Invasion Risk and Potential Impact of Alien Freshwater Fishes on Native Counterparts in Klang Valley, Malaysia

Simple Summary The mechanisms on how alien species naturally affect the native species in the real aquatic environment are infrequently studied. This study explores the potential effects of alien fishes on the native fish community, well-being, and trophic preferences in selected rivers of Klang Valley, Malaysia. We found that alien fishes benefited from the impacts of anthropogenic activities in their surrounding habitats, while their plasticity in feeding habits might help them to invade, survive, and dominate. This study revealed the natural mechanisms on the establishment of alien fish species and their potential ecological impacts on native fishes in the rivers of Klang Valley, Malaysia. Abstract This study explores the potential effects of alien fishes on the native fish community, well-being, and tropic preferences in selected rivers of Klang Valley, Malaysia. Following the Aquatic Species Invasiveness Screening Kit assessment, most of the alien fishes (80%) are invasive. The alien species occurrences correlated positively (p < 0.05) with poor water quality, such as rivers with high ammonia-nitrogen and nitrite, but negatively with phosphate and dissolved oxygen. Anthropogenic characteristics, such as rivers with high pollution levels and ease of accessibility to the fish habitat, are mainly associated positively (p < 0.05) with the occurrences of alien fish species. In general, the results of fish stomach contents analyses and their associated indices, together with stable carbon and nitrogen isotopes, revealed domination by alien fishes or diet overlaps between both alien and native fish species. This finding indicates that alien fishes benefited from the impacts of the anthropogenic activities in their surrounding habitats, while their plasticity in feeding habits might help them to invade, survive, and dominate in the rivers of Klang Valley, Malaysia.


Introduction
Alien fishes threaten native biodiversity and health through food and space competition, predation, hybridization, habitat and trophic modifications, and the introduction of diseases [1,2]. They are usually imported for beneficial purposes, including the need

Fish Sampling
A cast net of 150 cm long and 305 cm in diameter with a mesh size of 2 cm and scoop nets ranging from 2 mm to 3 mm mesh sizes were used for fish sampling. To ensure the uniformity of sampling efforts across sites, we used the same gear for a similar duration of four effort hours (10.00 a.m. to 2.00 p.m.) by four individuals. Along each 500 m stretch,

Fish Sampling
A cast net of 150 cm long and 305 cm in diameter with a mesh size of 2 cm and scoop nets ranging from 2 mm to 3 mm mesh sizes were used for fish sampling. To ensure the uniformity of sampling efforts across sites, we used the same gear for a similar duration of four effort hours (10.00 a.m. to 2.00 p.m.) by four individuals. Along each 500 m stretch, fish samples were collected at different points based on eye estimation for a similar duration and effort across the rivers. After collecting the fish samples, they were identified, enumerated, and distinguished as either native or alien by using a combination of keys from Kottelat et al. [35] and Zakaria-Ismail et al. [36]. The samples were also measured for standard length (SL) and total length (TL) to the nearest 0.1 mm, and body weight to the nearest 0.1 g of an individual fish specimen using a metal ruler and an electronic weighing scale (Camry, Guandong, China). The fishes used for further analysis were sedated with overdose tricaine methanesulphonate (MS-222 at 50 mg/L; Sigma-Aldrich, Kuala Lumpur, Malaysia) and later stored in ice packed styrofoam boxes for onward transportation to the laboratory, where they were immediately dissected, analyzed, or preserved.
The fish were sampled, handled, and sacrificed according to the methods approved by the Institutional Animal Care and Use Committee, Universiti Putra Malaysia. All procedures were carried out following relevant guidelines and regulations. No permit was required to conduct the present study, as none of the sampled fish species was considered endangered and protected by the government of Malaysia.
The anthropogenic characteristics of the sampling sites included; (1) accessibility (how easy people can enter and explore the waterbody); (2) level of protection (e.g., structure, such as gates, boundary); (3) pollution level (in the form of visible domestic and industrial waste); (4) usage for other purposes (e.g., domestic water source, recreation); and (5) distance from the human settlement, which were scored on a scale of 1 (very low susceptibility) to 5 (very high susceptibility) (Supplementary Material Table S2). Three different individuals carried out the assessment to reduce bias and increase accuracy at all the sampling points. After that, the average score from the three individuals was used to evaluate the relationship of fish occurrences with the scored anthropogenic factors.

Invasiveness Screening
Risk screening was carried out for the five main alien fishes recorded in this study: Oreochromis niloticus, P. pardalis, P. disjunctivus, P. reticulata, and Barbonymus gonionotus. The screening was achieved using the Aquatic Species Invasiveness Screening Kit (AS-ISK), a freely downloadable tool accessed on 17/05/2021 at www.cefas.co.uk/nns/tools/ [37]. This tool was an outcome of integrating the revised version of the generic screening module of the European Non-Native Species in Aquaculture Risk Analysis Scheme [38] into the Fish Invasiveness Screening Kit v2 [39].
Of the 55 questions in the AS-ISK, the first 49 questions, also referred to as basic risk assessment (BRA), relate to the taxon's biogeography/historical and biology/ecology aspects. The remaining six questions involve predicting the likely impacts of climatic conditions on the BRA, referred to as the Climate Change Assessment (CCA). Each question in AS-ISK requires the assessor to compulsorily respond, justify, and provide a level of confidence that ranges from 1 (very low) to 4 (very high). The resulting outcome gives a BRA and a BRA+CCA (composite) score ranging from −20.0 to 68.0 and from −32.0 to 80.0, respectively [40].
Similar to one of its parent tools, the FISK v2, AS-ISK scores below 1 indicate that the assessed species will likely not become invasive in the risk assessment (RA) area. Therefore, it is classified as 'low risk' [41,42]. On the other hand, scores higher than 1 indicate that the species poses either a 'medium risk' or a 'high risk' of invasion. Moreover, a calibration process is carried out specifically for the RA area to obtain the threshold value needed to differentiate between medium and high-risk species. An overall confidence factor (CF) was computed based on the assessor's level of confidence on each response for a given species as ∑ (CLQi)/(4 × 55) (i = 1, . . . , 55). CLQi is the certainty for question i, 4 is the Animals 2021, 11, 3152 5 of 22 maximum achievable certainty value (i.e. 'very certain'), and 55 is the total number of questions in the AS-ISK tool for each species. Hence, CLQi values range from 0.25 (for all 55 questions with a certainty score of 1) to 1 (with a certainty score of 4). Although multiple assessments are preferred, in this study, assessments were done only by the first author, who is knowledgeable of the biology and ecology of fish. The single evaluation was due to the difficulty of getting additional assessments. Moreover, risk screening studies based on a single assessor are not uncommon [40].
Calibration of the AS-ISK tool for Peninsular Malaysia was based on the Receiver Operating Characteristics (ROC) curve analysis using IBM SPSS, ver. 22.0 (IBM Corp., Chicago, IL, USA) [4]. The ROC evaluates the ability of AS-ISK to distinguish between invasive and non-invasive alien fishes assessed for the RA area, and the Area Under the Curve (AUC) values were recorded. After the ROC analysis, an AS-ISK value that maximizes and at the same time minimizes the false positive rate was selected for both BRA and BRA+CCA using Youden's J statistic [43,44].

Stomach Contents Analysis
The stomachs of randomly selected individuals of each fish species were removed and initially preserved in 10% formalin then 70% ethanol before examining the contents. Stomach contents examination was done under a stereomicroscope using a 0.2 mm deep, 16 by 16 Fuchs Rosenthal counting chamber. Where possible, a minimum of 30 individuals was used per fish species in each sampling site considered. The observation was done in triplicates after diluting the stomach contents with distilled water between 5 and 10 mL based on the size of the entire stomach contents. A pipette was used to place two drops of the diluted sample on the slide. Magnifications ranging from 4× to 40× were used to view the samples under a binocular Olympus CX 21 light microscope (Shinjuku, Tokyo, Japan) [17,51]. The fish stomach contents were identified using relevant identification keys [52][53][54].

Feeding Intensity, Stomach Fullness Index, Frequency of Occurrence, and Volumetric Measurement
The intensity of feeding as a measure of the degree of fullness of each fish stomach was observed and recorded as 0/4 full (empty), 1/4 full, 2/4 full, 3/4 full, and 4/4 full (full) [18]. Afterward, the feeding intensity (FI) was expressed as a percentage as follows: %FI = (the number of guts containing food/ total number of guts) × 100, where FI = feeding intensity [55].
For the stomach fullness index, prior to the examination under the microscope, the fish stomach was blotted to eliminate excess liquid. Further, food items from each stomach were emptied into a petri dish and weighed to the nearest 0.001 g using a Sartorius BP 221S digital scale (Sartorius, Gaithersburg, MD, USA) [56]. As a simple record of stomach contents data, the occurrence percentage of individual food items in the fish stomachs was assessed according to Hyslop et al. [57] as follows: %FO = (number of stomachs containing a prey item/ all non -empty stomachs) × 100, where FO = frequency of occurrence.
For the volumetric measurement, with the aid of the binocular Olympus CX 21 light microscope (Shinjuku, Tokyo, Japan), food items were identified and ranked depending on size and abundance by allotting points [18,55]. These points were summed and converted into corresponding volumes as follows: %V = (number of points allocated to the food component/ total points allocated to subsample) × 100.

Index of Preponderance, Diet Overlap, and Trophic Level
The index of preponderance was applied as follows: where I i = the index of preponderance, while v i and o i represent the volume and occurrence indices of food item i, respectively [58]. For diet overlap, it was assessed using the Morisita-Horn Index (C H ) in order to measure the potential for competition between alien and native fishes. The C H was determined using the equation as follows: C H = 2 ∑ pij × pik/ ∑ p 2 ij ± ∑ p 2 ik, where C H = diet overlap, pij = proportion of item i relative to the total resources used by species j, pik = proportion of i relative to all resources used by species k, and n = the total number of resource items [59].
Trophic levels (TROPH) of co-existing native and alien fishes in the rivers sampled were determined from the diet composition data. These were analyzed following the quantitative routine of TROPHLab software [60]. In this case, the contribution of each food item based on the relative volume derived from the points method was used. The equation was applied as follows: TLi = 1 ± ∑ j (TLj × DCij), where TLj = the fractional (i.e., non-integer) trophic level of the prey j [61] and DCij = the fraction of j in the diet of i.

Stable Isotopes Analysis
The preparation of samples for stable δ 15 N and δ 13 C analysis followed the method employed by Nakamura et al. [62] and Zulkifli et al. [63]. Muscle tissues from the dorsal parts of the fish were collected from selected representative fish sizes for each species in each of the three sites, such as the Gombak, Klang, and Langat Rivers. These were the sites from which sufficient numbers of native and alien fishes were obtained and initially subjected to stomach contents analysis. The muscle tissues were immediately stored at −20 • C until the final preparation. After that, the skin, bones, and scales were carefully separated from the muscle tissue and then washed with deionized water. The washed samples were then placed in Petri dishes before oven-drying at 60 • C for 24 to 48 h until constant weights were attained.
Excess lipids were removed from the dried and ground samples by treating them with a mixture of chloroform (analytical reagent grade; Fisher Chemical, Loughborough, UK) and methanol (AnaPur grade; Fisher Chemical, Hampton, NH, USA) (ratio 2:1) for 3 h. Following that, the samples were centrifuged at 2500 rpm for 10 min at a temperature of 4 • C with a high-speed refrigerated centrifuge (Sorvall, Ramsey, MN, USA). After discarding the supernatant, the resulting pellets were dried in a desiccator for at least 1 h. They were then fumed for 10 h with 12M HCl (analytical reagent (assay ≥ 37%); Sigma-Aldrich, St. Louis, MO, USA) to remove inorganic carbonates. Finally, excess acid was extracted using sodium hydroxide pellets for 3 h in a vacuum desiccator. The samples were dried for at least 1 h before sending for stable isotope analysis at the Department of Chemistry, Ministry of Science, Technology and Innovation, Petaling Jaya, Selangor, Malaysia.

Statistical Analyses
Microsoft Excel (Office 365, Version 2016, Microsoft Corp., Berkshire, UK) was used for descriptive statistics to reveal the overall occurrence of individual fish species, fish species by origin (native or alien), and the percentage occurrences of fish families from sampled locations. Measured water quality parameters were compared across sites with a one-way analysis of variances (ANOVA) with IBM SPSS, ver. 22.0 (IBM Corp., Chicago, IL, USA). Before analysis, data were log-transformed to normalize them since they did not satisfy the conditions for a parametric test. Graphical presentation of the result was performed using Microsoft Excel (Office 365, ver. 2016, Microsoft Corp., Berkshire, UK).
Principal Components Analysis (PCA) was used to extract the most crucial water quality parameters used to assess the relationships with fish occurrences using Canonical Correspondence Analysis (CCA). The IBM SPSS, ver. 22.0 (IBM Corp., Chicago, IL, USA) was used for this purpose. Further, the measured anthropogenic factors were related to fish occurrences using CCA. The analyses were done using the PAST (version 3.25) software.
Since the data were not normally distributed, non-parametric tests, such as Mann-Whitney U and Kruskal-Wallis H tests were used to compare stomach fullness indices, and δ 15 N and δ 13 C values of native and alien fishes. The closeness or overlap of the δ 15 N and δ 13 C values existing between the native and alien species was shown using the biplot of mean and standard deviations. Furthermore, to identify the trophic plasticity of the studied fishes, the δ 15 N and δ 13 C values of native and alien species, such as Mystacoleucus obtusirostris and O. niloticus, that occurred at least twice from the sampled rivers were compared. Except for TROPH, which was estimated using the TROPHLab software, all statistical analyses were done using Microsoft Excel 2016 (Office 365 ver. 2016) and IBM SPSS ver. 23 (IBM Corp., Chicago, IL, USA).

Fish Checklist and Community Structures
A total of 20 fish species were recorded, out of which six are aliens (Table 1). Of all the sampled fishes, O. niloticus, Poecilia reticulata, and Mystacoleucus obtusirostris were the most occurring by percentage, occurring at 45.9%, 15.4%, and 12.7%, respectively.
The Langat River recorded the highest number of species (10 species), and the lowest numbers were recorded in the Gombak and Tekala Rivers (five species). With 45% and 35% occurrence in the six rivers, the order Cypriniformes and family Cyprinidae were generally the most occurring. Furthermore, based on occurrence in the sampled rivers by origin, the Pusu River had the highest percentage occurrence (57%) of alien fishes, followed by the Langat (50%) and Gombak Rivers (40%) ( Figure 2).     Table 2).
The scoring of anthropogenic factors around the sampling sites revealed that the Langat River, with a mean score of 3.80 ± 1.12, is likely the most exposed to the human elements. Next was the Pusu River with a mean score of 3.60 ± 1.74, while the Semenyih, Klang, Gombak, and Tekala Rivers scored 3.50 ± 1.58, 3.10 ± 1.69, 3.00 ± 1.40, and 2.50 ± 0.93, respectively.

Relationships between Fish Occurrences vs. Water Quality Parameters and Anthropogenic Factors
Based on PCA and ordination plot analyses, three axes cumulatively explained 75.4% of the variation in the water quality parameters. Components with eigenvalues greater than one were considered as significant and thus extracted. Out of the 12 parameters measured, only six were retained based on the set criteria. Component one had strong loadings for temperature and DO, component two had strong loadings for PO 4 3− and NO 3 − , while component two had strong loadings for NH 3 -N and NO 2 − (Supplementary  Material Table S4 and Figure S1).

Feeding Intensity and Stomach Fullness Index
Oreochromis niloticus was the only species that occurred in abundant numbers for the three rivers assessed. Other alien species include P. reticulata, P. disjunctivus, and P. pardalis, and native species, such as B. banksi, O. vittatus, O. marmorata, T. vittata, and H. macro-   Table S6). High pollution levels and high ease of accessibility correlated positively with the occurrence of O. niloticus, P. pardalis, and P. disjunctivus, but negatively with native species, such as M. obtusirostris and P. normani, which also correlated with the usage for other purposes. The level of protection did not correlate with the occurrence of any of the species. In contrast, native species, such as O. vittatus, T. vittata, and O. marmorata, which were generally low in occurrences, did not show a clear pattern regarding the anthropogenic factors assessed ( Figure 3B).

Feeding Intensity and Stomach Fullness Index
Oreochromis niloticus was the only species that occurred in abundant numbers for the three rivers assessed. Other alien species include P. reticulata, P. disjunctivus, and P. pardalis, and native species, such as B. banksi, O. vittatus, O. marmorata, T. vittata, and H. macrolepidota, were the only ones subjected to stable isotope analysis.
Since other species were limited in numbers, only O. niloticus (alien) was suitable for comparison (using stomach contents analysis) with native species, such as M. obtusirostris, P. normani, R. vulgaris, and M. singaringan. A descriptive summary of the fish sizes (n = 183) from Gombak, Klang, and Langat Rivers is presented in Table 4 (Table 5).  For the Gombak River, the level of stomach fullness for the three species considered indicated that O. niloticus had the highest percentage of full (36.36%) and 3/4 full stomachs (36.36%), while P. normani had the highest percentage of empty (61.29%) and 1/4 full stomachs (20.03%) (Figure 4)  Oreochromis niloticus is the alien fish.
Detritus was generally the highest by occurrence, volume, and preponderance in the two fish species considered for the Langat River. Multicellular green algae and plant parts also occurred in all the non-empty stomachs (100%) of M. singaringan, while worms were

Rasbora vulgaris n = 30
Oreochromis niloticus * n = 28  Detritus was generally the highest by occurrence, volume, and preponderance in the two fish species considered for the Langat River. Multicellular green algae and plant parts also occurred in all the non-empty stomachs (100%) of M. singaringan, while worms were next in importance by volume (15.67%) and preponderance (14.25%). Mud occurred in all the non-empty stomachs (100%) of O. niloticus, while detritus was the most important by volume (24.76%) and by preponderance (29.01%), followed by worms by volume (18.17%) and preponderance (18.36%) ( Table 8). Some examples of the stomach contents encountered in fish samples are presented in the Supplementary Material Figure S2.

Diet Overlap and Trophic Level
Except for R. vulgaris vs. O. niloticus in the Klang River, the Morisita-Horn index (C H ) for alien vs. native species indicated a significant diet overlap (C H > 0.6) for all the pairs of species in each river (Supplementary Material Table S7). In the Gombak River, M. obtusirostris (2.12 ± 0.15) had the highest TROPH value, while R. vulgaris (2.6 ± 0.28) and M. singaringan (2.33 ± 0.20) had higher values for the Klang and Langat Rivers, respectively (Supplementary Material Table S8).

Stable Isotope Analysis
The positioning and overlap in the δ 15 N and δ 13 C for native and alien fish species are presented in Figure 5A For the Langat River, the Kruskal-Wallis test revealed the existence of significant differences in the δ 15 N (H = 15.339, df = 5, p = 0.009) and δ 13 C (H = 12.202, df = 5, p = 0.032) for fish species from the Langat River. For the δ 15 N values, pairwise comparisons showed that except for the significant differences observed between P. disjunctivus and M. singaringan (p = 0.01), there were no significant differences (p > 0.05) between all other pairs of fish species analyzed. For the δ 13 C values, pairwise comparisons showed that except for the significant differences observed between M. obtusirostris and P. disjunctivus (p = 0.019), there were no significant differences (p > 0.05) between all other pairs of fish species analyzed.
For M. obtusirostris sampled across the Gombak and Langat Rivers, the Mann-Whitney U test revealed no significant differences (p > 0.05) between their δ 15 N and δ 13 C values. According to the Kruskal-Wallis H test, O. niloticus sampled across the Klang, Gombak and Langat Rivers showed significant differences between their δ 15 N (H = 9.346, df = 2, p < 0.009) and δ 13 C (H = 9.379, df = 2, p < 0.009) values. For the δ 15 N values, pairwise comparisons showed a significant difference in the δ 15 N values between Gombak and Langat Rivers (p = 0.007). Moreover, a significant difference in the δ 13 C values was observed between the Gombak and Klang Rivers (p = 0.010).

Discussion
The present study set out with the aim of assessing the invasion risks of the identified alien fishes using AS-ISK and the fish community structures of selected rivers within the Klang Valley, Malaysia. More so, the fish occurrences were related to water quality parameters and anthropogenic factors. The stomach contents of co-existing native and alien fishes from three of the six rivers were also assessed, while stable isotope analyses, through the assessment of δ 15 N and δ 13 C values in fish muscle tissues, were carried out.
The results of this study revealed that, except for the Tekala River, alien fishes were recorded for all of the sites, with the most frequently occurring alien species being O. niloticus. Moreover, the sight of breeding nests belonging to this species in the Klang River confirms that this species had established a breeding population in this river. In line with the findings of this study, Shuai et al. [64] reported an increase in the relative abundance of O. niloticus, which is a non-native species, in a large subtropical river in China. Therefore, the ability of this alien species to successfully breed and multiply over time is confirmed, and this further explains the result of this study.

Discussion
The present study set out with the aim of assessing the invasion risks of the identified alien fishes using AS-ISK and the fish community structures of selected rivers within the Klang Valley, Malaysia. More so, the fish occurrences were related to water quality parameters and anthropogenic factors. The stomach contents of co-existing native and alien fishes from three of the six rivers were also assessed, while stable isotope analyses, through the assessment of δ 15 N and δ 13 C values in fish muscle tissues, were carried out.
The results of this study revealed that, except for the Tekala River, alien fishes were recorded for all of the sites, with the most frequently occurring alien species being O. niloticus. Moreover, the sight of breeding nests belonging to this species in the Klang River confirms that this species had established a breeding population in this river. In line with the findings of this study, Shuai et al. [64] reported an increase in the relative abundance of O. niloticus, which is a non-native species, in a large subtropical river in China. Therefore, the ability of this alien species to successfully breed and multiply over time is confirmed, and this further explains the result of this study. Similar to some other high-risk alien ornamental fish species recorded from pet stores in the Klang Valley, Malaysia, which had been earlier screened using the FISK v2 software [4], O. niloticus was labeled as invasive. Similarly, the high invasion risk of O. niloticus had been established in other regions too, including the Iberian Peninsula [65], South Africa [66], and China [64], where they have negatively impacted the native fish species and the environment. Consequently, the negative impacts that stem from the establishment of invasive species, such as O. niloticus, are also predicted for the water bodies that were the subject of this study.
The comparatively higher fish species richness, evenness, and lower dominance exhibited by the Semenyih and Tekala Rivers coincided with the low or no occurrence of alien fishes in these sites. More so, it indicates that the absence of alien fishes may have provided a better environment for native fishes to exist and flourish in these rivers. Generally, low fish diversity was commensurate with low richness and high dominance. Shannon's diversity index recorded in this study for the Pusu River is lower than that reported by Jalal et al. [31]. The number of alien fish species recorded in their study (n = 2) was lower than that recorded in the present study (n = 4), indicating that additional alien fishes may have been introduced from time to time. These species have succeeded in establishing breeding populations in the Pusu River. Specifically, O. niloticus was the dominant fish species in the Pusu River instead of B. schwanenfeldii, which is a native fish species that was earlier reported to be the most prevalent in the river.
This study discovered that alien fishes have shadowed the native ones in rivers with high NH 3 -N, TDS, conductivity and salinity, and low DO levels and water depths. More so, most of the native fishes assessed have shown the ability to thrive better in water bodies with higher DO levels, low levels of NH 3 -N, and the absence or low diversity of alien species as was observed in the Tekala and Semenyih Rivers. Moreover, the alien fish species displayed a comparatively higher abundance in rivers that recorded high TDS, conductivity, salinity, and NH 3 -N as observed in the Pusu and Klang Rivers. Fishes generally prefer water conditions of adequate physical and chemical characteristics [67]. However, many invasive fish species possess some special characteristics that make them highly sought after [68]. For example, the ability to survive and grow at low levels of dissolved oxygen and high levels of ammonia. More so, the characteristics of these alien fish species may contrast with those of their native counterparts, which may not be rugged enough to withstand a similar condition. Thus, according to the present study, the alien fishes tend to be more rugged and could flourish in these water bodies to the detriment of their native counterparts.
The anthropogenic factors indicate a possible contribution of human influence to the community and environmental indices such that the potential of alien fishes to invade the waterbodies is amplified. For example, the Langat River seems to be more exposed to anthropogenic influences. Furthermore, activities, such as housing construction, waste dumping, and land clearing, have been reported around the Langat and Pusu Rivers [69]. Public knowledge of the possible negative environmental impacts of human activities, including the introduction of alien fishes, is essential for effective conservation and management of inland freshwater [70]. In the present study, these rivers were characterized by high NH 3 -N and low DO concentrations, indicating that native species could be outcompeted since their alien counterparts are better able to withstand the poor water quality conditions.
Oreochromis niloticus, an omnivorous species, ingests zooplankton, phytoplankton, and debris present in rivers, resulting in competition for food and space with the native fishes [71]. Moreover, O. niloticus reportedly replaced native species in Thailand after excessive reproduction success [72]. They also predate on juveniles and eggs of native species and disrupt the habitat by grazing on benthic algae and detritus [73]. Paradoxically, due to its excellent culture characteristics, O. niloticus has been widely introduced to several countries across the globe for aquaculture improvement and to augment capture fisheries [74]. This fish was introduced into Malaysia for aquaculture and may have escaped from culture facilities into native waters due to natural disasters, such as flooding [3]. Similar to its other counterparts identified in this study, O. niloticus is invasive, and this study represents the first attempt to risk-assess this species for Peninsular Malaysia. Previous risk assessment studies, including Ellender et al. [75] for South Africa, and Perdikaris et al. [76] for Greece, have also found O. niloticus invasive. Pterygoplichthys disjunctivus was also found to pose a high invasion risk in South Africa [68], while P. pardalis, P. disjunctivus, and P. reticulata were invasive in Mexico [77].
With the highest percentage of full stomachs and significantly higher stomach fullness indices throughout the sampling sites, O. niloticus displayed the best feeding ability. Previous reports have shown that some native species exploit similar food with the O. niloticus, which can outcompete them, making them shift their preferences to less preferred resources [78]. This can consequently impact negatively on their growth and condition in the ecosystem [16,75,79].
Furthermore, diet overlap between pairs of alien and native fish species significantly showed that they essentially exploit similar food resources. This signifies competition between these fish species, with O. niloticus being more rugged and plastic [80]. Broad niche overlap of O. niloticus with native species in South Africa was also reported by Zengeya et al. [81,82]. However, in an experimental study, Ahmad et al. [83] recorded lower levels of overlap between O. niloticus and native small indigenous fish species of south Asia, such as mola (Amblypharyngodon mola), chela (Chela cachius), and punti (Puntius sophore). The omnivorous nature of the species considered in that study and the availability of alternative food resources may be responsible for the difference.
The TROPH values of both alien and native fishes indicated that they are all positioned within the same trophic level in the three rivers. From the TROPH values recorded in this study, O. niloticus from the Gombak and Klang Rivers are herbivorous. However, the values indicate that they are omnivorous with a preference for food of plant origin in the Langat River. In line with the outcome of the current study, O. niloticus had also been regarded as omnivorous and planktivorous, feeding mainly on green algae, diatoms, cyanobacteria, and detritus [84,85].
The results of this study indicate similarities in the trophic preferences of both native and alien fishes and the trophic plasticity of O. niloticus as opposed to its native counterpart M. obtusirostris [86]. Therefore, an alien species, such as O. niloticus, could explore food at a wider trophic range giving it the ability to outcompete the native species of narrower δ 15 N and δ 13 C values [87,88]. Diet overlaps between O. niloticus and the native fishes were found based on stomach contents analysis. The result from stable isotope analyses gave an insight into the statistical similarities and differences in the stable isotope signatures of the fish species; however, it may not be sufficient to conclude that the alien and native species have overlapping isotopic niches.

Conclusions
This study indicates that compared to native fish species, alien fish species likely benefited from the impacts of anthropogenic activities in their surrounding habitats, while their plasticity in feeding habits might help them to further invade, survive, and dominate. The potential ecological impacts on native fishes in the rivers of Klang Valley, Malaysia was, therefore, revealed.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ani11113152/s1, Figure S1: Ordination plot from Principal Components Analysis for water quality parameters from the sampling locations. Rotation Method: Varimax with Kaiser Normalization, Figure Table S1: Environmental conditions and characteristics of the sampling points. This file contains detailed information on the environmental conditions, characteristics and coordinates of the rivers, Table S2: Criteria for the measurement of anthropogenic characteristics at each sampling site within Klang Valley, Malaysia, Table S3: Minimum, maximum and mean ± SD values of measured water quality parameters from selected rivers within Klang Valley, Malaysia. Rows with different superscripts indicate significant difference (p < 0.05) between the means of the log-transformed data, Table S4: Loadings from Principal Components Analysis for water quality parameters from the sampled rivers. Extraction was based on eigenvalues > 1. Numbers in bold indicate parameters with high loadings, Table S5: Eigen values and percentage variance of Canonical Correspondence Analysis for water physicochemical parameters from the sampled rivers., Table S6: Eigenvalues and percentage variance of Canonical Correspondence Analysis for anthropogenic factors surrounding the sampled rivers, Table S7: Diet overlaps between fish species from Gombak, Klang, and Langat Rivers based on Morisita-Horn index. CH = Morisita-Horn's diet overlap index. * indicate alien species, Table S8: TROPH of native and alien fish species from Gombak, Klang, and Langat Rivers. *indicate alien species.  Institutional Review Board Statement: The fish were sampled, handled, and sacrificed according to the methods approved by Institutional Animal Care and Use Committee, Universiti Putra Malaysia. All procedures were carried out following relevant guidelines and regulations. No permit was required to conduct the present study, as none of the sampled fish species was considered endangered and protected by the government of Malaysia.

Data Availability Statement:
All data related to this study will be provided on a request sent to the corresponding or first author.