DOI: 10.5372/1905-7415.0502.

046

Asian Biomedicine Vol. 5 No. 3 June 2011; 353-360

Original article

Morphology and protein profiles of salivary glands of

filarial vector mosquito Mansonia uniformis; possible
relation to blood feeding process
Atchara Phumeea, Kanok Preativatanyoub, Kanyarat Kraivichainb, Usavadee Thavarac, Apiwat Tawatsinc,
Yutthana Phusupc, Padet Siriyasatienb
a
Medical Science Program, bDepartment of Parasitology, Faculty of Medicine, Chulalongkorn
University, Bangkok 10330; cNational Institute of Health, Department of Medical Sciences, Ministry
of Public Health, Nonthaburi 11000, Thailand

Background: Vector control is a key strategy for eradication of filariasis, but it is limited, possibly due to rapid
propagation from global warming. In Thailand, Mansonia mosquitoes are major vectors of filariasis caused by
Brugia malayi filarial nematodes. However, little is yet known about vector biology and host-parasite relationship.
Objectives: Demonstrate the preliminary data of salivary gland morphology and protein profile of human filarial
mosquitoes M. uniformis.
Methods: Morphology of M. uniformis salivary gland in both sexes was comparatively studied under a light
microscope. Total protein quantization and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) was performed to compare protein profile between male and female. In addition, quantitative analysis prior
to and after blood feeding was made at different times (0, 12, 24, 36, 48, 60, and 72 hours)
Results: Total salivary gland protein of males and females was 0.320.03 and 1.380.02 g/pair gland, respectively.
SDS-PAGE analysis of the female salivary gland protein prior to blood meal demonstrated twelve bands of major
proteins at 21, 22, 24, 26, 37, 39, 44, 53, 55, 61, 72, and 100 kDa. Compared to female, male salivary gland was
composed of seven major protein bands at 39, 44, 53, 55, 61, 83, and 100 kDa. Quantitative study after blood
feeding revealed that protein of 37 kDa decreased gradually whereas proteins of 61 and 83 kDa started to increase
dramatically at 24 hours. It was postulated that the 37 kDa band, found only in the female, might serve as a
candidate molecule for facilitating blood feeding.
Conclusion: Morphology and protein components of M. uniformis salivary glands might relate to blood feeding
process and filarial disease transmission.
Keywords: Filariasis, Mansonia uniformis, salivary gland protein, SDS-PAGE

Filariasis is caused by filarial nematodes, which

are transmitted to vertebrate hosts when female
mosquitoes take a blood meal. The third stage infective
larvae (L3) resided in the thoracic muscles, migrated
to proboscis of the mosquito, and eventually infected
into vertebrate host via a piercing wound [1, 2]. Even
though the pathogens are not directly transmitted from
salivary glands of infected mosquitoes into vertebrate
hosts, their saliva is believed to be an essential factor
Correspondence to: Dr. Padet Siriyasatien, Department of
Parasitology, Faculty of Medicine, Chulalongkorn University,
Bangkok 10330, Thailand. E-mail: padet.s@chula.ac.th

for facilitating the disease transmission by either

increasing parasites infectivity or suppressing host
immune responses.
The mosquito saliva contains -glucosidases,
-amylases that initiate the digestion of carbohydrates,
some other enzymes and peptides involving in
blood feeding process including anticoagulation,
platelet aggregation inhibitors, and vasodilators [3-5].
However, the ethiopathogenesis in the aspect of insect
host-parasite relationship has not been elucidated.
The challenge remains to identify and characterize
the vectors candidate molecules to create a logical
hypothesis for elucidating their precise role.

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In Thailand, Mansonia mosquitoes are major

vectors of lymphatic filariasis caused by Brugia
malayi, especially in the southern area. The World
Health Organization deliberately declared six species
in this subgenus including M. boneae, M. dives, M.
uniformis, M. indiana, M. annulata and M.
annulifera as natural vectors for the disease. To date,
morphology and protein analyses of other mosquito
vectors salivary gland have been reported including
Aedes aegypti [6], Anopheles stephensi [7], An.
gambiae [8], An. darlingi [9, 10], Culex pipiens [11],
Cx. quinquefasciatus [12], Ae. togoi [13], Armigeres
subalbatus [14] and An. dirus B [15]. Nevertheless,
the study has not been performed for M. uniformis
yet. In this study, we demonstrated the preliminary
data of salivary gland morphology and protein profiles
of human filarial mosquitoes M. uniformis.
Materials and methods
Mosquito rearing
M. uniformis mosquitoes were maintained in an
insectary of the Department of Medical Sciences,
National Institute of Health, Thailand. Conditions were
set at 281C and 805% relative humidity under
12/12 hours light/dark photo-period. Adults were
supplied with a damp cotton wool pad containing 10%
sucrose solution as a carbohydrate source. For blood
feeding, female mosquitoes were allowed to feed
on anesthetized mice for 60 minutes. Groups of
mosquitoes were reared simultaneously from the
same cohort of eggs. Mosquito larvae were reared in
water containing floating aquatic plant; creeping water
primrose (Jussiaea repens) to obtain oxygen through
roots of the aquatic plant. Adult mosquitoes aged
three to five days after emergence were used for the
experiments.
Mosquito salivary gland dissection
Mosquitoes were anesthetized by chilling on a
snap frozen tray. Salivary gland dissection was
performed as described by Suwan et al. [16] with
some modifications. Briefly, 10 pairs of female salivary
glands prior to and after blood feeding at 0, 12, 24,
36, 48, 60, and 72 hours were dissected under a
stereomicroscope (SZX9, Olympus, Tokyo, Japan) and
individually transferred into a micro-centrifuge tube
containing 10 L of ice cold 1X phosphate buffered
saline (PBS) solution. For the male, 50 pairs of salivary
glands were collected in 10 L of ice cold 1X PBS.
Then, the samples were stored at -80C prior to SDSPAGE.

Light microscopy
The salivary glands of adult mosquitoes were
dissected in 1X PBS onto slides without drying out
and verified under a light microscope with 100X
magnification. Photographs of the glands were taken
using a digital camera (Nikon, Tokyo, Japan) attached
to a light microscope.
Protein quantization
Ten pairs of 10% sucrose-fed female and male
M. uniformis salivary glands at age between three
and five days after emergence were used in this study.
The total salivary gland protein content was determined
using a Micro BCA Protein Assay Kit (Pierce,
Rockford, USA) according to the manufacturers
instruction. The protein concentration was quantitated
based on a bovine serum albumin (BSA) standard
curve. Each determination was repeated three times.
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and protein staining
SDS-PAGE was carried out according to standard
techniques [17]. Briefly, sample of 50 pairs of male
and 10 pairs of female salivary gland, prior to and after
blood feeding at different times, were individually boiled
in reducing SDS buffer at 95C for five minutes. Each
sample was resolved using the Hoefer miniVE system
(Amersham Pharmacia Biotech, San Francisco, USA)
containing 5% stacking gel and 12% resolving gel
condition. Proteins were silver-stained using a Silver
Stain kit (Amersham Pharmacia Biotech, San
Francisco, USA) following the manufacturers
instructions. PageRulerTM Prestained Protein Ladder
(Fermentas, Burlington, Canada) was used as a
standard marker.
Densitometric analysis
To quantify relative band intensity, the images of
SDS-polyacrylamide gel were analyzed using the
Quantity One quantification analysis software version
4.5.2 (Bio-Rad, California, USA). All values from
independent triplicate experiments are expressed in
arbitrary units as mean standard error of the mean
(SEM).
Results
Figures 1 and 2 show salivary gland morphology
of female and male M. uniformis (female and male)
under a stereomicroscope and light microscopy,
respectively. Apparently, the salivary glands of male
and female M. uniformis mosquitoes are paired organ

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Salivary gland morphology and protein profiles of filarial vector M. uniformis

located in the thorax and morphologically different.

Size of male salivary gland is approximately one-fifth
of the female. The female salivary gland is composed
of two identical lateral lobes and one shorter median
lobe. The lateral lobes are further divided into proximal,
intermediate, and distal regions. The median lobe
consists of two (neck and distal) regions. A simple

355

layer of epithelial cells surrounding a lumen of salivary

duct can be observed in each lobe. The intralobar ducts
from each lobe drain into the lateral interlobar ducts,
which further empty into a common salivary duct.
Compared to the female, the male salivary gland
consists of three small lobes, which appear
morphologically similar in size and shape.

Figure 1. Steromicrograph demonstrating salivary gland morphology of female M. uniformis (4X magnification) (A),
and light photomicrograph demonstrating key structure of female salivary gland (100X magnification) (B).
Morphology of both DR and ML are similar but different from PR. SD is lined through the end of DR.
ML=Median lobe, LL=Lateral lobe, PR=Proximal region, IR=Intermediate region, DR=Distal region,
SD=Salivary duct, LSD=Lateral Salivary duct, CSD=Common salivary duct.

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Figure 2. Steromicrograph demonstrating salivary gland morphology of male M. uniformis (4X magnification) (A) and
light photomicrograph demonstrating key structure of male salivary gland (100X magnification) (B). Morphology
of the three lobes is identical. SD is lined through the end of DL. ML=Median lobe, LL=Lateral lobe, SD=Salivary
duct, LSD=Lateral Salivary duct, CSD=Common salivary duct.

We determined total protein contents of male and

female salivary glands. The amount of salivary gland
proteins in the mosquitoes aged between three to
five days was 1.380.02 g/female gland pair and
0.320.03 g/male gland pair. Total proteins in male
and female salivary glands of M. uniformis were
also relatively examined in silver stained SDSpolyacrylamide gel. Interestingly, SDS-PAGE analysis
of the female salivary gland protein prior to blood meal
demonstrated twelve major protein bands at 21, 22,
24, 26, 37, 39, 44, 53, 55, 61, 72, and 100 kDa. Compared
to the female, male salivary gland protein profiles was
composed of seven bands of major proteins at 39, 44,
53, 55, 61, 83, and 100 kDa (Figure 3).
Interestingly, quantitative study of female salivary
gland protein revealed that protein of 37 kDa
decreased gradually after blood meal whereas
proteins of 61 and 83 kDa started to increase
dramatically during 12 to 24 hours post feeding
(Figures 4 and 5).

Discussion
Morphology of M. uniformis salivary glands are
similar to other mosquito species previously reported
[10, 13, 14, 16, 18-24]. Salivary glands of male and
female M. uniformis show differences in both size
and shape. The female salivary gland is larger than
the male. Salivary glands in both males and females
consist of three lobes, two lateral lobes and one median
lobe. However, the lateral lobe of female salivary gland
is divided into proximal, intermediate, and distal regions,
which have similar in structure to other species of
mosquito. Morphological variations of female salivary
glands which composed of one to six lobes were also
observed (data not shown), as previously reported in
An. darling [9] and Ae. aegypti [6].
The light microscopic study of the male
salivary gland of M. uniformis demonstrated that
cells of median lobe and lateral lobes have similar
characteristics and are similar to the proximal lobe

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357

Figure 3.Comparative profiles of M. uniformis salivary gland protein of male and female in the silver-stained 12% SDSpolyacrylamide gel. Male: fifty pairs of salivary glands, Female: ten pairs of female salivary glands. Size of
molecular weight marker is indicated on the left in kDa. Approximate size of salivary gland proteins are indicated
on the right.

Figure 4.Relative comparison of the unfed and blood-fed female salivary gland protein profiles in the silver-stained
12% SDS-polyacrylamide gel at 0, 12, 24, 36, 48, 60, and 72 hours. Size of molecular weight marker is indicated
on the left in kDa. Approximate size of salivary gland proteins are indicated on the right

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Figure 5.Relative profiles of the protein bands at 37, 61, and 83 kDa from unfed and blood-fed female salivary gland M.
uniformis. The bar graph indicates the relative intensity after blood feeding at 0, 12, 24, 36, 48, 60, and 72 hours.
Data are expressed in arbitrary unit as meanSEM (n=3).

of the female salivary glands. This finding has

been previously reported in An. gambiae [7], Ae. togoi
[13], An. dirus B [15] and Ae. aegypti [25]
mosquitoes and these cells are related to sugar
feeding. The median lobe and distal lobe of the female
salivary gland have similar cell types, which may
involve blood feeding [21]. The present comparative
study of protein content was similar to the previous
studies, i.e. Ae. togoi [13], An. dirus B [15] and Ar.
subalbatus [14]. This indicates that protein amount
of the male per pair was less than the female. In
addition, SDS-PAGE analysis illustrated the different
profiles between both sexes. Interestingly, protein of
37 kDa was predominantly expressed in the female,
but not found in the male. The protein profile after
blood feeding also showed that 37 kDa band promptly
decreased and later gradually increased to unfed level
in 60 hours. Thus, it is possible to be mentioned that
37 kDa polypeptide band was synthesized by female
salivary gland-specific cells and might involve in blood
feeding. Roditi et al. [26] and Arca et al. [27] reported
that approximately 37 kDa band in Ae. aegypti and
An. gambiae was characterized and identified as a
member of D7 salivary protein family. Specifically, it
was found in median lobe and distal region of lateral

lobe and believed to play a crucial role in blood feeding

process. This protein family is distantly related to the
odorant-binding protein (OBP) super-family that
specializes in small ligand binding function [28].
Recently, some mosquito D7 proteins were shown to
bind and inhibit the action of some biogenic amines,
such as serotonin, histamine, and norepinephrine.
Therefore, these might contribute in blood feeding
process [29]. Although D7 proteins are among the
most abundant salivary proteins in adult female
mosquitoes, their role in blood feeding remains elusive.
The present result in M. uniformis makes us
hypothesize that this polypeptide of 37 kDa may serve
as functional enzyme involving in blood feeding process
or reactive allergen. This study is report preliminary
information for better understanding of mosquito
salivary gland proteins and vector-borne disease
transmission. Two-dimensional gel electrophoresis and
other proteomics techniques will be required to
complete the knowledge jigsaw of mosquito salivary
gland protein.
In conclusion, morphology and protein components of M. uniformis salivary glands might relate
to blood feeding process and filarial disease
transmission. Further study of biological role of these

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Salivary gland morphology and protein profiles of filarial vector M. uniformis

female salivary gland proteins during blood feeding

would provide valuable data for effectively controlling
mosquito borne diseases.
Acknowledgement
We acknowledge the financial support from the
National Research University Project of CHE and
the Ratchadaphiseksomphot Endowment Fund
(HR1160A). This investigation was supported in part
by the Molecular Biology Project, Faculty of Medicine,
Chulalongkorn University.
The authors have no conflict of interest to declare.
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