Two serine proteases from Anopheles dirus haemocytes
exhibit changes in transcript abundance after
infection of an incompatible rodent
malaria parasite, Plasmodium yoelii
WenYue Xu*, Fu Sheng Huang*, Hong Xing Hao,
Jian Hua Duan, Zhong Wen Qiu
Department of Pathobiology, The Third Military Medical University, Chongqing 400038, PR China
Received 5 September 2005; received in revised form 15 February 2006; accepted 17 February 2006
Serine proteases are involved in regulation of innate immune responses, such as haemolymph coagulation, melanization
reaction and antimicrobial peptide synthesis. Although several serine proteases have been characterized in Anopheles gambiae
(A. gambiae), few were cloned from other malaria vectors. In this study, we identified three cDNA fragments of serine proteases
(AdSp1, AdSp2 and AdSp3) from haemocytes of an oriental malaria vector, Anopheles dirus (A. dirus), by cloning offragments
expressed in salivary gland. Basic local alignment search tool (BLAST) search found that both AdSp1 and AdSp3 were highly
similar in sequence to A. gambiae Sp14A and Sp14D2, insects prophenoloxidase activating enzyme (PPAE) and Drosophila
protease easter. Semi-quantitativeRT-PCR indicated the transcription level of both AdSp1 and AdSp3 in haemocytes of A. dirus
infected with Plasmodium yoelii (P. yoelii) was significant higher than that fed on 5% glucose or normal mouse blood at 7 days
after the infectious meal (p < 0.05), when P. yoelii oocysts began to be melanized by A. dirus. Our results indicated that both
AdSp1 and AdSp3 might play an important role during melanotic encapsulation of P. yoelii by A. dirus.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Plasmodium yoelii; Anopheles dirus; Serine protease; Melanization
Malaria is one of the most devastating infectious
diseases caused by protozoan parasites of the genus
Plasmodium. Plasmodium requires completion of their
sporogonic cycle within the Anopheles mosquitoes to
Veterinary Parasitology 139 (2006) 93–101
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E-mail addresses: firstname.lastname@example.org (W. Xu),
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be transmitted to vertebrate hosts. During their
development, however, Plasmodium will encounter
susceptible mosquito, Plasmodium experiences severe
losses due to the induced expression of antimicrobac-
terial peptides (Lowenberger et al., 1999) and nitric
oxide synthase (Luckhart et al., 1998). In genetically
selected mosquito, A. gambiae L35, development of
several species of Plasmodium can be completely
blocked due to lysis in midgut epithelium (Vernick
et al., 1995) and melanization of early oocysts (Collins
et al., 1986).
Melanization, also called melanotic encapsulation,
is characterized by isolation and killing of invaded
microorganisms in a layer of melanin produced by
cross-linking of host as well as microbial proteins.
Melanization is regarded as the main phenotype of the
refractory strain of A. gambiae, and three quantitative
trait locis (Pen1, Pen2 and Pen3) were reported to be
responsible for this phenotype (Zheng et al., 1997).
Although the specific genes that are responsible for
melanization of malaria parasites have not been
identified, sequencing of the Pen1 genomic region has
be related to the melanotic reaction of A. gambiae
(Thomasova et al., 2002). Broad physiological
differences between these refractory and other
susceptible mosquitoes were related to the production
and detoxification of reactive oxygen, and an
elevated level of reactive oxygen species was also
account for the Plasmodium melanotic encapsulation
(Kumar et al., 2003). Recently, dsRNA knock-out of a
complement-like protein TEP1 (thioeaster-containing
protein) was reported to reduce the melanization rate
of Plasmodium by the refractory A. gambiae,
indicating a pivotal role of recognition step preceding
melanization reaction (Blandin et al., 2004). The
Accumulating evidence demonstrates inverte-
brate defense responses, including haemolymph
coagulation, antimicrobial peptide synthesis, and
melanization of pathogen, were regulated by serine
proteases. For example, horseshoe crab haemo-
lymph coagulation (Iwanaga et al., 1998) was
initiated by autoactivation of serine protease Factor
C upon binding to LPS, and then followed by
activation of other two serine proteases, Factor B
and proclotting enzyme (PCE). The Toll pathway,
responsible for the induced synthesis of antimicro-
bial peptides, also involves cleavage activation of
spa ¨tzle by a cascade of serine proteases (Lemaitre
et al., 1996). During melanotic encapsulation
(Cerenius and Soderhall, 2004), prophenoloxiase
activating enzyme (PPAE) converts prophenolox-
idase (PPO) to active phenoloxidase (PO). Serine
proteases inhibitors (serpins) negatively regulate all
of the three defense responses. For example,
Limulus intracellular clotting inhibitors (LICIs)
inhibit horseshoe crab clotting pathway (Agarwala
et al., 1996), mutations of Spn43Ac result in
constitutive expression of drosomycin in Drosophila
melanogaster (D. melanogaster) (Levashina et al.,
1999), and serpin-3 through -6 regulate Manduca
sexta (M. sexta) PPO activation (Tong and Kanost,
2005; Zou and Jiang, 2005).
So far, five serine proteases have been character-
ized in a major Africa malaria vector, A. gambiae
(Gorman et al., 2000a; Paskewitz et al., 1999). Of
those, Sp14A, Sp14D2 and Sp22D exhibited change
in transcript abundance in response to bacteria
injections. Sp14A and Sp14D2, as well as Sp14D1,
were induced by infection with the malaria parasite,
Plasmodium berghei. Phylogenetic analysis grouped
and D. melanogaster easter. Interestingly, Sp14D1
was also identified as a refractory strain-specific
serine protease (Chun et al., 2000), mapped together
with one melanotic encapsulation phenotype-related
quantitative trait loci Pen3 (Zheng et al., 1997).
inducibility has suggested that Sp14A, Sp14D1 or
Sp14D2 might activate spa ¨tzle-like ligand or PPO in
melanization preventing factor, a putative serine
protease inhibitor in haemolymph of susceptible
mosquito, indicated that regulation of Plasmodium
melanization was also through serpin (Paskewitz and
Few serine proteases have been identified in other
malaria vectors so far. A. dirus is a major human
malaria vector in China and Southeast Asia, which is
naturally refractory to P. yoelii by melanization of
most oocysts on the midgut. Here, we reported the
cloning of three serine proteases from A. dirus
haemocytes and expression profiles of their transcript
W.Y. Xu et al./Veterinary Parasitology 139 (2006) 93–10194
abundances after infection of P. yoelii, especially
during melanization of malaria parasites.
2. Materials and methods
2.1. Mosquitoes rearing and infection of P. yoelii
A. dirus (Hainan strain) are routinely maintained in
cage at 27–28 8C, 70% humidity and then are fed on
5% glucose. For female adults to lay eggs, mosquitoes
are fed on mouse blood. Larvae are kept in water and
fed onyeast powder,and pupa emergences in the cage.
For infection experiment, mouse containing P. yoelii
gametocytes checked by Giemsa-staining (Payne,
1988) was selected to feed female adults (3–5 days
old) starved for 3 h.
2.2. Total RNA isolation
Haemolymph was collected through a centrifuga-
tion procedure described by (Sidjanski et al., 1997)
with slight modification. In brief, after anesthetiza-
tion by chilling on ice, the body wall of the last two
abdominal segments of female adults was pulled
open with a sterile 50 ml-microinjector. Twenty-five
killed mosquitoes were then placed in a pre-chilled
0.5 ml Eppendorf tube with four pores punctured
with 18-gauge needle at the bottom. The 0.5 ml tube
was inserted into a 1.5 ml Eppendorf tube containing
100 ml Tripure (Boehringer Mannheim, Germany)
with a pore on the cap. After centrifugation at
375 ? g for 10 min, haemolymph was collected in
the bottom of 1.5 ml Eppendorf tube. Four hundred
microliters of Tripure was then added to each tube,
and a total of 50 mosquitoes were proceeding to total
For isolation of midgut and salivary glands, Female
adults of 3–5 days old were anesthetized as described
under dissection microscope with sterile needle and
then transferred to 1.5 ml Eppendorf tube containing
1 ml phosphate-buffered saline (PBS). The tissues
were washed twice with PBS by centrifugation at
7500 ? g for 5 min to remove contaminating haemo-
cytes. A total of 50 midguts and salivary glands were
homogenized with a glass-Teflon in 800 ml Tripure
solution and used for total RNA isolation.
2.3. Reverse transcription-PCR and cDNA
Total RNA was isolated from haemocytes of 50 A.
dirus female adults with Tripure according to the
manufacturer’s instructions. About 0.6–0.8 mg total
RNA was used for first strand cDNA synthesis with
MMLV (Promega, USA). Three microliters of cDNA
(of a total of 20 ml) was used as PCR template to
amplify serine protease fragment usingExTaqenzyme
(TaKaRa, Japan). Degenerate PCR primers AdSpf:
CA(TC)TG-30) and AdSpr: (50-ATGAGCGG(AG)C-
designed based on conserved amino acid sequences
YIVTAAHC and GDSGGPLM of insect PPAEs. Each
cycle of PCR included 30 s at 94 8C, 30 s at 55 8C and
1 min at 72 8C. These steps were repeated 35 times,
followed by an extension at 72 8C for 5 min.
The PCR product was separated by 1% agarose gel
electrophoresis and 0.5 kb fragment was purified
using Silverbeads DNA Recovery Kit (Sangon,
China), and cloned into PinpointTMXa-1 T-vector
(Promega, USA), and sequenced. Similarly, a 0.46-kb
PCR fragment for ribosomal protein S7 was amplified
using degenerate primers AdS7f: (50-CTGGAGCTG-
GAGA(TC)(GA)AACTC(GCT)GA-30) and AdS7r:
30). The product was also gel-purified, cloned, and
2.4. In situ hybridisation
A digoxigenin (DIG)-labeled riboprobe of AdSp1
and a sense strand negative control probe were
synthesized using the DIG RNA Labeling Kit (Roche,
Germany). In situ hybridization to haemocytes was
done as described by Gorman et al. (2000c). In brief,
haemolymph was collected by cutting off the tip of the
proboscis of a newly enclosed female, pressing on the
thorax with a cover slip, and collecting the extruded
haemolymph into M199 medium on the slide.
Haemolymph from 10 female adults was immediately
put into 5 ml M199 medium. Cells were allowed to
settle on the slide for 5–10 min and fixed for 20 min in
10 ml 4% paraformaldehyde in PBS. Prehybridization
of cells was conducted for 1 h at 40 8C following by
washing with PBS twice. Cells then were incubated
W.Y. Xu et al./Veterinary Parasitology 139 (2006) 93–10195
with DIG-labeled riboprobe of AdSp1 and control
probe for 16 h, and anti-digoxigenin-AP (Roche,
Germany) for 2 h. Substrate containing NBT and
BCIP was finally added for color development.
2.5. cDNA library construction, screening
Poly (A)+RNA was prepared from 300 A. dirus
female adults using PolyATtract1mRNA isolation kit
(Promega, USA). Five micrograms of poly (A)+RNA
was used for library constructionwith ZAP ExpressTM
System according to the manufacturer’s instructions
(Stratagene, USA). DIG-labeled AdS7 probe was
prepared using DIG Easy Hyb (Roche, Germany) for
screening the cDNA library. Positive clones were
purified and subcloned by invivoexcision into pBKC-
MV phagemids using Exassist helper phage and E.
coli XLOLR (Stratagene, USA).
2.6. Reverse transcription-PCR analysis
The expression of AdSp1 and AdSp3 in midguts,
haemocytes and salivary glands pooled from 50 A.
dirus female were investigated by RT-PCR under the
conditions described above. For semi-quantitative RT-
PCR, ribosomal gene AdS7 was used as an internal
control. The effect of blood feeding (B) and infection
(I) of P. yoelii on both of AdSp1 and AdSp3 mRNA
levels in haemocytes pooled from 50 female
mosquitoes were analyzed at days 1, 3, 4, 7 and 11
after infection, with those from mosquitoes feed on
5% sugar as a control (N). The initial cDNA amounts
for PCR amplification were normalized with AdS7
signal. AdSp1 and AdSp3 cDNAs in the samples were
sequences were as following: S7A: 50-GATCAT-
CATCTACGTGCCGGTG-30; S7B: 50-TGGTGGT-
CTGCTGGTTCTTGTC-30; AdSp1A: 50-CTCACT-
GCGCCGTAGACAAACC-30; AdSp1B: 50-GACAT-
ACACCGAACCGCAAGC-30; AdSp3A: 50-CAAC-
AAGAGGCTGGATGAAGG-30; AdSp3B: 50-TGCT-
The specific primer
2.7. Determination of infection degree and
melanization rate of P. yoelii in A. dirus midgut
To determine whether A. dirus was successfully
infected with P. yoelii after infectious blood meal,
midguts from 30 A. dirus female adults were dissected
and oocysts on the midgut were visualized and
counted under light microscopy at 7 days post-
infectious blood meal. In our experiment, only
mosquitoes with infection rate (infected mosquitoes/
dissected mosquitoes) over 75% and infection degree
(oocysts/infected mosquitoes) over 20 oocysts per
mosquito were used for semi-quantitative RT-PCR
analysis. As melanization of oocysts is the main
manifestation of A. dirus naturally refractory to P.
yoelii, the percentage of melanized oocyst (melanized
oocysts/total of oocysts) on A. dirus midgut at 7, 11
and15daysafterinfection of P.yoeliiwere calculated,
respectively. A total of 30 female adults were
dissected at each time point.
2.8. Statistic analysis
Quantitative values of both AdSp1 and AdSp3
expression in haemocytes of mosquitoes in N, B and I
group at 1, 2, 3, 4, 7 and 11 days post-infection are
based on densitometry scanning of PCR bands. Both
AdSp1 and AdSp3 transcript abundance of individual
mosquito were calculated based on the densitometry
of PCR bands of corresponding pooled sample and
compared using Student’s t-test between the control
and experiment group. p < 0.05 was considered
3. Results and discussion
Using degenerate primers AdSpf and AdSpr, we
obtained a 0.5 kb cDNA fragment by PCR from
haemocytes of A. dirus (Fig. 1A). Sequencing of
plasmid clones containing PCR product resulted in
three unique clones, representing three different
partial serine protease cDNAs, AdSp1 (AF369386),
AdSp2 (AF369387) and AdSp3 (AY033140). Both
AdSp1 and AdSp3 were 512 bp long and encoded two
170-residue sequences, and AdSp2 was 488 bp long.
All three sequences contained the conserved amino
acid residues (His, Asp and Ser) of serine protease
catalytic domain. Conserved domain search showed
that all three serine proteases were belong to trypsin-
like serine protease.
The predicted amino acid sequences were used to
search for similar proteins. The partial cDNA of
W.Y. Xu et al./Veterinary Parasitology 139 (2006) 93–101 96
AdSp2 was similar with Sp2A (70 identities out of
190 residues), a possible coagulation factor regulat-
ing the immune response of A. gambiae against
infection (Gorman et al., 2000a). The partial
sequence of AdSp1 was most similar to A. gambiae
serine protease (127/171), followed by Sp14D2 (80/
195), D. melanogaster (81/197), and Bombyx mori
(B. mori) prophenoloxidase activating Factor 3 (77/
191). AdSp3 partial cDNA sequence shared great
similarity with A. gambiae Sp 14A (145/170),
followed by M. sexta PPAE (82/194), H. diomphalia
PPO activating enzyme-I precursor (85/203), B. mori
PPO activating Factor 3 (77/193) and D. melano-
gaster easter (82/200). All three insect PPAEs were
the major component of PPO cascade mediating
melanotic encapsulation response of insect against
invading microorganisms (Jiang et al., 1998; Kwon
et al., 1997; Satoh et al., 1999). Easter is involved in
Drosophila dorsal–ventral pattern formation through
Toll pathway by limited proteolysis of ligand
spa ¨tzle. Both Sp14D2 and Sp14A were located in
division 14, mapped together with the minor
quantities loci trait Pen3 (Zheng et al., 1997)
controlling the A. gambiae melanotic encapsulation
trait. Consequently, similarity of both AdSp1 and
AdSp3 with insect PPAEs, D. melanogaster easter
and A. gambiae Sp14D2 and Sp14A, suggested their
possible role as PPAE or spa ¨tzle-processing enzyme
in melanotic encapsulation of P. yoelii by an
incompatible mosquitoes, A. dirus.
W.Y. Xu et al./Veterinary Parasitology 139 (2006) 93–101 97
obtained with degenerate primers AdSpf and AdSpr. (B) S7 PCR fragment (S) was amplified with degenerate primers AdS7f and AdS7r.
It is interesting to us that both AdSp1 and AdSp3
shared high similarity to PPAEs, and their tissue-
specific expression was investigated. RT-PCR analysis
showed that both AdSp1 and AdSp3 also expressed in
salivary gland (Fig. 2). No expected size of PCR
fragment was amplified from the midgut extract.
However, multiple bands were obtained (Fig. 2),
which might be derived from alternatively spliced
It was reported that different type of haemocytes
might play distinct role in immune responses of
mosquito against invasions (Hillyer and Christensen,
haemocytes of A. dirus, and detected positive signals
in cells such as granular, plasmocytes and prohemo-
cytes, according to the morphological descriptions of
Aedes aegypti (Ae. aegypti), C. quinquefasciatus and
A. albimanus hemocytes (Hernandez et al., 1999;
Kaaya and Ratcliffe, 1982). Consistent with Sp22D of
A. gambiae (Gorman et al., 2000b), the signals were
of haemocytes (Fig. 3).
A. gambiae S7 cDNA was routinely used as an
internal control to analyses the transcription of
immune related genes upon infection (Dimopoulos
et al., 1997, 2000; Muller et al., 1999). Therefore, the
ribosomal protein S7 from haemocyte of A. dirus was
also cloned. Through screening 3 ? 105plaques from
an expression cDNA library of female adults using
DIG-labeled 465 bp PCR fragment (Fig. 1B) probe; a
positive clone (AdS7) encoding complete sequence of
S7 was obtained. AdS7 (AY369135) was 871 bp in
length and encodes a 192 amino acid residue
polypeptide. BLAST result showed AdS7 shared high
similarity with S7 from A. gambiae (186/192), Culex
pipiens (179/1920), Ae. aegypti (178/192), M. sexta
(149/188) and B. mori (148/188).
Compared to N group (mosquitoes fed on 5%
sugar), the transcript abundance of both AdSp1 and
AdSp3 gene in group B (mosquitoes fed on non-
infected mouse blood) and I (mosquitoes fed on P.
yoelii infected mouse blood) were obviously high at
24 h after infection, but no significant difference
between all three groups in the following 2, 3 and 4
days (Fig. 4). It was consistent with the report that
robust immune response occurs when ookinetes
began to traverse midgut epithelium, but subsided at
the late phase of Plasmodium development (Dimo-
poulos et al., 1998). While, expression of both
AdSp1 and AdSp3 gene in I group reached their
peak values at 7 days post-infection, markedly
higher than those in both group B and N (p < 0.05)
(Fig. 4). It was coordinated with the finding that
some oocyts on midgut were partially melanized at
W.Y. Xu et al./Veterinary Parasitology 139 (2006) 93–101 98
Fig. 2. Reverse transcript PCR analysis of distribution of both
AdSp1 and AdSp3 in main tissues of A. dirus. Both AdSp1 and
AdSp3 genes expressed in haemocytes (H), and salivary glands (S)
and midgut (M)from 50 mosquitoeswere analyzed by RT-PCR with
AdSp1 and AdSp3-specific primers, respectively.
Fig. 3. In situ hybridization of AdSp1 in haemocytes of A. dirus (1000?). Haemocytes such as granular cells (A), plasmatocytes (B), and
prohemocytes (C) have detectable levels of staining hybridized with a digoxigenin (DIG)-labeled riboprobe of AdSp1.
7 days after infection (Fig. 5). The result indicated
the important role of melanotic encapsulation in
suppression of P. yoelii development by A. dirus.
Likely, A. gambiae serine proteases Sp14D2 and
Sp14A were reported to be up-regulated upon
et al., 2000a). The data further supported the
possible role of AdSp1 and AdSp3 in melanization
of P. yoelii by A. dirus.
In conclusion, our results suggested that both
AdSp1, AdSp3 might act as a spa ¨tzle-processing
enzyme or as PPO activators during melanization of P.
yoelii by A. dirus. Reverse genetic analysis, however,
is still needed to elucidate the role of serine proteases
AdSp1 and AdSp3 in the immune response of A. dirus
against Plasmodium. Fortunately, RNA interference
has been successfully applied to functional analysis of
immune-related genes of A. gambiae invitro (Blandin
et al., 2002; Levashina et al., 2001) and in vivo
(Blandin et al., 2004; Brown et al., 2003), and dsRNA
knock-out of both AdSp1 and AdSp3 in A. dirus was
under investigation in our laboratory.
We are grateful to Dr. Jiang (Department of
Entomology and Plant Pathology, Oklahoma State
University) for his critical comments on our manu-
script, and thank Dr. Fei Jian (Shanghai Institute of
Cell Biology, Chinese Academy of Science) for his
assistance in cDNA library construction. This work
was supported by grant 30200237 and 30400363 of
National Natural Science Foundation of China.
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Fig. 4. Effects of blood feeding and Plasmodium yoelii infection on the transcript abundance of both AdSp1 and AdSp3 in A. dirus haemocytes.
Both AdSp1 and AdSp3 transcript level in haemocytes from 50 A. dirus female adults fed on 5% sugar (N), non-infected mouse blood (B) and P.
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