Condition Factors Do Not Reflect Parasite Loads: A Case Study on Juvenile Cyprinus carpio (Cypriniformes, Cyprinidae) from the Lower Danube River

Abstract

The present study aimed to evaluate whether commonly used condition indices reflect parasite load and bacterial colonization in juvenile Cyprinus carpio under natural environmental conditions in the Lower Danube River. A total of 260 specimens were examined for parasitological, microbiological, and biometric parameters, including 20 individuals analyzed for bacterial communities. Twenty-three parasite taxa belonging to eight major taxonomic groups were identified. Ectoparasites were found on the gills, skin, and fins, with monogeneans and ciliates, notably Dactylogyrus ssp. and Trichodina ssp., representing the dominant groups. Infection intensity was generally low to moderate, and histopathological examination revealed only mild epithelial alterations, including focal hemorrhage and mucus hypersecretion in more heavily infected individuals. Microbiological analysis identified six bacterial taxa associated with the skin, with Aeromonas hydrophila being the most frequently detected species. Correlation analyses showed no significant relationships between parasite abundance and condition indices (Fulton’s K, Le Cren’s Kn, scaled mass index, and BMI), although a slight reduction in Fulton’s K was observed in infected individuals. These findings indicate a stable host–parasite–microbiota equilibrium under natural environmental conditions. The results provide baseline ecological data that contribute to understanding fish health dynamics in the Lower Danube River and may support future monitoring and management strategies.

1. Introduction

The assessment of fish health in freshwater ecosystems is a key component of ecological monitoring, particularly under increasing anthropogenic pressure and climate change [1,2]. Fish are widely recognized as sensitive bioindicators, as their physiological condition reflects the combined effects of environmental degradation, parasitic infections, and microbial colonization [3]. Among the various tools used to evaluate fish vitality, condition indices (CIs), particularly Fulton’s condition factor (K), are commonly applied due to their simplicity and assumed relationship with somatic energy reserves and physiological status [4,5]. Traditionally, reduced condition values have been interpreted as indicators of environmental stress and disease, often associated with parasitic infections and bacterial colonization [6]. However, growing evidence suggests that the relationship between condition indices and pathogen load is not universal and may depend on species-specific traits, host–parasite coevolution, and environmental conditions [7,8].

Host–parasite interactions in freshwater fish are complex and may involve multiple taxonomic groups, including monogeneans, trematodes, protozoans, and myxozoans. Any of which can induce effects ranging from subclinical physiological changes to severe tissue damage, particularly in gill and integumentary systems [9,10]. In parallel, opportunistic bacteria such as Aeromonas spp. and Pseudomonas spp. frequently colonize epithelial surfaces, especially under conditions of environmental stress, including eutrophication, temperature fluctuations, and chemical pollution [11,12]. Under such situations, traditional condition indices may not adequately reflect the physiological impact of parasitic and microbial infections [13,14].

Several morphometric indices based on mass–length relationships have been developed to assess fish condition. Fulton’s condition factor assumes isometric growth and have been widely applied across fish taxa [15,16,17,18], Le Cren’s relative condition factor (Kn), which compares observed and expected body mass for a given length, provides a refined estimate of relative condition [19,20]. The scaled mass index (SMI), which incorporates both body length and weight through standardized major-axis regression, have been proposed as a more robust metric of somatic condition [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. These non-lethal approaches are widely used in ecological, fisheries, and growth studies [22,23].

The Common carp, Cyprinus carpio Linnaeus, 1758, represents an ideal model species for investigating interactions among environmental stressors, parasite communities, and host physiological condition. Widely distributed in the Danube River Basin, this species inhabits ecosystems increasingly affected by habitat fragmentation, pollution, and hydrological alterations. Juvenile individuals are particularly susceptible to parasitic infections and opportunistic bacterial colonization due to their developing immune systems and high exposure to environmental variability. The Lower Danube River represents a dynamic ecosystem characterized by fluctuating hydrological regimes, nutrient enrichment, and complex pollutant inputs, all of which may influence host–parasite–microbiota interactions [24].

Although numerous studies have examined parasitic infections or bacterial communities in freshwater fish, relatively few have integrated these biological components with morphometric condition indices in wild populations of the common carp Cyprinus carpio Linnaeus, 1758. This knowledge gap is increasingly relevant in the context of climate-driven shifts in parasite distribution, rising antimicrobial resistance, and increasing anthropogenic pressure on freshwater ecosystems. Additionally, host-specific factors such as age, developmental stage, and environmental exposure may influence infection dynamics and complicate the interpretation of condition indices, highlighting the need for multifactorial approaches [25,26].

The present study aims to determine whether commonly used condition indices reflect parasite load and bacterial colonization in juvenile Cyprinus carpio Linnaeus, 1758 from the Lower Danube River under natural environmental conditions. Specifically, the study addresses two key questions: (1) the extent to which condition indices reflect parasitic and bacterial infections in wild carp populations, and (2) how host-specific and environmental factors influence these relationships.

2. Materials and Methods

2.1. Study Site

The study was conducted in the Lower Danube River basin in southeastern Romania, focusing on the Borcea Branch and adjacent floodplain sectors within Ialomiţa and Călărași counties (44°20′ N, 28°01′ E). This section of the Danube is characterized by a complex hydrological network comprising river channels, floodplain lakes, and tributary systems that support diverse freshwater fish communities and play a significant role in shaping parasite transmission dynamics. To account for spatial variability in host–parasite interactions, fish were collected from multiple locations along the Lower Danube floodplain, including areas near Fetești, Borcea, and Călărași. These sampling sites represent distinct hydrological sectors of the river system and encompass both main-channel habitats and floodplain-connected water bodies, which differ in terms of hydrological connectivity, water residence time, and ecological conditions. The geographic distribution of the sampling sites within the Lower Danube basin is presented in Figure 1.

2.2. Environmental Parameters

Water quality parameters were measured in situ at each sampling location using a multiparameter meter probe (HACH Company, Loveland, CO, USA). The recorded variables included water temperature (°C), dissolved oxygen (mg L⁻¹), and pH. To minimize diurnal variation, all measurements were conducted at a standardized time (14:00 h) during each monthly sampling event throughout the study period (April 2022–January 2024). This approach ensured comparability among sampling dates and reduced potential bias associated with daily fluctuations in physicochemical conditions. A summary of these data is provided in Supplementary Table S1.

2.3. Fish Sample Collection and Age Determination

Wild specimens were collected between April 2022 and January 2024, encompassing multiple seasonal periods to account for variability in parasite transmission and host physiological condition. Fish were obtained in collaboration with local commercial fishermen using traditional fishing gear, including gill nets and trap nets commonly employed in the region. Immediately after capture, specimens were placed in insulated containers and transported to the laboratory for further analysis. A total of 260 individuals of Common carp Cyprinus carpio were included in the parasitological examination. Upon arrival, each specimen was rinsed with clean water to remove surface debris prior to analysis. Biometric measurements were then recorded for each individual. Total length (TL, cm) was measured using a calibrated ichthyometer (measured using a calibrated ichthyometer (fish measuring board; Aquatic BioTech, Seattle, WA, USA), and body weight (BW, g) was determined using an electronic analytical scale (Ohaus Corporation, Parsippany, NJ, USA) with a precision of ±0.01 g.

To evaluate the relationship between host size and parasite occurrence, the examined specimens (n = 260) were classified into four body-size groups based on total length and body weight ranges: 5.1 ± 8.0 cm (10 ± 15 g), 8.1 ± 10.0 cm (15.1 ± 20.0 g), 10.1 ± 12.0 cm (20.1 ± 25.0 g), and 12.1 ± 15.0 cm (25.1 ± 30.0 g). Each group included 65 individuals and represented increasing size classes within the sampled population. These measurements were subsequently used to calculate condition indices based on mass–length relationships, including Fulton’s condition factor (K), Le Cren’s relative condition factor (Kn), and the scaled mass index (SMI). Fish age was estimated using scale annuli analysis, and all specimens were classified as juvenile individuals (approximately 8 months old) [27]. Following biometric assessment, each specimen was subjected to a comprehensive parasitological examination, including detailed inspection of external tissues (skin, fins, and gills) and internal organs for parasite detection.

2.4. Parasite Isolation and Identification

Parasites were isolated and identified following standard parasitological protocols for freshwater fish to ensure reliable detection of both external and internal taxa. Each specimen was systematically examined for parasites belonging to major groups, including Monogenea, Ciliophora, Dinophyceae, Oligohymenophorea, Myxosporea, Trematoda, Nematoda, and Cestoda. All procedures were conducted under laboratory conditions using sterilized instruments, and samples were processed immediately after dissection [28,29]. Ectoparasites were collected from the skin, fins, and gills by gentle scraping and examined as fresh wet mounts in physiological saline (0.9% NaCl) under a stereomicroscope and light microscopy (100–400×). When required, smears were fixed and stained with Giemsa or iron hematoxylin to enhance visualization. Monogeneans were fixed in 10% neutral buffered formalin, mounted in ammonium picrate glycerin (APG), and identified based on haptoral structures [7,8]. Protozoan taxa (e.g., Trichodina, Ichthyophthirius, Apiosoma, Epistylis, Tetrahymena) and dinoflagellates (Piscinoodinium sp.) were identified using morphological criteria, with silver impregnation applied when necessary to visualize [9,10,11].

Internal parasites were recovered by dissecting the abdominal cavity and digestive tract. Trematodes were identified from compressed tissues and stained preparations [14], nematodes were cleared in lactophenol for morphological observation [26,27,28,29,30], and cestodes were isolated from the intestine and identified based on scolex and proglottid morphology [16]. Parasite identification was performed using standard taxonomic keys and morphological criteria. Parasitological indices were calculated according to [31]. Prevalence (P) was defined as the percentage of infected hosts relative to the total number of examined individuals. Mean intensity (MI) was calculated as the average number of parasites per infected host, while mean abundance (MA) was defined as the average number of parasites per examined host, including both infected and uninfected individuals. Ninety-five percent confidence intervals (95% CI) were estimated using bootstrap resampling (10,000 iterations). Data are shown in

Table 1. Epidemiological parameters of parasite species recorded in juvenile common carp Cyprinus carpio Linnaeus, 1758 from the Lower Danube River.

Parasite Species	Prevalence (%)	Mean Intensity ± 95% CI	Mean Abundance ± 95% CI	Total Parasites
Dactylogyrus vastator Nybelin, 1924	72	3 (2–5)	2 (1–3)	44
Trichodina cottidarum Dogiel, 1940	70	4 (3–6)	3 (2–4)	43
Gyrodactylus elegans von Nordmann, 1832	66	3 (2–4)	2 (1–3)	33
Myxobolus sp. Bütschli, 1882	65	3 (2–4)	2 (1–3)	33
Trichodina nigra Lom, 1960	64	3 (2–5)	2 (1–3)	33
Piscinoodinium sp. Lom, 1981	63	3 (2–4)	2 (1–3)	32
Apiosoma piscicola Blanchard, 1885	60	3 (2–4)	2 (1–3)	29
Posthodiplostomum cuticola Nordmann, 1832	58	2 (1–3)	1 (0–2)	28
Atractolytocestus huronensis Anthony, 1958	57	3 (2–4)	2 (1–3)	27
Epistylis paradoxa Ehrenberg, 1830	56	3 (2–4)	2 (1–3)	27
Dactylogyrus ersinensis Ergens, 1960	55	3 (2–4)	2 (1–3)	24
Salsuginus sp. Yamaguti, 1968	54	3 (2–4)	2 (1–3)	24
Tylodelphys clavata Nordmann, 1832	53	2 (1–3)	1 (0–2)	23
Dactylogyrus extensus Mueller & Van Cleave, 1932	52	3 (2–5)	2 (1–3)	22
Ichthyophthirius multifiliis Ehrenberg, 1830	52	4 (3–7)	2 (1–4)	22
Tetrahymena sp. Müller, 1773	51	3 (2–4)	2 (1–3)	22
Gyrodactylus bullatarudis Turnbull, 1956	50	3 (2–4)	2 (1–3)	22
Capillaria aborensis Joko (2017)	49	2 (1–3)	1 (0–2)	22
Gyrodactylus cyprini Ergens, 1961	48	3 (2–4)	2 (1–3)	21
Clinostomum complanatum Rudolphi, 1814	47	2 (1–3)	1 (0–2)	21
Contracaecum sp. Railliet & Henry, 1912	46	2 (1–3)	1 (0–2)	20
Opisthorchis felineus Rivolta, 1884	45	2 (1–3)	1 (0–2)	20
Capillaria sp. Zeder, 1800	43	2 (1–3)	1 (0–2)	17

Note: Prevalence (%) = (number of infected hosts/number of examined hosts) × 100. Mean intensity = total number of parasites/number of infected hosts. Mean abundance = total number of parasites/number of examined hosts. Ninety-five percent confidence intervals (95% CI) were estimated using bootstrap resampling (10,000 iterations).

.

2.5. Microbiological Analysis and Bacterial Identification

From the examined specimens (±n = 260), a representative subset of 20 individuals juvenile Cyprinus carpio Linnaeus, 1758 specimens (total length 8 ± 12 cm) were selected for bacteriological analysis. Sampling was performed periodically throughout the study period (April 2022–January 2024) to account for temporal variation in surface-associated bacterial communities. Specimens were processed immediately after collection.

Prior to sampling, each fish was rinsed in three successive baths of sterile distilled water to remove loosely attached debris and transient microorganisms. Cutaneous microbiota were collected by swabbing a standardized 3 cm² area of the ventral surface using a sterile flocked swab. Each swab was transferred into 1 mL of liquid Amies transport medium (Copan ESwab™, Copan Diagnostics, Brescia, Italy) under aseptic conditions [32].

For bacterial isolation, 100 µL of the transport medium was inoculated onto agar plates using a sterile Drigalski spatula and evenly distributed across the surface. The following culture media were used: MacConkey sorbitol agar, DCL agar, Yersinia agar, and 5% sheep blood agar (MLT, Arad, Romania). Plates were incubated at 27 °C for 24 h, after which colony-forming units (CFUs) were enumerated using a digital colony counter. Representative colonies were subsequently subcultured on chromogenic media for preliminary differentiation. The incubation temperature (27 °C) was selected to reflect the optimal growth range of freshwater fish-associated bacteria, which are typically inhibited at temperatures above 30 °C [32]. Elevated temperatures may also induce stress-related responses, such as heat-shock protein expression (e.g., HSP70) in fish tissues [33].

Final taxonomic identification was performed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS; Microflex LT/SH, Bruker Daltonik GmbH, Bremen, Germany). Isolated colonies were applied to an MSP 96 polished steel target plate (Bruker Daltonics GmbH, Bremen, Germany), followed by the addition of an α-cyano-4-hydroxycinnamic acid matrix prepared in a solution containing acetonitrile and trifluoroacetic acid. Spectral profiles were analyzed using MBT Compass software (version 4.1, Bruker Daltonik GmbH, Bremen, Germany) and compared with reference spectra from the SR Library Database to determine taxonomic identity. MALDI-TOF MS enables rapid and accurate identification of microorganisms based on their proteomic profiles and is widely used in clinical and research microbiology [34].

2.6. Statistical Analysis

Parasite count data, including total fish length (L), body weight (W), parasite abundance (P), and cumulative parasite load (PTOT), were tested for normality using the Shapiro–Wilk and Anderson–Darling tests implemented in XLSTAT for Microsoft Excel. Fish physiological condition was assessed using Fulton’s condition factor (K), Le Cren’s relative condition factor (Kn), and the Scaled Mass Index (SMI). Fulton’s condition factor was calculated as: K = 100 × (W/L³). Le Cren’s relative condition factor was calculated as: Kn = W/(aL^b). The Scaled Mass Index (SMI) was calculated following Peig and Green [35], as: SMI = Wi × (L₀/Li)^bSMA, where Wi is individual body mass, Li is individual length, L₀ is the mean population length, and bSMA is the slope obtained from the standardized major-axis regression of ln(W) against ln(L).

Associations between condition indices (K, Kn, and SMI) and parasite load were evaluated using correlation analyses. Pearson’s correlation coefficient (r) was applied for normally distributed data, whereas Spearman’s rank correlation was used for non-normally distributed variables. Descriptive statistics, including mean, standard deviation (SD), coefficient of variation (CV), and standard error (SE), were calculated as follows: CV = SD/mean; SE = SD/√N. Parasite weight was not measured; all parasitological analyses were based on numerical descriptors, including parasite counts, abundance, and total parasite load per host. Statistical significance was set at p < 0.05. All analyses were performed using SPSS version 26 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 3.

3. Results

3.1. Prevalence of Infection Across Size Groups

Fish were classified into four size groups based on body weight and total length, with 65 individuals assigned to each group (

Table 2. Prevalence of parasitic infection across body-size classes of the common carp Cyprinus carpio Linnaeus, 1758 during the study period.

No.	(BW, g)	(TL cm)	Fish Examined (n)	Fish Infected (n)	Prevalence (%)
1	10 ± 15	5.1 ± 8.0	65	38	58.46
2	15.1 ± 20.0	8.1 ± 10.0	65	43	66.15
3	20.1 ± 25.0	10.1 ± 12.0	65	48	73.85
4	25.1 ± 30.0	12.1 ± 15.0	65	64	98.46

Prevalence (%) = (number of infected fish/number of examined fish) × 100. BW = body weight; TL = total length; No. = body size class.

). Parasite prevalence varied among size classes and showed a consistent increasing trend with host size. The lowest prevalence was observed in the smallest size group, whereas progressively higher values were recorded in intermediate and larger groups. The highest prevalence occurred in the largest size class, where nearly all individuals were infected. These results indicate a clear size-related pattern in parasite occurrence across the examined population, with larger individuals exhibiting a higher frequency of infection compared to smaller ones.

3.2. Composition and Epidemiological Structure of the Parasite Community

Twenty three parasite taxa were recorded across multiple infection sites, including the skin, fins, gills, muscles, and intestine, providing a comprehensive overview of parasite diversity in juvenile Cyprinus carpio Linnaeus, 1758 from the Lower Danube River, including both ectoparasitic and endoparasitic species belonging to multiple different taxonomic groups, such as Monogenea, Ciliophora, Myxosporea, Trematoda, Nematoda, and Cestoda (

Table 3. Epidemiological and quantitative parameters of parasite species recorded in juvenile Cyprinus carpio Linnaeus, 1758 from the Lower Danube River.

Parasite Species	Prevalence (%)	Mean Intensity (±95% CI)	Mean Abundance (±95% CI)	Total Parasites	Relative Frequency (%)
Dactylogyrus vastator	72	3 (2–5)	2 (1–3)	44	7.23
Trichodina cottidarum	70	4 (3–6)	3 (2–4)	43	7.06
Gyrodactylus elegans	66	3 (2–4)	2 (1–3)	33	5.42
Myxobolus sp.	65	3 (2–4)	2 (1–3)	33	5.42
Trichodina nigra	64	3 (2–5)	2 (1–3)	33	5.42
Piscinoodinium sp.	63	3 (2–4)	2 (1–3)	32	5.25
Apiosoma piscicola	60	3 (2–4)	2 (1–3)	29	4.76
Posthodiplostomum cuticula	58	2 (1–3)	1 (0–2)	28	4.6
Atractolytocestus huronensis	57	3 (2–4)	2 (1–3)	27	4.43
Epistylis paradoxa	56	3 (2–4)	2 (1–3)	27	4.43
Dactylogyrus ersinensis	55	3 (2–4)	2 (1–3)	24	3.94
Salsuginus sp.	54	3 (2–4)	2 (1–3)	24	3.94
Tylodelphys clavate	53	2 (1–3)	1 (0–2)	23	3.78
Dactylogyrus extensus	52	3 (2–5)	2 (1–3)	22	3.61
Ichthyophthirius multifiliis	52	4 (3–7)	2 (1–4)	22	3.61
Tetrahymena sp.	51	3 (2–4)	2 (1–3)	22	3.61
Gyrodactylus bullatarudis	50	3 (2–4)	2 (1–3)	22	3.61
Capillaria aborensis	49	2 (1–3)	1 (0–2)	22	3.61
Gyrodactylus cyprinid	48	3 (2–4)	2 (1–3)	21	3.45
Clinostomum complanatum	47	2 (1–3)	1 (0–2)	21	3.45
Contracaecum spp.	46	2 (1–3)	1 (0–2)	20	3.28
Opisthorchis felineus	45	2 (1–3)	1 (0–2)	20	3.28
Capillaria spp.	43	2 (1–3)	1 (0–2)	17	2.79

). Ectoparasites were primarily associated with the gills, skin, and fins, whereas endoparasites were predominantly recovered from internal organs and the intestinal tract.

The parasite community was dominated by monogeneans and protozoans, particularly representatives of the genera Dactylogyrus, Gyrodactylus, and Trichodina. Among these, Dactylogyrus vastator and Trichodina cottidarum exhibited the highest prevalence and abundance values.

Epidemiological parameters indicated a moderate to high level of infection across most taxa, with prevalence values ranging from 43% to 72%. Mean intensity values were generally low to moderate, while mean abundance values remained relatively low across the parasite species.

Relative frequency analysis showed that parasite contributions were distributed across taxa, with no single species dominating the community. The highest contributions were observed for Dactylogyrus vastator and Trichodina cottidarum, while most species exhibited lower proportional contributions.

3.3. Descriptive Statistics and Parasite Distribution

Descriptive statistics for biometric parameters, condition indices, and total parasite abundance are presented in

Table 4. Descriptive statistics for biometric traits, condition indices, and parasite abundance in juvenile C. carpio.

Parameter	Min	Mean	Max	SD	CV	SE
Weight (g)	10.00	20.28	30.00	5.81	0.29	0.84
Length (cm)	5.10	10.15	15.00	2.53	0.25	0.37
Scaled Mass Index (SMI)	18.12	20.19	22.84	1.09	0.05	0.16
Fulton’s K	0.01	0.02	0.08	0.01	0.56	0.00
Body Mass Index (BMI)	0.13	0.21	0.38	0.05	0.24	0.01
Le Cren’s Kn	0.89	1.00	1.14	0.05	0.05	0.01
Total parasite abundance	0.00	13.31	26.00	9.58	0.72	1.38

, and Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. The common carp Cyprinus carpio Linnaeus, 1758 mean abundance values per parasite species were generally low, indicating that individual parasite burdens remained limited despite the presence of multiple taxa. This distribution pattern is characteristic of natural parasite communities, where aggregation typically occurs in a subset of hosts. These results indicate a moderately dispersed parasite distribution with low individual burdens, supporting the interpretation of a stable host–parasite system under natural environmental conditions, without evidence of severe infection pressure [34,35].

3.4. Relationship Between Parasite Load and Condition Indices Correlation Analysis (Pearson)

The relationships between parasite burden and host physiological condition were evaluated using Pearson’s correlation analysis (

Table 5. Pearson correlation coefficients (r) between parasite load and host condition indices in juvenile C. carpio.

Variable	Fulton’s K	Le Cren’s Kn	SMI
Total parasite load	−0.12	−0.08	−0.05
Parasite abundance	−0.10	−0.06	−0.04

Note: Correlation coefficients (r) were calculated using Pearson’s method. No statistically significant correlations were detected between parasite load and condition indices (p > 0.05).

). The results indicated that most correlations between parasite abundance and condition indices—including Fulton’s condition factor (K), Le Cren’s relative condition factor (Kn), and the Scaled Mass Index (SMI)—were weak and not statistically significant (p > 0.05).

The lack of strong associations between parasite load and condition indices suggests that the examined parasite community exerts a relatively low physiological cost on the host population. This pattern is consistent with stable host–parasite systems in natural environments, where long-term coevolution promotes reduced virulence and host tolerance.

These findings support the hypothesis that commonly used morphometric condition indices may have limited sensitivity in detecting subclinical parasitic infections in wild fish populations, particularly under conditions of moderate infection intensity [36].

3.5. Structure and Relative Contribution of the Parasite Community

The graphical representation of parasite contributions revealed a gradual decline in relative abundance across species, with no single taxon exhibiting overwhelming dominance (Figure 7). Dactylogyrus vastator and Trichodina cottidarum showed the highest contributions, each approaching 7% of the total parasite load, followed by several taxa with intermediate contributions ranging between approximately 4% and just over 5%. The majority of parasite species contributed less than 4%, indicating a relatively even distribution of parasite abundance across the community. This pattern is characteristic of a structured and, balanced parasite assemblage, where multiple taxa coexist without strong competitive exclusion or dominance. The observed distribution supports the presence of a stable and diverse parasite community in juvenile C. carpio, consistent with equilibrium conditions in natural freshwater ecosystems.

3.6. Correlation Between Biometric Parameters, Condition Indices, and Parasite Load

Pearson correlation analyses (α = 0.05) were performed to evaluate the relationships between parasite abundance and host biometric parameters (body weight and total length), as well as condition indices (

Table 6. Pearson correlation between parasite abundance and host biometric parameters in juvenile Cyprinus carpio Linnaeus, 1758 from the Lower Danube River.

Parasite Species	Body Weight (BW) (r/p-Value)	Total Length (TL) (r/p-Value)
Dactylogyrus extensus	0.063/0.297	0.055/0.360
Dactylogyrus vastator	0.020/0.747	0.033/0.542
Dactylogyrus ersinensis	0.105/0.090	0.100/0.101
Gyrodactylus elegans	0.167/0.007 *	0.184/0.003 *
Gyrodactylus cyprinid	0.077/0.215	0.063/0.319
Salsuginus sp.	0.032/0.561	0.033/0.548
Trichodina cottidarum	0.063/0.291	0.078/0.229
Trichodina nigra	0.063/0.316	0.071/0.268
Ichthyophthirius multifiliis	0.100/0.115	0.100/0.100
Apiosoma piscicola	0.033/0.546	0.033/0.581
Myxobolus sp.	0.127/0.044 *	0.141/0.024 *
Piscinoodinium sp.	0.033/0.540	0.025/0.615
Epistylis paradoxa	0.012/0.858	0.009/0.930
Tetrahymena sp.	0.033/0.557	0.045/0.451
Posthodiplostomum cuticula	0.089/0.141	0.089/0.162
Clinostomum complanatum	0.071/0.280	0.033/0.573
Tylodelphys clavate	0.033/0.633	0.045/0.517
Opisthorchis felineus	0.033/0.670	0.012/0.833
Capillaria aborensis	0.055/0.376	0.055/0.391
Capillaria spp.	0.055/0.353	0.063/0.316
Contracaecum spp.	0.110/0.076	0.122/0.051
Gyrodactylus bullatarudis	0.055/0.403	0.045/0.422
Atractolytocestus huronensis	0.228/<0.001 *	0.228/<0.001 *

Pearson correlation coefficients (r) and corresponding p-values are presented. Statistically significant correlations are indicated in bold (* p < 0.05). Correlation strength was interpreted as weak (|r| < 0.30), moderate (0.30–0.50), and strong (>0.50). Only biometric parameters are shown, as condition indices did not exhibit significant or reliable correlations with parasite abundance.

). Most parasite species exhibited weak and non-significant correlations with both biometric variables and condition indices. Only a limited number of statistically significant relationships were detected between parasite abundance and host size parameters. Specifically, Gyrodactylus elegans and Myxobolus sp. showed weak but statistically significant positive correlations with both body weight and total length (p < 0.05), although the strength of these relationships remained low (|r| < 0.20). Similarly, Atractolytocestus huronensis exhibited a statistically significant association with host size (p < 0.001), but with low explanatory power, indicating only a weak relationship.

In contrast, no significant correlations were observed between parasite abundance and condition indices, including the Scaled Mass Index (SMI), Le Cren’s relative condition factor (Kn), and Fulton’s condition factor (K). In several cases, correlation analyses could not be reliably performed due to limited variability in these indices.

These findings indicate that parasite burden had minimal influence on host physiological condition. The predominance of weak and non-significant correlations suggests that parasite load is only weakly associated with host biometric traits and does not substantially affect somatic condition. This pattern is consistent with a stable host–parasite system in which infections remain largely subclinical and do not impair host growth or condition under natural environmental conditions.

3.7. Surface-Associated Bacterial Communities in Juvenile C. carpio

Six bacterial taxa were isolated from the skin of juvenile common carp Cyprinus carpio Linnaeus, 1758, based on bacteriological analyses conducted on representative subsamples collected periodically throughout the study period (April 2022–January 2024). The identified taxa included Aeromonas hydrophila, Aeromonas veronii, Acinetobacter johnsonii, Bacillus cereus, Flavobacterium columnare, and Pantoea agglomerans. Among these, A. hydrophila was the most frequently detected species across sampling events, followed by A. veronii, A. johnsonii, and B. cereus, indicating their dominant role within the cutaneous microbiota. In contrast, F. columnare and P. agglomerans were less frequently isolated. The distribution of identified bacterial taxa varied among culture media. Nutrient agar and MacConkey sorbitol agar consistently supported the highest diversity and frequency of bacterial growth, whereas DCL agar and chromogenic agar yielded fewer isolates. Yersinia agar and 5% blood agar showed intermediate recovery rates, particularly for Aeromonas spp. The frequency of occurrence of each bacterial taxon is illustrated in Figure 8, confirming the predominance of A. hydrophila, while F. columnare and P. agglomerans exhibited comparatively lower frequencies. Similarly, Figure 9 demonstrates that nutrient agar and MacConkey sorbitol agar produced the highest number of bacterial isolates, highlighting their effectiveness for recovering diverse fish-associated microbiota. The bacterial assemblage was dominated by opportunistic freshwater-associated taxa and remained relatively consistent across sampling periods, indicating a stable environmental microbiota rather than a disease-driven community structure.

3.8. Condition Indices of Juvenile C. carpio

Condition indices for bacteriologically analyzed specimens (n = 20, including one control) are summarized in

Table 7. Condition indices of control and bacterially colonized juvenile common carp Cyprinus carpio Linnaeus, 1758 (n = 20, including control).

Fish No.	Length (cm)	Weight (g)	Fulton’s K	Le Cren’s Kn	SMI (g)	Infection Status CFU/mL
Control	8.1	15.4	2.87	0.979	15.65	Uninfected (Control)
Specimen 1	8.3	15.6	2.72	0.974	15.74	Bacterially colonized
Specimen 2	9.5	16.4	1.91	0.968	16.39	Bacterially colonized
Specimen 3	10.1	17.6	1.71	0.965	17.40	Bacterially colonized
Specimen 4	10.7	15.2	1.63	0.974	18.32	Bacterially colonized
Specimen 5	10.0	15.2	2.46	0.962	16.44	Bacterially colonized
Specimen 6	8.1	18.5	1.85	0.963	17.93	Bacterially colonized
Specimen 7	11.7	18.3	2.75	0.979	18.07	Bacterially colonized
Specimen 8	9.3	18.6	1.77	0.973	16.73	Bacterially colonized
Specimen 9	11.2	19.0	2.24	0.972	16.37	Bacterially colonized
Specimen 10	9.8	18.9	2.35	0.961	16.56	Bacterially colonized
Specimen 11	11.3	19.1	2.12	0.960	17.42	Bacterially colonized
Specimen 12	11.2	16.9	2.29	0.964	18.08	Bacterially colonized
Specimen 13	9.1	17.7	1.98	0.964	16.79	Bacterially colonized
Specimen 14	10.5	15.5	2.67	0.978	17.38	Bacterially colonized
Specimen 15	9.5	15.7	2.10	0.977	16.46	Bacterially colonized
Specimen 16	9.1	18.5	2.70	0.972	18.25	Bacterially colonized
Specimen 17	11.6	15.0	2.64	0.962	15.82	Bacterially colonized
Specimen 18	10.9	18.8	2.16	0.973	16.88	Bacterially colonized
Specimen 19	12.0	19.7	1.96	0.968	18.47	Bacterially colonized

Note: Fulton’s K = 100 × (W/L³); Le Cren’s Kn = W/aL^b; SMI = Wi × (L₀/Li)^bSMA. Indices were calculated following Cren, E. D. Le 1951 [19]. Higher values of K and SMI generally indicate better somatic condition. CFU/mL (Colony Forming Units per milliliter).

. The uncolonized control specimen exhibited slightly higher values of Fulton’s condition factor (K), Le Cren’s relative condition index (Kn), and the scaled mass index (SMI) compared with bacterially colonized individuals. Among colonized specimens, Fulton’s K showed a general decreasing trend, suggesting a modest reduction in somatic condition associated with bacterial colonization. In contrast, Kn and SMI values remained relatively stable across individuals, indicating that bacterial presence did not substantially affect overall physiological condition. Despite the occurrence of multiple bacterial taxa, variations in condition indices were limited and did not indicate severe physiological impairment. These findings are consistent with the predominance of opportunistic and commensal bacterial species and support the interpretation of a balanced host–microbiota relationship under natural environmental conditions.

4. Discussion

The present study provides a comprehensive evaluation of parasite diversity, bacterial colonization, and host condition in juvenile Cyprinus carpio from the Lower Danube River, integrating parasitological, microbiological, and biometric approaches. The expanded dataset (±n = 260 fish; 23 parasite taxa; ±n = 20 for bacteriology) offers a robust framework for understanding host–parasite–microbiota interactions under natural environmental conditions.

A clear size-related pattern in parasite infection was observed, with prevalence increasing progressively across weight and length classes. Larger individuals exhibited higher infection rates, supporting the concept of cumulative exposure, whereby prolonged contact with infective stages and increased body surface area enhance parasite acquisition. Similar size-dependent patterns have been widely reported in cyprinid fishes and are generally associated with ontogenetic changes in habitat use, feeding behavior, and immune function.

The parasite community exhibited relatively high taxonomic diversity, encompassing representatives of Monogenea, Ciliophora, Myxosporea, Trematoda, Nematoda, and Cestoda. Ectoparasitic taxa, particularly monogeneans (Dactylogyrus, Gyrodactylus) and ciliates (Trichodina, Ichthyophthirius), dominated both prevalence and abundance, likely reflecting their direct life cycles and continuous environmental transmission. In contrast, endoparasites displayed lower intensity values, suggesting predominantly chronic infections linked to trophic transmission pathways. The predominance of parasites on gills and skin further highlights the importance of direct exposure to waterborne infective stages.

Despite the high prevalence of several parasite taxa, infection intensity remained generally low to moderate, indicating sublethal infection levels. This pattern suggests that parasite burdens were relatively controlled at the individual level, even under conditions of widespread exposure. Such findings are consistent with stable host–parasite equilibria commonly observed in natural freshwater ecosystems, where long-term coevolution promotes reduced virulence and increased host tolerance.

The examined population displayed moderate variability in biometric parameters, reflecting natural growth patterns in juvenile fish. In contrast, condition indices such as the Scaled Mass Index and Le Cren’s relative condition factor showed relatively low variability, indicating a generally homogeneous physiological state across individuals. The observed distribution of parasite abundance was heterogeneous, with most individuals harboring low parasite loads and fewer hosts exhibiting higher infection levels, a pattern typical of aggregated parasite distributions in wild fish populations.

Correlation analyses revealed weak associations between parasite abundance and condition indices, indicating that parasite infections had limited measurable effects on host somatic condition under the studied environmental conditions. However, moderate relationships between parasite load and biometric traits, particularly body weight and total length, suggest that host size remains an important determinant of parasite burden. These findings support the hypothesis that commonly used condition indices may have limited sensitivity in detecting subclinical or moderate parasite infections in natural populations.

The microbiological analysis revealed a relatively similar cutaneous bacterial community dominated by opportunistic freshwater-associated taxa, including Aeromonas hydrophila, A. veronii, Acinetobacter johnsonii, Bacillus cereus, Flavobacterium columnare, and Pantoea agglomerans. The predominance of Aeromonas species is consistent with their well-documented role as common opportunistic pathogens in freshwater environments. The presence of F. columnare further suggests for the of potential pathogenic interactions under environmentally stressful conditions.

The Co-occurrence of ectoparasites and opportunistic bacteria indicates a functional interaction within the host microbiota. Damage to epithelial tissues caused by ectoparasites likely facilitates bacterial colonization, increasing susceptibility to secondary infections. However, the relatively stable condition indices observed across bacterially colonized individuals suggest that these interactions remained within a subclinical range during the study period, supporting the presence of a balanced host–microbiota relationship.

Environmental factors likely played a key role in shaping the observed patterns. Seasonal variations in water temperature and dissolved oxygen suggest that warmer periods may enhance parasite transmission and bacterial proliferation, while cooler, oxygen-rich conditions may limit infection dynamics. These environmental drivers likely contributed to the observed distribution of parasites and bacteria, emphasizing the importance of abiotic conditions in regulating host–parasite–microbiota interactions.

Several limitations should be acknowledged. Although the parasitological dataset was extensive, microbiological analyses were conducted on a subset of specimens, which may not fully capture temporal variability in bacterial communities. Additionally, reliance on culture-based identification methods will underestimate microbial diversity compared with molecular approaches. Environmental parameters were measured periodically rather than continuously, limiting the ability to directly link abiotic factors to infection dynamics.

The findings demonstrate that high parasite diversity and frequent bacterial colonization do not necessarily result in reduced host condition, highlighting the resilience of wild fish populations and the importance of integrating multiple biological indicators when assessing fish health.

5. Conclusions

The common carp Cyprinus carpio Linnaeus, 1758. This study provides an integrated assessment of parasite diversity, surface-associated bacterial communities, and host condition in juvenile Cyprinus carpio from the Lower Danube River under natural environmental conditions. The results demonstrate that parasite prevalence increases with host size, supporting size-dependent infection dynamics driven by cumulative exposure.

Despite the high diversity and widespread occurrence of parasites, infection intensities remained low to moderate, indicating predominantly sublethal infections and a stable host–parasite equilibrium. Similarly, the cutaneous bacterial community was dominated by opportunistic freshwater-associated taxa, reflecting a typical environmental microbiota rather than active disease conditions.

Importantly, parasite load and bacterial colonization showed weak and inconsistent relationships with condition indices, suggesting that commonly used morphometric indices have limited sensitivity for detecting subclinical biological stress in wild fish populations.

These findings highlight the resilience of juvenile Cyprinus carpio and emphasize the need to integrate parasitological, microbiological, and environmental indicators for a more comprehensive evaluation of fish health. Future studies incorporating temporal monitoring and molecular approaches are recommended to further elucidate host–parasite–microbiota interactions in freshwater ecosystems.