Access to this full-text is provided by PLOS.
Content available from PLOS Pathogens
This content is subject to copyright.
RESEARCH ARTICLE
Higher temperature accelerates the aging-
dependent weakening of the melanization
immune response in mosquitoes
Lindsay E. Martin, Julia
´n F. HillyerID*
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America
*julian.hillyer@vanderbilt.edu
Abstract
The body temperature of mosquitoes, like most insects, is dictated by the environmental
temperature. Climate change is increasing the body temperature of insects and thereby
altering physiological processes such as immune proficiency. Aging also alters insect physi-
ology, resulting in the weakening of the immune system in a process called senescence.
Although both temperature and aging independently affect the immune system, it is
unknown whether temperature alters the rate of immune senescence. Here, we evaluated
the independent and combined effects of temperature (27˚C, 30˚C and 32˚C) and aging (1,
5, 10 and 15 days old) on the melanization immune response of the adult female mosquito,
Anopheles gambiae. Using a spectrophotometric assay that measures phenoloxidase activ-
ity (a rate limiting enzyme) in hemolymph, and therefore, the melanization potential of the
mosquito, we discovered that the strength of melanization decreases with higher tempera-
ture, aging, and infection. Moreover, when the temperature is higher, the aging-dependent
decline in melanization begins at a younger age. Using an optical assay that measures mel-
anin deposition on the abdominal wall and in the periostial regions of the heart, we found
that melanin is deposited after infection, that this deposition decreases with aging, and that
this aging-dependent decline is accelerated by higher temperature. This study demon-
strates that higher temperature accelerates immune senescence in mosquitoes, with higher
temperature uncoupling physiological age from chronological age. These findings highlight
the importance of investigating the consequences of climate change on how disease trans-
mission by mosquitoes is affected by aging.
Author summary
Climate change is increasing global temperatures, and this is altering the ecosystems that
organisms inhabit. Insects are particularly susceptible to temperature changes because
their body temperature is dictated by the environmental temperature in which they reside.
Importantly, changes in temperature alter how their immune system combats infection.
Another factor that alters immunity is aging. Specifically, aging weakens immune profi-
ciency in a process called senescence. Although both temperature and aging affect the
PLOS PATHOGENS
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 1 / 27
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Martin LE, Hillyer JF (2024) Higher
temperature accelerates the aging-dependent
weakening of the melanization immune response in
mosquitoes. PLoS Pathog 20(1): e1011935.
https://doi.org/10.1371/journal.ppat.1011935
Editor: Elizabeth A. McGraw, Pennsylvania State
University - Main Campus: The Pennsylvania State
University - University Park Campus, UNITED
STATES
Received: November 2, 2023
Accepted: January 1, 2024
Published: January 10, 2024
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.ppat.1011935
Copyright: ©2024 Martin, Hillyer. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
immune system, an unanswered question is whether temperature alters the progression of
immune senescence. We tested this question in Anopheles gambiae, which is a major vec-
tor of malaria, by measuring the strength of the melanization immune response of mos-
quitoes reared at three different temperatures, at four different ages, under four different
infection conditions. We determined that, individually, higher temperature and aging
both weaken melanization, and importantly, that higher temperature accelerates the
senescence of the melanization immune response. These findings demonstrate a real-life
consequence of climate change on insect physiology and illustrate the need to holistically
scrutinize the impact of global warming on the ability of insects to transmit diseases to
humans, animals and plants, and on the ability of insects to serve as pollinators for our
food supply.
Introduction
Mosquitoes, like most insects, are both ectotherms and poikilotherms, meaning that their
body temperature is dictated by the environmental temperature in which they reside. Climate
change is causing an increase in environmental temperatures, and global temperatures are pre-
dicted to further rise by more than 1.5˚C between 2030 and 2052 [1]. Increasing environmen-
tal temperature is, in turn, raising the body temperature of insects, and this phenomenon is
more pronounced for species that inhabit the tropics [2–4]. This is particularly the case for
mosquitoes that transmit disease [5,6]; at higher temperatures the rate of development is faster
[7–10], the metabolic rate is higher [2,11], the body size is smaller [12,13], and the lifespan is
shorter [7,9,14].
Changes in environmental temperature also affect the immune system of mosquitoes [15–
17]. Higher temperature reduces the phagocytic activity of hemocytes, which are mosquito
immune cells [15]. Higher temperature also weakens melanization [15,18–20], which is a
humoral immune response that encases and kills bacteria, malaria parasites, fungi, viruses, and
filarial worms [20–25]. In contrast, higher temperature increases the transcription and activity
of nitric oxide synthase [15,26], which produces the antibacterial and antimalarial free radical,
nitric oxide [27,28]. Additionally, changes in temperature alter the expression of genes that
encode components of the Toll pathway, apoptosis pathways, antimicrobial peptides, and
other immune factors [15,19,29]. Because of these and other changes, the environmental tem-
perature affects the probability that a mosquito transmits disease [16,19,30–33].
Another factor that affects the immune system of mosquitoes is aging. Mosquitoes, like
most animals, undergo senescence, which is the gradual and irreversible deterioration of the
efficiency of physiological processes that occurs with aging [34–40]. Senescence includes an
aging-associated decline in immunity, leading to increased pathogen proliferation and
increased risk of mortality [41–47]. For example, aging weakens the melanization immune
response [47–52], and decreases the number of hemocytes available to quell an infection
[42,48,53,54].
Although the independent effects of temperature and aging on the immune system have
been investigated, it is unknown how temperature and age interact to shape the strength of the
immune response of a mosquito or any other insect. That is, we do not know whether the envi-
ronmental temperature influences the rate of immune senescence. We hypothesize that higher
temperature uncouples physiological age from chronological age, thereby accelerating the pro-
gression of immune senescence. To test this hypothesis, we evaluated the independent and
combined effects of temperature and aging on the melanization immune response of the mos-
quito, Anopheles gambiae, which is a major vector of malaria in sub-Saharan Africa (Fig 1)
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 2 / 27
Funding: This work was funded by National
Science Foundation (NSF) Grant IOS-1936843 to
JFH and NSF Graduate Research Fellowship to
LEM. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
[55]. We found that, individually, higher temperature and aging weaken the melanization
immune response, and that higher temperature accelerates the senescence of melanization.
Results
Melanization potential decreases with aging and higher temperature, and
the aging-dependent decline is accelerated by higher temperature
Phenoloxidase (PO) initiates the melanization immune response by catalyzing the hydroxyl-
ation of tyrosine to dopa and the oxidation of dopa to dopaquinone, which then spontaneously
converts to the melanin precursor, dopachrome [56,57]. To test the effects of higher tempera-
ture and aging on the melanization potential of hemolymph, we isolated hemolymph, incubated
it with the PO substrate, L-DOPA, and measured OD
490
30 min later (Fig 2). This assay captures
melanization potential because it measures the ability of hemolymph to melanize in the event
that the phenoloxidase-based cascade is turned on to its fullest. A higher OD
490
indicates a
higher melanization potential, or a greater ability to melanize a pathogen upon infection.
Mosquitoes at higher temperatures have a lower melanization potential regardless of age or
their infection status, with temperature accounting for 31% of the variation (Fig 3A and 3D).
Relative to mosquitoes at 27˚C, the melanization potential of mosquitoes at 30˚C and 32˚C
was 54% and 79% lower, respectively.
Aging reduces melanization potential regardless of temperature or infection status, with age
accounting for 10% of the variation (Fig 3B and 3D). Relative to 1-day-old mosquitoes, the mela-
nization potential of 5-day-old and 10-day-old mosquitoes was 36% and 52% lower, respectively.
The melanization potential of 15-day-old mosquitoes was 58% lower than 1-day-old mosquitoes,
indicating that aging beyond 10 days does not further reduce melanization potential.
Infection for 24 h reduces the melanization potential of the hemolymph regardless of tem-
perature or age, with infection status accounting for 13% of the variation (Fig 3C and 3D).
Fig 1. Experimental overview for investigating the effects of higher temperature, aging, and their interaction on the mosquito melanization immune
response. Figure created with BioRender.com.
https://doi.org/10.1371/journal.ppat.1011935.g001
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 3 / 27
Relative to naïve mosquitoes, the melanization potential was 62% and 36% lower in E.coli-
and M.luteus-infected mosquitoes, respectively, and injury also reduced the melanization
potential by 22%. The large reduction in melanization potential during an infection indicates
that PO is being depleted as it is used to fight the infection, and is an agreement with the find-
ings in an earlier study [51]. Moreover, the trend for lower melanization potential in injured
mosquitoes can be attributed to PO being used to heal the wound [58].
Temperature and age interact to reduce the melanization potential, with this interaction
accounting for 5% of the variation (Figs 3D and 4). Specifically, higher temperature accelerates
the aging-dependent decline in melanization potential. As an example using naïve mosquitoes
only, and relative to 1-day-olds maintained at 27˚C, 1-day-olds maintained at 32˚C had a mel-
anization potential that was 80% lower. As mosquitoes aged from 1 to 10 days old, the melani-
zation potential of mosquitoes at 27˚C decreased by 52%, but the melanization potential of
mosquitoes at 32˚C remained the same. As another example using M.luteus-infected mosqui-
toes only, and relative to 1-day-olds maintained at 27˚C, 1-day-olds maintained at 32˚C had a
melanization potential that was 82% lower. As mosquitoes aged from 1 to 10 days old, the mel-
anization potential of mosquitoes at 27˚C decreased by 53%, but the melanization potential of
mosquitoes at 32˚C remained the same. This signifies that at higher temperatures, the decrease
in melanization potential occurs earlier in life than at cooler temperatures. Other interactions,
such as the interaction between temperature and immune treatment, did not meaningfully
shape melanization potential, so they were excluded from the model-of-best-fit.
To confirm that the change in OD
490
was due to PO activity and not the auto-oxidation of
L-DOPA, every biological trial included a control composed of only L-DOPA and water.
Auto-oxidation of L-DOPA was not detected (S1 Fig). Furthermore, to determine whether the
change in OD
490
was enhanced by endogenous substrates in the hemolymph and not just the
exogenous L-DOPA that was added to the hemolymph, a subset of hemolymph samples was
Fig 2. Diagrammatic overview of the melanization biochemical cascade and the experimental workflow. A. Melanization biochemical cascade. B.
Workflow of the phenoloxidase spectrophotometric assay that measures melanization potential of the hemolymph and PO activity over time. C. Representative
images of melanization on the dorsal abdominal wall of 1-day-old mosquitoes reared at 27˚C, at 24 h following treatment. Melanin deposition results in dark
deposits. Panel B created with BioRender.com.
https://doi.org/10.1371/journal.ppat.1011935.g002
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 4 / 27
mixed with water that was devoid of exogenous L-DOPA. Without exogenous L-DOPA, a
change in OD
490
was not detected (S2 Fig).
In summary, the melanization potential of hemolymph significantly decreases with
higher temperature, aging, and infection. Furthermore, when the temperature is higher, the
aging-dependent decline in melanization begins at a younger age, and therefore, we conclude
that higher temperature accelerates the senescence-based deterioration of melanization
potential.
The speed of melanization decreases with aging and higher temperature,
and the aging-dependent decline is accelerated by higher temperature
Because the melanization potential described above only captures PO activity at a single point
in time, we next analyzed how higher temperature, aging, and their interaction alter the speed
Fig 3. Melanization potential decreases with higher temperature, aging, and infection. A. Melanization potential,
aggregated by temperature and immune treatment, irrespective of age. B. Melanization potential, aggregated by age
and immune treatment, irrespective of temperature. C. Melanization potential, aggregated by immune treatment,
irrespective of temperature or age. Column height marks the estimated marginal mean, and whiskers indicate the
standard error of the estimated marginal mean (S.E.M.). D. Statistical analyses of the data using a linear model and a
Type II ANOVA. The same measurements are plotted in Figs 3 and 4, but grouped or arranged differently, with
aggregated data shown in this figure.
https://doi.org/10.1371/journal.ppat.1011935.g003
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 5 / 27
of melanization over the time course of the 30 min assay. Similar to what we found for the
overall melanization potential, higher temperature reduces the speed of melanization. At every
timepoint in the assay, and regardless of age or infection status, mosquitoes at higher tempera-
tures had a slower pace of melanization, with temperature accounting for 25% of the variation
(Fig 5A and 5D). Combining the timepoint values for each individual temperature, and rela-
tive to mosquitoes at 27˚C, the PO activity of mosquitoes at 30˚C and 32˚C was 56% and 76%
lower, respectively.
Aging also reduces the speed of melanization regardless of temperature or infection status,
with age accounting for 11% of the variation (Fig 5B and 5D). The speed of melanization was
fastest in 1-day-old mosquitoes, and while the endpoint of PO activity (the melanization
potential at 30 min) was similar for 10- and 15-day-old mosquitoes, the oldest mosquitoes
reached their maximum PO activity later in the assay. Combining the timepoint values for
each individual age, and relative to 1-day-old mosquitoes, the PO activity of mosquitoes that
were 5, 10, and 15 days old was 32%, 53%, and 57% lower, respectively.
Infection for 24 h reduces the speed of melanization regardless of temperature or age,
accounting for 12% of the variation (Fig 5C and 5D). Combining the timepoint values for
each individual immune treatment, and relative to naïve mosquitoes, the PO activity of
hemolymph was 58% and 40% lower in E.coli-infected and M.luteus-infected mosquitoes,
respectively, whereas the PO activity of hemolymph was only 11% lower in injured
mosquitoes.
Fig 4. The aging-dependent decline in melanization is accelerated by higher temperature. Column height marks
the estimated marginal mean, and whiskers indicate the S.E.M. The same measurements are shown in Figs 3and 4, but
grouped or arranged differently, with unaggregated data shown in this figure. The outcomes of statistical analyses are
presented in Fig 3D.
https://doi.org/10.1371/journal.ppat.1011935.g004
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 6 / 27
Temperature and age interact to reduce the speed of melanization more rapidly, accounting
for 4% of the variation (Figs 5D and 6). Moreover, the reduction in PO activity over time with
higher temperature was more pronounced in infected mosquitoes. Altogether, these interac-
tions indicate that the shape of the increase in hemolymph melanization over the course of the
assay is governed by the combined effects of higher temperature, older age, and infection.
Mainly, the increase in PO activity over time is smallest in older mosquitoes that are at higher
temperatures. Other interactions did not meaningfully affect PO activity, such as the
Fig 5. Over time, melanization decreases with aging, higher temperature, and infection. A. Melanization activity over time, aggregated by temperature and
immune treatment, irrespective of age. B. Melanization activity over time, aggregated by age and immune treatment, irrespective of temperature. C.
Melanization activity over time, aggregated by immune treatment, irrespective of temperature or age. Each circle marks the estimated marginal mean, and
whiskers indicate the S.E.M. D. Statistical analyses of the data using a linear mixed-effects model and a Type II Wald Chi Square Test. The same measurements
are plotted in Figs 5 and 6, but grouped or arranged differently, with aggregated data shown in this figure.
https://doi.org/10.1371/journal.ppat.1011935.g005
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 7 / 27
interaction between immune treatment and age, the interaction between immune treatment
and temperature, and the three-way interaction between time, age, and temperature (Fig 5D).
In summary, the speed of melanization significantly decreases with higher temperature,
aging, and infection. Furthermore, higher temperature accelerates the aging-dependent deteri-
oration of the melanization immune response.
Fig 6. Over time, the aging-dependent decline in melanization is accelerated by higher temperature. Each circle
marks the estimated marginal mean, and whiskers indicate the S.E.M. The same measurements are shown in Figs 5
and 6, but grouped or arranged differently, with unaggregated data shown in this figure. The outcomes of statistical
analyses are presented in Fig 5D.
https://doi.org/10.1371/journal.ppat.1011935.g006
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 8 / 27
Melanin deposition on the dorsal abdominal wall decreases with aging, and
this decrease is accelerated by higher temperature
Microorganisms that enter the hemocoel circulate with the hemolymph, where they are often
melanized. Melanized microorganisms are subsequently phagocytosed by hemocytes that cir-
culate with the hemolymph or are attached to tissues [22,59–61]. Most attached hemocytes,
called sessile hemocytes, are attached to the dorsal abdominal wall [53], and therefore, we set
out to determine whether higher temperature, aging, and their interaction affect the melani-
zation of pathogens by examining melanin deposition on the dorsal abdominal wall (Fig 2).
Melanin deposition on the dorsal abdominal wall is not strongly shaped by temperature,
with this variable only accounting for 4% of the variation (Fig 7A and 7D). Melanin deposition
is greatest at 30˚C; relative to mosquitoes at 27˚C, melanin deposition was 25% and 8% greater
at 30˚C and 32˚C, respectively (Fig 7A and 7D).
Fig 7. Melanin deposition on the dorsal abdominal wall decreases with aging. A. Melanin deposition, aggregated by
temperature and immune treatment, irrespective of age. B. Melanin deposition, aggregated by age and immune
treatment, irrespective of temperature. C. Melanin deposition, aggregated by immune treatment, irrespective of
temperature or age. Column height marks the estimated marginal mean, and whiskers indicate the S.E.M. D. Statistical
analyses of the data using a linear model and a Type II ANOVA. The same measurements are plotted in Figs 7 and 8,
but grouped or arranged differently, with aggregated data shown in this figure.
https://doi.org/10.1371/journal.ppat.1011935.g007
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 9 / 27
Melanin deposition on the dorsal abdominal wall is predominantly shaped by aging, and
this accounted for 20% of the variation (Fig 7B and 7D). Melanin deposition decreased with
aging, with the greatest decrease occurring between 1 and 5 days post eclosion. Specifically, rel-
ative to 1-day-old mosquitoes, melanin deposition in 5-, 10-, and 15-day-old mosquitoes was
73%, 80%, and 82% lower, respectively (Fig 7).
With respect to immune treatment, melanin deposition on the dorsal abdominal wall of
naïve and injured mosquitoes is negligible because these mosquitoes are not actively melaniz-
ing pathogens. This was expected given prior observations [59,60,62]. Because infection
induces melanization, infection for 24 h increases melanin deposition, and this accounted for
14% of the variation (Fig 7C and 7D). Specifically, the melanized area of E.coli- and M.luteus-
infected mosquitoes was 217% and 225% greater, respectively, than the melanized area in
naïve mosquitoes.
Although higher temperature alone did not significantly affect melanin deposition on the
dorsal abdominal wall, the aging-dependent decrease in melanin deposition accelerated when
the temperature was higher, with the interaction between temperature and age accounting for
3% of the variation (Figs 7D and 8). As an example using E.coli-infected mosquitoes only, the
melanized area at 27˚C decreased by 70% when mosquitoes aged from 1 to 5 days old, but at
32˚C, this aging-based decrease was 75%, indicating a larger decline when the temperature is
higher.
Furthermore, temperature and immune treatment interact to shape melanin deposition,
and this accounted for 2% of the variation (Figs 7D and 8). This interaction is driven by the
Fig 8. The aging-associated decrease in melanin deposition is accelerated by higher temperature. Column height
marks the estimated marginal mean, and whiskers indicate the S.E.M. The same measurements are shown in Figs 7
and 8, but grouped or arranged differently, with unaggregated data shown in this figure. The outcomes of statistical
analyses are presented in Fig 7D.
https://doi.org/10.1371/journal.ppat.1011935.g008
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 10 / 27
presence or absence of infection. In the absence of infection, the amount of melanin deposition
at all temperatures is similar because pathogens are not being melanized. However, when a
mosquito is infected, the effect of higher temperature depends on the pathogen. Specifically,
higher temperature induced a decrease in melanin deposition following infection with M.
luteus, but an increase following infection with E.coli (Fig 7A). For example, in 1-day-old mos-
quitoes, as temperatures warmed from 27˚C to 32˚C, the melanized area decreased by 31% in
M.luteus-infected mosquitoes but increased by 57% in E.coli-infected mosquitoes.
The interaction between age and immune treatment also shapes melanin deposition, and
this interaction accounted for 6% of the variation (Figs 7D and 8). Specifically, the aging-
dependent decline in melanin deposition was only meaningful after infection, and the aging-
dependent decline was more pronounced in M.luteus-infected mosquitoes than in E.coli-
infected mosquitoes. For example, at 30˚C, as mosquitoes aged from 1 to 15 days old, melanin
deposition decreased by 87% and by 66% when infected with M.luteus and E.coli,
respectively.
Finally, temperature, age, and immune treatment interact to shape melanin deposition, and
this three-way interaction accounted for 5% of the variation (Figs 7D and 8). Similar to the
two-way interactions, this three-way interaction is driven by the infection status because in
uninfected mosquitoes, the deposition of melanin is similar at all temperatures and ages
because pathogens are not being melanized. As an example using M.luteus-infected mosqui-
toes only, and relative to 1-day-olds maintained at 27˚C, 1-day-olds maintained at 32˚C had
31% lower melanin deposition. As mosquitoes aged from 1 to 5 days old, the melanized area in
mosquitoes at 27˚C decreased by 96%, but the melanized area in mosquitoes at 32˚C decreased
by a smaller 76%.
In summary, melanin is deposited on the dorsal abdominal wall after an infection, but the
amount of melanin deposited decreases when the infection is initiated at an older age. Further-
more, higher temperature accelerates the aging-dependent decline in melanization.
Melanin deposition within the periostial regions decreases with aging but is
only marginally affected by higher temperature
Within the dorsal abdominal wall, an infection for 24 h induces the aggregation of hemocytes
around heart valves called ostia [61]. These hemocytes are called periostial hemocytes, and
they reside in the periostial regions of abdominal segments 2–7 [60]. Abdominal segment 8
contains the posterior excurrent opening, and few sessile hemocytes reside in this segment
[63,64]. Because the number of hemocytes, including periostial hemocytes, decreases with
aging [42,48,53,54], we hypothesized that melanin deposition in the periostial regions
decreases with aging and higher temperature, and that the effect of aging is amplified at higher
temperature. To test this hypothesis, we reanalyzed the same abdomens sampled above but
restricted the analysis to the periostial regions in abdominal segments 3–7 and the posterior
excurrent opening.
Although influential for the entire abdomen, temperature alone did not meaningfully affect
melanin deposition in the periostial regions, accounting for none (0%) of the variation (Fig 9A
and 9D). Aging, however, decreased melanin deposition within the periostial regions, with age
accounting for 10% of the variation (Fig 9B and 9D). Like for the entire dorsal abdominal wall,
the aging-related decrease in melanization was small when mosquitoes aged beyond 5 days.
Immune treatment accounted for 26% of the variation (Fig 9C and 9D); melanin deposition
was negligible in uninfected and injured mosquitoes but increased dramatically after infection.
Relative to naïve mosquitoes, melanin deposition was 13 times greater in E.coli-infected mos-
quitoes and 44 times greater in M.luteus-infected mosquitoes.
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 11 / 27
When examining interactions, the aging-associated decrease in melanization was depen-
dent on the infection status, with this interaction accounting for 17% of the variation (Figs 9D
and 10). Of interest, the aging-associated decrease in melanization was more pronounced in
M.luteus-infected mosquitoes than in E.coli-infected mosquitoes. As an example, at 27˚C the
melanized area in M.luteus-infected mosquitoes decreased by 99.8% between 1 and 15 days
after eclosion, but it only decreased by 84% in E.coli-infected mosquitoes. This is likely
because in 1-day-old mosquitoes, the melanization of M.luteus is much stronger than the mel-
anization of E.coli.
Although temperature did not independently affect melanin deposition within the perios-
tial regions, temperature interacted with age and infection status, with this three-way interac-
tion accounting for 4% of the variation (Figs 9D and 10). Similar to the whole abdomen, this
interaction was driven by the presence or absence of infection. Without infection, melani-
zation was negligible. In infected mosquitoes, however, the aging-dependent reduction in
Fig 9. Melanin deposition within the periostial regions and posterior excurrent opening decreases with aging but
is only marginally affected by higher temperature. A. Melanin deposition, aggregated by temperature and immune
treatment, irrespective of age. B. Melanin deposition, aggregated by age and immune treatment, irrespective of
temperature. C. Melanin deposition, aggregated by immune treatment, irrespective of temperature or age. Column
height marks the estimated marginal mean, and whiskers indicate the S.E.M. D. Statistical analyses of the data using a
linear model and a Type II ANOVA. The same measurements are plotted in Figs 9 and 10, but grouped or arranged
differently, with aggregated data shown in this figure.
https://doi.org/10.1371/journal.ppat.1011935.g009
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 12 / 27
melanin deposition was accelerated when the temperature was higher. This effect was more
pronounced when mosquitoes were infected with M.luteus, likely because this infection
induces the strongest melanization response. Additionally, similar to the entire dorsal abdomi-
nal wall, higher temperature had opposing effects depending on the pathogen, inducing a
decrease in melanin deposition following infection with M.luteus, but an increase following
infection with E.coli (Fig 9A).
In summary, melanin is deposited within the periostial regions only after infection. In
infected mosquitoes, melanin deposition within the periostial region decreases with aging.
Moreover, the aging-dependent decline in melanin deposition was more pronounced when
the temperature was higher, especially in M.luteus-infected mosquitoes.
Discussion
Although the independent effects of higher temperature and aging on the mosquito immune
system have been studied, it remained unknown whether temperature and age interact to
shape the immune response in mosquitoes or any other insect. We found that higher tempera-
ture and aging individually reduce melanization, and that higher temperature accelerates the
aging-induced weakening of the melanization immune response (Fig 11).
Using both an ex vivo biochemical assay and an in vivo optical assay, we showed that the
strength of the melanization response decreases with aging. This aging-dependent decline is in
agreement with prior studies on mosquitoes [47–52]. For example, Culex pipiens (order:
Fig 10. The aging-associated decrease in melanin deposition within the periostial regions and posterior excurrent
opening is only marginally accelerated by higher temperature. Column height marks the estimated marginal mean,
and whiskers indicate the S.E.M. The same measurements are shown in Figs 9and 10, but grouped or arranged
differently, with unaggregated data shown in this figure. The outcomes of statistical analyses are presented in Fig 9D.
https://doi.org/10.1371/journal.ppat.1011935.g010
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 13 / 27
Culicidae) loses over half of their PO activity by 14 days of adulthood [50], and aging reduces
PO activity in crickets (order: Orthoptera) [65]. This aging-related decrease in melanization
correlates with a decrease in the number of hemocytes [42,48,53,54], which is important
because hemocytes—mainly oenocytoids but also to some extent granulocytes—are major pro-
ducers of key melanization enzymes such as phenoloxidase and phenylalanine hydroxylase
[23,54,66,67].
Unexpectedly, aging beyond 10 days did not meaningfully alter the mosquito’s melani-
zation immune response. In insects, melanin is not only necessary for immune function, but it
is also required for the sclerotization and pigmentation of the cuticle during molting
[56,68,69]. Given that melanization is energetically costly [56,70], once the mosquito has
reached adulthood and the cuticle has fully hardened, we suspect that there is a lower need for
PO activity, and therefore, resources are deployed elsewhere. This is supported by mosquito
larvae having much greater PO activity than adults [51], and similar to our findings, the aging-
based weakening of the melanization of microfilaria is marginal beyond 5 days of age [48].
Thus, the plateau in the aging-associated decline of melanization is likely a consequence of
achieving an optimal investment in melanization potential during adulthood.
We also discovered that higher temperature weakens the melanization immune response.
Similar to our findings, the efficiency of C-type lectin 4-regulated melanization of Plasmodium
falciparum in A.gambiae is less efficient when the temperature warms from 19˚C to 27˚C [20].
Moreover, higher temperature reduces the melanization of Sephadex beads that have been
injected into A.gambiae or Anopheles stephensi [15,18]. Higher temperature also reduces the
strength of melanization in butterflies (order: Lepidoptera) and crickets [71–73], indicating
that this phenomenon transcends the mosquito taxon. Additionally, higher temperature
Fig 11. Summary of the effects of higher temperature, aging, and their interaction on the melanization immune
response. Graphic that includes cuvettes created with BioRender.com.
https://doi.org/10.1371/journal.ppat.1011935.g011
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 14 / 27
reduces the sclerotization and melanization of the cuticle, resulting in lighter colored insects
[74]. In moths, beetles (order: Coleoptera) and crickets, a lighter cuticle correlates with a
weaker melanization immune response [75–79], and in beetles, a lighter cuticle also correlates
with a lower hemocyte density [79]. Thus, higher temperature weakens both cuticular and
immune melanization across Insecta.
Prior to this study, it remained unknown whether or how temperature and age interact to
shape the immune response in insects. Without considering interactive effects, both higher
temperature and aging reduce mosquito survival and vector competence [19,31,32,35,80].
Recently, we reported that higher temperature and aging, individually and interactively,
reduce body size and deteriorate the body condition of A.gambiae [12]. Here, we discovered
that higher temperature and aging significantly interact to accelerate the weakening of the mel-
anization response. To our knowledge, our study provides the first evidence that higher tem-
perature accelerates immune senescence in mosquitoes—or any other insect—thereby
decoupling physiological age from chronological age. Previously, we reported that the melani-
zation of mosquito hemolymph is inhibited by a copper-specific enzyme inhibitor, diethyl-
dithiocarbamate (DETC), indicating that melanization is driven by PO [51], and we and
others have demonstrated an aging-related decline in the number of hemocytes [42,48,53,54].
Therefore, we conclude that the warming-based acceleration of the aging-dependent decline
in melanization is because of (i) an accelerated decline in PO availability, and (ii) an acceler-
ated decline in the number of hemocytes that produce PO and other melanization enzymes.
The aging-dependent weakening of melanization potential is less pronounced when the
temperature is higher. This is largely because mosquitoes reared at higher temperatures eclose
with a lower melanization potential, which resembles that of older adults that had been reared
at cooler temperatures. While this may seem to indicate a lack of aging-based weakening at
higher temperatures, it is more likely that the aging-based weakening at higher temperature
occurs during the immature stages and prior to adult emergence, reducing the initial adult
potential for melanization. This notion is supported by the findings that larvae have greater
melanization activity than adults [51], and that higher temperature reduces cuticular melani-
zation [73,78], which is needed during eclosion [68,69].
Infection for 24 h activates the immune system and increases melanin deposition on the
dorsal abdominal wall, so it may seem counterintuitive that infection for 24 h weakens the mel-
anization potential of hemolymph. We hypothesize that the reduction we observed is due to
PO enzyme depletion. PO enzymes are found in melanotic capsules following bacterial infec-
tion in Aedes aegypti [67], and upon wounding, PO enzymes also localize to the cuticle healing
sites in Armigeres subalbatus [58]. Moreover, the expression of PO genes is not significantly
upregulated following a hemocoelic infection in adult mosquitoes [51], and without a blood-
meal, the total protein content of a mosquito decreases with older age [12,49]. Therefore, we
suspect that the availability of PO in the hemolymph declines as PO is sequestered in the sites
of bacterial infection or wounding, reducing the remaining melanization potential. In our
spectrophotometric experiments, we provided a saturating amount of PO’s substrate,
L-DOPA, but inside the mosquito, it is likely that substrate reduction also contributes to
reduced melanization potential upon infection. This notion is supported by a study where
filarial worms were injected into the hemocoel of A.subalbatus, which found that the melani-
zation response is accompanied by a reduction in tyrosine, one of the initial substrates of the
melanization cascade [81]. Regardless of the specific reason for the reduction in melanization
potential at 24 h following infection, the melanization potential in naïve mosquitoes represents
the melanization activity that can take place at the initiation of any infection. The melanization
potential in mosquitoes infected for 24 h then informs on how the progression of an infection
affects this immune response.
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 15 / 27
Although infection for 24 h decreases melanization potential, it increases melanin deposi-
tion. This is because infection activates the melanization response, leading to the phagocytosis
and sequestration of melanized bacteria by sessile hemocytes on the abdominal wall [22,53,59–
61]. Interestingly, melanin deposition in mosquitoes infected with E.coli was greatest at the
highest temperature, even when melanization potential was not. This may at first suggest a
beneficial effect of higher temperature, but we do not believe that this is the case. Instead, we
hypothesize that because the highest temperature is closer to the optimal growth temperature
of E.coli, bacterial proliferation increases, leading to a greater melanization response. The opti-
mal temperature for the growth of M.luteus is lower than for E.coli, and hence, this rising tem-
perature trend is not observed for M.luteus.
With infection, the robustness of the melanization response was greater in M.luteus-
infected mosquitoes than in E.coli-infected mosquitoes, and the aging-dependent decline in
melanization was more pronounced in M.luteus-infected mosquitoes. Different types of bacte-
ria preferentially activate phagocytosis versus melanization responses [82]. In A.subalbatus
and A.aegypti,E.coli are primarily phagocytosed by hemocytes, whereas M.luteus are primar-
ily melanized [22,23]. Given that the weakening of melanization was more pronounced in M.
luteus-infected mosquitoes, we conclude that the warming-based acceleration of the aging-
dependent decline of melanization is more pronounced when melanization is the major
immune response elicited by the pathogen.
The experiments presented here were all conducted in the absence of a blood meal, and it is
possible that the absence of a blood meal exacerbates the senescence of the melanization
response. Upon ingesting a bloodmeal, mosquitoes synthesize cholesterol and convert it into
the hormone 20-hydroxyecdysone (20E), which is a pleiotropic steroid that regulates molting,
development, immunity, and longevity [83–85]. In A.gambiae, 20E primes and strengthens
the immune response against P.falciparum [86,87], including PO production [85,88]. How-
ever, given that the mosquitoes in our study did not ingest a bloodmeal, we hypothesize that
older adults may have less 20E, and therefore, weaker activation of hormone-regulated immu-
nity. Moreover, a blood meal induces hemocyte proliferation [89,90], and this opens the possi-
bility of an enhanced melanization response. Therefore, future studies will incorporate blood
feeding in the presence and absence of infection to test its effects on the senescence of the mel-
anization immune response.
This study did not consider mosquito survival as part of the analysis. However, the risk of
death increases with aging [36,41,42], higher temperature shortens lifespans [7,9,14], and
infection decreases survival [42,82]. How age, temperature and infection interact to shape
mosquito survival is the focus of ongoing experiments in our laboratory, but the rate of mos-
quito death does not affect our conclusions on melanization. Here, we only assessed melani-
zation in the surviving mosquitoes because these are the only mosquitoes that, under the
conditions tested, would be available for pathogen acquisition and transmission.
In summary, this study demonstrates that higher temperature accelerates immune senes-
cence in mosquitoes, with higher temperature uncoupling physiological age from chronologi-
cal age (Fig 11). These findings, which we believe are the first to show this interactive effect in
any insect, highlight the need to holistically examine the impact of warming global tempera-
tures on the ability of insects to transmit diseases to humans, animals and plants, or their abil-
ity to serve as pollinators for our food supply. Given that many abiotic and biotic factors—
such as temperature and age—alter mosquito physiology and vector competence [91], the
present study illuminates the importance of accounting for the interactive effects of the mos-
quito’s environment on internal physiology, which is inherently important when estimating
immune function, disease transmission dynamics, and other critical processes.
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 16 / 27
Materials and methods
Mosquito rearing, treatments, and experimental overview
A laboratory colony of Anopheles gambiae, Giles sensu stricto (G3 strain; Diptera: Culicidae)
was maintained at 27˚C, 75% relative humidity, and a 12h:12h light:dark photoperiod. Eggs
from this colony were collected and transferred to three environmental chambers held at
27˚C, 30˚C or 32˚C, where they were hatched and reared to adulthood, and then used for
experimentation. These temperatures were selected because they are experienced by A.gam-
biae in nature and represent warming global temperatures [1,92,93]. Larvae were fed a mixture
of 2.8 parts koi food to 1 part baker’s yeast daily, and pupae were separated daily. Upon eclo-
sion, adults were maintained in 2.4 L plastic buckets with a mesh marquisette top and were fed
10% sucrose solution ad libitum.
For all experiments, adult females at 1, 5, 10, and 15 days after eclosion were assessed in
each temperature (Fig 1). These ages were selected because substantial changes in mosquito
immunity occur with adult aging, and these ages encompass the timeline for Plasmodium para-
site development within this mosquito [30,42,47,94].
At each temperature and age, adult female mosquitoes were either (i) naïve (unmanipu-
lated), (ii) injured, (iii) infected with Escherichia coli (Gram-negative bacteria; modified
DH5α, GFP-expressing and tetracycline resistant), or (iv) infected with Micrococcus luteus
(Gram-positive bacteria; ATCC 4698). E.coli and M.luteus were grown overnight in Luria-
Bertani (LB) broth at 37˚C in a shaking incubator (New Brunswick Scientific, Edison, NJ,
USA), and the cultures were then normalized to OD
600
= 2. Mosquitoes were anesthetized on
ice and injected into the hemocoel with 69 nL of sterile LB (injured mosquitoes) or a bacterial
culture (infected mosquitoes) in the thoracic anepisternal cleft, using a Nanoject III Program-
mable Nanoliter Injector (Drummond Scientific Company, Broomall, PA, USA). Absolute
infection doses were determined by diluting the cultures, plating them on LB + tetracycline
agar plates (E.coli) or LB-only agar plates (M.luteus), and counting the colony forming units
(CFUs) that grew. Across experimental trials, the infection doses averaged at 12,203 E.coli per
mosquito and 6,865 M.luteus per mosquito. Throughout this study, the age of the mosquito
represents the age when the immune treatment was initiated.
Quantification of phenoloxidase activity in hemolymph
To quantify the melanization potential of hemolymph, or the ability of hemolymph to mela-
nize a pathogen upon infection using active PO, we used a spectrophotometric assay as previ-
ously described [51,95]. For each condition, hemolymph was extracted from 18–25
mosquitoes at 24 h after the immune treatment. Briefly, the lateral thorax of cold-anesthetized
females was punctured using a 0.20 mm diameter minutien insect pin and the mosquitoes
were placed inside a 0.6 mL microfuge tube that contained a small incision at the bottom. The
0.6 mL tube was nested inside a 1.5 mL microfuge tube and then centrifuged at 5000 RCF for 5
min at 4˚C. The ~1–2 μL of hemolymph that pooled at the bottom of the 1.5 mL tube was col-
lected and stored at -20˚C until further use.
The PO activity of hemolymph was quantified using a biochemical assay that measures the
conversion of 3,4-Dihydroxy-L-phenylalanine (L-DOPA, clear, λ
max
of 280 nm) to dopa-
chrome (reddish-brown, λ
max
of 475 nm) [51,96,97] (Fig 2A and 2B). For this assay, 1 μL of
hemolymph was added to 50 μL of deionized water. Then, 10 μL of that mixture was added to
a cuvette containing 90 μL of 4 mg/mL L-DOPA (Sigma, St. Louis, MO, USA), and the absor-
bance at OD
490
was measured every 5 min for 30 min using a BioPhotometer Plus spectropho-
tometer (Eppendorf AG, Hamburg, Germany). On average, 7 independent biological trials
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 17 / 27
were conducted for each temperature-age-immune treatment combination. In total, 317
hemolymph samples, derived from approximately 6800 mosquitoes, were assayed. A negative
control was conducted during each trial, where 10 μL of water was added to a cuvette contain-
ing 90 μL of 4 mg/mL L-DOPA. Additional controls were conducted using a subset of samples,
in which diluted hemolymph was added to 90 μL of water.
Quantification of melanization on the dorsal abdominal wall
To measure the melanization that occurs inside a mosquito, we imaged the dorsal abdominal
wall and quantified the dark melanin deposits using a light intensity method previously
described [51,59,60]. For each condition, mosquitoes were cold-anesthetized at 24 h after
immune treatment and fixed by injecting cold, 16% paraformaldehyde (Electron Microscopy
Sciences, Hatfield, PA) into the hemocoel. After 10 min on ice, abdomens were bisected along
the coronal plane and immersed in PBS containing 0.1% Triton X-100. Internal organs were
removed, and the dorsal abdomens were rinsed in PBS and mounted flat on glass slides with
coverslips using Aqua-Poly/Mount (Polysciences, Warrington, PA, USA).
Dorsal abdominal segments 3–8 were imaged under brightfield illumination using a 10x
objective on a Nikon Eclipse Ni-E compound microscope connected to a Nikon Digital Sight
DS-Qi1 monochrome digital camera and Nikon Advanced Research NIS Elements software
(Nikon, Tokyo, Japan). For each dorsal abdomen, two Z-stacks were captured using a linear
encoded Z-motor. The first stack contained abdominal segments 3–5 whereas the second stack
contained segments 6–8. Each Z-stack was then rendered into a focused, two-dimensional
image using the Extended Depth of Focus (EDF) function in NIS Elements (Fig 2C).
For each mosquito, each abdominal segment was delineated using the region of interest
(ROI) tool in NIS Elements, and each periostial region (the region surrounding the heart
valves in segments 3–7) and the excurrent opening (in segment 8) was further delineated.
Then, an intensity threshold was set such that it distinguished melanized areas (pixels below
the threshold) from non-melanized areas (pixels above the threshold). Three independent bio-
logical trials were conducted for each temperature-age-immune treatment combination, with
a minimum of three mosquitoes per group. On average, each combination contained 16 mos-
quitoes assayed across 3 independent biological trials, with 760 mosquitoes assayed in total.
Statistical analysis
Statistical analyses were completed using R Statistical Software, v4.2.2 [98]. The spectrophoto-
metric data were analyzed in two ways: (i) using the OD
490
reading at 30 min as a measure of
the final melanization potential, and (ii) using the change in OD
490
over the course of the 30
min experiment as a measure of PO activity over time. Data for each temperature-age-immune
treatment combination were first tested for normality using the Shapiro-Wilk test and were
found to be non-normal. Thus, the data were zero-adjusted and log-transformed to achieve
normality.
For data on the final melanization potential, we used a linear model to identify the main
effects of temperature, age, immune treatment, and the interaction between temperature and
age. Other interactions (e.g., temperature x immune treatment) did not meaningfully contrib-
ute to melanization potential, so they were excluded from the model.
For data on PO activity over time, we used a linear mixed-effects model, fit by maximum
likelihood, to identify the relationship between OD
490
and the main effects of time, immune
treatment, age, and temperature using the “lme4” package [99]. Two-way interactions among
the main effects (time x immune treatment;time x age;time x temperature;immune treatment
x age;immune treatment x temperature;age x temperature), three-way interactions among the
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 18 / 27
main effects (time x immune treatment x temperature;time x age x temperature), and the ran-
dom effect of the individual hemolymph sample were included as predictors in the model.
Melanin deposition on the dorsal abdominal wall was analyzed the same way for two sets of
data: (i) the melanized area on the entirety of dorsal abdominal segments 3–8, and (ii) the mel-
anized area within the periostial regions in segments 3–7 plus the excurrent opening of seg-
ment 8. Data were non-normal, so they were zero-adjusted and log-transformed to achieve
normality. Then, we used a linear model to identify the main effects of higher temperature,
aging, infection, and their interactions (temperature x age;temperature x immune treatment;
age x immune treatment;temperature x age x immune treatment) on the melanized area.
For all statistical analyses, final models were determined by a stepwise, multidirectional
selection method, comparing model residuals, log-likelihood ratios, and Akaike Information
Criterion (AIC) values. We then conducted type-II ANOVAs with Wald Chi Square Tests on
the final models using the “car” package [100]. Partial effect sizes and 95% confidence intervals
were calculated using the “effectsize” package [101]. To assess the effects of the interaction
between temperature and age, for each statistical model we calculated estimated marginal
means using the “emmeans” package on the response scale, also known as “least-square
means” [102,103]. Sidak-adjusted pairwise contrasts of the estimated marginal means were
then performed within each temperature-age-immune treatment combination to identify sig-
nificant differences between groups.
Estimated marginal means, ANOVA and Chi Square p-values, partial effect sizes, and effect
size confidence intervals are presented in the main figures. Observed means for main melani-
zation response variables and controls are presented in S1–S10 Figs. Additional information is
presented in the supplement, including the raw data, raw means, estimated marginal means,
model coefficients, and full ANOVA and Chi Square tables (S1–S3 Files).
Figures depicting methods were created with BioRender.com, whereas graphs depicting
data were created with R and assembled into figures using Adobe Illustrator. Graphics created
using BioRender.com are published under agreement numbers WV25ZSF5QR (Fig 1),
XC25ZSFWGC (Fig 2), and WG25ZSGDUE (Fig 11).
Supporting information
S1 Fig. Auto-oxidation of exogenous L-DOPA is negligible. Time course of OD
490
measure-
ments of L-DOPA plus water for 30 min. The lower end of the scale is amplified on the right.
No meaningful auto-oxidation of L-DOPA was detected. Each circle marks the mean, and
whiskers indicate the S.E.M.
(PDF)
S2 Fig. Melanization in isolated hemolymph is negligible without the addition of the phe-
noloxidase substrate, L-DOPA. Time course of OD
490
measurements of hemolymph for 30
min. The lower end of the scale is amplified on the right. No meaningful melanization was
detected in the absence of exogenous L-DOPA. Each circle marks the mean, and whiskers indi-
cate the S.E.M.
(PDF)
S3 Fig. Raw means of melanization potential, aggregated by temperature, age, and immune
treatment. A. Melanization potential, aggregated by temperature and immune treatment, irre-
spective of age. B. Melanization potential, aggregated by age and immune treatment, irrespec-
tive of temperature. C. Melanization potential, aggregated by immune treatment, irrespective
of temperature or age. Column height marks the raw mean, and whiskers indicate the S.E.M.
The same measurements are plotted in S3 and S4 Figs, but grouped or arranged differently,
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 19 / 27
with aggregated data shown in this figure. The estimated marginal means of these data, result-
ing from the linear model, are presented in Fig 3.
(PDF)
S4 Fig. Raw means of melanization potential. Column height marks the raw mean, and whis-
kers indicate the S.E.M. The same measurements are plotted in S3 and S4 Figs, but grouped or
arranged differently, with unaggregated data shown in this figure. The estimated marginal
means of these data, resulting from the linear model, are presented in Fig 4.
(PDF)
S5 Fig. Raw means of melanization over time, aggregated by temperature, age, and
immune treatment. A. Melanization activity over time, aggregated by temperature and
immune treatment, irrespective of age. B. Melanization activity over time, aggregated by age
and immune treatment, irrespective of temperature. C. Melanization activity over time, aggre-
gated by immune treatment, irrespective of temperature or age. Each circle marks the raw
mean, and whiskers indicate the S.E.M. The same measurements are plotted in S5 and S6 Figs,
but grouped or arranged differently, with aggregated data shown this figure. The estimated
marginal means of these data, resulting from the linear mixed model, are presented in Fig 5.
(PDF)
S6 Fig. Raw means of melanization over time. Each circle marks the raw mean, and whiskers
indicate the S.E.M. The same measurements are plotted in S5 and S6 Figs, but grouped or
arranged differently, with unaggregated data shown in this figure. The estimated marginal
means of these data, resulting from the linear mixed model, are presented in Fig 6.
(PDF)
S7 Fig. Raw means of melanin deposition on the dorsal abdominal wall, aggregated by
temperature, age, and immune treatment. A. Melanin deposition, aggregated by temperature
and immune treatment, irrespective of age. B. Melanin deposition, aggregated by age and
immune treatment, irrespective of temperature. C. Melanin deposition, aggregated by immune
treatment, irrespective of temperature or age. Column height marks the raw mean, and whis-
kers indicate the S.E.M. The same measurements are plotted in S7 and S8 Figs, but grouped or
arranged differently, with aggregated data shown in this figure. The estimated marginal means
of these data, resulting from the linear model, are presented in Fig 7.
(PDF)
S8 Fig. Raw means of melanin deposition on the dorsal abdominal wall. Column height
marks the raw mean, and whiskers indicate the S.E.M. The same measurements are plotted in
S7 and S8 Figs, but grouped or arranged differently, with unaggregated data shown in this fig-
ure. The estimated marginal means of these data, resulting from the linear model, are pre-
sented in Fig 8.
(PDF)
S9 Fig. Raw means of melanin deposition within the periostial regions and posterior excur-
rent opening, aggregated by temperature, age, and immune treatment. A. Melanin deposition,
aggregated by temperature and immune treatment, irrespective of age. B. Melanin deposition,
aggregated by age and immune treatment, irrespective of temperature. C. Melanin deposition,
aggregated by immune treatment, irrespective of temperature or age. Column height marks the
mean, and whiskers indicate the S.E.M. The same measurements are plotted in S9 and S10 Figs,
but grouped or arranged differently, with aggregated data shown in this figure. The estimated
marginal means of these data, resulting from the linear model, are presented in Fig 9.
(PDF)
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 20 / 27
S10 Fig. Raw means of melanin deposition within the periostial regions and posterior
excurrent opening. Column height marks the mean, and whiskers indicate the S.E.M. The
same measurements are shown in S9 and S10 Figs, but grouped or arranged differently, with
unaggregated data shown in this figure. The estimated marginal means of these data, resulting
from the linear model, are presented in Fig 10.
(PDF)
S1 File. Data and statistical information for Figs 3–6.
(XLSX)
S2 File. Data for Figs 3–6and S1–S6 and statistical information for S1–S6 Figs.
(XLSX)
S3 File. Data and statistical information for Figs 7–10 and S7–S10 Figs.
(XLSX)
Acknowledgments
We thank Drs. Courtney Murdock and Ann Tate for their helpful advice and discussion about
statistical analyses. We also thank Jordyn Barr, Cole Meier, Shabbir Ahmed, Tania Este
´vez-
Lao, and Tobias McCabe for offering comments and discussion on this manuscript.
Author Contributions
Conceptualization: Lindsay E. Martin, Julia
´n F. Hillyer.
Data curation: Lindsay E. Martin, Julia
´n F. Hillyer.
Formal analysis: Lindsay E. Martin, Julia
´n F. Hillyer.
Funding acquisition: Lindsay E. Martin, Julia
´n F. Hillyer.
Investigation: Lindsay E. Martin.
Methodology: Lindsay E. Martin, Julia
´n F. Hillyer.
Project administration: Lindsay E. Martin, Julia
´n F. Hillyer.
Resources: Julia
´n F. Hillyer.
Software: Lindsay E. Martin.
Supervision: Julia
´n F. Hillyer.
Validation: Lindsay E. Martin, Julia
´n F. Hillyer.
Visualization: Lindsay E. Martin, Julia
´n F. Hillyer.
Writing – original draft: Lindsay E. Martin, Julia
´n F. Hillyer.
Writing – review & editing: Lindsay E. Martin, Julia
´n F. Hillyer.
References
1. Po
¨rtner HO, Roberts DC, Tignor M, Poloczanska ES, Mintenbeck K, Alegrı
´a A, et al. IPCC, 2022: Cli-
mate Change 2022: Impacts, Adaptation, and Vulnerability. Contribution of Working Group II to the
Sixth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge, UK and
New York, USA: Cambridge University Press; 2022.
2. Dillon ME, Wang G, Huey RB. Global metabolic impacts of recent climate warming. Nature. 2010; 467
(7316):704–6. https://doi.org/10.1038/nature09407 PMID: 20930843
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 21 / 27
3. Gonza
´lez-Tokman D, Co
´rdoba-Aguilar A, Da
´ttilo W, Lira-Noriega A, Sa
´nchez-Guille
´n RA, Villalobos
F. Insect responses to heat: physiological mechanisms, evolution and ecological implications in a
warming world. Biol Rev. 2020; 95(3):802–21. https://doi.org/10.1111/brv.12588 PMID: 32035015
4. Deutsch CA, Tewksbury JJ, Huey RB, Sheldon KS, Ghalambor CK, Haak DC, et al. Impacts of climate
warming on terrestrial ectotherms across latitude. Proceedings of the National Academy of Sciences.
2008; 105(18):6668–72. https://doi.org/10.1073/pnas.0709472105 PMID: 18458348
5. Couper LI, Farner JE, Caldwell JM, Childs ML, Harris MJ, Kirk DG, et al. How will mosquitoes adapt to
climate warming? eLife. 2021;10. https://doi.org/10.7554/eLife.69630 PMID: 34402424
6. Paaijmans KP, Heinig RL, Seliga RA, Blanford JI, Blanford S, Murdock CC, et al. Temperature varia-
tion makes ectotherms more sensitive to climate change. Global Change Biology. 2013; 19(8):2373–
80. https://doi.org/10.1111/gcb.12240 PMID: 23630036
7. Agyekum TP, Botwe PK, Arko-Mensah J, Issah I, Acquah AA, Hogarh JN, et al. A systematic review of
the effects of temperature on Anopheles mosquito development and survival: Implications for malaria
control in a future warmer climate. International Journal of Environmental Research and Public Health.
2021; 18(14):7255. https://doi.org/10.3390/ijerph18147255 PMID: 34299706
8. Ciota AT, Matacchiero AC, Kilpatrick AM, Kramer LD. The effect of temperature on life history traits of
Culex mosquitoes. Journal of Medical Entomology. 2014; 51(1):55–62. https://doi.org/10.1603/
me13003 PMID: 24605453
9. Agyekum TP, Arko-Mensah J, Botwe PK, Hogarh JN, Issah I, Dwomoh D, et al. Effects of elevated
temperatures on the growth and development of adult Anopheles gambiae (s.l.) (Diptera: Culicidae)
mosquitoes. Journal of Medical Entomology. 2022; 59(4):1413–20. https://doi.org/10.1093/jme/
tjac046 PMID: 35452118
10. Bayoh MN, Lindsay SW. Effect of temperature on the development of the aquatic stages of Anopheles
gambiae sensu stricto (Diptera: Culicidae). Bulletin of Entomological Research. 2003; 93(5):375–81.
https://doi.org/10.1079/ber2003259 PMID: 14641976
11. Agyekum TP, Arko-Mensah J, Botwe PK, Hogarh JN, Issah I, Dadzie SK, et al. Relationship between
temperature and Anopheles gambiae sensu lato mosquitoes’ susceptibility to pyrethroids and expres-
sion of metabolic enzymes. Parasites & Vectors. 2022; 15(1). https://doi.org/10.1186/s13071-022-
05273-z PMID: 35527275
12. Barr JS, Estevez-Lao TY, Khalif M, Saksena S, Yarlagadda S, Farah O, et al. Temperature and age,
individually and interactively, shape the size, weight, and body composition of adult female mosqui-
toes. Journal of Insect Physiology. 2023; 148:104525. https://doi.org/10.1016/j.jinsphys.2023.104525
PMID: 37236342
13. Barreaux AMG, Stone CM, Barreaux P, Koella JC. The relationship between size and longevity of the
malaria vector Anopheles gambiae (s.s.) depends on the larval environment. Parasites & Vectors.
2018; 11(1). https://doi.org/10.1186/s13071-018-3058-3 PMID: 30157916
14. Christiansen-Jucht C, Parham PE, Saddler A, Koella JC, Basa
´ñez M-G. Temperature during larval
development and adult maintenance influences the survival of Anopheles gambiae s.s. Parasites &
Vectors. 2014; 7(1). https://doi.org/10.1186/s13071-014-0489-3 PMID: 25367091
15. Murdock CC, Paaijmans KP, Bell AS, King JG, Hillyer JF, Read AF, et al. Complex effects of tempera-
ture on mosquito immune function. Proc Biol Sci. 2012; 279(1741):3357–66. Epub 2012/05/16. https://
doi.org/10.1098/rspb.2012.0638 PMID: 22593107.
16. Mordecai EA, Cohen JM, Evans MV, Gudapati P, Johnson LR, Lippi CA, et al. Detecting the impact of
temperature on transmission of Zika, dengue, and chikungunya using mechanistic models. PLOS
Neglected Tropical Diseases. 2017; 11(4):e0005568. https://doi.org/10.1371/journal.pntd.0005568
PMID: 28448507
17. Murdock CC, Paaijmans KP, Cox-Foster D, Read AF, Thomas MB. Rethinking vector immunology:
the role of environmental temperature in shaping resistance. Nature Reviews Microbiology. 2012; 10
(12):869–76. https://doi.org/10.1038/nrmicro2900 PMID: 23147703
18. Suwanchaichinda C, Paskewitz SM. Effects of larval nutrition, adult body size, and adult temperature
on the ability of Anopheles gambiae (Diptera: Culicidae) to melanize sephadex beads. Journal of Medi-
cal Entomology. 1998; 35(2):157–61. https://doi.org/10.1093/jmedent/35.2.157 PMID: 9538577
19. Ferreira PG, Tesla B, Hora
´cio ECA, Nahum LA, Brindley MA, de Oliveira Mendes TA, et al. Tempera-
ture dramatically shapes mosquito gene expression with consequences for mosquito-Zika virus inter-
actions. Front Microbiol. 2020; 11:901. Epub 2020/07/01. https://doi.org/10.3389/fmicb.2020.00901
PMID: 32595607; PubMed Central PMCID: PMC7303344.
20. Simões ML, Dong Y, Mlambo G, Dimopoulos G. C-type lectin 4 regulates broad-spectrum melani-
zation-based refractoriness to malaria parasites. PLOS Biology. 2022; 20(1):e3001515. https://doi.
org/10.1371/journal.pbio.3001515 PMID: 35025886
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 22 / 27
21. Rodriguez-Andres J, Rani S, Varjak M, Chase-Topping ME, Beck MH, Ferguson MC, et al. Phenoloxi-
dase activity acts as a mosquito innate immune response against infection with Semliki forest virus.
PLoS Pathogens. 2012; 8(11):e1002977. https://doi.org/10.1371/journal.ppat.1002977 PMID:
23144608
22. Hillyer JF, Schmidt SL, Christensen BM. Rapid phagocytosis and melanization of bacteria and Plas-
modium sporozoites by hemocytes of the mosquito Aedes aegypti. Journal of Parasitology. 2003; 89
(1):62–9. https://doi.org/10.1645/0022-3395(2003)089[0062:RPAMOB]2.0.CO;2 PMID: 12659304
23. Hillyer JF, Schmidt SL, Christensen BM. Hemocyte-mediated phagocytosis and melanization in the
mosquito Armigeres subalbatus following immune challenge by bacteria. Cell and Tissue Research.
2003; 313(1):117–27. https://doi.org/10.1007/s00441-003-0744-y PMID: 12838409
24. Yassine H, Kamareddine L, Osta MA. The mosquito melanization response is implicated in defense
against the entomopathogenic fungus Beauveria bassiana. Plos Pathogens. 2012; 8(11):13. https://
doi.org/10.1371/journal.ppat.1003029 WOS:000311997100044. PMID: 23166497
25. Volz J, Muller H-M, Zdanowicz A, Kafatos FC, Osta MA. A genetic module regulates the melanization
response of Anopheles to Plasmodium. Cellular Microbiology. 2006; 8(9):1392–405. https://doi.org/
10.1111/j.1462-5822.2006.00718.x PMID: 16922859
26. Conde
´R, Hernandez-Torres E, Claudio-Piedras F, Recio-To
´toro B, Maya-Maldonado K, Cardoso-
Jaime V, et al. Heat shock causes lower Plasmodium infection rates in Anopheles albimanus. Frontiers
in Immunology. 2021; 12(2482). https://doi.org/10.3389/fimmu.2021.584660 PMID: 34248924
27. Hillyer JF, Este
´vez-Lao TY. Nitric oxide is an essential component of the hemocyte-mediated mos-
quito immune response against bacteria. Developmental & Comparative Immunology. 2010; 34
(2):141–9. https://doi.org/10.1016/j.dci.2009.08.014 PMID: 19733588
28. Luckhart S, Vodovotz Y, Cui L, Rosenberg R. The mosquito Anopheles stephensi limits malaria para-
site development with inducible synthesis of nitric oxide. Proceedings of the National Academy of Sci-
ences. 1998; 95(10):5700–5. https://doi.org/10.1073/pnas.95.10.5700 PMID: 9576947
29. Wimalasiri-Yapa BMCR Barrero RA, Stassen L Hafner LM, McGraw EA Pyke AT, et al. Temperature
modulates immune gene expression in mosquitoes during arbovirus infection. Open Biology. 2021; 11
(1):200246. https://doi.org/10.1098/rsob.200246 PMID: 33401993
30. Shapiro LLM, Whitehead SA, Thomas MB. Quantifying the effects of temperature on mosquito and
parasite traits that determine the transmission potential of human malaria. PLOS Biology. 2017; 15
(10):e2003489. https://doi.org/10.1371/journal.pbio.2003489 PMID: 29036170
31. Mordecai EA, Caldwell JM, Grossman MK, Lippi CA, Johnson LR, Neira M, et al. Thermal biology of
mosquito-borne disease. Ecology Letters. 2019; 22(10):1690–708. https://doi.org/10.1111/ele.13335
PMID: 31286630
32. Tesla B, Demakovsky LR, Mordecai EA, Ryan SJ, Bonds MH, Ngonghala CN, et al. Temperature
drives Zika virus transmission: evidence from empirical and mathematical models. Proc R Soc B-Biol
Sci. 2018; 285(1884):9. https://doi.org/10.1098/rspb.2018.0795 WOS:000441725900004. PMID:
30111605
33. Caldwell JM, Labeaud AD, Lambin EF, Stewart-Ibarra AM, Ndenga BA, Mutuku FM, et al. Climate pre-
dicts geographic and temporal variation in mosquito-borne disease dynamics on two continents.
Nature Communications. 2021; 12(1). https://doi.org/10.1038/s41467-021-21496-7 PMID: 33623008
34. Bowler K, Terblanche JS. Insect thermal tolerance: what is the role of ontogeny, ageing and senes-
cence? Biol Rev. 2008; 83(3):339–55. https://doi.org/10.1111/j.1469-185x.2008.00046.x
WOS:000257796900006. PMID: 18979595
35. Ryan SJ, Ben-Horin T, Johnson LR. Malaria control and senescence: the importance of accounting for
the pace and shape of aging in wild mosquitoes. Ecosphere. 2015; 6(9):art170. https://doi.org/10.
1890/es15-00094.1
36. Styer LM, Carey JR, Wang JL, Scott TW. Mosquitoes do senesce: departure from the paradigm of
constant mortality. Am J Trop Med Hyg. 2007; 76(1):111–7. Epub 2007/01/27. PMID: 17255238;
PubMed Central PMCID: PMC2408870.
37. Min K-J, Tatar M. Unraveling the molecular mechanism of immunosenescence in Drosophila. Interna-
tional Journal of Molecular Sciences. 2018; 19(9):2472. https://doi.org/10.3390/ijms19092472 PMID:
30134574
38. Zajitschek F, Zajitschek S, Bonduriansky R. Senescence in wild insects: Key questions and chal-
lenges. Functional Ecology. 2020; 34(1):26–37. https://doi.org/10.1111/1365-2435.13399
39. Mu¨ller L, Fu¨lo
¨p T, Pawelec G. Immunosenescence in vertebrates and invertebrates. Immun Ageing.
2013; 10(1):12. Epub 2013/04/04. https://doi.org/10.1186/1742-4933-10-12 PMID: 23547999;
PubMed Central PMCID: PMC3637519.
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 23 / 27
40. Gaillard JM, Lemaı
ˆtre JF. An integrative view of senescence in nature. Functional Ecology. 2020; 34
(1):4–16. https://doi.org/10.1111/1365-2435.13506
41. Sciambra N, Chtarbanova S. The impact of age on response to infection in Drosophila. Microorgan-
isms. 2021; 9(5):958. https://doi.org/10.3390/microorganisms9050958 PMID: 33946849
42. Hillyer JF, Schmidt SL, Fuchs JF, Boyle JP, Christensen BM. Age-associated mortality in immune
challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers. Cellular
Microbiology. 2005; 7(1):39–51. https://doi.org/10.1111/j.1462-5822.2004.00430.x PMID: 15617522
43. Styer LM, Carey JR, Wang J-L, Scott TW. Mosquitoes do senesce: departure from the paradigm of
constant mortality. The American journal of tropical medicine and hygiene. 2007; 76(1):111–7. PMID:
17255238.
44. Horn L, Leips J, Starz-Gaiano M. Phagocytic ability declines with age in adult Drosophila hemocytes.
Aging Cell. 2014; 13(4):719–28. https://doi.org/10.1111/acel.12227 PMID: 24828474
45. Prasai K, Karlsson B. Variation in immune defence in relation to age in the green-veined white butterfly
(Pieris napi L.). Journal of Invertebrate Pathology. 2012; 111(3):252–4. https://doi.org/10.1016/j.jip.
2012.08.004 PMID: 22903037
46. Ren C, Webster P, Finkel SE, Tower J. Increased internal and external bacterial load during Drosoph-
ila aging without life-span trade-off. Cell Metabolism. 2007; 6(2):144–52. https://doi.org/10.1016/j.
cmet.2007.06.006 PMID: 17681150
47. Chun J, Riehle M, Paskewitz SM. Effect of mosquito age and reproductive status on melanization of
sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae. Journal of
Invertebrate Pathology. 1995; 66(1):11–7. https://doi.org/10.1006/jipa.1995.1054 PMID: 7544819
48. Christensen BM, Lafond MM, Christensen LA. Defense reactions of mosquitoes to filarial worms:
Effect of host age on the immune response to Dirofilaria immitis microfilariae. The Journal of Parasitol-
ogy. 1986; 72(2):212. https://doi.org/10.2307/3281593 PMID: 3734989
49. Jianyong L, Tracy JW, Christensen BM. Relationship of hemolymph phenol oxidase and mosquito age
in Aedes aegypti. Journal of Invertebrate Pathology. 1992; 60(2):188–91. https://doi.org/10.1016/
0022-2011(92)90095-l PMID: 1401989
50. Cornet S, Gandon S, Rivero A. Patterns of phenoloxidase activity in insecticide resistant and suscepti-
ble mosquitoes differ between laboratory-selected and wild-caught individuals. Parasites & Vectors.
2013; 6(1):315. https://doi.org/10.1186/1756-3305-6-315 PMID: 24499651
51. League GP, Estevez-Lao TY, Yan Y, Garcia-Lopez VA, Hillyer JF. Anopheles gambiae larvae mount
stronger immune responses against bacterial infection than adults: evidence of adaptive decoupling in
mosquitoes. Parasites & Vectors. 2017;10. https://doi.org/10.1186/s13071-017-2302-6
WOS:000407054900004. PMID: 28764812
52. 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
(1):84–8. Epub 2002/04/05. https://doi.org/10.1603/0022-2585-39.1.84 PMID: 11931276.
53. King JG, Hillyer JF. Spatial and temporal in vivo analysis of circulating and sessile immune cells in
mosquitoes: hemocyte mitosis following infection. BMC Biology. 2013; 11(1):55. https://doi.org/10.
1186/1741-7007-11-55 PMID: 23631603
54. Castillo JC, Robertson AE, Strand MR. Characterization of hemocytes from the mosquitoes Anophe-
les gambiae and Aedes aegypti. Insect Biochemistry and Molecular Biology. 2006; 36(12):891–903.
https://doi.org/10.1016/j.ibmb.2006.08.010 PMID: 17098164
55. Sinka ME, Bangs MJ, Manguin S, Rubio-Palis Y, Chareonviriyaphap T, Coetzee M, et al. A global map
of dominant malaria vectors. Parasites & Vectors. 2012; 5(1):69. https://doi.org/10.1186/1756-3305-5-
69 PMID: 22475528
56. Christensen BM, Li J, Chen C-C, Nappi AJ. Melanization immune responses in mosquito vectors.
Trends in Parasitology. 2005; 21(4):192–9. https://doi.org/10.1016/j.pt.2005.02.007 PMID: 15780842
57. Hillyer JF. Insect immunology and hematopoiesis. Developmental & Comparative Immunology. 2016;
58:102–18. https://doi.org/10.1016/j.dci.2015.12.006 PMID: 26695127
58. Lai S-C, Chen C-C, Hou RF. Immunolocalization of prophenoloxidase in the process of wound healing
in the mosquito Armigeres subalbatus (Diptera: Culicidae). Journal of Medical Entomology. 2002;39
(2):266–74. https://doi.org/10.1603/0022-2585-39.2.266 PMID: 11931025
59. Yan Y, Hillyer JF. Complement-like proteins TEP1, TEP3 and TEP4 are positive regulators of periostial
hemocyte aggregation in the mosquito Anopheles gambiae. Insect Biochem Mol Biol. 2019; 107:1–9.
Epub 2019/01/29. https://doi.org/10.1016/j.ibmb.2019.01.007 PMID: 30690067.
60. Sigle LT, Hillyer JF. Mosquito hemocytes preferentially aggregate and phagocytose pathogens in the
periostial regions of the heart that experience the most hemolymph flow. Developmental & Compara-
tive Immunology. 2016; 55:90–101. https://doi.org/10.1016/j.dci.2015.10.018 PMID: 26526332
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 24 / 27
61. King JG, Hillyer JF. Infection-induced interaction between the mosquito circulatory and immune sys-
tems. PLoS Pathogens. 2012; 8(11):e1003058. https://doi.org/10.1371/journal.ppat.1003058 PMID:
23209421
62. Sousa GL, Bishnoi R, Baxter RHG, Povelones M. The CLIP-domain serine protease CLIPC9 regulates
melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gam-
biae. PLOS Pathogens. 2020; 16(10):e1008985. https://doi.org/10.1371/journal.ppat.1008985 PMID:
33045027
63. Sigle LT, Hillyer JF. Mosquito hemocytes associate with circulatory structures that support intracardiac
retrograde hemolymph flow. Front Physiol. 2018; 9:1187. Epub 2018/09/14. https://doi.org/10.3389/
fphys.2018.01187 PMID: 30210361; PubMed Central PMCID: PMC6121077.
64. Glenn JD, King JG, Hillyer JF. Structural mechanics of the mosquito heart and its function in bidirec-
tional hemolymph transport. J Exp Biol. 2010; 213(4):541–50. Epub 2010/02/02. https://doi.org/10.
1242/jeb.035014 PMID: 20118304.
65. Adamo SA, Jensen M, Younger M. Changes in lifetime immunocompetence in male and female Gryl-
lus texensis (formerly G. integer): trade-offs between immunity and reproduction. Animal Behaviour.
2001; 62(3):417–25. https://doi.org/10.1006/anbe.2001.1786
66. Hillyer JF, Christensen BM. Characterization of hemocytes from the yellow fever mosquito, Aedes
aegypti. Histochemistry and Cell Biology. 2002; 117(5):431–40. https://doi.org/10.1007/s00418-002-
0408-0 PMID: 12029490
67. Hillyer JF, Christensen BM. Mosquito phenoloxidase and defensin colocalize in melanization innate
immune responses. Journal of Histochemistry & Cytochemistry. 2005; 53(6):689–98. https://doi.org/
10.1369/jhc.4A6564.2005 PMID: 15928318
68. Sugumaran M, Barek H. Critical analysis of the melanogenic pathway in insects and higher animals.
International Journal of Molecular Sciences. 2016; 17(10):1753. https://doi.org/10.3390/ijms17101753
PMID: 27775611
69. Andersen SO. Insect cuticular sclerotization: A review. Insect Biochemistry and Molecular Biology.
2010; 40(3):166–78. https://doi.org/10.1016/j.ibmb.2009.10.007 PMID: 19932179
70. Nappi AJ, Christensen BM. Melanogenesis and associated cytotoxic reactions: Applications to insect
innate immunity. Insect Biochemistry and Molecular Biology. 2005; 35(5):443–59. https://doi.org/10.
1016/j.ibmb.2005.01.014 PMID: 15804578
71. Karl I, Stoks R, De Block M, Janowitz SA, Fischer K. Temperature extremes and butterfly fitness: con-
flicting evidence from life history and immune function. Global Change Biology. 2011; 17(2):676–87.
https://doi.org/10.1111/j.1365-2486.2010.02277.x
72. Ehrlich RL, Zuk M. The role of sex and temperature in melanin-based immune function. Canadian
Journal of Zoology. 2019; 97(9):825–32. https://doi.org/10.1139/cjz-2018-0323
73. Fedorka KM, Copeland EK, Winterhalter WE. Seasonality influences cuticle melanization and immune
defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects. J
Exp Biol. 2013; 216(Pt 21):4005–10. Epub 2013/07/23. https://doi.org/10.1242/jeb.091538 PMID:
23868839.
74. Clusella-Trullas S, Nielsen M. The evolution of insect body coloration under changing climates. Cur-
rent Opinion in Insect Science. 2020; 41:25–32. https://doi.org/10.1016/j.cois.2020.05.007 PMID:
32629405
75. Cotter SC, Myatt JP, Benskin CM, Wilson K. Selection for cuticular melanism reveals immune function
and life-history trade-offs in Spodoptera littoralis. J Evol Biol. 2008; 21(6):1744–54. Epub 2008/08/12.
https://doi.org/10.1111/j.1420-9101.2008.01587.x PMID: 18691239.
76. Wilson K, Cotter SC, Reeson AF, Pell JK. Melanism and disease resistance in insects. Ecology Let-
ters. 2001; 4(6):637–49. https://doi.org/10.1046/j.1461-0248.2001.00279.x
77. Bailey NW. A test of the relationship between cuticular melanism and immune function in wild-caught
Mormon crickets. Physiological Entomology. 2011; 36(2):155–64. https://doi.org/10.1111/j.1365-
3032.2011.00782.x
78. Fedorka KM, Lee V, Winterhalter WE. Thermal environment shapes cuticle melanism and melanin-
based immunity in the ground cricket Allonemobius socius. Evolutionary Ecology. 2013; 27(3):521–31.
https://doi.org/10.1007/s10682-012-9620-0
79. Armitage SAO, Siva-Jothy MT. Immune function responds to selection for cuticular colour in Tenebrio
molitor. Heredity. 2005; 94(6):650–6. https://doi.org/10.1038/sj.hdy.6800675 PMID: 15815710
80. Ariani CV, Juneja P, Smith S, Tinsley MC, Jiggins FM. Vector competence of Aedes aegypti mosqui-
toes for filarial nematodes is affected by age and nutrient limitation. Experimental Gerontology. 2015;
61:47–53. https://doi.org/10.1016/j.exger.2014.11.001 PMID: 25446985
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 25 / 27
81. Zhao X, Ferdig MT, Li J, Christensen BM. Biochemical pathway of melanotic encapsulation of Brugia
malayi in the mosquito, Armigeres subalbatus. Developmental & Comparative Immunology. 1995; 19
(3):205–15. https://doi.org/10.1016/0145-305x(95)00005-e PMID: 8595819
82. Hillyer JF, Schmidt SL, Christensen BM. The antibacterial innate immune response by the mosquito
Aedes aegypti is mediated by hemocytes and independent of Gram type and pathogenicity. Microbes
Infect. 2004; 6(5):448–59. Epub 2004/04/28. https://doi.org/10.1016/j.micinf.2004.01.005 PMID:
15109959.
83. Areiza M, Nouzova M, Rivera-Perez C, Noriega FG. 20-hydroxyecdysone stimulationof juvenile hor-
mone biosynthesis by the mosquito corpora allata. Insect Biochemistry and Molecular Biology. 2015;
64:100–5. https://doi.org/10.1016/j.ibmb.2015.08.001 PMID: 26255691
84. Ekoka E, Maharaj S, Nardini L, Dahan-Moss Y, Koekemoer LL. 20-Hydroxyecdysone (20E) signaling
as a promising target for the chemical control of malaria vectors. Parasites & Vectors. 2021; 14(1).
https://doi.org/10.1186/s13071-020-04558-5 PMID: 33514413
85. Ahmed A, Martı
´n D, Manetti AGO, Han SJ, Lee WJ, Mathiopoulos KD, et al. Genomic structure and
ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae. Pro-
ceedings of the National Academy of Sciences. 1999; 96(26):14795–800. https://doi.org/10.1073/
pnas.96.26.14795 PMID: 10611292
86. Werling K, Shaw WR, Itoe MA, Westervelt KA, Marcenac P, Paton DG, et al. Steroid hormone function
controls non-competitive Plasmodium development in Anopheles. Cell. 2019; 177(2):315–25.e14.
https://doi.org/10.1016/j.cell.2019.02.036 PMID: 30929905
87. Reynolds RA, Kwon H, Smith RC. 20-Hydroxyecdysone primes innate immune responses that limit
bacterial and malarial parasite survival in Anopheles gambiae. mSphere. 2020; 5(2). https://doi.org/
10.1128/mSphere.00983-19 PMID: 32295874
88. Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, et al. Immunity-related
genes and gene families in Anopheles gambiae. Science. 2002; 298(5591):159. https://doi.org/10.
1126/science.1077136 PMID: 12364793
89. Bryant WB, Michel K. Blood feeding induces hemocyte proliferation and activation in the African
malaria mosquito, Anopheles gambiae Giles. Journal of Experimental Biology. 2013; 217(8):1238–45.
https://doi.org/10.1242/jeb.094573 PMID: 24363411
90. Castillo J, Brown MR, Strand MR. Blood feeding and insulin-like peptide 3 stimulate proliferation of
hemocytes in the mosquito Aedes aegypti. PLoS Pathogens. 2011; 7(10):e1002274. https://doi.org/
10.1371/journal.ppat.1002274 PMID: 21998579
91. Chandrasegaran K, Lahondère C, Escobar LE, Vinauger C. Linking mosquitoecology, traits, behavior,
and disease transmission. Trends in Parasitology. 2020; 36(4):393–403. https://doi.org/10.1016/j.pt.
2020.02.001 PMID: 32191853
92. Sinka ME, Bangs MJ, Manguin S, Coetzee M, Mbogo CM, Hemingway J, et al. The dominant Anophe-
les vectors of human malaria in Africa, Europe and the Middle East: occurrence data, distribution
maps and bionomic pre
´cis. Parasites & Vectors. 2010; 3(1):117. https://doi.org/10.1186/1756-3305-3-
117 PMID: 21129198
93. Lindsay SW, Parson L, Thomas CJ. Mapping the range and relative abundance of the two principal
African malaria vectors, Anopheles gambiae sensu stricto and An. arabiensis, using climate data. Pro-
ceedings of the Royal Society of London Series B: Biological Sciences. 1998; 265(1399):847–54.
https://doi.org/10.1098/rspb.1998.0369 PMID: 9633110
94. Phillips MA, Burrows JN, Manyando C, Van Huijsduijnen RH, Van Voorhis WC, Wells TNC. Malaria.
Nature Reviews Disease Primers. 2017; 3(1):17050. https://doi.org/10.1038/nrdp.2017.50 PMID:
28770814
95. Meier CJ, Martin LE, Hillyer JF. Mosquito larvae exposed to a sublethal dose of photosensitive insecti-
cides have altered juvenile development but unaffected adult life history traits. Parasites & Vectors.
2023. https://doi.org/10.1186/s13071-023-06004-8 PMID: 37951916
96. Laughton AM, Siva-Jothy MT. A standardised protocol for measuring phenoloxidase and prophenolox-
idase in the honey bee, Apis mellifera. Apidologie. 2011; 42(2):140–9. https://doi.org/10.1051/apido/
2010046
97. Mason HS. The chemistry of melanin; mechanism of the oxidation of dihydroxyphenylalanine by tyrosi-
nase. J Biol Chem. 1948; 172(1):83–99. Epub 1948/01/01. PMID: 18920770.
98. R-Core-Team. R: A language and environment for statistical computing. Vienna, Austria: R Founda-
tion for Statistical Computing; 2022.
99. Bates D, Ma
¨chler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. Journal of
Statistical Software. 2015; 67(1):1–48. https://doi.org/10.18637/jss.v067.i01
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 26 / 27
100. Fox J, Weisberg S. An {R} Companion to Applied Regression. Third ed. Thousand Oaks, CA: Sage;
2019.
101. Ben-Shachar M, Lu¨decke D, Makowski D. effectsize: Estimation of effect size indices and standard-
ized parameters. Journal of Open Source Software. 2020; 5(56):2815. https://doi.org/10.21105/joss.
02815
102. Lenth RV. emmeans: Estimated marginal eans, aka least-squares means. R package version 1.8.3
ed2022.
103. Searle SR, Speed FM, Milliken GA. Population marginal means in the linear model: An alternative to
least squares means. The American Statistician. 1980; 34(4):216–21. https://doi.org/10.1080/
00031305.1980.10483031
PLOS PATHOGENS
Higher temperature accelerates immune senescence in mosquitoes
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1011935 January 10, 2024 27 / 27
Available via license: CC BY
Content may be subject to copyright.
