using elemental X-ray mapping to quantitatively determine leaf cell-specific nutrient concentrations. Many of the analysed species showed P-allocation patterns that differed substantially from that expected for eudicots. Instead, P-allocation patterns were strongly associated with the P availability in the natural habitat of the species, suggesting a convergent evolution of P-allocation patterns at the cellular level, with P limitation as selective pressure and without a consistent P-allocation pattern within eudicots. Here, we show that most eudicots from severely P-impoverished environments preferentially allocated P to their mesophyll. We surmise that this preferential P allocation to photosynthetically active cells might contribute to the very high photosynthetic P-use efficiency of species adapted to P-impoverished habitats., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Plants allocate nutrients to specific leaf cell types; eudicots are thought to predominantly allocate phosphorus (P) to epidermal/bundle sheath cells. However, three Proteaceae species have been shown to preferentially allocate P to mesophyll cells instead. These Proteaceae species are highly adapted to P-impoverished habitats, with exceptionally high photosynthetic P-use efficiencies (PPUE). We hypothesized that preferential allocation of P to photosynthetic mesophyll cells is an important trait in species adapted to extremely P-impoverished habitats, contributing to their high PPUE. We used elemental X-ray mapping to determine leaf cell-specific nutrient concentrations for 12 Proteaceae species, from habitats of strongly contrasting soil P concentrations, in Australia, Brazil, and Chile. We found that only species from extremely P-impoverished habitats preferentially allocated P to photosynthetic mesophyll cells, suggesting it has evolved as an adaptation to their extremely P-impoverished habitat and that it is not a family-wide trait. Our results highlight the possible role of soil P in driving the evolution of ecologically relevant nutrient allocation patterns and that these patterns cannot be generalized across families. Furthermore, preferential allocation of P to photosynthetic cells may provide new and exciting strategies to improve PPUE in crop species., (© 2018 John Wiley & Sons Ltd.)

Trypanosoma copemani has been found infecting several threatened/endangered marsupial species within Australia and is thought to be a key player in the rapid decline of the woylie (Bettongia penicillata). To better understand the biology and life cycle of this parasite, the growth requirements, and kinetics of infection of two newly described genotypes, T. copemani G1 and G2, were investigated and compared with the T. cruzi strain-10R26 in vitro. Both G1 and G2 were able to infect all four cell lines tested. The number of infected cells where at least one intracellular amastigote of T. copemani G1 and G2 was seen was below 7% and 15% respectively in most cell lines. However, in VERO cells the rate of infection for T. copemani G2 was 70%-approximately seven and two times higher than for G1 and T. cruzi respectively. Despite the higher infection rate, the number of intracellular forms of T. copemani G2 was lower compared with T. cruzi, and intracellular replicating forms were not observed. The capability of T. copemani G2 to infect cells may have important consequences for pathogenicity and suggests it might employ similar strategies to complete its life cycle in the vertebrate host to those seen in T. cruzi., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

To compare the crystalline microstructure of exsert septa, polyps of the scleractinian coral Galaxea fascicularis were sampled from shallow reef flat colonies, from colonies living at a depth of 9 m, and from colonies kept in a closed-circuit aquarium. Septal crystal structure and orientation was markedly different between corals in the field and in aquaria. In samples collected from deep water, acicular crystals were composed of conglomerates of finer crystals, and skeletal filling was considerably reduced when compared with samples collected from shallow water. Comparisons were also made between septa prepared in sodium hypochlorite (commercial bleach), sodium hydroxide (NaOH), hydrogen peroxide (H(2)O(2)), and distilled water (dH(2)O). Commercial bleach was the most effective solvent for tissue dissolution in investigations of skeletal structure. Samples prepared in NaOH commonly displayed crystalline artefacts, while the use of dH(2)O and H(2)O(2) was labor intensive and often resulted in unclean preparations. Fusiform crystals were seen only on G. fascicularis septa prepared in bleach and on Acropora formosa axial corallites prepared in either bleach or dH(2)O. We suggest that the mechanical agitation and additional washing necessary for samples prepared in dH(2)O, NaOH, or H(2)O(2) resulted in the loss of fusiform crystals from these preparations.