Introduction
The green dock leaf beetle, Gastrophysa viridula De Geer, 1775 (Coleoptera: Chrysomelidae), is a native and widely distributed species across Europe, typically inhabiting predominantly humid environments (Kwon, Reference Kwon2006; Voigt et al., Reference Voigt, Hosoda, Schuppert and Gorb2011; Özdikmen et al., Reference Özdikmen, Karsavuran, Bal and Pehlivan2021). This taxon, which is thought to be rare in Türkiye, has been recorded only in Erzurum, Trabzon and Gümüşhane provinces. As an oligophagous species, it primarily feeds on plants from the genera Rumex, Polygonum and Rheum within the family Polygonaceae (Aslan and Özbek, Reference Aslan and Özbek1999; Voigt et al., Reference Voigt, Hosoda, Schuppert and Gorb2011). In recent years, interest in the biology of G. viridula has grown, particularly due to its observed impact on rhubarb crops (Szwejda and Rogowska, Reference Szwejda and Rogowska2004). Conversely, this beetle has also been regarded as a potential biological control agent for dock plants (Rumex spp.), which are considered undesirable weeds in grazing lands (Aslan and Özbek, Reference Aslan and Özbek1999; Martinkova and Honek, Reference Martinkova and Honek2004; Zaller, Reference Zaller2004). Additionally, G. viridula has been extensively employed as a model organism in ecological research focusing on population dynamics (Smith and Whittaker, Reference Smith and Whittaker1980; Kozlowski, Reference Kozlowski2004) and in studies investigating insect biochemistry and behaviour (Kunert et al., Reference Kunert, Søe, Bartram, Discher, Tolzin-banasch, Nie, David, Pasteels and Boland2008). For these reasons, it is of utmost importance to accurately identify the pathogens and parasites affecting this beetle.
Microsporidia are obligate intracellular parasites known for causing persistent infections that result in significant deterioration of the host’s overall health. These parasites are highly effective in their transmission, occurring both horizontally and vertically (Becnel and Andreadis, Reference Becnel, Andreadis, Wittner and Weiss1999). Due to these characteristics, microsporidia can swiftly infiltrate and disseminate within colonies, often remaining undetected until nearly all individuals are infected, leading to substantial declines in productivity and lifespan (Becnel et al., Reference Becnel, Jeyaprakash, Hoy and Shapiro2002; Yıldırım and Bekircan, Reference Yıldırım and Bekircan2020). Entomopathogenic taxa within this group, recognized for their pronounced host specificity, are regarded as effective biological control agents capable of regulating insect populations. In this context, numerous studies have been conducted to identify and characterize novel microsporidian species, and to date, over 30 microsporidian species have been identified exclusively within chrysomelids (Toguebaye et al., Reference Toguebaye, Marchand, Bouix, Jolivet, Petitpierre and Hsiao1988; Bekircan et al., Reference Bekircan, Bülbül, Hİ and Becnel2016; Yıldırım and Bekircan, Reference Yıldırım and Bekircan2020). Two natural microsporidian infections have been identified in different populations of G. viridula: one is an uncharacterized record belonging to the genus Nosema (Nosema sp.), and the other is the species Vairimorpha gastrophysae (Wegensteiner et al., Reference Wegensteiner, Tkaczuk, Balazy and Kaiser2008; Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). In addition to these findings, several studies have demonstrated that G. viridula can be artificially infected with various microsporidian species, including Nosema algerae and Nosema gastroideae (Hostounsky, Reference Hostounsky1982, Reference Hostounsky1984).
In this study, the natural microsporidian parasites of the green dock leaf beetle, G. viridula, were investigated. This research provides a thorough examination of the morphology, ultrastructure and molecular phylogenetics of a newly identified microsporidian species. Additionally, the molecular phylogeny was constructed based on the analysis of partial sequences from the small subunit ribosomal RNA (SSU rRNA) and the largest subunit of RNA polymerase II (RPB1) genes to define this novel taxon. These findings offer valuable insights into the evolutionary trajectory of this microsporidian clade.
Material and methods
Sample collection and light microscopy
Between 2022 and 2023, adult specimens of G. viridula were collected from dock plants (Rumex spp.) in Trabzon (Tonya), Türkiye. Following macroscopic examination, the collected specimens were placed in sterile plastic containers and promptly transported to the laboratory for further analyses, ensuring that the cold chain was maintained throughout transportation. For light microscopy, specimens were dissected using Ringer’s solution (Baki and Bekircan, Reference Baki and Bekircan2018; Yıldırım and Bekircan, Reference Yıldırım and Bekircan2020). Wet smears were then prepared and examined under a light microscope (Nikon, Eclipse Ci) at various magnifications to detect potential microsporidian infections. Prevalence (%) was determined as the proportion of infected hosts among the examined individuals. To identify the life cycle stages, infection-positive smears were stained with Giemsa stain, as described in the literature (Bekircan et al., Reference Bekircan, Bülbül, Hİ and Becnel2016, Reference Bekircan, Tosun and Yıldırım2025; Bekircan, Reference Bekircan2020). Digital images and measurements were captured using a DS-Fi 2 digital camera mounted on a Nikon Eclipse Ci microscope and analysed with Nikon NIS Elements imaging software. Additionally, some infected tissue samples were preserved in 70% ethanol for subsequent PCR amplification and stored under refrigeration conditions.
Electron microscopy
Tissues fixed with 2.5% glutaraldehyde for electron microscopy were rinsed 3 times (15 minutes each) with 0.1 M cacodylate buffer prior to post-fixation. Post-fixation was conducted using 1% osmium tetroxide (OsO₄) for 2 hours. Subsequently, the post-fixed tissues were washed again with 0.1 M cacodylate buffer and dehydrated through a graded ethanol series. Spurr’s (Reference Spurr1969) resin was employed to embed the samples. Following ultramicrotomy using a Leica EM UC7, sections were stained with saturated uranyl acetate and Reynolds’ lead citrate (Reynolds, Reference Reynolds1963). Observations were performed with the JEOL JEM 1220 transmission electron microscope.
Genomic DNA extraction and phylogenetic analysis
Ethanol-fixed infected tissues were rinsed 3 times with distilled water to remove residual contaminants. Subsequently, the tissues were transferred to a new Eppendorf tube and mechanically homogenized using a micropestle. Genomic DNA was extracted from the resulting homogenate utilizing the QIAGEN DNA Isolation Kit (Cat. No. 69504), in accordance with the manufacturer’s protocol. Partial SSU rRNA gene amplification was carried out using primers 18 F and 1537 R (18 F/1537 R: 5′-CACCA GGTTG ATTCT GCC-3′/5′-TTATG ATCCTG CTAA TGGTT C-3′) (Vossbrinck and Debrunner-Vossbrinck, Reference Vossbrinck and Debrunner-Vossbrinck2005). PCR reactions (50 μl) were prepared with the QIAGEN Multiplex PCR Kit (Cat. No. 206143), and thermocycler conditions were applied according to the manufacturer’s instructions. Amplification of the RPB1 gene employed primers NosVar_RPB1f and NosVar_RPB1r (F/R:5′-CAARAA RAT CYT KGAA TGY RTTT-3′/5′-THYKATC AAA RAAWGMATC TCG-3′) (Bekircan et al., Reference Bekircan, Tosun and Yıldırım2025). The same amplification protocol used for SSU rRNA was consistently applied to RPB1. Amplicons from each gene were submitted to Macrogen, Netherlands, for sequencing.
Based on a BLAST search of the NCBI GenBank database and a review of the literature, sequences utilized in the phylogenetic analyses were selected (Table 1). All sequences were curated and aligned using BioEdit software and the CLUSTAL_W algorithm. Encephalitozoon cuniculi (Z19563) and Ordospora colligata (XM014708712) served as outgroups. Pairwise genetic distances and similarities were calculated using the Kimura-2 parameter model distance matrix. Phylogenetic analyses were conducted using the maximum likelihood (ML) method in MEGA version 12.0.10 and Bayesian inference (BI) in BEAST v1.10.4 (Kumar et al., Reference Kumar, Stecher, Suleski, Sanderford, Sharma and Tamura2024).
Accession codes for partial sequences of the small subunit (SSU) ribosomal RNA and the largest subunit of RNA polymerase II (RPB1), along with the corresponding host organisms of various microsporidian species, were utilized in the phylogenetic analysis
Table 1 Long description
Accession numbers are provided for two gene markers, SSU rRNA and RPB1, for multiple microsporidian taxa, alongside each taxon’s host organism and the host’s order and family. SSU rRNA entries include Encephalitozoon cuniculi from a rabbit host and many insect-associated records, especially Nosema and Vairimorpha. Lepidoptera hosts are most frequent across both markers, spanning families such as Noctuidae, Pyralidae, Bombycidae, Tortricidae, Crambidae, Geometridae, and Erebidae. Hymenoptera hosts appear repeatedly, including Apis cerana, Apis mellifera, and Bombus terrestris in Apidae, plus other hymenopterans such as Torymidae. Coleoptera hosts are also represented, notably Gastrophysa viridula and Leptinotarsa decemlineata in Chrysomelidae, and additional beetle families. Non-insect hosts occur in the RPB1 section, including Amphipoda, Decapoda, and Cladocera, indicating broader host coverage for some taxa. Several species have multiple accessions or appear under both markers, such as Nosema bombycis, Nosema fumiferanae, Nosema granulosis, Nosema pyrausta, Nosema tyriae, Vairimorpha lymantriae, and Vairimorpha rumexii sp. nov. Names include some genus-level identifications and potential spelling inconsistencies, so entries should be interpreted as reported rather than as standardized taxonomy.
The optimal evolutionary model, determined according to Akaike information criteria, was GTR + G + I. Bootstrap confidence values for ML analysis were calculated with 1000 repetitions. BI analysis involved 2 independent runs with 4 chains over 1 million generations, with phylogenetic tree sampling every 100 generations. The first 25% of samples were discarded as part of the burn-in process (burninfrac = 0.25).
Results
Light microscopy
Field studies conducted within the scope of this research resulted in the collection of 98 adult specimens, with 55 collected in 2022 and 43 in 2023. Dissection procedures and light microscopy examinations revealed microsporidian infections in 4 specimens from 2022 and 2 specimens from 2023. The prevalence of infection identified over the 2-year sampling period was 6.12%. Light microscopy examinations demonstrated that microsporidiosis was predominantly localized in the gut and gonads (Figure 1A, B). Two distinct spore types, designated as Types I and II, were identified during the examinations. Fresh Type I spores exhibited an ovocylindrical even rod-shaped morphology, with dimensions of 5.75 ± 0.39 μm in length (range: 5.00–6.69 μm; n = 100) and 2.16 ± 0.24 μm in width (range: 1.70–2.81 μm; n = 100). In contrast, fresh Type II spores were ovoid in shape, measuring 4.72 ± 0.16 μm in length (range: 4.45–4.96 μm; n = 100) and 2.22 ± 0.27 μm in width (range: 1.77–2.73 μm; n = 100). Microscopic examinations of Giemsa-stained preparations revealed 2 sporulation cycles. The first cycle, identified as a disporoblastic process, characteristic of the genus Nosema, was involved in the production of Type I spores, which were directly associated with the host cell cytoplasm (Figure 2A–D). In this sporulation cycle, binucleate meronts were generally spherical, with an average diameter of 6.49 ± 0.94 μm (n = 10) (Figure 2A). Disporous sporonts of varying shapes (oval or spherical) developed into sporoblasts. The oval sporonts measured 4.47 ± 0.85 μm, × 2.57 ± 0.43 μm (n = 10), while the spherical sporonts had an average diameter of 5.77 ± 1.27 μm (n = 6) (Figure 2B). Putative sporoblast stages were observed; however, their distinction from early mature spores was not always clearly resolved under light microscopy. These stages were identified based on their intermediate morphology between sporonts and fully developed spores. The observed sporoblasts were approximately 5.50 ± 1.10 μm in length and 1.78 ± 0.14 μm in width (n = 3) (Figure 2C). Measurements conducted on Giemsa-stained preparations, intended to analyse life cycle stages, showed that stained Type I spores were 5.10 ± 0.30 μm in length (range: 4.37–5.86 μm; n = 100) and 2.08 ± 0.14 μm in width (range: 1.53–2.33 μm; n = 100) (Figure 2D), whereas Type II spores were 4.30 ± 0.27 μm in length (range: 3.66–4.75 μm; n = 100) and 2.09 ± 0.31 μm in width (range: 1.35–2.90 μm; n = 100) (Figure 3F).
Light micrographs of Vairimorpha rumexii sp. nov. in G. viridula. (A) Severely infected gut tissue. Arrows indicate Types I and II spores. (B) Giemsa-stained gonad. Arrows highlight mature spores, while the arrowhead marks a sperm cell of the host insect (scale bars = 10 μm).
Light microscopy of Giemsa-stained Type I spores in the life cycle of Vairimorpha rumexii sp. nov., observed in gut and gonad. (A) Spherical binucleate meront. (B) Binucleate oval and spherical sporont. (C) Putative sporoblasts. (D) Mature spores (scale bars = 5 μm).
Giemsa-stained stages of Vairimorpha rumexii sp. nov. illustrating the sporulation sequence of meiospores (Type II). (A) Uninucleate meront. (B) Binucleate sporogonic proliferation. (C) Tetranucleate sporogonic proliferation. (D) Octonucleate sporogonic proliferation. (E) Type II spores (meiospores) within the sporophorous vesicle (SPV). (F) Mature meisospores (scale bars = 5 μm).
The second sporulation cycle, classified as the Thelohania type, was distinctly differentiated from the Nosema type life cycle by the presence of a sporophorous vesicle (SPV) containing meiospores. This cycle led to the formation of Type II spores. Within this type of sporulation, the merogonial stages exhibited no structural distinctions from those observed in the Nosema-type cycle. Sporogonic proliferation occurred within a sporophorous vesicle (SPV), presenting bi-, tetra- and octonucleate forms (Figure 3A–D). Measurements indicated that the diameters of these distinct nuclear types of sporogonic stages ranged from 5.41 to 9.58 μm (n = 14). Type II spores (meiospores) originated from multinucleate sporonts within the SPV (Figure 3E).
Electron microscopy
Transmission electron microscopy revealed the predominant presence of Type II spores, although spore types corresponding to both sporulation cycles were observed. The polar tube structure was isofilar in both spore types. In Type I spores, the polar tube measured from 95 to 114 nm and comprised 12 to 16 coils. Mature coils demonstrated a structure of 5 concentric layers, each varying in electron density and thickness (Figure 4). The thickness of the spore wall was homogeneous throughout the spore structure, and the spore wall was relatively thin (70–120 nm). The exospore was measured at 19–48 nm, and the endospore was 55–80 nm (Figure 4).
Transmission electron micrograph of a longitudinal section of a Type I spore and the fine structure of the polar tube (transversely sectioned). Numbers (1–5) indicate the most distinct layers and the trilaminar structure of the spore wall. Abbreviations: Ex: exospore, En: endospore, Pm: plasma membrane.
Electron microscopy observations conducted in this study reaffirmed the sporogonic development within sporophorous vesicles, a characteristic feature of the Thelohania-type sporulation cycle (Figure 5A, B). Furthermore, various stages of meiospore sporogony were identified, and sporoblasts were easily recognized by the formation of primordial polar tubes and anchoring discs (Figure 5C). The spore wall thickness ranged from 85 to 115 nm, with the exospore measuring between 24 and 46 nm and the endospore varying from 56 to 80 nm in thickness. The polar tube of mature meiospores comprised 9 to 11 coils and had a diameter ranging from 90 to 104 nm. Additionally, the spherical nucleus, with an average size of 600 nm, was centrally positioned within these spores (Figure 5D).
Transmission electron micrographs of meiospores (Type II) and sporogonic proliferations. (A, B) Sporogonic proliferations in the sporophorous vesicle (SPV). (C) Maturing sporoblast with a single layer of the coils of the primordial polar tubes and the formation of the anchoring disc. (D) Longitudinal section of mature meiospore with isofilar polar tubes. Abbreviations: Ex: exospore, En: endospore, Pm: plasma membrane. n: nucleus, ppt: primordial polar tube, ad: anchoring disc, m: manubrium.
Phylogenetic analysis
The SSU rRNA and the largest subunit of RPB1 sequences of the microsporidium infecting G. viridula collected from Trabzon (Tonya) were deposited in GenBank under accession codes PV627787-PV610738. The SSU rRNA gene partial sequence of the current microsporidium consisted of 1106 bp, and the GC content was 36.2% (GenBank Accession code: PV627787). BLAST searches using the obtained SSU rDNA sequence indicated that the closest relative to our finding was the unidentified record Microsporidium sp. (MH712042), infecting Megastigmus spermotrophus (Hymenoptera: Torymidae), a seed pest of Douglas-fir (Pseudotsuga menziesii) (with 99.80% identity). Sequence identities between the current microsporidium and other sequences included in the phylogenetic analysis ranged from 58.0% to 99.80% (Table 2). The pairwise distances calculated by the Kimura 2-parameter model between SSU rDNA sequences of this novel and 31 other related microsporidia ranged from 0.0020 to 0.4087. It has been determined that the type species of the genera Vairimorpha and Nosema, namely Vairimorpha necatrix (DQ996241 and U11051) and Nosema bombycis (D85503), are separated from novel species by pairwise phylogenetic distances of 0.0367 and 0.1710, respectively. Phylogenetic analysis using the ML method and BI of the obtained SSU rDNA sequence revealed that the novel microsporidium initially clustered with Microsporidium sp. (MH712042) and subsequently formed a sister group with an unidentified microsporidium, Vairimorpha sp. (MG907029), isolated from Amyelois transitella (Lepidoptera: Pyralidae). Together, these taxa established a distinct basal lineage (Figure 6).
Phylogenetic trees based on maximum likelihood and Bayesian inference analysis of the SSU rRNA gene illustrating the evolutionary relationship of Vairimorpha rumexii sp. nov. with other microsporidia. The numerical values above the branches represent bootstrap support percentages for the ML tree and posterior probability values for the BI tree.
Figure 6 Long description
Two phylogenetic trees, labeled as ML Tree and BI Tree, illustrate the evolutionary relationships of microsporidia. The ML Tree on the left uses Maximum Likelihood analysis, while the BI Tree on the right uses Bayesian Inference. Both trees depict the evolutionary placement of various microsporidia species, with branches labeled with numerical values indicating bootstrap support percentages for the ML Tree and posterior probability values for the BI Tree. The trees are color-coded, with the Nosema clade in red and the Vairimorpha clade in blue. A highlighted section in both trees marks the position of Vairimorpha rumexii sp. nov., indicating its evolutionary relationship within the Vairimorpha clade. The trees show the branching patterns and relationships among different species, with the ML Tree and BI Tree providing complementary views of the phylogenetic structure.
Comparative analysis of Vairimorpha rumexii sp. nov. and related microsporidian species, focusing on the small subunit ribosomal RNA (SSU rRNA) gene and the largest subunit of RNA polymerase II (RPB1) gene by % identity, pairwise distance analysis and GC% content
Table 2 Long description
Comparisons for Vairimorpha rumexii list, for two genes, each reference accession’s query coverage, percent identity, pairwise distance, and GC content relative to the V. rumexii sequence. For SSU rRNA (V. rumexii GC content 36.2 percent), the highest similarity is Microsporidium sp. MH712042 at 91 percent coverage with 99.80 percent identity and a very small pairwise distance of 0.0020; several other Microsporidium and Vairimorpha entries cluster around 96 to 98 percent identity with low distances near 0.03. Encephalitozoon cuniculi is listed with only 5 percent coverage and a much larger distance of 0.4087, so that high identity value is not directly comparable. Multiple Nosema species show much lower SSU rRNA identity around the mid 80 percent range with larger distances around 0.17. For RPB1 (V. rumexii GC content 30.9 percent), identities are generally lower, ranging from about 70.63 percent for Ordospora colligata to a maximum of 83.33 percent for Vairimorpha cerenae at 67 percent coverage, with distances commonly around 0.20 to 0.36. Query coverage varies widely in RPB1, including very low coverage entries such as Nosema pyrausta at 31 percent, so coverage should be considered when interpreting similarity and distance.
To further investigate the phylogenetic positioning of the current microsporidium, the RPB1 gene sequence was successfully amplified, and the resulting sequence was deposited in GenBank under accession code PV610738. The partial sequence of the RPB1 gene for the current microsporidium comprises 978 base pairs, with a GC content of 30.9%. The distances between the current microsporidium and the other 21 sequences ranged from 0.1911 to 0.4299. Analyses based on RPB1 gene region sequences have yielded results comparable to those obtained from SSU rRNA sequence analyses. Similarly, in both ML and BI analyses, the current microsporidium is positioned in the Vairimorpha group and diverges onto a distinct clade separate from the others (Figure 7).
Molecular phylogeny of Vairimorpha rumexii sp. nov. and related microsporidia based on the RPB1 gene. Phylogenetic trees were constructed using maximum likelihood and Bayesian inference analyses. The numerical values above the branches represent bootstrap support percentages for the ML tree and posterior probability values for the BI tree.
Figure 7 Long description
Two phylogenetic trees are presented side by side, labeled as Maximum Likelihood (ML) Tree on the left and Bayesian Inference (BI) Tree on the right. Both trees depict the evolutionary relationships of microsporidia, with branches colored red for Nosema clade and blue for Vairimorpha clade. Each tree includes various species with their respective accession numbers. The ML Tree shows bootstrap support percentages above the branches, while the BI Tree displays posterior probability values. A highlighted yellow box in both trees marks the position of Vairimorpha sp. nov., indicating its placement within the Vairimorpha clade. The trees are rooted with the outgroup labeled as Ostracoda outgroup at the bottom. The ML Tree and BI Tree show similar topologies, with Vairimorpha sp. nov. diverging onto a distinct clade separate from other species.
In conclusion, all microscopic and phylogenetic observations and analyses conducted in this study demonstrate that the microsporidium identified in Gastrophysa viridula (Coleoptera: Chrysomelidae) represents a novel species within the genus Vairimorpha, distinct from all previously known species. To refer to the primary dietary source of the host beetle, the genus Rumex L. (Caryophyllales: Polygonaceae), we designate this newly identified species as Vairimorpha rumexii sp. nov.
Taxonomic summary
Superphylum: Opisthosporidia Karpov et al., 2014
Phylum: Rozellomycota Tedersoo et al., 2018
Subphylum: Microsporidia Balbiani, 1882; Wijayawardene et al., 2020
Class: Terresporidia Vossbrinck et al., 2014
Order: Nosematida Wijayawardene et al., 2020; Bojko et al., 2022
Family: Nosematidae Labbe, 1899
Genus: Vairimorpha Pilley, 1976
Dimorphic developmental patterns predominate, encompassing both binary fission with disporoblastic sporogony and multiple fission with octosporoblastic sporogony. These developmental processes exhibit temperature-dependent variation. Spores derived from the disporoblastic sequence are binucleate, elongated, cylindrical and notably larger than those produced through octosporoblastic sporogony, which are mononucleate and oval in shape. Type species: Vairimorpha necatrix Pilley, 1976.
Vairimorpha rumexii sp. nov.
Type host: The green dock leaf beetle, Gastrophysa viridula De Geer, 1775 (Coleoptera: Chrysomelidae).
Site of infection: The infection primarily localizes within the gut and gonadal tissues.
Spores: Two distinct types of spores recognized; Type I: Binucleate mature spores exhibit an ovocylindrical to rod-shaped morphology, with an average length of 5.10 ± 0.30 μm and a width of 2.08 ± 0.14 μm (fixed, n = 100). Fresh spores vary in size, measuring 5.75 ± 0.39 (5.00–6.69) μm in length and 2.16 ± 0.24 (1.70–2.81) μm in width (n = 100). These spores contain an isofilar polar tube consisting of 12–16 coils, each with a diameter of 95–114 nm. The mature coils exhibit a structure comprising 5 concentric layers, each differing in electron density and thickness. The spore wall, characterized by a trilaminar structure, is relatively thin (70–120 nm) and maintains homogeneity throughout the spore body. The exospore measures 19–48 nm, while the endospore thickness ranges from 55 to 80 nm. Type II: Uninucleate spores (meiospores) exhibit an ovoid morphology, with an average length of 4.30 ± 0.27 μm and a width of 2.09 ± 0.31 μm (fixed, n = 100). Fresh spores range in size, measuring 4.72 ± 0.16 (4.45–4.96) μm in length and 2.22 ± 0.27 (1.77–2.73) μm in width. The isofilar polar tube undergoes 9–11 coils around the centrally positioned nucleus, with mature filament coils measuring 90–104 nm in diameter. The spore wall thickness varies between 85 and 115 nm, with the exospore ranging from 24 to 46 nm and the endospore measuring between 56 and 80 nm. Additionally, the exospore exhibits a relatively undulating structure compared to that of Type I.
Type locality: The samples of Gastrophysa viridula De Geer, the green dock leaf beetle, were collected from (Tonya) Trabzon, Türkiye.
Etymology: The species epithet ‘rumexii’ is derived from the primary dietary source of the host beetle, the genus Rumex L. (Caryophyllales: Polygonaceae).
Transmission: Horizontal transmission is likely; vertical transmission remains possible but unconfirmed.
Deposition of type specimens: Type slides and embedded resin specimens have been archived in the personal collection of Çağrı Bekircan under Catalogue No. VR-01. Partial gene sequences of Vairimorpha rumexii sp. nov. have been deposited in the NCBI GenBank under accession codes PV627787 and PV610738.
Discussion
This study examined microsporidiosis caused by Vairimorpha rumexii sp. nov. in the green dock leaf beetle (Gastrophysa viridula), collected from high-altitude regions of Tonya, Trabzon. Reports of microsporidiosis in G. viridula and other Gastrophysa species indicate that microsporidian parasites are more prevalent than previously recognized within this insect genus (Hostounsky and Weiser, Reference Hostounsky and Weiser1973; Hostounsky, Reference Hostounsky1982; Wegensteiner et al., Reference Wegensteiner, Tkaczuk, Balazy and Kaiser2008; Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). In G. viridula, both naturally occurring and experimentally induced microsporidiosis have been documented, with a total of 5 recorded microsporidian infections – 4 belonging to the genus Nosema and 1 to Vairimorpha (Hostounsky, Reference Hostounsky1982, Reference Hostounsky1984; Toguebaye et al., Reference Toguebaye, Marchand, Bouix, Jolivet, Petitpierre and Hsiao1988). Among these, Nosema sp., identified through light microscopy in the study published by Wegensteiner et al. (2008), and Vairimorpha gastrophysae, recently classified at the species level by Yaman et al., have been recognized as natural parasites of this beetle (Wegensteiner et al., Reference Wegensteiner, Tkaczuk, Balazy and Kaiser2008; Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). The possibility that the Nosema sp. identified through light microscopy in the study conducted by Wegensteiner et al. may belong to the genus Vairimorpha is a consideration that should not be overlooked, as noted in the research by Yaman et al. (Reference Yaman, Aydin, Linde and Radek2022).
Vairimorpha rumexii sp. nov. is a newly identified second microsporidium responsible for the natural microsporidian infection observed in G. viridula from Tonya, Trabzon. The first documented case was reported by Yaman et al. (Reference Yaman, Aydin, Linde and Radek2022), who identified the first Vairimorpha species as V. gastrophysae (Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). This identification was based on field studies conducted at various locations in Trabzon, including the Uzungöl, Hıdırnebi and Şimşirli plateaus. Building on this information, this article aims to compare the newly identified microsporidium with V. gastrophysae. The occurrence of more than 1 microsporidian species within a single host is not uncommon and has been reported in various insect–microsporidia systems. Such coexistence may reflect ecological differentiation, including variation in tissue tropism, transmission strategy or temporal dynamics of infection. In the case of Gastrophysa viridula, the presence of both V. gastrophysae and V. rumexii sp. nov. may indicate either geographic structuring of parasite populations or independent host colonization events (Quiles et al., Reference Quiles, Bacela-spychalska, Teixeira, Lambin, Grabowski, Rigaud and Wattier2019; Trzebny et al., Reference Trzebny, Mizera and Dabert2023). The taxonomy of Microsporidia is primarily based on fundamental characteristics, including life cycle, spore morphology and host specificity (Sprague et al., Reference Sprague, Becnel and Hazard1992; Wittner and Weiss, Reference Wittner, Weiss, Wittner and Weiss1999; Weiss and Becnel, Reference Weiss and Becnel2014; Han and Weiss, Reference Han and Weiss2017). The genus Vairimorpha is distinguished by 2 sporulation types: Nosema-like and Thelohania-like. The Thelohania-like sporulation is characterized by the proliferation of plasmodial multinucleate cells within a sporophorous vesicle, formed through plasmotomy, a process crucial for its development and differentiation (Pilley, Reference Pilley1976; Wittner and Weiss, Reference Wittner, Weiss, Wittner and Weiss1999; Weiss and Becnel, Reference Weiss and Becnel2014; Baki and Bekircan, Reference Baki and Bekircan2018; Bekircan, Reference Bekircan2020). Through comprehensive observations conducted using both electron and light microscopy, the newly identified microsporidium in this study was detected to exhibit all sporulation characteristics distinctive to the genus Vairimorpha (Figures 3A and 5A). These findings provide unequivocal evidence that the current microsporidium is a member of Vairimorpha. Spore morphometric characteristics, including shape and dimensions, serve as critical taxonomic parameters in the classification and delineation of microsporidian species (Lacey, Reference Lacey1997; Becnel et al., Reference Becnel, Jeyaprakash, Hoy and Shapiro2002; Yıldırım and Bekircan, Reference Yıldırım and Bekircan2020). The spore morphology of Vairimorpha rumexii sp. nov. is generally consistent with that of V. gastrophysae, with no clear differences in overall size ranges based on available data. However, spores of V. rumexii sp. nov. tend to be slightly larger. Due to the lack of reported measurements for the meiospores of V. gastrophysae, a direct comparison for this spore type remains limited. A comparative evaluation of the binucleate spore dimensions of Vairimorpha rumexii sp. nov. and V. gastrophysae, based on previously published data, indicates no clear differences in size ranges; however, mature spores of V. rumexii sp. nov. tend to be slightly larger (binucleate spore dimensions of V. gastrophysae: 5.02 ± 0.56 × 2.12 ± 0.23 μm; fixed, n = 50). Due to the absence of reported measurements for the meiospores of V. gastrophysae, a direct comparative evaluation remains unfeasible. In microsporidian taxonomy, the ultrastructural characteristics of the spore stage, particularly the number and structural organization of polar tube coils, constitute essential criteria for species differentiation and comparative analysis (Canning and Vávra, Reference Canning, Vávra, Lee, le GFL and Bradburry2000; Ovcharenko et al., Reference Ovcharenko, Świątek, Ironside and Skalski2013; Bekircan et al., Reference Bekircan, Bülbül, Hİ and Becnel2016). The mature binucleate spores and meiospores of V. rumexii sp. nov. possess an isofilar polar tube, consisting of 12–16 coils in binucleate spores (diameter range: 95–114 nm) and 9–11 coils in meiospores (diameter range: 90–104 nm). The binucleate spores of V. gastrophysae exhibit an isofilar polar tube structure, consisting of 9–21 coils with diameters ranging from 85 to 100 nm. Information on the polar tube structure of its meiospores is currently lacking (Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). Given the extensive range of polar tube coil numbers (9–21) reported in this study, a direct assessment of this characteristic between the 2 species remains inconclusive. A comparison of the binucleate spore wall thicknesses of V. rumexii sp. nov. and V. gastrophysae reveals that V. gastrophysae possesses a relatively thicker spore wall, measuring 90–140 nm. This structure comprises a distinct endospore (70–120 nm) and a uniform, electron-dense exospore (20–25 nm) (Yaman et al., Reference Yaman, Aydin, Linde and Radek2022). Although taxonomic characteristics identified through light and electron microscopy indicate distinctions between the 2 species, they may not provide sufficient evidence to justify the designation of a new species. Likewise, based on our initial observations, we initially classified the microsporidium identified in the early phase of this study as an isolate of V. gastrophysae. However, subsequent molecular analyses have conclusively demonstrated that this microsporidium constitutes a distinct new species, separate from V. gastrophysae. Although mixed infection by more than 1 microsporidian species is theoretically possible in naturally infected insects, the present observations do not support this interpretation. The 2 spore types were consistently associated with developmental stages corresponding to the dimorphic sporogony characteristic of Vairimorpha, including a Nosema-like disporoblastic cycle and a Thelohania-like octosporoblastic cycle within a sporophorous vesicle. Together with the ultrastructural consistency and the congruent placement of SSU rRNA and RPB1 sequences within the Vairimorpha lineage, these findings support the interpretation that Types I and II spores represent 2 developmental morphotypes of a single species, Vairimorpha rumexii sp. nov. Nevertheless, because molecular data were obtained from infected tissues rather than from individually isolated spores, the possibility of mixed infection cannot be excluded with absolute certainty. Future studies using single-spore PCR, laser microdissection or in situ hybridization would provide further confirmation of the genotype–morphotype association.
Evolutionary convergence in morphological traits, including nuclear organization and life cycle characteristics, arising from adaptation to diverse hosts and tissues in certain species, necessitates a molecular-based approach to microsporidian taxonomy (Franzen, Reference Franzen2008; Bekircan et al., Reference Bekircan, Tosun and Yıldırım2025). Molecular analyses conducted for V. rumexii sp. nov. included the examination of SSU rRNA and RPB1 gene regions. Phylogenetic reconstructions based on SSU rRNA gene sequences, employing both ML and BI methodologies, demonstrated a clear distinction between V. rumexii sp. nov. and V. gastrophysae, placing them in separate clades within the phylogenetic trees (Figure 6A, B). Pairwise distance analysis revealed a significant genetic difference of 0.0608 between V. rumexii sp. nov. (PV627787) and V. gastrophysae (ON391927). Upon comparison of the partial base sequences of 2 species using CLUSTAL_W, 72 substitutions and 32 deletions were identified between the sequences. In phylogenetic tree topologies, V. rumexii sp. nov. closely associated with Microsporidium sp. (MH712042) and Vairimorpha sp. (MG907029); however, their incomplete taxonomic characterization precludes a meaningful comparison based on ultrastructural and morphometric features. The high sequence similarity observed with incompletely characterized records such as Microsporidium sp. highlights a broader limitation in microsporidian systematics. Many sequences deposited in public databases remain taxonomically unresolved, which complicates species delimitation based solely on molecular similarity. These records may represent undescribed taxa, emphasizing the need for integrative approaches combining molecular, morphological and ultrastructural data. However, considering widely accepted orthodox taxonomic parameters – such as host species and geographical distribution – a comparative analysis remains possible (Sprague et al., Reference Sprague, Becnel and Hazard1992). The Microsporidium sp. (MH712042) record, documented in Canada, is associated with Megastigmus spermotrophus (parasitic wasp, Hymenoptera), whereas Vairimorpha sp. (MG907029) was found in Amyelois transitella (Navel orangeworm, Lepidoptera: Pyralidae) in the USA. Consequently, V. rumexii sp. nov. exhibits substantial divergence from these records in terms of its host species and recorded location. The molecular characterization of V. gastrophysae was conducted exclusively using the SSU rRNA gene region. As with all molecular analyses conducted for the SSU rRNA gene, studies performed on the RPB1 gene region have also confirmed that V. rumexii sp. nov. represents a novel species within the genus Vairimorpha. V. rumexii sp. nov. (PV610738) exhibits a nucleotide divergence of 0.2544 from V. necatrix (DQ996236), the type species of the genus, while displaying a difference of 0.1981 from its closest Vairimorpha member (MW415412) (Table 2). Additionally, in all phylogenetic tree topologies constructed based on this gene region, V. rumexii sp. nov. distinctly separated from all other Vairimorpha members, forming a separate clade that highlights its divergence (Figure 7A, B). From an evolutionary perspective, the observed divergence between closely related taxa infecting the same host may reflect processes such as host switching, geographic isolation or cryptic diversification. Microsporidia are known for their capacity to exploit new hosts and adapt to different ecological conditions, often without substantial morphological differentiation (Han and Weiss, Reference Han and Weiss2017; Quiles et al., Reference Quiles, Bacela-spychalska, Teixeira, Lambin, Grabowski, Rigaud and Wattier2019). This may explain the close phylogenetic relationship yet clear genetic separation observed between V. rumexii sp. nov. and related taxa.
In conclusion, all observations and analyses conducted on the microsporidium identified in Gastrophysa viridula De Geer (Coleoptera: Chrysomelidae) specimens collected from Tonya, Trabzon, confirm that this microsporidium represents a newly discovered species from Türkiye, distinct from Vairimorpha gastrophysae, despite sharing the same host insect.
Data availability statement
DNA sequences have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank), accession numbers PV627787 and PV610738. Sequence alignments will be made available upon request.
Acknowledgements
The transmission electron microscopy (TEM) analysis was conducted at the Central Research Laboratory, Research and Application Centre (ARUM) of Eskişehir Osmangazi University (ESOGU).
Author contributions
Ç.B.: Resources, Investigation, Supervision, Writing – original draft, Writing – review & editing. O.T.: Investigation, Resources, Visualization. H.Y.: Resources, Investigation.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors have declared that no competing interests exist.
Ethical standards
Not applicable.
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