Diapause and quiescence: Dormancy mechanisms that contribute to the geographical expansion of mosquitoes and their evolutionary success

Abstract

Mosquitoes are insects belonging to the order Diptera and family Culicidae. They are distributed worldwide and include approximately 3500 species, of which about 300 have medical and veterinary importance. The evolutionary success of mosquitoes, in both tropical and temperate regions, is due to the various survival strategies these insects have developed throughout their life histories. Of the many adaptive mechanisms, diapause and quiescence, two different types of dormancy, likely contribute to the establishment, maintenance and spread of natural mosquito populations. This review seeks to objectively and coherently describe the terms diapause and quiescence, which can be confused in the literature because the phenotypic effects of these mechanisms are often similar.

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R E V I E W Open Access
Diapause and quiescence: dormancy
mechanisms that contribute to the
geographical expansion of mosquitoes and
their evolutionary success
Diego Felipe Araujo Diniz
1
, Cleide Maria Ribeiro de Albuquerque
2
, Luciana Oliveira Oliva
2
,
Maria Alice Varjal de Melo-Santos
1
and Constância Flávia Junqueira Ayres
1*
Abstract
Mosquitoes are insects belonging to the order Diptera and family Culicidae. They are distributed worldwide and
include approximately 3500 species, of which about 300 have medical and veterinary importance. The evolutionary
success of mosquitoes, in both tropical and temperate regions, is due to the various survival strategies these insects
have developed throughout their life histories. Of the many adaptive mechanisms, diapause and quiescence, two
different types of dormancy, likely contribute to the establishment, maintenance and spread of natural mosquito
populations. This review seeks to objectively and coherently describe the terms diapause and quiescence, which
can be confused in the literature because the phenotypic effects of these mechanisms are often similar.
Keywords: Culicidae, Seasonality, Metabolism, Adaptation, Dispersion, Disease transmission
Background
Mosquitoes are arthropods that can cause considerable
nuisance and affect human health worldwide [1, 2]. They
are among the most prolific and invasive species, con-
tributing to the spread of endemic diseases [3, 4]. These
organisms are present in most places on the planet, from
the Arctic to the most remote desert oases, except Ant-
arctica due to its extremely low temperatures. Thus,
mosquitoes are widely diverse and can easily be found in
a wide variety of habitats, including forested, rural and
urban environments [2, 5].
These insects have been intensely studied since the
end of the nineteenth century due to their ability to act
as hosts for many pathogens, including helminths, pro-
tozoans and arboviruses, that cause disease in humans
and other vertebrates [2, 6]. However, only 10% of the
approximately 3500 mosquito species are medically rele-
vant [1, 7–11].
Mosquitoes, especially from the genera Anopheles,
Aedes and Culex, include vectors for three major groups
of human pathogens: parasites from the genus Plas-
modium, which cause malaria; filarial worms from the
genera Wuchereria and Brugia; and many arboviruses, in-
cluding the agents of dengue, yellow fever, chikungunya,
zika and others [12–14]. Estimates by the World Health
Organization (WHO) indicate that diseases transmitted by
mosquitoes are among the major causes of morbidity and
mortality in developing countries [15], and high densities
of mosquitoes severely challenge vector control programs
[16]. The explosive growth of natural mosquito popula-
tions is strongly related to the survival and dispersion
strategies that some species have acquired over the course
of their evolutionary history [17].
Dormancy is a biological trait that may play an import-
ant role in the maintenance of natural populations and
refers to a physiological phenomenon characterised by
the interruption or reduction of metabolic activity in an
organism. In mosquitoes, dormancy can occur at differ-
ent stages of the life-cycle [18]. Diapause and quiescence
represent different types of dormancy found in many
species of mosquitoes. In this review, these terms are
* Correspondence: tans@cpqam.fiocruz.br
1
Entomology Department, Aggeu Magalhães Institute, Oswaldo Cruz
Foundation, Av. Professor Moraes Rego, s/n –Cidade Universitária, Recife, PE,
Brazil
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Diniz et al. Parasites & Vectors (2017) 10:310
DOI 10.1186/s13071-017-2235-0
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analysed for their conceptual principles and their re-
spective delayed developmental effects; in addition, the
mosquito species found to exhibit these phenomena will
be noted.
Insect dormancy and its various types
Dormancy is a physiological phenomenon defined as a
state of suspended development or suppressed metabolic
activity in an organism [19]. Dormancy can occur in
both plants and animals; in insects, it can manifest in
the embryonic (pharate larvae), immature (larvae and
pupae) and adult stages [18]. This phenomenon can be
triggered by climactic signals, especially the photoperiod
for temperate climate insects and relative humidity for
tropical insects. This adaptation seeks to promote sur-
vival during and after unfavourable environmental con-
ditions and is known in the literature as heterodynamic
development [20]. In 1869, the term dormancy was first
described as a period of inactivity caused by low temper-
atures by the French researcher Duclaux,who was
studying silkworms (Bombyx mori) [20, 21]. According
to a literature review by Danks [20] on the definitions
and terminology of dormancy in insects, dormancy is di-
vided into two major categories: diapause and quies-
cence. The terms diapause and quiescence have been
reported to be synonymous in the literature [8, 22–27],
but these survival strategies arise from distinct signalling
pathways even though the strategies have the same goal:
to ensure survival during and after environmental stress.
Mosquitoes belong to one of the most well-adapted
taxa in the insect group; they are present across most of
the planet, they occupy diverse niches and are potential
disease vectors [2]. Diapause and quiescence are well
characterised in several stages of the mosquito life-cycle.
In the embryonic phase, for example, both strategies
have the same effect: the inhibition of larval hatching.
Conversely, only diapause drives dormancy in the larval
and adult stages of mosquitoes [28].
Diapause in mosquitoes
Diapause is a well-studied seasonal survival strategy and
is influenced by several factors, such as the species-
specific ecological interactions, biogeography, life history
and physiology of many insects [29]. The etymology of
the word “diapause”comes from the Greek diapausis
(pause), derived from the verb diapauein, which means
to stop or to decrease activity at a time of constant ac-
tion [30]. Biologically, Tauber et al. [31] defined the dia-
pause phenomenon as a dynamic state of low metabolic
activity that is genetically determined and mediated by
neurohormones that phenotypically affect individuals by
decreasing morphogenesis, blocking reproduction and
metamorphosis, and increasing tolerance to extreme en-
vironmental conditions
The first studies on diapause in mosquitoes coincided
with early studies of seasonality, diapause and photoperiod
in other insects [17]. Early reviews on the topic were per-
formed by Lees [32], Danilevskii [33], Tauber et al. [31]
and Danks [20]. Studies at the time were motivated by the
mosquito’s hematophagous habit, which is linked to its
ability to transmit the causative agents of several diseases
such as malaria, filariasis, and many arbovirus infections
(yellow fever, Western equine, St. Louis and Japanese en-
cephalitis, and West Nile fever) [34].
Diapause is common in insects and other arthropods,
especially in areas with harsh winters. Many aspects of
diapause are critical for understanding the transmission
cycle of vector-borne diseases, as this survival strategy
contributes to the maintenance, establishment, growth
and dispersion of natural vector populations after the
end of an unfavourable season to their development
[29]. The process of diapause seeks to reactivate devel-
opment via external signals that control the genetic fac-
tors underlying the dormant phenotype. This can occur
in several phases of the life-cycle, but most often only
one developmental stage enters diapause [34].
What is the environmental signal that induces diapause
in mosquitoes?
Species exhibiting the phenotypic plasticity to undergo
diapause have the required information encoded in their
genomes. The major stimuli inducing diapause in nat-
ural populations are changing photoperiod (short days
and long nights) and gradual decreases in temperature
[31, 35–40]. Mosquito species that use photoperiod to
signal diapause include Aedes albopictus,Aedes atropal-
pus,Aedes sollicitans,Aedes taeniorhynchus,Culex
pipiens and Culex restuans [38, 39, 41–44].
Preparation for diapause occurs in mosquitoes when
pupae and/or adult females, which are thought to be the
determining stages for this biological trait, are stimulated
by exposure to the seasonal changes that typically occur
during transitions between a favourable and unfavour-
able season [29, 39, 45–47]. For Ae. albopictus, for in-
stance, induced females develop their offspring for
diapause, which in turn present low metabolism in each
life-cycle stage during the winter [48, 49]. However, for
Cx. pipiens, the induced pupae females express diapause
when they become adults [50]. Therefore, this ecological
adaptation is indispensable for coordinating the growth,
development and reproduction of mosquito species
found in temperate zones [29].
Ecophysiological phases of diapause
The phenomenon of diapause consists of three ecophysi-
ological phases [51]. The first is the diapause preparation
or pre-diapause phase, which corresponds to the sensi-
tive stage in which the insect is exposed to one or more
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environmental signals (token-stimuli) that trigger and
initiate the phenomenon in the offspring in the following
season [19, 48]. In some species, this phase is favourable
for the storage of energetic reserves that will be used for
basal maintenance of the insect during dormancy and
the reinitiation of development at the end of the process.
In addition, morphophysiological, biochemical and be-
havioural changes can be observed in the individuals at
this phase [19, 29, 51, 52]. This occurs because some
mosquito species extend the developmental time of a
specific life-cycle stage (delayed developmental effects)
to increase their exposure to the stimulus, which is a
favourable event for ensuring that the dormancy pheno-
type occurs in the offspring [29].
Culex pipiens females programmed for adult diapause
have a longer larval phase, resulting in larger pupae and
adults that have more lipids than their non-diapausing
homologs [53]. The fat levels in females of this same
species destined for diapause continue to increase sig-
nificantly during the week following the emergence of
the adults, reaching twice the level observed in non-
diapausing females [54]. At the molecular level, this
increase in energetic reserves is accompanied by an in-
creased expression of genes associated with lipid reserve
synthesis [55]. In Ae. albopictus, eggs in diapause are lar-
ger and contain more lipids than non-diapausing eggs,
which is likely due to the increased expression of genes
involved in lipid storage during pre-diapause [56].
Diapause programming (Fig. 1) involves the capture of
photoperiod information by the central nervous system
(CNS) of gravid females, followed by a cascade of bio-
chemical events and culminating in the transfer of a
molecular diapause regulator that promotes a dormancy
state in embryos [29]. Thus, clock genes can reasonably
be assumed to be involved in the regulation of circadian
rhythms and, consequently, in the seasonal response
based on the length of day and night [57, 58]. The main
clock genes in mosquitoes that are involved in circadian
rhythm regulation but are not necessarily related to
diapause have been characterised in Ae. aegypti,Ae.
albopictus,Anopheles gambiae,Cx. quinquefasciatus and
Wyeomyia smithii [59–65].
Diapause specifically refers to the actual time when
development is interrupted or significantly slowed, and
the insect does not respond to environmental stimuli
[29]. This is the second phase and can be divided into
the following sub-phases: (i) the responsive phase-the be-
ginning of the process when development is stopped at a
specific life stage; (ii) the initiation stage-the phenomenon
is maintained and controlled by endogenous and/or ex-
ogenous factors, and (iii) termination-the time when the
individuals receive the signal to return to normal meta-
bolic activity [19]. During diapause, various endogenous
changes can be observed, but these depend on the species
studied. In Ae. albopictus embryos, Wy. smithii larvae and
Cx. pipiens adults, for example, lower lipid degradation
Fig. 1 Embryonic diapause induction in mosquitoes. 1 Exposure of pupae and/or adult females to short days, long nights and gradual
temperature drop, and abiotic factors that promote the preparation of the embryonic diapause. 2 Expression of specific genes transferred from
the female to the offspring allows diapause to be triggered and the embryos (pharate larvae) to become refractory to the hatching stimulus
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and higher tolerance to desiccation and low temperatures
are present [48, 66–70].
At the molecular level, a few genetic components that
mediate these adaptive physiological traits have been re-
ported in previous studies. In Ae. albopictus, resistance
to desiccation, promoted by diapause, results from an in-
crease in the external surface area of the egg with in-
creased hydrocarbon levels, and this is caused by an
overexpression of a transcript involved in lipid storage.
However, the mechanisms responsible for cold tolerance
in this species have not been determined [71]. In Cx.
pipiens, increased tolerance to desiccation during dia-
pause is primarily due to an increase in the hydrocarbon
layer on the cuticular body surface of adults and an in-
crease in trehalose production, which contribute to both
desiccation and cold tolerance [66]. In contrast, the mo-
lecular events that promote the effects of diapause in
Wy. smithii have yet to be discovered [29].
The last phase, termed post-diapause, is characterised
by the complete reactivation of metabolism and develop-
ment in the insect [51]. Although photoperiod is widely
used as an environmental stimulus for entering diapause,
it is less often used to signal the end of diapause;
however, some exceptions exist, such as, for example, in
Wy. smithii, where another change in photoperiod
causes diapause to end [29, 72]. In Ae. albopictus, the
termination of diapause in the eggs may be signalled by
changes in photoperiod and by increasing temperature
[73]. Another interesting characteristic, in addition to
post-diapause, is a phenomenon known as post-diapause
quiescence (Fig. 2), which is also present in Ae. albopic-
tus [49, 73]. This process is considered to be a phenotyp-
ically indistinguishable phase from diapause. The insect
remains in a state of dormancy, its metabolic rate con-
tinues to be low, and many of the same genes associated
with diapause continue to be expressed. Thus, diapause
and quiescence possibly have many molecular com-
ponents in common, although the components for initial
programming are exclusive to diapause [49]. Physio-
logically, the only difference is that during quiescence,
the insect remains fully capable of responding to envir-
onmental stimuli [29, 74, 75].
Diapause in different mosquito species/life-cycle stages
Diapause can occur in different phases of the mosquito
life-cycle, i.e. in the embryo (pharate larvae inside the
Fig. 2 Termination of the embryonic diapause in the mosquito (post-diapause). 1 Return to normal conditions (temperature and photoperiod)
that signal the end of diapause. 2 Post-diapause embryo under favorable conditions responds to the stimuli of relative humidity increase and
optimal temperature in the environment, resulting in larval hatching. 3 Post-diapause embryo under non-favorable abiotic conditions is sensitive
to environmental stimuli but remains dormant in a quiescent state (post-diapause quiescence) until the temperature and relative humidity
become ideal for larval hatching. The dynamics of quiescence are the same as post-diapause quiescence
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egg), larval and adult stages. However, this type of dor-
mancy tends to occur in a single stage of the life-cycle
for a given species [19, 29, 34]. Furthermore, in some
species, diapause can occur in more than one stage,
more precisely, between the embryonic and larval stages
[36, 62, 76–78].
Embryonic diapause
This is the most common type of dormancy and occurs in
the mosquito embryo. Using Ae. albopictus, a model or-
ganism for diapause, as a reference, the embryo is com-
pletely formed inside the egg chorion, but a metabolic
depression of post-embryonic development occurs due to
genetic programming; thus, the larva is unable to respond
to any abiotic signals, that is, the larva is refractory to
hatching stimuli [29, 45, 46, 79]. Embryos in diapause are
more tolerant to desiccation and tend to have a higher
total lipid content than normal embryos [57, 68, 80]. The
overexpression of ecdysteroid transcripts, found by tran-
scriptomic analysis of mature oocytes, likely regulates em-
bryonic diapause in Ae. albopictus and other mosquitoes
[48, 49]. The genera of mosquitoes with embryonic dia-
pause are Aedes,Anopheles,Psorophora and Ochlerotatus
[29, 34], and the major species reported in the literature
for each genus are listed in Table 1.
Diapause in larval stages
This physiological process is known in the literature as
the syndrome of larval diapause, which is characterized
in mosquitoes by the prolongation of the third- or
fourth-instar. The induction of diapause in larvae is dir-
ectly stimulated by a gradual decrease in environmental
temperature, and the metabolic activity rapidly returns
to normal in response to its normalisation in the wild,
although changes in photoperiod also play a role in its
induction [34]. The behaviour of the larvae is charac-
terised by reduced locomotor and feeding activities, con-
sequently promoting an increased accumulation of body
reserves that, in turn, provide increased cold tolerance
[34]. Under normal conditions, the progression of the
development of the larval stages occurs biochemically
through the periodic release of the steroid hormone ec-
dysone by the prothoracic gland, which culminates in
the moults. When the larvae are in diapause, ecdysone
release is lacking, and therefore, the larvae do not ad-
vance from one stage to the next [29]. Currently, no mo-
lecular studies have explained the hormonal basis for
diapause in mosquitoes, but some studies have reported
the absence of ecdysone as a major cause of larval dia-
pause in other insects, which is likely similar to mosqui-
toes [81]. Mosquito species in which this type of
dormancy has been observed are listed in Table 2.
Diapause in adult females
Diapause in adult female mosquitoes involves a set of
important characteristics, such as the interruption of go-
nadal development, reduced biting behaviour, negative
phototaxis and changes in total metabolism, leading to
the gradual accumulation of body fat. Mosquitoes can
enter diapause in many habitats, such as caves, soil cav-
ities, burrows, vegetable store-houses, empty sheds,
basements, and catacombs [34]. In adult females, a type
of dormancy occurs, known as reproductive diapause,
where sexual immaturity is prolonged because the ovar-
ian follicles do not differentiate completely and hence,
delay the blood feeding activity [8, 34, 74].
The majority of studies on diapause in adult mos-
quitoes has been performed on the species Cx. pipiens,
which is considered a model organism [29].Under nor-
mal conditions, after the emergence of the winged form,
juvenile hormone (JH) is synthesised and released,
Table 1 Embryonic diapause in different mosquito species
Species References
Aedes albopictus [46]
Aedes atropalpus [41]
Aedes campestris [150]
Aedes canadensis [151]
Aedes caspius [76]
Aedes dorsalis [152]
Aedes fitchii [153]
Aedes geniculatus [77]
Aedes hendersoni [78]
Aedes hexodontus [154]
Aedes impiger [129]
Aedes japonicus [155]
Aedes mariae [35]
Aedes nigripes [156]
Aedes nigromaculis [152]
Aedes sierrensis [36]
Aedes sticticus [157]
Aedes taeniorhynchus [44]
Aedes triseriatus [158]
Aedes vexans [159]
Anopheles walkeri [160]
Psorophora ferox [151]
Ochlerotatus dorsalis [152]
Ochlerotatus nigromaculis [152]
Ochlerotatus hexodontus [154]
Ochlerotatus flavescens [34]
Ochlerotatus triseriatus [75]
Ochlerotatus togoi [75]
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promoting ovarian follicle growth within 3 days, and
shortly after, the female is ready for its blood meal,
which will contribute to oocyte maturation. In contrast,
females programmed for diapause do not release JH im-
mediately after emergence, and the follicles remain atro-
phied. The females also have a reduced aggressiveness
[82]. Experiments on diapausing Cx. pipiens females
treated with JH have exhibited ovarian growth stimula-
tion, confirming the importance of inhibiting this hor-
mone to initiate diapause in adult mosquitoes [54, 83]. It
is important to highlight that in this species, males do
not undergo diapause, thus, they inseminate females and
then die, as they cannot overwinter [31, 33, 84]. The
Anopheles and Culex species reported as exhibiting adult
diapause are listed in Table 3 [38, 40, 75, 85–94].
The molecular biology of diapause in mosquitoes
Most studies on the genetic basis of diapause in mosqui-
toes have focused on two species, Ae. albopictus and Cx.
pipiens, which are considered model organisms for this
approach. Early studies were performed in the fly Dros-
ophila melanogaster; however, although this species is a
reference for basic genetic studies, it did not yield good
results in the gene expression studies, as the insect
showed highly variable responses and high variance be-
tween individuals [95–97].
Diapause in Cx. pipiens, according to breeding experi-
ments, is polygenetically regulated and involves genes on
all three chromosomes [98, 99]. A more detailed study on
the species using suppressive subtractive hybridization to
determine the expression profile of diapause genes re-
vealed that a set of 40 genes were differentially expressed.
Most of these genes were implicated in the expression of
structural components and responses to the environmen-
tal stress [100]. One of the upregulated genes was a stress
tolerance gene expressing a heat-shock protein (HSP70),
which functions as a chaperone to inhibit abnormal pro-
tein folding in harsh environmental conditions, including
desiccation and cold [101]. In addition, metabolic genes
are overexpressed in Cx. pipiens during diapause, includ-
ing the mitochondrial malate dehydrogenase (mmd),
methylmalonate-semialdehyde dehydrogenase and cyto-
chrome oxidase (cox) genes. These genes may be involved
in the specific metabolic events associated with diapause
and have been implicated in increased cold tolerance. The
expression of certain cytoskeletal genes was also upregu-
lated by preparation for diapause. The actin gene, for
example, is overexpressed during the diapause preparation
stage, likely due to increased flying activity before dor-
mancy begins, and the expression levels of this gene de-
crease gradually during diapause and are low at diapause
termination. Downregulated genes included the ribosomal
genes S3A, rpS6 and rpS24, which are involved in gene
regulation (translation initiation) and inhibit or reduce the
expression of several other metabolic genes [102].
Most information on changes in gene expression
associated with diapause in mosquitoes is based on re-
cent high-throughput sequencing studies (such as RNA-
seq) examining the transcriptome of Ae. albopictus at
Table 2 Larval diapause in different mosquito species
Species References
Aedes caspius [76]
Aedes geniculatus [77]
Aedes hendersoni [78]
Aedes sierrensis [36]
Aedes togoi [68]
Aedes triseriatus [159]
Anopheles barberi [161]
Anopheles plumbeus [162]
Anopheles pulcherrimus [85]
Armigeres subalbatus [163]
Culiseta melanura [164]
Orthopodomyia alba [165]
Orthopodomyia puchripalpis [166]
Orthopodomyia signifera [167]
Toxorhynchites rutilus [168]
Wyeomyia smithii [169]
Table 3 Adult diapause in different mosquito species
Species References
Anopheles atroparvus [85]
Anopheles earlei [86]
Anopheles freeborni [87]
Anopheles superpictus [85]
Anopheles gambiae [88]
Anopheles hyracanus [87]
Anopheles maculipennis [85]
Anopheles messeae [85]
Anopheles punctipennis [89]
Anopheles sacharovi [75]
Culex bitaeniorhynchus [75]
Culex apicalis [75]
Culex modestus [75]
Culex pipiens [39]
Culex restuans [39]
Culex tarsalis [90]
Culex tritaeniorhynchus [91]
Culiseta alaskaensis [92]
Culiseta impatiens [93]
Culiseta inornata [94]
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different stages [29, 48, 49, 102]. Early studies on the
differential expression of transcripts were performed in
the ovary cells of this mosquito (oocytes), and later, the
molecular mechanisms during embryogenesis were
investigated.
A study by Poelchau et al. [48], who sequenced the
oocyte transcriptome of diapausing Ae. albopictus females,
and another more recent similar study from the same
group, Poelchau et al. [49], who used diapausing embryos
from Ae. albopictus, revealed the overexpression of genes
involved in various biological processes. The following are
included among these genes: the gene ing1, which encodes
for the inhibitor of growth protein and is involved in the
interruption of the cell division cycle [52, 103]; the gene
rack1, a putative receptor for activated protein kinase C,
which may bind to several signaling molecules, including
transcription factors related to ecdysone (20-hydroxyecdy-
sone), and is probably associated with the preparation for
diapause [104, 105]; the gene pepck (phosphoenolpyruvate
carboxykinase), whose product participates in the glyco-
gen pathway to move from aerobic to anaerobic metabol-
ism in diapausing mosquitoes [106, 107]; and the gene
GPCR (G protein-coupled receptor), which is involved in
increased resistance to environmental stress [108].
Quiescence in mosquitoes
Quiescence is a type of irregular dormancy (non-seasonal)
characterised by slowed metabolism and directly resulting
from unfavourable environmental conditions, including
low humidity and high temperatures [22, 74, 109, 110].
This adaptive trait is often confused with diapause, espe-
cially when referring to embryonic dormancy, but quies-
cence is a less complex biological trait that does not
depend on endogenous control for its initiation. Stimuli
that trigger quiescence are referred to as acyclic envi-
ronmental changes [19]. Quiescence also differs from dia-
pause because it is neither a previously programmed
event, nor is it hormonally controlled; once the stimulus
that induces the process ceases, physiological activity is
restored [29, 34, 73]. Because quiescence is controlled
exogenously, it is possible that rapid gene activation and
macromolecule synthesis or degradation are not required
for entry into the quiescent state [109].
In mosquitoes, as in other organisms, the term quies-
cence is applied to various biological events. Most
commonly studied in the egg, quiescence in mosquitoes
can be stimulated in different stages or structures, enab-
ling the insect to attain favourable conditions for sur-
vival. In the mosquito Cx. quinquefasciatus, for example,
mature spermatozoids are maintained in quiescence in
the male reproductive tract and are activated in response
to specific chemical signals [111]. In this species, mo-
tility is stimulated by substances from the accessory
glands in males and is possibly controlled by protein
phosphorylation and Ca
2+
levels [111]. In addition, in fe-
males, degenerative dilations may develop in the ovary,
which contains granular material during winter, and the
presence of these expansions in the ovaries is thought to
be indicative of quiescence [112, 113].
In the family Culicidae, quiescence, unlike diapause,
has been primarily observed in the egg, reflected in the
resistance to desiccation that allows the embryo to
survive in dry conditions. The process begins when the
embryo (pharate larvae) receives an external stimulus,
such as a rapid drop in humidity or change in temperature,
which signals unfavourable environmental conditions and
impedes larval hatching [19, 34, 74]. In this case, the devel-
opmental arrest is temporary and immediately revers-
ible, as contact with water induces rapid hatching;
that is, the quiescent embryo is not refractory to
hatching stimuli as is found in diapausing embryos
[18, 19, 34, 49, 114]. As shown in Table 4, the genera re-
ported exhibiting quiescence are Aedes,Anopheles and
Culex [23, 49, 68, 80, 115–124]. The species Ae. aegypti is
prominent among mosquitoes due to its strategy of pro-
longed viability by embryonic quiescence, significantly
contributing to the constant expansion of populations in
Table 4 Embryonic quiescence in different mosquito species
Species References
Aedes aegypti [68]
[23]
[116]
[170]
[118]
[119]
[120]
[121]
[122]
[115]
[123]
Aedes albopictus [68]
[117]
[71]
[48]
Aedes flavopictus [68]
Aedes galloisi [68]
Aedes riversi [68]
Anopheles aquasalis [121]
[123]
Anopheles gambiae [124]
Culex quinquefasciatus [123]
[123]
Diniz et al. Parasites & Vectors (2017) 10:310 Page 7 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

the wild [74, 75]. However, several studies have errone-
ously reported this trait as diapause [8, 22–24, 26].
Egg quiescence or embryonic desiccation resistance
Egg quiescence is commonly referred to as “embryonic
desiccation resistance”(EDR) and depends on several
factors that range from differences in eggshell compos-
ition and structure to physiological changes, resulting in
reduced metabolism in the larvae contained within the
egg [22, 116, 121, 125]. However, because the ability to
resist desiccation is a property of the egg and not of the
embryo and because desiccation can occur at other
stages of development, the term “egg resistance to desic-
cation (ERD)”has been suggested as more appropriate
for referring to this phenomenon [123].
The three layers that form the eggshell, the exochorion,
endochorion and serosal cuticle, are particularly important
for ERD [116, 123]. The first two layers are produced in
the ovary, by females, and are, therefore, present at laying
[74, 123, 126]. The serosal cuticle (the innermost layer), in
turn, is an extracellular matrix produced by the extraem-
bryonic serosa during early embryogenesis. In Ae. aegypti,
secretion of the serosal cuticle occurs between 11 and
13 h after oviposition and approximately 8 h post-
fertilization in An. gambiae [115, 124].
This cuticle likely secretes a chitin-containing material
under the chorion, the external layer of the egg, making
it impermeable and protecting the embryo from desicca-
tion [116, 123]. Changes in the amounts of the eggshell
components are associated with water loss regulation
and are fundamental for determining the intensity of egg
dehydration. Aedes albopictus females exposed to short
day length in temperate regions produce eggs in photo-
periodic diapause, unlike populations in tropical regions,
which enter quiescence. One of the characteristics of the
egg that permits this adaptation is the high quantity of
fatty acyl-CoA elongase in the tissue of mature oocytes
responsible for producing hydrocarbons in the eggshell
[71]. These hydrocarbons regulate water loss in insect
eggs, and the abundance of this enzyme varies in the
eggs of Ae. albopictus exposed to long and short days in
temperate populations but is maintained at relatively
constant levels in tropical populations [80, 123]. In
addition to several hydrocarbons in the eggshell, the
amount of chitin is another factor involved in ERD in
mosquitoes, such as Cx. quinquefasciatus,An. aquasalis
and Ae. aegypti. Eggshells with higher amounts of chitin
are more resistant to desiccation [123].
Quiescence patterns in container-inhabiting mosquitoes
ERD has been more commonly studied in container-
inhabiting mosquitoes, including Ae. aegypti and Ae.
albopictus. In urban areas, females often lay their eggs in
containers with clean water, especially disposable con-
tainers, tires, plant pots and water storage containers
[127, 128]. Because the eggs are laid near the water sur-
face, this developmental phase is very susceptible to de-
hydration, particularly during the first few hours of
development [129].
First-instar larvae that remain inside quiescent eggs
have been referred to as pharate first-instar quiescence
[34, 74]. Normal development finishes approximately 3
days after oviposition and larval survival depends on ma-
ternal reserves [119]. Throughout the quiescent period,
the larval developmental period is significantly pro-
longed, and lipid reserves are reduced, incurring fitness
costs for larval viability, compromising the reproductive
performance of the adult [34, 74].
Minimally studied, quiescence in mosquito eggs does
not appear to have a uniform pattern, exhibiting variabil-
ity between species or even among populations of the
same species [69, 123, 130]. Under similar low-moisture
conditions, the pharate first instars of Cx. quinquefascia-
tus,An. aquasalis and Ae. aegypti can survive for a few
hours, 1 day or several months, respectively [123]. These
differences may be due to traits inherent to the eggs of
each species, such as size, the structure of the exochor-
ion and endochorion, differences in metabolite quantity
and formation of the serosal cuticle [68, 121, 131, 132].
Brazilian colonies of Ae. aegypti maintained at a
temperature of 28 ± 1 °C, a relative humidity of
80 ± 5% and a photoperiod of 12 h had a viability
period of up to 492 days, with high hatching rates be-
tween three and 121 days [23]. A similar pattern with
high larval hatching rates (80%) was reported by Diniz
et al. [115] in quiescent Ae. aegypti eggs that had been
stored for up to 150 days. The authors compared eggs
from laboratory and wild populations with different
susceptibilities to the insecticide temephos, which were
then maintained for up to 180 days at 26 °C, with a
photoperiod of 12 h and at 50–60% humidity. The high
viability of quiescent eggs from temephos-resistant fe-
males suggests a high contribution to the maintenance of
resistant individuals in the wild. Similarly, in Australia,
quiescent Ae. aegypti eggs remained viable for more than
a year with a hatching rate of approximately 2–15%, allow-
ing its dispersion to new locations [133]. Species inhabit-
ing forests have been shown to be less resistant to changes
in humidity. Aedes riversi,Ae. galloisi and Ae. flavopietus
eggs have different survivability rates in very humid
conditions but were less resistant than Ae. aegypti and Ae.
albopictus under low humidity. Intraspecific differences in
ERD were also observed among these species, as Ae.
riversi and Ae. flavopietus strains from subtropical re-
gions had lower viability than strains from temperate
regions [68].
Diniz et al. Parasites & Vectors (2017) 10:310 Page 8 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

The molecular biology of quiescence in mosquitoes
Although much is known about the metabolic mecha-
nisms and molecular biology of diapause, very little is
known about these aspects during quiescence in mos-
quitoes. A study by Poelchau et al. [49] that compared
the transcriptomes of quiescent and diapausing Ae. albo-
pictus eggs found that the genetic expression profile be-
tween these samples converged over time; that is, the
transcription profile in eggs during late diapause
(40 days) is similar to that in quiescent eggs [49]. An im-
portant aspect of this study is that expression levels of
genes related to lipid metabolism were always higher in
eggs in diapause, demonstrating the likely importance of
this reserve for maintaining embryonic diapause and
explaining why eggs in diapause have more lipid reserves
than quiescent eggs [49].
Currently, the metabolic pathways or hormones asso-
ciated with quiescence are unknown, and only the chitin
synthase (CHS) gene has been described as being related
to this phenomenon in mosquitoes. This gene promotes
the synthesis of chitin, which is then secreted into the
extracellular space of the egg, with direct implications
for the formation of the serosal cuticle and consequently
the resistance to desiccation [116, 124, 134]. Despite be-
ing primarily cited for An. gambiae (AgCHS), this gene
is highly conserved in two other species of mosquitoes,
An. quadrimaculatus and Ae. aegypti. The gene has two
variants, but only the allele AgCHS1 is involved in em-
bryogenesis. In Ae. aegypti, for example, the expression
of the gene peaks between nine and 12 h after ovipos-
ition, coinciding with the acquisition of resistance to
desiccation through the complete covering of the em-
bryo by the chitinized serosal cuticle [116, 124].
Eco-epidemiological importance of quiescence
In Europe, a considerable increase in invasive mosquito
propagation has been observed since the end of the
1990s, with the species Ae. albopictus,Ae. aegypti,Ae.
japonicus,Ae. atropalpus and Ae. koreicus already estab-
lished on the continent [131]. In addition to increased
population densities, the distribution of Ae. albopictus
has continued to increase, and several other species of
Aedes are being reported in new countries each year
[135]. For example, recently, a research group from
Brock University reported the detection of Ae. aegypti
for the first time in Canada [136]. In addition, Lima et
al. [137] reported a permanent Ae. aegypti local popula-
tion in the Capitol Hill neighbourhood in Washington
DC that can overwinter. This is contrary to the previous
hypothesis that different introductions of Ae. aegypti
every year maintain that local population. All these spe-
cies are well adapted to the urban environment, exploit-
ing a variety of container habitats that proliferate near
human settlements, and both quiescence and diapause
may be contributing to the maintenance of these popula-
tions. In addition to the annoyance of their bites, these
mosquitoes are potential vectors for agents that cause
tropical diseases, including Zika, dengue, chikungunya
and yellow fever [138]. Quiescence in Ae. aegypti may also
allow the survival of infected embryos, favouring virus
survival and its maintenance in nature [8, 122, 139]. For
example, DENV-1 was detected and isolated in 8.33% of
Ae. aegypti eggs in Florida, suggesting that maintenance of
dengue outbreaks in 2009 and 2010 in Key West may have
been facilitated by vertical transmission [140]. Transovar-
ian transmission of DENV in the field was also detected in
larvae and adults originating from larvae collected in do-
mestic containers in Rajasthan, India. Approximately
1.09% of the reservoirs contained larvae with the virus,
detected by the indirect fluorescence antibody test and
reverse transcriptase polymerase chain reaction. In this
case, dormant eggs may have contributed to prolonging
dengue epidemics [141]. Furthermore, Zika virus, a flavivi-
rus that has recently caused large outbreaks in several
countries and has been linked to microcephaly cases and
other neurological complications, has also been reported
as being transferred via transovarian transmission by Ae.
aegypti and Ae. albopictus [142–144, 145].
Although the implications of quiescence on the
ecology of mosquito vectors and public health are
well established, its effects on physiology, behaviour
and life history are less understood. Maternal reserves
accumulated in eggs directly influence the period of
dormancy in the first-instar larvae contained within
the eggs [8, 120]. Thus, quiescent eggs pose an im-
portant problem for vector control because these eggs
can directly contribute to the maintenance of mos-
quito populations in treated areas. Aedes aegypti eggs
fromasinglelaying,atthesameage,andmaintained
under the same environmental conditions had differ-
ent hatching rates during the same period of quies-
cence. Aedes aegypti embryos employ a hedge betting
mechanism not all eggs hatch at the first stimulus;
some need a second wetting stimulus to hatch [146].
This will ensure that in the event of sudden unfavourable
conditions, such as cold temperatures or a dry spell, fol-
lowing the oviposition of the egg batch, the entire batch is
not lost [34]. Another explanation could be that not all
larvae hatch simultaneously because of competition for
space and resources as noted by Livdahl et al. [147] for
Ae. triseriatus.Furthermore,Ebrahimietal.[148]showed
that the eggs of An. gambiae embryos are not stimulated
to hatch when the water surface is agitated, demonstrating
that environmental factors could indicate the best time for
hatching. Sota & Mogi [68] suggested that intraspecific
variation in the survival time of eggs is an inherited trait
dependent on environmental pressures. Variations in the
length of quiescence of eggs and variable hatching rates
Diniz et al. Parasites & Vectors (2017) 10:310 Page 9 of 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

may be mechanisms that Ae. aegypti employs to produce
continuous, although fluctuating, populations of adults in
the wild at various stages, depending on the existence of
favourable or unfavourable environmental conditions [23].
Therefore, quiescence provides a high adaptive poten-
tial to Ae. aegypti and Ae. albopictus populations, in-
creasing the viability of their eggs and the chances of
surviving in nature [148, 149]. This trait has contributed
to the geographical expansion of these two species at a
global level, an issue that is closely related to the spread
of diseases [122].
Conclusions
As presented in this review, dormancy, especially dia-
pause and quiescence, has a significant impact on the life
history of mosquitoes, as well as of many other arthro-
pods. Dormancy is part of the life history of many mos-
quito species, providing a mechanism to overcome
unfavorable seasons in tropical and temperate zones.
This trait may have independently evolved several times
in the family Culicidae, as the phenomenon occurs at
various developmental stages in different species. These
adaptive strategies provide, on an evolutionary scale,
mechanisms for species survival, as offspring continue to
be produced, even when exposed to the various types of
stress found in a habitat, and this, in turn, contributes to
the territorial expansion of natural populations, conse-
quently increasing their invasive potential. Diapause and
quiescence are not the same biological phenomenon but
have been treated as synonymous in previous studies. In
addition, these different types of dormancy likely aid the
propagation of the transmission cycles of diseases caused
by different types of arboviruses, as these etiological
agents can be transferred via the transovarian route.
Both of these biological phenomena could play import-
ant roles in the ecology and evolution of many insect
species, such as, for example, the mosquito Ae. albopic-
tus, which has both phenotypes. Thus, the phenotypic
plasticity generated by these intrinsic characteristics re-
sults in the reproductive success and survival of mosqui-
toes in the face of adverse environmental conditions and
the different control measures practised by humans.
These mechanisms are also fundamental for adapting to
more frequent changes in climate. These phenomena are
possibly still developing and need to be more thoroughly
studied, as the information generated from associated
research may be applied to innovative control strategies.
Abbreviations
AgCHS: Chitin synthase of Anopheles gambiae; CHS: Chitin synthase;
cox: Cytochrome oxidase; EDR: Embryonic desiccation resistance; GPCR: G
protein-coupled receptor; ing1: Inhibitor of growth protein; JH: Juvenile
hormone; MMD: Mitochondrial malate dehydrogenase;
pepck: Phosphoenolpyruvate carboxykinase; rack1: Receptor for activated
protein kinase C
Acknowledgements
Not applicable.
Funding
Not applicable.
Availability of data and materials
Not applicable.
Authors’contributions
DFAD, MAVMS and CFJA designed this study; DFAD conducted the review
and wrote the manuscript; CMRA and LOO participated in writing the
quiescence topic; and CFJA and MAVMS reviewed the manuscript. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Author details
1
Entomology Department, Aggeu Magalhães Institute, Oswaldo Cruz
Foundation, Av. Professor Moraes Rego, s/n –Cidade Universitária, Recife, PE,
Brazil.
2
Zoology Department, Federal University of Pernambuco, Av. Professor
Moraes Rego, 1235 –Cidade Universitária, Recife, PE, Brazil.
Received: 26 January 2017 Accepted: 8 June 2017
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