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Hydrobiologia (2015) 749:113123

DOI 10.1007/s10750-014-2154-4

PRIMARY RESEARCH PAPER

Behavioral changes in mosquito larvae induced by copepods


predation
Amit Kant Awasthi Juan Carlos Molinero
Cheng-Han Wu Kun-Hsien Tsai
Chwan-Chuen King Jiang-Shiou Hwang

Received: 5 September 2013 / Revised: 25 July 2014 / Accepted: 16 December 2014 / Published online: 15 January 2015
Springer International Publishing Switzerland 2015

Abstract In aquatic ecosystems, body size has a


remarkable influence on the hierarchical structure of
food webs and trophic interactions. The trophic
relationship between mosquito larvae and copepods
offers an exception to the conspicuous feature that the
bigger consumes the smaller. We here address this
ecological question by investigating the predation
impact of the epibenthic cyclopoid copepod Megacyclops formosanus on a 30-h old mosquito larvae Aedes
aegypti in a controlled environment. Our quantitative
assessment depicted the effect of M. formosanus on
30-h old mosquito larvae. We found that copepod

Handling editor: Boping Han

captured mosquito larvae on an average 1.6 0.20


larvae ind-1 h-1 with an attack speed
22.80 8.52 mm s-1. In addition, copepod predation
dramatically affected mosquito behavior. In the presence of predator, mosquito larvae significantly
decreased browsing and filtering paths, while using
mixed foraging tactics and shifting the habitat use
from the bottom to surfacing or airwater interfacing.
These results provide a mechanistic understanding of
the interaction between mosquito and cyclopoid
copepods, and show that copepod predation constrains
the habitat use of mosquito larvae. Copepod predation
pressure results in reduced facultative behavioral
responses that challenge mosquito life history and
ultimately percolate its population fitness.

A. K. Awasthi  J. C. Molinero  C.-H. Wu 


J.-S. Hwang (&)
Institute of Marine Biology, National Taiwan Ocean
University, 2 Pei Ning Rd., Keelung 202, Taiwan, ROC
e-mail: jshwang@mail.ntou.edu.tw

Keywords Predatorprey interaction  Behavior 


Video-recording experiment  Megacyclops  Aedes
aegypti

J. C. Molinero
GEOMAR Helmholtz Centre for Ocean Research Kiel,
Marine Ecology/Food Webs, Dusternbrooker Weg 20,
24105 Kiel, Germany

Introduction

K.-H. Tsai
Institute of Environmental Health, College of Public
Health, National Taiwan University, Taipei 100, Taiwan,
ROC
C.-C. King
Institute of Epidemiology, National Taiwan University,
Taipei 106, Taiwan, ROC

Copepods and insects are two of the most abundant


metazoans in aquatic and terrestrial ecosystems,
respectively. These groups generally inhabit different
realms, however, they coexist in freshwater systems
during the early life stages of some insect taxa, e.g.,
mosquito. This interaction yields a predatorprey
relationship where the relatively small cyclopoid

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copepods prey on the larger early mosquito larval


stages. In aquatic ecosystems, body size is an important determinant of the structure of communities, and
its influence is manifested in different ways within and
across multiple levels of ecosystem organization
(Barnes et al., 2010; Yvon-Durocher et al., 2011;
Wirtz, 2012). Size structure has a remarkable influence on trophic interactions, playing a role as important as the species identity (Brooks & Dodson, 1965;
Woodward & Warren, 2007; Barnes, 2008). Indeed,
the numerical abundance in aquatic food web components is hierarchically structured by their size, and
predators are generally larger than their prey (Woodward & Warren, 2007).
Predators may have strong prey selectivity and their
feeding behavior varies according to the prey (Kumar
et al., 2008; Hwang et al., 2009; Dhanker et al., 2012;
Klecka & Boukal, 2012). Most predators use hydrodynamics, chemosensory, and visual cues to detect the
prey (Trager et al., 1990; Hwang et al., 1994; Hwang
& Strickler, 2001; Aksnes et al., 2004). In turn, the
vulnerability of prey related to size may vary
throughout species ontogeny. For instance, some
prey are vulnerable to predation during early life
stages, but are not when they grow and become larger
than the predators (Nilsson & Bronmark, 2000), as it is
the case of the mosquito-copepod relationship.
In aquatic ecosystems, while larger predators
generally subdue their prey without using special
tactics, smaller predators use specialized tactics such
as poisoning, trapping, or social foraging to capture
larger or equally sized prey (Pough et al., 1990; Menin
et al., 2005). To date, most of the evidence on
behavioral changes promoted by predation relates to
the effect of larger predators on smaller prey (Nakazawa et al., 2013). However, less documented are the
behavioral changes in larger prey, e.g., mosquito
larvae, induced by smaller predators, e.g., copepods.
Here we address this ecological question by investigating the influence of the epibenthic cyclopoid
copepod Megacyclops formosanus, smaller predator,
on 30-h old mosquito larvae Aedes aegypti, larger
prey, in a controlled environment. A number of studies
advocate that cyclopoid copepods can reduce mosquito larvae, i.e., Anopheles, floodwater Aedes, and
Culex, from breeding habitats (Marten & Reid, 2007),
but the underlying mechanisms remain elusive. To
tackle this question, we used an experimental
approach to quantify the predatory impact of

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Hydrobiologia (2015) 749:113123

M. formosanus on the behavior and habitat use of


Ae. aegypti early instars. Resolving mortality causes in
early life stages might shed light on mosquito recruitment and ultimately on population fitness.

Materials and methods


Biological data
Both predator and prey organism used in this study coexist
in nature. Predatory copepods M. formosanus (length
1.16 0.077 mm and width 0.385 0.028 mm) were
sampled from a pond situated in Northern Taiwan, at
Keelung (2580 36.720 N 121460 54.7600 E) by means of
oblique hauls using a 153 lm plankton net with a 0.5 m
diameter. Ten gravid females were taken out under a
dissecting microscope and were inoculated into a glass
aquarium filled with 3 l of a culturing medium consisting
of ciliates, rotifers, and alga Chlorella vulgaris in
dechlorinated tap water. Copepods were raised at room
temperature (27 2C), at pH 7 and a photoperiod of
12:12 h (L: D) inside an incubator. Both, culturing
medium (ciliates, rotifers and algae) and mosquito larvae
were provided as food 3 days prior to the experiment to
familiarize the copepods with the variety of food types.
Copepods were starved during 12 h prior to experiments.
Eggs of Ae. aegypti were obtained from the Institute of
Environmental Health, College of Public Health, National
Taiwan University, and hatched in deoxygenated autoclaved tap water. Fifty 30-h old Ae. aegypti mosquito
larvae
(length
1.72 0.11 mm
and
width
0.32 0.05 mm) were used for each trial. The larvae
were kept at 27 2C and 75 10% relative humidity
in a lightdark cycle of 14:10 h. Likewise to predators,
prey larvae were not fed prior to the experiment. To
prevent any expansion of the disease vector, remaining Ae.
aegypti larvae were killed in boiling water after the
experiment.
Predation experiments and video recordings
Copepod predation rate (mosquito larvae copepod-1
h-1) was assessed by quantifying the feeding rates in a
glass aquarium before conducting video-recording
experiments. Larvae were counted in the aquarium
after 1 h. Eight trials were performed for treatment
(with predator) and the control (without predator). The
densities of prey and predator were set according to the

Hydrobiologia (2015) 749:113123

predation rate of M. formosanus prior to video


recording. Ten adult female copepods were gently
poured from a beaker into a transparent acrylic
aquarium (dimensions 5 9 5 9 6 cm) filled with
125 ml filtered autoclaved tap water. Fifty Ae. aegypti
larvae were added to the aquarium as prey after placing
it on a vibration-free bench top. For video-recording
experiments, each group of animals was independently
acclimatized to experimental conditions for 30 min
prior to video recording. Five trials with the predator
and 4 trials without the predator (control) were
recorded at a temperature of 27 2C in a dark room
by using a single fluorescent table lamp (1,400 lux).
One vertical and one horizontal camera were used for
video recordings, and one light lamp was placed at the
sidewall of the experimental aquarium at approximately 30 cm.
The mosquito larval behavior was digitally
recorded at 30 frames s-1 for 30 min for each trial
(Wu et al., 2010; Awasthi et al., 2012a). The recorded
videos were digitized, and mosquito larvae activity
was assessed using manual tracking software Track-It
(Iguana Gurus, Milwaukee, WI, USA) to transform
mosquito and copepod behaviors into x and y coordinates (Nihongi et al., 2004).
Behavioral descriptions of Aedes aegypti larvae
Four major activities were identified based on the
classification by Walker & Merritt (1991) and Juliano
& Reminger (1992) (1) Resting: the larvae stood still,
with their heads hanging downward at the airwater
interface and siphons touching the airwater interface
for respiration. (2) Browsing: The larvae cruised the
water column along the walls of the container, placing
their mouthparts against the walls to collect food. (3)
Filtering: The larvae floated in the open water column
and filtered the water with the help of their mouthparts
and the movements of mouth brushes. The body
posture remained steady and the siphon was held
opposite to the direction of movement without
abdominal flexion. (4) Flexing: The larvae used their
abdomen as a paddle, and they traveled the whole
water column by active paddling.
The four spatial locations of the mosquito larvae
were identified based on Juliano & Reminger (1992)
(1) Surface: The siphon of the mosquito larvae was in
contact with the airwater interface; (2) Walls: The
larvae browsed within 1 mm of the container wall; (3)

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Bottom: The larvae browsed within 1 mm of the


bottom; and (4) Middle: The larvae stayed away from
the surface, wall, and bottom.
Behavioral and kinematic data analysis
Two views of the experimental aquarium were used to
analyze both copepod and Aedes larval behaviors. The
horizontal camera was used to analyze the larval
behavior, whereas the vertical camera was used to
analyze copepod capturing. Observations started
2 min after the introduction of the prey and predator
in the experimental tank, and observations were taken
on a 2 min frequency basis for 30 min. The trajectories of 50 individuals were observed through recorded
video. The number of Aedes larvae at their respective
positions and activities was counted at each 2-min
intervals and larvae numbers were transformed into
proportions for further statistical analysis. Each 2-min
interval reading was treated as one independent
observation for the experiment. Fifteen observations
were obtained and analyzed from each trial. Seventyfive observations were made in 5 trials in the presence
of predator, and 60 observations were made in the
control group. To assess the behavioral changes in
mosquito larvae on a 2D scale, the 10th minute was
considered as the starting point for the principal
component analysis (PCA).
The average speed of mosquito larvae was assessed
as follows:
Speed

Total distance traveled


:
Total time taken

The Pythagorean theorem was used to obtain the


distance traveled by the mosquito larvae based on the
2-point coordinates traveled by larvae (x1, y1) and (x2,
y2). Thus, the distance traveled is
d 2 x2 y2
q
d x2  x1 2 y2  y1 2 ;
where x (x1 and x2) and y (y1 and y2) represent the
coordinates.
Attack speed and attack distance of Megacyclops
formosanus
The M. formosanus is an ambush predator, and its
attack speed was calculated when the copepod jumped

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directly onto the prey to capture. The attack distance


was measured from where the copepod aimed at the
prey and took the final jump to capture the mosquito
larvae. All the data for speeds and distances were
obtained from the recorded videos and pooled for
statistical analysis (Awasthi et al., 2012a, b).
Statistical analysis
PCA was performed to identify patterns in mosquito
larval behavior in the presence and absence of the
predator. PCA was performed with the software PAST
(Hammer et al., 2001). The best tracks for speeds and
distances traveled were identified, selected, and analyzed from each trial by using the recorded videos. The
speeds, travel distances, activities, and positions of the
mosquito larvae in the presence and absence of the
predators were compared using the non-parametric
KruskalWallis test. A Bonferroni correction was also
performed to reduce the probability of Type I errors
during multiple comparisons. Statistical tests were
performed at the 95% confidence interval.

Hydrobiologia (2015) 749:113123

in distances traveled were observed in the larvae


(KruskalWallis test, v2 (5, N = 84) = 56.87,
P \ 0.0001; Fig. 1b). The distances traveled were
higher in the control group. The browsing and filtering
distances were significantly different from the thrashing distance (Bonferroni-corrected P \ 0.0001). Figures 2a and 3a show that 66.37% of Aedes larvae
occupied the surface and bottom positions, and
33.63% of the larvae occupied the mid- and sidewalls
of the aquarium. The surface was the most preferred
place by the Aedes larvae in both conditions. The
sidewalls and middle did not differ significantly
(Bonferroni-corrected P = 1; Figs. 2a, 3a). Resting
and browsing were the most important activities for
mosquito larvae in the control group, and 74.18% of
the larvae performed resting and browsing, whereas
23.82% of the Aedes larvae performed thrashing and
filtering (Figs. 2b, 4a). The Aedes mosquito larvae
proportions for positions and activities demonstrated
that the larvae did not change their behavior temporally (Figs. 3, 4). Surface and bottom are the most
important positions in the mosquito larvae lifestyle.
Resting was the most favored activity for Aedes larvae
in both conditions.

Results
The M. formosanus predation rate on Aedes mosquito
larvae was 1.6 0.20 larvae ind-1 h-1. The M.
formosanus length and width were 1.16 0.077 and
0.385 0.028 mm, respectively, whereas the mean
length and width of (30-h old) Aedes larvae were
1.72 0.11 and 0.32 0.05 mm, respectively. The
copepods showed a significantly higher attack speed
compared to prey escape movements such as browsing, filtering, and flexing (P \ 0.001). The copepod
attack
speed
and
attack
distance
were
22.80 8.52 mm s-1 and 1.37 0.54 mm, respectively (Table 1).
Aedes aegypti mosquito larval behavior
in the absence of Megacyclops formosanus
The KruskalWallis test results were significant
(KruskalWallis test, v2 (6, N = 101) = 77.38,
P \ 0.0001) for traveled speeds between the prey
and predator movements (Table 1; Fig. 1). The predatory copepod attack speed was significantly higher
than the prey movements (Bonferroni-corrected
P \ 0.0001; Table 1; Fig. 1a), and significant changes

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Aedes aegypti mosquito larval behavior


in the presence of Megacyclops formosanus
The speed of mosquito larvae movements (e.g.,
browsing, filtering, and thrashing) did not show
substantial behavioral changes in response to a
predator (Fig. 1a). Only the filtering and thrashing
speeds were significantly different (Bonferroni-corrected P = 0.045; Fig. 1a). The distances traveled
were lower in mosquito larvae (KruskalWallis test,
v2 (5, N = 84) = 56.87, P \ 0.0001; Fig. 1b). The
thrashing distance was significantly higher than all
other distances traveled (Bonferroni-corrected
P \ 0.0001) except for thrashing in the control group
(Bonferroni-corrected P = 1; Fig. 1b). The browsing
distance was significantly lower (18.34 mm) in the
presence of a predator (Fig. 1b). Predatory copepods
enhanced mosquito larvae surfacing to 68.78%, making the bottom the least favored place to stay for larvae
(Figs. 2a, 3b). Predator presence had a significant
effect (KruskalWallis test, v2 (7, N = 396) =
336.36, P \ 0.0001) on larvae positions. Larvae
positions (i.e., surface, bottom, middle, and sidewalls)
were significantly compromised in the presence of

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Table 1 Key characters of M. formosanus as a predator


Characters

Number of
observations (n)

Measured values

Mean body length

15

1.16 0.07 mm

Mean body width

15

0.38 0.02 mm

Predation rate

1.6 0.20 larvae


ind-1 h-1

Attack speed

17

22.80 8.52 mm s-1

Attack distance

17

1.37 0.54 mm

M. formosanus (Figs. 2a, 3b). Mosquito larvae


increased surfacing significantly in the presence of
predatory copepods (Bonferroni-corrected P \ 0.0001;
Figs. 2a, 3b, d). Predator presence increased the
resting and thrashing and 86.35% of the larvae
performed these 2 activities, while filtering and
browsing were the two least favored activities in the
presence of a predator (Fig. 2b). Aedes larvae activities were significantly influenced by Megacyclops
(KruskalWallis test, v2 (7, N = 396) = 353.14,

Fig. 1 a Aedes aegypti mosquito larvae speed in the absence


and presence of Megacyclops formosanus shown using a bar
chart (Mean SD). b A. aegypti mosquito larvae distances
traveled in the absence and presence of a predator

(Mean SD). Figures B, F, and T represent browsing, filtering,


and thrashing, respectively. Bonferroni-corrected multiple
comparisons are denoted by a, b, and c at 95% confidence
intervals

Fig. 2 a Aedes aegypti mosquito larvae positions shown in the


absence and presence of M. formosanus during video recording,
where the characters S, M, SW, and BO respectively, represent
surface, middle, sidewall, and bottom. b Larvae activities in the

absence and presence of a predator where the characters R, T, B,


and F, respectively, represent resting, thrashing, browsing, and
filtering. Bonferroni-corrected multiple comparisons are
denoted by a, b, c, d, and e at 95% confidence intervals

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Fig. 3 Aedes aegypti (30-h old) larvae proportions of positions


displayed temporally during 30 min of video recording a control
(without predator); b experiment (with Megacyclops formosanus). Temporal changes in the profile of mosquito larvae
positions c control (without predator); d experiment (with

predator). The Ae. aegypti larvae position results show


logarithmic changes in surface and bottom positions in the
presence of a predator, and the coefficient of determinations (R2)
for the surface and bottom were 0.849 and 0.838, respectively

P \ 0.0001; Figs. 2b, 4b). Resting was significantly


higher than the other activities (Bonferroni-corrected
P \ 0.0001). Cyclopoid presence significantly
increased Aedes larvae resting (Bonferroni-corrected
P \ 0.0001: Figs. 2b, 4b, d). For mosquito larvae
positions, the surface position was positively correlated with predator presence, whereas the bottom
position was negatively associated with predator
presence (Fig. 3d). Aedes larvae showed positive and
negative correlations for resting and browsing activities, respectively (Fig. 4d). Aedes larvae increased

surfacing (P \ 0.0001 and R2 = 0.849) and reduced


bottoming (P \ 0.0001 and R2 = 0.838; Fig. 3d).
Aedes larvae reduced thrashing but increased their
resting logarithmically. The R2 values of both
activities were 0.84 (P \ 0.0001; Fig. 4d).
Principal components (PC) were used according to
the cumulative 75% of the total data variance. This
criterion conferred two PCs that summarized 80.59%
of the variation in activities and positions (Table 2;
Fig. 5). A positive PC1 score showed that Aedes larvae
preferred browsing at the bottom, whereas a negative

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Fig. 4 Aedes aegypti (30-h old) larvae proportions of activities


displayed temporally during 30 min of video recording a control
(without predator); b experiment (with Megacyclops formosanus). Temporal changes in the profile of mosquito larvae
activities c control (without predator); d experiment (with

predator). The Ae. aegypti larvae activity results show


logarithmic changes in thrashing and resting activities in the
presence of a predator, and the coefficient of determinations (R2)
for both the activities were 0.84

score indicated that more larvae preferred resting at


the surface. A greater positive PC2 score indicated that
more larvae preferred thrashing in the middle, whereas
a negative score indicated that more larvae preferred
bottoming behavior (Table 3). Some positive and
negative correlations appeared among these positions
and activity categories. Resting was positively correlated with the surface, yet negatively correlated with
thrashing (Table 4). Browsing was positively correlated with the bottom. PCA identified the behavioral
differences of these two mosquito larvae groups (with
and without a predator) on a 2D scale, clearly
indicating that the mosquito behavior in the control

and experiment groups exhibited two planes and


showed that these undersized predatory copepods
strongly affected larval behavior. The area covered by

Table 2 Principal components extracted by cumulative percent for the mosquito larval behavior in the presence and
absence of M. formosanus
PCs

Eigen value

Proportion

Cumulative

Percent

5.57

0.69

0.69

69.63

0.87

0.10

0.80

80.59

0.64

0.08

0.88

88.59

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Table 4 Frequencies of mosquito larvae for each activity and
position (proportion of observations) with and without predator
for all pooled treatments, correlation matrix obtained through
principal component analysis (N = 99); where R, T, B, F, S,
SW, M, and BO, respectively, correspond to resting, thrashing,
browsing, filtering, surfacing, side walls, middle and bottom

0.49

-0.92

-0.68

-0.73

T
B
F

SW

BO

0.92

-0.67

-0.51

-0.88

-0.67

0.56

-0.46

-0.22

-0.55

0.66

-0.90

0.64

0.45

0.88

-0.70

0.58

0.41

0.62

-0.76

-0.62

-0.91

0.46

0.53

S
SW
M

Fig. 5 Principal component analysis (PCA) characterizes two


groups with and without a predator; open and closed circles
represent the control and experiment. In PCA, PC1 explained
69.63% and PC2 explained 10.96% of the total variance. An
oval shape represents 95% of the group behavior of Aedes
mosquito larvae. C represents the control group (without
predator), and P represents the experimental group with a
predator. A positive PC1 score showed that Aedes larvae
preferred browsing at the bottom, whereas a negative score
indicated that more larvae preferred resting at the surface

0.32

Here we have shown that cyclopoid copepods,


smaller predators, substantially induce behavioral
changes in mosquito larvae, larger prey. Small predators typically use specific strategies such as poisoning,
trapping, or social foraging (Pough et al., 1990; Menin
et al., 2005). However, copepods used sudden bursts of
speed to attack larger prey (Table 1). Being an ambush
predator, the copepod uses a sit and wait tactics and
fast propulsive thrust to perform a sudden fast jump to
attack their prey. Their strong mandibles give them an
advantage over larger prey (Kirboe et al., 2009;
Awasthi et al., 2012b).
Copepods play a key role in structuring aquatic
invertebrate communities, and are commonly distributed in both benthic and pelagic environments (Kerfoot & Sih, 1997; Rao & Kumar, 2002; Marten &
Reid, 2007). In nature, the early four Ae. aegypti larval
instars are well adapted to benthos, which provide a
refuge against flushing out by rain events (Koenraadt
& Harrington, 2008). In our experiment, in the
presence of predatory copepods mosquito larvae
shifted toward the airwater interface, with a decline
of using the bottom habitat while avoiding encounters
with predators. The reduction in filtering and the
browsing associated with the presence of a predator

an oval in the PC graph represents 95% of the behavior


of the two groups (Fig. 5).

Discussion
Body size is arguably the most fundamental trait of
an organism as it determines essential processes
including foraging, growth, reproduction, and evolution (Peters, 1983; Brown et al., 2004). In recent
decades, most allometric principles on preypredator
interactions have focused on the size relationship of
the prey with predator. Allometric principles also
influence the range of potential behavioral responses
for a given organism to a specific size of the prey
and predator.

Table 3 Loadings of principal components for mosquito larval behavior with and without M. formosanus
R

PC1

-0.40

-0.30

0.40

0.34

PC2

-0.07

0.53

-0.31

-0.20

SW

BO

-0.41

0.33

0.25

0.40

-0.15

0.20

0.75

-0.63

Loadings stated in bold characters display the important loadings for the principal component analysis (PCs loadings [0.40); where
R, T, B, F, S, SW, M, and BO, respectively, represent resting, thrashing, browsing, filtering, surfacing, side walls, middle, and
bottom

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might hinder foraging, and therefore, may negatively


affect mosquito fitness (Fig. 1b). Indeed, filtering and
browsing are typically associated with foraging,
whereas increased resting is correlated with decreased
feeding (Jualiano & Reminger, 1992; Kesavraju et al.,
2007; Kesavaraju & Juliano, 2008).
Prey often counteracts a predator by reducing
predator capture efficiency or increasing escape
responses (McPeek, 1996; Van Buskirk & McCollum,
2000; Awasthi et al., 2012a, b). Aedes larvae did not
show anti-predator responses of increased speed, they
either increased the resting at the airwater interface
or increased flexing. Although Juliano & Reminger
(1992) considered thrashing/flexing at the bottom the
most vulnerable behavior for mosquito larvae, flexing
offers several advantages to mosquito larvae against
predators. The flexing larvae have a round body that
provides them a larger ring-shaped body, making it
difficult for copepods to grasp, handle, and capture
them because of feeding appendage limitations (gape
constraints). Ultimately, this offers more protection
against predators, although it is an energy-consuming
process. Survival is challenged by the presence of
predators, and to cope with this stress, mosquito larvae
use thrashing as a trade-off mechanism (Brackenbury,
2001; Ferrari et al., 2008).
The selectivity pattern of copepods is associated
with the energy costs of prey capture, and is also
related to prey size. The key components of predation,
i.e., handling time, encounter rate, and attack rate,
often scale with the body sizes of the interacting
organisms (Rao & Kumar, 2002; Mahjoub et al.,
2011). Prey selectivity in cyclopoid copepods is based
on several prey factors, including size, shape, external
setae or spines, taste, and behavior (Kumar & Rao,
2003; Kumar et al., 2008; Mahjoub et al., 2011). Prey
handling time generally increases exponentially with
increasing prey size (Rao & Kumar, 2002). Previous
studies on immature mosquitoes have shown that
copepods actively select and prey on instar-I larvae
and avoid instar-IV larvae, regardless of the species
(Kumar & Rao, 2003). By increasing resting at the air
water interface (Figs. 3, 4), larvae reduce hydrodynamic cues to predatory copepods (Hwang et al., 1994;
Hwang & Strickler, 2001).
In nature, flexibility in anti-predator behavior
enables prey to cope with various types of predators
(Krupa & Sih, 1998), before and after the encounter
(Scharf et al., 2003). Avoidance behavior reduces the

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probability of encounters with a predator, whereas


escape behavior increases the probability of surviving
after an encounter (Mathis & Vincent, 2000; Scharf
et al., 2003; Gilbert & Buskey, 2005). Prey constantly
faces a trade-off between foraging to gain energy for
fitness and avoiding predation. The mosquito larvae in
this study showed dichotomous anti-predator
responses to avoid predator escape either by fast
flexing or resting at the surface, and also by shifting
locations to avoid encounters with predators. Mosquito larvae displayed such dichotomous strategies in
response to predator or predation cues, either by
fleeing or freezing (Ferrari et al., 2008). Furthermore,
freezing behavior might help mosquito larvae be less
detected and likely increases the chance of survival in
the presence of predators (Ferrari et al., 2008). The
deflective behavior of prey frequently focuses on
escaping from predators that are poised to attack at a
close distance with a fast speed or in the presence of
ambush predators, such as cyclopoid copepods (Cooper, 1998; Awasthi et al., 2012b). A fast and vigorous
response by flexion is likely because of late detection
of a predatory threat by Ae. aegypti larvae (Ferrari
et al., 2008). Reactive deflection is effective when the
predator is close to the prey, whereas if the predator is
detected from far enough, the best option is to flee
without displaying reactive deflection (Ydenberg &
Dill, 1986).
Although the predatory copepods M. formosanus
are smaller than the Ae. aegypti larvae, Megacyclops
predation pressure forces Aedes larvae to modify their
behavior. Under these challenges, prey requires optimizing foraging strategies, and fulfilling their nourishment to complete their life cycle. Our results
consistently showed that copepod predation appears as
a natural potential biocontrol of mosquito larvae
reducing their facultative behavioral responses. The
observed shift in behavioral patterns might affect
mosquito population fitness and potentially have
implications for disease expansion in terrestrial ecosystems. The mechanistic understanding we provide
on the predatorprey relationship of cyclopoid copepods and mosquito larvae sheds light on a potentially
important component of the mosquito-related disease
dynamics.
Acknowledgments This study was supported by the funding
provided by projects NSC 98-2621-B-019-001-MY3, NSC
99-2611-M-019-009, NSC 100-2611-M-019-010, NSC

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101-2611-M-019-011, NSC 102-2611-M-019-003, and NSC
102-2923-B-019-001-MY3 from Ministry of Science and
Technology of Taiwan, ROC. JCM was supported by the NSC
grant 102-2811-B-019-002.

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