VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Predation and Control Efficacies of Misgurnus mizolepis
(Cypriniformes: Cobitidae) Toward Culex pipiens molestus (Diptera:
Culicidae) and Fish Toxicity of Temephos in Laboratory and Septic
Tank Conditions
SEONG CHUN CHAE,1 YOUNG HYUN KWON,2 KYUNG IL MIN,2 HYUNG SOO KIM,2
NAM-JIN KIM,3 JUN-RAN KIM,4 BONG GI SON,1 AND YOUNG-JOON AHN5,6
J. Med. Entomol. 51(4): 817Ð823 (2014); DOI: http://dx.doi.org/10.1603/ME13203
ABSTRACT Culex pipiens molestus Forskal (Diptera: Culicidae) is the dominant mosquito species
in septic tanks in South Korea. An assessment was made of the biological control potential of mud
loaches, Misgurnus mizolepis Günther (Cypriniformes: Cobitidae), toward Cx. p. molestus larvae in
laboratory and septic tanks. Results were compared with those of temephos 20% emulsiÞable concentrate. In laboratory tests, all mud loaches survived on sedimentation chamber- and efßuent
chamber-collected water of aerobic septic tanks (ASTs), whereas all mud loaches died within 3Ð12
h after introduction into sedimentation chamber- and efßuent chamber-collected water of anaerobic
septic tanks. Gill hyperplasia and hemorrhages at the bases of pectoral Þns were detected in all dead
mud loaches. These appeared to have been caused by bacterial disease, rather than the physical and
chemical characteristics of the septic tank water. A mud loach consumed an average range of
1,072Ð1,058 larvae of Cx. p. molestus in the AST water at 24 h. At the manufacturerÕs recommended
rate (10 ml/ton) in the AST water, the temephos formulation did not cause Þsh mortality. In the AST
experiment, predation of mosquito larvae by mud loaches at a release rate of one Þsh per 900 mosquito
larvae resulted in complete mosquito control from the third day after treatment throughout the 18-wk
survey period, compared with temephos 20% emulsiÞable concentrate-treated AST water (reduction
rate, 40% at 28 days after treatment). Reasonable mosquito control in aerobic septic tanks can be
achieved by mosquito breeding season stocking of a rate of one mud loach per 900 mosquito larvae.
KEY WORDS Misgurnus mizolepis, Culex pipiens molestus, biological control, temephos, septic tank
In South Korea, the mosquitoes in the Culex pipiens
complex mostly consist of Cx. p. pallens Coquillett and
Cx. p. molestus Forskal (Sohn 2005, 2007). In particular, Cx. p. molestus occurs mostly in underground
structures where it was the dominant species, with
84 Ð97% of the Cx. pipiens complex (Sohn 2007). Cx.
pipiens complex have been found to infest an average
of 10.9% of septic tanks in urban residential areas (Lee
2006). In general, there are the two types of septic
tanks, aerobic septic tank (AST) and anaerobic septic
tank (AnAST) systems. Unlike anaerobic treatment,
aerobic treatment units use a mechanism to inject and
1 Interdisciplinary Program in Agricultural Biotechnology Major,
Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea.
2 Health Management Division, Seocho Public Health Center, Seocho 137-704, South Korea.
3 ICU Laboratory, Henkel Home Care Korea, Ansan 426-901, Gyeonggi, South Korea.
4 Research Institute of Agriculture and Life Sciences, College of
Agriculture and Life Sciences, Seoul National University, Seoul 151921, South Korea.
5 Biomodulation Major, Department of Agricultural Biotechnology,
Seoul National University, Seoul 151-921, South Korea.
6 Corresponding author, e-mail: [email protected].
circulate air inside the treatment tank. AnAST makes
up 70 Ð 80% of all waste disposal systems (Anonymous
2009). However, the AST is usually used for large
buildings, which serves as a more typical large habitat
of mosquito larvae, compared with AnAST in urban
area. Adults move indoors through passages connected to ventilators or entrances of septic tank systems (Lee 2006) and cause persistent complaints of
mosquito nuisance, particularly in late autumn and in
winter (Sohn 2007). However, no clear distinction
between the two types of septic tanks was given in
their studies. Mosquito larval abatement in septic tank
systems is mainly provided using residual larvicides
such as temephos, insect growth regulators such as
methoprene and dißubenzuron, and bacterial larvicides such as Bacillus thuringiensis israelensis (Korea
Centers for Disease Control and Prevention [KCDC]
2008). Increasing public concern about environmental
effects of mosquito larvicides, groundwater contamination, human health effects, and undesirable effects
on nontarget organisms intensiÞes when continued or
repeated applications of conventional larvicides to
septic tank systems become necessary. In addition,
factors such as increased costs of labor and larvicide
0022-2585/14/0817Ð0823$04.00/0 䉷 2014 Entomological Society of America
818
JOURNAL OF MEDICAL ENTOMOLOGY
application and safety issues have made mosquito control difÞcult in the septic tank system. There is, therefore, a critical need for the development of ecologically friendly control alternatives for mosquitoes.
Larvivorous Þshes are used as biological control
agents for mosquitoes largely because they are cost
effective and environmentally sound (Sharma and
Ghosh 1994, Rozendaal 1997, Ghosh and Dash 2007).
It is possible to rear them in large quantities in special
breeding ponds and distribute these widely in endemic areas without harmful effects on the indigenous
Þsh fauna (Sharma and Ghosh 1994, Rozendaal 1997).
Recently, the use of larvivorous Þshes in mosquito
control is generating renewed interest as an alternative or complement to conventional insecticides in
South Korea. An indigenous Þsh, the mud loach, Misgurnus mizolepis Günther, is one of the most efÞcient
biological control agents for different mosquito species in Korean rice Þelds because this species is widely
distributed and abundant in rice Þelds (Lee et al. 1997;
Lee 1998, 2000). The Þsh can tolerate a wide range of
temperatures and low dissolved oxygen levels because
it can obtain oxygen from intestinal respiration, in
addition to ordinary gill respiration (Park and Kim
2001, Cho et al. 2012). No information is available
concerning the potential of mud loaches for managing
Cx. pipiens complex and the toxicity of temephos to
mud loaches in septic tank water.
In the current study, an assessment is made of the
biological control potential of mud loaches toward Cx.
p. molestus in septic tank water. Control efÞcacy of
mud loaches was compared with that of the commercial larvicide temephos 20% emulsiÞable concentrate
(EC) because the larvicide is known to have very low
toxicity to mammals and aquatic organisms and is less
persistent in the environment (Opong-Mensa 1984).
The toxicity of the temephos formulation to mud loaches was examined in AST water. Some physical,
chemical, and biological parameters of AST and
AnAST water also are discussed in relation to survival
of mud loaches.
Materials and Methods
The ethical approval for use of mice and mud loaches was permitted by the Seoul National University
Institutional Animal Care and Use Committee (SNU
IACUC) (SNU-130703-2).
Chemicals. The organophosphorus larvicide temephos 20% EC was obtained from Pharmcle (Seoul,
South Korea). The manufacturerÕs recommended rate
in septic tank is 10 ml/ton (http//www.pharmcle.
com). All of the other chemicals used in this study were
of reagent-grade quality and available commercially.
Mosquitoes. Larvae of Cx. pipiens were collected
separately from water of two ASTs in Seocho Borough
(Seoul) from early June to mid-July 2011. The collected larvae were immediately transferred to an insect rearing room (Seoul National University). They
were then separately maintained in plastic trays (27 by
15 by 4 cm) containing 0.5 g of sterilized diet (40-mesh
chick chow powder:yeast, 1:1 by weight). Adult mos-
Vol. 51, no. 4
quitoes were maintained on a 10% sucrose solution
and blood fed on live mice. All stages were held at 27 ⫾
1⬚C and 65Ð75% relative humidity under a photoperiod
of 12:12 (L:D) h. Species identiÞcation based on polymerase chain reaction (Kasai et al. 2008) conÞrmed
that larvae from the wild collections were Cx. p. molestus.
Mud Loaches. Mud loaches used in both laboratory
and septic tank tests were purchased from the Garakdong Agricultural & Marine Products Wholesale
(Seoul) 1 wk before the beginning of each experimental series. They were reared in outdoor aquaria
containing underground water with proper aeration.
Mud loaches were fed daily with dog chow ßake diet
during acclimation (Lee 2000). Medium-sized mud
loaches (⬇1 yr old, body length ⬇10.6 cm, body
weight ⬇8.5 g) were used for the experiments.
Septic Tank Design. In total, six AST, three AnAST,
and one control AST systems of apartments in Seocho
Borough were selected for the study due to persistent
complaints by residents of mosquito nuisance in the
area. A diagram showing the layout of the general
design of the septic system is provided (Fig. 1). Detailed information on the efßuent chambers of ASTs
and AnASTs is recorded in Table 1.
Sampling. Collection and preservation of water
samples were performed in accordance with the OfÞcial Test Methods of Water Quality ES 04130.1
(Anonymous 2011). Samples were taken during the
laboratory test. Surface water (from a depth of 30 cm)
was collected aseptically from sedimentation chamber
(SE) and efßuent chamber (EF) of the three ASTs
(AST-1, AST-2, and AST-5) and the three AnASTs
using thoroughly cleaned 1-liter low density polyethylene collection bottles because Cx. p. molestus larvae
have been found in various types of breeding places
between depths of 5 and 50 cm (Salit et al. 1996).
Collections occurred between 9:30 a.m. and 11:30 a.m.
The water samples were transported immediately to
the laboratory in portable ice boxes.
Physical, Chemical, and Microbiological Measurements. All samples were analyzed according to the
OfÞcial Test Methods of Water Quality (Anonymous
2011). The pH was measured on site with a portable
pH meter (ES 04306.1). Dissolved oxygen (DO) and
biological oxygen demand (BOD) were measured by
a modiÞed Winkler-azide method (ES 04308.1 and ES
04305.1). Chemical oxygen demand (COD) was determined by an acidic permanganate titrimetric
method (ES 04315.1). Chlorine and total organic carbon (TOC) were determined by chlorine titration (ES
04309.2) and UV-persulfate oxidation (ES 04311.1),
respectively. Turbidity was measured using a Hach
model 2100AN turbidimeter (Loveland, CO; ES
04313.1). Total coliform bacteria were measured using
a pour plate method (ES 04701.3).
Laboratory Test Procedure. Groups of Þve healthy,
unsexed mud loaches were placed separately into 400by 470- by 781-mm high density polyethylene containers containing 50 liter water collected from SE and
EF of the three ASTs and the three AnASTs stated
previously. Continuous aeration was provided using a
July 2014
CHAE ET AL.: CONTROL OF Cx. p. molestus BY M. mizolepis
819
Fig. 1. General design of anaerobic septic tank system (A) and aerobic septic tank system (B). Abbreviations: SC, settling
chamber; AC, aeration chamber; SE, sedimentation chamber; EF, efßuent chamber; I, inlet; O, outlet; VO, ventilating opening.
Shinhwa Hitech Zephyros ZP-40 air pump (Gwangju,
Gyeonggi, South Korea). Distilled water served as a
reference standard. Mud loaches were fed daily as
stated previously. The high density polyethylene containers were held at 25 ⫾ 2⬚C in darkness, which is the
same conditions as those of septic tank systems. Mortalities were determined at 3, 6, and 12 h posttreatment
and then at every 12-h interval until 14 d posttreatment. A Þsh was considered dead if it did not move
when prodded with a wooden dowel. The dead Þsh
were used for pathological analysis of gills and bases
of the pectoral Þns because these organs come into
immediate contact with surrounding water and therefore are responsive to water quality (Syasina et al.
Table 1. Size and capacity of effluent chamber test sites used
in this study
Systema
Size (m3)
Capacity (ton)
AST-1
AST-2
AST-3
AST-4
AST-5
AST-6
AnAST-1
AnAST-2
AnAST-3
Control AST
12.0 by 3.3 by 1.6
3.9 by 2.1 by 1.6
1.2 by 2.3 by 1.2
3.8 by 1.2 by 1.8
2.0 by 1.8 by 1.6
2.1 by 2.0 by 1.8
3.6 by 3.5 by 1.6
3.2 by 3.6 by 2.6
3.2 by 2.4 by 1.8
2.7 by 1.9 by 2.8
63.4
13.1
3.3
8.2
5.8
7.6
Ð
Ð
Ð
14.4
a
AST, aerobic septic tank; AnAST, anaerobic septic tank.
2012). All treatments were replicated four times using
Þve mud loaches per replicate.
Predation of Cx. p. molestus larvae by a mud loach
was examined using SE- and EF-collected water of the
three ASTs and distilled water. A single healthy, unsexed medium-sized mud loach was placed into a 300by 300- by 350-mm glass aquarium containing 10 liters
of test water. Based on the preliminary test results,
groups of 1,500 third-instar Cx. p. molestus larvae were
added. The mosquito larvae in each test aquarium
were examined subsequently to determine the number surviving at 24 h. The aquaria were held under the
same conditions as those used for mud loach survival.
A direct-contact mortality bioassay was used to
evaluate the toxicity of temephos 20% EC to mediumsized mud loaches. The manufacturerÕs recommended
rate (10 ml/ton) and twofold rate of the formulation
were separately applied to 300- by 300- by 350-mm
glass aquaria containing 18 liter EF-collected water of
the three ASTs. Groups of 10 mud loaches were separately put into the aquaria. Treatment and control
mud loaches were held under the same conditions as
stated previously. Fish mortalities were determined at
12-h interval for 6 d. All treatments were replicated
three times using 10 mud loaches per replicate.
Field Evaluation Procedure. Because Cx. p. molestus larvae bred only in EF water (Lee 2006), a Þeld
assessment was conducted in the chambers of the six
ASTs (Table 1) over a period of 18 wk from 05 October
Means within a column followed by the same letter are not signiÞcantly different (P ⫽ 0.05, Bonferroni method).
a
AST-SE, sedimentation chamber of aerobic septic tank; AST-EF, efßuent chamber of aerobic septic tank; AnAST-SE, sedimentation chamber of anaerobic septic tank; AnAST-EF, efßuent chamber of
anaerobic septic tank.
b
The mean values ⫾ standard errors are given.
c
Dissolved oxygen.
d
Biological oxygen demand.
e
Chemical oxygen demand.
f
Total organic carbon.
10.7 ⫾ 2.70b
8.7 ⫾ 1.52b
25.8 ⫾ 1.07a
20.3 ⫾ 0.19a
1.4 ⫾ 0.01c
F ⫽ 49.54; df ⫽ 4, 10;
P ⬍ 0.0001
4.7 ⫾ 1.64b
4.7 ⫾ 1.39b
29.5 ⫾ 0.88a
28.5 ⫾ 5.04a
0.1 ⫾ 0.01c
F ⫽ 52.47; df ⫽ 4, 10;
P ⬍ 0.0001
0.04 ⫾ 0.000a
0.04 ⫾ 0.003a
0.07 ⫾ 0.012a
0.07 ⫾ 0.012a
0.01 ⫾ 0.000b
F ⫽ 18.36; df ⫽ 4, 10;
P ⫽ 0.0001
12.8 ⫾ 3.72a
12.1 ⫾ 2.74a
26.6 ⫾ 4.57a
22.0 ⫾ 4.12a
0.8 ⫾ 0.33b
F ⫽ 15.42; df ⫽ 4, 10;
P ⫽ 0.0003
6.0 ⫾ 1.02a
5.9 ⫾ 0.94a
7.1 ⫾ 0.27a
7.1 ⫾ 0.27a
6.6 ⫾ 0.01a
F ⫽ 0.85; df ⫽ 4, 10;
P ⫽ 0.5243
AST-SE
AST-EF
AnAST-SE
AnAST-EF
Distilled
ANOVA
7.0 ⫾ 0.07b
7.1 ⫾ 0.00b
7.6 ⫾ 0.06a
7.5 ⫾ 0.03a
7.4 ⫾ 0.01a
F ⫽ 33.45; df ⫽ 4, 10;
P ⬍ 0.0001
11.3 ⫾ 5.53ab
10.2 ⫾ 4.57ab
26.5 ⫾ 4.94a
21.3 ⫾ 1.96a
0.3 ⫾ 0.05b
F ⫽ 10.85; df ⫽ 4, 10;
P ⫽ 0.0012
Turbidity
(NTU)b
Chlorine
(%)b
CODe
(mg/liter)b
BODd
(mg/liter)b
DOc
(ml/liter)b
pHb
Test
watera
Results
Water quality data indicated that the mean physical
and chemical parameter values varied according to the
test water (Table 2). The water from the AnASTs had
signiÞcantly higher pH, turbidity, and TOC than the
water from the ASTs. No signiÞcant difference in DO,
BOD, COD, and chlorine concentration was observed
among the septic tank-collected water. SigniÞcant difference in bacterial counts was observed among the
septic tank-collected water (Table 3). The bacterial
counts were signiÞcantly higher in the AnAST-collected water than in the AST-collected water (F ⫽
206.44; df ⫽ 4, 10; P ⬍ 0.0001).
Physical and chemical characteristics of aerobic septic tank-collected water, anaerobic septic tank-collected water, and distilled water
2011 to 07 February 2012. Based on the laboratory test
results and a previous feeding study in which a medium-sized mud loach consumed an average of 1,193
Cx. p. pallens larvae in 24 h when contained in a 1-m2
rice plot (Lee 2000), mud loaches were released at a
rate of one Þsh per 900 Cx. p. molestus larvae into
AST-3 EF, AST-4 EF, and AST-6 EF water. The manufacturerÕs recommended rate (10 ml/ton) of temephos 20% EC was applied to AST-1 EF, AST-2 EF, and
AST-5 EF water based on capacity of EF. Because of
the complaints of mosquito nuisance, mud loaches
were introduced in the three temephos-treated ASTs
28 d posttreatment. Samples were taken through the
hole of EF with 350-ml long-handled dippers. Five dips
(each 300 ml) were taken from a depth of 30 cm of
each septic tank before treatment and on days 3, 7, 14,
21, 28, 42, 56, 84, 112, 119, and 126.
Data Analysis. For water quality, the data obtained
through analysis for various parameters in control
(distilled water) and septic tank collected water were
subjected to analysis of variance (ANOVA). Data pertaining to the concentrations of coliform bacteria and
the number of mosquito larvae consumed per Þsh per
day in control and septic tank-collected water were
transformed using log (␹ ⫹ 1). Mud loach mortality
percentages in control and septic tank-collected water
and % reduction of larvae in septic tanks were transformed to arcsine square root. ANOVA was performed
on transformed data. The Bonferroni multiple-comparison method was used for comparison of means
(SAS Institute 2004). Difference between the treatments with two rates of temephos in percent mortality
was evaluated after transforming data into arcsine
square root values followed by a StudentÕs t-test (SAS
Institute 2004). Mosquito reduction was calculated
according to the formula: % reduction ⫽ [(C2 ⫻ T1/
C1) ⫺ T2)/(C2 ⫻ T1/C1)] ⫻ 100, where C1 is number
of mosquito larvae sampled from EF water of control
AST before treatment, C2 is number of mosquito larvae sampled from EF water of control AST, T1 is
number of mosquito larvae sampled from EF water of
AST before treatment with temephos 20% EC or mud
loaches, and T2 is number of mosquito larvae sampled
from EF water of AST after treatment with the test
materials (Lee 2002). Means ⫾ SE of untransformed
data are reported.
Vol. 51, no. 4
TOCf
(mg/liter)b
JOURNAL OF MEDICAL ENTOMOLOGY
Table 2.
820
July 2014
CHAE ET AL.: CONTROL OF Cx. p. molestus BY M. mizolepis
Table 3. Mean coliform counts for aerobic septic tank water,
anaerobic septic tank water, and distilled water
Test watera
Coliform counts, cfub/ml
(mean ⫾ SE)
AST-SE
AST-EF
AnAST-SE
AnAST-EF
Distilled
1.456 ⫻ 10 ⫾ 84.7b
1.897 ⫻ 103 ⫾ 1286.0b
8.104 ⫻ 104 ⫾ 8078.9a
6.700 ⫻ 104 ⫾ 30197.6a
0c
3
Means within a column followed by the same letter are not significantly different (P ⫽ 0.05, Bonferroni method).
a
AST-SE, sedimentation chamber of aerobic septic tank; AST-EF,
efßuent chamber of aerobic septic tank; AnAST-SE, sedimentation
chamber of anaerobic septic tank; AnAST-EF, efßuent chamber of
anaerobic septic tank.
b
Colony-forming unit.
All mud loaches survived in SE- and EF-collected
water of the three ASTs. However, all mud loaches
died within 3Ð12 h after introduction in SE- and EFcollected water of the three AnASTs. Dead mud loaches showed the signs of pathology in the gills and at
the bases of the pectoral Þns, compared with the gills
and the bases of the pectoral Þns of survived mud
loaches in the ASTs (Fig. 2). Gill and pectoral Þn
damage were found in all mud loaches studied (20
loaches) and characterized by hyperplasia and hemorrhages, respectively.
The number of Cx. p. molestus larvae consumed by
a mud loach was determined using only SE- and EFcollected water of the three ASTs and distilled water
24 h posttreatment because all mud loaches died in the
AnAST water. The mean numbers of larvae consumed
were 1,072 larvae in SE-collected water, 1,058 larvae in
EF-collected water, and 1,376 larvae in distilled water.
There was no signiÞcant difference in larval consumption among the treatments (F ⫽ 4.65; df ⫽ 2, 6; P ⫽
0.06).
821
Table 4. Percentage reduction of Cx. p. molestus larvae after
treatment with temephos 20% emulsifiable concentrate, M. mizolepis in effluent chamber water of aerobic septic tanks, or both
DATa
0
3
7
14
21
28
42
56
84
112
126
No. larvae/300 ml (% reduction of mosquito larvae)
Control
AST-EF-T/Mb
AST-EF-Mc
21.2
20.2
21.4
24.1
22.4
20.4
19.1
21.2
17.1
15.2
14.1
15.2
0.8 (94 ⫾ 1.2)ab
1.2 (91 ⫾ 2.5)b
2.0 (82 ⫾ 8.5)bc
4.6 (66 ⫾ 5.2)cd
7.9 (40 ⫾ 5.6)d
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
11.6
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
0 (100)a
Means within a column followed by the same letter are not significantly different (Bonferroni method).
a
Days after treatment.
b
The efßuent chamber (EF) water of aerobic septic tank (AST)
treated with 10 ml/ton of temephos 20% emulsiÞable concentrate (T).
The mud loach (M) introduction into efßuent chamber water of two
ASTs was performed 28 d posttreatment.
c
The efßuent chamber water of AST treated with mud loaches (M).
After 6 d of exposure to the 10 ml/ton formulation
of temephos, no mortality of loaches was observed.
However, exposure to the 20 ml/ton rate resulted in
signiÞcantly greater mortality (73 ⫾ 3.3%) toward
mud loaches (P ⬍ 0.0001, StudentÕs t-test).
The mean larval populations had averages of 21.2,
15.2, and 11.6 larvae per dip in the three AST-EFtemephos per mud loach (T/M), three AST-EF-mud
loach (M), and control AST, respectively, before
treatment (Table 4). In the AST-EF-M, no mosquito
larvae were observed from the third DAT through the
end of the survey period for 18 wk, documenting that
the mud loach introduction provided complete suppression of Cx. p. molestus. In the AST-EF-T/M, there
was signiÞcant difference in % reduction of mosquito
larvae (F ⫽ 28.10; df ⫽ 5, 12; P ⬍ 0.0001). Treatment
with 10 ml/ton of temephos 20% EC resulted in sharp
decrease in numbers of the mosquito larvae at 3 DAT.
However, the mosquito larvae increased from an average of 1.2 larvae per dip at 7 DAT to an average of
7.9 larvae per dip at 28 DAT. The average mosquito
reduction rates by the formulation treatment were 91
and 40% at 7 and 28 DAT, respectively. After the
introduction of mud loaches into the AST-EF-T/M at
28 DAT, complete mosquito suppression was maintained until the termination of the study.
Discussion
Fig. 2. Pathologic symptoms of a mud loach sampled
from the anaerobic septic tanks. Dead mud loaches in water
collected from sedimentation chamber and efßuent chamber
of anaerobic septic tanks showed pathological changes in the
gills (a) and at the bases of the pectoral Þns (b), compared
with the gills (c) and the bases of the pectoral Þns (d) of
survived mud loaches in sedimentation chamber- and efßuent chamber-collected water from the aerobic septic tanks.
(Online Þgure in color.)
The current laboratory and septic tank studies
clearly indicate that the mud loach has the potential
for biological control of Cx. p. molestus in the aerobic
septic tank systems based on the predation of mosquito larvae, larval reduction, and mud loach survival
data. Of the Þsh species bred in rice Þelds in South
Korea, the weather loach, Mizolepis anguillicaudatus
(Cantor), and mud loach are two of the most promising biological control agents (Kim et al. 1994; Lee
822
JOURNAL OF MEDICAL ENTOMOLOGY
1998, 2000). It has been reported that predation of Cx.
p. pallens larvae by a weather loach varied according
to the developmental stage of the Þsh. Two mature,
two medium-sized (⬇1 yr old), and two immature
weather loaches consumed 168, 144, and 113 thirdinstar Cx. p. pallens larvae per day in a 10-liter aquarium, respectively (Kim et al. 1994). Lee (2000) studied
predation of third-instar Cx. p. pallens larvae by a
medium-sized mud loach in semiÞeld condition. He
reported that a mud loach consumed an average of
1,193 of 1,200 third-instar larvae offered daily in a 1-m2
rice plot. In the current study, a mud loach consumed
an average range of 1,072, 1,058, and 1,376 of 1,500
third-instar Cx. p. molestus larvae offered daily in an
18-liter aquarium containing SE- and EF-collected water of the ASTs and distilled water, respectively, but
there was no signiÞcant difference in the number of
larvae consumed among the treatments.
After introduction of mud loaches either with or
without temephos, complete mosquito reduction was
observed, indicating the importance of mud loaches as
a biological control agent. This Þnding indicates that
mud loaches may serve as an effective mosquito control agent in an AST system. The reduced control
efÞcacy observed in the temephos treatment may
be attributable to a dilution effect of the larvicide,
rather than physical and chemical characteristics of
septic tank water. This effect occurs when water containing temephos is continuously discharged out of the
EF tank. In an earlier study, temephos was reported to
be undetectable in AST water at 30 min after treatment (Anonymous 2012). It has been suggested that
the chemical characteristics of septic tank water, such
as DO and turbidity, did not seem to affect the abundance or the control of Cx. pipiens larvae in septic
tanks (no description of septic tank systems) treated
with temephos (Kang et al. 2011). Temephos 5% granule applied at 10 ppm active ingredient (AI) to scrap
tires was reported to provide 100% mortality of Aedes
triseriatus (Say) larvae for 14 mo with a drop to 21%
mortality after 21 mo (Beehler et al. 1991). However,
application rate of 2 ppm (AI) of temephos 1% water
soluble granule to EF of septic tanks resulted in 100,
68, and 43% mortality of Cx. pipiens larvae at 8, 29, and
50 DAT, respectively (Kang et al. 2011), suggesting a
need for repeated treatments for the effective mosquito control.
Numerous factors can inßuence the condition of
Þsh in different water systems: high sediment load,
water quality, high or elevated concentrations of contaminating organic chemicals in water and bottom
sediments, accumulation of pollutants in Þsh tissues,
bacterial impurities, predators, parasites, and competition (Jackson et al. 2001, Syasina et al. 2012). Both
pathogenic and nonpathogenic microorganisms are
present in human feces. Pathogenic bacteria in septic
tank systems have been well described by Geldreich
and LeChevallier (1999) and United States Environmental Protection Agency (USEPA; 2006). Histopathological changes and morphological alterations in
Þsh by microbial pathogens or pollutants, such as
harmful chemicals or heavy metals, have been well
Vol. 51, no. 4
described by Woo and Bruno (2011) and Syasina et al.
(2012), respectively. Of the Þve categories of pathologic changes in gill tissue observed, bacterial gill disease is most lethal (Bullock 2012). The disease is characterized by the presence of large numbers of the long,
thin gram-negative bacteria Flavobacterium branchiophila (causing hyperplasia) or Cytophaga columnaris
(causing gill necrosis) on the gills of a broad range of
cultured coldwater and warmwater Þshes (Starliper
and Schill 2011, Bullock 2012).
No information is available concerning the survival
of larvivorous Þshes in septic tank systems. In the
current study, gill hyperplasia and hemorrhages at the
bases of the pectoral Þns were detected in all dead
mud loaches in SE- and EF-collected water of the
AnASTs. The coliform counts were signiÞcantly
higher in the AnAST water than in the AST water.
Based on the current and previous studies, impaired
gill circulation and hemorrhages at the bases of Þns
might result in the acute death of mud loaches. However, an involvement of the physical and chemical
characteristics of septic tank water cannot be ruled
out, although mud loach is one of the Þsh species most
resistant to impacts of environmental factors and contamination in aquatic environments (Park and Kim
2001, Cho et al. 2012). In anaerobic systems, one would
think that it was oxygen deprivation because BOD
increases with the increase in the bacteria colonies
(Kim et al. 1996). However, the three parameters that
seem to have major differences between AST and
AnAST were pH, turbidity, and TOC. Detailed tests
are needed to understand relationship between the
mud loach death and these parameters. Based on the
poor performance, mud loaches are not appropriate
for use in AnAST systems for management of Cx. p.
molestus. Aerobic digestion is less sensitive to household chemicals than anaerobic digestion, and aerobic
systems break down human waste at a rate much faster
and clean water than anaerobic systems, resulting in
less odor and environmental impact (Marchaim 1992).
In conclusion, reasonable mosquito control in aerobic septic tanks can be achieved by mosquito breeding season stocking of a rate of one mud loach per 900
Cx. p. molestus larvae. Use of temephos can be limited
because of continuous discharge of the larvicide out of
the septic tank. The causes of the histopathological
changes (gill hyperplasia or hemorrhage at the bases
of pectoral Þn) occurring in mud loaches in AnAST
water needs to be examined in greater detail.
Acknowledgments
This research was supported by the Mosquito Control
Program funded by Seocho Borough and Research Institute
of Agriculture and Life Sciences (to Y.-J.A.).
References Cited
Anonymous. 2009. Sewerage statistics: single process septic
tank. The Statistics Korea, Ministry of Land, Transport
and Maritime Affairs, Daejeon, South Korea. (http://
kostat.go.kr).
July 2014
CHAE ET AL.: CONTROL OF Cx. p. molestus BY M. mizolepis
Anonymous. 2011. OfÞcial test methods of water quality.
Ministry of Environment, Gwacheon, Gyeonggi, South
Korea. (http://www.law.go.kr).
Anonymous. 2012. Study on water pollution standard limits
guideline of insecticides used in septic tank. National
Institute of Food and Drug Safety Evaluation, Korea Food
and Drug Administration, Osong, Chungbuk, South Korea.
Beehler, J. W., T. C. Quick, and G. R. Defoliart. 1991. Residual toxicity of four insecticides to Aedes triseriatus in
scrap tires. J. Am. Mosq. Control Assoc. 7: 121Ð122.
Bullock, G. L. 2012. Bacterial gill disease of freshwater
Þshes. United States Department of the Interior, Fish and
Wildlife Service. (http://www.koiÞshponds.com/bgd.
htm).
Cho, Y. S., B. S. Kim, D. S. Kim, and Y. K. Nam. 2012.
Modulation of warm-temperature-acclimation-associated 65-kDa protein genes (Wap 65–1 and Wap 65–2) in
mud loach (Misgurnus mizolepis, Cypriniformes) liver in
response to different stimulatory treatments. Fish ShellÞsh Immunol. 32: 662Ð 669.
Geldreich, E. E., and M. LeChevallier. 1999. Microbiological quality control in distribution systems, pp. 18.1Ð18.49.
In R. D. Letterman (ed.), Water quality and treatment Ð
a handbook of community water supplies, 5th ed.
McGraw-Hill, New York, NY.
Ghosh, S. K., and A. P. Dash. 2007. Larvivorous Þsh against
malaria vectors: a new outlook. Trans. R. Soc. Trop. Med.
Hyg. 101: 1063Ð1064.
Jackson, D. A., P. R. Peres-Neto, and J. D. Olden. 2001. What
controls who is where in freshwater Þsh communitiesÐthe
roles of biotic, abiotic, and spatial factors. Can. J. Fish
Aquat. Sci. 58: 157Ð170.
Kang, J. O., D. K. Jeong, D. K. Lee, H. Kang, and M. KiarieMakara. 2011. Field efÞcacy and non-target effects of
temephos granules against Culex pipiens (Diptera: Culicidae) and microorganisms in septic tanks, Republic of
Korea. Entomol. Res. 41: 60 Ð 65.
Kasai, S., O. Komagata, T. Tomita, K. Sawabe, Y. Tsuda, H.
Kurahashi, T. Ishikawa, M. Motoki, T. Takahashi, T. Tanikawa, et al. 2008. PCR-based ideniÞcation of Culex pipiens complex collected in Japan. Jpn. J. Infect. Dis. 61:
184 Ð191.
(KCDC) Korea Centers for Disease Control and Prevention.
2008. Guideline for the use of insecticide, fungicide, and
rodenticide Ð Control of medical insects. Korea Centers
for Disease Control and Prevention, Seoul, South Korea.
Kim, H. C., M. S. Kim, and H. S. Yu. 1994. Biological control
of vector mosquitoes by the use of Þsh predators, Moroco
oxycephalus and Misgurnus anguillicaudatus in the laboratory and semiÞeld rice paddy. Korean J. Entomol. 24:
269 Ð284.
Kim, D. H., W. A. Scak, and D. Gera. 1996. Measurement of
fecal coliform bacteria from domestic waste. Toxicol.
Environ. Chem. 56: 249 Ð257.
Lee, D. K. 1998. Effect of two rice culture methods on the
seasonal occurrence of mosquito larvae and other aquatic
larvae and other aquatic animals in rice Þelds of southwestern Korea. J. Vector Ecol. 23: 161Ð170.
Lee, D. K. 2000. Predation efÞcacy of the Þsh muddy loach,
Misgurnus mizolepis, against Aedes and Culex mosquitoes
in laboratory and small rice plots. J. Am. Mosq. Control
Assoc. 16: 258 Ð261.
Lee, D. K. 2002. Biological control of Culex pipiens pallens
(Diptera: Culicidae) by the release of Þsh muddy loach,
823
Misgurnus mizolepis in natural ponds, Korea. Korean J.
Entomol. 32: 43Ð 47.
Lee, D. K. 2006. Occurrence of Culex pipiens (Diptera, Culicidae) and effect of vent net sets for mosquito control
at septic tanks in south-eastern area of the Korean peninsula. Korean J. Appl. Entomol. 45: 51Ð57.
Lee, D. K., J. H. Jeon, H. S. Kang, and H. S. Yu. 1997.
Analyses of aquatic ecosystems in organic and conventional farming rice Þelds and mosquito larval populations.
Korean J. Entomol. 27: 203Ð214.
Marchaim, U. 1992. Biogas processes for sustainable development. FAO Agricultural Services Bulletin 95, Food and
Agriculture Organization of the United Nations, Rome,
Italy.
Opong-Mensa, K. 1984. A review of temephos with particular reference to West Africa onchocerciasis control program. Residue Rev. 91: 47Ð 69.
Park, J. Y., and I. S. Kim. 2001. Histology and mucin histochemistry of the gastrointestinal tract of the mud loach,
in relation to respiration. J. Fish Biol. 58: 861Ð 872.
Rozendaal, J. A. 1997. Vector controlÑmethods for use by
individuals and communities. World Health Organization, Geneva, Switzerland.
Salit, A. M., S. S. Al-Tubiakh, S. A. El-Fiki, and O. H. Enan.
1996. Physical and chemical properties of different types
of mosquito aquatic breeding places in Kuwait State, pp.
185Ð193. In K. B. Wildey (ed.), Proceedings of the 2nd
International Conference on Insect Pests in the Urban
Environment, 7Ð10 July 1996, Exeter, BPC Wheatons Ltd,
Exeter, Devon, United Kingdom.
SAS Institute. 2004. Online Doc䉸, version 9.2. SAS Institute,
Cary, NC.
Sharma, V. P., and A. Ghosh. 1994. Larvivorous Þshes of
inland ecosystems. In Proceedings of the MRC-CIFRI
workshop New Delhi, 27Ð28 September 1984. India Malaria Research Centre (ICMR) Publication, Delhi, India.
Sohn, S. R. 2005. The distribution of Culex pipiens group at
the mid-southern area of Korea. J. Daegu NatÕl Univ. Ed.
28: 1Ð9.
Sohn, S. R. 2007. Seasonal prevalence of the Culex pipiens
group (Diptera: Culicidae) larvae occurring in a puddle
in the basement of an apartment building. Entomol. Res.
37: 95Ð99.
Starliper, C. E., and W. B. Schill. 2011. Flavobacterial diseases: columnaris disease, coldwater disease and bacterial
gill disease, pp. 606 Ð 631. In P.T.K. Woo and D. W. Bruno
(eds.), Fish diseases and disorders: viral, bacterial and
fungal infection, vol. 3. CAB International, Wallingford,
Oxfordshire, United Kingdom.
Syasina, I. G., A. V. Khlopova, and L. M. Chukhlebova. 2012.
Assessment of the state of the gibel carp Carassius auratus
in the Amur river basin: heavy-metal and arsenic concentrations and histopathology of internal organs. Arch.
Environ. Contam. Toxicol. 62: 465Ð 478.
(USEPA) United States Environmental Protection Agency.
2006. Occurrence and monitoring document for the Þnal
ground water rule. (http://www.epa.gov/safewater).
Woo, P.T.K., and D. W. Bruno (eds.). 2011. Fish diseases
and disorders: viral, bacterial and fungal infection, vol. 3.
CAB International, Wallingford, Oxfordshire, United
Kingdom
Received 7 October 2013; accepted 31 March 2014.