Seasonal Dynamics and Spatial Distribution of the Dactylogyrus Species on the Gills of Grass Carp (Ctenopharyngodon idellus) from a Fish Pond in Wuhan, China

The grass carp, Ctenopharyngodon idellus, native to China, has been introduced to over 100 countries since the 1960s, mainly for aquatic vegetation control and aquaculture (Cudmore and Mandrak, 2004). In China, with total production of 5.07 million tons in 2013 (20.4% of the total annual output), it is the leading cultured freshwater fish species (Zhao, 2014). However, grass carp are susceptible to various kinds of pathogens (Nie and Pan, 1985). Among the most prevalent diseases are hemorrhagic disease caused by grass carp reovirus (Chen and Jiang, 1984) and gill-rot disease caused by bacteria and parasites (He and Deng, 1987), both of which often result in very high mortality (Lu et al., 1975). Dactylogyrus spp. are one of the most important parasitic pathogens causing gill-rot disease of grass carp.

Only 2 species of Dactylogyrus are known to parasitize on the gills of grass carp: Dactylogyrus lamellatus and Dactylogyrus ctenopharyngodonis. Even though D. lamellatus was found in all grass carp age groups during the entire year in Hungary (Molnár, 1971b), it predominantly occurs on fry and fingerlings. Dactylogyrus ctenopharyngodonis, however, parasitizes mainly on larger fish (Schäperclaus, 1992). The maximum intensity of D. ctenopharyngodonis among the 4–5-yr-old fish can be as high as 12,000–17,000 in ponds with high stocking density (Bauer et al., 1973). Dactylogyrus lamellatus is more pathogenic and its infection can induce severe damage to the gill filaments by causing hyperplastic proliferation, vacuolar degeneration, edema, and necrosis (Molnár, 1971c; Ramadan et al., 1995). Hence, in some cases, mortality may be caused by ensuing secondary bacterial, viral, and fungal infections (Thoney and Hargis, 1991).

Regarding the seasonality and niche preference of D. lamellatus, the parasite manifested a high level of occurrence and reproductive activity at the beginning of spring; its infection level was relatively low in summer and autumn (Pojmańska, 1995; Yao and Nie, 2004). Also, it appears to exhibit a preference for the second gill arch (Yao and Nie, 2004). As for D. ctenopharyngodonis, no information is available about the population dynamics and microhabitat distribution.

Many studies have already reported the coexistence of potentially competing congeneric species of monogeneans on host gills (Buchmann, 1988a; Dzika and Szymański, 1989). Certain species exhibited different preferences when they co-occurred on the same host, and occupied an adjacent microsettlement site, which resulted in antagonism and clearly defined site segregation (Paperna, 1964). However, some species occupied similar niches, and competitive interaction was not evident, or was even absent (Dzika and Szymański, 1989). The coexistence of congeneric monogeneans on grass carp has not been studied yet. Therefore the objective of this study was to investigate the seasonal occurrence and spatial distribution of the 2 monogenean species, D. lamellatus and D. ctenopharyngodonis, on the gills of cultured grass carp.

MATERIALS AND METHODS

Parasitological examination

Fish were transferred to the laboratory alive and examined after anesthetization with MS-222. The fork length of each fish was measured and the gills excised. Left and right arches were both numbered I to IV (I closest to the operculum, transverse partition); then each gill arch was divided into 3 sectors (horizontal partition) and 3 zones (vertical partition) according to Lo and Morand (2001), with some modifications (Fig. 1). Thus, 9 subequal sections were defined. The number and location of parasites on the gill hemibranches were recorded. Dactylogyrus species were identified by observing the hard parts of the haptor (anchors, dorsal and ventral connective bars, marginal hooks) and reproductive organs (male copulatory organ and vaginal armament) (Gusev, 1985) under a stereomicroscope and a light microscope.

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RESULTS

A total of 1,274 grass carp specimens was examined in 28 mo of the investigation. The mean fork length was 19.02 (±4.57) cm. A significant difference in the mean fish length was found among months (F = 47.41, df = 1,273, P < 0.01). A significant negative correlation was found between fish length and abundance of D. lamellatus (R = −0.19, n = 1,274, P < 0.01), but a significant positive correlation for D. ctenopharyngodonis (R = 0.11, n = 555, P < 0.01). There was no significant correlation between the abundance of D. lamellatus and D. ctenopharyngodonis (R = 0.05, n = 555, P = 0.25).

Seasonal changes in prevalence and mean intensity of D. lamellatus

The overall prevalence and mean intensity of D. lamellatus were 90.5% (84.0–95.0%) and 36.79 ± 60.50, respectively. The prevalence was high (more than 75%) throughout the experiment, with the exceptions of 38% (24.5–52.8%) in July 2013 and 43.6% (30.2–57.7%) in November 2013 (Fig. 2). No significant difference was found in prevalence among different months (G = 25.81, df = 27, P > 0.05).

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The mean intensity increased from November 2012 (3.47 ± 2.64) gradually and peaked in March 2013 (92.88 ± 96.48). Subsequently it declined sharply to the lowest point in July (1.89 ± 0.74), and remained low afterward (Fig. 2). A similar seasonal pattern was observed in 2014, but the highest value was recorded in February (215.26 ± 102.57), and the lowest in October (4.54 ± 2.74) (Fig. 2). Significant differences in the mean intensity were observed among different months (F = 82.24, df = 27, P < 0.01). In brief, the prevalence of D. lamellatus was high throughout the year, whereas the mean intensity peaked in late winter and spring, and dropped to the lowest point in summer and autumn.

Seasonal changes in prevalence and mean intensity of D. ctenopharyngodonis

The prevalence of D. ctenopharyngodonis was higher than 95% throughout the experiment, with the exception of 66.7% (49.0–81.4%) in January 2014 (Fig. 3). There was no significant difference in the prevalence among different months (G = 2.21, df = 11, P > 0.05).

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The mean intensity of D. ctenopharyngodonis was 47.22 ± 48.93, which increased in April, followed by relatively high levels until November, with the exception of a slight decrease in October 2014. The highest value was found in June (97.71 ± 63.27) and the lowest in March (12.25 ± 9.32) (Fig. 3). There were significant differences in the mean intensity among different months (F = 24.24, df = 11, P < 0.01). In short, the prevalence of D. ctenopharyngodonis was high throughout the year, whereas the mean intensity was higher in summer and autumn, and lower in winter and spring.

Spatial distribution of D. lamellatus

During period I (from December 2012 to December 2013), when the grass carp were infected only with D. lamellatus, there was no significant difference in the mean intensity of the species between the right and the left gills (t = −1.45, df = 596, P = 0.15) (Table I). The numbers of parasites on left and right gills were then pooled together according to the corresponding gill arches and locations. However, there were significant differences in the mean intensity among the 4 gill arches (one-way ANOVA: F = 6.63, df = 3, P < 0.01), 3 sectors (nested ANOVA: F = 115.68, df = 8, P < 0.01), and 3 zones (nested ANOVA: F = 73.55, df = 8, P < 0.01) (Table II). The highest mean intensity was found on the second gill arch (6.86), the medial sector (11.87), and the distal zone (12.84) (Table I). During period II (from January 2014 to December 2014), when the grass carp were infected with both Dactylogyrus species, spatial distribution of D. lamellatus was similar to that in period I: there were significant differences in the mean intensity among the 4 gill arches (one-way ANOVA: F = 10.76, df = 3, P < 0.01), 3 sectors (nested ANOVA: F = 61.62, df = 8, P < 0.01), and 3 zones (nested ANOVA: F = 587.82, df = 8, P < 0.01) (Table II). The highest mean intensity was recorded on the second gill arch (14.79), the medial sector (22.37), and the distal zone (39.45) (Table I). No significant differences in the percentage of mean intensity on each gill arch were found among the low-, medium-, and high-intensity groups (Table III; ANCOVA: P > 0.29).

Table I Mean intensity (±SD) of Dactylogyrus lamellatus and D. ctenopharyngodonis on the 4 gill arches, 3 sectors, and 3 zones of grass carp Ctenopharyngodon idellus in period I (December 2012 to December 2013) and period II (January to December 2014). R, right gill; L, left gill.

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Table II Results of the nested analysis of variance (ANOVA) on the number of Dactylogyrus lamellatus and Dactylogyrus ctenopharyngodonis on the gill arches, zones, and sectors of Ctenopharyngodon idellus in period I (December 2012 to December 2013) and period II (January to December 2014) (log-transformed data). R, right gill; L, left gill; MS, mean square; F, ANOVA; df, degrees of freedom; P, probability.

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Spatial distribution of D. ctenopharyngodonis

Mean intensity of D. ctenopharyngodonis on the right gill was significantly higher than on the left (t = 8.26, df = 554, P < 0.05). On both right and left gills, significant differences in mean intensity were found among the 4 gill arches (one-way ANOVA: F = 87.10, df = 3, P < 0.01), 3 sectors (nested ANOVA: P < 0.01), and 3 zones (nested ANOVA: P < 0.01) (Table II). The highest mean intensity was found on the second gill arch, the medial sector, and the distal zone (Table I). No significant differences in the percentage of mean intensity on each gill arch were found among the 3 intensity groups (Table III; ANCOVA: P > 0.34).

Table III Significant differences in the percentage of mean intensity of Dactylogyrus lamellatus (20–21-cm fork length) and Dactylogyrus ctenopharyngodonis (19–21-cm fork length) on each gill arch among low-, medium-, and high-intensity groups (<10, 10–30, >30 for D. lamellatus; <20, 20–100, >100 for D. ctenopharyngodonis) using analysis of covariance (ANCOVA) with total intensity of the 4 gill arches as the covariate. MS, mean square; F, analysis of variance; df, degrees of freedom; P, probability.

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DISCUSSION

Water temperature is considered to be the most important factor regulating the occurrence and abundance of monogeneans by means of directly acting upon their development and reproduction, or indirectly increasing the immunological resistance or vulnerability of the hosts (Chubb, 1979; Valtonen et al., 1990; Šimková et al., 2001). Generally, the reproduction rate of Dactylogyrus increases as water temperature rises in spring and summer, and then decreases as water temperature declines in autumn and winter. However, the effect of temperature differs among parasite species (Chubb, 1979; Pojmańska, 1995). Some species prefer low temperatures, such as Dactylogyrus extensus, which flourishes at 16–17 C, whereas others prefer high water temperatures, such as Dactylogyrus vastator, which prefers the warmer 20–30 C range (Zhang et al., 2015). Dactylogyrus lamellatus and Dactylogyrus nobilis can produce eggs at the end of winter and have a high level of occurrence and reproductive activity at the beginning of spring, which shows a strong preference for colder seasons. However, Dactylogyrus aristichthys, Dactylogyrus hypophthalmichthys, and Dactylogyrus suchengtaii probably do not reproduce in the cold season; instead they develop fast in high water temperatures in summer (Pojmańska, 1995). In the present study, D. lamellatus exhibited a high mean intensity in late winter and early spring, unlike D. ctenopharyngodonis, which was abundant in summer and autumn. This result suggests that low water temperatures are more suitable for the development and transmission of D. lamellatus, whereas high water temperatures are more appropriate for D. ctenopharyngodonis.

The highest infection level of D. lamellatus in spring was also observed in a field investigation in Poland (Pojmańska, 1995). However, D. lamellatus hardly laid any eggs, and those eggs did not hatch below 14 C under experimental conditions (Musselius and Ptashuk, 1970; Molnár, 1971a). The water temperature is generally not above 10 C in late winter in Central China and in spring in Poland and Hungary. Therefore, the massive newly infected worms in the cold season could be the larvae that hatched in autumn, and maintained viability during the winter months (Molnár, 1971a). However, a report showed that the longevity of the oncomiracidia of Heterobothrium ecuadori was 4–7 days (Grano-Maldonado et al., 2011). Furthermore, the infectivity (for host gills) declined sharply with time, so most oncomiracidia lost infectivity 2 days after hatching at room temperature (Chigasaki et al., 2000). These results indicate that it might also be difficult for oncomiracidia to retain infectivity for several months in winter. Our experiments (unpublished) on D. vastator have shown that 2-wk exposure of eggs to 5 C had significantly negative impacts on hatching success at 20 C, but low temperature had little impact on hatching success when the eggs were first exposed to 20 C for 12 hr and then to 5 C. Therefore, the eggs produced in autumn might be hatched in winter with relatively high hatching success. This indicates that the observed increase in the mean intensity of D. lamellatus in late winter probably resulted from a large egg production in autumn, followed by egg hatching and the infection of oncomiracidia in winter.

Regarding the spatial distribution of D. lamellatus and D. ctenopharyngodonis, the highest mean intensity of the 2 species was found on the medial and distal parts of the second gill arch. Microhabitat preference for particular gill arches or certain parts of the gill arches has been often reported for monogenean parasites (Buchmann, 1988b; Dzika and Szymański, 1989; Lo and Morand, 2000). For example, Pseudodactylogyrus anguillae preferred arches 3 and 4, whereas Pseudodactylogyrus bini and Ancyrocephalus mogurndae preferred arches 1 and 2 (Buchmann, 1988b; Nie, 1996). Dactylogyrus amphibothrium occurred mostly on the distal and dorsal part of the hemibranch; Dactylogyrus falcatus, Dactylogyrus wunderi, and Dactylogyrus auriculatus occurred on the proximal and medial parts; D. auriculatus on the medial and distal parts; and D. vastator on the proximal and ventral parts (Wootten, 1974; Dzika and Szymański, 1989; Vinobaba and Wootten, 1996). Differences in water circulation or surface area among the different parts of the gill arches are considered to be important factors in determining the distribution of parasites on the gills (Wootten, 1974; Gutiérrez and Martorelli, 1999). Some monogeneans prefer gill arches with minimum water flows, usually first and fourth, to avoid washing away (Tripathi et al., 2010). However, the middle 2 gill arches are often the ones with the highest parasite abundance (Wootten, 1974; Yao and Nie, 2004), which is believed to be in connection with the surface area (the 2 middle gills usually have the largest) and the volume of the passing water, which is in positive correlation with dissolved oxygen (Wootten, 1974; Turgut et al., 2006). Therefore, the higher mean intensity of the 2 monogenean species observed in the middle 2 gills perhaps indicates their preferences for better-oxygenated conditions.

The ventral segment of the gill arch is the most exposed to respiratory current, although its surface area is smaller than in either of the other 2 segments (Wootten, 1974; Lo and Morand, 2000). In the present study, both of the 2 Dactylogyrus spp. showed a fairly similar and clearly defined niche preference, with a majority occupying the distal zone of the medial sector of the second gill arch, and a minority occupying the ventral sector. Thus, the site preference of the 2 monogeneans on the medial sector may be influenced more by surface area of the arch rather than the hydrodynamic constraints. Dactylogyrids regularly relocate themselves on the gill surface to obtain fresh epithelium and avoid ruining the surface on which they depend (Roubal, 1986; Kearn, 1994). Therefore, staying on the medial sector, with moderate water current, can help them avoid being washed away during locomotion for food. In addition, monogenean aggregation at infracommunity level is important for successful mating and cross-fertilization (Bagge et al., 2005; Karvonen et al., 2007). The aggregation of the majority of the 2 Dactylogyrus spp. on the distal zone of the medial sector of the second gill arch, observed in this study may promote cross-fertilization and ensure genetic diversity (Kearn, 1994).

Although the 2 Dactylogyrus spp. almost always occupied the same niche, no interspecific competition was observed. First, no significant negative correlation was found between mean intensity of the 2 species on the gill arches. Second, the spatial distribution of D. lamellatus did not change after the infection with D. ctenopharyngodonis. Finally, the 2 species showed a high niche overlap in their spatial distribution, which suggests a lack of interspecific competition (Tripathi et al., 2010). The absence of competition was also observed in other monogeneans (Bagge and Valtonen, 1999; Morand et al., 2002; Bagge et al., 2005). It can be explained by unsaturated gill niches, mainly because of the low diversity and low infection level (Rohde, 1977, 1991), particularly as the maximal parasite carrying capacity is rarely reached in natural conditions (Rohde, 1979). The size discrepancy between small-sized monogeneans and large host gills may also render vacant niches more easily available and competition over resources unnecessary (Dzika and Szymański, 1989). The site preference of the 2 Dactylogyrus species at gill arch level did not vary with increasing intensity, which also suggests unsaturated niches.

The lack of niche segregation between congeneric monogenean species may also be related to reproductive barriers and differentiation of the attachment organs (Šimková et al., 2002; Jarkovský et al., 2004), whereas the site preference could be primarily the result of adaptation to the structure of host gills or water currents crossing the gills (Adams, 1986; Tripathi et al., 2010). Dactylogyrus lamellatus and D. ctenopharyngodonis exhibit different shape and size of copulatory organs, but high similarity in the morphology of attachment organs; perhaps these characteristics facilitate coexistence of the congeneric species.

Dactylogyrus lamellatus exhibits a preference for smaller grass carp, whereas D. ctenopharyngodonis mainly prefers larger fish (Schäperclaus, 1992). Infection of D. lamellatus is regarded to be responsible for high mortalities observed in fry and fingerling production (Shamsi et al., 2009). Our study corroborated this, as there was a weak but significant negative correlation between fish length and abundance of D. lamellatus, and a significant positive correlation for D. ctenopharyngodonis. Therefore, D. lamellatus is probably more harmful than D. ctenopharyngodonis because of high intensity on smaller grass carp.