ORIGINAL RESEARCH
published: 24 June 2021
doi: 10.3389/fimmu.2021.584660
Heat Shock Causes Lower
Plasmodium Infection Rates in
Anopheles albimanus
Renaud Condé 1, Erika Hernandez-Torres 1, Fabiola Claudio-Piedras 1,
Benito Recio-Tótoro 1,2, Krystal Maya-Maldonado 1, Victor Cardoso-Jaime 1
and Humberto Lanz-Mendoza 1*
1
2
Edited by:
Massimo E. Maffei,
University of Turin, Italy
Reviewed by:
Nora Kristin Elisa Schulz,
Vanderbilt University, United States
Maria Luisa Simões,
Johns Hopkins University,
United States
*Correspondence:
Humberto Lanz-Mendoza
humberto@insp.mx
Specialty section:
This article was submitted to
Molecular Innate Immunity,
a section of the journal
Frontiers in Immunology
Received: 17 July 2020
Accepted: 08 June 2021
Published: 24 June 2021
Citation:
Condé R, Hernandez-Torres E,
Claudio-Piedras F, Recio-Tótoro B,
Maya-Maldonado K, Cardoso-Jaime V
and Lanz-Mendoza H (2021) Heat
Shock Causes Lower Plasmodium
Infection Rates in Anopheles albimanus.
Front. Immunol. 12:584660.
doi: 10.3389/fimmu.2021.584660
Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Mexico,
Instituto de Biotecnologı´a, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
The immune response of Anopheles mosquitoes to Plasmodium invasion has been
extensively studied and shown to be mediated mainly by the nitric oxide synthase
(NOS), dual oxidase (DUOX), phenoloxidase (PO), and antimicrobial peptides activity.
Here, we studied the correlation between a heat shock insult, transcription of immune
response genes, and subsequent susceptibility to Plasmodium berghei infection in
Anopheles albimanus. We found that transcript levels of many immune genes were
drastically affected by the thermal stress, either positively or negatively. Furthermore, the
transcription of genes associated with modifications of nucleic acid methylation was
affected, suggesting an increment in both DNA and RNA methylation. The heat shock
increased PO and NOS activity in the hemolymph, as well as the transcription of several
immune genes. As consequence, we observed that heat shock increased the resistance
of mosquitoes to Plasmodium invasion. The data provided here could help the
understanding of infection transmission under the ever more common heat waves.
Keywords: Anopheles albimanus, Plasmodium berghei, heat shock, immunity, infection resistance
INTRODUCTION
Insects are commonly stressed by pathogens (1), toxic compounds, dietary factors, temperature (2),
and hypoxia (3). These stressors share commonalities in their effects on the molecular components
of cells, and therefore much of the transcriptional response they elicit is conserved (4). There is
significant crosstalk in the signalling cascades that regulate each of the various stress-specific
transcription factors (5, 6) and so any stressor has the potential to, and generally does, affect the
transcription of genes that are not directly related to the specific stress applied (7). When
microorganisms challenge insects, molecular patterns on the pathogen trigger alterations in
transcription via Toll, IMD, and the JAK-STAT pathway, ultimately activating the Rel1, Rel2,
and STAT transcription factors. These transcriptional factors control the expression of hundreds of
genes, including many immune response genes. The immune response has been shown to be
intertwined with the general stress response in several insect models, and a number of genes
activated by pathogen invasion are not directly linked to anti-pathogenic functions. For instance,
Frontiers in Immunology | www.frontiersin.org
1
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
LPS injection of the Tribolium castaneum beetle also induces
heat shock stress response genes (HSP 68 and HSP 27) and
hypoxia-inducible gene transcription (7). Conversely, in the
same insect, heat shock triggers the transcription of the
immune genes tlr6, pgrp2, defensin1, and defensin2 (8). In
Galleria mellonella larvae, heat shock alters the expression of
antimicrobial peptide genes. For example, apoLp-III gene
expression is transiently inhibited after heat shock but, when
an infection occurs 96 h after the heat shock, it is induced at
higher levels than in the absence of prior heat shock (9). Some
Drosophila melanogaster immune genes are regulated by the heat
shock factor, and in fact, most Heat shock Factor Binding Sites in
the genome are found in Non-Heat shock Genes (10). The
regulatory cascades of Heat shock Factor 1 and NF-kB share
some components, such as Droj2 (HSP40) that functions
downstream of or directly regulates Relish and DNAJA3 which
are required for IkB phosphorylation (11).
Interestingly, heat shock-mediated immune activation can be
transgenerational, hence affecting immune homeostasis over a long
period of time (12). Upon stress, the beetle Nicrophorus vespilloides
initiates a transposon-mediated genomic reorganization of its
immune genes, suggesting that this reorganization might
constitute a common pathway to enhance survival during protein
damage (13). This reorganization could occur because stress or
infection mobilizes HSP 90 protein, impairing its mutationdampening function (14). Aside from heat shock direct
transcriptional effects, or genomic reorganization, the temperature
also affects the function of enzymes that modify genomic DNA such
as the Ten eleven translocation (TET) dioxygenase, providing
another level of transcriptional regulation (15).
HSP70 also protects the midgut of Aedes aegypti mosquitoes
against the dramatic increase in body temperature after ingesting
a hot blood meal (16), considering that the mosquito ingests its
own weight of blood at 37°C (17). Though immune signaling
pathways of anopheline mosquitoes have been scrutinized in
great detail, little is known about their interactions with hypoxia
(HIF) and heat shock factor (HSF) mediated transcriptional
regulation. The blood-feeding of female mosquitoes implies
that a heat shock is produced in the midgut and that HSP70
proteins are transiently expressed (16). In anopheline
mosquitoes, Toll and Imd pathways show a certain amount of
cross signaling (18); opening the possibility of a cooperative
effect of bacterial infection response and Plasmodium mediated
response, as shown in Anopheles gambiae by Ramirez et al. (19).
The gut tissues of hematophagous insects are subjected to large
temperature changes when feeding. As a consequence, HSP 82,
HSP 90, and HSP 105 of A. gambiae are up-regulated during
blood-feeding (20, 21). Later, ex vivo analysis of the A. gambiae
HSC70B promoter revealed that the transcription was influenced
by immune activation (5), proving the existence of a cross-talk
between immune and heat shock activation cascades. In
Anopheles stephensi, the rearing temperature alters the
transcription levels of immune effectors differentially. While
the nitric oxide synthase (NOS) expression peaked at 30°C, the
humoral melanization, phagocytosis, and defensin expression
were maximum at 18°C (22), though the melanization reaction
Frontiers in Immunology | www.frontiersin.org
has been described as a minor factor in resistance to P.
falciparum (23). In general, phenoloxidase (PO) and NO have
been implicated in the immune response to Plasmodium
infection in Anopheles. In A. stephensi, the metabolites of NOS
activity increase nitric oxide concentrations and subsequent
ookinete death (24). The enzymatic cleavage regulating NOS
activity is temperature-dependent (25). Some heat shock
proteins (HSPs), such as A. gambiae HSC70B have shown
antiviral activities capable of suppressing O’nyong’nyong viral
infection (26). In turn, this observation opens the possibility that
the HSPs could affect the microorganisms directly, once released
in the hemolymph. In particular, heat shock triggers a general
damage response in mosquitoes, including immune gene
transcription. Though P. berghei does not constitute a natural
A. albimanus parasite, this model has proven an invaluable tool
for the study of the immune response of this insect. Here, we use
this model to investigate the effects of heat shock on the
expression of immune-related genes and the subsequent effect
on susceptibility to infection.
MATERIALS AND METHODS
Mosquito Rearing and Infection
With P. berghei
A Plasmodium-susceptible strain of A. albimanus females (27)
were obtained from the insectary of the National Institute of
Public Health (INSP) in Cuernavaca, Mexico. Mosquitoes were
bred under a 12:12 photoperiod at 28°C and 70–80% relative
humidity. At four-days post-emergence, mosquitoes were
infected with P. berghei ANKA strain expressing the green
fluorescent protein (GFP) (28) (kindly donated by Robert E.
Sinden, Imperial College, U.K.). Ookinetes were produced by
culturing gametocyte-infected mouse blood, as described
previously (29). Groups of 300 female mosquitoes were fed for
1 h using artificial membrane feeders with: (i) mouse blood +
GFP ookinetes (infected group, with approximately 900
ookinetes per ml), or (ii) mouse blood only (control group).
Unfed mosquitoes were removed, and the engorged ones
maintained at 21°C to allow for parasite invasion and
interaction with the mosquito midgut. Three experimental
repetitions were performed.
Mosquito Heat Shock
Since in Drosophila HSF binding reaches a maximum level
following a 30-minute heat shock at 36.5°C (30), and
mosquitoes endure a thermic shock while feeding on mammals
blood (16), we exposed the mosquitoes to 30 min of 37°C heat
shock, with 80% humidity and availability of 10% sugared water.
In nature, the blood feeding is concomitant to the heat shock in
naturally infected mosquitoes, and lead to the infection of the
mosquito (16). Since the objective of our experiments is to test
the effect of abiotic stress on the susceptibility to P. berghei
infection of A. albimanus, we challenged them thermally 6 h
before the infective blood meal.
2
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
Mosquito Protein Extract Preparation for
Acrylamide Electrophoresis
TABLE 1 | Oligonucleotides used to amplify the mRNA transcripts of A.
albimanus genes.
About 10 mosquitoes per sample were homogenized in 100 µl of
lysis solution (8 M urea, 2 M thiourea, 1% Chaps, 13 mM DTT,
and 4 µl of protease inhibitor cocktail (Sigma, P2714)). The
resulting solution was cleared by 15 min centrifugation at
14,000g and 4°C. The total protein content of the supernatant
was determined according to Lowry et al. (31). Samples were
obtained before the heat shock, immediately after, and at 2 and
6 h after 30 min of exposure at 37°C.
ppo1 F
ppo1 R
tet 2 F
tet 2 R
dnmt2 F
dnmt2 R
CECA-R
CECA-F
Gam F
Gam R
GNBPA F
GNBPA R
duox F
duox R
hsp70 F
hsp70 R
frep3 F
frep3 R
S7 F
S7 R
Acrylamide Electrophoresis and
Western Blot Assay of the Mosquito
Protein Extracts
Some 25 mg of mosquito protein extract were separated in SDSPAGE (10% acrylamide) and transferred to Immobilon-P
membrane. The protein transfer on the membrane was
assessed by Ponceau S (P7170 Sigma-Aldrich) staining. For the
Hsp-70 immunodetection, anti-Hsp70 monoclonal antibody
[3A3] Thermo Scientific MA3-006 was used as primary
antibody at a final dilution of 1:1,000, and goat anti-mouseIgG-horseradish peroxidase (Abcam) diluted 1:1,000 as
secondary antibody. Development of immunoblots was
performed with an ECL kit from Amersham. Fluorescence was
developed on a Kodak BioMax ML-2 film for capturing
chemiluminescent data (Catalog Number Z370428) using
Kodak Developer (Catalog Number P7042) and Kodak Fixer
(Catalog Number P7167).
ppo1, Prophenol oxidase 1; tet2, ten-eleven translocation methylcytosine dioxygenases;
dnmt2, DNA methyltransferase 2; duox, Dual oxidase; hsp70, heat shock protein 70;
frep3, Fibrinogen related protein 3, cecropin, gambicin and GNBP4.
set to a volume of 20 ml with water free of nucleases (Thermo
Scientific). The relative expression was quantified by normalizing
the expression of immune response genes with the S7
ribosomal gene.
Assays were performed three times in different batches of 10
mosquitoes and three times in different batches of five
mosquitoes’ midguts. The control and experimental tests were
made at the same time. For real-time PCR, 2.5 µl of cDNA was
used in SYBR Green I Kit (Applied Biosystems) following the kit
instructions. The primers used are described in Table 1. The fold
changes in expression were calculated using the comparative
“delta delta Ct” (Ct) method against the blood-fed control (33)
using three replicates per sample. Three independent
experiments were done. The data represents the average foldchange relative to the control group. The amplification efficiency
was similar between the test and control genes.
Mosquito Hemolymph Collection
Hemolymph was obtained by perfusion from 30 control and
heat-shocked mosquitoes (at six hours post-heat shock), and 24hours post-P. berghei infection (30-hours post-heat shock) as
described elsewhere (32). Three experimental repetitions
were performed.
Mosquito RNA Extraction
Total RNA from 10 whole female mosquitoes (without head) and
midguts only, were obtained by Trizol method (Invitrogen) and
then re-purified using RNA Clean-Up Kit (Zimo Research).
cDNA was synthesized by reverse transcription using 1 µg of
RNA, 100 ng of oligonucleotide dT, and 200 U of the enzyme
reverse transcriptase RNase H-SuperScript II (Gibco BRL). Three
experimental repetitions were performed, each counting three
samples per conditions.
Phenoloxidase (PO) Activity
PO activity was measured as described (34). Three pools of 30
female mosquitoes were macerated and centrifuged at 10,000g
for 10 min at 4°C. L-DOPA was used as the substrate for PO,
which is transformed into the dye dopachrome. Auto-oxidation
controls (L-DOPA only) and blanks (macerated mosquitoes)
were included. PO activity was measured every minute for
30 min at 490 nm in a microplate reader (ELISA iMark,
BIO-RAD).
RT-qPCR Amplification
The amplification of genes of the An. albimanus immune
response was carried out with previously recovered genetic
material. Specific primers were used for each gene (Table 1).
The samples were run in a real-time thermal cycler (viiA7;
Applied Biosystems) under optimal running conditions,
according to the manufacturer’s recommendations. Samples
were incubated at 60°C in a master mix containing SYBR
Green (Maximum SYBR Green/Rox qPCR Master Mix;
Thermo Scientific), primers, and cDNA of each of the samples,
Frontiers in Immunology | www.frontiersin.org
5’-GGCGGACCAAATCAAGCAG-3’
5’-CGATTGCCCGATTCGTCAAC-3’
5’-TCCTCCGATCCGAGGATCAGGT-3’
5’-GTACCTTGCTGTTGCTGGGCA-3’
5’-GAGCCATCTTTTCCGATTCGTC-3’
5’-GAGCCATCTTTTCCGATTCGTC-3’
5’-ATTTGCCAAGTGCCTTCAC-3’
5’-AGTGGACGCTGGTTTTCTCAAG-3’
5’-CGCTTATGCTTCGACTTGC-3’
5’-AATCATCGTCTGACCATCGC-3’
5´-CACTCGATACGGAGTCGGC-3´
5’-AACTAATCTGGGCTCATCGTG-3’
5’-CTCTCTCTGTTGCAGAATCCAG-3’
5’-TGGTGTGAGATGGTTATCGACT-3’
5’-CCAGCATGGAAAGGTGGAGA-3’
5’-CCATCCATCAGGGCGTCAAT-3’
5’-CAGTGCGTGTCGTGCAAT-3’
5’-AACCGTTTGAGAATCTGTAGCA-3’
5’-AACAACAAGAAGGCCATCGTC-3’
5’-GGCTTGGGCAGAATACGA-3’
NO Quantification
Nitrites (NO−2 ) and nitrates (NO−3 ) were evaluated by the Griess
assay (35). Pools of 30 female mosquitoes per treatment were
macerated and centrifuged twice at 10,000g for 10 min at 4°C.
Proteins were eliminated with ZnSO4. Nitrates were reduced into
nitrites using VCl3 immediately followed by the addition of
sulfanilamide and NED. The reaction was incubated for
3
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
15 min at R.T., and the absorbance was measured at 490 and 630
nm in a microplate reader.
Plasmodium (36). The effect of heat shock upon the enzyme
activity could be key to understanding the reduction of the
mosquito’s susceptibility to the parasite.
Therefore, we measured the PO activity in the hemoplymph
and full mosquito body during the heat shock. In three separate
experiments, the hemolymph from 30 female A. albimanus
susceptible strain that were heat-shocked at 37°C for 30 min
was collected. The sampled hemolymph was obtained at 6 h postheat shock, and 24 h post-P. berghei infection (30 h post-heat
shock). Samples were tested for phenoloxidase activity using the
colorimetric L-DOPA assay. As can be seen in Figure 2,
phenoloxidase activity was altered by the heat shock regime. The
heat-shock by itself did not increase the phenoloxidase activity
significantly in the hemolymph. As shown previously (37, 38),
P. berghei infection increased hemolymph phenoloxidase activity,
mainly through enzymatic activation by proteolysis and secretion
of prophenoloxidase. While heat shock alone did not increase
hemolymph phenoloxidase activity, heat shock increased the
hemolymph phenoloxidase activity (Figure 2) in the P.berghei
infected mosquito hemolymph when compared to the heatshocked non infected mosquitoes hemolymph and relative to
non-heat shocked infected mosquitoes.
The phenoloxidase activity encountered in the whole
mosquito body increased upon heat shock. However, when
comparing both control/Pb and HS/HS Pb conditions, the
activity diminishes upon infection. When considering the
whole mosquito body mRNA, the heat shock did alter the PPO
gene transcription at 6 h post-heat shock (Figure 4), and showed
a tendency to diminish in the midgut (Figure 5). Considering
that, in insects, the central organ of phenoloxidase production
are the hemocytes (39) this result was expected.
Statistical Analysis
Data were analyzed and graphed in Prism v6.01 statistical
software. qPCR results were evaluated by one way ANOVA
followed Tukey’s test (Whole body RT-qPCR), and unpaired t
test with Welch’s correction (Midgut RT-qPCR). The infection
parameters were analyzed through Mann–Whitney. Considering
that three independent repetitions of the experiment were
performed, we applied a log-like generalized lineal model with
random effect to determine the difference in ookinete prevalence
between control and heat shocked mosquitoes. We used the
individual experimental repetitions as categorical variable, this
with the objective of measuring the effect of the individual
repetitions on the mean differences between the two conditions.
PO and NO results were analysed by Student’s t-test comparing the
heat shock and control groups for the non-infected and
infected mosquitoes.
RESULTS
Heat Shock Diminishes Infection of an
A. albimanus Susceptible Strain
To assess the global effects of heat shock response on parasite
development, female A. albimanus (susceptible strain)
mosquitoes were heat-shocked at 37°C for half an hour, 6 h
before infective blood-feeding (900 ookinetes/µl) in three
separate experiments with three replicates. Oocyst numbers
were assessed five days post blood meal. The prevalence and
the intensity were significantly diminished in the heat-shocked
mosquitoes (control 77.27%, n = 198 vs HS 67%, n = 184 with
X2 = 4.7 and p = 0.0102). A median of three oocyst per mosquito
midgut were found in control mosquitoes while a median of two
oocysts per midgut were found in the heat shocked mosquitoes
midguts (Figure 1A). A log-like generalized lineal model with
random effect showed that oocyst prevalence is 83% smaller in
the heat-shocked mosquitoes than in the control (95% IC 80–
87%) with p = 0.00. The individual experiments performed
(considering each experiment separately) do not affect the
outcome of the analysis (P = 0.98).
Anti-HSP-70 western-blot analysis of the mosquito protein
extracts shown in Figure 1B reveal an increase in expression of
HSP 70 at both 2 and 6 h after heat shock. The protein profile of
the mosquito’s midguts was altered by the treatment,
demonstrating the impact of the heat stress on the mosquito
cells 2 and 6 h post heat shock (Figure 1C, black arrows).
Hemolymph From Heat-Shocked
A. albimanus susceptible Strain
Mosquitoes Present Higher Nitric
Oxide Concentration
Another key factor involved in the immune response of A.
albimanus to Plasmodium infection occurs through NO
synthesis (32). The only stable product of NO, which decays in
seconds, is nitrite (NO−2 ), however, the more oxidized
nitrate (NO−3 ) can also be produced. Therefore, to measure the
total NO production, one must measure the total NOx
derivatives. The NO−2 found in the hemolymph of the heatshocked mosquitoes diminished (Figure 3A) while NO−3 levels
increased when compared with their control (Figure 3B),
indicating that total NOx production was not changed. This
may, however, indicate more oxidizing conditions in the
hemolymph after heat shock. In whole body extracts, we observed
that the combination of HS and infection increased NO−2
production above either heat shock or infection alone
(Figure 3). NO−3 production in the body of mosquitoes was
decreased by heat shock both in uninfected and those infected by
P. berghei. From these results we suggest that P. berghei infection
creates reducing conditions in the mosquito body that limits the full
oxidation of nitrites into nitrates (Figure 3B).
Hemolymph and Body From Heat-Shocked
A. albimanus Susceptible Strain
Mosquitoes Present Higher
Phenoloxidase Activity
Phenoloxidase activity has been previously described as an
important factor limiting the Anopheles infection by
Frontiers in Immunology | www.frontiersin.org
4
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
A
B
C
FIGURE 1 | Effect of Heat shock on P. berghei infection in A. albimanus. (A) A. albimanus susceptible strain oocyst infection prevalence expressed as the ratio of
infected mosquitoes over total mosquitoes sampled with (0.67 on average) and without previous heat shock (0.77 on average). (B) Western blot using the antiHSP70 antibody of a 10% acrylamide gel of control 0, 2, and 6 h post-heat shock A. albimanus body protein extracts. (C) Protein profile of the A. albimanus body
control T0C (control), T0P (Time zero post heat-shock), 2H (2 h post-heat shock), 6H (6 h post-heat shock). Black arrows indicate differential bands appearing in the
ponceau red staining of protein profile of the 2 h post heat shock and 6 h post heat shock sample.
Heat Shock Response Affects Gene
Transcription in the Whole Mosquito Body
ppo, also increased following heat shock. ppo transcription
increased significantly 6 h post-heat shock, with duox
increasing slightly (Figure 4, Supplementary Information).
To follow the transcription of inducible heat shock response, RTqPCR was performed on female An. albimanus cDNA. Three
independent experiments were performed with three groups of
five mosquitoes per condition, per experiment, and the S7 gene
cDNA was used to normalize the RT-qPCR. We observed an
increase of ppo and hsp70 transcripts upon heat shock. Decreases
in tet gene transcription were observed immediately after heat
shock while dnmt2 increased two hours post-heat shock, and
continued to increase thereafter. The hsp70 gene showed a
transcriptional upsurge after the heat shock, as expected. The
transcription of effector molecules commonly considered central
to the early response to P. berghei infection, such as duox and
Frontiers in Immunology | www.frontiersin.org
Heat Shock Alters Antimicrobial Peptide
Genes Transcription in the Mosquito Midgut
Control and heat-shocked mosquito midguts were extracted 6 h
post-treatment, mRNA was extracted and cDNA synthetized.
Cecropin, gambicin, GNBP4B and ppo genes were analyzed by
RT-qPCR. Transcription of ppo was diminished at 6 h post-heat
shock (Figure 5). This observation is in contradiction with whole
body ppo transcription results. Inhibition of ppo transcription in
the midgut could result from a specific sensitivity of this organ to
heat shock or may involve a negative feedback loop resulting
5
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
FIGURE 2 | Effect of heat shock on the phenoloxidase response of female A. albimanus during a P. berghei infection. Phenoloxidase activity in macerated body
extracts (right) and hemolymph (left). Control, Mosquitoes without heat shock; HS, heat shock mosquitoes; Pb, Plasmodium berghei fed mosquitoes. Data of three
independent experiments were analyzed using Student’s t-test and are represented with their standard deviations.
DISCUSSION
A
Stress history drastically influences the mosquito immune system.
The response to heat shock is generally characterized by heat shock
protein expression. This response is, in the case of A. gambiae,
protective from O’nyong’nyong virus infection (26). The
experimental heat shock scheme chosen allowed us to track the
effect of heat stress upon the mosquito immune system, although
without a direct relation with the natural circumstances
encountered by the mosquito in the wild, with the exception of
the ever more common heat waves that are occurring worldwide
due to climate change. Heat shock induces a state of increased
resistance that lasts for at least 6 h. The heat shock possibly poises
the immune system of the insect to respond faster and more
intensively to the infection. For this to happen, the relevant
changes must persist through the subsequent changes in the
cellular environment. Methylation, demethylation, and other
alterations to DNA and chromatin constitute a good theoretical
mechanism for this ongoing physiological change. In mammals, the
differentiation and specification of hematopoietic stem cells is
mediated by tet2 gene transcription. This gene codes for a
methylcytosine dioxygenase that is required for activation of
genes associated with differentiation (40, 41). In A. albimanus, the
tet2 gene could be involved in alteration of transcriptional activity
upon P. berghei exposure (42). In this article, it was demonstrated
that P. berghei infection of A. albimanus mosquitoes results in
alterations in both DNA and mRNA methylation. The resulting
methylation in turn alters the transcriptional pattern of the insect
cells, suggesting longer term biological accommodation when
challenged with P. berghei (42). In Tribolium beetles, general
stress conditions do also lead to alterations in DNA methylation,
allowing for wide spread transcriptional reprogramming (15). In
insects, heat shock leads to substantial changes in the transcription,
particularly in the expression of the Bt DNMT2 (DNA
methyltransferase) gene, a part of the DNA methylation system.
In the white fly Bemisia tabaci DNMT3 inhibition leads to an
B
FIGURE 3 | Effect of heat shock on the nitric oxide derivatives of A.
albimanus during a P. berghei infection. (A) NO−2 production (B) NO−3
production. Nitric oxide derivatives present in the hemolymph and body of A.
albimanus female mosquitoes upon P. berghei (Pb) infection and 6 h post
30 min at 37°C heat-shock (heat shock) and the respective control samples.
Data of three independent experiments were analyzed using Student’s t-test.
from proteolytic phenoloxidase activation. Transcription of the
cecropin, gambicin, and GNBP4B genes increased upon heat
shock (Figure 5, Supplementary Information) while ppo
transcription slightly decreased, indicating again a crosstalk
between heat shock and the immune response.
Frontiers in Immunology | www.frontiersin.org
6
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
FIGURE 4 | Gene expression at different times post-heat shock in whole body. Gene expression was evaluated immediately (0 h), at 2, and 6 h after 30 min of
exposure at 37°C (heat shock) in whole mosquitoes body. Data are indicated as mean ± SD. Data of three independent experiments were analyzed using ANOVAone way followed by Tukey’s test. Different letters indicate statistical significance.
FIGURE 5 | Gene expression at 6 h post heat shock in midguts. Gene expression was evaluated at 6 h after 30 min of exposure at 37°C (heat shock-HS) in
midguts. Data of three independent experiments were analyzed using unpaired t test with Welch’s correction. Data are indicated as mean ± SD. Cecropin 6.2 ± 1.4,
Gambicin 3.2 ± 1.2, PPO 0.5 ± 0.6, and GNBP4B 9.6 ± 7.7.
observed (Figure 6). When analyzing the effect of heat shock on
immunity gene transcription, we observed that genes related to
DNA methylation modification (tet, dnmt2) showed the largest
effect. In summary, heat shock alters the expression of many genes
and induces the activation of phenoloxidase enzyme as well as
increase in heat susceptibility (43). Here, we observed that when
aseptic stress in the form of heat shock is applied to A. albimanus, it
enhances resistance to P. berghei infection. After heat shock, the
activity of oxidative enzymes present in the mosquito hemolymph
increased, potentially providing an explanation for the resistance
Frontiers in Immunology | www.frontiersin.org
7
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
shock is sufficient to decrease P. berghei infection without requiring
a prerequisite bacterial immune challenge as reported by Dieme
et al. (44). The resistance of Anopheles to Plasmodium infection
induced by high temperature has been reported elsewhere (45),
though the mechanism underlying the phenomenon was not
described. Here we observed that thermal stress affected the
transcription of both heat shock proteins and elements of the
anti-pathogenic immune response. Given the recent increases in
temperature in the tropical regions, the effect of heat shock on
malaria transmission is relevant to future disease tends.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found in the article/Supplementary Material.
FIGURE 6 | Schematic summary. After a 6 h of a 30 min heat shock at 37°C,
several immune genes are upregulated in the mosquitoes. These include
genes that have been shown responsible for limiting a Plasmodium infection
such as ppo, duox, frep3 in the whole mosquito, and antimicrobial peptides in
the midgut. hsp70 is also upregulated along with dnmt2, a gene responsible
for the methylation of DNA and RNA in mosquitoes. This might indicate an
increment in methylcytosines in the nucleic acids, which is also supported by
the down regulation of the enzyme that removes these epigenetic marks, tet2
(not shown). The PO activity also increases in the hemolymph of the
Plasmodium-challenged and heat-shocked mosquitoes, although the total
amount of NO produced did not changed (not shown). This overall response
caused the mosquitoes to become less infected with the parasite. Only
statistically significant results are portrayed.
AUTHOR CONTRIBUTIONS
RC and HL-M conceived the presented idea. RC developed the
theory and performed the statistics. EH-T, FC-P, BR-T, KM-M,
and VC-J carried out the experiments. RC and HL-M wrote the
manuscript with support from BR-T and FC-P. FC-P, KM-M,
and VC-J fabricated the mosquito samples. EH-T, FC-P, BR-T,
KM-M, and VC-J processed and analyzed the samples. HL-M
supervised the project and planned the experiments. All authors
contributed to the article and approved the submitted version.
increasing transcription of its gene. Infection also increased the
activation of phenoloxidase.
The heat shock had little effect on NO−2 production, a species
resulting from oxidation of the highly reactive NO. A slight
increase in NO−3 production was detected. Altogether, the results
obtained demonstrate that immune alteration induced by heat
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.
584660/full#supplementary-material
REFERENCES
7. Altincicek B, Knorr E, Vilcinskas A. Beetle Immunity: Identification of
Immune-Inducible Genes From the Model Insect Tribolium Castaneum.
Dev Comp Immunol (2008) 32:585–95. doi: 10.1016/j.dci.2007.09.005
8. Xiong W, Zhai M, Yu X, Wei L, Mao J, Liu J, et al. Comparative RNAsequencing Analysis of ER-based HSP90 Functions and Signal Pathways in
Tribolium Castaneum. Cell Stress Chaperones (2018) 23:29–43. doi: 10.1007/
s12192-017-0821-x
9. Vertyporokh L, Taszlow P, Samorek-Pierog M, Wojda I. Short-Term Heat
Shock Affects the Course of Immune Response in Galleria Mellonella
Naturally Infected With the Entomopathogenic Fungus Beauveria Bassiana.
J Invertebr Pathol (2015) 130:42–51. doi: 10.1016/j.jip.2015.07.001
10. Gonsalves SE, Moses AM, Razak Z, Robert F, Westwood JT. Whole-Genome
Analysis Reveals That Active Heat Shock Factor Binding Sites Are Mostly
Associated With Non-Heat Shock Genes in Drosophila Melanogaster. PloS
One (2011) 6:e15934. doi: 10.1371/journal.pone.0015934
11. Momiuchi Y, Kumada K, Kuraishi T, Takagaki T, Aigaki T, Oshima Y, et al.
The Role of the Phylogenetically Conserved Cochaperone Protein Droj2/
DNAJA3 in NF-kappaB Signaling. J Biol Chem (2015) 290:23816–25.
doi: 10.1074/jbc.M115.664193
1. Janeh M, Osman D, Kambris Z. Comparative Analysis of Midgut
Regeneration Capacity and Resistance to Oral Infection in Three DiseaseVector Mosquitoes. Sci Rep (2019) 9:14556. doi: 10.1038/s41598-019-50994-4
2. Harvey JA, Heinen R, Gols R, Thakur MP. Climate Change-Mediated
Temperature Extremes and Insects: From Outbreaks to Breakdowns. Glob
Chang Biol (2020)26:6685–701. doi: 10.1111/gcb.15377
3. Suzuki J, Imamura M, Fujita M. Oxidative Stress Response of Caddisfly
Stenopsyche Marmorata Larvae to Abrupt Hypoxia–Normoxia Shift. Aquat
Toxicol (2019) 211:66–72. doi: 10.1016/j.aquatox.2019.04.001
4. Smith JA. Regulation of Cytokine Production by the Unfolded Protein
Response; Implications for Infection and Autoimmunity. Front Immunol
(2018) 9:422. doi: 10.3389/fimmu.2018.00422
5. Kang S, Sim C, Byrd BD, Collins FH, Hong YS. Ex Vivo Promoter Analysis of
Antiviral Heat Shock Cognate 70B Gene in Anopheles Gambiae. Virol J
(2008) 5:136. doi: 10.1186/1743-422X-5-136
6. Zhao L, Jones WA. Expression of Heat Shock Protein Genes in Insect Stress
Responses. Invertebr Surviv J (2012) 9:93–101.
Frontiers in Immunology | www.frontiersin.org
8
June 2021 | Volume 12 | Article 584660
Condé et al.
Anopheles Heat Shock Response
31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Lowry Protein Assay. J Biol
Chem (1951) 193(1):265–75.
32. Luckhart S, Vodovotz Y, Cui L, Rosenberg R. The Mosquito Anopheles
Stephensi Limits Malaria Parasite Development With Inducible Synthesis of
Nitric Oxide. Proc Natl Acad Sci USA (1998) 95:5700–5. doi: 10.1073/
pnas.95.10.5700
33. Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative Real-Time RT-PCR–A
Perspective. J Mol Endocrinol (2005) 34:597–601. doi: 10.1677/jme.1.01755
34. Li X, Ma M, Liu F, Chen Y, Lu A, Ling QZ, et al. Properties of Drosophila
Melanogaster Prophenoloxidases Expressed in Escherichia Coli. Dev Comp
Immunol (2012) 36(4):648–56. doi: 10.1016/j.dci.2011.11.005
35. Miranda KM, Espey MG, Wink DA. A Rapid, Simple Spectrophotometric
Method for Simultaneous Detection of Nitrate and Nitrite. Nitric Oxide (2001)
5:62–71. doi: 10.1006/niox.2000.0319
36. Ahmad S, Leake CJ, Ketterman AJ. Qualitative Differences Occur in
Phenoloxidase Activity in Anopheles Mosquitoes Refractory to
Plasmodium. Biochem Soc Trans (1995) 23:110S. doi: 10.1042/bst023110s
37. Herrera-Ortı́z A, Lanz-Mendoza H, Martı́nez-Barnetche J, Herná ndezMartı́nez S, Villarreal-Treviño C, Aguilar-Marcelino L, et al. Plasmodium
Berghei Ookinetes Induce Nitric Oxide Production in Anopheles
Pseudopunctipennis Midguts Cultured In Vitro. Insect Biochem Mol Biol
(2004) 34(9):893–901. doi: 10.1016/j.ibmb.2004.05.007
38. Simões ML, Mlambo G, Tripathi A, Dong Y, Dimopoulos G. Immune Regulation
of Plasmodium Is Anopheles Species Specific and Infection Intensity Dependent.
MBio (2017)8(5):e01631–17. doi: 10.1128/mBio.01631-17
39. Ribeiro C, Brehé lin M. Insect Haemocytes: What Type of Cell Is That? J Insect
Physiol (2006) 52(5):417–29. doi: 10.1016/j.jinsphys.2006.01.005
40. Ko M, Bandukwala HS, An J, Lamperti ED, Thompson EC, Hastie R, et al.
Ten-Eleven-Translocation 2 (TET2) Negatively Regulates Homeostasis and
Differentiation of Hematopoietic Stem Cells in Mice. Proc Natl Acad Sci USA
(2011) 108:14566–71. doi: 10.1073/pnas.1112317108
41. Ko M, An J, Pastor WA, Koralov SB, Rajewsky K, Rao A. TET Proteins and 5Methylcytosine Oxidation in Hematological Cancers. Immunol Rev (2015)
263(1):6–21. doi: 10.1111/imr.12239
42. Claudio-Piedras F, Recio-Tó toro B, Condé R, Herná ndez-Tablas JM,
Hurtado-Sil G, Lanz-Mendoza H. DNA Methylation in Anopheles
Albimanus Modulates the Midgut Immune Response Against Plasmodium
Berghei. Front Immunol (2020) 10:3025. doi: 10.3389/fimmu.2019.03025
43. Dai TM, Lu ZC, Wang YS, Liu WX, Hong XY, Wan FH. Molecular
Characterizations of DNA Methyltransferase 3 and its Roles in
Temperature Tolerance in the Whitefly, Bemisia Tabaci Mediterranean.
Insect Mol Biol (2018) 27:123–32. doi: 10.1111/imb.12354
44. Dieme C, Rotureau B, Mitri C. Microbial Pre-exposure and Vectorial
Competence of Anopheles Mosquitoes. Front Cell Infect Microbiol (2017)
7:508. doi: 10.3389/fcimb.2017.00508
45. Lambrechts L, Chavatte JM, Snounou G, Koella JC. Environmental Influence
on the Genetic Basis of Mosquito Resistance to Malaria Parasites. Proc Biol Sci
(2006) 273:1501–6. doi: 10.1098/rspb.2006.3483
12. Eggert H, Diddens-de Buhr MF, Kurtz J. A Temperature Shock can Lead to
Trans-Generational Immune Priming in the Red Flour Beetle, Tribolium
Castaneum. Ecol Evol (2015) 5:1318–26. doi: 10.1002/ece3.1443
13. Vogel H, Badapanda C, Vilcinskas A. Identification of Immunity-Related
Genes in the Burying Beetle Nicrophorus Vespilloides by Suppression
Subtractive Hybridization. Insect Mol Biol (2011) 20:787–800. doi: 10.1111/
j.1365-2583.2011.01109.x
14. Peub R, Eggert H, Armitage SA, Kurtz J. Downregulation of the Evolutionary
Capacitor Hsp90 Is Mediated by Social Cues. Proc Biol Sci (2015) 282(1819)
1819:20152041. doi: 10.1098/rspb.2015.2041
15. Feliciello I, Parazajder J, Akrap I, Ugarkovic D. First Evidence of DNA
Methylation in Insect Tribolium Castaneum: Environmental Regulation of
DNA Methylation Within Heterochromatin. Epigenetics (2013) 8:534–41.
doi: 10.4161/epi.24507
16. Benoit JB, Lopez-Martinez G, Patrick KR, Phillips ZP, Krause TB, Denlinger
DL. Drinking a Hot Blood Meal Elicits a Protective Heat Shock Response in
Mosquitoes. Proc Natl Acad Sci USA (2011) 108(19):8026–9. doi: 10.1073/
pnas.1105195108
17. Jové V, Venkataraman K, Gabel TM, Duvall LB. Feeding and Quantifying
Animal-Derived Blood and Artificial Meals in Aedes Aegypti Mosquitoes.
J Vis Exp (2020) 164:10.3791/61835. doi: 10.3791/61835
18. Luna C, Hoa NT, Lin H, Zhang L, Nguyen HL, Kanzok SM, et al. Expression of
Immune Responsive Genes in Cell Lines From Two Different Anopheline Species.
Insect Mol Biol (2006) 15:721–9. doi: 10.1111/j.1365-2583.2006.00661.x
19. Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A,
et al. The Role of Hemocytes in Anopheles Gambiae Antiplasmodial
Immunity. J Innate Immun (2013) 6:119–28. doi: 10.1159/000353765
20. Ribeiro JM. A Catalogue of Anopheles Gambiae Transcripts Significantly
More or Less Expressed Following a Blood Meal. Insect Biochem Mol Biol
(2003) 33(9):865–82. doi: 10.1016/S0965174803000808
21. Raddi G, Barletta ABF, Efremova M, Ramirez JL, Cantera R, Teichmann SA,
et al. Mosquito Cellular Immunity at Single-Cell Resolution. Science (2020)
369(6507):1128–32. doi: 10.1126/science.abc0322
22. Murdock CC, Paaijmans KP, Bell AS, King JG, Hillyer JF, Read AF, et al.
Complex Effects of Temperature on Mosquito Immune Function. Proc Biol Sci
(2012) 279:3357–66. doi: 10.1098/rspb.2012.0638
23. Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, et al. Genetic
Selection of a Plasmodium-Refractory Strain of the Malaria Vector Anopheles
Gambiae. Science (1986) 234:607–10. doi: 10.1126/science.3532325
24. Peterson TML, Gow AJ, Luckhart S. Nitric Oxide Metabolites Induced in
Anopheles Stephensi Control Malaria Parasite Infection. Free Radic Biol Med
(2007)42(1):132–42. doi: 10.1016/j.freeradbiomed.2006.10.037
25. Schwartz A, Koella JC. Melanization of Plasmodium Falciparum and C-25
Sephadex Beads by Field-Caught Anopheles Gambiae (Diptera: Culicidae)
From Southern Tanzania. J Med Entomol (2002) 39:84–8. doi: 10.1603/00222585-39.1.84
26. Sim C, Hong YS, Tsetsarkin KA, Vanlandingham DL, Higgs S, Collins FH.
Anopheles Gambiae Heat Shock Protein Cognate 70B Impedes O’nyong-Nyong
Virus Replication. BMC Genomics (2007) 8:231. doi: 10.1186/1471-2164-8-231
27. Narang S, Seawright JA. Genetic and Physiochemical Studies on BetaHydroxy Acid Dehydrogenase in Anopheles Albimanus. Biochem Genet
(1983) 21:885–93. doi: 10.1007/bf00483947
28. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der
Linden R, et al. A Plasmodium Berghei Reference Line That Constitutively
Expresses GFP at a High Level Throughout the Complete Life Cycle. Mol
Biochem Parasitol (2004) 137:23–33. doi: 10.1016/j.molbiopara.2004.04.007
29. Rodrı́guez MC, Margos G, Compton H, Ku M, Lanz H, Rodrı́guez MH, et al.
Plasmodium Berghei: Routine Production of Pure Gametocytes, Extracellular
Gametes, Zygotes, and Ookinetes. Exp Parasitol (2002)101(1):73–6.
doi: 10.1016/S0014-4894(02)00035-8
30. Wu C. Heat Shock Transcription Factors: Structure and Regulation. Annu Rev
Cell Dev Biol (1995) 1(11):441–69. doi: 10.1146/annurev.cellbio.11.1.441
Frontiers in Immunology | www.frontiersin.org
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Conde,́ Hernandez-Torres, Claudio-Piedras, Recio-Tot́ oro,
Maya-Maldonado, Cardoso-Jaime and Lanz-Mendoza. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that
the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does
not comply with these terms.
9
June 2021 | Volume 12 | Article 584660