Introduction
The large yellow croaker (Larimichthys crocea) is a high-value species in China’s mariculture industry, with an annual production exceeding 281 000 tons in 2023 (Liu, Reference Liu2024). However, the sustainable development of L. crocea aquaculture is hindered by several diseases (Tang et al. Reference Tang, Jiang, Li, Li and Li2022), particularly parasitic pathogens, such as Cryptocaryon irritans (Zuo et al., Reference Zuo, Ai, Mai, Xu, Wang, Xu, Liufu and Zhang2012), Neobenedenia melleni (Yang et al., Reference Yang, Li and Wang2004) and Trypanosoma larimichthysi (Yang et al., Reference Yang, Qi, Tao, Zhang, Wang, Zhu, Yan, Fu and Guo2025).
Material and method
Sample collection
During a helminthological survey of the large yellow croaker, Longicollum pagrosomi (Yamaguti, Reference Yamaguti1935) was collected from the hindgut of cage-cultured L. crocea in the Sanduao Bay in Ningde, Fujian Province. A total of 11 acanthocephalans, including 6 males and 5 females, were fixed in 70% ethanol for morphological identification.
Morphological identification
The parasite samples were processed following the protocol described by Fu et al. (Reference Fu, Li, Zou, Zhang, Wu, Li, Wang and Xi2019). After hydration, the specimens were stained with ferric hydrochloric acid carmine, differentiated in 70% acid ethanol, dehydrated through a graded ethanol series and clarified in xylene. The samples were mounted with Canada balsam and examined under a light microscope (Olympus, DP72, Japan). Images were captured for further analysis, and morphological measurements (Table S1) were conducted using ImageJ software. All measurements were expressed in millimetres unless otherwise specified.
DNA extraction, primer designed, PCR amplification, sequencing of mitogenome and sequence annotation
To obtain the mitogenome of L. pagrosomi, genomic DNA was extracted from a single specimen using the Tissue Cell Genome Kit. Primers (Table S2) targeting conserved mitochondrial regions were used to amplify short fragments of 16S, nad4, nad5, cytb, and 12S. Specific primers were then designed to amplify the remaining sequence. PCR conditions followed those described in a previous study (Song et al., Reference Song, Zhang, Gao, Cheng, Xie, Li and Wu2019). The PCR products were sequenced using a primer-walking strategy at Sangon Biotech. The mitogenomic sequences were manually assembled using DNASTAR v7.1 software (Burland, Reference Burland2000) and annotated according to the procedures described by Li et al. (Reference Li, Zhang, Fu, Song, Zou, Li, Wu and Wang2019). Raw sequences were imported into the online software MITOS (http://mitos.bioinf.uni-leipzig.de) to determine approximate gene boundaries. The precise positions of protein-coding genes (PCGs) were identified by searching for open reading frames (ORFs) using genetic code 5 (invertebrate mitochondrion). A majority of transfer RNAs (tRNAs) were identified using MITOS and RNAfold WebServer (http://rna.tbi.univie.ac.at), with the remaining tRNAs identified by alignment with other acanthocephalan species. The boundaries of the 2 ribosomal RNAs (rRNAs), rrnL and rrnS, were determined by comparing them with homologous sequences. Codon usage and relative synonymous codon usage (RSCU) for the 12 PCGs were calculated using PhyloSuite software (Zhang et al., Reference Zhang, Gao, Jakovlic, Zou, Zhang, Li and Wang2020). AT and GC skew values were calculated using the following formulas: AT-skew = (A – T)/ (A + T) and GC-skew = (G – C)/(G + C). The circular map of the L. pagrosomi mitogenome was visualized using the CGView server (http://cgview.ca). The secondary structure of tRNAs and rRNAs were displayed using Adobe Photoshop CC (Figure S1 and S2).
Phylogenetic analyses
Phylogenetic analyses based on the newly sequenced mitogenome and 23 acanthocephalan mitogenomes available in GenBank (Table S3) were conducted. Rotaria rotatoria Pallas, 1776 (NC013568.1) and Philodina citrina Lansing, 1947 (FR856884.1) were selected as outgroups. Fasta files for the sequences, including 12 PCGs, 22 tRNAs and 2 rRNAs, were retrieved from GenBank using PhyloSuite, followed by multiple sequence alignment in MAFFT (Katoh et al., Reference Katoh, Misawa, Kuma and Miyata2002) and sequence concatenation. The optimal partitioning schemes and models were determined using PartitionFinder 2 (Lanfear et al., Reference Lanfear, Frandsen, Wright, Senfeld and Calcott2017). Bayesian inference (BI) analysis was conducted using MrBayes 3.2.7 (Ronquist et al., Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard and Huelsenbeck2012) with default settings and 2, 000, 000 metropolis-coupled MCMC generations. Maximum-likelihood (ML) analysis was performed using IQ-TREE (Nguyen et al., Reference Nguyen, Schmidt, von Haeseler and Minh2014) with 50 000 ultrafast bootstraps.
Result
Morphological description
The morphology of the acanthocephalan was shown in Figure S3. The presoma and metasoma (trunk) of the parasite were creamy white and taupe, respectively. The trunk was cylindrical, with an elongated neck exhibiting distinct expansion. The proboscis was short, club-shaped and gradually widened toward the anterior region, adorned with 10–14 spiral, longitudinal rows, each containing 11–16 hooks.
Chracterization of the mitochondrial genome of L. pagrosomi
The circular duplex mitogenome of L. pagrosomi was 14 632 bp in size (GenBank accession number: OR215045) and contained all 36 typical metazoan genes, including 12 PCGs, 22 tRNAs and 2 rRNAs, but lacked the atp8 gene (Figure 1). All genes were transcribed from the same strand, and the genome featured 11 overlapping regions (Table 1). The base composition of the mitogenome was as follows: A: 21.63%, T: 34.16%, C: 10.91% and G: 33.30%, indicating an AT bias. The overall nucleotide composition of the complete mitogenome was skewed away from A in favour of T, and strongly biased toward G, with an AT skew of −0.225 and a GC skew of 0.672 (Table 2). The concatenated length of the 12 PCGs was 10 405 bp, with an average A + T content of 54.79%, ranging from 51.34% in atp6 to 56.51% in cytb (Table 2). The most frequently used start codon was GTG (observed in eight PCGs), followed by TTG (in two PCGs). The most common stop codon was TAG (found in five PCGs), while TAA was used by nad2, nad6 and cox3 (Table 1). Codon usage and RSCU were presented in Figure 2 and Table 3. Among the PCGs of L. pagrosomi, valine (16.86%), leucine (15.22%), glycine (13.12%) and serine (10.39%) were the most abundant amino acids, while cysteine (1.04%), glutamine (1.09%) and asparagine (1.15%) were relatively rare. The higher T content (34.19%) in the 12 PCGs correlated with a higher frequency of T-rich codons, including TTA for leucine (6.07%), GTT for valine (5.06%) and TTT for phenylalanine (4.75%). All 22 tRNAs were identified in the L. pagrosomi mitogenome, with a total length of 1255 bp, and sizes ranging from 44 bp (trnC) to 72 bp (trnL1) (Table 1). The 2 rRNAs, rrnL and rrnS, were 908 bp and 615 bp in length, respectively, with A + T contents of 61.46% and 63.25%. The rrnL (16S) gene was located between trnY and trnL1, while rrnS (12S) was positioned between trnF and trnR. This genes arrangement was consistent with other members of the Pomphorhynchidae family (Figure 3).
Figure 1. Map of the complete mitogenomes of longicollum pagrosomi. 12 protein-coding genes (12) are shown in blue, rRNAs (2) in pink, and tRNAs (22) in yellow.
Table 1. The organization of the mitochondrial genome of L. pagrosomi
Table 2. Nucleotide composition and skewness of different elements of the mitogenome of L. pagrosomi
Figure 2. Relative synonymous codon usage (RSCU) of the complete mitogenomes of L. pagrosomi. Codon families are labelled on the x-axis. Values on the top of the bars refer to amino acid usage.
Table 3. The codon number and relative synonymous codon usage in the mitochondrial genomes of the L. pagrosomi
Figure 3. Phylogeny and gene order of the acanthocephalans. (A) Phylogenetic tree was constructed using the Bayesian inference method for almost complete genomic datasets (36 genes: 12 PCGs, 2 rRNAs and 22 tRNAs). (B) Linear comparison of genome order.
Phylogeny and gene order
Phylogenetic analysis of the concatenated 22 mitochondrial genes, using both ML and BI methods, produced identical topologies with strong statistical support for most nodes. Therefore, only the BI tree was shown (Figure 3). The results indicated that the newly sequenced mitogenome of L. pagrosomi from large yellow croaker formed a sister clade with Pomphorhynchus species, supporting the monophyly of the Pomphorhynchus genus. The mitochondrial gene arrangement in acanthocephalan species was generally conserved, with the arrangement of the 12 PCGs and 2 rRNAs being consistent (Figure 3).
Discussion
In this study, we collected an acanthocephalan species from the large yellow croaker. Morphological analysis clearly identified the specimens as L. pagrasomi (Yamaguti, Reference Yamaguti1935; Wang et al., Reference Wang, Wang and Wu1993; Li et al., Reference Li, Yang and Zhang2017a; Cheng et al., Reference Cheng, Rao, Wang and Chen2022), based on the number of longitudinal rows of proboscis hooks, the number of hooks per longitudinal row, the shape and length of the proboscis hooks, trunk size, cement glands and proboscis receptacles (Table S1). However, the color of the samples was taupe, differing from previous descriptions (Kim et al., Reference Kim, Lee, Kim, Oh, Kim, Park and Park2011; Cheng et al., Reference Cheng, Rao, Wang and Chen2022). The body colour of this parasite has been reported to vary, including white, orange, red, green and black (Ha et al., Reference Ha, Hong, Ryu, Sim, Chae, Kim, Park, Choi, Yu and Park2017; Cheng et al., Reference Cheng, Rao, Wang and Chen2022), and the variation in colour could be attributed to differences in the host type, possibly related to host-derived pigments and dietary composition.
The genus Longicollum includes 13 nominal species, with only 2 species, Longicollum alemniscus and L. pagrosomi, reported from Chinese waters (Wang et al., Reference Wang, Wang and Wu1993). Longicollum pagrosomi parasitizes the intestine of marine fish, with a broad host range that includes Sparidae (Yamaguti, Reference Yamaguti1935; Wang et al., Reference Wang, Wang and Wu1993), Oplegnathidae (Li et al., Reference Li, Yang and Zhang2017a) and Lutjanidae (Cheng et al., Reference Cheng, Rao, Wang and Chen2022). In this study, L. pagrosomi was reported from a new host, Sciaenidae, L. crocea. The 4 species of Perciformes fish hosting L. pagrosomi were all collected from the East China Sea and Japan, indicating that L. pagrosomi has a wide spectrum of definitive hosts.
In the order Echinorhynchida, only 14 acanthocephalan species from 8 different families have their mitogenomes sequenced (Steinauer et al., Reference Steinauer, Nickol, Broughton and Ortí2005; Weber et al., Reference Weber, Wey-Fabrizius, Podsiadlowski, Witek, Schill, Sugár, Herlyn and Hankeln2013; Song et al., Reference Song, Zhang, Gao, Cheng, Xie, Li and Wu2019; Muhammad et al., Reference Muhammad, Li, Suleman, Bannai, Mohammad, Khan, Zhu and Ma2020; Gao et al., Reference Gao, Yuan, Jakovlić, Wu, Xiang, Xie, Song, Xie, Wu and Ou2023; Zhao et al., Reference Zhao, Yang, L, Ru, Wayland, Chen, Li and Li2023; Xie et al., Reference Xie, Chen, Kuzmina, Lisitsyna and Li2024). No mitogenomic data for Longicollum species had been reported previously. This study presented the first mitogenome of L. pagrosomi, exhibiting several common features of Acanthocephala. All genes in the mitogenomic structure were encoded on the same strand, a characteristic typical of Acanthocephala (Song et al., Reference Song, Zhang, Gao, Cheng, Xie, Li and Wu2019). The newly sequenced mitogenome lacks the atp8 gene, a trait common to parasitic flatworms (Le et al., Reference Le, Blair and McManus2002). Additionally, the mitogenome of L. pagrosomi exhibited several unique features, including an overall A + T content of 55.79%, the lowest reported among mitogenomes of the Echinorhynchida (Xie et al., Reference Xie, Chen, Kuzmina, Lisitsyna and Li2024). Leucine is typically the most abundant amino acid in the PCGs of fish acanthocephalan mitogenomes (Song et al., Reference Song, Zhang, Gao, Cheng, Xie, Li and Wu2019; Muhammad et al., Reference Muhammad, Li, Ru, Suleman, Alvi and Li2023; Xie et al., Reference Xie, Chen, Kuzmina, Lisitsyna and Li2024). While, L. pagrosomi predominantly uses valine (16.86%). The gene order of 12 PCGs and 2 rRNA in L. pagrosomi matches that observed in other fish acanthocephalans, including cox1, rrnL, nad6, atp6, nad3, nad4L, nad4, nad5, ctyb, nad1, rrnS, cox2, cox3 and nad2 (Song et al., Reference Song, Zhang, Gao, Cheng, Xie, Li and Wu2019; Muhammad et al., Reference Muhammad, Li, Ru, Suleman, Alvi and Li2023; Zhao et al., Reference Zhao, Yang, L, Ru, Wayland, Chen, Li and Li2023; Xie et al., Reference Xie, Chen, Kuzmina, Lisitsyna and Li2024). Only a few tRNAs translocations (trnR, trnM and trnI) were detected in the mitogenome of L. pagrosomi (Figure 3), and the arrangement of tRNA gene in this mitogenome differs from all known acanthocephalan mitogenomes.
Phylogenetic analysis based on mitochondrial genes (12 PCGs + 22 tRNA + 2 rRNA) from this study, using both ML and BI methods, supported the monophyly of the genus Pomphorhynchus, consistent with the current taxonomy of the genus (Amin Reference Amin2013). However, the tree topology based on the 18S, ITS and cox1 sequences of Pomphorhynchus zhoushanensis Li, Chen, Amin & Yang, 2017, using maximum parsimony (MP) and ML methods showed that Pomphorhynchus was a paraphyletic group (Li et al., Reference Li, Chen, Amin and Yang2017b). The discrepancy between these results indicates that the taxonomic status of P. zhoushanensis remains uncertain and requires further investigation.
Conclusion
In summary, L. crocea represents a new host for L. pagrosomi, thereby expanding its host range within Perciformes. This study provides the first mitochondrial genome of Longicollum and supports the monophyly of Pomphorhynchus while raising doubts about the classification of P. zhoushanensis. The findings contribute significantly to the genetic data available for acanthocephalans.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S003118202510036X.
Data availability statement
The newly generated mitochondrial genome of Longicollum pagrosomi have been submitted to the NCBI GenBank database with accession numbers OR215045.
Acknowledgements
We would like to thank Rong Chen of the BT Lab (Wuhan, China) for helping us with mitochondrial genome sequencing and annotation.
Author contributions
All authors designed and conducted laboratory work and all of them were involved in the manuscript and approved the final version.
Financial support
This study was supported by the Earmarked Fund for the National Natural Science Foundation of China (32173020), Special Grant of Zhoushan for Breeding Aquatic Animals (2025Y001) and the General Research Project of Zhejiang Provincial Department of Education (Special Project for Reforming the Training Mode of Professional Degree Graduate Students) (Y202352327).
Competing interests
The authors declare that they have no competing interests.
Ethics standards
Not applicable.
Open access
