INTRODUCTION
Dengue fever is considered as a rapidly emerging ar- thropod-borne viral disease with about 50 million den- gue infections occurring worldwide annually1. The trans- mission of this disease is achieved through a bite of an infected female mosquito specifically Aedes aegypti and Ae. albopictus. These mosquito species are incriminated as the principal vectors for the transmission of dengue virus in urban and sub-urban areas2–4. Aedes aegypti has a greater chance of transmitting dengue virus (DENV) and remains as one of the most important vector of DENV5. As there is no available vaccine available at present, the primary control in the transmission of the disease is by targeting its principal mosquito vector.
Hence, the need to study and understand the mosquito’s biology of dengue vectors are crucial towards an effec- tive vector control programme.
The life-cycle of Ae. aegypti mosquitoes has two phases, the aquatic phase (larval and pupal stages) and the terrestrial phase (adult and egg stages)6. The comple- tion of their development is strongly associated with hu-
mans because anthropophilic female adults require blood meals to be able to lay eggs7. Humans also provide artifi- cial water-holding containers (AWHCs) that serve as the mosquito’s niche for completing its life-cycle. Ae. aegypti are urban dwelling mosquitoes and the presence of AWHCs in these areas benefits this mosquito in its pro- liferation, population productivity and drive dengue dis- ease outbreaks. It has been reported that Ae. aegypti can breed in 37 kinds of man-made water-holding contain- ers8–16. The developmental stages are determined by the genetic programme of the organism and influenced by prevailing environmental conditions which may include temperature, photoperiod, diet gradient and density gra- dient17. However, the developmental rate in AWHCs has always been assumed and never been tested, thus we con- sidered it as an important perspective to investigate the variations in developmental rate.
The objective of our study is rather straightforward,
“What is the influence of different AHWCs (glass, por- celain, plastic, clay, concrete, etc.) on the development rate and ecdysis periods of Ae. aegypti?” Knowing which AWHCs encourage development of Ae. aegypti could be
Ecdysis period and rate deviations of dengue mosquito vector, Aedes aegypti reared in different artificial water-holding containers
Beatriz Louise J. Almanzor
1, Howell T. Ho
2& Thaddeus M. Carvajal
31Natural Science Department, St. Scholastica’s College, Manila, Philippines; 2Department of Biological Sciences and Biotechnology, Hannam University, Daejeon, South Korea; 3Department of Civil and Environmental Engineering, Ehime University, Matsuyama, Japan and Biology Department, De La Salle University, Manila, Philippines
ABSTRACT
Background & objectives: Artificial water-holding containers (AWHCs) have been well-documented in many Aedes aegypti studies for dengue surveillance and developmental research. Hence, we investigated the role of different AHWCs on the development and ecdysis period of Ae. aegypti dengue vector, a container breeding mosquito.
Methods: Nine types of AWHCs, namely glass, polystyrene foam, rubber, steel, porcelain, plastic, aluminum, clay and concrete, were chosen for the study. All AWHCs were subjected to the developmental assay for an observation period of 10 days. Regression and hazard analyses were employed to the developmental stages and the characteristics of the AWHCs.
Results: The observations revealed that Ae. aegypti development is fastest in glass and polystyrene containers while slowest in concrete containers. Moreover, pupal ecdysis appears to be the most affected by the characteristics of the AWHCs based on regression and hazard analyses.
Interpretation & conclusion: Characteristics of the container that can regulate water temperature seem to be the driving force with regards to the slow or fast development of Ae. aegypti, more notably in pupal ecdysis. The results of the study further strengthen our understanding on the dynamics of Ae. aegypti’s developmental biology to different characteristics of artificial water containers. This, in turn, would aid in devising vector control strategies against dengue especially in endemic areas.
Key words Aedes aegypti; artificial water-holding containers; dengue; ecdysis period
used for information campaigns regarding proper disposal of specifically hazardous AWHCs. Determining the type of container that would serve as the best environment for the developing mosquito could also give an idea to lay- man as to what containers should be “searched and de- stroyed”18. This, in turn, may lead to proper waste man- agement practices that could be modified to pay special attention to those hazardous AWHCs.
MATERIAL & METHODS Mosquito samples and hatching
Laboratory cultured Ae. aegypti eggs were obtained from the Research Institute for Tropical Medicine, Alabang, Mutinlupa City, on pieces of filter paper. At least 600 eggs were submerged in three plastic pans with 24 h standing water. Upon hatching, I instar larvae were transferred using medicine droppers to their respective containers for the experimentation.
Preparation of artificial water-holding containers (AWHCs)
A total of nine AWHCs differing in material type, were identified based on exhausted literature search. The types of AWHCs used in this study included glass, poly- styrene foam, rubber, steel, porcelain, plastic, aluminum, clay and concrete. Each AWHC is also described based on their colour darkness, opacity, relative illumination, and dimensions (Table 1). The colour darkness, opacity relative illumination were qualitatively measured; dimen- sions such as length, width, height, volume, mouth pe- rimeter and surface area were quantitatively measured using measuring devices and computed, while the spe-
cific heat capacity and thermal conductivity were deter- mined from Engineering tool box19. Room temperature and humidity were controlled using a digital temperature and humidity monitor.
Experimental proper
A total of 27 set-ups were prepared with three repli- cates per container type. Each AWHC was filled with 200 ml of the 24 h standing clean water before placement of the mosquito larvae. Maintenance and care for each set-up was done by feeding the mosquito larvae with equal amounts of crushed dog food slurry. The 20 I instar lar- vae were placed in each set-up with a total of 60 larvae for each container type. A 10-day observation period was fixed wherein mosquitoes were observed periodically at 0.5 day intervals. Due to time intervals between observa- tions and setup of experiment, all periods had ± 0.25 day of variation to the actual occurrence or time. Three peri- ods of the developmental cycle (Fig. 1) were determined and expressed in units of hours namely; late instar ecdysis period [from hatching to late instar (III/IV)], pupal ecdysis period [from late instar (III/IV) to sighting of pupa], and imaginal ecdysis period (from pupa to emergence of imago). The study also defined, total pupal ecdysis pe- riod (from hatching to sighting of pupa) and total imagi- nal ecdysis (from hatching to emergence of imago). All set-ups were regulated to similar environmental condi- tions such as water quality and temperature. An identifi- cation key was used for the identification of the different larval stages20.
Statistical analysis
Analysis of variance (ANOVA) was employed to
Table 1. Characteristics of the nine artificial water-holding containers (AWHCs)
Artificial water- Colour Opacity Relative Length Width Height Volume Mouth Surface Specific Thermal holding containers darkness illumination (cm) (cm) (cm) (ml) perimeter area heat conductivity
(reflection (cm2) (cm2) capacity (W/mK)
of light in (kJ/kgK)
container)
Glass container Light Clear Very good 10.50 10.50 19.50 350 32.97 86.55 0.84 1.05
Polystyrene foam Light Opaque Good 9.10 9.10 6.50 250 28.57 65.01 1.30 0.03
container
Rubber tyre Dark Opaque Poor 61 9 9 500 140 549 2.01 0.13
Steel pot container Light Opaque Good 12.70 12.70 11.80 500 39.88 126.61 0.49 43
Porcelain container Light Opaque Good 5.50 5.50 10.50 300 17.27 23.75 1.07 1.50
Plastic container Dark Opaque Slight 6.20 6.20 11.80 400 19.47 30.18 1.67 0.47
Aluminum container Light Opaque Good 5.80 5.80 11.20 300 18.21 26.41 0.87 205
Clay container Dark Opaque Slight 9.60 9.60 14 500 30.14 72.35 0.92 0.17
Concrete container Dark Opaque Slight 10 10 14.60 400 31.40 78.50 0.75 1.70
compare the AWHCs based on each ecdysis period. More- over, ecdysis periods of different AHWCs were corre- lated to characteristics of containers using Pearson’s cor- relation. Afterwards, models were derived using multiple regression. Hazard analysis was employed to determine differences in the ecdysis rate at which the mosquito samples reared in various AWHCs for late instar, pupa and imago. Results of hazard analysis were expressed as hazard ratios to indicate whether the AHWC tended to promote development of late instar, pupa or imago using concrete container as point comparison. Higher hazard ratios demonstrated greater tendency to develop. Further- more, the tendency for late instar, pupal or imaginal ecdysis was compared using Log Rank test (to determine differences in the late stages), Breslow test (to compare differences in early stages) and Taron-wane test (to com- pare differences in mid-stages). These tests demonstrated which AWHCs tended to promote development in the early, mid or late stages of late instar, pupal or imaginal ecdysis. Significant testing was achieved using an alpha value of 0.5. All computations were done using IBM SPSS Statistics version 20.
RESULTS
The Fig. 2 and Table 2 summarize the three ecdysis periods, i.e. late instar, total pupal and total adult ecdysis period; wherein they are ordered from shortest to longest period (days): glass, polystyrene, rubber, steel, porcelain, plastic, clay and concrete. Total ecdysis period was short- est for glass and polystyrene (average of 7.2 days) con- tainers. Midrange total ecdysis period was observed in rubber, steel, porcelain, plastic and aluminium the aver-
age ranging from 7.53 to 7.77 days and were non-signifi- cantly different from each other. The clay pot and con- crete containers showed/demonstrated longest periods for total ecdysis with an average of 8 and 8.8 days, respec- tively. Among all the containers, the concrete showed the most variable results. Total adult ecdysis period demon- strated similar patterns to that of total ecdysis period. Most notable difference was for the aluminium containers which ranked midrange in total imaginal ecdysis and low in to- tal pupal ecdysis. Total pupal ecdysis periods were short- est in glass, aluminium and polystyrene containers with average of 5.43, 5.58, and 5.6 days and were significantly different from each other. Midrange total pupal ecdysis period was seen in rubber and steel containers with an
Fig. 1: The developmental life cycle of Aedes aegypti which defines operationally the different observation periods of the study.
Fig. 2: Summary of total ecdysis periods (days) from egg to late instar, egg to pupa and egg to adult from the nine artificial water- holding containers (AHWCs): (GC—Glass container; PFC—
Polystyrene foam container; RT—Rubber tyre; SPC—Steel pot container; PoC—Porcelain container; PIC—Plastic container; AC—Aluminum container; CIC—Clay container;
CoC— Concrete container).
Table 2. Analysis of variance (ANOVA) of the different containers for three ecdysis periods.
Ecdysis period (Days) Container p-value Remarks
Glass Polystyrene Rubber Steel Porcelain Plastic Aluminum Clay Concrete
cup foam tyre pot cup cup can pot pot
cup Imaginal ecdysis period
Minimum 2 2 2.50 2 2 2 2 2.50 1.50
Average 2.77 2.78 2.83 2.82 2.78 2.71 3.17 2.97 2.55
Maximum 3.50 3.50 3.50 4 4 3.50 4.50 4 4.50 Very different
95% Confidence 2.66- 2.67- 2.73- 2.71- 2.67- 2.60- 3.06- 2.86- 2.29- 0 significantly
interval 2.88 2.89 2.94 2.92 2.88 2.82 3.28 3.07 2.81
p-value (diffrence 0 0 0 0 0 0 1 0.18 0
from aluminum)
Average rank 4.50 4.50 4.50 4.50 4.50 4.50 9 8 1
Pupal ecdysis period
Minimum 2 2 2 2.50 2.50 2.50 2 2 3
Average 2.59 2.72 2.87 3.03 3.00 3.18 2.73 3.16 5.20
Maximum 3 3 3.50 4 3.50 4 3.50 4.50 6 Very
95% Confidence 2.50- 2.63- 2.77- 2.94- 2.91- 3.08- 2.63- 3.07- 5.09- 0 significantly
interval 2.68 2.81 2.96 3.13 3.09 3.27 2.82 3.25 5.31 different
p-value (Diffrence 0 0 0 0 0 0 0 0 1
from concrete)
Average rank 1 2.50 4 5.50 5.50 7.50 2.50 7.50 9
Late instar ecdysis period
Minimum 3 3 3 3 3 3 3 3 3
Average 3.84 3.88 3.83 3.77 3.86 3.81 3.85 3.88 3.89 Non-
Maximum 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 4.50 0.94 signficiantly
95% Confidence 3.71- 3.75- 3.69- 3.64- 3.73- 3.68- 3.72- 3.74- 3.76- different
interval 3.97 4.01 3.96 3.90 3.99 3.94 3.98 4.01 4.02
Table 3. Correlation analysis of the characteristics of AWHCs to the different ecdysis periods measured
Ecdysis period Colour Opacity Relative Length Width Height Volume Mouth Surface Specific Thermal Interpretation (Days) darkness illumination (cm) (cm) (cm) (ml) perimeter area heat conductivity
(reflection (cm2) (cm2) capacity (W/mK)
of light in (kJ/kgK)
container) Late instar ecdysis period
Correlation coefficient 0.01 0 0 – 0.02 – 0.02 0 – 0.03 – 0.02 – 0.02 0 – 0.01 Not
p-value 0.82 0.96 0.97 0.72 0.68 0.99 0.43 0.70 0.68 0.95 0.87 correlated
Pupal ecdysis period
Correlation coefficient 0.44 0.24 – 0.31 – 0.09 0.12 0.15 0.17 – 0.08 – 0.09 – 0.17 – 0.18 Low to
p-value 0 0 0 0.03 0.01 0 0 0.06 0.05 0 0 moderate
Total pupal ecdysis period
Correlation coefficient 0.35 0.19 – 0.24 – 0.08 0.08 0.11 0.12 – 0.07 – 0.08 – 0.14 – 0.15 Low
p-value 0 0 0 0.07 0.06 0.01 0.01 0.09 0.08 0 0 correlation
Imaginal ecdysis period
Correlation coefficient – 0.04 0.06 0 – 0.02 – 0.07 – 0.01 – 0.01 – 0.02 – 0.02 – 0.08 0.27 Generally
p-value 0.35 0.17 0.92 0.66 0.12 0.79 0.84 0.59 0.60 0.07 0 not correlated
Total imaginal ecdysis period
Correlation coefficient 0.16 0.18 – 0.14 – 0.05 – 0.05 – 0.01 0.10 – 0.05 – 0.05 – 0.05 0.06 Generally not
p-value 0 0 0 0.26 0.23 0.75 0.02 0.24 0.24 0.27 0.19 correlated
average duration of 5.69 and 5.8 days respectively. Long- est total pupal periods were recorded for porcelain, plas- tic, clay and concrete containers with averages of 5.86, 5.97, 6.03 and 8 days respectively.The shortest observed total pupal ecdysis (seen in glass, aluminum and polysty- rene) is significantly different from longest observed to- tal pupal ecdysis (seen in porcelain, plastic, clay and con- crete). It was notably observed that concrete containers showed dramatically delayed total adult ecdysis period (~2.5 days) and total pupal ecdysis period (~1.5 days).
Variations in the total adult ecdysis were almost the same with the exception of concrete containers which had the highest and most different variability in period. The ecdysis period from egg to late instar was not signifi- cantly different among AHWCs with averages ranging from 3.77 to 3.89 days. In terms of variability, no differ- ence was seen among AHWCs.
Correlation analysis revealed that the characteristics of the AHWCs were not correlated to the late instar, adult and total ecdysis period, except for the thermal conduc- tivity for all container types (Table 3). It is noteworthy to mention that thermal conductivity of the containers had a very significant low positive correlation to imaginal ecdysis, while colour darkness, opacity and relative illu-
mination of the containers had a very significant low cor- relation to total imaginal ecdysis period. There was a low to moderate correlation of the characteristics of the AHWCs to pupal and total pupal ecdysis period. The pat- tern of correlation for the pupal ecdysis period and total pupal ecdysis was same and very significantly correlated to colour darkness, opacity, relative illumination, specific heat capacity and thermal conductivity.
Because pupal ecdysis period showed low to moder- ate correlation in most AHWC characteristics, further analysis was done using multiple regression. Of four mod- els were constructed based on the characteristics of the different containers and all were considered equally good, with a correlation coefficient of 0.875 and predictive value of 76.6% (Supplemental Table 1). All four models shared four variables in common-specific heat capacity, colour darkness, volume and thermal conductivity. Models 1, 2 and 3 were similar differing only in one variable, namely contact area, relative illumination and mouth perimeter respectively. On the other hand, Model 4 included addi- tional factors, namely relative illumination, mouth perim- eter, length and surface area.
Hazard analysis (Table 4 and Supplemental Table 2) showed that there was no significant difference in the
Supplemental Table 2. Hazard function comparisons of the different development stages of Aedes aegypti from all artificial water holding containers (AWHCs)
Comparisons Late instar ecdysis period Pupal ecdysis period Imaginal ecdysis period
Chi-square p-value Chi-square p-value Chi-square p-value
Log rank (Mantel-Cox) 3.325 0.912 369.658 0 55.037 0
Breslow (Generalized Wilcoxon) 2.765 0.948 277.948 0 34.056 0
Tarone-Ware 2.955 0.937 317.692 0 41.586 0
Supplemental Table 1. Multiple regression model predicting pupal ecdysis period
AWHC characteristics Model No.
1 2 3 4
R (Correlation coefficient) 0.875 0.875 0.875 0.875
R-square 0.766 0.766 0.766 0.766
p-value 0 0 0 0
Coefficients
Constant (Y-intercept) – 19.868 – 15.761 – 20.255 16.992
Specific heat capacity 3.401 3.375 3.375 – 2.057
Colour darkness – 1.829 – 3.328 – 1.831 3.104
Volume – 0.018 – 0.018 – 0.018 – 0.015
Thermal conductivity – 0.002 – 0.002 – 0.002 – 0.003
Height 0.979 1.022 1.022 –
Width 0.546 0.596 0.554 –
Opacity 10.966 9.433 10.931 –
Contact area 0.001 – – –
Relative illumination – – 1.498 – – 0.176
Mouth perimeter – – 0.013 – 0.526
Length – – – – 0.261
Surface area – – – 0.153
probability of entering late instar ecdysis for all AHWCs.
All the hazard ratios were near 1.0 and showed that haz- ard rates were almost same to that of the concrete con- tainer. Though, there was greater variability of occurrence between the different containers in the later stage of late instar ecdysis period as evidenced by a lower p-value for the Log Rank test. Pupal hazard rate was most affected by the different AHWCs, wherein glass containers showed shortest pupal ecdysis rate. Rubber containers showed a slower rate as compared to polystyrene and aluminium containers, but the differences were primarily seen in the later stages as evidenced with a lower p-value for the Breslow test. Porcelain and steel containers demonstrated similar hazard rates but slower as compared to rubber containers. These differences were observed mainly in the middle stages (~3 days) as evidenced by the low p-value in the Tarone-Ware test. Plastic and clay pot con- tainers showed similar hazard rates but they were slower as compared to porcelain and steel containers. The dif- ferences in their rates reflect in the late stages with the p-value being lowest in the Log Rank test. Concrete con- tainers showed the slowest hazard rate with the most atypi- cal hazard function as compared to the other AHWCs.
Imaginal hazard ratios for the different containers dem- onstrated that plastic containers have the highest hazard ratio. Plastic, porcelain, foam, glass, steel and rubber con- tainers showed most similar hazard rate with fastest de- velopment, while clay containers are midrange in hazard rate. Aluminium and concrete containers have similar hazard rate with almost the same hazard ratio demonstrat- ing the slowest rate of development.
Limitations
The study only focused on the differences of the ecdysis periods and could not consider specific morpho- logical and physiological effects of the different contain- ers. Furthermore, it did not focus on the preference of the mosquito to lay eggs in specific containers.
DISCUSSION
All containers used in the study were conducive for mosquito growth and development in the laboratory set- ting. Furthermore, it was clearly observed that there were differences in the developmental rates in specific periods in different AHWCs. The results of the study emphasized that total ecdysis period was fastest in glass and polysty- rene containers and slowest in concrete container, and pupal ecdysis development appeared to be most affected by the AHWCs based from regression analysis and haz- ard ratios.
Several studies have utilized glass containers for labo- ratory rearing of Ae. aegypti 21–22. Ho et al23 showed that larval development of Ae. aegypti was significantly faster in laboratory-reared glass jars than tyres placed in the field.
Both of these containers were deemed to affect pupal ecdysis based on hazard ratios. Despite of the observed fast developmental rate in glass, wild Ae. aegypti mos- quitoes do not prefer glass containers that much15–24. Poly- styrene containers have been identified to be a breeding site of Ae. aegypti25. However, there is scarcity of litera- ture on the development of the mosquito species in this type of container. On the other hand, cement containers, deemed to take longest period for total ecdysis in the study, is noteworthy because water levels tend to decrease dras- tically due to absorption of water by the container. Hence, frequent maintenance of water levels might have induced larval or pupal stress, thus, an observed slow develop- ment. It was initially expected by the present researchers that either plastic or rubber tyre would provide the fastest developmental rate or ecdysis period. Plastic containers are used in rearing Ae. aegypti mosquitoes in the labora- tory and frequently used for developmental studies26. Gardner et al27 emphasized that higher level of soluble reactive phosphorus and ammonium in plastic water drums favoured larval production of Ae. aegypti in the urban ecosystem. However, despite being a very produc-
Table 4. Hazard ratio comparison of all artificial water holding containers to the concrete container in different development stages Compared to concrete Late instar ecdysis period Pupal ecdysis period Imaginal ecdysis period
container Hazard ratio p-value Hazard ratio p-value Hazard ratio p-value
Glass container 1.074 0.695 125.195 0 1.918 0.059
Polystyrene foam container 1.040 0.828 87.451 0 1.938 0.056
Aluminum container 1.113 0.558 85.688 0 1.020 0.954
Rubber tyre 1.149 0.448 63.815 0 1.801 0.088
Porcelain container 1.053 0.778 54.700 0 1.962 0.050
Steel container 1.209 0.300 49.390 0 1.838 0.077
Plastic container 1.070 0.712 39.644 0 2.175 0.024
Clay container 1.078 0.680 37.735 0 1.482 0.253
tive container for Ae. aegypti mosquitoes, plastic has been observed to be easily susceptible to heat and dryness28. Rubber tyres, on the other hand, has thoroughly been in- vestigated in hastening the development of mosquitoes in the natural environment23, 29 and it was reported by many researchers to be the preferred breeding site.
In regards to the different AHWCs, Chan et al29 showed that Ae. aegypti in clay jars had slower develop- mental rates when compared with those reared in tyres.
There is a scarcity of literature on the influence of metal containers such as steel and aluminum in the develop- ment of the mosquito vector. However, these containers were able to substantially support Ae. aegypti growth and development. Medronho et al15 showed the presence of larvae and pupa in metal containers, but these are not con- sidered to be a preferred breeding site. The reason is that these containers are easily susceptible to heat and dry- ness, making it a less conducive for mosquito develop- ment. Metals have also been reported to release ions that are toxic to mosquitoes30. Gardner et al27 discussed that substances which are said to favour larval production like soluble reactive phosphorus and ammonium were signifi- cantly lower in these type of containers.
Regression and hazard ratio analyses revealed that pupal ecdysis was affected by the AHWCs. In all models of regression, the characteristics of specific heat capac- ity, colour darkness, volume and thermal conductivity were common characteristics in affecting the pupal ecdysis. Three out of the four characteristics might be associated with temperature regulation of the container.
A more extensive study of containers in affecting the de- velopment of Ae. aegypti have been done by Richardson et al31. They reported that both the size and height of the container affect the thermal conductivity regimen in the temporal scale. However, they observed only minor dif- ferences in the development of Ae. aegypti affected by the different containers. Temperature is still considered to be the main driving force in the developmental rate of this mosquito vector32–34.
In our study, tyres, clay and cement had water tem- peratures ranging from 29–30°C, while the temperatures of remaining containers ranged from 30–32°C. Also, the temperature and relative humidity of the room were mea- sured which ranged from 30–32°C and 63–79%, respec- tively. Our study conforms to the study of Mohammed and Chadee35 that showed high pupation rates in a con- stant temperature of 30°C and diurnal temperature that ranged from 26–33°C. The characteristics of the differ- ent containers may regulate the water temperature as ob- served by Richardson et al31. The pupa of Ae. aegypti has been considered to be an important developmental stage
in entomological surveys because of its practicality and consistency in estimating adult abundance, dengue out- breaks and evaluation of control interventions36–39. How- ever, the physiological conditions to which pupal devel- opment may be regulated due to temperature changes or other factors are yet to be explored or investigated.
CONCLUSION
Our study demonstrated that AHWCs might affect the ecdysis development of the dengue mosquito vector, Ae. aegypti. The findings of our study can further strengthen and add to the complexity of the interaction of its preferred breeding containers towards its developmen- tal consequences. Also, our results may broaden the per- spective of dengue risk modeling in developing urban areas in relation to their population abundance and dis- persal for an efficient vector control. Nonetheless, cur- rent vector control strategies should be reinforced such as environmental management procedures that would pre- vent or lessen vector proliferation40. Ae. aegypti breed- ing site elimination has been broadly adopted worldwide to significantly lessen vector population density by tar- geting the most productive containers9. According to Williams et al12, the ecology of Ae. aegypti is regionally variable, especially with respect to the nature of its breed- ing habitat (i.e. water-filled containers). Thus, the role of AHWCs that promote the mosquito vector’s development could aid as a form of educational material to households or communities and provide strategies that focus on dis- posal of these AHWCs that supports breeding of Ae.
aegypti.
Conflict of interest
The authors declare that there is no conflict of inter- est in this study.
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Correspondence to: Mr. Thaddeus M. Carvajal, Department of Civil and Environmental Engineering, Ehime University, Matsuyama, Japan and Biology Department, De La Salle University–Manila, Philippines.
E-mail: [email protected]
Received: 27 August 2015 Accepted in revised form: 17 November 2015
