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Effects of Different pH levels on the Viability, Metamorphosis Rate and

Morphology of Aedes Mosquitoes

Conference Paper · November 2016

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 Effects of Different pH levels on the Viability, Metamorphosis Rate and Morphology of
Aedes Mosquitoes
Irish Magallanes Sequihod
St. Paul University Dumaguete
iseq22@gmail.com

OBJECTIVES

This study determined the effects of pH levels on the viability and metamorphosis rate of mosquito

Aedes sp. larvae. Furthermore, the study explored the morphological changes of the pupa when

subjected to different pH levels. This study offers a significant contribution in understanding more

deeply the physiological responses of mosquito larvae on the impact of climate change on its

aquatic habitat. This will also provide support for studies on climate change and its effect on our

biodiversity, which may result to alteration of the food chain in a freshwater ecosystem. Since this

study also examined whether a geometric-morphometric change exists in the pupa, the result may

inform readers regarding the effects of climate change on the morphology of Aedes sp. larvae.

REVIEW OF RELATED LITERATURE

Mosquitoes are a highly diverse group of insects that belong to the Diptera family. There are over

2500 species of mosquitoes located in every continent except Antarctica (Connecticut Mosquito

Management Program, 2015). Most studies on mosquitoes in the Philippines deal with listings

and distributions of the species (Almarinez & Claveria, 2014; Edillo, Roble, & Otero, 2012;

Tsukamato, Miyagi, & Toma, 1985; Edillo et al., 2002; & Edillo et al., 2004). In a study conducted

by Ramos et al. (1985), 279 species of mosquitoes, including its subspecies and varieties, exist in

the Philippines. Other studies delve on how they transmit diseases as vectors and how they can be

1
controlled as medically important vectors of many diseases (Edillo, Sayson, & Sarcos, 2015;

Santiago & Claveria, 2012; & Cabrera, 1966).

A 2-year survey on the species of mosquitoes in the Duamguete City was conducted by Nakao and

Valder (1976) from October 1968 to October 1970. It revealed that 32 species of mosquitoes can

be found in Dumaguete City and that they belong to five genera: Aedes, Anopheles, Armigeres,

Culex, and Uranotaenia.

The Provincial Health Office (PHO) of Negros Oriental gathered a 2007-2008 data on incidences

of Dengue fever/Dengue hemorrhagic fever in Negros Oriental. These were collected from the

Silliman Medical Center, Holy Child Hospital, and Negros Oriental Provincial Hospital. The PHO

drew a comparison using a line and bar graph to show the pattern of a yearlong weekly morbidity

of Dengue fever cases based on hospital admissions (Figure 1).

The total number of Dengue fever cases admitted in Dumaguete City in 2007 and 2008 was nearly

equal with 1204 and 1201, respectively. The highest number of incidences was from July to

October; while the lowest number of incidences was from March to June. The peak of Dengue

fever was at approximately 50 to 60 cases, and this was from August to September. The prevalence

of Dengue fever, therefore, is at its peak during the dry-to-wet season shift in Dumaguete City.

The August-to-September peak of Dengue fever revealed in this study falls within the recognized

June-to-November wet season period of the country (Provincial Health Office, 2009). This data

shows that during this time of the year, mosquitoes breed highly.

2
Figure 1. A graph showing the weekly Dengue fever/Dengue Hemorrhagic fever Morbidity of
2008 vs. 2007

Mosquitoes develop through four stages: egg, larva, pupa, and adult. Most of its life cycle stages

are associated with water; only the adult flies actively. Different mosquito groups have different

oviposition strategies. Some species would lay their eggs on water surfaces and deposit them either

singly (e.g. Anopheles) or in rafts (e.g. Culex). Some would lay them in the surface of vegetation,

which is often below the water surface (e.g. Mansonia).

In the case of Aedes, eggs are not laid on the surface of water but on substrates that are subject to

recurrent flooding such as on leaf litters, soil at edges of ponds, tree holes, or on walls of man-

made containers. They can produce an average of 100 to 200 eggs per batch. They do not lay all

their eggs at one time. The female can lay its eggs everywhere and depending on the availability

of suitable substrate (Dengue Virus Net, 2015). Its eggs hatch in less than a day when under a

3
conducive condition, that is, when in contact with water. The larvae feed on organic matter, algae,

and other microorganisms in the water. The development of larvae is temperature dependent and

passes through instars: 1st, 2nd, 3rd, 4th instar. It takes about four days for the larvae to develop into

its next stage – the pupa (National Environmental Agency, 2015). The pupa stage is a resting

stage. The pupa does not feed for about two days for it to develop. Then finally, the adult emerges

when the pupa ingests air to expand the abdomen which causes it to split (Dengue Virus Net,

2015). The adult mosquito then flies and finds a terrestrial habitat (Nature Education, 2011).

Mosquitoes live in a variety of habitats, although there are some species that thrive on specific

habitats for specific requirements for its metamorphosis. A mosquito habitat is influenced by 4

factors: i) water movement, ii) the amount of floating and emergent vegetation, iii) water quality,

and iv) the number and type of natural predators (Sacramento & Yolo Mosquito & Vector Control

Unit, 2006). In a study conducted by Vasudevan et al. (1999), they have checked the chemical

analysis of waters that support the breeding sites of different genus of mosquitoes in Kalkota,

India. In their results, Aedes group thrives at the range of 8.1-8.5 and average of 8.26 pH level.

pH and its Effects on mosquito larvae

In a Technical Memorandum prepared by Robertson-Bryan, Inc. (2004), they examined the pH

Requirements of Freshwater Aquatic Life. They were able to make a table that shows the effects

of limiting pH values on different aquatic organisms. At a pH range of 3.3 to 4.7, mosquito larvae

could thrive; but at pH 1, mosquito larvae died. A study by Adebote et al. (2008) on mosquito

breeding in rock pools on Inselbergs around Zaria, Northern Nigeria found that pH level of pools

4
supportive for Aedes vittatus ranges from 5.97 to 9.85. From this result, we can deduce that this

species has wider pH level tolerance compared to other mosquito species (Anopheles ardensis,

Anopheles distinctus, Anopheles wilsoni, and Culex ingrami) that dwell in pools.

Clark et al. (2004) also found that Aedes aegypti could complete its larval development at pH 4 to

11, but the larvae that were unable to acclimatize die at pH 3 or 12. Moreover, when pH was at 3

or 12, only a minimal percentage of the larvae succeeded to pupate. These pH levels are considered

extremes for these larvae.

METHODOLOGY
Collection of Larvae

Five egg raft pans were set-up Aedes mosquito adults to lay their eggs (Silver, 2008). The egg raft

pans were placed strategically, mostly under a shady area, to ensure that water from the pans would

not dry out quickly and that enough eggs would be collected. A preliminary egg collection was

done to check whether the eggs collected were really from the Aedes group of mosquitoes. The

manner by which the eggs laid was documented and a sample of eggs was examined under the

light microscope to check the egg’s anatomical characteristics. These were facilitated to ensure

that the eggs collected were from the Aedes group of mosquitoes.

The egg raft pans were left undisturbed for one to two weeks to allow the mosquitoes to lay enough

eggs. These were checked daily to ensure that a sufficient number of eggs would be collected for

5
the set-up. Eggs were collected by scraping the container carefully using a fine bristled brush. The

eggs were then transferred to another container that contained distilled water to allow them to

hatch into larvae (see Figure 2 below).

Figure 2. Egg collection. A. The mosquito egg raft pans that were set-up. B. The egg raft pans were placed
strategically (under the shade of vegetation) to ensure collection of enough Aedes eggs. C. The
egg raft pans were checked every day. D. The eggs from the egg raft pans were collected carefully
using a fine bristled brush and were stored in a container.

6
Preparations of water media with different levels of pH

Acidic Medium

In this study three pH treatments, pH 4, pH 7, and pH 8, were used in each set-up (A and B). Each

treatment had five replicates. For the acidic media (pH 4), Acetic acid (CH3COOH) and Sodium

bicarbonate (NaCHO3) were used. These compounds were allowed to react with each other in an

improvised CO2 generator (Beran, 2011). To acquire the desired pH level, monitoring of the

carbonated distilled water using a pH meter was. When the desired pH was already achieved, 150

ml of the medium was contained in each container. A total of 1500 ml of medium was prepared to

fill in the 10 glass containers (at 150 ml each) for pH 4. There were 5 replicates of pH 4 treatment

per set-up.

Neutral Medium

For the control treatment (pH 7), an Absolute distilled water was used. A volume of 150 ml of

distilled water was contained in each container. A total of 1500 ml of distilled water was prepared

to fill in the 10 glass containers (at 150 ml each). There were 5 replicates of pH 7 treatment per

set-up. The pH level of the distilled water used in the set-up was also checked using a pH meter.

Alkaline Medium

To prepare the alkaline medium, Sodium hydroxide (NaOH) crystal was used to produce the water

basic. Three to five pellets of (NaOH) was added and dissolved into a 1000 ml of distilled water.

To check whether the desired pH level was already reached, a pH meter was used. A volume of

7
150 ml of the medium was contained in each glass container. A total of 1500 ml of the medium

was prepared to fill in the 10 glass containers (at 150 ml each) for pH 8. There were 5 replicates

of pH 8 treatment per set-up.

Experimental Set-ups

Each pH treatment had five replicates per set-up. Five newly-hatched eggs (1st instar larvae) were

placed in each replicate. The larvae were allowed to metamorphose. Fish pellets (no.1 granule

sinker) of about 10 pieces were placed inside the container to provide the larvae food. The glass

containers that contained the larvae were organized, following the Randomized Complete Block

Design (RCBD) technique (see Figure 3). The temperature of the set-up is reliant on the heat

emitted by the halogen lamp in the closed system.

Figure 3. The replicates. A. Replicates were placed inside the set-ups and were arranged using the
RCBD technique. B. This was how each set-up looked like inside the aquarium. The tip
of the pocket thermometer probe (encircled in red) and the light bulb (encircled in yellow)
used in the set-up were both attached to the lid cover.

8
Monitoring for Larvae Metamorphosis

Daily monitoring of the set-ups was done to check whether the metamorphosis of the larva from

the different instars to pupa occurred. The larvae were checked only before turning off the light.

The number of larvae that survived and those that expired were monitored and recorded daily. The

pH of each replicate per treatment was recorded only after the experimental set-ups were

terminated. The set-ups were terminated only when the larvae were on their pupa stage. The final

pH level of all media was recorded after the termination of the set-ups.

Morphology data Analysis

The larvae that developed into pupa in the experiment were photo-documented. In describing the

morphology of the pupa, the most distinctive feature given focus was its head. Morphometric

measurements were recorded to determine whether changes in the morphology of the larvae

occurred. With the use of a stereomicroscope and a mounted camera, photos of the pupae were

taken. The pupae were mounted on a depression slide and were placed on the stereomicroscope’s

stage. Images were taken to compare the geometric-morphometric characteristics of the each pupa.

A total of 16 anatomical landmarks were established around the head of the pupa and were

digitized using tpsDig 2.17 software (Rolf, 2015). The landmarks were then established and

analyzed (see Figure 4) using the software MorphoJ (Klingerberg Lab, 2011). This is a free and

downloadable computer program under the Apache License, Version, 2.0.

9
Figure 4. The anatomical landmarks were established around the head of the pupa and digitized
using tpsDig 2.17 software. A sample and its 16 anatomical landmarks.

RESULTS AND DISCUSSIONS

Mosquito eggs collected from the egg raft pans were examined to determine whether they belong

to the Aedes species. The way the eggs were laid in the pans was observed. Figure 5 shows that

the eggs collected were laid individually over the water line at the sides of the pans; this

demonstrates a typical behavior of the Aedes group. The eggs were collected and a sample was

observed under the microscope to ensure that the eggs had floaters. A sample of an egg was

examined under the light microscope at low power objective (100x magnification) to verify

whether air floaters were present. The egg showed no paired air floaters which is another

characteristic of an Aedes group. Eggs were hatched into larvae to also check its behavioral

characteristic in the water when in resting position. Figure 6 shows a picture of the larvae resting

at an angle when in water. This is another characteristic of Aedes group (“Mosquitoes”, 1997).

10
 A B

Figure 5. The replicates. A. The eggs were laid on the sides of the egg raft pans, an egg laying
behavior of Aedes group B. A sample of an egg was examined under the light microscope
at low power objective (100x magnification) to ensure that air floaters were present. The
egg showed no paired air floaters which is another characteristic of an Aedes group.

Figure 6. Behavior of the larvae when in water. Resting at an angle to the water surface
(encircled in red).

11
pH Monitoring

The pH of all set-ups was monitored. Fluxes were recorded, presented in a table and graphed. The

mean average of all pH treatments (pH 4, 7, 8) decreased slightly at the end of the experiment: pH

4 from 4.2 to 4.08; pH 7 from 7.28 to 7.08; and pH 8 from 8.22 to 8.06. Tables 1 and Figures 7

show the fluxes of across all the pH levels.

Table 1. The pH measurements at the initial and final stages of the experiment.

END
pH START MEAN S.D. C.I.
pH4 4.22 4.12 0.268328 0.235196
pH7 7.26 6.92 0.109545 0.096018
pH8 8.3 7.6 0.2 0.175305

12
 START FINAL

5
PH LEVEL+ C.I.

0
pH4 pH7 pH8
PH TREATMENTS

Figure 7. The graph of pH measurements at the initial and final stages of the experiment.

Two factors could cause the decrease of the pH levels across all the media in both set-ups at the

end of the experiment. First, the decrease could be due to the pH regulation of the larvae and

bacterial activity in the water. Mosquito larvae can regulate its pH when in different environments

(alkaline or acidic). They achieve this by using ion exchange in maintaining its hemolymph pH

13
and by increasing their uptake of water. Mosquito larvae can use Na+ to maintain the alkalinity of

their alimentary canal which is necessary for their electrophoretic amino acid uptake needed for

nerve function. This process helps them conserve Na+ and, at the same time, excrete H+ and HCO-

3 (Okech, et. al. 2007). These excretions are contributory to the decreasing pH of the three media

in both set-ups.

The fish pellets (sinker) that the larvae in all the media in fed on primarily composed a mixture of

vegetable product and animal visceral by-products (Tacon, 1990), which are a good source of

organic material. This organic material can be acted by bacteria through decomposition. In this

process, CO2 is released and, in effect, carbonic acid is formed – this can further cause the

fluctuation of the pH levels of all media.

Light and Temperature monitoring

Light intensity was recorded using the HOBO Pro Light meter. The light intensity of the set-up

was 5965 Lux. The temperatures of the set-ups were monitored daily. The temperature of the set-

up was measured before turning the light on and after turning it off. The light was turned on for

12 hours. The average temperatures of the set-up was initially 27.3 oC and the final temperature is

33 oC. Table 2 shows the change of temperature of the set-up. A bar graph shown in Figure 8

shows the average initial and final temperature of the set-up (+C.I.).

14
Table 2. The average temperature of the two set-ups before the lights were turned on (initial) and
after the lights were turned off (final).

AVE. AVE.
S.D. S.D.
SET UP INITIAL FINAL C.I.
INITIAL FINAL
TEMP. TEMP.
12 hours light 27.3 oC 1.25356634 33 oC 0.816497 0.604858

Average Temperature of the Set-up

40

35

30

25
TEMPERATURE +C.I.

20

15

10

0
1

Initial Final

Figure 8. The graph showing the average temperature (+C.I.) of the set-up before the lights were
turned on (initial) and after the lights were turned off (final).

15
The flux of temperature is due to the heat coming from the light turned on for 12 hours. Halogen

bulb is characterized as illuminated at temperature higher than any other lamps. The surface

temperature of a halogen bulb is higher than that of LED bulbs (257.4 oC > 78.3oC) and it has a

great impact at enclosed system temperature. A longer running time of the light leads to an

increasing temperature in an enclosed system (“Halogen vs. LED Spotlights Comparison”, 2010).

Viability

Most of the larvae at pH 4 failed to metamorphose into the second instar. In terms of the onset of

mortality, the larvae across all pH treatments in the experiment started expiring on the second day

(see Table 3) with pH 4 having the largest mortality (84%), pH 8 was second (60%), and pH 7 had

the lowest (48%). Figure 9 shows that there is a decreasing percentage of surviving larvae per

day.

Table 3. Average of larvae viability at the end of the experiment.

pH % % Onset of
Viability Mortality Mortality
pH4 16 84 2nd day
pH7 52 48 2nd day
pH8 40 60 2nd day

One Way ANOVA was employed to compare the daily viability of the mosquito larvae across the

pH levels in the experiment. The results show that there is a significant difference in the viability

of the mosquito larvae at different pH levels in the experiment. The F value is 9.279 and p value

is 0.001. A Post Hoc analysis was conducted and the results show that the viability of the larvae at
16
pH 4 was significantly different from the viability of the larvae at pH 7 and 8. Comparatively, pH

4 has the least mean of 28. 50.

% Viability of Different pH per Day

pH4 pH7 pH8

120

100

80
% Viability

60

40

20

0
DAY1 DAY2 DAY3 DAY4 DAY5 DAY6 DAY7 DAY8
DAY

Figure 9. Graph showing the percentage viability of larvae per day.

The temperature of the experimental set-up provided by the halogen lamp could also possibly

contribute to the high mortality of the larvae, hence affecting its viability. Bayoh and Lindsay

(2004) in their study entitled Temperature-related duration of aquatic stages of the Afrotropical

malaria vector mosquito Anopheles gambiae, showed that larval mortality was higher when

temperature was high, from 30 to 32 oC. Couret et al. (2014) reported the same. He found that high

mortality of mosquito larvae occurred at 30 oC; it was lowest at temperature 24 oC.

17
Metamorphosis Rate

It took eight days for the larvae to metamorphose into pupa. A line graph is shown in Figures 10

to illustrate the metamorphosis rate of the 1st instar larvae to pupae in the experiment. Although

the line graph shows that pH 4 has the highest rate (80%) of metamorphosis compared to pH 7

(69.23%) and pH 8 (60%); statistics shows that there is no significant difference in the larvae-to-

pupae metamorphosis rate across pH treatment (F value = 0.374 and p value =0.710). A summary

of the number of larvae that succeeded to pupate, expressed in percentage, at experiment-final

across all pH treatments is shown Table 4. Not all larvae in the experiment succeeded to pupate

even if they survived.

The study of Clark et al. (2004) posits that Aedes aegypti can tolerate and successfully develop

within the pH ranges of 4 and 11. They also found that the development of Aedes aegypti larvae

is influenced greatly by sex and species and is less influenced by pH. Devi et al. (2014), however,

adds that Aedes mosquitoes are positively affected by temperature and dissolved oxygen and

negatively by pH. They also found that this group of mosquitoes could acclimatize extreme pH

levels; thus, it could survive at another pH extreme.

The ability of Aedes to acclimatize in extreme pH has implications on its survival in habitats that

will be affected greatly by climate change. As a result of climate change, the projected pH drop

is approximately 0.14 to 0.35 units lower (GRID-Arendal, 2014) in freshwater habitats in the 21st

century; this may influence the adaptability of this group to extreme pH levels.

18
 pH4 pH7 pH8

120%

Percentage that metamorphose

100%

80%

60%

40%

20%

0%
Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8
1st instar 1st instar 2nd instar 3rd instar 3rd instar 4th instar 4th instar Pupa
Days with Life Stages

Figure 10. A graph showing the metamorphosis rate of 1st instar to pupa.

Table 4. Summary of percentage of surviving larvae that pupate at the end of the experiment.

Replicate pH4 pH7 pH8

1 100.00
2
3 100.00 100.00
4 50.00 33.33
5 100.00 100.00

GEOMETRIC AND MORPHOMETRIC ANALYSIS

All the larvae that had succeeded to pupate after being subjected to their respective pH treatments

were photographed for geometric-morphometric analysis. The head of the pupa which appears as

the temporary fusing of the head and thorax of the larvae is the main focus of analysis. Samples of

photographs of pupa are shown in Figure 11.

19
Pupas exhibited allometric relationship between the pupa head shape and the log centroid with p

value of <.001. Component analysis was done to determine indications of shape variation of the

pupa head. Illustrations of the shapes are shown in Figures 12. Pairwise permutation test

Canonical Variate Analysis was employed with 10, 000 permutations to compare the differences

among the pupa heads from the different pH treatments. The analysis (results shown in Table 5)

shows indications of differences in the head shapes of all pupae. All pupa head shapes are

significantly different from each other.

Table 5. Summary of p values from the Canonical Variate Analysis (CVA) on the head shapes
of pupae from different pH levels.

p values on the Head shapes

pH 4 pH 7

pH 7 0.0020

pH 8 0.0053 0.0112

The thorax part of the pupa head subjected to pH 7 was broader compared to that subjected to pH

4 and pH 8. The first abdominal segment of the pupa covering the metathoracic wing pads was

angled for those subjected to pH 7 and pH 8 and rounded for those subjected to pH 4. This is shown

between landmark no.1 and no. 16.

20
Becker et al. (2010), in his publication entitled Biology of Mosquito, argues that the head of larvae

molts four times and the head size increases in the process. Thus, the head capsule makes a good

morphometric indicator. Shami (2010) studied the morphological deformities of the head capsule

of Chironomus spp. larvae inhabiting polluted rivers in Malaysia. It revealed that in the

morphology of various head capsules, the mentum of the larvae proved to be strongly affected by

environmental stress. They analyzed the water quality by measuring its total suspended solids,

ammonium and phosphate levels, temperature, and pH. They found that low pH is greatly

correlated with the deformities in the body structure of the larvae, particularly in the mentum and

epipharyngis.

In the published worked of Snodgrass (1959) entitled The Anatomical Life of the Mosquito, the

pupa is the non-feeding stage of the mosquito because its mouth parts are not fitted for any feeding

activity. Moreover, this stage has to keep itself afloat for respiration. The pair of respiratory

trumpets that is just above the surface of the water and the two small brushes of hairs on the back

of the first abdominal segment will help the pupa float in the water. This is critical for the future

emergence of the adult. Its suspension in the water is maintained by the bubbles of air trapped in

the folds of the legs and beneath the wings. Furthermore, this trapped air is from the pair of large

open spiracles on the sides of the first abdominal segment covered by the metathoracic wing pads,

which prevents it from drowning. Figure 13 illustrates the parts of the pupa head of Aedes aegypti.

21
Figure 13. Illustration of the anatomy of the pupa’s head of Aedes aegypti adopted from Snodgrass
(1959). br, suspensory brusg of first abdomen tergum; W2, mesothoracic wing; W3, metathoracic
wing pad; Tmp, repiratory trumpet; N3, metanotum; I-II, segments.

The exoskeleton of the pupa is made of chitin. The pH coupled with a relatively high temperature

of the experiment may have caused the altered shape of the head of the pupa. Rojas et al. (2015),

in their study entitled Effect of Acid Hydrolysis on Tableting Properties of Chitin Obtained from

Shrimp Heads, found out that the combined effect of high temperature (>80oC), HCl concentration

(>2M) and reaction time can result to high depolymerization of chitin. Furthermore, it can also

cause reduction in degree of acetylation, crystallinity, and chitin yield. These can decrease tensile

strength and can accelerate compact disintegration time, therefore causing it to be fragile.

The cuticle covers the head of the pupa as it continues to develop into adult. The sturdiness of the

cuticle is critically important at this stage because it will protect the developing head and thorax.

Furthermore, the changed shape of the metathoracic wing could be detrimental to the pupa since

this is an essential part of the head to keep the pupa suspended in the water and prevents it from

drowning.

22
SCALE: M1 = 66.01 pix

pH 4 pH 7 pH 8

Figure 26. Sample of photos of pupa per pH level and scale.

Figure 26. Grids representing the variation in the mean shape and size of the pupa heads from
different pH levels.

23
CONCLUSIONS AND IMPLICATIONS

The viability of mosquito larvae was greatly affected by pH. Furthermore, mortality rate of larvae

was higher in pH 4, although the temperature of the set-up may also contribute to it. Most of them

survived for only two days and only a few succeeded to pupate on the 8th day. While pH

significantly affects the viability of the larvae, it has no significant effect on the metamorphosis

rate of the larvae. The morphology (shape and size) of the pupa’s head was affected by pH. The

geometric-morphometric analysis results showed that the first abdominal segments that cover the

metathoracic wing pads of the pupae subjected to pH 4 were round in shape and those subjected

to pH 7 and 8 were angled.

The shape change of the head of Aedes pupa when subjected to different pH levels shows that this

group of mosquito is highly sensitive to changes in their habitat. With the occurrence of climate

change, deformities in the larvae’s body structure is detrimental to Aedes in its aquatic stage would

arise (e.g. thinning of or having fragile exoskeleton).

The ecology and biology of Aedes can be greatly affected by climate change. The following could

potentially occur if climate change persists: 1) possible exploration of new niche in new habitat

conditions which include tolerance on extreme pH levels and temperatures; 2) occurrence of

deformities in their morphology which can be detrimental to larvae in their aquatic stages; 3)

possible implications on the spread and incidences of vector borne diseases (e.g. Dengue).

24
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