ORIGINAL ARTICLE Aquaculture

Use of a Noctiluca-killing bacterium Marinobacter salsuginis strainBS2 to reduce shrimp mortality caused by Noctiluca scintillans

Teeyaporn Keawtawee • Kimio Fukami •

Putth Songsangjinda

Received: 26 September 2011 / Accepted: 15 March 2012 / Published online: 13 April 2012

� The Japanese Society of Fisheries Science 2012

Abstract The ability of an algicidal bacterium Marin-

obacter salsuginis strain BS2, isolated from shrimp pond

water, to reduce shrimp mortality was investigated

under laboratory conditions. When two species of shrimp

(Penaeus monodon and Litopenaeus vannamei) (body

length 1.5–1.8 cm) were cultured together with the dino-

flagellate Noctiluca scintillans, nearly 80 % of the shrimps

died within 7 days. However, when bacterial strain BS2

was also added to the culture, N. scintillans was killed

within 48 h, and shrimp survival rates on the 7th day

improved from 23 to 87 % for both P. monodon and

L. vannamei. The bacterium BS2 alone had no effect on

shrimp condition. Under conditions of increased dissolved

oxygen, the effect of using BS2 was greater, and shrimp

survival rates improved even more dramatically, from 26 to

98 %. These studies provide the first evidence that the use

of killing bacteria, isolated from shrimp culture water, can

suppress harmful algal blooms (HABs) and thus restore the

efficiency of shrimp production. The control of HABs in

this way in shrimp culture farms would be a major benefit

for shrimp production.

Keywords Killing bacteria � Noctiluca scintillans �HABs � Shrimp culture

Introduction

Shrimp aquaculture is one of the fastest developing eco-

nomic activities in the Asia–Pacific region, where almost

80 % of the world’s production of farmed shrimp occurs

[30, 33]. In an intensive shrimp culture system, the concen-

tration of ammonia, resulting from excretion by the cultured

animals and mineralization of organic detritus, such as

unconsumed feed and feces, can reach toxic levels. The

accumulation of ammonia in pond water may cause the

deterioration of water quality, reduced growth, increased

oxygen consumption and ammonia–N excretion by shrimps,

and even high mortality [5, 32]. The lethal effects of

ammonia on juveniles of penaeid shrimps have been reported

for Penaeus chinensis, P. penicillatus [9], P. monodon [8],

P. paulensis [24], P. semisulcatus [31], P. japonicas juve-

niles [7], and Metapenaeus ensis [23].

Dinoflagellate blooms also cause disease and increased

mortality of cultured shrimp [2, 6]. Noctiluca scintillans is

a large, luminescent, heterotrophic dinoflagellate which

commonly forms red tides in temperate and subtropical

coastal regions. N. scintillans blooms have been reported in

New Zealand [4], Japan, China, Hong Kong [6, 16],

Indonesia, Malaysia, and Thailand [28, 29], providing

evidence that such bloom events are often associated with

massive fish and marine invertebrate mortality events.

Extensive blooms have been reported on the east and west

coasts of India, where they have been implicated in the

decline of fishery resources [1, 3]. The dense blooms of

green Noctiluca sp. are occasionally a stress factor for

shrimps, with a lower or even total loss of production

reported in Thailand [27] and mass mortality of shrimp

populations in China [6]. Although Noctiluca does not

produce toxins, it is catalogued as a harmful algal bloom

(HAB) species on the basis of its association with mass

T. Keawtawee (&) � K. Fukami

Graduate School of Kuroshio Science, Kochi University,

200 Monobe, Nankoku, Kochi 783-8502, Japan

e-mail: teeya19@hotmail.com

P. Songsangjinda

Department of Fisheries, Marine Shrimp Culture Research

Institute, Coastal Fisheries Research and Development Bureau,

Kaset-Klang, Chatuchak, Bangkok 10900, Thailand

123

Fish Sci (2012) 78:641–646

DOI 10.1007/s12562-012-0497-1

mortalities in farmed finfish due to oxygen depletion, gill

clogging, and high ammonia levels [25] resulting from the

high levels of ammonium and phosphate in N. scintillans

cells [15, 22, 25].

Previous studies have shown that various bacterial spe-

cies in the natural environment are capable of inhibiting or

controlling the growth of HAB [11, 13, 17]. Other studies

have shown that naturally occurring bacteria are also

capable of controlling the growth of phytoplankton [10, 12,

18, 20] and playing important roles during the development

and decay periods of red tide by influencing the initiation,

growth, maintenance and termination of phytoplankton

populations [14, 19, 21, 26]. In a recent study carried out

by our group, we found that some bacteria, which had been

isolated from a shrimp culture pond and identified as

Marinobacter sulsuginis, Vibrio sp., and Pseudoaltero-

monas sp. could suppress the growth of N. scintillans [20].

We also found that several strains of killing bacteria iso-

lated from shrimp culture water showed species-specific

inhibition against the growth of N. scintillans by either

direct or indirect attack [20]. However, to the best of our

knowledge, there have been no published reports on the

role of algicidal bacteria on shrimps in culture systems. It is

therefore essential to study the effect of isolated bacterial

strains that inhibit the growth of HABs, N. scintillans in

particular, in shrimp culture systems. If it is possible to use

killing bacteria to control the formation of N. scintillans

bloom, this would represent a promising approach by

which to improve the efficiency of shrimp farming man-

agement and shrimp production.

The aim of this study was to determine the ability of a

killing bacterium to suppress N. scintillans growth in a

shrimp culture system under laboratory conditions as a first

step in evaluating the potential applications of the isolated

bacterium for suppressing N. scintillans and controlling

HABs in shrimp farms.

Materials and methods

Cultures of N. scintillans and killing bacteria

Noctiluca scintillans was isolated from seawater at the

Sriracha Station (13.12�N, 100.56�E), Mueang Chon Buri,

Chonburi Province in June–July 2009 and from Don Hoy

Load Station, Samut Songkhram Province, Thailand in

January–February 2010.

The most effective killing bacterium (Marinobacter sul-

suginis strain BS2) was isolated from a pond used for the

culture of the shrimp P. monodon in June to July 2008 during

60–110 days of shrimp stocking at the Domrong shrimp farm

(6.845�N, 101.00�E), Thepha, Songkhla Province, Thailand

[20]. The ranges of salinity, temperature, and pH in the

shrimp pond were 23–26 ppt, 29–30 �C and 7.8–8.8,

respectively [20].

Shrimp culture experiments

The bacterium Marinobacter sulsuginis BS2 strain was

incubated in FeTY liquid medium at 28 ± 1 �C for 48 h in

preparation for the experiment. The cell suspension was

then centrifuged at 4500 rpm for 10–15 min, and the cells

were rinsed with sterile seawater and resuspended in fil-

tered seawater. The initial bacterial density was determined

by the DAPI method.

The first experiment was carried out to compare the

sensitivity of shrimps of different species and sizes to

N. scintillans in shrimp culture under laboratory condi-

tions. Ten individuals of each shrimp species, i.e., Penaeus

monodon (Black tiger shrimp) and Litopenaeus vannamei

(White shrimp), with a body length of about 1.5–1.8 cm

(small size) and about 3.5–4.0 cm (larger size), were reared

in 500 ml seawater in 1-l glass bottles (Fig. 1). The first

control (S) system contained only shrimps without any

addition of N. scintillans or bacterial strain BS2. The sec-

ond control (SB) system contained shrimps and BS2, but

no N. scintillans. The first treatment (SN) system contained

shrimps and N. scintillans without BS2, and the test system

contained shrimps, N. scintillans, and bacterial strain BS2

(SNB). The initial N. scintillans density was 10 cells/ml,

which had increased to 20 cells/ml with the addition of N.

scintillans cells after a 12-h incubation, followed by the

addition of BS2 (concentration approx. 103 cells/ml) in the

SB and SNB treatments. Each treatment was carried out in

Fig. 1 Shrimp rearing procedure under laboratory conditions.

S shrimp, SB shrimp ? Noctiluca-killing bacteria strain BS2, SNshrimp ? N. scintillans, SNB shrimp ? N. scintillans ? Noctiluca-

killing bacteria strain BS2

642 Fish Sci (2012) 78:641–646

123

triplicate. The supply of dissolved oxygen (DO) was con-

trolled at more than 4–5 mg O2/l by means of an air pump.

Observations were made at 12-h intervals for 168 h.

Shrimp were assumed to be dead when they were immobile

and showed no response when touched with a glass rod.

Dead shrimps were removed daily from the containers.

N. scintillans abundance, total bacteria density, and shrimp

survival were monitored every day until the 7th day

according to the procedure described in Fig. 1.

For the second experiment, the effect of DO on shrimp

survival rates was investigated. P. monodon (Black tiger

shrimp) juveniles (body length about 2.8–3.2 cm) were

used in treatments with and without DO [concentration of

DO in the treatment with DO: 4–5 mgO2/l (controlled by

an air pump)] . The DO concentration was determined

using a DO meter twice daily (7:00 a.m. and 3:00 p.m.)

The same series of treatments was used, in triplicate, as in

the first experiment. For each treatment, 30 shrimps were

placed in a 20-l aquarium tank. The initial cell density was

20 cells/ml for N. scintillans and approximately 103 cells/

ml for bacterial strain BS2. The abundance of N. scintillans

and the shrimp survival were monitored every day for

7 days. Water samples (30 ml) were collected and used for

determining ammonia concentration on days 0, 1, 3, 5, and

7 of the experiment by means of TRACCS-800.

Statistical analysis

The significance of variations in shrimp mortality among

treatments was tested by one-way analysis of variance

(ANOVA). The mean comparison was performed by

Duncan’s multiple range tests. All of the statistical tests

were considered to be significant at the 95 % significant

level of confidence.

Results

In the first experiment, we investigated the effects of

N. scintillans on small and larger P. monodon (Black tiger

shrimp) and L. vannamei (White shrimp). The results show

that the survival rate of the smaller sized P. monodon in the

SN treatment (N. scintillans without adding BS2) was 87 %

on the third day, decreasing to 23 % by the seventh day

(Fig. 2a); in comparison, the survival rate of L. vannamei

was 83 % on the second day, decreasing to 23 % by the

seventh day (Fig. 2c). In contrast, survival rates of the larger

sized P. monodon and L. vannamei on the seventh day were

47 and 87 %, respectively (Fig. 2b, d). These results indicate

that smaller shrimp of both P. monodon and L. vannamei

were more sensitive to N. scintillans than the larger shrimp,

while among the larger sized shrimp tested, P. monodon was

more sensitive to N. scintillans than L. vannamei (Fig. 2b, d).

The bacterial strain M. sulsuginis BS2 was tested against

N. scintillans under shrimp rearing conditions (SNB) along

with a control (SN). The results show that following the

addition of bacteria to the culture (treatment SNB), the

survival by the seventh day was 87 % for the smaller sized

P. monodon (Fig. 2a) and 93 % for the larger sized ones

(Fig. 2b), and 87 % for the smaller sized (Fig. 2c) L. van-

namei and 94 % for the larger sized ones (Fig. 2d). Clearly,

shrimp survival was much higher in the SNB treatment

than in the SN treatment, and there were no significant

differences between the S and SB treatments.

Fig. 2 Percentage (%) of

shrimp survival after

inoculation of N. scintillans and

algicidal bacteria strain BS2.

Data are means of replicates

with standard deviations (SD).

For abbreviations of treatment

groups, see Fig. 1

Fish Sci (2012) 78:641–646 643

123

In the case of smaller sized P. monodon, the cell

abundance of N. scintillans in the SN treatment (without

added BS2) increased slightly from 20 cells/ml after 12 h

of incubation to 23 cells/ml after 7 days (Fig. 3a). In

contrast, when bacterium BS2 was present (SNB), there

were relatively fewer N. scintillans cells, and these had

almost disappeared after 2 days (Fig. 3a). Similar results

were obtained with the larger sized P. monodon and both

sizes of L. vannamei (data not shown).

Fluctuation of the abundance of total bacterial cells is

illustrated in Fig. 3b for smaller sized P. monodon culture.

The results show that the highest abundance of

1.06 9 107 cells/ml was observed in SNB after 2 days,

decreasing gradually to 2.11 9 106 cells/ml at the end of

the experiment. Total bacteria cell abundance was not

significantly different among the four treatments on the

seventh day of shrimp rearing (Fig. 3b).

In the second experiment, the effect of DO on the sur-

vival of P. monodon was investigated. When additional DO

was introduced, the DO concentration was [5 mg O2/l

(Fig. 4a), in contrast to the lower concentration

(\1.5 mgO2/l) during all experiments (Fig. 4d) in the

absence of additional oxygenation. At the end of the

experiment, shrimp survival in the SN group decreased to

77 and 43 % in the treatments with and without additional

DO, respectively (Fig. 4b, e). However, shrimp survival in

SNB was 98 and 72 % in the treatments with and without

added DO, respectively. As in the first experiment, the

number of N. scintillans cells decreased and disappeared

after the second day in the SNB treatment but slowly

increased in the SN treatment (data not shown). Shrimp

survival in treatments with additional DO was always

higher than when no additional DO was introduced

(Fig. 4b, e, respectively).

Fluctuations in the concentration of total ammonia are

shown in Fig. 4c, f. The ammonia concentration increased

from 16 to 93 lM in the SNB treatment with added DO

and from 9 to 72 lM in the SNB treatment without added

DO. In SN treatments, the ammonium concentration also

increased, from 14 to 48 lM and from 6 to 84 lM in

treatments with and without added DO, respectively. The

results indicate that the ammonia concentration in SNB

treatments with and without added DO was not signifi-

cantly different from that in the controls S and SB.

Discussion

In a previous study, we reported the isolation of new

Noctiluca-killing bacteria from a shrimp culture pond [20]

and noted that among these algicidal bacterial isolates,

Marinobacter salsuginis strain BS2 had a strong effect on

the growth of the dinoflagellate N. scintillans, which is

notorious for its adverse effects on shrimp culture. In the

study reported here, we examined whether Noctiluca-

killing bacteria could be used to reduce shrimp mortality

caused by N. scintillans. The results show that strain BS2

was able to inhibit the growth of N. scintillans in shrimp

culture within 2 days of inoculation (Fig. 3a), resulting in

improved shrimp survival rates. The survival rates of the

smaller sized shrimps improved from 23 to 87 % in both P.

monodon and L. vannamei (Fig. 2a, c, respectively), while

those of the larger sized P. monodon and L. vannamei were

also improved, from 47 to 93 and from 87 to 93 %,

respectively (Fig. 2b, d). Moreover, the algicidal bacterial

strain BS2 did not itself have any effect on growth and

mortality of either shrimp species (Fig. 2, SB treatment).

Although the smaller shrimps were more sensitive to the

effects of N. scintillans than the larger ones, the shrimp

survival rates improved considerably when bacterial strain

BS2 was added into the shrimp culture, (Fig. 2). The

improvement can thus be attributed to the inhibitory effect

of strain BS2 on N. scintillans growth, thereby limiting the

worst effects of N. scintillans on the shrimps.

Shrimp survival rate was also improved when the level

of DO in the shrimp culture was increased (Fig. 4e).

Fig. 3 N. scintillans abundance (a) and total bacteria count (b) after

the inoculation of N. scintillans and algicidal bacteria strain BS2 in

treatments with smaller sized P. monodon treatment. Data are means

of replicates with standard deviations (SD). For abbreviations of

treatment groups, see Fig. 1

644 Fish Sci (2012) 78:641–646

123

However, also at the normal concentration of DO, the

effect of N. scintillans on the survival rate of smaller sized

shrimp (SN) was greater (Fig. 2a, b) than that of larger

sized shrimp (Fig. 4b). A substantial portion of the nitrogen

inside the N. scintillans cells was in the form of ammonium

ion [22, 25]. Noctiluca sp. is not known to produce toxins

but has been catalogued as a harmful algal species because

it has been associated with the mass mortality of finfish in

fish farms, caused by oxygen depletion, gill clogging, and

high ammonia levels [25]. As such, its adverse effects on

shrimp, in particular under conditions of DO deficiency,

could be due to the release of ammonia and the accumu-

lation of ammonium ions in the water. In this study,

however, ammonia concentrations were not significantly

different between the SN and SNB treatments, either with

or without additional DO, after 7 days of shrimp rearing

(Fig. 4c, f), suggesting that shrimp mortality during

N. scintillans blooms in the pond was not due to increased

ammonia concentration. In our study, however, we were

able to restore shrimp survival by adding the algicidal

bacterial isolate, Marinobacter salsuginis strain BS2, to the

shrimp culture to suppress or kill N. scintillans.

In conclusion, the results of this study provide the first

evidence to support the use of killing bacteria, isolated

from shrimp culture water, to suppress HAB and thus

restore the efficiency of shrimp production. The control of

HABs, in particular those of dinoflagellate N. scintillans, in

shrimp culture farms using killing bacteria would be a

major benefit for shrimp farming.

Acknowledgments This study was supported by the Ministry of

Education, Culture, Sports, Science, and Technology (Mon-

bukagakusho), Japanese Government, JSPS, and Fund of the Presi-

dent of Kochi University. We are gratefully to the staff of the Coastal

Aquaculture Research Institute, Songkhla, the Research Food Aquatic

Animal Department of Fisheries, Chonburi, and Dumrong farm for

their valuable help with fieldwork. We thank G. Britton for his helpful

reading and comments.

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