Transcript Malaria Transmission Seminar Slides

Differences in the behavior organization structure of Aedes
albopictus, Anopheles stephensi & Culex pipiens fourth
instar larvae; an insight into potential ways for developing a
larvacide.
Itaki. R, Suguri. S, Arif. H, Fujimoto. C, Harada. M
Department of International Medical Zoology, Faculty of
Medicine, Kagawa University.
Itaki. R, Suguri. S, Arif. H, Fujimoto. C, Harada. M
Department of International Medical Zoology, Faculty of
Medicine, Kagawa University.
Introduction
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Mosquitoes as vectors transmit several human parasitic
and viral diseases.
Certain species that were previously regarded as nonvectors have become a threat to humans as they find
new habitats to establish themselves. Example Aedes
albopictus in North America (Durhopf & Benny 1990).
Emergence of potential vectors has been attributed to
changes in environment and influence of modern life
(Gould 2006).
Malaria entomology & vector control, Learner’s guide, WHO.
Introduction
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Indeed the ability to establish itself in a new
environment and change in host feeding behavior
patterns have resulted in emergence of new diseases
among humans (Gould 2006).
Therefore the ability of the mosquito to establish itself
in a new environment is directly affected by the ability
of the larva to exploit its aquatic habitat.
‘Airport malaria in non-endemic countries is due to lack
of mosquito to establish resident population.
Malaria entomology & vector control, Learner’s guide, WHO.
Previous studies on larva behavior
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Jones (1954) listed Anopheles quadrimaculatus behaviors.
Walker & Merritt (1991) developed a catalogue of
behaviors for Aedes triseriatus.
Durhopf & Benny (1990) looked at the alarm
responses of several strains of Aedes albopictus and Aedes
aegypti.
Tuno et al (2004) examined the submergence time of
Anopheles gambiae Giles larva.
Response to visual stimuli, to waves at water’s surface,
to falling water droplets and to vibration have all been
studied to some degree (Clements 1999).
Previous studies on larva behavior
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The different behaviors observed have been
proposed to be survival mechanisms to escape
predators or from being washed away in
container breeding mosquitoes during rainfall.
In a controlled environment larva become
habituated to the stimulus. This habituation is
lost with a new stimulus.
Studies on feeding behavior & larval toxins
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Development of bacterially (B. thuringiensis israelensis;
B.sphaericus) derived stomach toxins as control agents
for mosquito larva (Lacey & Undeen 1986) resulted in
an increased interest in their feeding behavior.
Difference in feeding behavior may result in different
abilities to exploit their habitat (Duhrkopf & Benny
1990).
Species –specific differences in larval behavior may
allow certain species to adapt more quickly to new
environment and establish themselves more rapidly
(Workman & Walton 2003, Yee et al 2004).
Previous studies on larva behavior
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Aedes species tend to use their entire habitat for feeding
(Walker & Merritt 1991).
Anopheles species generally feed at the air-water interface
but can dive and feed at the bottom of their aquatic
environments (Tuno et al 2004).
Culex species forage primarily by filtering the water
column and tend to concentrate their effort near the
water’s surface (Yee et al 2004).
Yee et al (2004) found out that mosquito larvae tend to
modify their feeding behavior according to the food
environment.
Rationale for this study
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General differences in the larval behavior of Anopheles
species compared to Aedes and Culex species can be
deduced from other studies but the details are obscure.
Few studies on Anopheles larva behavior.
Aims & objectives
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To observe and compare the behaviors of
Anopheles, Aedes and Culex mosquito larvae.
Determine the differences in the organizational
structure of their behavior.
Determine the differences in the amount of
time larvae spent in each behavioral state.
Malaria entomology & vector control, Learner’s guide, WHO.
Materials & method – species & larvae
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Aedes albopictus, Anopheles stephansi & Culex pipiens.
Larvae & adults maintained using standard protocols in
an insectary (temp. 26oC, RH 65%) with a 15 hrs, 8 hrs
day-night cycle.
Light provided by x4 40-watt fluorescent light bulbs.
Eggs hatched in 250ml of de-chlorinated tap H2O in
plastic cups.
2-3 instar stage larvae transferred to a 33x24x7-cm
pans.
Larvae fed on Tetra Min baby fish food.
4th instar larvae used in experiment.
Materials & methods - observation
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All observations done in a 59x28x35-cm glass
aquarium.
Aquarium filled with de-chlorinated tap H2O &
incubated for 1 week to permit growth of
microorganisms on walls, floor, water column and airwater interface (Walker & Merritt 1991).
4th instar larvae pipetted into aquarium and allowed to
acclimatized for 1 hr prior to behavior recording.
Total of 40 4th instar larvae used in experiment.
Definitions of behaviors same as Walker & Merritt
(1991) and Clements (1999) with minor modifications.
Observation methods
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20 4th instar larvae were placed in the aquarium and
behavior recorded after 1hr using handy cam (Sony Co.
Japan) video recorder (Brackenbury 2001).
An individual was chosen at random an its behavior
recorded for 5 mins. After filming 10 larvae, video
taping experiment repeated with a new batch of 20 4th
instar larvae.
This “focal-individual sampling” method (Altmann
1974) possible due to low larval density and tempo of
larval behavior allowing observer to track an individual
larva.
Great care taken not to record a larva twice.
Data analysis
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Video tape recordings converted to DVD and viewed
on a Mac Os X version 9.3 personal computer.
Sequential organization & relationship among behaviors
- data of transitions from one behavior to another for
all observations were collated into a matrix of
preceding and succeeding behaviors
Diagonal of matrix held at logical zero, assuming a
behavior can not follow itself.
Comparison of behaviors – kinematograph constructed
from transitions matrix showing frequencies of
significant non-random transitions between behavioral
states.
Data analysis – transition matrix
Succeeding behavior
Preceding
behavior
Dive
Rise
Floor feed
Allogroom
etc
Dive
Rise
Floor feed Allogroom etc
0
0
0
0
0
Data analysis
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Time budget also created for each behavior.
Transition matrix collapsed about the cell of interest to
form a 2x2 contingency table for significant testing.
Significant testing for first-order transitions done by G
statistics (P<0.005) on Microsoft excel.
Mean time spent for each behavior compared by oneway ANOVA for 3 independent samples.
Significant differences in mean time further analyzed by
pair-wise comparison using Tukey HSD test.
Results – activity
Mosquito species
Mean behavior per
observation
Mean behavior per
larva observed
Aedes albopictus
(n=22)
18.4
31.82
Culex pipiens
(n=20)
9.83
11.30
Anopheles stephansi
(n=22)
9.18
13.77
Results - activity
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Aedes albopictus was the most active species compared to
Culex pipiens & Anopheles stephansi.
Culex pipiens was slightly more active overall when
compared with Anopheles stephansi.
Individually, Anopheles stephansi was more active than
Culex pipiens.
Aedes albopictus 4th instar larvae kinematograph
float/
interfacial
feed (3.28)
35.68
98.31
float/
suspension
feed (19.16)
95
3.24
1.62
29.19
float/
brushwall
(9.0)
30.27
44.74
rise
(17.13)
23.73
brushwall (14.0)
48.05
32.20
71.43
dive
(11.63)
91.67
1.75
31.63
autogroom (1.81)
40.26
74.77
18.69
bottom feed (17.68)
wriggle
swim
(2.68)
Culex pipiens 4th instar larvae kinematograph
float/
interfacial
Feed (1.08)
100
30.68
float/
suspension
feed (118)
22.72 float/
75 brushwall
(5.75)
18.18
95.62
27.27
78.13
rise (8.09)
45
90
dive (23.30)
30
bottom feed (73.10)
wriggle-swim
(2.08)
Anopheles stephensi 4th instar larvae kinematograph
float/
98.15
interfacial
feed (21.61) 46.30
2.63
float/
suspension
feed (5.71)
6.48
85.71
82.35
4.17
float/
brushwall (1.44)
97
34.26
allogroom/
feed (1.33)
dive (23.78)22.22
rise (15.0)
brushwall (1.50)
29.17
54.55
50
33.33
wriggleswim (4.20)
50
45.83
underwater/still (11.33)
20.33
bottom feed (40.52)
Float/suspension feed
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Common behavior among all three species.
Significant difference in the mean time (F=55.94, df=2,
P<0.0001).
Significant difference between Aedes albopictus & Culex
pipiens (P<0.01, HSD [0.01]=30.59), between Anopheles
stephansi & Culex pipiens (P<0.01, HSD [0.01)=30.59).
Not significant between Aedes albopictus & Anopheles
stephansi.
Float/interfacial feed
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Common behavior among all three species.
Significant difference in the mean time
(F=11.16, df=2, P<0.0001).
Significant difference between Anopheles stephansi
& Aedes albopictus and between Anopheles stephansi
& Culex pipiens (P<0.01, HSD[0.01]=17.24).
Not significant between Aedes albopictus & Culex
pipiens.
Dive
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Common behavior among all three species.
Aedes albopictus dived frequently & vertically.
Culex pipiens & Anopheles stephansi did not dive
frequently.
Culex pipiens dived at an angle while Anopheles
stephansi dived in a zigzag manner & sometimes
passively.
Brushwall
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Common behavior in Aedes albopictus and
Anopheles stephansi but not in Culex pipiens.
Aedes albopictus brushed the wall while at the
surface as well as after diving.
Anopheles stephansi brushed the wall only after
diving.
No significant difference in the mean time spent
among the three species (F=0.22, df=2,
P=0.81).
Float/brushwall
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Common behavior among all three species.
No significant difference in mean time spent
performing this behavior.
Wriggle swim
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Common behavior among all three species.
Culex pipiens performed this behavior near the
surface, Anopheles stephansi at the bottom of the
water column.
Aedes albopictus used this behavior both near the
surface and the bottom.
No significant difference in the mean time
(F=0.93, df=2, P=0.4).
Bottom feed
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Common behavior among the three species.
Significant difference in the mean time
(F=21.28, df=2, P<0.0001).
Aedes albopictus significantly difference to both
Culex pipiens (P<0.01, HSD[0.01]=25.67) &
Anopheles stephansi (P<0.05, HSD[0.05]=20.42).
Significant difference between Culex pipiens &
Anopheles stephansi (P<0.01, HSD[0.01]=25.67).
Autogroom
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Common only in Aedes albopictus & Anopheles
stephansi. Not Culex pipiens.
No significant difference in mean time (F=0.06,
df=2, P=0.81).
Allogroom
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Common in Anopheles stephansi but not in Aedes
albopictus & Culex pipiens.
Commence when larvae bumped into each other
while in the “float/suspension feed” state.
Larvae moved away from each other upon
touching.
Rise
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Common in all three species.
No significant difference in mean time (F=4.38,
df=2, P=0.02.
Underwater still
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Only observed in Anopheles stephansi.
Not observed in Aedes Albopictus or Culex pipiens.
Underwater mouth-swim not observed in all
three species.
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Percentage time (%).
Percentage of time spent spent by larvae in specific behavior.
100
90
80
70
60
50
40
30
Anopheles
stephensi
Aedes
albopictus
Culex
pipiens
20
10
0
Larval behavior.
Conclusion
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There is significant difference in the behavioral
organization of Aedes albopictus, Anopheles stephansi and
Culex pipiens larvae.
Some larval behaviors that are common in one species
are not observed in other species.
There is significant difference in the mean time spent
performing each behavior.
These differences in larval behavior could be attributed
to differences in feeding behavior as well as physiology.
These differences can be exploited to design new larval
control methods.
Thank you
Thank you