Parasites Infestation on Cultured Barramundi (Lates calcarifer) and the Impact of Fish Health

Barramundi Lates calcarifer is one of the leading marine aquaculture commodities in Indonesia, therefore fish health should be observed. This study was conducted to determine the type and number of parasites present in barramundi, the prevalence, mean intensity and mean abundance of parasite and the number of leukocytes in each individual, based on the level of infestation. This study used a quantitative descriptive method, using a total of 30 fish with two different sizes of host of ± 20 cm (large) and ±5 cm (small) each as many as 15 individuals. The results showed that size of host samples influenced the number of parasites infested with the prevalence of 40%, while ectoparasite 20%. This may due to differences in the living environment as well as the larger host provides more space to the parasites. The ectoparasites found in barramundi, namely Trichodina sp, Cryptocaryon sp, Benedenia sp, Diplectanum sp and endoparasites, namely Camallanus sp. Ectoparasites Trichodina sp. has the highest mean intensity of 20%. The highest mean abundance on large fish was 3.2 ind/fish on ectoparasite and endoparasites was 0.07 ind/fish. The lowest leukocytes were 179.82 103/μl (19.6 cm) and the highest in sample number three was 218 103/μl (20 cm), all exceeded the normal amount of leucocytes cells for fish was uninfected.


Introduction
The activities in the floating net cages farming business are considered one of the efficient and effective methods for starting an aquaculture venture [1].One of the commonly raised fish in floating net cages is barramundi, nowadays is a leading marine aquaculture commodity in Indonesia, known for its high market value both locally and globally [2].As an aquaculture species, the white snapper offers several advantages, including suitability for farming in marine, brackish, and freshwater environments; the potential to grow to a large size; a relatively large market demand; and rapid growth [3].
Aquaculture activities are closely related to water quality and diseases that often affect cultured organisms, leading to disease outbreaks due to the incompatible interaction between the host, pathogens, and suboptimal environmental conditions.Diseases affecting fish are the result of interactions between the host and the environment [4].This incompatible interaction induces stress in fish, weakening their immune defences, making them susceptible to diseases, which can easily enter their bodies and lead to illness [5].
Parasites are one of the causes of disease in barramundi.Fish infected by parasites are more susceptible to other diseases caused by bacteria, fungi, and viruses [6].Based on their microhabitat, parasites can be external (ectoparasites), found on parts of the fish organs such as fins, body surface, and gill lamellae.Internal parasites (endoparasites) live inside the fish's organs, including the liver, kidneys, spleen, brain, digestive, circulatory, and respiratory systems, abdominal cavity, muscles, meat, and other internal tissues [7].Fish production can sometimes halt in the cultivation process due to parasite infections in both juvenile and adult barramundi.Additionally, the number of parasites can depend on the age of the host, the larger the fish, the more parasite infestation is found [8].The ecological index of barramundi fish parasites and the impact on leukocyte production are important to study in relation to the health of the fish.Parasite infections can cause losses to the definitive host, such as stunting growth and reduced production.The objectives of this study is to determine the presence of ectoparasites and endoparasites, the prevalence, mean intensity, mean abundance of parasites occupation 1329 (2024) 012010 IOP Publishing doi:10.1088/1755-1315/1329/1/012010 2 of fish, the influence of host size on parasite presence and the number of leukocytes as part of immune system of the host.

Material and Method
This research was conducted in April 2023, with the location of sampling in the floating net cages of Poka, Waiheru and Lateri.Samples were observed thoroughly for parasites infestation in the Cultivation Laboratory University of Pattimura, while blood samples were taken to The Special Regional Hospital of Nania Ambon.The equipment used in this study includes a microscope, pH meter, DO meter, thermometer, refractometer, scale, dissecting set and camera.The fish samples used in this study were 30 barramundi of two different sizes group, 15 fish measuring between 17-20 cm and 15 fish approximately 4-5,5 cm.The method used in this study is the descriptive method, while the sampling of barramundi uses the purposive sampling method.
The examination of fish ectoparasites includes scales, fins, gills, and eyes by cutting these parts and then observing them on a glass slide under the microscope.Parasite examination on fins is carried out on the dorsal fin, pectoral fin, pelvic fin, anal fin, and caudal fin using a magnifying glass, and each fin is separated with tweezers.Each part of the fin is cut and then placed in a petri dish containing water, and then the liquid in the petri dish is observed under a microscope.Parasite examination on gills is done by removing the gills using tweezers and scissors, then transferred to a petri dish containing water, and each gill filament is scraped and then observed.Eye examination is done by lifting the eye and then examined under a microscope [9].
Examination of endoparasites is done on the liver, stomach, intestines, kidneys, and swim bladder.Changes in colour and surface of the liver are observed.Small pieces of liver are placed on a slide and observed under a microscope.The intestines and stomach are opened with scissors and a parasite examination is conducted.Changes in colour, size, and the presence of lumps in the intestines are observed by taking small pieces of the kidney placed on a slide and observed under a microscope.The swim bladder is opened and the appearance of the inner surface and parasite examination are observed [10].The parasites observed were then identified according to the guidelines in the books Aquatic Parasitology [11] and Parasites in Fish [12].
The measurement of water quality parameters is done in situ.The observed parameters include physical aspects such as temperature, clarity, and chemical aspects such as pH, Dissolved Oxygen and salinity.
Some ecological parameters were calculate to measure prevalence, mean intensity and mean abundance.Prevalence is the percentage of infected fish with a particular parasites species compared to the total number of fish samples examined for that parasite species [13].Calculation was using formula [14] as follows: where P is the prevalence (%); n is the number of infected fish; N is the total number of fish examined.Mean intensity is defined as the average intensity of a particular parasite species residing on an infected host.Parasite mean intensity is calculated using the formula [15] as follows:

𝑁
where I is the mean intensity (ind of parasite/individuals of fish observed); P is the number of particular parasites found (individuals); N is the number of fish observed (individuals).Mean abundance is the total number of individuals of a particular parasites species in a sample of a particular host species divided by the total number of hosts of that species examined.Mean abundance is calculated by counting each individual found from several host samples that have been examined [16] using the formula as follows:

𝐴 = ∑𝑛𝑖 𝑁
where A is mean abundance (ind/fish); ni number of particular parasite found (ind.);N is number of fish examined.

Presence of parasites.
The presence of parasites in smaller sizes of host only in one host, three individual of one type of parasite, whereas, seven host for larger sizes had six types of parasite with the total number of 48 individuals.This is in line with the findings that the longer the body size of the fish is directly proportional with the prevalence rate of parasites [17] and also there was a tendency to increase in parasite and bacterial numbers with the increase of the host size [18].The presence of parasites from its hosts taken from three different sites showed more parasites presence in Poka, lesser in Waiheru and none in Lateri.This condition probably due to the sites of fish living in floating net cages has to exposed to trash carried by currents, the environment condition of the fish habitat in Poka are more susceptible make them being exposed to stress.A study about micro plastic from trash existence in the related area shows the floating net cages of Poka already contaminated [19].Conversely, by looking at the presence of parasite in this study, the fish from Waiheru location is relatively safe from stress, but Lateri is the best location for placing floating net cages in term of no trash in the surrounding area (Table 1).The ectoparasites types was found in barramundi as the host taken from floating net cages in this study includes Trichodina sp, Cryptocaryon sp, Benedenia sp,and Diplectanum sp (Figure 1), while the endoparasites found only the species of Camallanus sp.Compare to ectoparasites attacking marine aquaculture in Indonesia include parasites from the genera Benedenia, Neobedenia, Diplectanum, Pseudorhabdosynochus, and Trichodina, while the genus of ectoparasite often found on barramundi is Halioterma, which attacks the gills [20][21] [22].
Parasites in this study were frequently found in larger host, occupied target organs such as the dorsal fin, scales, eyes, and gills.Camallanus sp found in barramundi occupy the intestinal organs, Tricodina sp on dorsal fin, Crytocaryon sp found in scales or skin, while Benedenia sp and Diplectanum sp was at the eye and gills, respectively (Table 2).One of the common parasites affecting barramundi is Trichodina, an ectoparasite that attacks the gills or the surface body of fish.This parasite infection is often occurred and considered highly pathogenic because it may cause significant mortality as reported in Indonesia where L. calcarifer widely cultured [23].Trichodina is a type of parasite known for causing Trichodiniasis [24].This parasites attaches to the body surface and rotates 360° using cilia, damaging the surrounding cells and consuming destroyed epithelial cells, leading to irritation on the body surface [25].
Ectoparasites with short life cycles, such as ciliates, monogeneans, and crustaceans (copepods, isopods), often attack fish in densely populated cages.Cryptocaryon irritans is a pathogenic parasite from the Ciliata protozoa group causing the white spot disease (white spots) on the surface of infected fish.This parasite can cause mass mortality in marine aquaculture [26].Benedenia sp. is also known as skin worm because it usually attaches to the skin of fish and can cause skin irritation as an entry point  for secondary infections.Common signs when fish are infected with this parasite include decreased appetite, weak swimming, and at severe levels, it can cause wounds on the skin and open up opportunities for secondary infections by bacteria [27].The Diplectanum parasite, also known as gill worm, is a dangerous parasite often found in marine fish [28].Infected gills usually appear pale, and there is excessive mucus production [29].Diplectanum infection is closely related to systemic diseases like vibriosis.The only endoparasites in barramundi L. calcarifer were found in sample taken from the floating net cages in Waiheru and Poka are Camallanus sp.This worm parasite is a non-specific parasite that generally infects specific organs such as the intestines, stomach, rectum, and anus [30] [31].Fish infected with the Camallanus sp.parasite become weak, exhibit wounds, and their bodies become emaciated [32], this parasite does not infect directly but can kill its host [33].

Ecological Parameters of Parasites
The values of prevalence, mean intensity and mean abundance of parasites during this study can be seen in Table 3.The prevalence of parasites infestation on large hosts was 4o% and on small host 6.7%.Based on prevalence criteria, the category fall in 'commonly' and 'sometimes' parasite attack level as in Table 4.It is clear that increase of parasite prevalence in line with the increase in fish body size.The mean abundance also shows the larger fish had the highest number or parasite infestation.This is presumed to be the larger the size and the older the age of the fish, the larger the surface area of the body, including the longer the duration of interaction between the fish and the parasites [34].This is also in line with the statement that the longer the body size of the fish, the higher the prevalence rate of parasites [35].The increase in the prevalence rate of parasites is presumed because smaller fish have lesser cross-sectional area than larger fish, hence fewer parasites live and attach, and there is also less opportunity for contact between parasites and hosts.
High prevalence rates can indicate a significant health problem ecologically, particularly in fish.However, using criteria of the attacking level of parasites in this study mostly the level often which means frequent with low prevalence levels between 10-29% [36].All types of parasites infestation was between 3,3 -6,7%.In this study (Table 3) Tricodina sp has prevalence of 6,7% far less compare to the previous study the prevalence was 90% [37], while mean intensity 20 ind. of parasites/n much more to the same study of 3,56 ind. of parasites/n of host infested.This is shows that this parasite infestation on captivated barramundi more on the host individually than in the whole population.Maintaining fish in open water conditions such as in floating net cages means as an environment with water quality that cannot be fully controlled, since it depending on seasons, waves and the environment.Moreover, the fish can get stress in the floating net cages with high density of host, allowing the parasites to develop rapidly where the high stocking density of fish causes competition for IOP Publishing doi:10.1088/1755-1315/1329/1/0120106 space, food and oxygen.Fish infested by monogeneans usually show behavioural changes such as lethargic and floating near the surface of the water.This infested fish have to tolerate to low oxygen condition as the respiratory rate increase.Fish with severe respiratory distress will be seen swimming to the surface of the water to breathe [38].Monogeneans parasites tend to have a direct cycle, which means that no intermediate host is needed to reproduce, therefore the parasites can easily infest fish.The mean intensity level of ectoparasites in larger barramundi was at 8,2 which corresponds to a moderate infection level.In contrast, the intensity of endoparasites is at a low infection level of 1,0 individual of parasite.Smaller barramundi have both ectoparasites and endoparasites at a low infection level as in Table 5 the intensity criteria [36].
Factors affecting the intensity value of parasites include not paying attention to environmental conditions and fish density in the maintenance pond [38].This is also in line with a statement that the condition of fish can be disturbed due to high density, lack of nutrition, and poor water quality, thus making the fish weak and easily susceptible to diseases [39].Fish rearing uncontrolled water quality because it depends on the season, and the environment of the water.
Tricodina sp has the highest mean intensity of parasites more likely happened because of life cycle of this parasite.The life cycle of Trichodina is relatively straightforward, direct cycle, have a single host and do not use alternation of generations or mass asexual replication off the host.Instead, they reproduce by cell-splitting or binary fission, which is essentially produces daughter cells with half the number of denticles of the parent cell [40].Super infection >1000 Abundance is the total number of individuals of a particular parasite species in a sample of a particular host species divided by the total number of hosts of that species examined (including both infected and uninfected hosts).This study shows the highest abundance level is in larger barramundi of 3,2 while the small size has 0,20 ind. of parasite/ind. of host (n).This is consistent with the statement that older fish have a larger body size and therefore have more time for contact with parasites in the water, making them more vulnerable to parasite infections [5].The abundance of parasites would be influenced by host population density, host health status, environmental factors and interaction with other organisms [39].This study has found a relatively less parasites infestation in barramundi means density of fish in the cage and its health status is already maintain in maximal level, especially in Lateri.Density of the host has negative associated with parasite abundance especially with a complex life cycle [41].

Total Leukocytes
Leukocytes are one of the blood components that function as a non-specific defense mechanism, localizing and eliminating pathogenic agents through the process of phagocytosis [42].One of the characteristics of a non-specific response is marked by the migration of leukocytes into tissues.The results of the leukocyte count in this study can be seen in Table 6.

Table 6. Leucocytes count of barramundi infested parasites
The research results in Table 4 show that the leukocyte values range from 192.53-218,00x10 3 cells/mm3.The normal leukocyte count in fish ranges from 20x10 3 to 150x10 3 cells/mm3 [43].The results of this study indicate that the leukocyte count in barramundi is above the normal value.This increase in leukocytes is called leukocytosis [44].The high number of leukocytes is likely due to infection by pathogens (parasites).Similar situation was reported that the heavily infected Nile tilapia juveniles (>400 zooids per fish) reared in cages in Brazil by Epistylis sp showed higher lymphocyte and lower neutrophil numbers than the less infected group (<400) [45].Changes in the number of leukocytes, in particular, can be used as an indicator of the health status of fish.An increase in leukocyte count a response in the form of protection against foreign cells, including infections by bacteria [46].A research stated that the number of leukocytes will increase if there is a change in the maintenance environment or an infection caused by viruses, bacteria, parasites or fungi, disease infection, and stress factors [47].When fish defences attack by pathogens, inflammatory responses are triggered and flow of blood with leucocytes increases to the infected area that attempt to destroy the pathogen.

Water Quality Parameters
All measurements of water quality was appropriate considerably (Table 7) because they were within the range established by National Standardization Agency of Indonesia [48].The optimal pH for seawater during the production stage of adult barramundi is between 7-9, with water temperatures ranging from 28.6-29.2°Cand salinity of 31-31.3ppt [49].Barramundi (L.calcarifer) is a fish that has a high tolerance for salt levels (euryhaline) and is a catadromous fish (raised in freshwater and mating in the sea), as well as being a carnivorous fish [50].Generally, Dissolved Oxygen (DO) should be around 5 ppm or more and should not be less than 4 ppm for pelagic or 3 ppm for demersal fish cultured in floating net cages.were in the range of 5.2-6.2mg/l.Oxygen consumption varies by species, with pelagic fish like red and white snapper requiring higher DO levels compared to demersal fish [51].

Conclusion
This study result concluded that the size of the fish is closely related to the presence of parasites, means the larger the fish, the higher the prevalence, mean intensity and mean abundance of parasite in barramundi.The parasites found in this study are five genera including Trichodina sp., Cryptocaryon sp., Benedenia sp., Diplectanum sp., and Camallanus sp.The health condition of barramundi Lates calcarifer raised in floating net cages is based on the ecological index and it was common infestation prevalence, moderate intensity category, and moderate abundance category.The total leukocyte value of barramundi was very high, which is a response of the fish body to the stress although water quality was optimum.

Table 1 .
Precence of parasites in two group of host sizes from three sites

Table 3 .
Ecological parameter values of particular parasites species, two different group of parasites and two different sizes of barramundi Particular

Table 7 .
Water quality parameter The research results show that the DO values