A reassessment of testate amoebae diversity in Tierra del Fuego peatlands: Implications for large scale inferences.

Testate amoebae are a diverse group of shelled protists frequently used as model organisms in microbial biogeography. Relatively few species have been reported for the Southern Hemisphere, however, it remains unclear whether this lower diversity is real or an artifact of under-sampling or misidentifications, which would reduce their potential to address macroecological questions. We evaluated testate amoebae diversity from the full range of habitats occurring within two Tierra del Fuego peatlands and compared it with the reported diversity for the area and from the Northern Hemisphere peatlands. We recorded 87 species, of which 69 are new for the region and 45 of them probably new to science and likely to have restricted geographical distributions. Combined with previous studies, the total diversity of testate amoebae only from Tierra del Fuego peatlands now reaches 119, as compared with 183 reported from all Northern Hemisphere peatlands. Our results demonstrate that the number of Gondwanian and Neotropical endemic testate amoeba may be substantially higher than currently known. Previous reports of Holarctic taxa in Tierra del Fuego may result from forcing the identification of morphotypes to the descriptions in the most common literature (force-fitting) South American species into species common in literature from other regions.

Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

PCR-based identification of thermotolerant free-living amoebae in Italian hot springs.

Several thermal areas, also used for leisure purposes, may represent suitable habitats for free-living amoebae (FLAs), but few studies have been carried out in search for these organisms. The aim of this study was to assess the presence and distribution of FLAs by culture detection and molecular identification, over a one year-round sampling of two sites in Central Italy. Two geothermal springs (Site A and Site B) were investigated for a total of 36 water samples. Four sets of primers were used to amplify FLA DNA from all cultures positive for amoebic growth at both 37 °C and 45 °C. Overall, 33 (91.6%) water samples produced PCR amplification. Eleven taxa were identified. The array of identified species varied over the sampling period, and differed between the two hot springs, Site A harbouring 11 taxa compared to 5 of site B. However, both sites were characterized by the most common species Vermamoeba vermiformis and Naegleria australiensis. Acanthamoeba genotypes T4 and T15 were found at low frequency. Differences in the composition between the two sites could reflect environmental changes in biotic and chemical/physical parameters. From a public health perspective, the detection of potentially pathogenic amoebae could unveil a potential risk for humans.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Morphology and Ecology of Two New Amoebae, Isolated From a Thalassohaline Lake, Dziani Dzaha.

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus.

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha.

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.

Identification of Free-Living Amoebas in Tap Water of Buildings with Storage Tanks in Korea.

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

Vermamoeba vermiformis CDC-19 draft genome sequence reveals considerable gene trafficking including with candidate phyla radiation and giant viruses.

Vermamoeba vermiformis is a predominant free-living amoeba in human environments and amongst the most common amoebae that can cause severe infections in humans. It is a niche for numerous amoeba-resisting microorganisms such as bacteria and giant viruses. Differences in the susceptibility to these giant viruses have been observed. V. vermiformis and amoeba-resisting microorganisms share a sympatric lifestyle that can promote exchanges of genetic material. This work analyzed the first draft genome sequence of a V. vermiformis strain (CDC-19) through comparative genomic, transcriptomic and phylogenetic analyses. The genome of V. vermiformis is 59.5 megabase pairs in size, and 22,483 genes were predicted. A high proportion (10% (n = 2,295)) of putative genes encoded proteins showed the highest sequence homology with a bacterial sequence. The expression of these genes was demonstrated for some bacterial homologous genes. In addition, for 30 genes, we detected best BLAST hits with members of the Candidate Phyla Radiation. Moreover, 185 genes (0.8%) best matched with giant viruses, mostly those related to the subfamily Klosneuvirinae (101 genes), in particular Bodo saltans virus (69 genes). Lateral sequence transfers between V. vermiformis and amoeba-resisting microorganisms were strengthened by Sanger sequencing, transcriptomic and phylogenetic analyses. This work provides important insights and genetic data for further studies about this amoeba and its interactions with microorganisms.

Testate amoebae taxonomy and trait diversity are coupled along an openness and wetness gradient in pine-dominated Baltic bogs.

Sphagnum peatlands host a high abundance of protists, especially testate amoebae. Here, we designed a study to investigate the functional diversity of testate amoebae in relation to wetness and forest cover in Baltic bogs. We provided new data on the influence of openness/wetness gradient on testate amoebae communities, showing significant differences in selected testate amoebae (TA) traits. Three key messages emerged from our investigations: 1) we recorded an effect of peatland surface openness on testate amoebae functional traits that led us to accept the hypothesis that TA traits differ according to light intensity and hydrology. Mixotrophic species were recorded in high relative abundance in open plots, whereas they were nearly absent in forested sites; 2) we revealed a hydrological threshold for the occurrence of mixotrophic testate amoebae that might be very important in terms of peatland functioning and carbon sink vs. source context; and 3) mixotrophic species with organic tests were nearly absent in forested sites that were dominated by heterotrophic species with agglutinated or idiosomic tests. An important message from this study is that taxonomy of TA rather indicates the hydrological gradient whereas traits of mixotrophs the openness gradient.

Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila.

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea.

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

Isolation and Phylogenetic Analysis of Free-Living Amoebae (Acanthamoeba, Naegleria, and Vermamoeba) in the Farmland Soils and Recreational Places in Iran.

PURPOSE: Free-living amoeba (FLA) including Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria are among the soil-born parasites. There are reports of FLA-related keratitis with a history of contact with soil and dust sources, particularly among the farmers. Due to lack of the previous studies on the farmland soils and a limited number of researches conducted on recreational soils in Iran, the present study was conducted. METHODS: A total of 93 soil samples including farming lands and recreational places were tested for the presence of Acanthamoeba spp. Balamuthia mandrillaris, Naegleria, and Vermamoeba using morphological key and sequencing-based tools. Pathogenicity of Acanthamoeba positive strains was also evaluated. To verify genetic associations and taxonomic status of isolated amoeba, a phylogenetic tree was built by MEGA 5.05 software inferred by the 18S rRNA gene based on maximum likelihood algorithm. RESULTS: Overall, 28 samples (30%) were contaminated with potentially pathogenic FLA, and according to the sequencing data, 17 strains were successfully sequenced. The isolated Acanthamoeba belonged to T2, T4, T5, mixed T4 and T5 contaminations, and T11. ITS sequencing revealed the occurrence of one strain of Naegleria canariensis. Four strains of Vermamoeba vermiformis were also confirmed. Morphological survey and PCR assay failed to show any positive results for Balamuthia mandrillaris. Pathogenic potential of the Acanthamoeba strains showed that T2, T4, and T11 genotypes were highly pathogenic, whereas T5 genotypes demonstrated lower pathogenic potential. CONCLUSION: The results indicate that soil could be a serious hazard to human health, and therefore, further studies are expected to investigate the source of infection in patients developing FLA-related diseases. The present study is the first to investigate FLA in the farmland soils in Iran and the first to report the presence of N. canariensis in the country.

Novel data from Italian Vermamoeba vermiformis isolates from multiple sources add to genetic diversity within the genus.

Vermamoeba vermiformis represents one of the most common free-living amoebae identified in worldwide environmental surveys. We analyzed 56 water samples with varying characteristics, including temperature and the particular settings in which humans may be exposed to water, plus one corneal scraping from a keratitis patient, with the following aims: (i) to investigate the presence of V. vermiformis; (ii) to identify the isolate subtypes; (iii) to place the Italian isolates in the broader picture of the genetic diversity within V. vermiformis. Twenty-two isolates were identified upon culturing and sequencing of > 600 bp in the 18S ribosomal RNA (rRNA) gene sequence, bringing to 27 the number of sequences recovered from Italian sources. By adding deposited sequences, we assembled a dataset of 74 isolates. Three of our isolates were characterized by allelic code 7-5-1-1, never reported before, and two showed 100% identity with an uncultured eukaryote and carried the 719T>C variant. We show that the variable segments E5, E3, F, and G convey most of the information on diversity, enabling the clustering of the isolates in a replicable fashion. The presence of different strains in natural thermal waters and in distribution systems indicated heterogeneity of the amoebic populations. Also, ours and the only other sequence from human infection were mapped in different clades. Overall, we enlarged the repertoire of single nucleotide and indel variants and the list of allelic codes, proceeding one step further in the description of the diversity within the genus.

Isolation and molecular identification of free-living amoebae from dishcloths in Tenerife, Canary Islands, Spain.

In this work, the presence of free-living amoebae (FLA) in dishcloths collected from human activity related places was evaluated. Once in the laboratory, 6 cm2 pieces of each dishcloth were cut and washed with Page's Amoeba Solution (PAS) in sterile tubes. After washing, the dishcloth pieces were removed, and the tubes were centrifuged (1500 rpm for 10 min). The obtained pellets were seeded onto 2% non-nutrient agar (NNA) plates, incubated at room temperature and were monitored daily an inverted microscope. Once clonal cultures were obtained (only one type of FLA observed), molecular analyses were carried out in order to characterize the isolated FLA strains at the genus/genotype level. From the 31 dishcloths which were processed, FLA strains were isolated in NNA plates in 13 the samples (13/31, 42%). However, and due to bacterial overgrowth, only six strains were characterized at the molecular level (PCR and sequencing). Among the PCR positive strains, 83.33% (5/6) of the PCR positive samples belonged to Acanthamoeba genus (80% (4/5) to genotype T4 and 20% (1/5) to genotype T11). Furthermore, one strain was identified as a member of Allovahlkampfia genus using both morphological and molecular approaches. To the best of our knowledge, this is the first report on the isolation of Allovahlkampfia genus from dishcloths and in the Spanish territory. The presence of FLA in dishcloths should raise awareness to improve hygienic strategies in food- and domestic-related environments, in order to prevent contamination with these protozoa, which are able to be pathogenic and even to act as vehicles of other pathogenic agents.

Why Do Testate Amoeba Optima Related to Water Table Depth Vary?

This study focusses on the ecology of testate amoeba species in peatlands of the southern taiga of Western Siberia. To estimate the influence of the trophic state of mires on species optima related to water table depth, a separate study of three calibration datasets including ombrotrophic, minerotrophic and the combined habitats was conducted. In the datasets obtained separately from ombrotrophic and minerotrophic mires, the water table depth was the main factor affecting testate amoeba assemblages. However, the trophic state (specifically pH and ash content) was more important factor in the combined dataset, including all of the studied mires. For 36 testate amoeba species, which were found in the ombrotrophic and minerotrophic mire habitats, their species optima, obtained separately in ombrotrophic and minerotrophic datasets, differed significantly from each other. Some of these species preferred minerotrophic conditions, while others preferred ombrotrophic ones. For all species, the trophic state of the mires affected the values of the species optima related to water table depth, as revealed in the form of a threshold effect. In extreme conditions, the species were more sensitive to the trophic status than to the water table depth, and their optimum related to water table depth was distorted. Variation of the optimum was observed in those species that inhabited both ombrotrophic and minerotrophic mires due to the fact that mires with a different trophic status were included in the training sets. The optima did not vary for species inhabiting only ombrotrophic or only minerotrophic mires.

Five new species of dictyostelid social amoebae (Amoebozoa) from Thailand.

BACKGROUND: Dictyostelid cellular slime molds (dictyostelids) are common inhabitants of the soil and leaf litter layer of fields and forests, along with animal dung, where they feed mostly on bacteria. However, reports on the species diversity of dictyostelids in South Asia, particularly Thailand, are limited. The research reported in this paper was carried out to increase our knowledge of the species diversity of this group of organisms in northern Thailand. RESULTS: Forty soil samples were collected at four localities in northern Thailand to assess the species richness of dictyostelids. These samples yielded five dictyostelid isolates that were not morphologically consistent with any described species. Based on molecular signatures, all five of these isolates were assigned to the family Cavenderiaceae, genus Cavenderia. All five share a number of morphological similarities with other known species from this family. The new taxa differ from previously described species primarily in the size and complexity of their fruiting bodies (sorocarps). This paper describes these new species (Cavenderia aureostabilis, C. bhumiboliana, C. protodigitata, C. pseudoaureostipes, and C. subdiscoidea) based on a combination of morphological characteristics and their phylogenetic positions. CONCLUSIONS: At least 15 taxa of dictyostelids were obtained from the four localities in northern Thailand, which indicates the high level of species diversity in this region. Five species were found to be new to science. These belong to the family Cavenderiaceae, genus Cavenderia, and were described based on both morphology and phylogeny.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring.

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Successional change of testate amoeba assemblages along a space-for-time sequence of peatland development.

It is well established that in ombrotrophic bogs, water-table depth (WTD) is the primary environmental control on testate amoeba distribution. However, the environmental controls on testate amoebae in minerotrophic fens are less well known and successional change in their assemblages associated with fen-bog peatland development has been scarcely investigated. Here we investigate a peatland space-for-time sequence resulting from postglacial rebound on the west coast of Finland, to assess successional patterns in testate amoeba communities and their relationships with environmental variables during peatland development. Sample sites along a 10-km transect from coast to inland ranged from a recently emerged wet meadow to a mature bog. Environmental variables (e.g., peat thickness, carbon and nitrogen content, pH, WTD and vegetation) were measured alongside testate amoeba samples. Results showed that even though the distribution of testate amoebae was to some extent determined by the succession stage, many taxa had wide WTD and pH ranges. The primary environmental control for many taxa changed along the succession. In conclusion, the ecological constraints on testate amoebae in minerotrophic systems are more complex than in bogs. The detected patterns also complicate the use of testate amoebae as a primary proxy in palaeoecological reconstructions where fen-to-bog shifts occur.

Evaluation of Morphological Characteristics to Delineate Taxa of the Genus Trigonopyxis (Amoebozoa, Arcellinida).

Morphological features are often the only characteristics suitable for identification of taxa in testate amoebae, especially in ecological and palaeoecological studies. However, whereas the morphology of some species is rather stable it may vary considerably in others. Within the order Arcellinida the genus Trigonopyxis with the type species Trigonopyxis arcula is morphologically highly variable. To identify reliable characteristics for morphology-based taxon delineation we investigated variations in shell size, pseudostome diameter and pseudostome form in T. arcula from three different sites of the Ecuadorian Andes, where these characteristics vary even more than previously described. Further, we investigated if morphological characteristics in Trigonopyxis varied with changes in environmental factors. We studied 951 shells of Trigonopyxis collected along an altitudinal gradient with varying abiotic factors. We established a method for characterization of the pseudostome form, which lead to five different morphotypes. Our results suggest that shell size alone is not an appropriate character for taxon delineation but can be used as an indicator for changes in environmental conditions. In contrast, the pseudostome form might be used for taxon delineation, but likely also varies considerably within taxa. Overall, the study provides an overview of the morphological variability of the genus Trigonopyxis.