Self-activated arsenic manganite nanohybrids for visible and synergistic thermo/immuno-arsenotherapy.

Arsenotherapy has been clinically exploited to treat a few types of solid tumors despite of acute promyelocytic leukemia using arsenic trioxide (ATO), however, its efficacy is hampered by inadequate delivery of ATO into solid tumors owing to the absence of efficient and biodegradable vehicles. Precise spatiotemporal control of subcellular ATO delivery for potent arsenotherapy thus remains challengeable. Herein, we report the self-activated arsenic manganite nanohybrids for high-contrast magnetic resonance imaging (MRI) and arsenotherapeutic synergy on triple-negative breast cancer (TNBC). The nanohybrids, composed of arsenic­manganese-co-biomineralized nanoparticles inside albumin nanocages (As/Mn-NHs), switch signal-silent background to high proton relaxivity, and simultaneously afford remarkable subcellular ATO level in acidic and glutathione environments, together with reduced ATO resistance against tumor cells. Then, the nanohybrids enable in vivo high-contrast T1-weighted MRI signals in various tumor models for delineating tumor boundary, and simultaneously yield efficient arsenotherapeutic efficacy through multiple apoptotic pathways for potently suppressing subcutaneous and orthotopic breast models. As/Mn-NHs exhibited the maximum tumor-to-normal tissue (T/N) contrast ratio of 205% and tumor growth inhibition rate of 88% at subcutaneous 4T1 tumors. These nanohybrids further yield preferable synergistic antitumor efficacy against both primary and metastatic breast tumors upon combination with concurrent thermotherapy. More importantly, As/Mn-NHs considerably induce immunogenic cell death (ICD) effect to activate the immunogenically "cold" tumor microenvironment into "hot" one, thus synergizing with immune checkpoint blockade to yield the strongest tumor inhibition and negligible metastatic foci in the lung. Our study offers the insight into clinically potential arsenotherapeutic nanomedicine for potent therapy against solid tumors.

Transcriptomic Insights into the Diversity and Evolution of Myxozoa (Cnidaria, Endocnidozoa) Toxin-like Proteins.

Myxozoa is a speciose group of endoparasitic cnidarians that can cause severe ecological and economic effects. Their cnidarian affinity is affirmed by genetic relatedness and the presence of nematocysts, historically called "polar capsules". Previous studies have revealed the presence of toxin-like proteins in myxozoans; however, the diversity and evolution of venom in Myxozoa are not fully understood. Here, we performed a comparative analysis using the newly sequenced transcriptomes of five Myxobolidae species as well as some public datasets. Toxin mining revealed that myxozoans have lost most of their toxin families, while most species retained Kunitz, M12B, and CRISP, which may play a role in endoparasitism. The venom composition of Endocnidozoa (Myxozoa + Polypodium) differs from that of free-living cnidarians and may be influenced by ecological and environmental factors. Phylogenetic analyses showed that toxin families of myxozoans and free-living cnidarians were clustered into different clades. Selection analyses showed that purifying selection was the dominant evolutionary pressure in toxins, while they were still influenced by episodic adaptive selection. This suggests that the potency or specificity of a particular toxin or species might increase. Overall, our findings provide a more comprehensive framework for understanding the diversity and evolution of Myxozoa venoms.

A myxozoan genome reveals mosaic evolution in a parasitic cnidarian.

BACKGROUND: Parasite evolution has been conceptualized as a process of genetic loss and simplification. Contrary to this model, there is evidence of expansion and conservation of gene families related to essential functions of parasitism in some parasite genomes, reminiscent of widespread mosaic evolution-where subregions of a genome have different rates of evolutionary change. We found evidence of mosaic genome evolution in the cnidarian Myxobolus honghuensis, a myxozoan parasite of fish, with extremely simple morphology. RESULTS: We compared M. honghuensis with other myxozoans and free-living cnidarians, and determined that it has a relatively larger myxozoan genome (206 Mb), which is less reduced and less compact due to gene retention, large introns, transposon insertion, but not polyploidy. Relative to other metazoans, the M. honghuensis genome is depleted of neural genes and has only the simplest animal immune components. Conversely, it has relatively more genes involved in stress resistance, tissue invasion, energy metabolism, and cellular processes compared to other myxozoans and free-living cnidarians. We postulate that the expansion of these gene families is the result of evolutionary adaptations to endoparasitism. M. honghuensis retains genes found in free-living Cnidaria, including a reduced nervous system, myogenic components, ANTP class Homeobox genes, and components of the Wnt and Hedgehog pathways. CONCLUSIONS: Our analyses suggest that the M. honghuensis genome evolved as a mosaic of conservative, divergent, depleted, and enhanced genes and pathways. These findings illustrate that myxozoans are not as genetically simple as previously regarded, and the evolution of some myxozoans is driven by both genomic streamlining and expansion.

Quantitative Insights into the Contribution of Nematocysts to the Adaptive Success of Cnidarians Based on Proteomic Analysis.

Nematocysts are secretory organelles in cnidarians that play important roles in predation, defense, locomotion, and host invasion. However, the extent to which nematocysts contribute to adaptation and the mechanisms underlying nematocyst evolution are unclear. Here, we investigated the role of the nematocyst in cnidarian evolution based on eight nematocyst proteomes and 110 cnidarian transcriptomes/genomes. We detected extensive species-specific adaptive mutations in nematocyst proteins (NEMs) and evidence for decentralized evolution, in which most evolutionary events involved non-core NEMs, reflecting the rapid diversification of NEMs in cnidarians. Moreover, there was a 33-55 million year macroevolutionary lag between nematocyst evolution and the main phases of cnidarian diversification, suggesting that the nematocyst can act as a driving force in evolution. Quantitative analysis revealed an excess of adaptive changes in NEMs and enrichment for positively selected conserved NEMs. Together, these findings suggest that nematocysts may be key to the adaptive success of cnidarians and provide a reference for quantitative analyses of the roles of phenotypic novelties in adaptation.

Molecular and light microscopy evidence for the transfer of Myxobolus honghuensis from Carassius auratus gibelio broodfish to progeny.

Myxozoans usually have a complex life cycle involving indirect transmission between vertebrate and invertebrate hosts. The vertical transmission of these parasites in vertebrate hosts has not been documented so far. Here, we assessed whether the myxozoan parasite Myxobolus honghuensis is vertically transmitted in naturally infected allogynogenetic gibel carp Carassius auratus gibelio (Bloch). M. honghuensis infection of broodfish, fertilized eggs and laboratory-cultured progeny was monitored in 2018 and 2019. The presporogonic stage was microscopically observed in the pharynx of broodfish and their progeny. In situ hybridization confirmed the presence of M. honghuensis presporogonic stage in the pharynx of broodfish and progeny. Nested PCR results showed that M. honghuensis was present in tissues and eggs of broodfish, fertilized eggs and their corresponding progeny. The sequences obtained from broodfish and progeny showed 98.0-99.8% similarity with ITS-5.8S rDNA of M. honghuensis. This study provides molecular and light microscopy evidence for the transfer of M. honghuensis from broodfish to progeny via the eggs, but it is insufficient to assert that M. honghuensis can transmit vertically in naturally infected allogynogenetic gibel carp. This is the first record about vertical transfer of myxozoan in the vertebrate host.

CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

Protein reference databases are a critical part of producing efficient proteomic analyses. However, the method for constructing clean, efficient, and comprehensive protein reference databases of nonmodel organisms is lacking. Existing methods either do not have contamination control procedures, or these methods rely on a three-frame and/or six-frame translation that sharply increases the search space and the need for computational resources. Herein, we propose a framework for constructing a customized comprehensive proteomic reference database (CCPRD) from draft genomes and deep sequencing transcriptomes. Its effectiveness is demonstrated by incorporating the proteomes of nematocysts from endoparasitic cnidarian: myxozoans. By applying customized contamination removal procedures, contaminations in omic data were successfully identified and removed. This is an effective method that does not result in overdecontamination. This can be shown by comparing the CCPRD MS results with an artificially contaminated database and another database with removed contaminations in genomes and transcriptomes added back. CCPRD outperformed traditional frame-based methods by identifying 35.2-50.7% more peptides and 35.8-43.8% more proteins, with a maximum of 84.6% in size reduction. A BUSCO analysis showed that the CCPRD maintained a relatively high level of completeness compared to traditional methods. These results confirm the superiority of the CCPRD over existing methods in peptide and protein identification numbers, database size, and completeness. By providing a general framework for generating the reference database, the CCPRD, which does not need a high-quality genome, can potentially be applied to nonmodel organisms and significantly contribute to proteomic research.

Controllable coupling between an ultra-high-Q microtoroid cavity and a graphene monolayer for optical filtering and switching applications.

Whispering-gallery-mode optical microresonators have found impactful applications in various areas due to their remarkable properties such as ultra-high quality factor (Q-factor), small mode volume, and strong evanescent field. Among these applications, controllable tuning of the optical Q-factor is vital for on-chip optical modulation and various opto-electronic devices. Here, we report an experimental demonstration with a hybrid structure formed by an ultra-high-Q microtoroid cavity and a graphene monolayer. Thanks to the strong interaction of the evanescent wave with the graphene, the structure allows the Q-factor to be controllably varied in the range of 3.9 × 105 ∼ 6.2 × 107 by engineering optical absorption via changing the gap distance in between. At the same time, a resonant wavelength shift of 32 pm was also observed. Besides, the scheme enables us to approach the critical coupling with a coupling depth of 99.6%. As potential applications in integrated opto-electronic devices, we further use the system to realize a tunable optical filter with tunable bandwidth from 116.5 MHz to 2.2 GHz as well as an optical switch with a maximal extinction ratio of 31 dB and response time of 21 ms.

A fast and effective method for dissecting parasitic spores: myxozoans as an example.

Disassembling parasitic spores and acquiring the main subunits for analysis is a prerequisite for a deep understanding of the basic biology of parasites. Herein, we present a fast and efficient method to dissect myxospores in a few steps, which mainly involves sonication, and sucrose and Percoll density gradient ultracentrifugation. We tested our method on three myxozoan species and demonstrate that this method allows the dismembering of myxospores, and the isolation of intact and clean nematocysts and shell valves within 2 h at low cost. This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.

A fast and effective method for dissecting parasitic spores: myxozoans as an example.

Disassembling the parasitic spores and acquiring the main subunits is a prerequisite for deep understanding of the basic biology of parasites. Herein we present a fast and efficient method to dissect the myxospores in a few steps, which mainly involved sonication, sucrose density gradient and Percoll density gradient. We tested our method on three myxozoans species and demonstrated this method allows the dismembering of myxospores, isolation of intact and clean nematocysts and shell valves within 2h by low-cost. This new tool will facilitate subsequent analyses and enable a better understanding of the ecological and evolutionary significance of parasitic spores.

Anti-Parity-Time Symmetric Optical Four-Wave Mixing in Cold Atoms.

Non-Hermitian optical systems with parity-time (PT) symmetry have recently revealed many intriguing prospects that outperform conservative structures. The previous works are mostly rooted in complex arrangements with controlled gain-loss interplay. Here, we demonstrate anti-PT symmetry inherent in the nonlinear optical interaction based upon forward optical four-wave mixing in a laser-cooled atomic ensemble with negligible linear gain and loss. We observe that the pair of frequency modes undergo a nontrivial anti-PT phase transition between coherent power oscillation and optical parametric amplification in presence of a large phase mismatch.

Morphological, histological and molecular characterization of Myxobolus kingchowensis and Thelohanellus cf. sinensis infecting gibel carp Carassius auratus gibelio (Bloch, 1782).

A Myxobolus species and a Thelohanellus species infecting Carassius auratus gibelio (Bloch, 1782) were redescribed by their morphological, histological and molecular characterization. In the present study, the Myxobolus species infecting the muscle was identified as Myxobolus kingchowensis Chen et Ma, 1998 by the morphological and molecular data. Histologically, mature spores of M. kingchowensis were observed in the intercellular and connective tissue of muscle, though the plasmodia were not found. In addition, scattered spores also occurred in the intercellular of haematopoietic cells, intraepithelial of the renal tubules and interior of the melano-macrophage centres. Phylogenetic analysis showed that M. kingchowensis clustered in the clade of muscle-infecting Myxobolus species, further supporting muscle as the infection site of M. kingchowensis. The present Thelohanellus species infecting the gills was identified conspecific as Thelohanellus sinensis reported in Sun (2006) (mark it as T. sinensis-Sun)based on spore morphology, biological traits (host specificity and organ specificity), and molecular data. However, compared with the original description of T. sinensis Chen et Hsieh, 1960, the present Thelohanellus species and T. sinensis-Sun both infecting the gills of gibel carp are distinguishable from the original description in the host and infection site, which made the validity of T. sinensis-Sun dubious. Due to the absence of molecular data in the original description of T. sinensis, we suggest marking the present species and T. sinensis-Sun as T. cf. sinensis to avoid the confusion until T. sinensis is obtained from the type host and type infection site.

Whole-transcriptome splicing profiling of E7.5 mouse primary germ layers reveals frequent alternative promoter usage during mouse early embryogenesis.

Alternative splicing (AS) and alternative promoter (AP) usage expand the repertories of mammalian transcriptome profiles and thus diversify gene functions. However, our knowledge about the extent and functions of AS and AP usage in mouse early embryogenesis remains elusive. Here, by performing whole-transcriptome splicing profiling with high-throughput next generation sequencing, we report that AS extensively occurs in embryonic day (E) 7.5 mouse primary germ layers, and may be involved in multiple developmental processes. In addition, numerous RNA splicing factors are differentially expressed and alternatively spliced across the three germ layers, implying the potential importance of AS machinery in shaping early embryogenesis. Notably, AP usage is remarkably frequent at this stage, accounting for more than one quarter (430/1,648) of the total significantly different AS events. Genes generating the 430 AP events participate in numerous biological processes, and include important regulators essential for mouse early embryogenesis, suggesting that AP usage is widely used and might be relevant to mouse germ layer specification. Our data underline the potential significance of AP usage in mouse gastrulation, providing a rich data source and opening another dimension for understanding the regulatory mechanisms of mammalian early development.

ERK inhibition promotes neuroectodermal precursor commitment by blocking self-renewal and primitive streak formation of the epiblast.

BACKGROUND: Pluripotent stem cells hold great promise for regenerative medicine. However, before clinical application, reproducible protocols for pluripotent stem cell differentiation should be established. Extracellular signal-regulated protein kinase (ERK) signaling plays a central role for the self-renewal of epiblast stem cells (EpiSCs), but its role for subsequent germ layer differentiation is still ambiguous. We proposed that ERK could modulate differentiation of the epiblast. METHODS: PD0325901 was used to inhibit ERK activation during the differentiation of embryonic stem cells and EpiSCs. Immunofluorescence, western blot analysis, real-time PCR and flow cytometry were used to detect germ layer markers and pathway activation. RESULTS: We demonstrate that the ERK phosphorylation level is lower in neuroectoderm of mouse E7.5 embryos than that in the primitive streak. ERK inhibition results in neural lineage commitment of epiblast. Mechanistically, PD0325901 abrogates the expression of primitive streak markers by ß-catenin retention in the cytoplasm, and inhibits the expression of OCT4 and NANOG during EpiSC differentiation. Thus, EpiSCs differentiate into neuroectodermal lineage efficiently under PD0325901 treatment. These results suggest that neuroectoderm differentiation does not require extrinsic signals, supporting the default differentiation of neural lineage. CONCLUSIONS: We report that a single ERK inhibitor, PD0325901, can specify epiblasts and EpiSCs into neural-like cells, providing an efficient strategy for neural differentiation.

Myxobolus pseudowulii sp. n. (Myxozoa: Myxosporea), a new skin parasite of yellow catfish Tachysurus fulvidraco (Richardson) and redescription of Myxobolus voremkhai (Akhmerov, 1960).

Two species of Myxobolus Bütschli, 1882 were found in yellow catfish Tachysurus fulvidraco (Richardson). A species of Myxobolus infecting the gills was morphologically identified as Myxobolus voremkhai (Akhmerov, 1960) and it was characterised here with additional morphological and molecular data. The other species of Myxobolus infecting the host's skin did not conform to any known myxosporean species. It is characterised by the presence of round, black or milky white plasmodia with black spots. Myxospores are pyriform in frontal view and lemon-shaped in lateral view, measuring 12.9-16.2 µm (14.6 ± 0.7 µm) in length, 8.1-10.8 µm (9.4 ± 0.5 µm) in width, and 6.1-8.1 µm (7.0 ± 0.4 µm) in thickness. Two ampullaceous polar capsules are slightly unequal in size, larger polar capsule 7.2-9.5 µm (7.9 ± 0.4 µm) long by 3.0-3.9 µm (3.5 ± 0.2 µm) wide, smaller capsule 6.9-8.0 µm (7.4 ± 0.3 µm) long by 2.9-3.9 µm (3.4 ± 0.2 µm) wide. Polar filaments are coiled with seven to nine turns. Histologically, the plasmodia develop in the stratum spongiosum of skin dermis, resulting in epithelial cell shedding and immunological cell infiltration. Given the morphological and molecular differences between this species and other species of Myxobolus, we proposed the name of Myxobolus pseudowulii sp. n. for this parasite from the skin of yellow catfish. Interestingly, some spores of the new species possess Henneguya-like caudal appendages. Phylogenetically, M. pseudowulii sp. n. and M. voremkhai infecting yellow catfish group together in one clade with other parasites of Siluriformes, indicating that parasites clustering according to the fish host order may be an important factor affecting the evolution of species within the Myxobolus clade.

Development of a multiplex PCR method for the simultaneous detection of four myxosporeans infecting gibel carp Carassius auratus gibelio.

Gibel carp Carassius auratus gibelio (Bloch), a commercially important freshwater-cultured fish in China, is threatened by myxosporeans, particularly Thelohanellus wuhanensis, Myxobolus honghuensis, M. wulii and M. turpisrotundus. Here, we developed a multiplex PCR assay for simultaneous detection of these 4 myxosporeans. The specific primers for each species were designed based on the 28S rDNA gene of T. wuhanensis, the ITS-5.8S rDNA of M. honghuensis and M. wulii, and the 18S rDNA gene of M. turpisrotundus. Specificity testing confirmed that the 4 primer sets have no cross-reactivity with other related myxosporean species tested. Detection limits of the multiplex PCR assay were 0.2, 0.3, 3.1 and 3.8 spores for T. wuhanensis, M. honghuensis, M. wulii and M. turpisrotundus, respectively. Following screening of 104 field samples, the analytical sensitivity of the present multiplex PCR assay was found to be similar to the sensitivity obtained by the singleplex PCR assays and was higher than that of microscopic examination. Moreover, Kappa analysis showed a strong agreement between the results of the singleplex and multiplex PCR assays, indicating that the developed multiplex PCR assay was an efficient approach for the diagnosis of the 4 myxosporeans infecting gibel carp.

Histopathological and ultrastructural studies of Myxobolus turpisrotundus from allogynogenetic gibel carp Carassius auratus gibelio in China.

During an ongoing systematic survey on species diversity of myxozoans parasitising allogynogenetic gibel carp Carassius auratus gibelio (Bloch) in China, plasmodia were detected in the fins, lip, jaw, gill chamber, gill arches, operculum and oral cavity of infected fish. Combining the morphological and molecular data, the present species was identified as Myxobolus turpisrotundus Zhang, Wang, Li et Gong, 2010. Histopathological examination revealed that despite infecting different organs, M. turpisrotundus always occurred in dermis, demonstrating its affinity to this tissue. Histopathological effect of M. turpisrotundus on the host is relatively mild except parasites in the gill arches producing compression of the adipose tissue and heavy adductor muscles deformation with lymphohistiocytic infiltrates. In addition, the plasmodia in different sites were with the same complex structure arrangement: cup-like cells with unknown derivation, a thin collagenous fibril layer, areolar connective tissue, basement membrane and host epithelial cell. Ultrastructural analysis showed that the parasite has monosporic pansporoblast and sporogenesis followed the usual pattern of most of the myxosporeans.

TSC1 controls IL-1ß expression in macrophages via mTORC1-dependent C/EBPß pathway.

The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR), which serves as a key regulator of inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Previous studies have shown that TSC1 knockout (KO) macrophages produced increased inflammatory responses including tumor necrosis factor-α (TNF-α) and IL-12 to pro-inflammatory stimuli, but whether and how TSC1 regulates pro-IL-1ß expression remains unclear. Here using a mouse model in which myeloid lineage-specific deletion of TSC1 leads to constitutive mTORC1 activation, we found that TSC1 deficiency resulted in impaired expression of pro-IL-1ß in macrophages following lipopolysaccharide stimulation. Such decreased pro-IL-1ß expression in TSC1 KO macrophages was rescued by reducing mTORC1 activity with rapamycin or deletion of mTOR. Rictor deficiency has no detectable effect on pro-IL-1ß synthesis, suggesting that TSC1 positively controls pro-IL-1ß expression through mTORC1 pathway. Moreover, mechanism studies suggest that mTORC1-mediated downregulation of the CCAAT enhancer-binding protein (C/EBPß) critically contributes to the defective pro-IL-1ß expression. Overall, these findings highlight a critical role of TSC1 in regulating innate immunity by control of the mTOR1-C/EBPß pathway.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

Intraspecific morphometric variation in myxosporeans.

Morphometric data from spores of ten myxosporean species were statistically analysed to explore myxosporean intraspecific variation in measurements when obtained from a sample from: (1) the same plasmodium, (2) different plasmodia from the same host and (3) different host individuals and localities. In some cases, significant differences in spore dimensions were found between samples from the same plasmodium, highlighting the difficulty of obtaining representative measurements of myxosporean spore. In addition, significant differences in spore dimensions were found when plasmodia from the same site of infection were compared, suggesting that measurements of spores should come from several different plasmodia of the sampling to increase the reliability of the morphology data. Moreover, significant differences in spore dimensions were observed for most spore dimensions when data were compared between localities. In all cases, there was clear overlap in ranges of dimensions even when means differed significantly. The present statistical analysis shows that intraspecific morphometric variation of myxosporean species commonly occurs, highlighting the importance of reporting ranges of measurements for a species, not just the mean dimensions, and taking into account all evidence when assigning or describing myxosporean species.

Morphological and molecular characterization of Thelohanellus macrovacuolaris n. sp. (Myxosporea: Bivalvulida) infecting the palate in the mouth of common carp Cyprinus carpio L. in China.

Thelohanellus macrovacuolaris n. sp. is described during a survey on myxozoan diversity of common carp Cyprinus carpio L. in China. It is characterized by the presence of round or ellipsoidal plasmodium in the palate in the mouth of host. Mature spores were pyriform in frontal view, lemon shaped in lateral view, measuring 21.6±0.9 (19.3-23.8) long, 12.5±0.7 (10.3-13.6) wide, and 10.2±0.4 (9.8-11.8) thick. Most spores were surrounded by the membrane sheath. Single polar capsule was round with an apophysis at its top end presented close to apex of spore, measuring 9.1±0.6 (8.0-10.0) in length, 8.6±0.5 (7.8-9.6) in width. Polar filaments coiled, with 7 to 8 turns. A large, round iodinophilous vacuole was present, with 5.8-7.5 in diameter. The present species is morphologically distinct from all other Thelohanellus species. The BLAST search indicated that the newly obtained small subunit ribosomal RNA (ssrRNA) gene sequence of T. macrovacuolaris n. sp. did not match any available sequences in GenBank. Phylogenetically, T. macrovacuolaris n. sp. was placed sister to Thelohanellus wangi in the Thelohanellus clade. Both morphology and ssrRNA gene sequence data revealed that the present parasite is a new species of genus Thelohanellus.