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Abstract Malaria is caused by parasites of the genus
Plasmodium that are transmitted through the bite of female
mosquitoes of the genus Anopheles. Responsible for high
mobility and mortality rates around the world, this
protozoan disease is most common in the tropical and
sub-tropical regions. Herein, using a pure transcriptomic
data analysis approach on mosquito salivary glands we
have identified, compiled and compared immune-related
transcripts and their levels of expression in A. gambiae and
A. stephensi after P. berghei infection. Focusing in immune
mechanisms such as recognition of the parasite, signal
modulation by serine protease cascades and effector
mechanisms, several subclasses of proteins were
investigated, including thioester-containing proteins,
leucine-rich domain-containing proteins, C-type lectins,
galactoside-binding lectins, clip-domain serine proteases,
serine protease inhibitors, and antimicrobial peptides. The
anti-vector vaccine potential of key-molecules that have
exert an action in regulating parasite development have
been considered thus, targeting highly conserved antigenic
molecules can be effective to control arthropod-borne
diseases, including malaria. This study constitutes the first
comparative sialotranscriptomic analysis between these
two mosquito vectors upon pathogen invasion, focusing
solely specific subclasses of immune-related transcripts.
Lastly, in order to search for new targets with potential to
become pan-arthropod vaccines, we provide potential
candidate genes with interest to be further investigated for
malaria control
Keywords Malaria, Anopheles gambiae, Anopheles stephensi,
sialotranscriptome, RNA-seq, innate immune response.
This paragraph of the first footnote will be completed by the Editor and will
contain the date on which you submitted your paper for review.
1Instituto de Higiene e Medicina Tropical (IHMT), Rua da Junqueira 100,
1349-008 Lisboa, Portugal
2SaBio - Instituto de Investigación de Recursos Cinegéticos,
IREC-CSIC-UCLM-JCCM, Ciudad Real, Spain
3Department of Veterinary Pathobiology, Center for Veterinary Health
Sciences, Oklahoma State University, Stillwater, USA
4Global Health and Tropical Medicine - Instituto de Higiene e Medicina
Tropical (GHMT-IHMT), Rua da Junqueira 100, 1349-008 Lisboa, Portugal
*Correspondence to Domingos A. (e-mail: adomingos@ihmt.unl.pt).
DOI: 10.18281/iti.2016.2.4
I. INTRODUCTION
alaria is a protozoan disease caused by parasites of the
genus Plasmodium mostly spread in the tropical and
sub-tropical regions, transmitted through the bite of
female mosquitoes from the genus Anopheles [1, 2]. At present,
five species of the genus Plasmodium are known to cause
malaria in humans, resulting in 198 million cases and 584000
deaths in 2013 [2]. In areas such as sub-Saharan Africa, the
majority of the infections are caused by P. falciparum,
responsible for cerebral malaria, the most malignant form of the
disease [3, 4]. P. vivax and P. ovale, together with P.
falciparum, are the most widespread species but P. malariae
and P. knowlesi have also been associated with cases of malaria
in Southeast Asia [5, 6].
The biological cycle of these protozoa is very complex,
including several developmental stages that have to
sequentially occur both in the invertebrate definitive host, the
anopheline vector, and in the vertebrate intermediate host,
humans or others [3]. During its life span, a female Anopheles
mosquito feeds on an infected vertebrate host repeatedly for
protein and ion intake to promote egg development [7]. During
the blood meal, gametocytes are ingested and mobile zygotes,
the ookinetes, penetrate the mosquito midgut further
developing into oocysts [8]. The disruption of the oocysts
origins up to thousands of new sporozoites that are released and
spread to the haemocoel [9]. Subsequently, these parasitic
stages migrate to the salivary glands (SGs) and, during a new
blood meal, the parasite, in its infectious form, is transmitted
along with the saliva to the vertebrate host [8, 10]. Through the
bloodstream, sporozoites rapidly reach the liver, forming the
schizonts, which rupture releasing merozoites into the blood
stream [11]. This form is able to adhere to and infect red blood
cells (RBC), developing into the trophozoite stage [11]. After
dividing asexually into mature schizonts (erythrocytic
schizogony), RBC lysis occurs and the merozoite form is
released into the blood stream infecting other RBC, thus
perpetuating the infection [12]. Some of the merozoites may
develop into male and female gametocytes, the sexual
erythrocytic stages [13].
The mosquito SGs play an important role in transmission of
malaria due to the close contact and interaction with
Plasmodium sporozoites. This interaction is likely to be
Anopheles Gambiae and A. Stephensi Immune
Response during Plasmodium Berghei Infection
Uncovered by Sialotranscriptomic Analysis
Couto J. 1 MSc, Ferrolho J.1 DVM, MScVetSc, MScRes, de la Fuente J. 2,3 PhD and Domingos A.1,4*, BSc, PhD
M
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receptor-mediated [14, 15] reinforcing the importance of the
identification of the membrane proteins, the genes encoding
these molecules and investigation of their effect on parasite
transmission. New molecular advances in areas such as
transcriptomics and proteomics, alongside with complete
genome sequencing, and a robust method for interference RNA
(RNAi) in adult Anopheles mosquitoes [16] have brought the
possibility of investigate the role of genes and proteins that
might be involved in pathogen survival and transmission
mechanisms [17].
Invertebrates, as mosquitos, do not possess an adaptive
immune system but, instead, a potent innate immune system
[18]. Invading microorganisms must overcome several barriers
that are initially physical, such as the midgut wall, and after the
immune responses triggered by this epithelium, including those
activated systemically by the hemocytes and the fat body
culminating in the release of immune effectors into the
haemolymph [19]. The few microorganisms that successfully
evade these immune defenses are able to progress their
developmental cycle and become infective, to be later
transmitted to another host during a new blood meal. One such
example is the interaction between the Plasmodium parasite
and its invertebrate host. After the female mosquito blood meal,
the ookinetes have to penetrate the midgut epithelium to
develop into oocysts in the haemocoel [20]. Thus, successful
parasite transmission is dependent on several factors at the
molecular and cellular level and these interactions occur at
different stages of the invertebrate host and the parasite life
cycle. Sequencing of the complete A. gambiae genome has
identified 242 genes potentially encoding components of the
mosquito innate immune system [21] and recently, several
specific mosquito gene products that function as antagonists or
agonists of parasite development related to the immune
response have been successfully identified [19, 22].
Comparative transcriptomics can expand our understanding
on malaria vectors bionomics by revealing differences on the
expression of genes depending of the Plasmodium infection.
Mohien and colleagues (2013) developed an integrated
approach for transcriptome assembly that facilitated an analysis
of the midgut brush borders of the two malaria vectors A.
gambiae and A. albimanus [23]. Another comparative analysis
was performed correlating Drosophila melanogaster and A.
gambiae transcriptomes revealing an overall strong and
positive correlation of developmental expression between
orthologous genes [24].
Since Anopheles immune response clearly determines its
vectorial capacity, understanding how vector and parasite
interact at different stages of parasite development is vital.
Identification of the molecules that have an important role in
regulating parasite development will inform and direct the
design of new strategies to control and block malaria parasite
progress in the mosquito. These elements have been targeted to
evaluate their potential role as anti-vector vaccines, once they
can be species-specific or conserved between species.
Targeting highly conserved antigenic molecules can cause
cross-reactions against several species [25]. Based on this,
pan-arthropod vaccines can be useful to control
arthropod-borne diseases, being for this reason a more
amplified approach. In order to search for new targets with
potential to become pan-arthropod vaccines, we have analyzed
and compared the sialotranscriptome of A. gambiae and A.
stephensi during P. berghei infection focusing solely in the
transcripts that participate in the immune response. A refined
shortlist of potential candidate genes was further examined to
guide for new approaches to malaria control.
II. MATERIALS AND METHODS
Sialotranscriptome analysis of A. gambiae and A. stephensi
were carried out as described by Pinheiro-Silva et al. (2015)
[26] and Couto et al. (2015) [27]. RNA sequencing (RNA-seq)
data can be found in the database: ArrayExpress
(www.ebi.ac.uk/arrayexpress) with the Accession no.
E-MTAB-3415 and E-MTAB-3964, for A. gambiae and A.
stephensi, respectively.
Both groups of RNA-seq data were analysed and organized
into subclasses according to their functions in the immune
response and transcripts that are known to have a crucial impact
in the innate immune response of Anopheles mosquitoes were
selected. Seven subclasses were then selected for further
analysis and comparison: thioester-containing proteins (TEPs),
leucine-rich immune proteins (LRRs), C-type lysozymes or
lectins (CTLs), galectins (GALEs), Clip-domain serine
proteases (CLIPs), serine protease inhibitor - serpins (SRPNs)
and antimicrobial peptides (AMPs).
III. RESULTS
Effector molecules of the mosquito innate immune system
that are involved in the elimination of Plasmodium parasites are
potential targets for disease control [28], and their function can
be determined by transcriptomic and RNAi-based studies [29].
Immune reactions start when microorganisms are detected and
recognized by the host germline-encoded pattern recognition
receptors (PRRs) that can be cell-bound or circulating in the
haemolymph [30]. PRRs include several classes of molecules
[31], and from these we have focused on the TEPs, LRRs,
C-type lectins CTLs and GALEs, whose transcripts that
correspond to the genes encoding these proteins were found in
the sialotranscriptomes. Our selection was based on the
importance of each of these molecules in the mosquito innate
immune response and their role during pathogen infection.
Furthermore, for the same reason key components of the serine
protease cascades that act in signal modulation, such as CLIPs
and SRPNs, and those recognized as AMPs, were also selected
for comparison and analysis.
During the A. gambiae and A. stephensi transcriptomic
analysis, a total of 154 and 79 transcripts belonging to the
immunity class were found differentially expressed,
respectively (Fig. 1-A). Our results have shown that both
anophelines had more transcripts upregulated (63.6% in A.
gambiae and 77.2% in A. stephensi) than downregulated
(36.4% in A. gambiae and 22.8% in A. stephensi) (Fig. 1-B).
However, within the immunity class only 104 and 39 of these
transcripts of A. gambiae and A. stephensi belonged to the
aforementioned selected subclasses (Fig. 1-B and 1-C). Levels
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of expression for each of the transcripts can be observed in
Appendix I - Table 1.
Fig. 1. Global comparative sialotranscriptome profiles of Anopheles gambiae
and Anopheles stephensi during Plasmodium berghei infection. (A)
Distribution of differentially expressed transcripts per functional class. Green
and red arrows indicate genes up and downregulated, respectively, of each
functional class. (B) Functional classification and comparison of A. gambiae
and A. stephensi transcripts. Each area represents the percentage (%) of
transcripts within each functional class per mosquito species. (C) Number of
transcripts significantly differentially expressed for A. gambiae and A.
stephensi. The area of intersection corresponds to sequences with 80% of
homology between these two species. Green and red arrows indicate genes up
and downregulated, respectively, of each functional class. TEPs -
Thioester-containing proteins; SRPNs - Serpins; LRRs - Leucine-rich
domain-containing proteins; GALEs - Galactoside-binding lectins; CTLs -
C-type lectins; CLIPs - Clip-domain serine proteases.
A. Pattern recognition receptors
When triggered by the pathogen, PRRs recognize the invader
and can induce direct anti-pathogen defense mediating
microbial destruction by encapsulation or phagocytosis, or
indirect triggering of intracellular signaling pathways leading
to gene activation [32, 33]. Contrasting with vertebrates, the
invertebrates’ innate immune system seems to be deficient in a
memory and adaptive response, which is thought to be
compensated by specific activation of anti-pathogen immune
response pathways by class-specific PPRs [32]. To date, only
approximately 150 predicted PPRs genes have been found in
the A. gambiae genome [21]; but interestingly the wide variety
of these receptors gives the insects the capacity and plasticity of
recognizing a number of different of pathogens originated by
V(D)J recombination, as well as somatic hyper-mutation of the
antibody immunoglobulin (Ig) domains [32]. One such
example is the response of A. gambiae mosquitoes against the
challenge with P. berghei or P. falciparum with the splice
variants of the A. gambiae Down syndrome cell adhesion
molecule that is coded by AgDscam gene [32]. Our data
analysis regarding the A. gambiae sialotranscriptome alone has
revealed 51 transcripts differentially expressed that encode
PRR proteins, suggesting an intense immune response by this
mosquito species. From these, 26 were genes encoding LRRs,
12 related to TEPs functions, 8 and 5 were representative of
CTLs and GALEs (Fig. 1-A). In the A. stephensi
A
B
B
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sialotranscriptome 29 transcripts were found, and this lower
number might be due to its weaker immune response, as already
described [34]. From these, 23 transcripts were related to
LRRs, 5 were genes encoding TEPs, and only one was included
in CTLs subclass (Fig. 1-A). Each group of proteins will be
characterized in detail below.
A.1. Thioester-containing proteins
Leucine-rich repeat domain-containing proteins are a family
of proteins of the mosquito immune system with an important
role against pathogens, including Plasmodium parasites [29].
Two putative PRRs of this family, leucine-rich immune protein
1 (LRIM1) and Anopheles-Plasmodium-responsive
leucine-rich repeat 1 (APL1C), present in the mosquito
haemolymph in a form of a complex were found to be necessary
for P. berghei destruction during midgut invasion of
susceptible A. gambiae. After gene knockdown, a parasite
infection intensity increase was observed [35]. Dimopoulos and
colleagues (2002) have found that upon P. berghei infection
LRIM1 gene was strongly upregulated [36]. In a different
study, it was found that LRIM1 plays a role as an antagonist of
ookinetes development P. berghei in A. gambiae midgut. The
absence of LRIM1 induced approximately a fourfold increase
in the number of parasites in susceptible mosquitoes [19]. In
this study, we have found that the family of LRRs was one of
the most represented subclass within this immunity subclass in
both mosquito species, with 16.8% and 29.1% of transcripts
found in A. gambiae and A. stephensi, respectively (Fig. 1-B).
Also, it was observed that in A. gambiae and A. stephensi the
genes encoding these proteins were mostly upregulated,
corresponding to 76.9% and 60.9% of transcripts from the
LRRs subclass (Fig. 1-B and Appendix I - Table 1). Our
findings reinforce that LRRs are present and expressed to
antagonize the malaria parasite infection as part of a
complement-like system [37]. RNA-seq results are in line with
previous findings regarding the silencing of LRIM1 during a
medium-level infection of P. falciparum in A. gambiae
mosquitoes, which resulted in a 3.1-fold increase of infection
[38]. The overall results suggest a crucial role of these proteins
in the intensity of infection.
A.2. C-type lectin-like and galactoside-binding lectin
proteins
Dependent of Ca2+ C-type lectins like (CTLs) can recognize
and bind to a pathogen through the C-terminal carbohydrate
recognition domain [29]. This family of extracellular or
membrane-bound proteins, act as regulators of the mosquito
immune response focused in pathogen recognition [29]. Osta
and colleagues (2004) identified via gene silencing two
important CTLs, CTL4 and CTLMA2, as genes whose
products protect P. berghei ookinetes against an A. gambiae
innate immune response, namely melanization [20]. This
process is part of the humoral immune response of insects, that
takes place in the extracellular space between the midgut
epithelial cells and the basal lamina, and one of its purposes is
the sequestration of invading microorganisms in a dense
melanin coat [20]. These vector proteins seem to have been
somehow modified to protect the parasite, once their functional
silencing in a susceptible (G3) strain lead to the melanization of
the parasites. The knockout of CTL4 caused practically a
complete melanization of the developing ookinetes (97%) on
the basal site of the midgut epithelium, whereas the knockout of
CTLMA2 led to a partial melanization (48%). Nonetheless, the
total number of Plasmodium parasites that had penetrated the
epithelium was not significantly affected [20]. This partial
melanization can be justified by a possible inactivation of
LRIM1 that might defend the Plasmodium parasite from the
melanization response, leading to parasite development [39].
Our analysis has shown that the genes encoding CTLs were
mostly upregulated in both mosquito species after P. berghei
infection (Fig. 1-A and Appendix I - Table 1); although, more
transcripts that were found were expressed in A. gambiae (8
transcripts) in comparison with A. stephensi (1 transcript) (Fig.
1-A).
As members of the lectins family, GALEs have a C-terminal
carbohydrate recognition domain [40]. By recognizing specific
glycoconjugates, it is now suggested that they might act in
pathogen recognition or in phagocytosis [41], although the
knowledge regarding their precise role and abundance in
insects is still limited [42]. A novel putative secreted GALE has
been identified from the SGs transcriptome and proteome of A.
stephensi, showing an 81% identity at the amino acid level with
a protein of similar size from A. gambiae (agCP6926) [43]. A
putative infection-responsive GALE (IGALE20) was found to
be expressed in the midgut of A. gambiae after P. berghei
challenge, revealing their important role in insect innate
immunity during midgut invasion [44]. After a deep analysis of
both sialotranscriptome catalogues, we were able to verify that
genes encoding for GALEs (5 transcripts) were only present in
A. gambiae (Fig. 1-A) and, interestingly, these genes were
found downregulated (Fig. 1-A and Appendix I - Table 1). This
decrease in the expression level can be explained by the ability
of some parasites to alter the capacity of A. gambiae GALEs to
recognize them, enabling the attachment and invasion by
Plasmodium [40]. On the other hand, an absent differential
expression of GALE genes in the SG of A. stephensi during P.
berghei infection does not invalidate the presence of these
genes in this vector [42].
B. Signal modulation
The recognition of invading microorganisms activates
proteolytic cascades of serine proteases to trigger effector
responses, for example, the synthesis of AMPs to eliminate the
attacker [45, 46]. Serine proteases belong to a wide family of
endopeptidases which have a residue of serine that is
catalytically active. Some of these proteins have a particular
domain between 30 to 60 amino acids that can be identified by
their N-terminal, and due to their configuration, these are
named as CLIPs [47]. Phylogenetic studies have showed that
Anopheles and Drosophila genomes present four sub-families
of CLIPs (CLIPs A, B, C and D) [21], that can be divided in two
categories, positive regulators and negative regulators [48].
During the serine protease cascade, the signal amplification is
regulated by SRPNs, serine protease inhibitors. SRPNs inhibit
serine proteases through an irreversible covalent bind to the
center of these enzymes [19]. In terms of the immune response,
serine proteases play an essential role through melanization.
For this process to occur, the proteolytic activation of inactive
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prophenoloxidases (PPOs) zymogens to active phenoloxidases
(POs) is necessary and closely regulated through a balance
between positive and negative regulators, CLIPs, and their
inhibitors, SRPNs [19]. In our sialotranscriptomic results, A.
gambiae expressed more transcripts related to serine proteases
(46 transcripts) than A. stephensi (9 transcripts), which
represents 29.9 and 11.4% of differential expressed genes
within the immunity class, respectively (Fig. 1-B). From this 46
A. gambiae transcripts, 35 were related to CLIPs functions and
11 encoded for SRPNs. In the A. stephensi mosquito, 8 and 1
transcripts corresponded to CLIPs and SRPNs, respectively,
included in serine proteases category.
B.1. CLIP-domain serine proteases
The melanotic encapsulation of parasites can be the cause of
mosquito refractoriness against Plasmodium. In Aedes aegypti,
CLIPA14 and CLIPB6 are homologous of the PPO activation
cofactors, enriched by different transcriptional factors such as
REL2 and REL1, respectively [49]. Interestingly, these two
CLIPs can be found both upregulated in susceptible strains of
A. gambiae [50]. Christophides and colleagues (2002) have
found that A. gambiae CLIPB14 was persistently upregulated
when the midgut invasion by Plasmodium ookinetes occurs
[21]. Also, that these infections led to a transient induction of
CLIPB15. A different study has characterized molecular and
functionally A. gambiae CLIPB14 and CLIPB15 expressed in
the hemocytes [51], demonstrating that, after gene knockdown,
these two CLIPs are involved in parasite destruction. In
susceptible and refractory strains of mosquitoes it was observed
that the number of developing oocysts and melanized ookinetes
had a significant increase in comparison with the controls and
with the individual CLIP knockdowns [51]. Nevertheless, when
silencing CLIPB14 or CLIPB15 in refractory mosquitoes,
ookinete melanization was not prevented [51]. These results
suggest that CLIPB14 and CLIP15 play a role as negative
regulators of ookinete melanization. The same negative
feedback of suppressing melanization process is also mediated
by CLIPA2, 5 and 7, allowing the development of malaria
parasites in A. gambiae mosquitoes [52]. On the other hand,
different CLIPs, such as CLIPB17 and CLIPA8, have been
found to be positive regulators of mosquito melanization. In A.
gambiae, the depletion of CLIPB17 and CLIPA8 led to a higher
or absolute elimination of melanization, respectively [48].
When analyzing the A. gambiae sialotranscriptome alone, it
was observed that the majority of CLIPs found and that were
differentially expressed were included within the sub-families
A and B. CLIPA2, 5 and 7 serine proteases associated with the
melanization suppression [52] were found upregulated in our
results, suggesting a similar mediations process to the one that
occurs in the mosquito midgut. The gene encoding CLIPA8
was found upregulated, probably because is acting in the
stimulation of melanization process. Although our targets were
the SGs, the result is in agreement with previous findings
reported for the midgut in that CLIPA8 activates the PO
cascade and its knockdown blocks melanization, promoting the
oocysts development [48, 52]. Similarly, CLIPB14 and
CLIPB15 were also found upregulated in our analysis. Studies
conducted in the midgut reported that CLIPB14 is only found
enriched 28 hours post-infection, particularly during midgut
invasion; whilst CLIPB15 is continuously upregulated having a
mild increase during Plasmodium infection [21]. When
analyzing the A. stephensi sialotranscriptome, we have found
that the sub-families A and B were less represented within the
CLIPs subclass. Only the three transcripts encoding for
CLIPA4, CLIPA14 and CLIPB6 were found and were related
to these two sub-families. Interestingly, CLIPA14 and CLIPB6
were exclusively found in this mosquito data and they have
been implicated in the PPO activation [49]. Finally, we have
found that TEPs, LRIMs and CLIPs transcripts in both
mosquitoes SGs infected by P. berghei were predominantly
upregulated (Fig. 1-A and Appendix I - Table 1). It has been
found that during midgut pathogen invasion, these proteins
form the complex LRIM1/APL1C/TEP to interact with the
ookinetes, being therefore, as in our results, their genes also
upregulated [46]. The same was verified previously with APL
and LRIM in A. funestus [53]. Moreover, previous studies
indicate that in the midgut, silencing of a determined TEP leads
to the inhibition of CLIP cleavage activity. This confirms that
the accumulation of TEP on malaria parasites and bacteria is
regulated by CLIPs, which leads to distinct defense reactions,
including lysis and melanization of the pathogen [54].
B.2. Serpins
The haemolymph of insects is rich in serine protease
inhibitors. As an important class of negative regulators, SRPNs
belong to this large family of proteases with an intra or
extra-cellular conserved structure [31]. In insects, SRPNs can
be produced in response to physiological or pathological
stimulus and regulate immune protease pathways that prevent
damaging effects of uncontrolled immune responses [21, 54,
55]. They are substrate-specific and their inhibitory function is
mediated by a reactive center loop (RCL) [31]. The first
inhibitory SRPN has been reported and designated as SRPN2,
and its presence was found required for successful P. berghei
oocyst development in A. gambiae. The knockdown of SRPN2
affected the invasion of A. gambiae midgut by the parasite
through oocyst number reduction, due to increased ookinete
lysis and melanization [31]. The serpin SRPN6 has been
studied in A. stephensi and A. gambiae after infection with P.
berghei, revealing that its gene expression is strongly induced
in both mosquitos’ midguts during ookinete invasion. This
SRPN has been associated with the outcome of infection in A.
stephensi in that its gene knockdown increased the number of
oocysts; whereas the knockdown of A. gambiae SRPN6 had no
effect on the oocyst load or prevalence of infection in the
midgut [9]. A different study has suggested that the transcripts
encoding four intracellular serine protease inhibitors of
SRPN10 isoforms (KRAL, RCM, FCM and CAM) in A.
gambiae females are induced as a response to midgut invasion
by P. berghei ookinetes and might regulate the apoptosis of
invaded cells that leads to epithelial damage [56, 57]. The
upregulation of each isoform, under different physiological
conditions, suggests that alternative splicing has a fundamental
role for the regulation of serpins’ function [56]. Our data
analysis has shown that the number of transcripts encoding for
SRPNs was higher for A. gambiae (11 transcripts) in
comparison with A. stephensi (1 transcript) (Fig. 1-B), of which
the genes differentially expressed included SRPN2, SRPN10
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and SRPN6. Levels of expression for each of the transcripts can
be observed in Appendix I - Table 1. In A. gambiae, SRPN2
was upregulated, while SRPN10 was downregulated
(Appendix I - Table 1). The transcript corresponding to the
protein SRPN6 was not found in this mosquito. In the A.
stephensi sialotranscriptome, only SRPN6 was identified and
was upregulated (Appendix I - Table 1).
C. Effector mechanisms
Mosquito immunity includes several effector mechanisms,
such as AMPs, melanization, phagocytosis and cellular
immune response [46]. In this study the main focus will be on
the AMPs because after pathogen invasion, the synthesis of
small mostly positively charged AMPs is the last step of the
mosquito inducible humoral response by different pathways
[19]. In our sialotranscriptome analysis most of them were
found in A. gambiae (7 transcripts) in comparison with A.
stephensi (1 transcript) (Fig. 1-B).
C.1. Antimicrobial peptides
After microorganism recognition by PRRs, AMPs are
quickly synthesized by the fatty body, hemocytes and epithelia,
and secreted into the haemolymph where they accumulate in
high concentrations [58-60]. In A. gambiae they are also
synthesized locally by the barrier epithelia [36, 61, 62]. The A.
gambiae full genome sequencing has revealed genes that
encode for several of these proteins, including defensins
(DEFs), cecropins (CECs), attacins and gambicin (GAM) [21].
As reported by Luna and collegues (2006), the expression of
some AMPs can be influenced by Toll and Imd pathways [63].
After silencing components of the Imd pathway, such as the
NF-κB transcription factor REL2, the expression of DEF1 and
a GAM1 was reduced in A. gambiae cell lines. Besides, the
over expression of either NF-κB transcription factor REL1 or
REL2 induced the expression of CEC1, GAM1 and DEF1,
implicating them in the expression of AMPs. In contrast, in the
A. stephensi cell line MSQ43 the transcription factors were
downregulated [63]. In other studies, AMPs such as CEC1,
CEC3, GAM1, are REL2-influenced in A. gambiae cells lines
[64, 65]. Kim and colleagues (2004) have shown in vivo that
CEC1 transgene expression in transgenic A. gambiae reduced
the number of P. berghei oocysts in the midgut [66]. Other
authors demonstrated that CEC1 was found upregulated in
mosquitoes infected by Plasmodium sp. [21], and that CEC2 is
significantly enriched in the transcriptome of midgut,
especially in a compartment named cardia [67]. At the moment,
DEF3, DEF2 and CEC2 still have unidentified functions;
though, clustering studies suggest that DEF3 exerts an action in
immunity during blood meal inducing cuticle expansion, and
perhaps against fungal infection [68]. In our results,
AMPs-related transcripts that were identified in A. gambiae
sialotranscriptome included CEC1, CEC2, CEC3 and GAM1.
These revealed a low expression at the transcriptional level
(Appendix I - Table 1) probably because the mosquito immune
response is being affected by the parasite. The transcripts
referring to the proteins DEF1 and DEF3 were also found.
Finally, when the SG transcriptome of A. stephensi was
analyzed, only DEF1 was found in the AMPs subclass. The
absence of others AMPs transcripts in the analyzed data may be
related to a non-differential expression of AMP genes by
different pathways, such as Imd and Toll pathway.
Our overall results have identified the transcripts that codify
for IGL-1, TEP4, TEP12, TEP14, CLIPA4, CLIPA14 and
CLIPC4 (Fig. 2) as good targets for future studies to evaluate
their potential as pan-Anopheles vaccines, due to the high
homology and conservation between of these genes in the two
species. Fig. 2 shows the conserved immunity genes that are
common between A. gambiae and A. stephensi with at least
80% homology, and the ones only found in one of the species.
Also, the immune mechanism in which they are involved is
highlighted. Interestingly, with a similar expression and high
homology of sequences between both species we were able to
identify IGL-1, TEP4, TEP12, TEP14, CLIPA4, CLIPA14 and
CLIPC4.
Fig. 2. Immune-related transcripts of Anopheles gambiae and Anopheles
stephensi salivary glands during Plasmodium berghei infection. Selected
subclasses of proteins involved in pattern recognition, signal modulation, and
effector mechanisms. AMPs - Antimicrobial peptides; APLs -
Anopheles-Plasmodium-responsive Leucine-Rich Repeat; CECs - Cecropins;
CLIPs - Clip-domain serine proteases from signal modulation; CTLs - C-type
lectins; DEFs - Defensins; GALEs - galectins; GAMBs - Gambicins; LRIMs -
Leucine-rich immune protein; PRRs - Pattern recognition receptors; SRPNs -
serpins; TEPs - Thioester containing proteins. Differential expression is
represented by the green and red arrows, indicating up or downregulated,
respectively.
IV. CONCLUSIONS
To date, several efforts have been made to control human
malaria worldwide but, nonetheless, this disease burden is still
increasing, not only due to socio-economic and politic factors
that are not positively contributing for its reduction, but also
because the control measures that have been directed to the
mosquito vector with insecticides, environment control and
insecticide-impregnated bed nets are not completely effective.
These strategies have faced major drawbacks regarding
insecticide-resistance of the vector and drug-resistance of the
parasite, which led to the malaria reassurance. For this, new
approaches and the design of new strategies are urgently
needed. One strategy is to unravel the key-molecules and
mechanisms, particularly the ones present in the mosquito
immunity that are essential for the Plasmodium successful
development in the mosquito vector and cause their blockage to
interrupt parasite transmission to the vertebrate host. Here, we
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64
have compared sialotranscriptome of A. gambiae and A.
stephensi during P. berghei infection, focusing in transcripts
that are known to have a crucial impact in innate immune
response of Anopheles mosquitoes. In this study, we conclude
that A. gambiae and A. stephensi have similarities and
differences in the expression of genes related to innate immune
system, since initial immune response to the production of
antimicrobial peptides. The understanding of this interplay may
be conducted for the development of the new strategies of
malaria control.
ACKNOWLEDGMENTS
The authors would like to acknowledge Renato
Pinheiro-Silva for the scientific advice and constructive
comments regarding the A. gambiae transcriptomic analysis.
The authors declare no competing personal or financial
interests.
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APPENDIX
Appendix A
Table 1. List of immune-related transcripts of Anopheles gambiae and Anopheles stephensi salivary glands during Plasmodium
berghei infection. Selected transcripts grouped by immune subclasses are identified by their Gene ID from the database:
VectorBase (www.vectorbase.org) and gene description. The levels of expression in fold-change are described. AMPs -
Antimicrobial peptides; CLIPs - Clip-domain serine proteases from signal modulation; CTLs - C-type lectins; DEFs - Defensins;
GALEs - galectins; LRRs - Leucine-rich repeat; SRPNs - serpins; TEPs - Thioester containing proteins.
Mosquito
Subclass
Gene ID
Gene description
Fold-change
Anopheles
gambiae
AMPs
AGAP000692
CEC3: cecropin anti-microbial peptide C
-1,124
AGAP000693
CEC1: cecropin anti-microbial peptide A
-1,005
AGAP000694
CEC2: cecropin anti-microbial peptide B
-1,111
AGAP004632
DEF2: defensin anti-microbial peptide 2
1,141
AGAP007199
DEF3: defensin anti-microbial peptide 3
2,368
AGAP008645
GAM1: gambicin anti-microbial peptide
-1,030
AGAP011294
DEF1: defensin anti-microbial peptide 1
-1,052
CLIPs
AGAP000315
CLIPC6: Clip-Domain Serine Protease
1,259
AGAP000573
CLIPC4: Clip-Domain Serine Protease
1,083
AGAP001648
CLIPB17: Clip-Domain Serine Protease
1,639
AGAP002270
CLIPB7: Clip-Domain Serine Protease
2,270
AGAP003057
CLIPB8: Clip-Domain Serine Protease
1,175
AGAP003246
CLIPB2: Clip-Domain Serine Protease
1,508
AGAP003247
CLIPB19: Clip-Domain Serine Protease
1,198
AGAP003249
CLIPB3: Clip-Domain Serine Protease
1,833
AGAP003250
CLIPB4: Clip-Domain Serine Protease
1,270
AGAP003251
CLIPB1: Clip-Domain Serine Protease
1,256
AGAP003689
CLIPC7: Clip-Domain Serine Protease
1,700
AGAP004148
CLIPB5: Clip-Domain Serine Protease
-1,087
AGAP004317
CLIPC2: Clip-Domain Serine Protease
1,110
AGAP004318
CLIPC3: Clip-Domain Serine Protease
1,073
AGAP004719
CLIPC9: Clip-Domain Serine Protease
1,230
AGAP004855
CLIPB13: Clip-Domain Serine Protease
1,107
AGAP008091
CLIPE1: Clip-Domain Serine Protease
-1,463
AGAP009215
CLIPB18: Clip-Domain Serine Protease
1,643
AGAP009217
CLIPB12: Clip-Domain Serine Protease
2,267
AGAP009844
CLIPB15: Clip-Domain Serine Protease
1,405
AGAP010530
CLIPE4: Clip-Domain Serine Protease
1,289
AGAP010547
CLIPE5: Clip-Domain Serine Protease
1,093
AGAP010731
CLIPA8: Clip-Domain Serine Protease
1,240
AGAP010833
CLIPB14: Clip-Domain Serine Protease
1,303
AGAP010968
CLIPA9: Clip-Domain Serine Protease
1,052
AGAP011780
CLIPA4: Clip-Domain Serine Protease
1,077
AGAP011781
CLIPA12: Clip-Domain Serine Protease
1,338
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Mosquito
Subclass
Gene ID
Gene description
Fold-change
AGAP011787
CLIPA5: Clip-Domain Serine Protease
1,268
AGAP011788
CLIPA14: Clip-Domain Serine Protease
1,277
AGAP011789
CLIPA6: Clip-Domain Serine Protease
1,244
AGAP011790
CLIPA2: Clip-Domain Serine Protease
1,369
AGAP011791
CLIPA1: Clip-Domain Serine Protease A1
1,302
AGAP011792
CLIPA7: Clip-Domain Serine Protease
1,315
AGAP013184
CLIPB36: Clip-Domain Serine Protease
1,234
AGAP013442
CLIPB10: Clip-Domain Serine Protease
1,064
CTLs
AGAP000007
IGL1: contactin-like putative cell adhesion molecule
1,087
AGAP002625
CTL9: C-type lectin enzyme
-1,218
AGAP005334
CTLMA2: C-Type Lectin (CTL) - mannose binding
1,075
AGAP005335
CTL4: C-type lectin enzyme
1,093
AGAP006430
CTLGA2: C-Type Lectin (CTL) - galactose binding A2
1,557
AGAP007407
CTLMA4: C-Type Lectin (CTL) - mannose binding
1,310
AGAP010193
CTLGA3: C-Type Lectin (CTL) - galactose binding A3
-1,099
AGAP010196
CTLGA1: C-Type Lectin (CTL) - galactose binding A1
-1,552
GALEs
AGAP000341
GALE2: galectin2
-1,171
AGAP004806
GALE6: galectin6
-1,155
AGAP004807
GALE7: galectin 7
-1,216
AGAP004934
GALE3: galectin3
-1,130
AGAP011287
GALE5: galectin5
-1,078
LRRs
AGAP001127
P37NB protein
1,157
AGAP003878
n/a
1,086
AGAP004405
glucose-repressible alcohol dehydrogenase transcriptional effector homolog
1,522
AGAP005496
LRIM12
-1,256
AGAP005693
LRIM17/LRRD7
1,256
AGAP005962
n/a
1,134
AGAP006183
slit protein
1,529
AGAP006348
LRIM1
1,457
AGAP006408
n/a
-1,158
AGAP006643
n/a
-1,070
AGAP007033
APL1C: Anopheles Plasmodium-responsive Leucine-Rich Repeat 1C
1,383
AGAP007035
APL1B: Anopheles Plasmodium-responsive Leucine-Rich Repeat 1B
1,688
AGAP007036
APL1A: Anopheles Plasmodium-responsive Leucine-Rich Repeat 1A
1,641
AGAP007039
LRIM4
1,274
AGAP007453
LRIM9: leucine-rich immune protein (Short)
1,263
AGAP007454
LRIM8A: leucine-rich immune protein (Short)
1,186
AGAP007455
LRIM10
1,304
AGAP007456
LRIM8B
1,220
AGAP007758
n/a
1,156
AGAP008927
protein TILB homolog
2,734
AGAP009924
n/a
-1,121
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Mosquito
Subclass
Gene ID
Gene description
Fold-change
AGAP010180
centrosomal protein CEP97
2,734
AGAP012317
n/a
-1,261
AGAP012425
n/a
-1,070
AGAP013059
n/a
1,145
AGAP013186
n/a
1,164
OTHERS
AGAP000016
SCRB10: Class B Scavenger Receptor
-1,211
AGAP000536
PGRPS1: Peptidoglycan Recognition Protein (Short) 1
1,130
AGAP000631
integrator complex subunit 6
1,189
AGAP000999
TOLL5A: Toll-like protein 5A
1,261
AGAP001182
n/a
-1,141
AGAP001212
PGRPLB: peptidoglycan recognition protein (Long)
1,088
AGAP003141
Insulin-related peptide binding protein
1,179
AGAP003354
Venom allergen
-1,322
AGAP003428
speckle-type POZ protein
1,167
AGAP004170
n/a
-1,348
AGAP004455
GNBPB1: 3-Glucan Binding Protein
-1,091
AGAP004774
host cell factor
1,137
AGAP004845
scavenger receptor class B member
1,120
AGAP004846
SCRB9: Class B Scavenger Receptor
-1,074
AGAP004847
SCRB7: Class B Scavenger Receptor 7
1,094
AGAP004916
n/a
-1,023
AGAP004920
CASPS6: caspase (short class) 6
-1,181
AGAP004921
CASPS5: caspase (short class) 5
-1,082
AGAP004922
CASPS11: caspase (short class) 11
-1,401
AGAP004977
PPO6: Prophenoloxidase enzyme
-1,056
AGAP005203
PGRPLC: Peptidoglycan Recognition Protein (Long) Transcript 2
-1,050
AGAP005252
MYD: TOLL pathway signalling
-1,128
AGAP005716
SCRB16: Class B Scavenger Receptor
-1,159
AGAP005725
SCRB3: Class B Scavenger Receptor 3
-1,583
AGAP005933
NFkappaB essential modulator
-1,128
AGAP006342
PGRPS3: Peptidoglycan Recognition Protein (Short) 3
-1,115
AGAP006343
PGRPS2: Peptidoglycan Recognition Protein (Short) 2
-1,068
AGAP006419
Venom allergen
1,256
AGAP006421
Venom allergen
1,178
AGAP006974
TOLL9: Toll-like protein 9
-1,081
AGAP007293
IAP7: Inhibitor of Apoptosis 7
-1,275
AGAP007294
IAP1: Inhibitor of Apoptosis 1
-1,111
AGAP007343
LYSC2: C-Type Lysozyme
3,365
AGAP007347
LYSC1: Lysozyme c-1
1,119
AGAP007385
LYSC4: C-Type Lysozyme
1,177
AGAP007386
LYSC7: C-Type Lysozyme
-1,089
AGAP007394
protein AATF/BFR2
1,237
INTERNATIONAL TRENDS IN IMMUNITY VOL.4 NO.2 APRIL 2016
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70
Mosquito
Subclass
Gene ID
Gene description
Fold-change
AGAP007612
n/a
-1,172
AGAP007679
n/a
-1,081
AGAP007684
Tubulointerstitial nephritis antigen
1,202
AGAP007821
n/a
-1,124
AGAP008004
n/a
1,114
AGAP008113
n/a
-1,022
AGAP009166
IKK1: IMD pathway signalling IKK-beta
1,127
AGAP009651
CD63 antigen
-1,036
AGAP009653
n/a
-1,038
AGAP010186
echinoid
1,227
AGAP010773
n/a
1,933
AGAP011693
CASPL1 : caspase (long class) 1
1,162
AGAP011952
CASPS3: caspase (short class) 3
-1,276
SRPNs
AGAP001375
SRPN12: serine protease inhibitor (serpin) homologue - unlikely to be
inhibitory
-2,637
AGAP001377
SRPN11: serine protease inhibitor (serpin) homologue - unlikely to be
inhibitory
1,236
AGAP003139
SRPN9: serine protease inhibitor (serpin)
-1,092
AGAP005246
SRPN10: serine protease inhibitor (serpin) homologue - unlikely to be
inhibitory
-1,153
AGAP006910
SRPN3:serine protease inhibitor (serpin)
1,182
AGAP006911
SRPN2: serine protease inhibitor (serpin)
1,221
AGAP007693
SRPN7: serine protease inhibitor (serpin)
1,115
AGAP009212
SRPN6: serine protease inhibitor (serpin)
-1,226
AGAP009213
SRPN16: serine protease inhibitor (serpin)
1,164
AGAP009221
SRPN5: serine protease inhibitor (serpin)
-1,111
AGAP009670
SRPN4: serine protease inhibitor (serpin)
1,068
TEPs
AGAP008364
TEP15: thioester-containing protein
1,103
AGAP008366
TEP2: thioester-containing protein
-1,093
AGAP008368
TEP14: thioester-containing protein
1,262
AGAP008654
TEP12: thioester-containing protein
1,585
AGAP010812
TEP4: thioester-containing protein
1,375
AGAP010814
TEP6: thioester-containing protein
1,601
AGAP010815
TEP1 (CM000358 11202090..11205737) thioester-containing protein
1,522
AGAP010815
TEP1 (CM000358 11206189..11206882): thioester-containing protein
1,830
AGAP010816
TEP3: thioester-containing protein
1,518
AGAP010818
TEP11: thioester-containing protein
1,651
AGAP010830
TEP9: thioester-containing protein
1,597
AGAP010831
TEP8: thioester-containing protein
2,618
Mosquito
Subclass
Gene ID
Gene description
Fold-change
Anopheles
stephensi
AMP
AGAP011294
DEF1: defensin anti-microbial peptide 1
1,860
CLIP
AGAP000572
CLIPC10: Clip-Domain Serine Protease
1,053
AGAP000573
CLIPC4: Clip-Domain Serine Protease
3,478
AGAP002422
CLIPD1: Clip-Domain Serine Protease
2,905
INTERNATIONAL TRENDS IN IMMUNITY VOL.4 NO.2 APRIL 2016
ISSN 2326-3121 (Print) ISSN 2326-313X (Online) http://www.researchpub.org/journal/iti/iti.html
71
Mosquito
Subclass
Gene ID
Gene description
Fold-change
AGAP002813
CLIPD6: Clip-Domain Serine Protease
2,123
AGAP003252
CLIPB6: Clip-Domain Serine Protease
5,220
AGAP004719
CLIPC9: Clip-Domain Serine Protease
-0,840
AGAP011780
CLIPA4: Clip-Domain Serine Protease
3,617
AGAP011788
CLIPA14: Clip-Domain Serine Protease
1,241
CTL
AGAP000007
IGL1: contactin-like putative cell adhesion molecule
1,360
LRR
AGAP000601
n/a
1,892
AGAP001127
P37NB protein
-1,874
AGAP002007
n/a
-1,906
AGAP004405
glucose-repressible alcohol dehydrogenase transcriptional effector homolog
-2,277
AGAP004458
n/a
-1,081
AGAP005496
LRIM13
1,074
AGAP005693
n/a
-0,957
AGAP005744
n/a
1,995
AGAP006644
n/a
1,161
AGAP006647
n/a
-1,074
AGAP007059
n/a
-2,449
AGAP007384
n/a
2,061
AGAP007454
LRIM8A: leucine-rich immune protein (Short)
-1,435
AGAP008611
n/a
0,963
AGAP008785
n/a
2,953
AGAP009839
n/a
1,216
AGAP009924
n/a
2,055
AGAP010770
n/a
1,556
AGAP011292
n/a
2,618
AGAP012317
n/a
2,647
AND_003345
Leucine rich repeat containing protein
1,915
AND_005520
Membrane glycoprotein LIG-1
-3,871
AND_006574
Leucine rich repeat protein
3,052
OTHERS
AGAP000247
n/a
3,509
AGAP000536
PGRPS1: Peptidoglycan Recognition Protein (Short) 1
3,619
AGAP000904
n/a
3,181
AGAP000999
TOLL5A: Toll-like protein 5A
1,256
AGAP001212
PGRPLB: peptidoglycan recognition protein (Long)
2,277
AGAP001954
psidin: Phagocyte signaling-impaired protein
1,218
AGAP002731
n/a
1,452
AGAP002738
SCRB5
1,658
AGAP002790
n/a
-1,822
AGAP003428
speckle-type POZ protein
1,056
AGAP003521
n/a
3,510
AGAP004455
GNBPB1: 3-Glucan Binding Protein
-0,973
AGAP004643
SCRB6: Class B Scavenger Receptor 6
-2,613
INTERNATIONAL TRENDS IN IMMUNITY VOL.4 NO.2 APRIL 2016
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72
Mosquito
Subclass
Gene ID
Gene description
Fold-change
AGAP004845
scavenger receptor class B member
-1,803
AGAP004847
SCRB7: Class B Scavenger Receptor 8
1,749
AGAP004918
n/a
-2,205
AGAP005252
MYD: TOLL pathway signalling
2,352
AGAP005713
n/a
3,079
AGAP005901
n/a
1,485
AGAP005901
n/a
2,217
AGAP006328
n/a
4,216
AGAP006747
REL2
2,117
AGAP006771
n/a
1,506
AGAP006974
TOLL9: Toll-like protein 10
2,688
AGAP007294
IAP1: Inhibitor of Apoptosis 2
1,385
AGAP007563
n/a
1,828
AGAP007684
Tubulointerstitial nephritis antigen
-1,568
AGAP007809
n/a
1,024
AGAP008412
n/a
3,249
AGAP008813
n/a
2,381
AGAP009143
SCRAC1
2,378
AGAP009166
IKK1: IMD pathway signalling IKK-beta
1,733
AGAP010186
echinoid
1,271
AGAP011119
Lysozyme i-1
2,395
AGAP013186
n/a
2,621
AND_003269
Pellino
2,076
AND_004871
Interleukin enhancer binding factor
0,989
n/a
Immune response-related protein
-1,271
n/a
Prophenol oxidase (EC 1.14.18.1)
1,743
n/a
Atlastin
2,297
SRPN
AGAP009212
SRPN6: serine protease inhibitor (serpin)
1,496
TEP
AGAP008368
TEP14: thioester-containing protein
1,071
AGAP008407
TEP13
3,230
AGAP008654
TEP12: thioester-containing protein
2,943
AGAP010812
TEP4: thioester-containing protein
2,287
AGAP010815
TEP-I
-0,882
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