Tissue metabarcoding identifies diverse microsporidian lineages in invertebrates from three South Bohemian fish ponds

A tissue-based metabarcoding study of chironomid larvae and oligochaetes from three carp-production ponds in South Bohemia found 66 microsporidian operational taxonomic units, four of the five principal clades recognized in the study’s classification framework, and strong differences in prevalence and community composition among ponds and operational host groups. The findings indicate substantial microsporidian diversity in the sampled benthic invertebrates, but the molecular detections identify candidate host associations rather than confirming active infection or complete life cycles.

The investigators sampled sediments on 9 June 2024 at two sites in each of the Malá Outrata, Velký Ústavní and Doktorovský ponds, which are hydrologically connected to the Blanice River and used for common carp production. They extracted 2,028 invertebrates and processed them as 579 samples: 214 individually processed large chironomid larvae longer than 7 mm, 145 pools of five small chironomid larvae shorter than 7 mm, and 220 pools of five oligochaetes. Large and small chironomids were operational morphogroups based on size, colouration and appearance rather than formal taxonomic groups.

Each sample was homogenised, and DNA was extracted with a modified protocol using bead beating and silica beads to disrupt resistant microsporidian spores. The researchers amplified approximately 400 base pairs of small-subunit ribosomal DNA with V1F and 530R primers, generated paired-end 250-base-pair Illumina MiSeq reads, removed chimeras and clustered sequences into OTUs at 97% similarity. To reduce possible false positives, they retained only OTUs represented by at least 50 reads, a threshold that may also exclude genuine low-abundance taxa. Their phylogenetic analysis combined 66 newly obtained sequences with 565 published sequences in a maximum-likelihood analysis of 2,648 nucleotide positions.

Among the 66 detected OTUs, 38 belonged to Enterocytozoonida, 12 to Nosematida, 11 to Amblyosporida and five to Neopereziida; Glugeida was not detected. Enterocytozoonida accounted for more than 62% of reads, including 57% from the Arlequin group. Nosematida represented more than 30%, driven by Encephalitozoonidae, while Amblyosporida and Neopereziida accounted for approximately 7% and 0.4%, respectively. The analysis found 51 OTUs without close matches in reference databases or previous studies. Three OTUs matched described species, four matched previously reported freshwater environmental sequences and five corresponded to undescribed lineages or undescribed species.

Sample-level microsporidian prevalence varied substantially among sites and host groups. It was highest overall in oligochaetes, at 69 positive samples out of 220, or 31%, compared with 37 of 145, or 26%, for small chironomids and 21 of 214, or 19%, for large chironomids. Site-level prevalence ranged from 0% to 75% among small chironomids and from 6% to 57% among large chironomids, while oligochaete values ranged from 17% to 69%. Velký Ústavní generally had the highest prevalence, including 75% for small chironomids and 69% for oligochaetes at site 2. The sampling design could not distinguish temporal variation from small-scale spatial effects.

Within-sample diversity also differed among operational groups. Small chironomids had the highest Shannon and Simpson values, 1.89 and 0.741, while oligochaetes had the lowest, 1.11 and 0.506. COI barcoding showed that the small-chironomid fraction was compositionally broader, including tube-building Tanytarsini, vegetation- and detritus-associated larvae and predatory Tanypodinae, whereas the large fraction consisted of Chironomus species. The groups overlapped because smaller samples included early instars of Chironomus. These ecological and developmental differences may help explain the observed diversity patterns, although metabarcoding cannot by itself establish infection or causality.

Read distributions were generally concentrated in one host group. For 49 of 66 OTUs, more than 90% of reads came from a single morphogroup, indicating a dominant read association rather than confirmed host specificity. Encephalitozoonidae OTUs were predominantly associated with oligochaetes, whereas Amblyosporida OTUs were mainly associated with chironomids. Only four OTUs were shared by all three ponds, while 14 were shared by all three host groups. Enterocytozoonida and Neopereziida included OTUs distributed across multiple groups, showing that apparent host association varied within the detected lineages.

Two OTUs previously reported from Daphnia were concentrated in chironomids. OTU2 was assigned to Conglomerata obtusa, with more than 99% of its reads associated with large and small chironomids, although it was detected rarely in the oligochaete Limnodrilus hoffmeisteri. COI barcoding identified Chironomus plumosus, C. annularius and C. curabilis in positive samples. OTU8 matched Microsporidium sp. MIC1, previously reported from Daphnia cucullata, and 99% of its reads were associated with chironomids, particularly C. plumosus and C. curabilis. These patterns are consistent with possible additional hosts in indirect life cycles, but they do not prove active infection; the authors note that low read counts could reflect ingested spores or exposure rather than infection.

The principal limitations are the short SSU rDNA target, which could not confidently resolve OTU39 to a described Nosema species, and the absence of direct parasite localisation or transmission experiments. Read abundance was used for relative comparisons, and pooled samples limited certainty about which individual host carried a detected lineage. The study therefore documents broad microsporidian diversity and candidate host associations in a geographically restricted set of ponds, while infection status, transmission routes and the ecological roles of the detected lineages remain unresolved.

Subject of Research: Microsporidian diversity and candidate host associations in benthic invertebrates from South Bohemian fish ponds

Article Title: Microsporidian diversity and host associations in aquatic invertebrate communities of South Bohemian fish ponds revealed by metabarcoding

Article References: Lisnerová, M., Nayak, S., van Beest, G. S., Bürgerová, M., Lövy, A., Pecková, H., Kyslík, J., Bartošová-Sojková, P., & Fiala, I. (2026). Microsporidian diversity and host associations in aquatic invertebrate communities of South Bohemian fish ponds revealed by metabarcoding. Metabarcoding and Metagenomics, 10, Article e201781. https://doi.org/10.3897/mbmg.10.201781

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.201781

Keywords: microsporidia, metabarcoding, fish ponds, chironomids, oligochaetes, environmental DNA, SSU rDNA, host associations, South Bohemia, freshwater parasites, Microsporidian, diversity