Mosquito Diapause. David L. Denlinger 1, and Peter A. Armbruster 2. Keywords. Abstract

Transcription

1 Annu. Rev. Entomol :7393 First published online as a Review in Advance on October 18, 2013 The Annual Review of Entomology is online at ento.annualreviews.org This article s doi: /annurev-ento Copyright c 2014 by Annual Reviews. All rights reserved Corresponding author Mosquito Diapause David L. Denlinger 1, and Peter A. Armbruster 2 1 Departments of Entomology and Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, Ohio 43210; denlinger.1@osu.edu 2 Department of Biology, Georgetown University, Washington, DC 20057; paa9@georgetown.edu Keywords dormancy, aestivation, insect seasonality, global warming, disease transmission, population variation Abstract Diapause, a dominant feature in the life history of many mosquito species, offers a mechanism for bridging unfavorable seasons in both temperate and tropical environments and serves to synchronize development within populations, thus directly affecting disease transmission cycles. The trait appears to have evolved independently numerous times within the Culicidae, as exemplified by the diverse developmental stages of diapause in closely related species. Its impact is pervasive, not only influencing the arrested stage, but also frequently altering physiological processes both before and after diapause. How the diapause response can be molded evolutionarily is critical for understanding potential range expansions of native and newly introduced species. The study of hormonal regulation of mosquito diapause has focused primarily on adult diapause, with little current information available on larval diapause or the intriguing maternal effects that regulate egg diapause. Recent quantitative trait locus, transcriptome, and RNA interference studies hold promise for interpreting the complex suite of genes that subserve the diapause phenotype. 73

2 Diapause: an environmentally preprogrammed period of arrested development that can occur during any stage of insect development and has a fixed period of latency that must be completed before development resumes Quiescence: a form of dormancy that is an immediate response to the prevailing environmental conditions INTRODUCTION Winters in temperate zones and dry seasons in the tropics impose dramatic seasonal constraints on mosquito development. For blood-feeding insects such as mosquitoes, this constraint is imposed by a physical environment that is either too cold or too dry to sustain continuous development, rather than by a lack of food, as seen for plant-feeding insects. In response, mosquitoes and other insects have repeatedly evolved a programmed dormancy known as diapause. Diapause encompasses a whole suite of characteristics often referred to as the diapause syndrome, with pervasive impacts on biogeography, ecological interactions, life history, and physiology. Many aspects of diapause are critical for understanding the dynamics of disease transmission by mosquitoes, thus underscoring the importance of understanding diapause in this taxon. Major segments of the year may be spent in diapause, and the success of diapause has a direct effect on the size of mosquito populations the following season. The diapausing stage frequently serves as a reservoir, enabling disease pathogens to re-establish in the next favorable season. Diapause also has a profound impact on species distributions. Species that lack diapause, such as Aedes aegypti, are restricted to subtropical and tropical habitats. In addition, like that of many other insects, mosquito distribution has been influenced significantly in recent years by commerce, resulting in frequent invasions of vector species into new geographic areas. The increased stress resistance associated with diapause is likely to enhance propagule survival during long-distance transport; thus, diapause responses are critical for understanding how mosquitoes will adapt to new environments. In a similar fashion, global climate change opens new opportunities for range expansion, and diapause is a key element in understanding the capacity for invasions into new territories. In spite of huge, recent investments in mosquito research, relatively little work has focused on mosquito diapause, a stage that is enormously important for understanding the success of disease vectors in the natural world. The insect diapause literature offers comprehensive reviews of diapause ecology (25, 111), clock mechanisms (97), hormonal regulation (32), energetics (43), and cold tolerance mechanisms (31). Previous reviews of mosquito diapause focus primarily on ecological aspects of diapause and its environmental control (74, 115). Our understanding of the molecular and physiological underpinnings of mosquito diapause, as well as the critical role of diapause in mosquito range expansion and response to global climate change, has progressed substantially over the past decade. This review aims to provide an overview of mosquito diapause, building on previous summaries of diapause ecology and integrating recent advances in the evolutionary dynamics and molecular physiology of diapause in mosquitoes. What Is Diapause? The answer to this question is more complicated for mosquitoes than it is for most other insect taxa because mosquitoes display several forms of dormancy. Classically, diapause is defined as a form of dormancy that is hormonally programmed in advance of its onset and is not immediately terminated in response to favorable conditions. The advanced programming of diapause is critical for enabling an insect to enter diapause in autumn and bridge the warm days until the cold of winter arrives. This advanced programming distinguishes diapause from quiescence, a dormancy that is elicited in direct response to unfavorable environmental conditions and is immediately terminated upon the return of favorable environmental conditions. For example, the failure of hatching in Ae. aegypti eggs that have been deposited above the water line is considered quiescence because development can be prompted immediately upon immersion in water (81). In contrast, when adult females of Ae. albopictus are exposed to short day lengths, they also lay eggs that fail to hatch, but 74 Denlinger Armbruster

3 this developmental arrest is considered a diapause because it is programmed environmentally in advance of its onset (during the mother s life) and will persist even if the eggs are submerged in water. Only after a fixed interval has elapsed are the diapause eggs of Ae. albopictus capable of responding to the water stimulus. Little is known about the molecular distinctions between these two forms of developmental arrest (84). Possibly diapause and quiescence have much in common at the molecular level, except for the fixed latency and early programming components that are unique to diapause. Diapause does not necessarily imply a complete cessation of development, but if development does occur, it progresses at a much slower rate than in the nondiapause state. Annu. Rev. Entomol : Downloaded from WHY STUDY MOSQUITO DIAPAUSE? For many mosquito vectors, diapause is the primary mechanism for survival in environments with seasonal periods of winter or drought. Vast gaps in our understanding of major diseases such as malaria are evident because basic questions about the existence of diapause in Anopheles gambiae and other malaria vectors still remain largely unresolved. Diapause not only affects survival during the unfavorable seasons, but also serves to synchronize population dynamics and thus may directly impact disease transmission cycles (10) and can influence the seasonal abundance of vector species (53). For diseases such as West Nile virus and LaCrosse virus, transmitted by Culex pipiens and Ae. triseriatus, respectively, diapausing stages are critical for harboring the viruses during winter and subsequently reintroducing them as disease agents the following summer (78). Vertical transmission appears to be the mechanism by which the overwintering mosquitoes are infected (3, 95). Failure of the virus to replicate while the mosquito host is in diapause implies an intriguing interaction between host and virus. Diapause also offers a unique tool for probing the molecular and physiological bases of specific behaviors and physiological conditions. For example, diapause in Cx. pipiens provides an opportunity to explore the dramatic behavioral distinction between choosing nectar versus a vertebrate host as a food source (94). Likewise, the huge fat accumulation evident in diapausing Cx. pipiens suggests this species will be particularly useful for deciphering regulatory and metabolic pathways linked to decisions to store or utilize energy reserves (43). Pathways that generate enhanced stress tolerance are also prominent in diapausing mosquitoes; e.g., diapausing eggs of Ae. albopictus are more desiccation resistant than nondiapausing eggs (107), and fatty acyl-coa elongase, a key mrna transcript mediating this process (113), is highly conserved across mosquito taxa. Desiccation resistance is a crucial ecological trait related to invasion success (55), drought tolerance (56), and competitive outcomes (54), so it will be exciting to know whether the same fatty acyl-coa elongase also mediates egg desiccation resistance under nondiapause conditions and/or in species such as Ae. aegypti that lack diapause. Ultimately, identifying conserved pathways underlying the molecular regulation of diapause, or diapause-associated physiological processes such as energy storage and egg desiccation resistance, may provide a basis for novel forms of vector control based on genetic or chemical disruption of such key traits. EVOLUTION OF DIAPAUSE WITHIN AND AMONG CULICID SPECIES Diapause is widespread in insects, having been described in hundreds of species across most insect orders (2, 25, 111). Within Insecta, diapause can occur in all developmental stages embryo, larva, pupa, and adult but for most species, diapause is restricted to a single stage. Pupal diapause, common in other Diptera, is conspicuously absent in the Culicidae; however, all other stages of diapause are represented. Egg, or more appropriately embryonic, diapauses in insects can occur Mosquito Diapause 75

4 GENUS Armigeres Life cycle stage of diapause EGG LARVA ADULT 1 Aedes * 18 6 (egg and larva) Psorophora 1 Orthopodomyia 3 Wyeomyia * 1 Toxorhynchites 1 Culiseta 1 3 Culex * 8 Annu. Rev. Entomol : Downloaded from Figure 1 Pharate first instar larva: a fully developed first instar larva that has completed embryonic development but remains within the chorion of the egg Hibernal diapause: an overwintering diapause common to temperate zones Anopheles * Substantial information on molecular basis of diapause available Cladogram based on a phylogenetic analysis adapted from Reidenbach et al. (91) for nine culicid genera for which information on the life cycle stage of diapause could be obtained. Numbers indicate the number of species within each genera that diapause in a specific life cycle stage (egg, larva, adult), and red asterisks indicate the genera for which substantial information on the molecular basis of diapause is available (see text for details). in nearly any stage of embryonic development, but all reported cases of embryonic diapause in mosquitoes occur at the stage of the pharate first instar larva, i.e., the nearly completely formed first instar larva encased within the chorion. As with other insects, larval diapause in mosquitoes is most common in the final (fourth) larval instar, but diapause can also occur in other larval stages (see below). Mosquitoes that diapause as adults do so soon after adult eclosion, prior to taking a blood meal and producing eggs. Blood feeding and egg production are initiated only after diapause is terminated. In the case of Cx. pipiens, only females undergo diapause. Mating occurs in the autumn and males then die, leaving only inseminated females to overwinter. To investigate the evolution of diapause life histories within and among culicid species, we identified diapause reports on 58 species from nine genera (Figure 1). A full summary of the species included in our analysis and the relevant primary literature is available in Supplemental Table 1 (follow the Supplemental Material link from the Annual Reviews home page at This sample likely represents a small fraction of the species that actually undergo diapause, but several general patterns emerge from this dataset. The cladogram in Figure 1 representing the life cycle stage of diapause is based on a phylogenetic analysis of the Culicidae adapted from Reidenbach et al. (91). Although the relatively strong phylogenetic conservation illustrated in Figure 1 implies an evolutionary constraint affecting the diapause stage among genera, the variation among species within genera clearly indicates a degree of evolutionary flexibility in terms of how diapause can be integrated into species-specific life histories. Presumably, this variation is related to selective pressures caused by species-specific ecology. For example, Ae. sierrensis is a tree-hole-breeding mosquito that is widespread across the Pacific slope of North America, from British Columbia to Southern California. This region is characterized by mild and moist autumn and spring conditions, winters that are restrictive to growth due to low temperature, and summers that are restrictive due to low water availability. Ae. sierrensis larvae overwinter in a fourth instar hibernal diapause induced by short-day photoperiods Denlinger Armbruster

5 (52). This larval diapause is terminated in the spring, leading to adult reproduction in early summer, and the eggs then enter a long-day-induced aestivation that is terminated in the early fall (51). Thus, by evolving diapause at two stages of the life cycle, one initiated by short day lengths and one initiated by long day lengths, this mosquito is able to exploit the temporally discontinuous availability of environmental conditions suitable for growth and development. The evolved differences in diapause life histories illustrated in Figure 1 can have important ecological consequences related to differences in the seasonal abundance of specific life cycle stages. In North America, the native container-breeding mosquito Ae. triseriatus, which can overwinter as either an egg or a larva (47), terminates diapause earlier than the invasive container-breeding Ae. albopictus, leading to differences in seasonal abundance of larvae in tire habitats (53). This temporal habitat segregation likely contributes to coexistence despite the competitive superiority of Ae. albopictus and predicted competitive displacement of Ae. triseriatus by Ae. albopictus (66). In British tree-hole communities, diapause-related differences in seasonal abundance also lead to temporal partitioning of shared larval habitats and may thereby contribute to species coexistence (18). In addition to the interspecific variation illustrated in Figure 1, intraspecific variation in the occurrence and/or life cycle timing of diapause has been documented. For a number of species with broad geographic ranges that include both temperate and tropical habitats, such as Ae. albopictus and Ae. togoi, photoperiodic diapause has been well characterized in temperate populations but does not occur in tropical populations (46, 106), although tropical populations may undergo an aestivation that is initiated by environmental factors other than photoperiod. In the pitcherplant mosquito, Wyeomyia smithii, ancestral populations from the Gulf Coast enter diapause as fourth instar larvae, whereas derived northern populations enter diapause as third instar larvae but can also enter a vernal backup fourth instar larval diapause (22, 67). PHASES OF DIAPAUSE Some features of diapause are evident before the entry into diapause (prediapause). The diapause stage itself is characterized by a conspicuous progression of physiological and molecular changes, and certain consequences may become evident only long after diapause has been terminated (postdiapause). Prediapause Most mosquitoes undergo facultative diapause, a plastic response in which diapause initiation is determined by the receipt of specific environmental signals during prediapause. The stage sensitive to the environmental programming of diapause frequently occurs well in advance of the actual onset of diapause and may even occur in the maternal generation (as discussed below). Many mosquitoes undergo a photoperiodic hibernal diapause in which the shortening days of late summer provide a reliable environmental token signaling the advent of winter. How day length is measured and how this information is translated into the diapause program remain among the challenging questions in diapause physiology. Although it is tempting to assume that the clock genes involved in regulating circadian (daily) rhythms would also be involved in photoperiodism (seasonal response to day length), there may very well be differences in these two timing systems (42, 61, 98). The major clock genes involved in circadian rhythms have been characterized in Ae. aegypti (41, 85), An. gambiae (26, 96), and Culex quinquefasciatus (41). Select clock genes, period and timeless, have been characterized in Ae. albopictus (110) and timeless has been characterized in Wy. smithii (71). However, the role of these genes in photoperiodism is Aestivation: a form of dormancy commonly used to bridge tropical dry seasons; can be a diapause or may be a form of quiescence Photoperiodism: a response such as diapause that is evoked in response to predictable seasonal changes in day length Mosquito Diapause 77

6 Critical photoperiod (CPP): the day length that marks the transition between a diapause and a nondiapause response in a population Quantitative trait locus (QTL): a genomic region that contains one or more genes affecting variation of a quantitative trait Diapause incidence: proportion of a population entering diapause under specific conditions Maternal effect: the common case in which the mother receives the environmental signals that result in the programming of diapause in her progeny just beginning to be explored. In Wy. smithii, timeless expression is correlated with geographical differences in critical photoperiod (CPP) (71), and although the timeless gene does not map directly to a quantitative trait locus (QTL) affecting CPP, it does interact epistatically with a CPP QTL (72). These results suggest that timeless in Wy. smithii may affect day length measurement independently of its role in regulating the circadian clock (72). Both cryptochrome 1 and cryptochrome 2 are present in mosquitoes. cryptochrome 1 is a candidate photoreceptor pigment and is likely the conduit for reception of the photoperiodic information. A functional clock is likely essential for mosquito diapause, as suggested by evidence from other insects (73). Although low temperature by itself is usually not sufficient to induce diapause, in most cases it enhances the photoperiodic response, generating a higher diapause incidence at any given diapause-inducing photoperiod. Examples of this effect can be found in Ae. albopictus (87), Ae. atropalpus (1), Ae. sollicitans (80), Ae. taeniorhynchus (79), Cx. pipiens (33, 109), and Cx. restuans (34). How this is achieved may be rather simple. In most cases, low temperature delays development and thereby increases the number of short days the insect receives during the photosensitive stage, thereby increasing the diapause incidence. Decreases in relative humidity or increases in solute content within water pools would seem to be likely cues foretelling a pending dry season, but we are unaware of any evidence implicating such cues for the programming of mosquito diapause. As noted, Ae. sierrensis undergoes aestivation in the egg stage during the dry summers common to western North America, but photoperiod serves as the token cue for diapause initiation (51). In Southern California, populations of Culiseta inornata enter an adult aestivation but the token cue initiating aestivation is not known (4). Similarly, as discussed further below, the M-form of An. gambiae appears to survive the 4- to 8-month dry season of the Sahel region in sub-saharan Africa in an adult aestivation (64), but the environmental factor(s) that prompts this aestivation has not been determined. In Aedes and Psorophora species, the photoperiod received by the mother is a common determinant of the diapause fate of her embryos (Figure 1). Maternal programming implies receipt of photoperiodic information within the brain of the mother and an ensuing cascade of events culminating in the transfer of a diapause determinant that affects the diapause status of her embryos. There is no known role for diapause hormone, the determinant of embryonic diapause in the silkworm Bombyx mori, in any of the Diptera (32). How maternal effects are regulated remains a fascinating, yet unresolved, issue in mosquito diapause. Other environmental factors impinging upon the mother can also affect the diapause characteristics of her offspring. For example, in both Ae. albopictus (87) and Ae. atropalpus (5), reduced larval nutrition in the maternal generation increases the diapause incidence of resulting eggs. Some mosquitoes undergo an obligatory diapause, implying that they enter diapause at a specific stage each generation, regardless of the environmental conditions. Obligatory diapauses are usually restricted to populations at high latitudes where the favorable season can accommodate only a single generation each year. Examples include mosquitoes from northern continental United States such as Ae. stimulans (48), and species from Canada and Alaska such as Ae. hexondontus (7), Cs. impatiens (39), and Cs. alaskensis (40). Development time during prediapause is frequently extended beyond what is observed in nondiapause-destined individuals. This delay enables diapausing individuals to accumulate more energy reserves and attain a larger size. In Cx. pipiens programmed for adult diapause, the final larval instar is extended by one day at 18 C, resulting in pupae and adults that are larger and contain more fat than their nondiapausing counterparts (119). Fat content of diapause-destined females continues to increase dramatically during the week following adult eclosion, reaching twice the level noted in nondiapausing females (101). This switch to high fat accumulation is accompanied by increased expression of genes associated with synthesis and sequestration of fat reserves 78 Denlinger Armbruster

7 (94, 103). In Ae. albopictus, diapause eggs are larger and contain more total lipids than nondiapause eggs, presumably due at least in part to an increased expression of genes involved in lipid storage and a decrease in lipid mobilization genes during prediapause (92). In Wy. smithii, ancestral Gulf Coast populations diapause in the fourth larval instar, and diapause-destined larvae have a prolonged third instar relative to nondiapause-destined larvae. The prolonged third instar is proposed to be an evolutionary precursor of the derived third instar diapause of northern populations (22). Behavioral changes are frequently associated with diapause. One conspicuous aspect of diapause in Cx. pipiens is the lack of blood-feeding activity (11, 12, 75, 76) in females programmed for diapause. Diapause-destined females selectively feed on nectar and are not attracted to vertebrate hosts. Additionally, at the onset of diapause females of Cx. pipiens seek a dark, humid, protected hibernaculum, a site buffered from the full brunt of winter (114). In Ae. mariae, which undergoes an embryonic diapause, short-day photoperiods cause females to switch their oviposition preferences from exposed to protected microhabitats (24). Diapause Diapause refers to the actual time when development is halted or greatly retarded and the insect is unresponsive to environmental signals that prompt development. Metabolic depression is a key feature of diapause. The rate of fat utilization, an indirect index of metabolism, is lower in Cx. pipiens under diapause conditions (9). Stress tolerance commonly increases during diapause. Diapausing embryos of Ae. albopictus (44, 107) and adults of Cx. pipiens (9, 93) are considerably more tolerant of desiccation and low temperatures than their nondiapausing counterparts. In Ae. albopictus, enhanced desiccation resistance is associated with increased egg surface hydrocarbons and expression of a fatty acylcoa elongase transcript during prediapause (113), but mechanisms of increased cold tolerance have not been determined. In Cx. pipiens, elevated desiccation tolerance during diapause is due at least partially to an increased layer of hydrocarbons on the cuticular surface of diapausing adults (9), and higher trehalose levels present during diapause may enhance both cold tolerance and desiccation tolerance (9). Diapausing larvae of Wy. smithii are also more cold tolerant than their nondiapausing counterparts (37), but the physiological basis for this difference remains unknown. Diapause is not a complete biological stasis, but rather a period in which the insect progresses toward being able to respond to diapause-terminating signals. In Cx. pipiens this progression can be noted in the gradual increase in length of the ovarian follicles (90). The dynamics of diapause can also be seen by monitoring changes in responsiveness to exogenous hormones, in responses to environmental stresses, and in energy resources (30, 121). At the molecular level, in Cx. pipiens, genes involved in fatty acid synthesis and sequestration are highly upregulated at the onset of diapause, whereas those involved in beta-oxidation are downregulated (103). Later in diapause, this profile changes: Genes involved in fatty acid synthesis are downregulated, whereas those involved in beta-oxidation are upregulated, thus reflecting a fundamental switch in how lipids are stored and utilized during diapause. Postdiapause Although photoperiod is widely used as a cue for entry into diapause, it is less commonly used to signal diapause termination. There are, however, notable exceptions. In Wy. smithii, photoperiod does terminate diapause, and the CPP is the same for both initiation and termination of diapause (21). In Toxorhynchites rutilus, which diapauses as a larva, autumnal and vernal chilling increases Mosquito Diapause 79

8 sensitivity to photoperiod, so that it is the interaction between these two factors that terminates diapause (17). Chilling also appears to be a common factor promoting termination of egg diapause in aedine species (48, 49, 115). The termination of diapause is followed by a period of postdiapause quiescence, a stage morphologically indistinguishable from diapause. The insect is still in an arrested state, its metabolic rate remains low, and many of the same diapause-associated genes continue to be expressed. The distinction is that at this time the insect is fully capable of responding to environmental signals that prompt development. Mosquitoes may terminate diapause at different times during the winter, but suppression of development by low winter temperatures results in the stockpiling of individuals that are all primed to start developing, and then, when temperatures rise above a certain threshold, the whole population can initiate development simultaneously, leading to synchronous springtime emergence. By analogy, a tropical dry season can serve the same role of stockpiling aestivating individuals as they await the onset of the rains. Energetic reserves are significantly diminished during diapause (43). This has the potential to incur substantial reproductive costs once diapause is terminated. The most thorough examination of this issue comes from a study on postdiapause inclusive fitness (replacement rate, R 0 ) in Wy. smithii (14). This study documented a 60% reduction in R 0 of postdiapause cohorts relative to nondiapause cohorts. The decrease in postdiapause fitness was due to a reduction of multiple fitness components, including mass-specific fecundity, fertility, and adult longevity. These results emphasize that diapause is much more than an arrest in development and in fact is more akin to an alternative developmental pathway. Diapause encompasses a whole suite of responses and metabolic adjustments that subserve the diapause state, some of which are noted before the actual onset of diapause, whereas others are postdiapause repercussions. TROPICAL ENVIRONMENTS The common perception that diapause is an event restricted to temperate zone species is not well founded. Many tropical insects enter diapause or some other form of dormancy (28). The question of tropical diapause is especially noteworthy for mosquitoes owing to the preponderance of tropical species that transmit human diseases. From the broader insect literature we can predict that mosquitoes living within 5 of the equator will not be using photoperiodic cues for diapause induction. The seasonal changes in day length at such latitudes are simply too modest to be detected. But, within 10 of the equator, where seasonal changes in day length are greater, photoperiodism has been demonstrated in several insect species (28). Mosquito vectors thrive in such areas and hence may be capable of using photoperiodic signals. The absence of photoperiodism near the equator, however, does not imply the absence of diapause, but simply that, if a diapause occurs, it is likely regulated by some other environmental cue. For example, flesh flies, such as Sarcophaga inzi and Poecilometopa spilogaster living in East Africa at latitude 1 south, experience a 7 min difference in day length throughout the year; they do not respond to day length, yet they have a robust pupal diapause cued by low daytime temperatures that occur in July-August (27). Whether such alternative cues are used by mosquitoes is unknown. The recent discovery of aestivation in the malaria vector An. gambiae (M-form) in the Sahel (latitude 15 north) nicely highlights both the significance and unresolved questions concerning aestivation in mosquitoes. Understanding how An. gambiae survives the 4- to 8-month dry season in arid regions of sub-saharan Africa remains one of the outstanding questions in medical entomology. Two hypotheses have been proposed. The first is that mosquitoes undergo long-distance migration to wet refugia during the dry season, and the second is that they undergo aestivation. 80 Denlinger Armbruster

9 This question has important public health implications, because if mosquito vectors survive the dry season in aestivation, populations are likely at seasonal lows and/or may be concentrated in specific microhabitats and thus be particularly susceptible to control efforts. Data showing a tenfold increase in adult An. gambiae (M-form) abundance within five days of the first rain following a seven-month dry season, and the recovery of an adult female that had been marked and released seven months earlier in the same village, are strong evidence that aestivation is the mechanism of dry season survival (64), at least for some populations. Understanding the molecular and physiological mechanisms of aestivation in this and other tropical vector species remains a major challenge. A fivefold decrease in flight activity of mosquitoes captured in village huts during the dry season relative to the wet season is consistent with the reduction of activity expected during diapause (50). However, the resting metabolic rate was not lower in the dry-season mosquitoes but rather exhibited a complex pattern of change during the course of the dry season (50). Reproductive physiology was generally depressed during the dry season, with reduced oviposition rates, reduced egg batch sizes, and an increase in the proportion of females exhibiting gonotrophic dissociation. However, there was no suppression of blood feeding during the dry season nor did all females exhibit gonotrophic dissociation, and mating swarms were still observed (118). These results point to potentially important differences between common physiological themes of diapause and physiological processes during aestivation. The high temperatures encountered during aestivation may impose unique physiological constraints or selective pressures relative to diapause (118). We are unaware of any additional studies examining physiological mechanisms of aestivation in mosquitoes, and relatively few studies have examined this issue in other insects (8, 28). POPULATION VARIATION OF DIAPAUSE RESPONSE Geographic Variation Diapause timing varies among geographic populations of mosquitoes, consistent with a wide range of studies showing that an increase in CPP with increasing latitude (and altitude) is a robust biogeographic trend (111). Furthermore, the selective mechanism giving rise to this pattern is well understood. In the Northern Hemisphere, the length of the growing season declines as one moves north (or higher in altitude), and the optimal timing of entry into diapause occurs earlier in the year owing to the earlier arrival of winter at northern (or high altitude) relative to southern (or low altitude) locations. An especially compelling example is that of Wy. smithii populations across the eastern United States, where a 1-h increase in CPP corresponds roughly to a 5.5 increase in latitude or a 775-m increase in altitude, and the combined effects of latitude and altitude explain 96% (= R 2 ) of the geographic variation in CPP among populations (13). Several additional mosquitoes, including Ae. sierrensis (52) and Ae. triseriatus (100), show a similar increase in CPP with latitude, although the R 2 is considerably lower in both cases than in Wy. smithii. In populations of Ae. albopictus from Japan and the United States, CPP also increases with latitude, although the rate of increase is less than in the above examples: approximately 0.5-h increase in CPP per 5 C increase in latitude (112). CPP can also be influenced by temperature and nutritional conditions (87). A recent study of the northern tamarisk beetle, Diorhabda carinulata, introduced into North America as a biological control agent against tamarisk (Tamarix), indicates that fluctuating rather than constant temperatures generate a more accurate estimate of the CPP (6). Thus, common garden experiments that measure CPP of multiple geographic populations under constant temperatures or other artificial laboratory conditions can provide valuable information on genetically based relative differences Gonotrophic dissociation: when oocyte maturation does not occur in response to a blood meal; considered a key indication of diapause in mosquito species that enter diapause as adults Mosquito Diapause 81

10 among populations, but such results must be interpreted cautiously when one is trying to predict the exact seasonal timing of diapause initiation in the field. Annu. Rev. Entomol : Downloaded from Response of Invasive Species Rapid evolution of CPP in Ae. albopictus during invasion and range expansion in North America indicates the crucial ecological significance of diapause timing. Ae. albopictus invaded the eastern United States in 1985 and subsequently spread across approximately 15 of latitude. Populations collected across the eastern United States in 1988 showed a weak increase of CPP with latitude, a response that was significantly more shallow than the slope of CPP versus latitude in Japan (38, 86). However, populations collected from the United States and Japan only 20 years later exhibited parallel slopes of CPP versus latitude owing to a rapid evolutionary shift in United States populations (112). Diapause incidence also shifted subsequent to the 1985 United States invasion. By 2000, subtropical populations of Ae. albopictus from peninsular Florida exhibited a slight decline in diapause incidence from north to south (68), but by , the distinction was more pronounced, ranging from >90% in northern Florida to 2030% in Ft. Lauderdale and Miami (69, 112). In contrast, a suite of life-history traits implicated in climatic adaptation in other Diptera, including wing length, pupal mass (body size), and egg volume, show no evidence of consistent latitudinal variation in either the United States or Japan (112). Consequences of Climate Change The rapid evolution of CPP across the spatial climatic gradient of eastern North America in Ae. albopictus suggests that evolution of CPP is also likely to occur in response to temporal climate change. This expectation is confirmed by an elegant study (19) that measured latitudinal variation in CPP of Wy. smithii over 30 years and demonstrated genetically based shifts toward shorter, more southern CPPs in northern populations. Some shifts were apparent within just 5 years. The shift toward shorter, more southern CPPs is consistent with an adaptive response to longer growing seasons caused by global warming. The magnitude of the shift in CPP corresponds to entering diapause nine days earlier in the fall, a remarkably close match to seasonal shifts in egg-laying of birds and spawning of frogs in Britain (19). An important implication of these results is that evolutionary responses to global climate change are likely to impose selection on traits that mediate seasonal timing of life-history events such as diapause initiation and termination (i.e., CPP) rather than overt thermal stress tolerance (20). Indeed, when northern populations of Wy. smithii were reared under a carefully simulated southern climate, the populations suffered a dramatic decrease in fitness (23); 88% of this fitness decline was attributed to inappropriate seasonal timing and none was attributed to the direct effects of warmer summer temperatures. Rapid evolution of CPP in both Ae. albopictus and Wy. smithii emphasizes the important role of diapause in mediating how mosquitoes will respond to future climate change. These results imply that mosquitoes will likely be able to quickly adjust their phenology to expand their geographic range and take advantage of extended growing seasons associated with global warming. Models that seek to predict the future of vector-borne disease dynamics should incorporate these phenological effects. HORMONAL REGULATION Endocrine experiments on mosquito diapause have focused mainly on adults; thus, the sections below on embryonic and larval diapauses rely primarily on studies discussing how these diapause stages are mediated in nonmosquito systems (32). 82 Denlinger Armbruster

11 Embryonic Diapause The challenge of working with endocrine events in an embryo has resulted in a limited understanding of the hormonal basis for diapause in this stage. As indicated above, many embryonic diapauses are regulated by maternal factors, and diapause hormone in Bombyx mori is the beststudied maternal regulator of an embryonic diapause. However, diapause hormone has no known role in Diptera, and diapause in B. mori occurs quite early in embryonic development (late gastrula stage), in contrast to the pharate larval stage of developmental arrest in all mosquito egg diapauses. Thus, other pharate larval diapauses may offer a better model for the hormonal control of embryonic diapause in mosquitoes. For example, in the gypsy moth, Lymantria dispar, the pharate first instar larval diapause is linked to the presence of ecdysteroids (63). As long as the ecdysteroid titer remains high, diapause persists; a drop in the titer is essential for breaking diapause. Transcriptome sequencing of mature oocytes and developing embryos also suggests a role for ecdysteroid signaling in the regulation of the pharate larval diapause in Ae. albopictus and potentially other mosquitoes (82, 83). Larval Diapause The normal progression of larval development, culminating in larval molts and eventually pupation, is achieved by periodic release of the steroid hormone ecdysone. In turn, the release of ecdysone from the prothoracic gland is regulated by synthesis and release of the neuropeptide prothoracicotropic hormone from the brain. In larval diapause, the brain/prothoracic gland axis is shut down. The consequence is a failure of the prothoracic gland to release the ecdysone required to prompt a molt to the next instar; hence, the larva remains locked in its current developmental stage. Although the hormonal basis for larval diapause has not been explicitly examined in mosquitoes, lack of ecdysone is a feature consistent with reports on larval diapause in diverse insect taxa (32). Adult Diapause Most work on adult mosquito diapause is restricted to a single species, Cx. pipiens. Shortly after eclosion in nondiapausing adult females, the corpora allata synthesize and release juvenile hormone ( JH). This early pulse of JH ( JH-III is the dominant form present in mosquitoes) triggers previtellogenic growth of the ovarian follicles. Within 3 days the previtellogenic growth phase is completed, and the nondiapausing female is primed to seek a blood meal, after which the egg dramatically increases in size as it reaches maturity by incorporating the newly synthesized vitellogenins produced by the fat body. In contrast, females programmed for diapause do not release JH immediately upon eclosion (90), follicles remain small, and the female does not seek a blood meal. Application of exogenous JH stimulates follicle growth in diapausing females (101, 108); thus, all evidence indicates that a lack of JH is one of the essential regulators of diapause in Cx. pipiens, as in adult diapauses of other insects (32). Insulin signaling is also implicated in the regulatory scheme for diapause in Cx. pipiens. Knocking down the insulin receptor in nondiapausing females with RNA interference (RNAi) simulates diapause, an effect that can be reversed with the application of JH (101). The absence of insulinlike peptide 1 appears to be particularly critical for the manifestation of diapause (102). Forkhead transcription factor (FOXO), which is downstream of insulin, is thought to be suppressed in the presence of insulin and activated in its absence. FOXO is a potentially important regulator of diverse characteristics (e.g., increased longevity, stress tolerance, fat accumulation) that make up the diapause phenotype (101, 102, 104). Several lines of evidence suggest interplay between the Prothoracicotropic hormone: the neuropeptide produced in the brain to stimulate synthesis of ecdysone by the prothoracic gland Juvenile hormone ( JH): a family of sesquiterpenoids produced by the corpora allata of insects that act together with ecdysteroids to regulate insect molting, metamorphosis, and reproduction Insulin signaling: a signal transduction pathway affecting growth, reproduction, and metabolism that is activated by the presence of insulin or an insulin-like peptide RNA interference (RNAi): a method of gene silencing using double-stranded RNA to suppress targeted genes Forkhead transcription factor (FOXO): a downstream target of insulin signaling Mosquito Diapause 83

12 a Nondiapause Insulin-like peptide 1 b Diapause Insulin-like peptide 1 InR JH synthesis InR JH synthesis FOXO βftz-f1 FOXO βftz-f1 Annu. Rev. Entomol : Downloaded from Figure 2 No fat accumulation Oocyte development Fat hypertrophy No oocyte development The role of the insulin signaling pathway in regulating adult diapause in the mosquito Culex pipiens. (a) The nondiapause phenotype is prompted by long day length, which promotes synthesis of insulin-like peptide 1, leading to stimulation of juvenile hormone ( JH) synthesis and concurrent suppression of forkhead transcription factor (FOXO). (b) The diapause phenotype is promoted by short day length and results in a shutdown of the insulin signaling pathway, failure to stimulate JH synthesis, and concurrent release of the suppression of FOXO. Based on References 101, 102, and 105. Abbreviations: InR, insulin receptor; βftz-f1, beta nuclear receptor fushi tarazu factor 1. insulin signaling pathway and JH (104, 105), observations consistent with the model proposed in Figure 2. GENETIC BASIS OF DIAPAUSE Isolated components of the genetic basis for diapause have been reported (29, 70), but a comprehensive understanding of the genetic basis for diapause in any single organism remains elusive. In part, this is because Drosophila melanogaster, the insect model for molecular biology, has a weak and highly temperature-dependent diapause response that varies substantially among individuals (36, 99). Thus, as predicted by Saunders (97), D. melanogaster has not been an effective organism in which to address this issue (c.f. 116). Mosquitoes have the potential to emerge as an exciting model system for determining the genetic underpinnings of diapause. Line-Crossing Experiments and QTL Mapping Early crossing experiments are qualitatively consistent with the hypothesis of polygenic control of adult diapause in Cx. pipiens (117). More recent results of line-crossing experiments that simultaneously measure the CPP of parental and F 1,F 2, and backcross populations consistently indicate a complex genetic architecture underlying differences in CPP between populations and variation of CPP within populations of Wy. smithii (16, 45, 62). Several studies have used QTL mapping to identify specific regions of the genome involved in diapause regulation. Mori et al. (77) crossed a diapausing population of Cx. pipiens and a nondiapausing strain of Cx. quinquefasciatus to map QTLs for follicle size (an indicator of adult diapause) and several additional life-history traits (wing length, wing shape, larval development time). Composite interval mapping (CIM) followed by a less conservative analysis based on all possible subsets supported four QTLs affecting diapause, located on all three chromosomes. However, it is important to recognize that QTLs represent chromosomal regions that often harbor multiple causative genetic loci. Because this study utilized a relatively small number of molecular 84 Denlinger Armbruster

13 markers (9 and 13 per cross), the QTL intervals ranged from 6 to 30 cm, and the estimate of 4 QTLs is likely an underestimate of the number of genes controlling diapause. Several QTLs affecting other life-history traits also colocalized to diapause QTLs, suggesting linkage between genes controlling diapause and other life-history traits or pleiotropic effects. A QTL analysis of CPP in Wy. smithii that crossed populations from Florida and Alberta, Canada, identified 9 QTLs, explaining 62% of the variation in CPP (72). Only 2 QTLs were of moderate effect; the remaining 7 QTLs explained less than 10% of the variation. There was also evidence for pervasive epistasis among the QTLs. One QTL also affected the stage of diapause (third versus fourth instar larva); the orthologue of this QTL in An. gambiae is tightly linked with orthologues of two D. melanogaster genes involved in ecdysteroid signaling, consistent with the previous argument that ecdysteroid signaling is likely to be important in regulating larval diapause in mosquitoes. A subsequent study highlighted further challenges (15). Additional QTL analyses of CPP in Wy. smithii were performed on reciprocal crosses between Alabama (AL) and Maine (ME) populations and between New Jersey (NJ) and ME populations. When RADtag markers were used, the average QTL intervals were cm. CIM identified only two significant QTLs in the NJ male ME female cross, three different QTLs in the AL male ME female cross, and another two different QTLs in the ME male AL female cross. A less conservative analysis based on individual markerregression identified a large number of additional candidate QTLs, but similar to the CIM results, the clustering of candidate QTLs was not concordant among the crosses. The lack of concordance between the reciprocal FL ME crosses, the NJ ME crosses, and previous results (72) was interpreted as a consequence of variation in the genetic basis of CPP both within and among populations. Lack of a complete genome sequence for Wy. smithii has slowed progress in moving from QTL to specific candidate diapause genes; however, as more mosquito genomes and transcriptomes become available, identifying the molecular basis for diapause by combining QTL mapping with additional molecular approaches should be feasible. Transcriptional Basis of Mosquito Diapause The most extensive information on transcriptional changes associated with mosquito diapause is based on recent high-throughput RNAseq studies that examined the transcriptome at different stages in the developmental trajectory of Ae. albopictus from prediapause to postdiapause (82, 83, 84) (BLAST query at A 454 sequencing platform used to examine mature oocytes from females programmed to lay diapausing eggs (82) revealed 10 upregulated transcripts, including genes involved in DNA replication and transcription (inhibitor of growth protein, ing1, and bhlhzip transcription factor bigmax), 2 genes related to ecdysone signaling (rack1 and ecdysone inducible protein, eip2), a component of the gluconeogenesis pathway (phosphoenolpyruvate carboxykinase, pepck), a transcript from the methuselah family, and a transcript previously associated with diapause in this species (113) as well as plant photoperiodism ( pepb). A subsequent study of Ae. albopictus used Illumina paired-end sequencing to examine early and late stages of embryonic development (83). The number of differentially expressed genes increased dramatically from 7178 h to h postoviposition. Functional enrichment analyses indicated that early prediapause embryos differentially expressed genes related to cell cycle, DNA repair, cytoskeleton, and DNA binding, and that the later stage of embryonic development was enriched for gene expression involved in energy production, metabolism, cuticle formation and metabolism, membrane and extracellular matrix formation, and transport. The increased expression of lipid storage transcripts and the decrease in lipid mobilization and components of the citric acid cycle phosphoenolpyruvate carboxykinase ( pepck): a gene encoding an enzyme associated with the transition from aerobic to anaerobic metabolism, an event common to diapause in diverse species Mosquito Diapause 85

14 likely reflect the general shutdown of metabolism associated with diapause. Many of these diapauseassociated changes appear to be due to pathway-specific developmental delays in diapause-destined embryos. A search for diapause-specific gene expression in Wy. smithii, using microarrays, examined changes in response to short or long day lengths (the diapause-terminating trigger in this species) and found 25 upregulated and 5 downregulated transcripts at diapause termination (35). Included among the upregulated transcripts at diapause termination were paxillan, which encodes a cytoskeletal protein involved in cell adhesion; seven cuticular proteins; two ribosomal transcripts; pepck (paradoxically, this gene regulating gluconeogenesis is upregulated in several other species during diapause); and a transcript known as WsPpdrg1, which intriguingly maps to a major QTL for CPP (72). In addition, the allele frequency of WsPpdrg1 is associated with CPP in experiments examining hybrid recombination in crosses between populations from Florida and Alberta (35), so three separate lines of evidence point to a role for this transcript in diapause regulation of Wy. smithii. This gene is a member of a gene family of cuticular proteins of low complexity that are within a larger gene family in D. melanogaster that includes retinin, a gene encoding a signal protein expressed only in the cornea. WsPpdrg1 may be a cuticular protein with a pleiotropic function in photoperiodism, or possibly it has a novel photoperiodic function. Although no transcriptome sequencing or microarray studies have addressed diapause in Cx. pipiens, expression profiling and RNAi have been directed against targeted genes. Knocking down insulin receptor (101) or insulin-like peptide 1 (102) in nondiapausing females recapitulates the arrested ovarian development characteristic of diapause. Conversely, knocking down the transcription factor FOXO in diapause-programmed females prevents accumulation of fat (101), reduces longevity (101), and reduces tolerance of oxidative stress (104), all of which are features of the diapause syndrome. These results underscore the role of the insulin signaling/foxo pathway (Figure 2) as a key regulator of diapause. Future work designed to track genes downstream of FOXO can be expected to reveal other elements of the pathway that generate the diapause phenotype. Other genes likely to be downstream elements of the diapause syndrome in Cx. pipiens include genes involved in regulating the huge accumulation of fat reserves associated with diapause. The switch to feeding on sugar rather than blood in diapause-programmed females is reflected in the upregulation of fatty acid synthase, a gene encoding an enzyme that converts sugars to fats, and the concurrent downregulation of genes encoding proteases needed to digest a blood meal, e.g. trypsin and chymotrypsin-like protease (94). Profiling of an additional 31 genes associated with lipid metabolism indicates an early diapause focus on lipid accumulation and downregulation of lipid utilization, whereas in late diapause, as the female prepares to initiate reproductive activity, the reverse is true (103). Several genes associated with structural modifications also have distinct diapause profiles in Cx. pipiens. Two actin genes, upregulated in early diapause, likely contribute to redistribution of polymerized actin during diapause (59). Conversely, beta-tubulin is downregulated in early diapause, a response correlated with the decrease in microtubules in flight muscles and reduced flight activity of diapausing females (57). Transcripts associated with ovarian development, such as ribosomal protein S3a (60) and ribosomal protein S2 (58), are reduced in diapausing females, as is the gene encoding prothoracicotropic hormone (119). Paradoxically, couch potato, a gene linked to diapause in D. melanogaster, is expressed more highly in the early diapause of Cx. pipiens (120). Taken together, a number of commonalities underlie the transcriptional studies of diapause in Ae. albopictus, Wy. smithii, and Cx. pipiens, despite differences in the stages of diapause in these three species. Differential expression of genes encoding cell structural components, especially cytoskeletal elements, is common to diapause in all these species. Genes encoding cuticular proteins 86 Denlinger Armbruster

15 have been implicated in diapause in both Ae. albopictus and Wy. smithii, and differences in cuticular protein abundance have been noted in Cx. pipiens (65), differences that may contribute to such features as the enhanced desiccation resistance associated with diapause. Transcriptional regulation of lipid metabolism in general, and beta-oxidation in particular, is common to the embryonic diapause of Ae. albopictus and adult diapause of Cx. pipiens but has not been investigated in larval diapause of Wy. smithii. Another intriguing commonality is the differential expression of pepck, as noted in both Ae. albopictus (82) and Wy. smithii (35), as well as in pupal diapauses of the flesh fly Sarcophaga crassipalpis (88) and apple maggot, Rhagoletis pomonella (89). pepck mediates a key step in gluconeogenesis and thus is associated with the transition from aerobic to anaerobic metabolism. One feature common to diapause in many other insects is the upregulation of genes encoding heat shock proteins (93), but this has not been observed in the culicids. As the database for diapause expands, it will be interesting to determine whether some of the same genes emerge as potential players among additional species. The fact that, thus far, relatively few genes in common are differentially expressed during diapause suggests that the diapause phenotype can be attained by using diverse molecular pathways. SUMMARY POINTS 1. Diapause is more than a simple arrest in development; it represents a complex alternative developmental pathway with distinct molecular, physiological, developmental, and behavioral features that precede the onset of diapause, are manifested in dynamic changes throughout diapause, and exert an influence on postdiapause development and reproduction. Numerous life-history traits, including those related to disease transmission, are affected by the diapause pathway. 2. Embryonic, larval, and adult diapauses are common within the Culicidae, and the documentation of all three stages of diapause within a single genus suggests that diapause has evolved independently numerous times in mosquitoes. Pupal diapauses are conspicuously absent in this taxon. 3. The diapause response in mosquitoes, especially diapause timing (CPP), has evolved rapidly during invasion and range expansion across broad climatic gradients and in response to global climate change, indicating that diapause is a crucial ecological adaptation to spatiotemporal climatic variation. Understanding this response is essential for predicting the spread of mosquito-borne diseases. 4. The molecular basis of mosquito diapause is increasingly well characterized, and rapid advances in DNA sequencing technology will likely lead to exciting progress in the near future. Emerging discoveries in this area are expected to generate desperately needed novel approaches to vector control based on chemical or genetic disruption of diapause. FUTURE ISSUES 1. How diapause-mediated phenology affects species interactions deserves more attention. The current literature examining competitive interactions between mosquito species is based mostly on experiments with uniformly aged cohorts that do not reflect the asymmetries caused by diapause-mediated phenological differences. Mosquito Diapause 87

16 2. A more detailed understanding of diapause termination would increase the accuracy of models predicting seasonal abundance of mosquitoes. Determining the environmental cues that both initiate and terminate aestivation, especially for disease vectors in the tropics, is a high priority. 3. Recent progress has enhanced our understanding of the molecular physiology of mosquito diapause in temperate zones, but we know little about either the molecular physiology of aestivation or the quiescent stage of nondiapausing embryos. Probing similarities and differences between these different forms of dormancy is expected to reveal exciting insights into the evolution of these diverse types of developmental arrest. 4. Understanding the molecular mechanisms by which photoperiod is interpreted and subsequently integrated with downstream regulatory elements to initiate the diapause phenotype is a high priority. 5. Most embryonic diapauses in mosquitoes appear to be maternally regulated, but what information is stored and how it is transferred from mother to offspring remain unknown. 6. Viruses harbored by overwintering mosquitoes in diapause contribute to the resurgence of vertebrate diseases the following spring, yet little is known about the impact of the mosquito s diapause on regulation of viral replication and survival. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS While preparing this review we were supported by funds from NIH R01-AI and NSF IOS to DLD and NIH 5R21AI to PAA and DLD. LITERATURE CITED 1. Anderson JF Influence of photoperiod and temperature on the induction of diapause in Aedes atropalpus (Diptera, Culicidae). Entomol. Exp. Appl. 11: Andrewartha HG Diapause in relation to the ecology of insects. Biol. Rev. 27: Baqar S, Hayes CG, Murphy JR, Watts DM Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes. Am. J. Trop. Med. Hyg. 48: Barnard DR, Mulla MS The ecology of Culiseta inornata in the Colorado desert of California: seasonal abundance, gonotrophic status, and oviparity of adult mosquitoes. Ann. Entomol. Soc. Am. 71: Beach RF The required day number and timely induction of diapause in geographic strains of the mosquito, Aedes atropalpus. J. Insect Physiol. 24: Bean DW, Dalin P, Dudley TL Evolution of critical day length for diapause induction enables range expansion of Diorhabda carinulata, a biological control agent against tamarisk (Tamarix spp.). Evol. Appl. 5: Beckel WE Investigations of permeability, diapause, and hatching in eggs of the mosquito Aedes hexodontus Dyar. Can. J. Zool. 36: Denlinger Armbruster

17 8. Benoit JB Water management by dormant insects: comparisons between dehydration resistance during summer aestivation and winter diapause. In Aestivation: Molecular and Physiological Aspects, ed. CA Navas, JE Caralho, pp Berlin: Springer 9. Benoit JB, Denlinger DL Suppression of water loss during adult diapause in the northern house mosquito, Culex pipiens. J. Exp. Biol. 210: Black WC, Moore CG Population biology as a tool to study vector-borne disease. In Biology of Disease Vectors, ed. WC Marquardt, pp Burlington, MA: Elsevier Academic 11. Bowen MF Patterns of sugar feeding in diapausing and nondiapausing Culex pipiens (Diptera: Culicidae) females. J. Med. Entomol. 29: Bowen MF, Davis EE, Haggart DA A behavioral and sensory analysis of host-seeking behavior in the diapausing mosquito Culex pipiens (Diptera: Culicidae) females. J. Insect Physiol. 34: Bradshaw WE Geography of photoperiodic response in a diapausing mosquito. Nature 262: Bradshaw WE, Armbruster P, Holzapfel CM Fitness consequences of hibernal diapause in the pitcher-plant mosquito, Wyeomyia smithii. Ecology 79: Bradshaw WE, Emerson KJ, Catchen JM, Cresko WA, Holzapfel CM Footprints in time: comparative quantitative trait loci mapping of the pitcher-plant mosquito, Wyeomyia smithii. Proc. R. Soc. B 279: Bradshaw WE, Haggerty BP, Holzapfel CM Epistasis underlying a fitness trait within a natural population of the pitcher-plant mosquito, Wyeomyia smithii. Genetics 169: Bradshaw WE, Holzapfel CM Interaction between photoperiod, temperature and chilling in dormant larvae of the tree-hole mosquito, Toxorhynchites rutilus Coq. Biol. Bull. 152: Bradshaw WE, Holzapfel CM Resource limitation, habitat segregation, and species interactions of British tree-hole mosquitoes in nature. Oecologia 90: Bradshaw WE, Holzapfel CM Genetic shift in photoperiodic response correlated with global warming. Proc. Natl. Acad. Sci. USA 98: Bradshaw WE, Holzapfel CM Light, time, and the physiology of biotic response to rapid climate change in animals. Annu. Rev. Physiol. 72: Bradshaw WE, Lounibos LP Photoperiodic control of development in the pitcher-plant mosquito, Wyeomyia smithii. Can. J. Zool. 50: Bradshaw WE, Lounibos LP Evolution of dormancy and its photoperiodic control in pitcher-plant mosquitoes. Evolution 31: Bradshaw WE, Zani PA, Holzapfel CM Adaptation to temperate climates. Evolution 58: Coluzzi M, Di Deco M, Gironi A The influence of photoperiod on the selection of oviposition sites in Aedes mariae (Diptera: Culicidae). Parassitologia 17: Danks HV Insect Dormancy: An Ecological Perspective. Ottawa: Biol. Surv. Can. (Terrestrial Arthropods) 26. Das S, Dimopoulos G Molecular analysis of photic inhibition of blood-feeding in Anopheles gambiae. BMC Physiol. 8: Denlinger DL Diapause potential in tropical flesh flies. Nature 252: Denlinger DL Dormancy in tropical insects. Annu. Rev. Entomol. 31: Denlinger DL Regulation of diapause. Annu. Rev. Entomol. 47: Denlinger DL, Giebultowicz J, Adedokun T Insect diapause: dynamics of hormone sensitivity and vulnerability to environmental stress. In Endocrinological Frontiers in Physiological Insect Ecology, ed. F Sehnal, A Zabza, DL Denlinger, pp Wroclaw, Poland: Wroclaw Tech. Univ. Press 31. Denlinger DL, Lee RE Low Temperature Biology of Insects. Cambridge, UK: Cambridge Univ. Press 32. Denlinger DL, Yocum GD, Rinehart JP Hormonal control of diapause. In Insect Endocrinology, ed. LI Gilbert, pp San Diego, CA: Academic 33. Eldridge BF The effect of temperature and photoperiod on blood-feeding and ovarian development in mosquitoes of the Culex pipiens complex. Am. J. Trop. Med. Hyg. 17: Eldridge BF, Johnson MD, Bailey CL Comparative studies of two North American mosquito species, Culex restuans and Culex salinarius: response to temperature and photoperiod in the laboratory. Mosq. News 36: Demonstrates genetically based change in CPP in response to climate warming. Mosquito Diapause 89

18 35. Emerson KJ, Bradshaw WE, Holzapfel CM Microarrays reveal early transcriptional events during the termination of larval diapause in natural populations of the mosquito, Wyeomyia smithii. PLoS One 5:e Emerson KJ, Uyemura AM, McDaniel KL, Schmidt PS, Bradshaw WE, Holzapfel CM Environmental control of ovarian dormancy in natural populations of Drosophila melanogaster. J. Comp. Physiol. A 195: Evans KW, Brust RA Induction and termination of diapause in W. smithii (Diptera: Culicidae), and larval survival studies at low and sub-zero temperatures. Can. Entomol. 104: Focks DA, Linda SB, Craig GBJ, Hawley WA, Pumpuni CB Aedes albopictus (Diptera: Culicidae): a statistical model of the role of temperature, photoperiod, and geography in the induction of egg diapause. J. Med. Entomol. 31: Frohne WC Natural history of Culiseta impatiens (Wlk.), (Diptera, Culicidae) in Alaska. Trans. Am. Microsc. Sci. 72: Frohne WC Biology of an Alaskan mosquito, Culiseta alaskaensis (Ludl.). Ann. Entomol. Soc. Am. 47: Gentile C, Rivas GBS, Meireles ACA, Lima JBP, Peixoto AA Circadian expression of clock genes in two mosquito disease vectors: cry2 is different. J. Biol. Rhythms 24: Goto SG, Shiga S, Numata H Photoperiodism in insects: perception of light and the role of clock genes. In Photoperiodism: The Biological Calendar, ed. RJ Nelson, DL Denlinger, DE Somers, pp Oxford, UK: Oxford Univ. Press 43. Hahn DA, Denlinger DL Energetics of insect diapause. Annu. Rev. Entomol. 56: Hanson SM, Craig GBJ Aedes albopictus (Diptera: Culicidae) eggs: field survivorship during northern Indiana winters. J. Med. Entomol. 32: Hard JJ, Bradshaw WE, Holzapfel CM Epistasis and the genetic divergence of photoperiodism between populations of the pitcher-plant mosquito, Wyeomyia smithii. Genetics 131: Hawley WA, Reiter P, Copeland RS, Pumpuni CB, Craig GB Jr Aedes albopictus in North America: probable introduction in used tires from northern Asia. Science 236: Holzapfel CM, Bradshaw WE Geography of larval dormancy in the tree-hole mosquito, Aedes triseriatus (Say). Can. J. Zool. 59: Horsfall WR, Fowler HWJ Eggs of floodwater mosquitoes. VIII. Effect of serial temperatures on conditioning of eggs of Aedes stimulans Walker (Diptera: Culicidae). Ann. Entomol. Soc. Am. 54: Horsfall WR, Trpis M Eggs of floodwater mosquitoes. X. Conditioning and hatching of winterized eggs of Aedes sticticus (Diptera: Culicidae). Ann. Entomol. Soc. Am. 60: Huestis DL, Yaro AS, Traore AI, Dieter KL, Nwagbara JI, et al Seasonal variation in metabolic rate, flight activity and body size of Anopheles gambiae in the Sahel. J. Exp. Biol. 215: Jordan RG Embryonic diapause in three populations of the western tree hole mosquito, Aedes sierrensis. Ann. Entomol. Soc. Am. 73: Jordan RG, Bradshaw WE Geographic variation in the photoperiodic response of the western tree-hole mosquito, Aedes sierrensis. Ann. Entomol. Soc. Am. 71: Joy JE, Sullivan SN Occurrence of tire inhabiting mosquito larvae in different geographic regions of West Virginia. J. Am. Mosq. Control Assoc. 21: Juliano SA Species interactions among larval mosquitoes: context dependence across habitat gradients. Annu. Rev. Entomol. 54: Juliano SA, Lounibos LP Ecology of invasive mosquitoes: effects on resident species and human health. Ecol. Lett. 8: Juliano SA, O Meara GF, Morrill JR, Cutwa MM Desiccation and thermal tolerance of eggs and the coexistence of competing mosquitoes. Oecologia 130: Kim M, Denlinger DL Decrease in expression of the beta-tubulin and microtubule abundance in flight muscles during diapause in adults of Culex pipiens. Insect Mol. Biol. 18: Kim M, Denlinger DL A potential role for ribosomal protein S2 in the gene network regulating reproductive diapause in the mosquito Culex pipiens. J. Comp. Physiol. B 180: Denlinger Armbruster

19 59. Kim M, Robich RM, Rinehart JP, Denlinger DL Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens. J. Insect Physiol. 52: Kim M, Sim C, Denlinger DL RNA interference directed against ribosomal protein S3a suggests a link between this gene and arrested ovarian development during adult diapause in Culex pipiens. Insect Mol. Biol. 19: Kostal V Insect photoperiodic calendar and circadian clock: independence, cooperation, or unity? J. Insect Physiol. 57: Lair KP, Bradshaw WE, Holzapfel CM Evolutionary divergence of the genetic architecture underlying photoperiodism in the pitcher-plant mosquito, Wyeomyia smithii. Genetics 147: Lee K-Y, Denlinger DL A role for ecdysteroids in the induction and maintenance of the pharate first instar diapause of the gypsy moth, Lymantria dispar. J. Insect Physiol. 43: Lehmann T, Dao A, Yaro AS, Adamou A, Kassogue T, et al Aestivation of the African malaria mosquito, Anopheles gambiae in the Sahel. Am. J. Trop. Med. Hyg. 83: Li AQ, Denlinger DL Pupal cuticle protein is abundant during early adult diapause in the mosquito Culex pipiens. J. Med. Entomol. 46: Livdahl TP, Willey MS Prospects for an invasion: competition between Aedes albopictus and native Aedes triseriatus. Science 253: Lounibos LP, Bradshaw WE A second diapause in Wyeomyia smithii: seasonal incidence and maintenance by photoperiod. Can. J. Zool. 53: Lounibos LP, Escher RL, Lourenco-de-Oliveira R Asymmetric evolution of photoperiodic diapause in temperate and tropical invasive populations of Aedes albopictus (Diptera: Culicidae). Ann. Entomol. Soc. Am. 96: Lounibos LP, Escher RL, Nishimura N Retention and adaptiveness of photoperiodic egg diapause in Florida populations of invasive Aedes albopictus. J. Am. Mosq. Control Assoc. 27: MacRae TH Gene expression, metabolic regulation and stress tolerance during diapause. Cell. Mol. Life Sci. 67: Mathias D, Jacky L, Bradshaw WE, Holzapfel CM Geographic and developmental variation in expression of the circadian rhythm gene, timeless, in the pitcher-plant mosquito, Wyeomyia smithii. J. Insect Physiol. 51: Mathias D, Jacky L, Bradshaw WE, Holzapfel CM Quantitative trait loci associated with photoperiodic response and stage of diapause in the pitcher-plant mosquito, Wyeomyia smithii. Genetics 176: Meuti ME, Denlinger DL Evolutionary links between circadian clocks and photoperiodic diapause in insects. Integr. Comp. Biol. 53: Mitchell CJ Occurrence, biology and physiology of diapause in overwintering mosquitoes. In The Arboviruses: Epidemiology and Ecology, Vol. 1, ed. TP Monath, pp Boca Raton, FL: CRC Press 75. Mitchell CJ, Briegel H Fate of the blood meal in force-fed, diapausing Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 26: Mitchell CJ, Briegel H Inability of diapausing Culex pipiens (Diptera: Culicidae) to use blood for producing lipid reserves for overwinter survival. J. Med. Entomol. 26: Mori A, Romero-Severson J, Severson DW Genetic basis for reproductive diapause is correlated with life history traits within the Culex pipiens complex. Insect Mol. Biol. 16: Nasci RS, Savage HM, White DJ, Miller JR, Cropp BC, et al West Nile virus in overwintering Culex mosquitoes, New York City, Emerg. Infect. Dis. 7: Parker BM Effects of photoperiod on the induction of embryonic diapause in Aedes taeniorhynchus (Diptera: Culicidae). J. Med. Entomol. 22: Parker BM Photoperiod-induced diapause in a North Carolina strain of Aedes sollicitans: photosensitivity of fully formed and developing embryos. J. Am. Mosq. Control Assoc. 4: Perez MH, Noriega FG Aedes aegypti pharate 1st instar quiescence: a case for anticipatory reproductive plasticity. J. Insect Physiol. 59: Provides evidence that adults of Anopheles gambiae M-form survive the dry season in the Sahel by aestivation. 78. Describes overwintering of West Nile virus in diapausing mosquitoes. Mosquito Diapause 91

20 83. Provides a comprehensive transcriptome analysis of diapause preparation in Aedes albopictus Proposes the insulin signaling pathway as a key regulator of adult diapause in Culex pipiens. 82. Poelchau M, Reynolds J, Denlinger D, Elsik C, Armbruster P A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation. BMC Genomics 12: Poelchau MF, Reynolds JA, Elsik CG, Denlinger DL, Armbruster PA Deep sequencing reveals complex mechanisms of diapause preparation in the invasive mosquito, Aedes albopictus. Proc. R. Soc. B 280: Poelchau MF, Reynolds JA, Elsik CG, Denlinger DL, Armbruster PA RNA-Seq reveals early distinctions and late convergence of gene expression between diapause and quiescence in the Asian tiger mosquito, Aedes albopictus. J. Exp. Biol. 216: Ptitsyn AA, Reyes-Solis G, Saavedra-Rodriguez K, Betz J, Suchman EL, et al Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito. BMC Genomics 12: Pumpuni CB Factors influencing photoperiodic control of egg diapause in Aedes albopictus (Skuse). PhD Thesis. Univ. Notre Dame, Notre Dame, Indiana. 148 pp. 87. Pumpuni CB, Knepler J, Craig GB Influence of temperature and larval nutrition on the diapause inducing photoperiod of Aedes albopictus. J. Am. Mosq. Control Assoc. 14: Ragland GJ, Denlinger DL, Hahn DA Mechanisms of suspended animation are revealed by transcript profiling of diapause in the flesh fly. Proc. Natl. Acad. Sci. USA 17: Ragland GJ, Egan SP, Feder JL, Berlocher SH, Hahn DA Developmental trajectories of gene expression reveal candidates for diapause termination: a key life-history transition in the apple maggot fly Rhagoletis pomonella. J. Exp. Biol. 214: Readio J, Chen MH, Meola R Juvenile hormone biosynthesis in diapausing and nondiapausing Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 36: Reidenbach KR, Cook S, Bertone MA, Harbach RE, Wiegmann BM, Besansky NJ Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and morphology. BMC Evol. Biol. 9: Reynolds JA, Poelchau MF, Rahman Z, Armbruster PA, Denlinger DL Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus. J. Insect Physiol. 58: Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SAL, Denlinger DL Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proc. Natl. Acad. Sci. USA 104: Robich RM, Denlinger DL Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony. Proc. Natl. Acad. Sci. USA 102: Rosen L Further observations on the mechanisms of vertical transmission by Aedes mosquitoes. Am. J. Trop. Med. Hyg. 39: Rund SS, Lee SJ, Bush BR, Duffield GE Strain- and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. J. Insect Physiol. 58: Saunders DS Insect Clocks. Amsterdam: Elsevier 98. Saunders DS Controversial aspects of photoperiodism in insects and mites. J. Insect Physiol. 56: Schmidt PS, Paaby AB, Heschel MS Genetic variance for diapause expression and associated life histories in Drosophila melanogaster. Evolution 59: Shroyer DA, Craig GB Egg diapause in Aedes triseriatus (Diptera, Culicidae): geographic variation in photoperiodic response and factors influencing diapause termination. J. Med. Entomol. 20: Sim C, Denlinger DL Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens. Proc. Natl. Acad. Sci. USA 105: Sim C, Denlinger DL A shut-down in expression of an insulin-like peptide, ILP-1, halts ovarian maturation during the overwintering diapause of the mosquito Culex pipiens. Insect Mol. Biol. 18: Sim C, Denlinger DL Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens. Physiol. Genomics 39: Sim C, Denlinger DL Catalase and superoxide dismutase-2 enhance survival and protect ovaries during overwintering diapause in the mosquito Culex pipiens. J. Insect Physiol. 57: Denlinger Armbruster

21 105. Sim C, Denlinger DL Juvenile hormone III suppresses forkhead of transcription factor in the fat body and reduces fat accumulation in the diapausing mosquito, Culex pipiens. Insect Mol. Biol. 22: Sota T Larval diapause, size and autogeny in the mosquito Aedes togoi (Diptera, Culicidae) from tropical to subarctic zone. Can. J. Zool. 72: Sota T, Mogi M Survival time and resistance to desiccation of diapause and non-diapause eggs of temperate Aedes (Stegomyia) mosquitoes. Entomol. Exp. Appl. 63: Spielman A Effect of synthetic juvenile hormone on ovarian diapause of Culex pipiens mosquitoes. J. Med. Entomol. 11: Spielman A, Wong J Environmental control of ovarian diapause in Culex pipiens. Ann. Entomol. Soc. Am. 66: Summa K, Urbanski JM, Zhao X, Poelchau MF, Armbruster P Cloning and sequence analysis of the circadian clock genes period and timeless in Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 49: Tauber MJ, Tauber CA, Masaki S Seasonal Adaptations of Insects. New York, NY: Oxford Univ. Press 112. Urbanski J, Mogi M, O Donnell D, DeCotiis M, Toma T, Armbruster P Rapid adaptive evolution of photoperiodic response during invasion and range expansion across a climatic gradient. Am. Nat. 179: Urbanski JM, Benoit JB, Michaud MR, Denlinger DL, Armbruster P The molecular basis of increased desiccation resistance during diapause in the Asian tiger mosquito, Aedes albopictus. Proc. R. Soc. B 277: Vinogradova EB Culex pipiens pipiens Mosquitoes: Taxonomy, Distribution, Ecology, Physiology, Genetics, Applied Importance and Control. Sofia, Bulgaria: PenSoft 115. Vinogradova EB Diapause in aquatic insects, with emphasis on mosquitoes. In Diapause in Aquatic Invertebrates, ed. VR Alekseev, B De Stasio, JJ Gilbert, pp London: Springer 116. Williams KD, Schmidt PS, Sokolowski MB Photoperiodism in insects: molecular basis and consequences of diapause. In Photoperiodism: The Biological Calendar, ed. RJ Nelson, DL Denlinger, DE Somers, pp New York, NY: Oxford Univ. Press 117. Wilton DP, Smith GC Ovarian diapause in three geographic strains of Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 22: Yaro AS, Traore A, Huestis DL, Adamou A, Timine S, et al Dry season reproductive depression of Anopheles gambiae in the Sahel. J. Insect Physiol. 58: Zhang Q, Denlinger DL Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding. Insect Mol. Biol. 20: Zhang QR, Denlinger DL Elevated couch potato transcripts associated with adult diapause in the mosquito Culex pipiens. J. Insect Physiol. 57: Zhou GL, Miesfeld RL Energy metabolism during diapause in Culex pipiens mosquitoes. J. Insect Physiol. 55: A foundational introduction to the ecological significance of insect diapause Demonstrates rapid adaptive evolution of CPP during invasion and range expansion of Aedes albopictus in North America. Mosquito Diapause 93

22 Contents Annual Review of Entomology Volume 59, 2014 Annu. Rev. Entomol : Downloaded from Nancy E. Beckage ( ): Pioneer in Insect Host-Parasite Interactions Lynn M. Riddiford and Bruce A. Webb 1 Emerald Ash Borer Invasion of North America: History, Biology, Ecology, Impacts, and Management Daniel A. Herms and Deborah G. McCullough 13 Invasion Biology of Aedes japonicus japonicus (Diptera: Culicidae) Michael G. Kaufman and Dina M. Fonseca 31 Death Valley, Drosophila, and the Devonian Toolkit Michael H. Dickinson 51 Mosquito Diapause David L. Denlinger and Peter A. Armbruster 73 Insect Mitochondrial Genomics: Implications for Evolution and Phylogeny Stephen L. Cameron 95 Response of Native Insect Communities to Invasive Plants T. Martijn Bezemer, Jeffrey A. Harvey, and James T. Cronin 119 Freshwater Biodiversity and Aquatic Insect Diversification Klaas-Douwe B. Dijkstra, Michael T. Monaghan, and Steffen U. Pauls 143 Organization and Functional Roles of the Central Complex in the Insect Brain Keram Pfeiffer and Uwe Homberg 165 Interactions Between Insect Herbivores and Plant Mating Systems David E. Carr and Micky D. Eubanks 185 Genetic Control of Mosquitoes Luke Alphey 205 Molecular Mechanisms of Phase Change in Locusts Xianhui Wang and Le Kang 225 vii