A high prevalence of myxosporean infection (78.7%, 85/108) was determined in the kidney of silver carp. Two dominant myxosporeans were found under microscope and were identified as Myxobolus lieni and Myxobolus varius. The infection prevalence of M. lieni and M. varius were 60.2% (65/108) and 35.2% (38/108), respectively; meanwhile, a co-infection of the two species were found in 18 silver carp (16.7%, 18/108). Morphological, histological, and molecular characteristics of the two species were described as follows.
Morphological characteristics of Myxobolus lieni (Nie & Li, 1973)
Mature spores (Fig. 1a–d) appeared suborbicular and slightly flat in frontal view, fusiform shaped in sutural view, measuring 7.33 ± 0.22 (6.62–7.96) in length, 7.51 ± 0.14 (6.93–8.23) in width (n = 50), and 5.78 ± 0.13 (5.5–6.08) in thickness (n = 10). Two equal polar capsules, pear-shaped with apophysis at top end, measuring 4.29 ± 0.34 (3.81–4.82) in length, 2.92 ± 0.13 (2.56–3.29) in width, and polar tubules coiled 4–5 turns.
Type host
Silver carp Hypophthalmichthys molitrix (Valenciennes, 1844)
Host size
22.0–35.0 cm
Site of infection
histozoic, plasmodia found in the intraepithelium of renal tubular (Fig. 2a–c)
Prevalence
60.2% (65/108)
Locality
Lake Taihu, Wuxi, China.
Vouch specimens
Infected tissues fixed in 4% buffered paraformaldehyde were deposited in Freshwater Fisheries Research Center, Chinese Academy of Fishery Science (accession no. MTR20181116)
Morphological characteristics of Myxobolus varius (Achmerov, 1960)
Mature spores (Fig. 1e–h) appeared to be ellipse in frontal view, with wider anterior than posterior, and shuttle shaped in sutural view, measuring 11.03 ± 0.005 (10.24–12.11) in length, 7.34 ± 0.055 (6.36–7.67) in width (n = 30), and 5.49 (5.19–5.71) in thickness (n = 5). Two equal polar capsules are pyriform with apophysis at top end, measuring 4.66 ± 0.08 (4.32–4.94) in length, 2.64 ± 0.03 (2.40–2.98) in width, polar tubules coiled 4–5 turns.
Type host
Silver carp Hypophthalmichthys molitrix (Valenciennes, 1844)
Host size
20.1–36.4 cm
Site of infection
histozoic, renal interstitium, plasmodia not found
Prevalence
35.2% (38/108)
Locality
Lake Taihu, Wuxi, China
Vouch specimens
Infected tissues fixed in 4% buffered paraformaldehyde were deposited in Freshwater Fisheries Research Center, Chinese Academy of Fishery Science (accession no. MTR20181117)
Histology
No significant pathological changes were observed in the sections of infected fish kidney. Small roundish plasmodia of M. lieni, containing several mature myxospores, were found within the epithelial cells of the renal tubules (Fig. 2a–c). Dispersed myxospores of M. varius were found in the renal interstitium without forming plasmodia structures and enclosed within melano-macrophage centers (Fig. 2d).
Molecular data
Partial SSU rDNA sequences of M. lieni (1994 bp, GenBank accession MK371243) and M. varius (2019 bp, MK371244) were obtained; and in the 20 PCR clones of kidney tissues, eight clones showing sequence similarity 99.5–99.9% were identified as M. lieni, six with 99.9–100% similarity were identified as M. varius. Meanwhile, six clones showing only 88.8–97.2% similarity, which significantly beyond the common intraspecific variation < 2% (Table 2). Here we tentatively assigned these sequences as Myxobolus sp1 (1 clone, MK371245), Myxobolus sp2 (1 clone, MK371246), Myxobolus sp3 (4 clones, MK371247 and MK371248).
Table 1
Comparative data for spore measurements (means ± standard deviation in micrometres) of Myxobolus lieni and Myxobolus varius and morphologically similar species
Species | Host | Site in host | SL | SW | PCL | PCW | TS | PTC | Reference | |
M. lieni | Hypophthalmichthys molitrix | Kidney | 7.33 ± 0.22 (6.62–7.96) | 7.51 ± 0.14 (6.93–8.23) | 4.29 ± 0.34 (3.81–4.82) | 2.92 ± 0.13 (2.56–3.29) | 5.78 ± 0.13 (5.5–6.08) | 4–5 | Present study | |
M. lieni Nie et Li, 1973 | Hypophthalmichthys molitrix Hypophthalmichthys nobilis(Richardson, 1845) | Kidney, gills, Urinary bladder | 7.2–7.4 | 7.2–7.4 | 3.8(3.6–4.2) | 2.8 (2.6–3.0) | 4.8–5.0 | 5 | Chen and Ma, 1998 | |
M. artus | Hypophthalmichthys molitrix | Spleen, Intestine, Kidney | 6.0 (5.4–6.6) | 8.1 (7.2–8.4) | 3.4 (3.1–3.6) | 2.7 (2.6–2.8) | 5.6 (5.5–5.7) | 4–5 | Chen and Ma, 1998 | |
M. parvus | Hypophthalmichthys molitrix Hypophthalmichthys nobilis | Almost all organ | 6.1 (5.8–6.4) | 6.0 (5.6–6.2) | 3.2 (3.0–3.4) | 2.3 (2.2–2.4) | 4.2 | 4–5 | Chen and Ma, 1998 | |
M. varius | Hypophthalmichthys molitrix | Kidney | 11.03 ± 0.005 (10.24–12.11) | 7.34 ± 0.06 (6.36–7.67) | 4.66 ± 0.08 (4.32–4.94) | 2.64 ± 0.03 (2.40–2.98) | 5.49 (5.19–5.71) | 5 | Present study | |
M. varius Achmerov, 1960 | Hypophthalmichthys molitrix | Kidney, Intestine | 10.9(9.0–13.0) | 7.3(6.0–8.5) | 5.2 (4.1–6.0) | 2.7 (2.2–3.0) | 5.6 (4.5–6.5) | 5–6 | Chen and Ma, 1998 | |
M. ellipsoides | Hypophthalmichthys molitrix Hypophthalmichthys nobilis | Almost all organ | 11.0 (9.6–12.0) | 7.3 (7.0–7.8) | 4.9 (4.5–5.4) | 3.0 (2.4–3.2) | 6.0 | 5 | Chen and Ma, 1998 | |
M. pronini | Carassius auratus gibelio | Mesentery, Abdominal cavity | 14.7 ± 0.24 (13.8 − 15.6) | 9.6 ± 0.65 (9.0 − 13.3) | 5.3 ± 0.16 (4.8 − 5.6) | 3.0 ± 0.12 (2.9 − 3.4) | 6.6 ± 0.16 (6.2 − 7.2) | 5–6 | Liu et al. 2016 |
Abbreviations: SL spore length, SW spore width, ST spore thickness, PCL polar capsule length, PCW polar capsule width, PTC polar tubules coil. |
Table 2
SSU rDNA sequence similarity (%) between Myxobolus spp. isolated from kidney of silver carp (p-distance).
| M. lieni | Myxobolus sp1 | Myxobolus sp2 | Myxobolus sp3 | M. varius |
M. lieni | 99.5–99.9 | | | | |
Myxobolus sp1 | 96.7–97.2 | - | | | |
Myxobolus sp2 | 89.2–89.5 | 88.4 | - | | |
Myxobolus sp3 | 89.1–89.6 | 87.5–87.8 | 89.2–89.5 | 99.2–100 | |
M. varius | 90.3–90.7 | 88.8–88.9 | 90.7–90.6 | 93.1–93.8 | 99.9–100 |
BLASTn analyses showed the new obtained sequences were not identical to any sequences available in GenBank. The SSU rDNA sequence of M. lieni has a relative higher identity with Myxobolus intimus (JF311899), Myxobolus pronini (MH329619), Myxobolus pavlovskii (MG520368), Myxobolus abitus (MG520367), Myxobolus linzhiensis (KY965935), 86.8–83.5%; M. varius has a relative higher identity with Myxobolus pavlovskii (MG520368), Myxobolus kiuchowensis (MG520366), Myxobolus abitus (MG520367), Myxobolus drjagini (MH119078), 86.2–85.9%.
Bayesian inference (BI) and maximum likelihood (ML) analyses conducted with SSU rDNA all demonstrated similar phylogenetic topology of myxozoans examined (Fig. 3). M. lieni and M. varius, as well as the 3 unidentified Myxobolus in the kidney of silver carp, formed a separate clade of closely related to cyprinid-infecting myxozoans. However, this clade was away from the species recently detected from the other organs of silver carp and bighead carp, such as M. pavlovskii, M. kiuchowensis, M. drjagini, M. paratypicus and M. abitus (Chen and Ma 1998).