J Arthropod-Borne Dis, June 2013, 7(1): 23–30 M Koosha et al.: Lectin Activity in…

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Original Article

Lectin Activity in Gut Extract of Culex pipiens
Mona Koosha 1, Mohammad Reza Abai 1, Mandan Abolhasani 1, Soroor Charedar 2,

*Hamid Reza Basseri 1

1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of
Medical Sciences, Tehran, Iran

2Department of Medical Parasitology, School of Public Health, Tehran University of Medical Sciences,
Tehran, Iran

(Received 15 June 2011; accepted 13 June 2012)

Abstract
Background: The role of lectins is important in interaction between pathogens and mosquito vectors. This study was
performed to identify agglutinin activities of protein molecules on the midgut of Culex pipiens.
Methods: Culex pipiens was reared in insectray condition and the midguts of males and females (blood fed and un-
fed) were dissected separately in Tris-HCl buffer. The extracts of midguts were applied for hemagglutinin assay
against red blood cells of rabbit, mouse, rat, dog, horse, sheep, guinea pig, cow, human (A, B, AB, O groups). Then,
the RBCs with relatively high agglutinin activity were chosen for carbohydrate inhibition assay. D (+) glucose, D (+)
galactose, D (+) mannose, D (-) fructose, D (-) arabinose, L (-) fucose, lactose, N-acetyl-D-glucosamine, N-acetyl-D-
galactosamine, sialic acid were used to specify carbohydrate binding lectin.
Results: The highest agglutinin activities were found against sheep and rabbits RBCs. Sexual diversity of agglutinin
activities was observed among midgut extraction of males and females. In addition, variation in agglutinin activity of
blood fed and unfed female mosquitoes were detected. The lectin activity was inhibited highly with glucose,
galactose, fucose and fructose but less inhibitor activities was observed by arabinose, N-acetyl-D-galactosamine, n-
acetyl-d-glucosamine, lactose and mannose.
Conclusion: The secretion of hemagglutinins (lectins or lectin-like molecules) in the digestive system depends on
the type of food in the gut. This suggests that emptying of the gut in preparation for protein rich food probably starts
the secretion of hemagglutinins.

Keywords: Mosquitoes, Culex pipiens, Lectin, Hemagglutination activity, Midgut

Introduction

Lectins are defined as carbohydrate-binding
proteins or glycoproteins of non immune
origin which agglutinate cells and/or precip-
itate glyco-conjugates (Goldstein and Poretz
1986). Lectins in insects with distinct sugar
specificities involve in recognition and protec-
tive roles in immune defense against microbial
pathogens. The agglutinins against vertebrate
erythrocytes have been reported in various in-
sects (Basseri 2002). Insect hemagglutinins
are lectin or lectin-like molecules that are
ubiquitous, non-enzymatic carbohydrate bind-
ing proteins or glycoproteins and once bound

to erythrocytes or other cells, usually cause
their agglutination, and may also precipitate
glycol conjugates (Stebbins and Hapner1986,
Ingram 1997).

Most studies have concentrated on the
hemagglutination activity (HA) in the gut of
hematophagous insects especially Dipteran
(Rudin and Hecker 1989, Volf 1993,
Grubhoffer et al. 1994, Volf et al. 1994, 1995,
1998). The role of lectins are important in the
life cycle of some parasitic protozoa carried
by mosquitoes, sand flies, tsetse flies, and
many other bloodsucking insects (Ingram

*Corresponding author: Dr Hamid Reza Basseri, E-
mail: basserih@tums.ac.ir

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and Molyneux 1991, Basseri 2002, Basseri
et al. 2008). These molecules in the gut and
hemolymph cause the establishment of in-
fection and parasitic development (Ibrahim
et al. 1984, Mello et al. 1999).

In addition, anti-parasite agglutinins have
been detected in midgut extracts of trypano-
some vectors (Ibrahim et al. 1984, Wallbanks
et al. 1986). Unfortunately there is not enough
information about the nature of lectin- car-
bohydrate in insect-vector–parasite interac-
tion. In spite of the fact that there are spe-
cific mechanisms between lectin receptors
on surface of parasites of vector tissue which
can ensure the success or failure of infection
(Rudin 1991, Billingsley 1997, Basseri et al.
2008), some investigators suggest that inter-
actions between parasites and vector gut
walls may be mediated by the carbohydrates
on the surface of parasites. Besides the lectins
in the vector gut, as characteristic carbohy-
drate markers have been identified on the sur-
face of parasites such as Trypanosoma and
Leishmania (Schottelius 1982a, 1982b, 1982c).
The midgut lectins have been identified from
such vectors as Rhodnius prolixus (Pereira et al.
1980) Glossina austain (Ibrahim et al. 1984)
Phlebotomus papatasi (Wallbanks et al. 1986)
and Anopheles gambiae (Mohamed and Ingram
1994).

Culex pipiens is usually the most common
pest mosquito in urban as well as rural areas
of Iran (Azari-Hamidian 2007, Dehghan et
al. 2010). The purpose of present study was
to detect the hemagglutinin activity in the
midgut extraction of males and females of
Cu. pipiens. Furthermore, to find changes in
hemagglutination activity in blood fed and
unfed female mosquitoes. Moreover, spe-
cific carbohydrates of lectins were surveyed
to characterize the lectins in the midgut. The
mosquito lectin has been partially charac-
terized for further study and their functional
roles.

Materials and Methods

Insect rearing and sample preparation
The laboratory strain of the Cx. pipiens was

used. The mosquitoes were reared for more
than 48 years in the insectray. During the
present study they were occasionally al-
lowed to feed on laboratory guinea pigs and
reared under a photoperiod of 12:12
day/night at 28±2 ºC and 50–60% relative
humidity. The adult females including fed
and unfed females as well as males were
then applied separately for midgut dissec-
tions.

Preparation of gut
Mosquitoes gut were dissected separately

in TN buffer (20mM Tris - HCl, 0.15M
NaCl, pH=7, 5mM CaCl2). The guts were
collected and washed with the buffer and
homogenate using mechanical homogenizer
in cold condition. Then, the homogenate
samples were centrifuged at 10,000g for
15min, three times. The supernatant were
kept in -80 °C until use.

Protein Assay
The concentration of midgut proteins was

estimated as discussed by Bradford (1976)
and in order to obtain standard curve, serial
dilution of different concentrations of bovine
serum albumin (BSA) was used.

Preparation of erythrocyte
Blood from rabbit, mouse, rat, dog, horse,

sheep, guinea pig, cow, human (A, B, AB, O
groups) were prepared in 3.8% (w/v)
trisodium citrate. In order to prepare red
blood cells, whole bloods were washed three
times in TN buffer at 1500g for 5min each to
remove serum and gain RBCs.

Finally a 2% (v/v) suspension of RBC was
prepared and kept at +4 °C until use for
hemagglutination assay and also hemagglu-
tination inhibition assay.

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J Arthropod-Borne Dis, June 2013, 7(1): 23–30 M Koosha et al.: Lectin Activity in…

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Published Online: April 10, 2013

Hemagglutination assay
Five microlitre of each midgut extract was

serially diluted in TN buffer (as suggested
by Uhlir et al. 1996) in the v-bottom wells of
micro titration plates. Then 5 microlitre of
2% mentioned erythrocytes suspension was
added to each well. The titer of
hemagglutination activity was determined
under stereomicroscope after 60 min incu-
bation at room temperature. Unagglutination
described as RBCs with clear dot on the bot-
tom of the well, and agglutinatinated targets
formed a diffuse mat.

All experiments were repeated three times.
The controls contained TN buffer and 2%
BSA. Finally, the erythrocyte which had the
highest dilution activities visually was cho-
sen for next experiments. The reciprocal
value of the highest dilution with positive
reaction was scored as the titer.

Hemagglutination inhibition assay (HIA)
The HIA was performed to determine the

inhibitory activities of different carbohy-
drates against the midgut lectin activities as
follows: D (+) glucose, D (+) galactose, D
(+) mannose, D (-) fructose, D (-) arabinose,
L (-) fucose, lactose, N-acetyl-D-glucosa-
mine, N- acetyl –D-galactosamine, sialic ac-
id (all form sigma). The stock solutions of car-
bohydrates were prepared in NaCl/Tris/Ca
2+ buffer at 0.2M stored at -20 ºC until use.
For each inhibition (5μl) aliquots of buffer
contain carbohydrate was added in each
microlitre wells, followed by 5 microlitre of
gut extracted adjusted subsequently to titer
of 1: 4096. These were mixed gently by
shaking and incubated for 60 min at room
temperature. Finally, 5 microlitres of 2%
RBCs of sheep or rabbit suspension was
added into each well and left the microplates
for 1 h at room temperature to incubate the
mixtures. These tests were done three times.
Well without lectin or inhibitors were con-
sidered as control.

Enzyme treatment of RBCs
In order to expose more receptors of RBCs

to extracted lectins, all RBCs were treated
with trypsin. Equal volume of 2% above
RBCs, trypsin (2 mg/ml) solution prepared
in tris-HCl, were mixed together and incu-
bated at 37 ºC for 25 min.

The RBCs were then washed three times
with buffer and adjusted to 2% suspensions.
Treated or untreated RBCs were incubated
with gut extract at room temperature for 30
min. subsequently, hemagglutination titers
were assessed.

Results

Hemagglutination assay
The hemagglutination patterns of whole

proteins from the midguts of Cx. pipiens
against a range of erythrocytes were deter-
mined (Fig. 1). The highest activates oc-
curred against sheep erythrocytes (titer of
256) followed by rabbits (titer of 64). The
less activity was observed against Guinea
pig erythrocytes (titer≤ 16). Thus, sheep
erythrocyte was used for inhibition assay.

Trypsinization assay
Modification of rabbit’s erythrocyte mem-

branes by trypsin treatment increased the
agglutinin activities more than eight times
indicating more lectin molecules present in
the extracted proteins.

Appearance of hemagglutinating activity
at sex and fed condition

The hemagglutination activity of midgut
extracts of male and female mosquitoes was
different (Table 1) while the proteins of
males' midgut showed less agglutinin activi-
ties rather than females. In addition, fed fe-
males of Cx. pipiens demonstrated two times
more agglutinin activity than unfed females
(Table 1).

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J Arthropod-Borne Dis, June 2013, 7(1): 23–30 M Koosha et al.: Lectin Activity in…

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Published Online: April 10, 2013

Carbohydrate inhibition assay
As shown in Table 2, the hemagglutination

inhibition of sugars for extracted proteins of
midgut was dissimilar. The lectin activity
was inhibited highly by glucose followed by

galactose, fucose and fructose but less in-
hibitor activities were observed by arabi-
nose, N-acetyl-D-galactosamine, N-acetyl-
D-glucosamine, lactose and mannose.

Table 1. Effect of induction and blood meal feeding
and compartment with mosquito sex on Lectin
Hemagglutination Activity (HA)

Protein Con-
centration

(mg/ml)

HA (with candi-
date RBCs)

(Titer)
Fed 0.17 32

Unfed 0.06 16
Male 0.15 8

The number in last column represents endpoint
titers expressed as the reciprocals of the dilutions
(1/n). All samples were prepared three times and
the assay repeated

Table 2. Effects of inhibitory sugars on agglutinin ac-
tivities of protein on midgut extract of Culex pipiens

Inhibitor Titer

D(+) glucose >256
D(+) galactose >128
D(+) mannose >64

L(+) fucose >128

Lactose >64
N-acetyl-D-glucosamine >64

N-acetyl-D-galactoseamine >64
L(+) Arabinose >64
D(+) Fructose >128

Fig. 1. Agglutination activities of midgut extract of
Culex pipiens against different erythrocytes

Fig. 2. Comparison between Trypsin enzyme treated
and untreated RBC agglutinin activities against
midgut extract of Culex pipiens

Discussion

The present study shows that hemagglutination
activity exists in midgut of adults of both sexes.
In addition, this activity was higher among
fed females. The hemagglutination activities

were described in digestive systems of six
species of phlebotomine sand flies (Volf and
Killick-Kendrick 1996). They showed that
higher hemagglutination activity in the gut of

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females and characterized the lectins in the
females' midgut. The results of their study
showed that, the agglutinin activity was more
than 50 times higher in unfed females than
in males. Similarity, blood meal increased
agglutinin levels in the midgut extraction of
Cx. quinquefasciatus against Escherichia coli
as well as rabbit RBCs (Ayaad 2009).

Presence of variation in lectin activity be-
tween males and females is typical only for
hematophagous Nematocera (suborder of dip-
terous insects), presumably because only the
females of these insects take blood meals.
This presumption is supported by findings in
Glossinae, in which both sexes take blood
and where there are no significant differ-
ences between lectin activities of males and
females (Ingram and Molyneux 1988, 1991).

However, our results indicated that the se-
cretion of hemagglutinins (lectins or lectin-
like molecules) in the digestive system de-
pends on the type of food in the gut. Inter-
estingly, trypsinization increased binding of
agglutinin of midgut extracted proteins with
rabbit erythrocytes indicating there are more
hidden lectins molecule receptors.

Though, fasting adults have very high
hemagglutination activity, which may be due
to the degradation of gut cells and presence
of innate lectin molecules in the epithelial
cells. On the other hand, the high activity in
the rest of abdomen may be correlated with
the presence of symbionts.

Generally, in adults of mosquitoes, symbionts
might use ‘host’ reserves for their own growth
and start to act like pathogens inside the body.
In this case, haemocytes, which presumably
produce the lectins or hemagglutinins, act to
eliminate infection. This concept is supported
by the observation the levels of hemagglutinins
in females taking blood meals are uncharged.

However, our results is different with those
presented by Gelbic and Olejnicek (2004)
though they stated that hemagglutination ac-
tivity in the midgut of Cx. pipiens complex
is not dependent on a blood meal or the up-

take of protein food. Apparently, the variation
in results may be due to different population
used. We showed the effect of geographical
populations of Anopheles stephensi on ag-
glutinin activities of the mosquitoes' midgut
(Basseri et al. 2004). Similarly, Grubhoffer and
Noriega (1995) and also Grubhoffer et al.
(1997) reported significant increases in
hemagglutination activity in female Aedes
aegypti after the ingestion of protein food and
Volf and Palanova (1996) obtained similar re-
sults in phlebotomids. Mohamed et al. (1992)
did not find differences either between sexes
or after blood fed of An. gambiae. According
to the present results, hemagglutination ac-
tivity correlates with some digestive pro-
cesses and sex of the mosquitoes.

It has been shown that haemagglutinins in
the gut of Cx. pipiens may separate food
from liquid of blood meal or nectar and re-
tard the passage food through the gut (Gelbic
and Olejnicek 2004).

Interestingly, significant increase of
hemagglutination titer was observed in fe-
males after blood meal. This suggests that the
empty gut probably starts the secretion of
hemagglutinins to prepare for protein-rich
food. Male mosquitoes do not suck blood, and
therefore the filling of the gut does not start the
reaction in the same way as in the females.

Hemagglutination assay on the midgut of Ae.
aegypti showed that the  agglutinin might not
be protein (lectin) but a glycan (Olejnıcek et
al. 2000). However, further species of Culicinae
need to be investigated for induction of gut
hemagglutination activity because different re-
sponses in different mosquito genera or even
species can be expected (Nayar and Knight
1997).

The mosquito midgut represents one of the
most challenging environments for many mi-
croorganism born diseases such as Plasmo-
dium, arboviruses and fungus (Tajedin et al.
2009, Chugh et al. 2011, Cox et al. 2011). The
ability of a panel of lectins to potentiate the
uptake of a variety of microorganisms is espe-

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cially important in terms of nonself recognition
in invertebrate immune system (Rattclife and
Whitten 2004, Yoshida et al. 2007).

However, agglutinin activity may be im-
portant for the elimination of infections, as
well as for the processing of food and the
utilization and transportation of nutrients. In
conclusion, the secretion of hemagglutinins
(lectins or lectin-like molecules) in the diges-
tive system of Cx. pipiens depends on the
type of food as well sex.

Acknowledgements

We received no external funding sources
for this study. The authors would like to ap-
preciate very much for kind collaboration of
staff in the Department of Medical Entomol-
ogy and Vector Control, School of Public
Health, Tehran University of Medical Sci-
ences. The authors declare that there is no
conflict of interest.

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