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401

LEUKOCYTE DIFFERENTIAL OF ANGUILLID EEL, Anguilla bicolor

McClelland, EXPOSED TO VARIED SALINITIES
FITA FATIMAH, FARIDA NUR RACHMAWATI, EKO SETIO WIBOWO
Fakultas Biologi, Universitas Jenderal Soedirman, Jalan dr. Suparno 63 Purwokerto 53122

ABSTRACT
The anguillid eel is a catadromous eel capable of inhabiting freshwater growth habitat and seawater spawning habitat throughout their life
cycle. At the juvenile to mature stage, they inhabit freshwater then migrate to marine water to spawn. Changes in salinity, which is one of the
stressful environmental factors for the eel, affect their physiological condition by increasing the leukocytes number. This increase is an
adaptation method to improve their immune system as a response to salinity change. This study intended to evaluate the leukocyte
differential of anguillid eel (Anguilla bicolor McClelland) exposed to various salinities. This research applied a Completely Randomized
Design. The treatment was three levels of saline media including 4 ppt, 15 ppt, and 30 ppt with five replicates. The independent variable was
the different salinity, and the dependent variable was the leukocyte differential. The parameters measured consisted of the different
percentage of neutrophils, lymphocytes, monocytes, and eosinophils in which the measurements administered after two months of the eel
exposure. We analyzed the data with ANOVA at the confidence level of 95%. The results showed that exposure of salinity significantly
affected the percentage of leukocyte differential (P < 0.05). The increase in salinity decreased the neutrophils and monocytes, but increased
the lymphocytes, and showed no effect on eosinophils.
KEY WORDS: Anguilla bicolor McClelland, leukocyte differential, salinity

Corresponding Author: FARIDA NUR RACHMAWATI | email: faridanur12@gmail.com

According to Herianti (2005), extreme exposure to

INTRODUCTION
high salinities can affect the physiological condition of
The anguillid eel is a potential fish to cultivate. In fish. They need time to adapt, survive, and perform
Indonesia, it is known as Sidat, according to Haryono physiological processes. Alteration of the fish
(2008) it is also known as moa, lubang, and uling (in physiological conditions might cause changes in blood
West Java), pelus (in Central Jawa), and ‘Eared- Eel’ components.
because of the pectoral fin which resembles an Salinity affects the osmoregulation system in fish.
earlobe structure. It has a high economic value and is Osmoregulation is the process of regulating osmotic
an export commodity in the fishery sector. The pressure between the organism body and the media.
request for anguillid eel in markets can reach up to Marine organisms require to adapt to the osmotic
500.000 tons per year and is mostly from Japan and pressure of their environment, and those do not adapt
Korea (Purwanto, 2007). The anguillid eel has a wide encounter stress and die (Sobirin et al., 2014).
distribution range, for they are divided into tropical, According to Royan et al. (2014), different salinities
and subtropical anguillid eel. There are at least 17 cause an alteration in the blood profile (decrease in
species of anguillid eel, and six species are in hematocrit, hemoglobin, total erythrocyte, increase in
Indonesia. They include the Anguilla marmorata, A. blood glucose and leukocyte) and decrease fish
celebensis, A. ancentralis, A. borneoensis, A. bicolor survival as the salinity increase.
bicolor and A. bicolor pacifica. The A. bicolor is the only Stress animals adapt various ways to survive, for
species that successfully cultivated in Indonesia example, by increasing the amount of leukocyte as a
(Affandi, 2005). response to salinity changes. Rise in leukocyte due to
The anguillid eel cultivation in Indonesia is limited stress might serve as adaptation indicator and
to grow out culture because their unique life cycle resistance towards external disturbances (Isroli,
hampers the reproduction technique to be completely 2002).
understood. The anguillid eel is a catadromous fish The response of blood profile is to increase the fish
inhabiting two different waters, in term of salinity, immunity to which adjusting of leukocytes increases
throughout its life cycle. Throughout its juvenile and body defense mechanism (Suprayudi et al., 2006). The
mature stage, it inhabits fresh waters. Whereas during effect of salinity towards the fish physiological
spawning stage, it migrates to the marine waters condition can be observed from the leukocyte
(Sudo et al., 2013). According to Haryono (2008), differential (Verdegem et al., 2008). According to
spawning occurs at depths of 400–500 m with Gross & Siegel (1983), the comparison between
temperatures of 16–17°C and salinities of 35 ppt. The monocytes, neutrophils, and lymphocytes, is an
migration of anguillid eel from fresh waters to the sea excellent indicator to measure the organism stress
with a high difference salinity indirectly affects the level. The purpose of this research was to evaluate the
physiological condition of the fish. differential in leukocytes of anguillid eel (A. bicolor
The anguillid eel used in this research was those in McClelland) due to the exposure of different salinities.
the silver eel stadium which inhabited fresh waters
and was ready to migrate to the sea to spawn.

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 FITA FATIMAH, FARIDA NUR RACHMAWATI, EKO SETIO WIBOWO

METHODS % neutrophil = number of neutrophil counted (100) x 100%

% lymphocyte = number of lymphocyte counted (100) x 100%
Tools used in this research were tarpaulin cloth tanks, fiber % monocyte = number of monocyte counted (100) x 100%
tanks, aerator, light microscope, object glass, scissors, scale, % eosinophil = number of eosinophil counted (100) x 100%
and hand refractometer. Materials consisted of silver stage
anguillid eel Anguilla bicolor McClelland, seawater, The data was analyzed with ANOVA at 5% degree of
freshwater, labels, tissue, distilled water, earthworms, error to measure the significant difference of salinity on the
Giemsa 7% and methanol PA. We conducted the anguillid eel Anguilla bicolor McClelland leukocyte
experiments at the Experimental Station, and the differential. If ANOVA showed a significant difference, then
Laboratory of Animal Physiology, Faculty of Biology, the analysis was continued to the LSD (Harjosuwono et al.,
Universitas Jenderal Soedirman from October 2015 until 2011).
March 2016.
This research applied a Complete Randomized Design
RESULTS AND DISCUSSION
with three salinity treatments and five replicates (15 units). Figure 1. represented the differential leukocyte of
Salinity levels of the growth media were based on Royan et
anguillid eel (A. bicolor). The fish neutrophil reached
al. (2014) as follows:
25 ± 4.30%, 21.8 ± 2.39% and 9.8 ± 1.79% for salinity
A = 4 ppt (control) of 4, 15, and 30 ppt respectively. The fish neutrophil
B = 15 ppt
levels of 4 and 15 ppt coincided with Utami et al.
C = 30 ppt
(2013), who reported the neutrophil of Nile tilapia
The observation of leukocyte profile was conducted fish of 18–27% when exposed to freshwater salinity.
after two months exposure. The independent variables were
The neutrophil decreased when the fish were in 30
the media of various salinities, whereas the dependent
variable was the leukocyte differential. The parameters ppt salinity. However, this neutrophil amounts fell
measured were the percentage of leukocyte differential of between the normal range of marine fish. Johnny et al.
the anguillid eel. (2003) claimed that the neutrophil of marine fish such
The fish were placed in three units of tarpaulin cloth as grouper was between 6 and 11%.
tanks with the size of 130 x 100 x 100 cm3 equipped with
aerators. Each tank was cleaned before use to prevent the 100
growth of molds and fungi. The preparation of media 90
78,2
Leucocyte Diferential (%)

salinity (4 ppt, 15 ppt, and 30 ppt) followed Setyo (2006): 80

S1. V1 + S2. V2 70
𝑆𝑛 = 58,8
𝑉1 + 𝑉2 60
49,2
where, 50
Sn = Salinity level intended 40
S1 = Salinity of stock water 25 23,8
30 21,8
S2 = Salinity of mixed freshwater 15
V1 = Volume of stock water 20 9,8 10,2
V2 = Volume of mixed freshwater 10 4,4
2 1,8
The fish silver stadium (15 individuals) with an average 0
Neutrofil Limfosit Monosit Eosinofil
body length of 60 cm and an average weight of 480 g were
A B C
selected. The fish were from Pesanggrahan Village, District
of Cilacap, and were healthy, had no disabilities, and active. Figure 1. The average percentage of leukocyte differential
The fish were acclimated for at least one week in a fiber of anguillid eel exposed to (A) 4 ppt salinity (control), (B) 15
tank measuring 70 cm in height and 125 cm in diameter. ppt salinity, (C) 30 ppt salinity. The different alphabet
The fish exposed to high salinities required to acclimate notation above the numbers showed a significant difference
gradually from 4 ppt, 10 ppt, 15 ppt, and 30 ppt for one (P < 0.05).
week in each level of salinity. As many as eight fish were
placed in one tube containing media. The fish were fed with The ANOVA showed a significant difference of
earthworms once a day on ad libitum at 16.00 pm. The neutrophil in the fish exposed in three different
exposure allowed for two months. The aeration and salinity salinities (P < 0.05). This result indicated that salinity
in each tub were regularly controlled to ensure the efficient affected the amount of neutrophil. Neutrophil
oxygen circulation and maintain the right level of salinity.
decreased as the salinity increased. According to Davis
The fish weight was measured with a scale, whereas the
length was measured using a measuring tape. The blood was
et al. (2008), the neutrophil decline might be due to
taken by cutting the caudal fin and placed in on an object fish adaptation to their environmental alteration.
glass to smear. The blood smear method followed Anderson When the fish adapted to the change, cortisol levels
& Siwicki (1993). The object glass used was immersed in within the blood decreased, resulted in the neutrophil
methanol to remove any excess fat. The blood was dropped decrease.
on the object glass and smeared using another object glass The LSD revealed the fish neutrophil of 4 and 15
with an angle of 45° and dried. The dried smear was then ppt salinities was not significantly different (P < 0.05),
fixed with methanol for 3–5 minutes then dyed with Giemsa whereas the salinity of 30 ppt showed a significant
for 20–30 minutes, washed and dried. We observe the
difference (P < 0.05) between the salinities. The
smear under a microscope at 400x magnification. Each type
of leukocyte was calculated up to the 100th cells
decrease of neutrophil in the 30 ppt salinity most
(Kurniawan et al., 2013). likely was because of the lymphocyte increase during
the treatment. The neutrophil level within each

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salinity was relatively similar to that of other fish The LSD demonstrated significant result for
inhabiting the given salinity. Suprayudi et al. (2006) lymphocyte of each treatment (P < 0.05). This result
stated that salinity was one of many abiotic factors suggested that each salinity had a possible effect on
affecting the leukocyte differential in fish. Salinity lymphocyte amount. As explained by Davis et al.
stress might cause a high increase in leukocyte and (2008) that to overcome stress, fish had to adapt. One
result in the increase of lymphocyte. The lymphocyte type of which was to increase their lymphocyte level
increase stimulated neutrophil decrease. This result as a response to environment stressors such as
coincided with Erika (2008) who stated that the changes in temperature, salinity, and density.
neutrophil decrease might occur due to the balancing Leukocyte increase was related to the decrease in
mechanism of other proportions of leukocytes such as cortisol levels in the body. When cortisol level
the lymphocyte. decreased, DNA synthesis of the lymphocyte cells
occurred and led to the high amount of lymphocyte.
According to Inoue et al. (2008), in several aquatic
animals, increase in the ratio of lymphocyte and
neutrophil could be used as an indicator of long-term
stress levels of the animal (chronically). Fujaya (2004)
mentioned that lymphocytes in the fish body were not
phagocytic but hold a major role in the formation of
antibodies. The decrease in lymphocyte amount could
lead to a reduction in antibodies and prone to disease.

Figure 2. Neutrophil (N) (arrow) of anguillid eel Anguilla

bicolor McClelland (400x)
The anguillid eel lymphocyte exposed to salinities
of 4, 15, and 30 ppt were 49.2 ± 5.93%, 58.8 ± 3.42%
and 78.2 ± 2.77% respectively. The lymphocyte
increased as the salinity increased. These results were
similar to Sugito et al., (2014). They measured the
lymphocyte level of the freshwater fish that ranged
between 42 and 51%. Further, brackish fish showed
Figure 4. Monocyte (arrow) of anguillid eel Anguilla bicolor
the lymphocyte between 60 and 70%, and marine fish
McClelland (400x)
between 60 and 80% (Hartika et al., 2011). The
ANOVA showed a significant difference in the The average monocyte in 4, 15, and 30 ppt
lymphocyte percentage between anguillid eel exposed salinities were 23.8 ± 2.59%, 15 ± 4.30% and 10.2 ±
to different salinities (P < 0.05). This result 2.28% respectively. Monocyte decreased with the
demonstrated that salinity most likely influenced the increase of salinity. Sugito et al. (2014) claimed that
lymphocyte amount. The increase in lymphocyte monocyte percentage of Nile tilapia fish ranged
suggested adaptation towards the salinity change in between 17 and 25% when presented in freshwater.
the fish environment. Lymphocyte of the anguillid eel Monocyte of marine fish such as groupers was 9–15%
resembled the lymphocyte amount of other fishes in (Agung et al., 2013). According to Kurniawan et al.
the given waters. Moyle & Cech (2004), reported that (2013), a decrease in monocyte could be caused by
the immune system responded to external the increase in lymphocyte amount that produced
interferences, such as alteration in salinity, with the antibodies, therefore led to an obstruction of the
increase of lymphocyte amount. According to Sakai monocyte production. The production of antibodies
(1999), the amount of lymphocyte could increase was crucial for the immune system.
during stress. Stress interfered non-specific immune ANOVA demonstrated a significant difference (P <
responses, such as lymphocyte proliferation (increase 0.05) between anguillid eel exposed to different
in cell amount and alterations into T cells and B cells). salinities. The monocyte decreased when the salinity
increased. According to Moyle & Cech (2004), a
decline in monocyte possibly was because monocytes
were short-lived (14–36 hours in the blood system).
They moved to the tissues and differentiated into
macrophages. Putri (2003) stated that phagocytic
activity of macrophages was the initial step of the next
phase in immunity response, the antibody production.
LSD showed no significant difference (P < 0.05) in
monocyte level of 15 and 30 ppt salinities. However,
salinities 15 and 30 ppt differed significantly (P <
Figure 3. Lymphocyte (arrow) of anguillid eel Anguilla 0.05) from the 4 ppt salinity. Monocyte amount in
bicolor McClelland (400x) salinities 15 and 30 ppt was smaller than that of 4 ppt.

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 FITA FATIMAH, FARIDA NUR RACHMAWATI, EKO SETIO WIBOWO

According to Utami et al. (2006), a decrease in CONCLUSION

monocyte might occur because of the blood
It was concluded that salinity influenced the leukocyte
equilibrium response to the increase of lymphocyte.
differential of the anguillid eel Anguilla bicolor
The monocyte level showed similar values for fish in
McClelland. Increased salinity led to decrease the
the given environment. This result indicated that
percentage of neutrophil and monocyte, increase the
anguillid eel adapted to a wide range of salinity.
percentage of lymphocyte, but no effect on eosinophil
percentage that fell within the normal range.
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