A survey of gyrodactylid parasites on the fins of Homatula variegata in central China.

In this study, two parasites on the fins of Homatula variegata were recorded from March to September 2016. A dissection mirror was used to examine the distribution and quantity of the ectoparasitic Gyrodactylus sp. and Paragyrodactylus variegatus on the host Homatula variegata in different seasons. The present study explored possible explanations for the site specificity of gyrodactylid parasites in 442 Homatula variegata infected with 4307 Gyrodactylus sp. (species identification is incomplete, only characterized to the genus level) and 1712 Paragyrodactylus variegatus. These two gyrodactylid parasites were collected from fish fins, and the fish were harvested in China's Qinling Mountains.The results indicated that the highest number of Gyrodactylus sp., which was numerically the dominant species, appeared on the fish fins in April, while the highest number of Paragyrodactylus variegatus was found on the fish fins in March. The two parasite species appeared to be partitioned spatially, with Gyrodactylus sp. occurring more frequently on pectoral and pelvic fins, and P. variegatus occurring more frequently on caudal fins. However, Gyrodactylus sp. appeared to occur on fish of all lengths, while P. variegatus tended to occur more abundantly on shorter fish rather than on longer fish. At lower Gyrodactylus sp. infection levels (<100), the pelvic and pectoral fins were the main locations of attachment, followed by the dorsal fin. For infections of more than 100 parasites, more samples of Gyrodactylus sp. were located on the pectoral fin. For a low number of Paragyrodactylus variegatus infections (<100), the pelvic and pectoral fins were the preferred locations of attachment, followed by the caudal fin. Between April and September, there were many monogenean parasites on fish fins, and the fish size was within the range of 5-10 cm. However, when a fish was longer than 10 cm long, the number of parasites on its fins greatly decreased.

Introduction

Among the members of the class Monogenea, viviparous gyrodactylids are one of the most common parasites in wild and cultured fish, causing great ecological and economic harm [1]. Some gyrodactylids show significant microhabitat specificity, but this is highly variable among species. Some researchers focused on the site preference of Gyrodactylus turnbulli on Poecilia reticulata (guppy) in an experimental environment. Studies have found that lymphocytes in fish epithelial tissues have a direct effect on parasites after they come into contact with a host. The host's innate and adaptive immune system determines where the parasite lives [2-4].

However, the distribution of parasites on fish is strongly correlated with the age of infection. Water quality and water nutrition are factors that determine the abundance of fish parasites. However, changes in water temperature and seasons are also determinants of parasite abundance [5-6]. Other groups have reached similar conclusions by studying the behavior of parasites (G. colemanensis) on Salvelinus fontinalis fry [7]. Parasites attached to any part of fish epithelial tissue. In particular, many parasites occur on the edges of the tail, pectoral and peritoneal fins. Parasites periodically migrate to the edges of the fins and can travel through the body to reach other fins [8]. Recently, the fish Homatula variegata (Dabry de Thiersant, 1874) has become an increasing concern due to its potential aquaculture in China [9]. Gyrodactylus sp. and Paragyrodactylus variegatus (You, King, Ye and Cone, 2014) [10] are two parasites that are found on the fins and, occasionally, the body surface, of H. variegata in Xunyangba. However, gyrodactylids that live on the surface of fish appear to be less specific in terms of their environmental requirements and therefore occur in a variety of locations. This assumption, has led to a lack of information on the positioning of parasites on this fish; most authors locate parasites on the major branches of the body of a fish, such as the gills or torso [11]. However, no specific studies on the distribution of gyrodactylid parasites in Homatula variegata have been performed. Therefore, this study attempts to describe the position specificity of Gyrodactylus sp. in Homatula variegata.

Materials and methods

Ethical note

This study was approved by the Animal Care and Use Committee of Shaanxi Normal University.

Study area and sample collection

The fish (Homatula variegata) were collected (n = 442) with seine nets from late March to late September 2016 in Xunyangba (33.33°N, 108.33°E), Ningshan, County located on the southern slopes of the Qinling Mountains in Shaanxi Province, central China. The water temperatures on the collection days were recorded (Table 1).

Table 1

Morphometrics of the collected Homatula variegata.

Month	Water temperature (°C)	Number of Fish	Body length (cm)	Average body length mean± SE (cm)
March	7	43	3.60–13.60	5.70±0.372
April	13	40	3.80–13.80	8.43±0.425
May	17.5	42	4.10–11.70	7.15±0.303
June	21	46	4.50–14.60	8.37±0.370
July	23	85	4.50–14.00	9.10±0.211
August	17	100	3.10–13.90	9.36±0.218
September	15	86	3.40–13.80	7.91±0.287

Each fish was individually placed in a plastic tank filled with filtered river water and was transported to a field laboratory and examined within one hour. The fish were euthanized with excessive eugenol anesthetic fluid and fixed with 5–10% formalin. The total length of each fish was recorded, and the fins were examined for the presence of parasites that were removed and immediately identified on temporary wet mounts.

These two species of Gyrodactylus were found under a dissecting microscope (OLYMPUS, SZ61, 45X), Then they were placed on glass slides that had drops of glycerin-water with pointed ophthalmic forceps. If there was a cap-like bone piece structure covering the base of the central hook, then it was recorded as Paragyrodactylus variegatus; otherwise, it was recorded as Gyrodactylus sp.

The parasites were stored in formalin. Almost all the parasites stuck to the skin after immobilization. Voucher specimens of the parasites and host were deposited in the Fish Disease Laboratory, Shaanxi Normal University (Accession number: H. variegata: Acc.HV20160012; Gyrodactylus sp.: Acc.GS20160001 and P. variegatus: Acc.PV20160001).

Analysis of parasite location

The different numbers of parasites on each fin corresponding to different fish body lengths were examined, from March to September, 2016. The parasite species and location of parasites on the host’s fins were examined by using a two-way ANOVA, with the number of parasites on different fins used as the dependent variable. The microhabitat occurrence for each gyrodactylid species was determined by observing its position on the fins. The distributions of each gyrodactylid species on each of the different fins were compared by two-way ANOVA with multiple comparisons (Tukey's HSD test) to assess the significance of the difference. The significance level was set at p < 0.05.

Relationships between water temperature, parasite load location and fish size

To test for the overall effects of water temperature and fish body length on the distribution of the number of parasites on the host fins, a generalized linear model (GLM) was also built using water temperature or fish body length as predictors. To further explore the relationships of fish size (length) and water temperature with the number of parasites on different fins, Spearman correlation statistical analysis was conducted using SPSS (Statistical Package for Social Sciences, v21).

Results

The number dynamics of gyrodactylid parasites and distribution of parasites on fish fins

The fish (Homatula variegata) were collected (n = 442) with seine nets from late March to late September 2016 in Xunyangba (33.33°N, 108.33°E), Ningshan County located on the southern aspects of the Qinling Mountains in Shaanxi Province. The water temperatures on the collection days were recorded. We measured the body length of each host. We specifically collected two gyrodactylid parasites from the host fins. The species and number of parasites on different fins were recorded in detail. Tukey's HSD test was used to analyze the significance of parasite distribution on the fins, and GLM was used to analyze the effect of host length and water temperature on the location of parasites. To explore the specific effect of water temperature and length on the number of parasites on fins, a Spearman correlation analysis was performed.

Temporal changes in the number of parasites on the fins were analyzed. The highest number of Gyrodactylus sp. on the fish fins appeared in April, while the highest number of P. variegatus on the fish fins occurred in March. The number of Gyrodactylus sp. and P. variegatus showed roughly similar trends, with the number of the two parasites (mean ± SE) on fins decreasing in May (4.17 ± 0.74 and 2.6 ± 0.43, respectively); the number of parasites on the fins decreased in August (2.09 ± 0.31 and 2.17 ± 0.27) and increased in September (5.53 ± 0.43 and 5.23 ± 0.45) (Fig 1). Of the 7 months, the number of the two gyrodactylid parasites on the fins were lowest in August. There was no significant correlation between number of parasites and the water temperature, which ranged from 7°C (in March) to 23°C (in July) (Gyrodactylus sp.: r = - 0.149, p = 0.751; P. variegatus: r = 0.090, p = 0.847).

Fig 1

Parasites number (mean±SE) of Gyrodactylus sp. and Paragyrodactylus variegatus on total fish fins by month.

On the other hand, the number of P. variegatus on the pectoral fin was relatively high in May and July. The number of parasites detected on the dorsal fins and anal fins was relatively low (Fig 2A). The number of parasites on different fins decreased significantly during April and May. In June, the number of Gyrodactylus sp. increased on the pectoral fin (Fig 2B).

Fig 2

Parasites number (mean±SE) on different fish fins monthly.

A. Paragyrodactylus variegatus, B. Gyrodactylus sp.; the abbreviations Ana., Cau., Dor., Pec., and Pel., indicate, anal fin, caudal fin, dorsal fin, pectoral fin and pelvic fin, respectively.

For Gyrodactylus sp., there was a significant difference (two-way ANOVA, F = 11.97, df = 4, p < 0.001) among the mean number (mean ± SE) of parasites that were distributed on the five fins. All data were examined from a total of 442 specimens of Homatula variegata from Xunyangbain the Qinling Mountains of Shaanxi Province, central China, which were collected from March to September 2016. The total length of the host (± 0.1 cm) ranged from 3.1 to 14.6 cm. The number of fish and the water temperature for each sampling period are recorded in Table 1. For P. variegatus, there was also a significant difference (two-way ANOVA, F = 30.94, df = 4, p < 0.001) among the mean numbers (mean ± SE) of parasites that were distributed on the five fins. In general, the mean number (mean ± SE) of Gyrodactylus sp. infecting different fin parts was higher than that of P. variegatus (Fig 2) by month. However, we also detected a contrasting pattern between the densities of specific parasitic infections of the fins. Although there was no difference between the pectoral fins and the pelvic fins of the two gyrodactylid parasites (Tukey’s HSD, df = 4, p > 0.05), and the number of parasites on these two fins was higher than that on any other fin (Tukey’s HSD, df = 4, all p < 0.001; Fig 3). The patterns of parasite number were comparable in Gyrodactylus sp. and P. variegatus but were clearly different from those on respective fins (two-way ANOVA, estimated marginal mean test).

Fig 3

Number of Gyrodactylus sp. and Paragyrodactylus variegatus on different fish fins, the abbreviations Ana., Cau., Dor., Pec., and Pel., indicate, anal fin, caudal fin, dorsal fin, pectoral fin and pelvic fin, respectively.

*p < 0.05; **p < 0.01.

By comparison, the number of Gyrodactylus sp. was significantly higher on pelvic fins and pectoral fins than on other fins (test of between-subjects effects, F (4, 4410), p < 0.001). Both parasite species appeared in moderate amounts on the dorsal fins (Gyrodactylus sp.) and caudal fins (P. variegatus), respectively (Fig 3). On the other hand, the number of P. variegatus was also significantly higher on pelvic, pectoral and caudal fins than that on anal and dorsal fins (Tukey’s HSD, df = 4, both p < 0.001). No other significant differences in the number of specific parasitic infections among host fins were detected. At lower levels of infection, parasites preferentially colonized the pelvic, pectoral and dorsal fins (Table 2). Most fins were infected with a small number of parasites. The number of P. variegatus present in fish was less than 100. As the number of parasites on each fish fin increased by 11 to 100, pelvic fins continued to be the main area of attachment, followed by the pectoral fin. However, at a level of 100 or more parasites per fish, relatively few Gyrodactylus sp. were observed on some fins, such as caudal fins. The total number of Gyrodactylus sp. was higher than that of P. variegatus on all fins per fish. The cause of this phenomenon needs further study.

Table 2

The site specificity of Gyrodactylus sp. and Paragyrodactylus variegatus on four different range of infection of Homatula variegata (the average number of infections is on each fin).

Gyrodactylus sp.							Paragyrodactylus variegatus
Nop	Nof	Cau	Ana	Dor	Pel	Pec	Nop	Nof	Cau	Ana	Dor	Pel	Pec
0	53	0	0	0	0	0	0	79	0	0	0	0	0
1~10	271	0.727	0.539	0.738	0.86	1.251	1~10	331	0.925	0.378	0.347	0.876	1.187
11~100	114	3.07	3.693	5.579	6.342	6.781	11~100	35	3.457	1.571	1.657	4.057	3.657
101~200	3	3	16	29	36.667	41.667	101~200	0	N/A	N/A	N/A	N/A	N/A

Nop: Range of parasites number; Nof: number of fish; Cau: caudal fin; Ana: anal fin; Dor: dorsal fin; Pel: pelvic fin; Pec: pectoral fin.

Relationships between the number of parasites on the fins and the body length of the fish and water temperature

Of the 442 fish collected, 135 were less than 7 cm in length, accounting for 30.5% of the total; 183 fish had a body length of more than 7 cm but less than 10 cm, accounting for 41.4% of the total;124 fish had a body length of more than 10 cm, accounting for 28.1% of the total. Interestingly, throughout the study period (March to September 2016), host fish were infected with the two gyrodactylid parasites at significantly fluctuating levels (Table 3). By examining the fins of all the fish samples, a large number of the two parasites were observed on the fins of the fish hosts with relatively shorter body lengths (1–5 cm). There were fewer of these two parasites on the fins of fish hosts with relatively larger body lengths (greater than 5 cm).

Table 3

Parasitism by Gyrodactylus sp. and Paragyrodactylus variegatus on Homatula variegata.

Gyrodactylus sp.								Paragyrodactylus variegatus
Month	Fs (cm)	Nof	Cau	Ana	Dor	Pel	Pec	Month	Fs (cm)	Nof	Cau	Ana	Dor	Pel	Pec
March 2016	1–5	25	85	97	125	134	68	March 2016	1–5	25	22	6	20	20	12
	5–10	14	68	104	102	118	134		5–10	14	31	19	28	47	23
	10–15	4	6	28	51	90	90		10–15	4	20	6	12	16	16
April 2016	1–5	7	9	16	31	32	29	April 2016	1–5	7	0	0	0	2	2
	5–10	19	48	71	145	168	192		5–10	19	7	9	13	12	13
	10–15	14	10	71	141	125	184		10–15	14	2	12	5	22	15
May 2016	1–5	8	9	12	19	15	12	May 2016	1–5	8	2	3	1	4	6
	5–10	32	23	17	22	20	23		5–10	32	29	9	8	26	22
	10–15	3	2	0	1	1	1		10–15	3	0	0	0	1	0
June 2016	1–5	6	8	2	9	3	29	June 2016	1–5	6	11	1	2	1	13
	5–10	31	67	40	45	77	116		5–10	31	46	20	9	54	98
	10–15	10	9	3	11	15	21		10–15	10	7	2	2	11	15
July 2016	1–5	1	0	0	1	1	0	July 2016	1–5	1	2	3	1	0	0
	5–10	57	52	40	52	52	85		5–10	57	31	14	14	25	67
	10–15	27	19	18	23	24	30		10–15	27	16	7	5	17	23
Aug. 2016	1–5	4	1	0	0	0	0	Aug. 2016	1–5	4	0	0	0	0	3
	5–10	57	29	15	19	31	52		5–10	57	27	6	8	35	82
	10–15	39	16	7	21	9	8		10–15	39	15	5	3	13	20
Sep. 2016	1–5	19	27	11	13	24	32	Sep. 2016	1–5	19	60	11	6	17	8
	5–10	47	54	45	42	66	86		5–10	47	84	34	19	77	51
	10–15	20	10	4	29	16	17		10–15	20	11	14	3	7	16

Fs: Range of fish body size; Nof: number of fish; Cau: caudal fin; Ana: anal fin; Dor: dorsal fin; Pel: pelvic fin; Pecf: pectoral fin.

GLM analysis showed that host body length had a significant effect on the number of Gyrodactylus sp. on the pectoral fins. Host body length had a significant effect on the number of P. variegatus on the caudal and anal fins. Water temperature had a significant effect on the number of these two gyrodactylid parasites on all fins, and the specific differences need further analysis (Table 4).

Table 4

Results of the generalized linear models (GLM) used to determine the body length and water temperature on the total gyrodactylid parasite number on the different fins of Homatula variegata.

Dependent variable a	Degrees of freedom b	Coefficient	p value
Total fins (Gyrodactylus sp.)	105	Intercept	<0.001
		Host body size(length)	0.006*
		water temperature	<0.001**
Cau. fins (Gyrodactylus sp.)	82	Intercept	<0.001
		Host body size(length)	0.692Δ
		water temperature	<0.001**
Ana. fins (Gyrodactylus sp.)	85	Intercept	<0.001**
		Host body size(length)	0.101Δ
		water temperature	<0.001**
Dor. fins (Gyrodactylus sp.)	92	Intercept	<0.001**
		Host body size(length)	0.130Δ
		water temperature	<0.001**
Pel. fins (Gyrodactylus sp.)	95	Intercept	<0.001**
		Host body size(length)	0.066Δ
		water temperature	<0.001**
Pec. fins (Gyrodactylus sp.)	93	Intercept	<0.001**
		Host body size(length)	<0.001**
		water temperature	<0.001**
Total fins (Paragyrodactylus variegatus)	98	Intercept	<0.001**
		Host body size(length)	<0.001**
		water temperature	<0.001**
Cau. fins (Paragyrodactylus variegatus)	75	Intercept	<0.001**
		Host body size(length)	0.005*
		water temperature	0.003*
Ana. fins (Paragyrodactylus variegatus)	62	Intercept	<0.001**
		Host body size(length)	0.032*
		water temperature	0.038*
Dor. fins (Paragyrodactylus variegatus)	58	Intercept	<0.001**
		Host body size(length)	0.139Δ
		water temperature	0.006*
Pel. fins (Paragyrodactylus variegatus)	80	Intercept	<0.001**
		Host body size(length)	0.085Δ
		water temperature	<0.001**
Pec. fins (Paragyrodactylus variegatus)	90	Intercept	<0.001**
		Host body size(length)	0.357Δ
		water temperature	0.001*

a. The number of parasites in the corresponding location

b. Sig. of Omnibus test

Δ. No Significance (p > 0.05)

* Significance (p < 0.05)

**Extremely Significance (p < 0.001)

After statistical analysis, water temperature and host body length had a high negative correlation (spearman) with the number of Gyrodactylus sp. on the fins (Table 5). However, these factors were relatively less relevant to the number of P. variegatus on the fins. Independently, the correlation between water temperature and the number of Gyrodactylus sp. on the fins was higher. Interestingly, the water temperature was negatively related to not only the number of Gyrodactylus sp. on the fins but also the host body length but to a lesser extent. These results are essentially consistent with the HSD analysis and GLM analysis (Table 4).

Table 5

Spearman coefficients between body length of fish and water temperature with gyrodactylid parasites on the fins.

Parasite	Water temperature vs Number of parasites on fins. Spearman correlation coefficient and significance.	Host body length vs Number of parasites on fins. Spearman correlation coefficient and significance.
Gyrodactylus sp.	r = -0.351, p < 0.001	r = -0.269, p < 0.001
Paragyrodactylus variegatus	r = -0.183, p < 0.001	r = -0.197, p < 0.001

Host samples = 442

Discussion

Although it has been reported in China, little research has been conducted on the survival of gyrodactylids on Homatula variegata, for which a total of two genera were found: Gyrodactylus (von Nordmann, 1832) and Paragyrodactylus (Gvosdev et Martechov, 1953). Our investigation revealed that Gyrodactylus sp. and P. variegatus survive on this host. The microhabitat of monogeneans living on fins has been investigated by many authors [12-14]. Monogeneans exhibit the characteristics of aggregate parasitism. For example, benedeniines are significantly parasitic on specific fins [12]. Studies have found that parasites that are attached to the dorsal fins or pelvic fins of fish may be designed to evade host predation, competition, and local immune responses [15, 16]. In addition, each developmental cohort that inhabits different fish fins can receive exclusive food and spatial resources [14].

In the present study, the two species of parasites appear to have subtle spatial partitions in their common resources. Gyrodactylus sp. occurred most frequently on the pectoral and pelvic fins, while P. variegatus occurred on the caudal fins.

In this study, we investigated the average water temperature of the sampling points during sampling. Water temperature is thought to be a factor affecting a parasite's ability to reproduce [17-19]. Moreover, there is a certain degree of correlation between the water temperature and number of parasites on fins, but the influence of water temperature is distinct in different parasite species [20]. The relationship between temperature and parasite reproduction is complex. Some literature has noted that the number of parasites increases with increasing water temperature [21, 22]. On the other hand, for some species, elevated temperatures can be a limiting factor for survival and reproduction [23, 24]. In our study, we found that the number of Gyrodactylus sp. and P. variegatus on the fins of H. variegata showed a different trend; specifically, the number of Gyrodactylus sp. number on the fins reached its highest point in April, gradually declined in summer and increased again in autumn (Fig 1). Some previous findings support the results of increased numbers in summer [18, 25], which is consistent with our findings. In addition, although the water temperature in July (23°C) was higher than that in June (21°C), the volumes of the two fin parasites in July were lower than those in June. The reason may be that the immunity of the host fish increases with increasing water temperature, thus leading to a decline in the number of parasites. Previous studies have demonstrated the same results in higher levels of infection and with weaker host immunity [20, 26]. Another reason for this outcome may have been the changes in aquatic environmental factors that are were caused by the increased water temperatures in July, which led to a decrease in the number of aquatic environmental factors. In addition to temperature, photoperiod, salinity and water flow can influence the success of infection. Studies have found that host fish are only infected by monogenean parasites during the day, and low-temperature and high-salt waters are more conducive to parasitic infections of fish [27]. Interestingly, the number of Gyrodactylus sp. on the fish fins decreased significantly relative to that of P. variegatus, in May. The cause for this is unknown at this time but could involve interspecific competition. The number of Gyrodactylus sp. rose again in September, possibly due to changes in the water flow rate. More detailed work on this topic is clearly required. We found a negative correlation between the number of Gyrodactylus sp. and the body length of H. variegata, and there was a relatively weak negative correlation between the number of P. variegatus and the body length of the fish. This finding is similar to the results of some previous studies. Thus, there was a negative correlation between parasite species richness and fish body size [28-30]. Due to the increase in parasite amount, in comparison to larger fish, smaller fish hosts may be more susceptible to disease [1]. However, some previous literature observed the opposite, in which there was a positive correlation between the number of parasites on the fins and the size of the host [31-34]. Some researchers have noted that the relationship between the number of parasites on the infected sites of fish and the length of the host should also be highlighted. This effect is more pronounced in small fish, which have a higher number of parasites on the body surface [35, 36]. Another possible reason might be that fish with a longer body length may be found in microhabitats with less exposure to parasitic infections [30]. Fish use group behavior and immune responses to reduce the risk of parasites [37]. Perhaps large fish are better suited to finding groups. The current scope of the study reveals that there was less aggregation of parasites on large Homatula variegata fins, which is consistent with the idea that the chances of avoiding infection are enhanced. It is important to emphasize, however, that not only the host size but also the ecology of each host species affects the species richness of the parasite [34].

Different kinds of Gyrodactylus are parasitic to different parts of the host. Gyrodactylus masu is found on the body surface of salmonids, and the fins, gill arches and gill filaments are the main locations [38]. By observing the parasitic behavior of the five species of Gyrodactylus parasites, researchers found that four of the parasites prefer to parasitize the fish surface and fish gills. When studying two parasites (G. colemanensis and G. salmonis) on the surface of the salmonids, most of the G. colemanensis were attached to the edge of the fin. Gyrodactylus salmonis attached to the head and body surface of the fish [7]. Studies have found that different types of Gyrodactylus have different haptor shapes, which may lead to differences in their habitats [7, 39]. The morphology of the haptors of each Gyrodactylus species is probably adapted to the surface of their host.

Conclusions

In summary, through the investigation of two parasites that infect Homatula variegata, we found that (1) the highest number of Gyrodactylus sp. on the fins appeared in April and March, whereas the number of Paragyrodactylus variegatus on the fins appeared in June. That is, the peak number of the two parasites on the fins showed a time niche separation. However, the trend in the number of parasites on the fins was similar during from May to September. The number of the two parasites on the fins of the host rise again in the autumn. (2) The two gyrodactylid parasites seemed to partition their common resources spatially. Gyrodactylus sp. preferred to parasitize the pectoral and pelvic fins, while P. variegatus preferred to parasitize the caudal fins. This may be explained by the avoidance of predation competition and the host's local immune response. This selection mode can be used as a potential delivery policy in gyrodactylids. The main factors leading to the preference for specific habitats have not yet been determined and may be linked to physiological, environmental, ecological and physical factors. More research is required to clarify this preference.

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17 Jan 2020

PONE-D-19-26341

A survey of gyrodactylid parasites on the fins of Homatula variegata in central China

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Reviewer #2: No

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Reviewer #2: Yes

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Reviewer #1: The authors did a great work in the manuscript and the analysis of the specimens collected. But I consider that this paper should be submitted to another journal rather than PlosOne. Although, the data and the species reported are something new for China, I would identify the Gyrodactylus species to give a better approach to the study, it is been demostrated that a single fish species can be infected by different gyrodatylid species and this can give different results, and the numbers in this study, for example can be mistaken. The infection dynamics of different species are very different, and this can be mistaken (in the order to imply, that is only one species that they found in the present study).

Abstract:

The authors mentioned that they collected the Gyrodactylus specimens with fishing nets and scorpions. I think they collected the fish with these nets, rather than the parasites. Please change.

Introduction:

The geograhical locality is mentioned with the coordinates, please delete this, that should be mentioned in the Material and Methods (M&M) subject. Delete it and move it to this section.

Materials and Methods:

The authors did not mentioned the number of fish they sampled (this is mentioned in Abstract, but not in M&M, please do so.

The accession numbers should be written as follows: H. variegata Acc. No. HV20160012 or Accession numbers H. variegata HV20160012, but is better the first one.

The animal processing must go with the name of the institution, number of permission, city and country.

Results:

Please check the manuscript for comments on this part.

How can the authors be sure that they only have 1 species of Gyrodactylus infecting the host?

Discussion:

Please check the manuscript for comments on this part.

Conclusions:

The authors have some works wrongly spelled. Please check and change.

Acknowledgements:

The research was funded by the National Natural Science Foundation of China (Project number 31872203) and the Natural Science Foundation of Shaanxi Province (Project number 2017JM3014).

References:

Most of the references are miss spelled and don’t have a unified style. Please change it in the correct journal form.

Tables:

Table 2. I think that if the authors have shown the data in table1 per month, they should do the same here with the parasite data. They must add the abbreviations here of the different fins names rather than in Table 3.

Figures:

Figure 2. The names on axis X there is a missing space between parasites the brackets and on, please change it.

Figure 5. I don’t see the point of adding this figure, it doesn’t show anything.

Reviewer #2: The ms covers a potentially important topic well worth publication, but as presented, has several important shortcomings. Even though original, unpublished experimental data are presented, the methods used to generate and interpret the results are insufficient to replicate the work and use the data to their full potential. Additionally, inconsistent and not widely accepted parameters, not fully described in the materials and methods are used throughout the text. Conclusions are not really well supported by the data as presented, and are nevertheless used as a base for unnecessary, non-related speculation that could be omitted.

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-19-26341_reviewer.pdf

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Submitted filename: PONE-D-19-26341_reviewer.pdf

Click here for additional data file.

26 Feb 2020

Dear Dr. Ulrike Gertrud Munderloh,

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled ‘A survey of gyrodactylid parasites on the fins of Homatula variegata in central China’ (PONE-D-19-26341). The comments were very helpful for revising our paper. We have studied the comments carefully and have made revisions accordingly, which we hope meet with approval. Revisions have been made using the Track Changes feature in MS Word. The revisions to the paper are described in our responses to the academic editor and reviewers’ comments below.

Thank you in advance.

Cheers,

Ping You

Notes on content modification

Thank you very much for your noble opinions and suggestions. According to the requirements, we have revised the full manuscript peer-to-peer. The main content is as follows

Abstract

Question：

Not clear what this is; please specify, “scorpions”

Answer：

It is a pair of tweezers used to clamp the parasite from the fish's body. Revised the description of the sentence.

Question：

Please revise/rephrase: if the species identity was not established, and parasites were only characterized to the genus level, this should be reflected in the text.

Answer：

The description of the species name has been revised.

The present study will explore possible explanations for the site specificity of gyrodactylidae parasites in 442 Homatula variegata infected with 4307 Gyrodactylus sp. (Species identification is incomplete, only characterized to the genus level) and 1712 Paragyrodactylus variegatus.

Question：

A bit confusing, if both taxa occur more frequently on the same two areas...

Answer：

Through GLM analysis, it was further found that the fin positions of the two parasites were not exactly the same. Revised the description of the sentence

Question：

Are you sure that you use fish nets to collect the parasites, rather than the fish??

Answer：

The original sentence was deleted. Replace with the following sentence.

These two gyrodactylidae parasites were collected from the fish fins, and these fish were harvested in China's Qinling Mountains.

Question：

Add space

Answer：

Added space in numbers betweeen unit symbols. “10 cm”

Introduction

Question：

Significant “bit” specificity, microhabitat?

Answer：

Revised this word as “microhabitat”.

Question：

Rephrase? The cited styudy shows that parasites occur more frequently/abundantly in those fins, but does not really address differences in susceptibility. In contrast, work by Buchmann et al., Lindenström at al, and by Rubio-Godoy et al, does address this topic by analyzing potential immune differences between fish fins/body regions.

Answer：

This sentence has been modified to increase the impact of immunity. References have been added. See references 2, 3, and 4 for details

Question：

True; but very important to also consider the well-documented effect of temperature on population dynamics of ectoparasites such as those studied here

Answer：

Water temperature does have an effect on the number of fish parasites. We have added quotations in this regard.

Question：

Better: on Salvelinus fotinalis fry

Answer：

The original sentence was deleted. Replace with the following sentence.

on Salvelinus fotinalis fry

Question：

attached

Answer：

Modified word, new one is “Parasites attached”

Question：

Add space

Answer：

Added spaces in symbol between word. The new format is “parasites (G. colemanensis)”.

Question：

Delete this sentence. If you are already denoting that is Gyrodactylus sp., of course means that the species is not described, other wise you will add the species name as you are doing with P. variegatus

Answer：

A newly discovered parasite has not been given a formal name. So, the original text was deleted.

Question：

Delete: which is located by the…, and the geographical position of the locarion. This shouldnt be in the introduction, just only in the materials and methods or results.

Answer：

Removed this geographic information. It is introduced in the methods and materials section.

Question：

Gyrodctylus sp. add sp.

Answer：

Modified latin name format. Gyrodctylus sp.

Materials & Methods

Question：

Collection permit obtained? It was not stated in the submission format

Answer

We have obtained permission for field experiments. We recapitulate in the first paragraph of the Methods and Materials section.

Question：

microscope; please state if thus was the case , the type of microscope used and what magnification was used - Materials and methods should enable replication of the work reported and this important information is missing.

Answer

We have rewritten this part. The method of selecting parasites using a microscope is detailed. The type and magnification of the microscope are also described. GLM was used to analyze the effect of host length and water temperature on the location of parasites. In order to explore the specific effect of water temperature and length on the number of parasites on fins, a Spearman correlation analysis was performed.

Question：

it would be helpful if brief mention were made on how easy/complicated it is to differentiate between these two genera, as both are tiny organisms. This would also be relevant to be able to replicate this study.

Answer

We have rewritten this part. The method of selecting parasites using a microscope is detailed.

Question：

Density of infection is an unusual way to describe ecological parameters of infection; and insufficiently described here as the surface area of the fins is not considered to truly reflect density.

Answer：

Our experiments used a direct parasite counting method. Change the density to the number of parasites throughout.

Question：

How many fish were collected?

Answer：

The number of captured hosts is hereby marked. Fish (Homatula variegata) were collected (n = 442)

Question：

You should put Acession numbers or After is species name the prefix Acc. Nos.

Answer：

We have revised numbers format. Voucher specimens of the parasites and host were deposited in the Fish Disease Laboratory, Shaanxi Normal University (Accession number: H. variegata: Acc.HV20160012; Gyrodactylus sp.: Acc.GS20160001 and P. variegatus: Acc.PV20160001).

Results

Question：

Before addressing density of infection on fins, it would be interesting/relevant, to describe overall infection trends; and only later describe dynamics on the fins.

Answer：

Our experiments used a direct parasite counting method. Change the density to the number of parasites throughout.

Question：

a bit confusing to present abundance, as this has not been presented in Materials and Methods and the first part of this paragraph mentions density.

Answer：

Question：

this whole section is very confusing, and should be carefully revised as basic concepts are not applied correctly: e.g., fins do not infect parasites, and parasites are not susceptible to specific microhabitats on the host

Answer：

Our experiments used a direct parasite counting method. The descriptions of density, intensity, and abundance have been omitted throughout the text, and the number of parasites has been unified. In addition, the influence of two factors (water temperature, host length) on the number of parasites on fins was analyzed using GLM.

Question：

check: these statements are contradictory

Answer：

We have rewritten this sentence.

Question：

I would move this after the next title, or combine this one with the next title. This part of the Material and methods are vry messy, please rewrite. Always write the name of the fih species, number of fish, place where they were collected, what did you find and then you stablish which statistical methods you used for your study

Answer：

We added content. The specific experimental contents are described, such as the fishing geographic information of the host, the body length of each host sample, the types of two parasites, and the statistical analysis method. See the first paragraph in the results section for details.

Question：

Between the Mean and SE must and space before the simbol, please add it and make it in the whole document.

Answer：

We adjusted all the formats.

Question：

Delete Figure 2A and move it after anal fins was relatively small (Figure 2A).

Answer：

We adjusted the position of the image name after this sentence. The details are as follows

The density of the infection of parasites detected on the dorsal fins and anal fins was relatively small (Figure 2A).

Question：

Put it at the end of the sentence. The number of paraaites on different fins decreased significantly during April and May. In June, the mumber of Gyrodactylus sp. increased on the pectoral fin (Figure 2B).

Answer：

We adjusted the position of the image name after this sentence.

Question：

This should be first or put it together with the previous subject

Answer：

We merged these two paragraphs

Question：

The case is different than the rest of the MS. You can not cite first a higer Fig and then a lower one, pease change this

Answer：

This section adds GLM analysis to explore the significant effects of water temperature and host length on the number of parasites on different fins. Figure 5 in the original manuscript is similar to other diagrams, so it was deleted.

Question：

If you put this in this way, it means you did the samplin on September the following year. Otherwise you should put March-September 2016

Answer：

We have corrected the date expression. Interestingly, throughout the study period (March to September, 2016),

Discussion

Question：

Gyrodactylus sp. occurred most frequently on the pectoral and pelvic fins, while P. variegatus occurred on the pectoral fin, pelvic fin and caudal fins. contradiction

Answer：

We have modified this sentence. On the basis of HSD analysis, further analysis by GLM revealed that the two parasites were not distributed in the same position on the fins.

Question：

irrelevant infromation, not directly related to the hypotheses tested in this study

Answer：

We removed irrelevant references

Question：

The correct authority is von Nordmann, 1832, please correct.

Answer：

We have corrected the author's name. Gyrodactylus (von Nordmann, 1832)

Question：

Delete it “Fish also often appear to compete for territory [2].”

Answer：

Removed this sentence in the manuscript

Question：

This is not completely true, “It has been reported that some Monogenea parasite species have the longest life span at 21°C, and are not able to survive at 30°C [16].”

Answer：

Removed this sentence in the manuscript

Question：

References [18]

Answer：

This reference has been deleted

Conclusions

Question：

I think is rise??? please change

Answer：

Deleted rose and changed it to rise. The number of the two parasites on the fins of the host rise again in the autumn.

References

We have modified the reference format of the manuscript with reference to the Plos ONE format. Examined and corrected species names, author names in references.

Table

Due to the similar content to Table 1 and 3, we deleted Table 2.

Added Table 4, corresponding to GLM analysis.

Added Table 5, corresponding to Spearman analysis.

Figure

We changed the name of the Y axis in Figure 2.

Figure 4 was deleted because Spearman analysis was used, and the new content is in Table 5.

Deleted Figure 5, because its content is similar to Table 3.

Thanks again for your review

Sincerely

All authors

27 Feb 2020

A survey of gyrodactylid parasites on the fins of Homatula variegata in central China

PONE-D-19-26341R1

Dear Dr. You,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ulrike Gertrud Munderloh, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

3 Mar 2020

PONE-D-19-26341R1

A survey of gyrodactylid parasites on the fins of Homatula variegata in central China

Dear Dr. You:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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With kind regards,

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on behalf of

Dr. Ulrike Gertrud Munderloh

Academic Editor

PLOS ONE