Infection, Genetics and Evolution: Diana L. Huestis, Tovi Lehmann

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Ecophysiology of Anopheles gambiae s.l.: Persistence in the Sahel
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6 Diana L. Huestis
Q1 , Tovi Lehmann
7 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
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a r t i c l e i n f o
12 Article history:
13 Received 21 March 2014
14 Received in revised form 21 May 2014
15 Accepted 26 May 2014
16 Available online xxxx
17 Keywords Q2 :
18 Aestivation
19 Diapause
20 Dormancy
21 Dry season
22 Geographic variation
23 Malaria
24 Physiology
25 Vector biology
26 Vector ecology
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a b s t r a c t
29 The dry-season biology of malaria vectors is poorly understood, especially in arid environments when no
30 surface waters are available for several months, such as during the dry season in the Sahel. Here we reap-
31 praise results on the dry-season physiology of members of the Anopheles gambiae s.l. complex in the
32 broad context of dormancy in insects and especially in mosquitoes. We examine evidence on seasonal
33 changes in reproduction, metabolism, stress tolerance, nutrition, molecular regulation, and environmen-
34 tal conditions and determine if the current results are compatible with dry-season diapause (aestivation)
35 as the primary strategy for persistence throughout the dry season in the Sahel. In the process, we point
36 out critical gaps in our knowledge that future studies can ll. We nd compelling evidence that members
37 of the An. gambiae s.l. complex undergo a form of aestivation during the Sahelian dry season by shifting
38 energetic resources away from reproduction and towards increased longevity. Considering the differ-
39 ences between winter at temperate latitudes, which entails immobility of the insect and hence reliance
40 on physiological solutions, as opposed to the Sahelian dry season, which restricts reproduction exclu-
41 sively, we propose that behavioral changes play an important role in complementing physiological
42 changes in this strategy.
43 2014 Elsevier B.V. All rights reserved.
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47 1. Introduction
48 The burden of malaria has lessened over the past decade, yet it
49 is still very high with hundreds of millions of cases and over half a
50 million deaths annually, most of which occur in sub-Saharan Africa
51 (WHO, 2013). The principal malaria vectors in Africa are members
52 of the Anopheles gambiae s.l. complex, which includes seven or
53 eight sibling species. Three brackish-water species are conned
54 to a relatively narrow range (Anopheles bwambae, Anopheles melas,
55 and Anopheles merus) and four or ve freshwater species that
56 include Anopheles arabiensis, Anopheles gambiae s.s., and Anopheles
57 coluzzii (which was recently split from An. gambiae s.s., formerly
58 known as the S and M molecular forms, respectively; Coetzee
59 et al., 2013). Malaria is vectored by all sibling species except the
60 two freshwater species that typically feed on animals: Anopheles
61 quadriannulatus and Anopheles amharicus (formerly known as spe-
62 cies B of An. quadriannulatus; Coetzee et al., 2013). Unlike the
63 brackish-water vectors, An. gambiae s.s., An. coluzzii, and An. arabi-
64 ensis transmit malaria over vast ranges of sub-Saharan Africa,
65 including dry savannahs and semi-arid areas of the Sahel. The
66 means by which these mosquitoes (and malaria) persist in areas
67 without surface waters for three to eight months a year has been
68 one of the long-standing questions in malariology because no stage
69 of the vector is known to survive for over 12 months under such
70 conditions (Coluzzi, 1964; Davidson, 1964; Donnelly et al., 2002;
71 Gillies and De Meillon, 1968; Omer and Cloudsley-Thompson,
72 1968). The rapid build-up of mosquito density after the rst rains
73 indicates that they persist locally, possibly via aestivation (summer
74 diapause), or arrive shortly after the rains by long-distance migra-
75 tion (Adamou et al., 2011; Lehmann et al., 2010, 2014). Here we
76 review the current knowledge of the eco-physiological mecha-
77 nisms that allow persistence of mosquitoes, and thus malaria, in
78 such dry habitats and identify key gaps that future research might
79 ll.
80 The African Sahel is a belt 1000 km wide and 5400 km long
81 between the Sahara desert in the north and the Sudan Savannah in
82 the south. It has a short wet season (JuneOctober), when 90% of
83 the annual rain (500 mm) falls, and a long dry season (Novem-
84 berMay, Fig. 1). The ephemeral surface waters that abound from
85 June to October may last until December, based on local conditions.
86 From November to May, rainfall is negligible and no surface waters
87 are available over vast expanses of land. The conditions in most of
88 the Sahel are drier than those depicted in Fig. 1, which illustrates
89 conditions around the Sahels southern border (Segou, Mali). The
90 dry season is divided into cold (NovemberFebruary) and hot
91 (MarchMay) periods, but outdoor temperatures rarely fall below
http://dx.doi.org/10.1016/j.meegid.2014.05.027
1567-1348/ 2014 Elsevier B.V. All rights reserved.
Corresponding author. Address: 12735 Twinbrook Pkwy, Room 2W-13A,

Rockville, MD 20852, USA. Tel.: +1 301 496 4880.
E-mail address: diana.huestis@nih.gov (D.L. Huestis).
Infection, Genetics and Evolution xxx (2014) xxxxxx
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Please cite this article in press as: Huestis, D.L., Lehmann, T. Ecophysiology of Anopheles gambiae s.l.: Persistence in the Sahel. Infect. Genet. Evol. (2014),
92 15 C or above 40 C (Fig. 1) and the temperature range is consid-
93 erably narrower indoors, in tree holes, or in burrows underground.
94 The daily uctuation in air temperature is greater during the dry
95 season (Fig. 1). Air moisture (measured as dew point) is elevated
96 from late April to October, but is very low (RH 20%) during much
97 of the dry season (Fig. 1). For mosquito activity, the absence of
T
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Fig. 1. Climatic conditions recorded from 2008 to 2013 by a governmental weather station at Segou, Mali (13.45 N, 6.26 W), a village at the southern edge of the Sahel;
north of this location, conditions are drier. The period dened as the wet season (June through mid-October) is shaded in light green throughout. (A) Total monthly rainfall (in
mm), shown as a box-whisker plot (box stretches from the 1st to the 3rd quartiles and whiskers extend to the extreme values up to 1.5 times the inter-quartile range). Mean
monthly rainfall depicted with diamonds; mean annual rainfall is 570 mm with a range of 225964 over the 5-year period. Dotted line marks 30 mm rain, representing the
minimal single rain event providing enough water to keep some larval sites for the duration of complete development of the aquatic stages of Anopheles gambiae s.l. (B) Daily
maximum temperature (red; measured at 15:00), minimum temperature (dark blue; measured at 06:00), and dew point (light blue; measured at 06:00). Shaded bands
represent the range between 1st and 3rd quartiles. (C) Daily photoperiod, with the number of daylight hours (sunrise to sunset) shown.
2 D.L. Huestis, T. Lehmann/ Infection, Genetics and Evolution xxx (2014) xxxxxx
18 June 2014
98 surface water is by far the most restrictive element in this environ-
99 ment. The low humidity during the dry season probably connes
100 activity to short ights during more humid nights, but water is
101 available in every house (e.g., pots with water, fruits), in wells
102 and seepages nearby, and in owers nectar and woody-plant
103 juices. Without suitable larval sites, the eggs, larvae, and pupae
104 cannot survive beyond a few days (Beier et al., 1990; Minakawa
105 et al., 2001). The adults typically survive for only a few weeks,
Conditions which are similar between seasons:
1) Host for blood-feeding: Readily available Host for blood-feeding: Readily available
2) Plants for sugar-feeding: Available Plants for sugar-feeding: Semi-available
3) Water pots in and around houses: Available Water in houses and in wells: Available
Conditions which differ but may be accommodated behaviorally:
4) Temperature: Moderate/stable (25-35 C) Temperature: High/unstable (20-40 C)
5) Humidity: Typically high (70-95%) Humidity: Very low (15-25%)
6) Sunlight: 12.5-13 hours/day Sunlight: 11.5-12 hours/day
7) Wind: Mild except when raining Wind: Intense hot, dry Harmattan winds
Conditions which differ and require physiological changes:
8) Larval sites: Readily available Larval sites: Not available up to 50 km
9) Rainfall: Frequent (daily to weekly) Rainfall: None to infrequent
6 6
9
9
8 8
4 4
5 5
2
2
1 1
Sahelian Wet Season Sahelian Dry Season
20 80
60
20 80
60
3 3
7
7
Fig. 2. Conditions experienced by mosquitoes during the Sahelian wet season as compared with the Sahelian dry season. Conditions 13 are very similar between the wet and
dry seasons. Conditions 47 are different between the seasons, but presumably mosquitoes could accommodate them behaviorally by seeking cool, humid shelters, only
ying at night, etc. Conditions 8 and 9 are the critical differences between the seasons and are why aestivation is required.
Table 1
Summary of physiological comparisons relevant to the dry-season persistence of the members of Anopheles gambiae s.l. complex.
Diapause trait Appearance in An. gambiae s.l. complex Field/laboratory Agree
a
Reference
Longevity extension >7 months in the dry season in Sudan An. arabiensis Field: insectary Yes Omer and Cloudsley-Thompson
(1968)
7 months in the dry season in Mali An. coluzzii Field: mark
recapture
Yes Lehmann et al. (2010)
Reproductive arrest Gonotrophic dissociation An. arabiensis Field Yes Omer and Cloudsley-Thompson
(1970)
70% reduction in female reproductive output An. coluzzii Field Yes Yaro et al. (2012)
Continued male presence and swarming An. coluzzii Field No Yaro et al. (2012)
Suppression of activity Suppression of ight An. coluzzii Field Yes Huestis et al. (2012)
Metabolic suppression Elevated in the late dry season An. coluzzii Field No Huestis et al. (2012)
Photoperiod induction 1.6-fold extension of life in fresh laboratory colony An.
coluzzii
Laboratory Partly Huestis et al. (in prep)
1.7-fold extension of life of wild mosquitoes in eld
insectary
Field: insectary Partly Kassogue et al. (unpublished)
Stress tolerance: desiccation 1.3-fold higher in An. coluzzii vs. An. gambiae Laboratory (F1s) No Lee et al. (2009)
1.3-fold higher in <24 h-old 2La vs. 2La+ homozygotes Laboratory No Fouet et al. (2012), Gray et al.
(2009)
Stress tolerance: high
temperature
1.5-fold higher in dry-season vs. wet-season An. coluzzii Field Partly Dao et al. (unpublished)
1.8-fold increase in An. arabiensis vs. An. gambiae Laboratory Yes Kirby and Lindsay (2004)
No difference in larvae and pupae of 2La vs. 2La+
homozygotes
Laboratory No Rocca et al. (2009)
No increased tolerance to high temperature An. coluzzii Field No Dao et al. (unpublished)
Cuticular hydrocarbons 28% increase in G3 females (virgin only) under short
photoperiod
Laboratory No Wagoner et al. (2014)
5-fold increase in dry-season vs. wet-season An. coluzzii in
Mali
Field Yes Huestis et al. (in prep)
a
Indicates overall agreement with aestivation predictions in direction, biological magnitude (arbitrarily dened as >1.5-fold), and statistical signicance. Partly refers to
cases where the effects direction and statistical signicance were in agreement with aestivation, but the magnitude of the effect was below this expectation.
D.L. Huestis, T. Lehmann/ Infection, Genetics and Evolution xxx (2014) xxxxxx 3
18 June 2014
106 which would spell doom to the population, unless a different strat-
107 egy can be used by mosquitoes to cope with the 57 month-long
108 dry season. The only strategy that has been supported by direct
109 evidence is a 7-fold extension of (normal) adult life span
110 (Holstein, 1954; Lehmann et al., 2010; Omer and Cloudsley-
111 Thompson, 1968, 1970). This life-span extension has often been
112 referred to as aestivation (Box 1), but, until recently, had not been
113 subjected to rigorous physiological analysis. The main aim of this
114 review is to evaluate how new results (mostly over the last decade)
115 on the ecophysiology of Anopheles gambiae t within the frame-
116 work of aestivation as a mechanism of dry-season survival.
118
119
120 Box 1. Terminology.
121 1. Dormancy is a broad term used for the ephemeral state of
122 depressed growth, development, reproduction (in repro-
123 ductively mature organisms), metabolic rate, and activity
124 linked to unfavorable environmental conditions. Both qui-
125 escence and diapause are different forms of dormancy
126 which are well known in insects.
127 1.1 Diapause is a pre-programmed dormant state, initiated
128 by token stimuli (e.g., photoperiod) in anticipation of
129 future unfavorable environmental conditions and
130 requires a minimum period of latency before termina-
131 tion. The term diapause syndrome expresses its
132 many phenotypic and physiological manifestations.
133 1.1.1 Overwintering diapause (hibernal diapause) is the
134 diapause syndrome associated with cold tempera-
135 tures typical of the winter in temperate latitudes
136 and high altitudes. It involves a build-up of nutri-
137 tional reserves ahead of time, reduced metabolism,
138 little or no feeding, cessation of reproduction (in
139 reproductively mature adults), and increased desic-
140 cation- and cold-tolerance.
141 1.1.2 Aestivation (summer diapause) is the form of dia-
142 pause associated with low humidity and/or high
143 temperatures prevailing through the dry season in
144 tropical and sub-tropical climatic zones. It may
145 involve some or all of the following: a build-up of
146 nutritional reserves ahead of time, reduced metabo-
147 lism, little or no feeding, cessation of reproduction
148 (in reproductively mature adults), and increased
149 desiccation- and heat-tolerance.
150
151 1.2 Reproductive diapause is the seasonality-related arrest
152 of ovarian development in an early previtellogenic
153 stage, used as an indicator of diapause in adult insects
154 and typically accompanied by depressed metabolism,
155 activity, and feeding. In diapausing (overwintering)
156 mosquitoes, two forms of this condition are often cited
157 (gonotrophic dissociation and gonotrophic
158 concordance).
159 1.2.1 Gonotrophic dissociation is a form of reproductive
160 diapause in which females continue to blood-feed,
161 but eggs remain undeveloped.
162 1.2.2 Gonotrophic concordance is a form of reproductive
163 diapause in which females stop blood-feeding and
164 eggs remain undeveloped.
165 166
167
168
169
171
172 1.3 Quiescence is a dormant state that is initiated as a direct
173 response to unfavorable environmental conditions
174 (e.g., desiccation, extreme temperatures) and ends
175 when favorable conditions resume (e.g., the sleeping
176 midge, Polypedilum vanderplanki, can survive extreme
177 desiccation and lose >95% of its water, yet resume its
178 life processes upon rehydration; Keilin, 1959).
179 2. Migration is the intentional, directional movement away
180 from an area in anticipation of future unfavorable condi-
181 tions (similar to those which initiate diapause) and into a
182 favorable environment, often located a considerable dis-
183 tance away.
184 3. Seasonal polyphenism refers to seasonal phenotypic
185 changes in morphology, coloration, physiology, and/or
186 behavior without the dramatic suppression of metabo-
187 lism, activity, growth, development, or reproduction that
188 characterize dormancy (above). Although distinct from
189 acclimation, some of the physiological changes might be
190 augmented by acclimation.
191
192 Seasonality in insects (and invertebrates) refers to suites of
193 phenotypic changes that increase the prospects of survival through
194 an inhospitable period until favorable conditions resume
195 (Andrewartha, 1952; Denlinger and Armbruster, 2014; Dingle,
196 1996; Kostal, 2006; Masaki, 1980; Tauber et al., 1986). These
197 include migration away from the inhospitable environment and
198 forms of dormancy such as quiescence and diapause (Box 1).
199 The characteristic diversity in the expression of diapause
200 (Andrewartha, 1952) led to the term diapause syndrome, which
201 highlights the challenge in classifying these physiological states.
202 Whether the physiology of aestivation is similar to that of overwin-
203 tering diapause is difcult to resolve, because very few studies
204 have addressed this topic in aestivating insects and less than a
205 handful have addressed this subject in mosquitoes. Thus, in recent
206 reviews of diapause in mosquitoes, aestivation was mentioned
207 with respect to a single (or two) species as opposed to over 50 with
208 respect to hibernation (Denlinger and Armbruster, 2014;
209 Vinogradova et al., 2007). This is surprising, given the overwhelm-
210 ing evidence that seasonal adaptations are critical for mosquitoes
211 and thus for disease transmission (Denlinger and Armbruster,
212 2014; Jetten and Takken, 1994). Here we will draw broadly on
213 the ecophysiology of mosquitoes living through inhospitable sea-
214 sons, and consider strategies relevant for mosquito survival of
215 the Sahelian dry season in situ. We emphasize recent ndings on
216 the seasonal ecophysiology of anophelines, and (i) explore the
217 physiological mechanisms used to survive the long dry season,
218 (ii) assess whether these known mechanisms sufciently explain
219 mosquito persistence, (iii) evaluate if these strategies are in accor-
220 dance with different forms of dormancy, and (iv) propose, based on
221 a synthesis of i-iii above, mechanisms used by African anophelines.
222 In the process, we identify key gaps in our knowledge of the dry-
223 season ecophysiology of African anophelines, which, if lled, could
224 yield novel vector control methods in these seasonal habitats.
225 2. Environmental cues used to initiate diapause
226 A key hallmark of diapause, whether overwintering diapause or
227 aestivation, is that it is a pre-programmed suite of physiological
228 changes in response to one or more token external stimuli that pre-
229 dict the onset of future environmental changes which will require
230 these changes for survival (Box 1). However, the diapause-inducing
18 June 2014
231 cues for many insects remain unknown. Here we will describe
232 some cases in which the induction cues have been well-studied,
233 and propose some that could be utilized by An. gambiae s.l. in the
234 Sahel.
235 2.1. Cues used by insects to induce winter diapause
236 Depending on latitude, extreme cold and lack of nutritional
237 resources during winter are predictable by decreasing photoperiod
238 (Danilevskii, 1965). As such, much research has explored the role of
239 photoperiod in the induction, maintenance, and termination of
240 winter diapause in a wide variety of taxa, including several mos-
241 quito species. For example, in Culex tarsalis, decreased photoperiod
242 induces adult females to shift into a hibernal diapause initiation
243 phase by accumulating fat reserves while simultaneously decreas-
244 ing ovary size (Harwood and Halfhill, 1964). These ndings were
245 further supported by a study comparing the simultaneous effects
246 of photoperiod and temperature on Cx. tarsalis and Culex restuans,
247 which found that short-day photoperiod was more inuential than
248 temperature in determining (adult) winter diapause for both spe-
249 cies (Buth et al., 1990). However, temperature was found to be of
250 secondary importance, as under long days, the coolest temperature
251 produced diapausing females as compared with the 2 warmer
252 treatments (Buth et al., 1990). Similarly, short-day photoperiod
253 stimulated the production of winter-diapausing eggs of Aedes alb-
254 opictus both in its native habitat in Japan and across its newly
255 acquired range in the United States (Focks et al., 1994; Lounibos
256 et al., 2003, 2011; Urbanski et al., 2012). This response varied sig-
257 nicantly with latitude, such that northern populations had a
258 higher diapause incidence in response to differences in photope-
259 riod than southern populations (Focks et al., 1994; Lounibos
260 et al., 2003, 2011; Urbanski et al., 2012). Similarly, populations of
261 introduced Ae. albopictus in southern Brazil have evolved photope-
262 riod-induced diapause despite having arisen from non-diapausing
263 colonizers (Lounibos et al., 2003). Geographic variation in photope-
264 riodic induction of winter diapause was also found across the range
265 of the mosquito Aedes atropalpus in North America (Beach, 1978).
266 Photoperiod as a cue to enter winter diapause has also been
267 studied in several anopheline species. For example, short photope-
268 riod was found to cue overwintering diapause in adult Anopheles
269 freeborni (Depner and Harwood, 1966). Importantly, the critical
270 photoperiod for entering winter diapause varied among popula-
271 tions from different latitudes (Depner and Harwood, 1966;
272 Washino, 1970), providing appropriate, ne-tuned diapause
273 responses across the geographic range of the species. Photoperiod
274 has also been implicated as one of the key factors to stimulate win-
275 ter diapause in Anopheles punctipennis (Washino and Bailey, 1970)
276 and Anopheles messeae (Jaenson and Ameneshewa, 1991).
277 Other environmental factors, including temperature and nutri-
278 tional quality, are also used by arthropods to indicate oncoming
279 seasonal environmental changes (Danilevskii, 1965). In deep aqua-
280 tic environments, photoperiod may not be as detectable as temper-
281 ature, and temperature was found to be an important cue for
282 winter diapause in aquatic copepods (Hairston and Kearns,
283 1995). However, for the terrestrial environments that adult mos-
284 quitoes inhabit, photoperiod appears to be the most common cue
285 for initiating winter diapause. It is not clear if studies on cues of
286 diapause induction in mosquitoes ignored other factors, such as
287 larval nutrition quality/composition, water salinity, acidity, and
288 hormonal changes in their vertebrate hosts; however, the consen-
289 sus from these studies remains that short photoperiod (with length
290 depending on latitude) is the key signal used to induce overwinter-
291 ing diapause in mosquitoes.
292 2.2. Cues used by insects to induce aestivation
293 For insects in tropical and sub-tropical climates, where varia-
294 tion in daylength is smaller, several environmental conditions
295 (not excluding photoperiod) can be reliable cues used to initiate
296 aestivation. For example, summer diapause in some lepidopterans
297 is presumed to have evolved in anticipation of a decrease in food
298 availability, and is triggered in response to long photoperiods
299 and high temperatures. Specically, long daylengths induce aesti-
300 vation of the cabbage buttery, Pieris brassicae, from southern
301 Spain, while short daylengths cause other populations from north-
302 ern Europe to enter winter diapause (Held and Spieth, 1999). Thus,
303 long and short daylengths induce summer diapause and overwin-
304 tering diapause, respectively, across the large geographic range of
305 this species and these phenomena serve to synchronize the result-
306 ing generation while protecting them from inhospitable condi-
307 tions. Similarly, a short summer aestivation is induced by long
308 daylength in the tiger moth Cymbalophora pudica, a species which
309 also undergoes a long winter diapause in response to short day-
310 lengths (Kostal and Hodek, 1997; Kostal et al., 1998). In another
311 moth species, Helicoverpa armigera, summer diapause is induced
312 by high summer temperatures, not long photoperiods; however,
313 winter diapause in this species is induced by short photoperiods
314 (Liu et al., 2006). The manifestation of both summer and winter
315 diapause in the same species (by the same or different populations)
316 has contributed to the presumption that similar physiological pro-
317 cesses are involved in each case. Yet, empirical evidence to com-
318 pare these forms of diapause within species and with others
319 manifesting diapause only in one season is still lacking.
320 The buttery Bicyclus anynana in sub-Saharan Africa experi-
321 ences distinct wet and dry seasons, and reproduction is suppressed
322 during the dry season, described as reproductive summer diapause
323 by researchers. Temperature was found to affect reproductive out-
324 put while food availability affected both reproduction and longev-
325 ity of these butteries (Brakeeld et al., 2007). Additionally, those
326 butteries reared in cool and dry conditions are more stress-resis-
327 tant than those that are not; combined, this species appears to use
328 several environmental cues (temperature, humidity, and food
329 availability) to initiate diapause. Similarly, tropical grasshoppers
330 which experience a dry season reduce their fecundity in response
331 to a low-quality diet, presumably as a mechanism to enhance their
332 own survival and that of the few offspring produced (Luker et al.,
333 2002). In this study, both shortened photoperiod and lower food
334 quality were required to stimulate this reproductive change
335 (Luker et al., 2002), possibly to ensure that aestivation would only
336 be entered at the appropriate season. High temperatures, low plant
337 nutrients, and crowding have been shown as cues used by the syc-
338 amore aphid, Drepanosiphum platanoidis, to shift into aestivation
339 expressed as reproductive suppression, low activity, and changes
340 in nutrient composition (Chambers, 1982; Dixon, 1966; Dixon
341 et al., 1993; Douglas, 2000).
342 Aestivation by adult female Culiseta inornata mosquitoes has
343 been observed in southern California, presumably as a mechanism
344 to avoid the hottest part of the summer (Barnard and Mulla, 1977,
345 1978). This species also undergoes winter diapause in response to
346 shortened photoperiod (Buth et al., 1990; Hudson, 1977). Although
347 long daylengths and short daylengths induced fat body hypertro-
348 phy and hypotrophy, respectively, the cues which initiate aestiva-
349 tion of Cs. inornata are not as clear (Barnard and Mulla, 1977, 1978;
350 Denlinger and Armbruster, 2014) and have not yet been fully mod-
351 eled in the laboratory setting (Reisen et al., 1989).
352 Moisture availability is a critical, but often overlooked, factor in
353 studies investigating the environmental triggers of diapause, but
354 often is more reliable than either temperature or photoperiod in
18 June 2014
355 tropical and sub-tropical environments (Tauber et al., 1998), such
356 as that experienced by mosquitoes in the Sahel. However, changes
357 in moisture (in the form of surface-water availability and/or
358 humidity) may act as a cue to reinforce or maintain diapause,
359 rather than initiate it, because it lacks the predictive properties
360 of photoperiod, given that a decrease in moisture means the envi-
361 ronmental stress has already begun. Several studies have indirectly
362 measured the effects of changing oviposition habitat on life-history
363 traits of An. gambiae. Female An. gambiae avoid oviposition in
364 otherwise acceptable larval sites which contained eggs or larvae
365 of Culex quinquefasciatus (Wachira et al., 2010). Therefore, the dis-
366 appearance of typical larval sites and use of potential alternative,
367 albeit atypical, larval sites (e.g., wells, pit latrines) by competitors
368 could act as another possible cue to initiate aestivation, although,
369 like moisture, oviposition-deprivation is a result of the dry season
370 rather than a predictor of it. Contrary to expectations, oviposition-
371 deprivation of female mosquitoes of a laboratory colony of An.
372 gambiae (G3), with or without multiple bloodmeals, did not
373 increase female longevity and instead reduced it by a few days rel-
374 ative to females with the opportunity for regular gonotrophic
375 cycles (Artis et al., 2014) Q3 . Notably, this strain has been in colony
376 for about 60 years, and therefore may not reect the behavior of
377 wild mosquitoes from areas with an annual dry season.
378 In the case of An. coluzzii, aestivation has been proposed as a
379 mechanism for adult mosquitoes to extend their longevity over
380 the long Sahelian dry season in order to reproduce at the begin-
381 ning of the next rainy season (Adamou et al., 2011; Huestis
382 et al., 2012; Lehmann et al., 2010, 2014; Yaro et al., 2012). Our
383 recent studies revealed that this species declines rapidly about a
384 month before larval sites disappeared (Lehmann et al., 2010,
385 2014; Dao et al., in prep), in accordance with anticipation of the
386 coming dry season based on an unknown) token stimulus.
387 Although Sahelian anopheline populations are located within trop-
388 ical latitudes, photoperiod may still act as an important cue for
389 aestivation; previous studies have suggested that any latitude
390 above 10 has a large enough seasonal change in photoperiod to
391 signicantly affect the life-history traits of insects (Denlinger,
392 1986; Denlinger and Armbruster, 2014). The seasonal change in
393 photoperiod experienced by these mosquito populations is around
394 two hours (Fig. 1) and the daily rate of change in photoperiod may
395 also be used as a cue. Just as a shortened photoperiod precedes the
396 oncoming winter and can act as a cue for winter-diapausing insect
397 species, photoperiod decreases at the end of the wet season in the
398 Sahel and could be used by anophelines to predict the upcoming
399 dry season. Previous research has shown that decreased daylength
400 increased longevity of both Anopheles crucians (Lanciani, 1993) and
401 Anopheles quadrimaculatus (Lanciani and Anderson, 1993) from
402 Florida, despite being nondiapausing in their native habitat.
403 Research recently completed in our lab indicated a modest
404 increase in longevity under a photoperiod which mimicked dry-
405 season conditions in the eld (Huestis et al., in prep). Additionally,
406 under short photoperiod (11.5 h), mosquitoes from a well-known
407 laboratory colony of An. gambiae (G3; a mix of M- and S-forms)
408 exhibited a larger body size and a greater total amount of cuticular
409 hydrocarbons than mosquitoes under long photoperiod (13.5 h;
410 Wagoner et al., 2014). Therefore it seems possible, if not likely,
411 that a decreased photoperiod, perhaps in conjunction with lower
412 humidity and/or other factors, acts as a cue to initiate aestivation
413 of Sahelian anophelines. However, much future research in this
414 area is required.
415 3. Changes in reproduction and blood-feeding
416 Another key hallmark of mosquito diapause (overwintering or
417 aestivation) is a dramatic shift in feeding (sugar vs. bloodmeals,
418 or avoiding both) and a decrease in reproduction (for those species
419 which diapause as adults; Box 1).
420 3.1. Reproduction and blood-feeding during insect winter diapause
421 Other than extended survival, reproductive arrest is arguably
422 the ultimate hallmark of diapause in adult mosquitoes (Bates,
423 1949; Clements, 1963; Rao, 1947; Swellengrebel, 1929; Washino,
424 1977). Typically, both mosquito reproduction and blood-feeding
425 are depressed during diapause (Bates, 1949; Clements, 1992;
426 Washino, 1977). Blood-feeding during or prior to diapause is an
427 avenue that pathogens taken in the bloodmeal may use to persist
428 in diapausing mosquitoes throughout the winter (or the summer),
429 especially if transmission halts and the pathogen is cleared from
430 vertebrate hosts, as has been suggested for certain viruses (WEE,
431 JE, and SLE) in Culex spp. For example, in Cx. tarsalis, decreased pho-
432 toperiod induces adult females to shift into the hibernal-diapause
433 initiation phase by accumulating fat reserves while simultaneously
434 decreasing ovary size (Harwood and Halfhill, 1964). Notably, feed-
435 ing response prior to diapause did not change with photoperiod,
436 but rather the way in which nutritional resources are used; this
437 shift in resource distribution was reinforced and strengthened by
438 lower temperatures (Harwood and Halfhill, 1964). The terms
439 reproductive- or ovarian-diapause typically refer to arrested ovar-
440 ian development in an early previtellogenic stage as an indicator of
441 diapause (Vinogradova, 1960; Vinogradova et al., 2007). However,
442 it also implies that other indicators of diapause such as depressed
443 metabolism, ight activity, sugar-feeding and even blood-feeding,
444 may not be manifested by females in reproductive diapause. Egg
445 size (e.g., follicular length) below a species-specic threshold has
446 often been used as a marker of diapause especially in Culex spp.
447 (Eldridge, 1987; Washino, 1977). Female mosquitoes in winter dia-
448 pause may (i) avoid feeding altogether (e.g., An. maculipennis mes-
449 seae), (ii) feed on sugars exclusively (e.g., Culex pipiens), or (iii) take
450 blood-meals with or without sugars (e.g., Anopheles labranchiae
451 atroparvus). In diapausing anautogenous mosquitoes (which
452 require a bloodmeal to mature even their rst egg batch), gono-
453 trophic concordance, refers to state i or ii (above) where eggs
454 remain undeveloped because no blood-meal is taken, whereas
455 gonotrophic dissociation, only known in anophelines, refers to
456 state iii, where eggs remain undeveloped even after taking
457 blood-meals (Vinogradova et al., 2007; Washino, 1977; Box 1).
458 3.2. Reproduction and blood-feeding during insect aestivation
459 For aestivating mosquitoes, the situation is less clear. Labora-
460 tory studies on Cs. inornata showed that under long photoperiod
461 (summer daylight), blood-feeding rates in females from southern
462 populations dropped from 60% to 20% (Barnard and Mulla, 1977),
463 suggesting that gonotrophic concordance was the primary mecha-
464 nisms of aestivation. All females that blood-fed developed eggs
465 normally, but, unlike females raised under short photoperiod, most
466 retained their eggs (for over 2 weeks) when offered water for ovi-
467 position, suggesting that gravid females may also aestivate
468 (Barnard and Mulla, 1977, 1978). Results of eld studies suggested
469 that all aestivating females took at least one blood-meal and were
470 parous (Barnard and Mulla, 1978). There is large variation in mode
471 and degree of diapause among populations of Cs. inornata. In
472 southern California, this species is active during the cool winter
473 and it aestivates during the summer as described above, while in
474 central California, populations exhibited no (reproductive) dia-
475 pause during the hot summer although their activity was markedly
476 depressed (Reisen et al., 1989). In contrast, Canadian populations
477 of Cs. inornata are active through the summer yet undergo winter
478 diapause (Buth et al., 1990; Hudson, 1977).
18 June 2014
479 Populations of An. arabiensis in the Sudan, over 20 km away
480 from the Nile with no surface water available, exhibited gono-
481 trophic dissociation, whereas females from populations along the
482 Nile developed eggs normally and larvae were found throughout
483 the dry season in puddles along the river (Omer and Cloudsley-
484 Thompson, 1970). When raised under conditions similar to the
485 natural environment away from the river, survival was extended
486 up to 206 days, providing further evidence for aestivation (Omer
487 and Cloudsley-Thompson, 1968; Table 1). However, results of sim-
488 ilar studies in other parts of Africa have not corroborated these
489 ndings (Charlwood et al., 2000; Ramsdale and Fontaine,
490 1970a,b). Recent studies on An. coluzzii (previously known as the
491 M molecular form of An. gambiae; Coetzee et al., 2013) showed that
492 in a Sahelian population, reproduction was depressed sharply dur-
493 ing the dry season, while the blood-feeding response did not
494 change seasonally (Yaro et al., 2012; Table 1). Furthermore, a high
495 rate of blood feeding in this population was noted among
496 mosquitoes collected indoors throughout the dry season
497 (Adamou et al., 2011; Huestis et al., 2012; Lehmann et al., 2010).
498 The oviposition rate dropped from 70% (wet season) to 20% (dry
499 season), and in those females that actually laid eggs, the mean
500 number of eggs per female fell signicantly, from 173 to 101
501 (Yaro et al., 2012). Correspondingly, the fraction of females that
502 exhibited gonotrophic dissociation increased over the dry season
503 from 5% to 45%, while a similar fraction of the population retained
504 developed eggs despite having access to water (Yaro et al., 2012).
505 Notably, less extreme changes were measured in a population from
506 along the Niger River. Finally, articial larval sites constructed and
507 maintained during the Sahelian dry season were used by culicine
508 mosquitoes but not by An. coluzzii or other Anopheles spp.), consis-
509 tent with latency of this physiological state which is not easily bro-
510 ken (Lehmann et al., 2010). As noted above, studies using the G3
511 colony of An. gambiae showed that oviposition-site deprivation
512 alone was insufcient to shift mosquitoes into reproductive quies-
513 cence and extended longevity (Artis et al., 2014; Dieter et al.,
514 2012). During the dry season, Anopheles funestus exhibited a
515 reduced proportion of gravid females and a higher proportion of
516 partly blood-fed females compared with wet season, suggesting
517 that reproduction is suppressed in at least a fraction of the popula-
518 tion (Charlwood et al., 2013).
519 As far as we know, aestivation in mosquitoes has been studied
520 only in Cs. inornata, An. arabiensis, and An. coluzzii. The degree of
521 reproductive inhibition exhibited by apparently aestivating An.
522 arabiensis, An. coluzzii, and Cs. inornata varied greatly between pop-
523 ulations only tens of kilometers apart, but was evident during the
524 dry season in at least one population of each species. Gonotrophic
525 dissociation and gonoactive females that retained eggs (when
526 offered water for oviposition) were observed under eld conditions
527 even within the same population (Barnard and Mulla, 1978; Omer
528 and Cloudsley-Thompson, 1970; Yaro et al., 2012). Such heteroge-
529 neity may represent different bet-hedging strategies with respect
530 to reproductive-diapause strength and duration. Heterogeneity
531 along similar lines was noted in overwintering Cx. pipiens, Cx. tar-
532 salis, and Culex tritaeniorhynchus (Reisen et al., 2010; Spielman,
533 2001; Spielman and Wong, 1973; Tsuda and Kim, 2008), suggest-
534 ing that such variation is not unique to aestivation. Yaro et al.
535 (2012) proposed that the composition of weak aestivators and
536 strong aestivators differs among populations, reecting the
537 severity and prospects of reproductive opportunities during the
538 dry or cold seasons. These ndings parallel earlier studies on over-
539 wintering larval diapause of Wyeomia smithii, in which the depth
540 of diapause was found to vary latitudinally among populations,
541 such that southern populations entered dormancy later in develop-
542 ment and could emerge from diapause faster and with less
543 environmental input than northern populations (Bradshaw and
544 Lounibos, 1977).
545 Unlike Cs. inornata and even An. arabiensis, An. coluzzii males
546 were detected throughout the dry season, albeit in very small
547 numbers (Adamou et al., 2011; Huestis et al., 2012; Lehmann
548 et al., 2010; Yaro et al., 2012). Small swarms were occasionally
549 observed with 16 males per swarm, indicating that male repro-
550 ductive activity is not arrested (Yaro et al., 2012; Table 1), although
551 it might be greatly reduced. The presence of males throughout the
552 dry season is a radical deviation from expectations for mosquito
553 overwintering diapause. The signicance of this is unclear, as is
554 whether males were missed in studies of aestivation of Cs. inornata
555 or An. arabiensis. An. coluzzii females exhibited a lower rate of gono-
556 trophic dissociation (Yaro et al., 2012) compared with that of An.
557 arabiensis (Omer and Cloudsley-Thompson, 1970), although both
558 species exhibited high rate of blood-feeding. It is unclear if these
559 are species-specic or population-specic differences.
560 4. Metabolic rate, nutritional reserves, and activity level during
561 diapause
562 For many species, a decrease in metabolic rate is a key trait of
563 overwintering diapause, often accompanied by an increase in
564 nutritional reserves to sustain the individual during the diapause
565 period and a decrease in activity level so that these reserves will
566 last longer.
567 4.1. Metabolic rate during diapause
568 During winter diapause, a dramatic decrease in metabolic rate
569 occurs in many insects (Clarke and Fraser, 2004; Denlinger, 2002;
570 Guppy and Withers, 1999; Hahn and Denlinger, 2011). However,
571 the magnitude of the decrease cannot be attributed fully to low
572 temperatures alone; rather it is lowered by 5090% beyond what
573 is expected by the scaling with temperature (Guppy, 2004;
574 Guppy and Withers, 1999; Storey and Storey, 1990, 2004), not
575 including those organisms which enter a cryptobiotic state
576 (extreme slow down/arrest of life processes that accompanies sur-
577 vival over many years). A few well-documented examples include
578 a decrease in metabolic rate in the y Rhagoletis pomonella
579 (Ragland et al., 2009) and in adult female Cx. pipiens mosquitoes
580 (Benoit and Denlinger, 2007). The decrease in metabolic rate dur-
581 ing insect winter diapause has been previously reviewed thor-
582 oughly (e.g., Clarke and Fraser, 2004; Denlinger and Armbruster,
583 2014; Guppy, 2004; Hahn and Denlinger, 2011; MacRae, 2010);
584 therefore, we will focus on metabolic processes during aestivation
585 and use winter diapause for comparative purposes only.
586 Some species undergo both summer and winter diapause. For
587 example, adults of the alfalfa weevil Hypera postica aestivate in
588 the summer, actively feed and reproduce in the fall, and their eggs
589 undergo winter diapause until the next spring (Tombes, 1964).
590 During adult aestivation, the mean metabolic rate drops to about
591 one-quarter of its mean during the active phase, and remains low
592 for 3 months (Tombes, 1964). This drop in metabolic rate is also
593 associated with a linear decrease in the size of the fat body during
594 aestivation and a decrease in water content; together, these results
595 suggest that these aestivating weevils are inactive and conserve
596 resources similar to winter diapause. Likewise, the leaf beetle
597 Zygogramma suturalis has a short aestivation in summer and a long
598 winter diapause, both in the adult phase and found at different fre-
599 quencies in different years (Vinogradova and Pantyuchov, 1995).
600 The metabolic rate (measured as oxygen consumption) of female
601 beetles was approximately 25 times lower in both aestivating
602 and diapausing individuals compared with the spring and fall
603 active phases (Vinogradova and Pantyuchov, 1995), indicating that
604 the physiological mechanisms between the two types of dormancy
605 may be similar. For males, the metabolic rate during aestivation
18 June 2014
606 was not as low as that during diapause, but both were lower than
607 normal active levels (Vinogradova and Pantyuchov, 1995). Insects
608 may undergo aestivation at different developmental stages, which
609 may also impact what physiological changes occur. For example,
610 during pupal aestivation in the tiger moth C. pudica, the metabolic
611 rate decreased to 515% of its normal level, and morphological
612 development is halted (Kostal et al., 1998). In contrast, two species
613 of tropical buttery in the genus Euploea undergo adult aestivation
614 in the cool dry season, characterized by an increased fat body and a
615 lack of egg production (Canzano et al., 2006). For both of these
616 species, the reduction in metabolic rate during this state was esti-
617 mated at about 28% (Canzano et al., 2006), a less dramatic decline
618 than the range reported above for winter-diapausing insects.
619 Although metabolic rate was measured in An. coluzzii during the
620 dry season, it is unknown if these mosquitoes were actually in aes-
621 tivation because they were found indoors and most had recently
622 blood-fed (Huestis et al., 2012). However, some factors which
623 affect metabolic rate have been previously studied in other anoph-
624 elines and may allow us to make additional predictions about sce-
625 narios during dry-season aestivation. For example, metabolic rate
626 of An. quadrimaculatus adults varied with photoperiod: metabolism
627 was higher under long days than under short days, but for mosqui-
628 toes collected in the fall, the reverse was found (Lanciani and
629 Anderson, 1993). Furthermore, although this study was presum-
630 ably conducted using a non-aestivating Floridian population (see
631 above), the short-day photoperiod was always associated with
632 increased longevity, regardless of collection time (Lanciani and
633 Anderson, 1993). For wet-season anophelines in the Sahel, meta-
634 bolic rate was signicantly affected by body size, female gono-
635 trophic status, ight activity, and temperature (Huestis et al.,
636 2011). Huestis et al. (2012) measured seasonal variation in meta-
637 bolic rate of An. coluzzii and compared a Sahelian population with
638 a riparian population; we found signicant seasonal variation at
639 the Sahelian site but not at the riparian site after adjusting for
640 the effect of temperature (Huestis et al., 2012). Surprisingly, meta-
641 bolic rate at the Sahelian site was the highest in the late dry season,
642 even after accounting for temperature and other factors (Huestis
643 et al., 2012), indicating that these mosquitoes which are hypothe-
644 sized to be undergoing aestivation do not have a reduced metabolic
645 rate (Table 1). However, because the mosquitoes used in this
646 experiment were collected inside houses and most were freshly
647 blood-fed, they may represent mosquitoes which have temporarily
648 suspended or totally broken aestivation, and thus may not
649 display the same physiological characteristics as mosquitoes which
650 are in shelters. Previous studies have shown that starvation does
651 not decrease metabolic rate in ies (Djawdan et al., 1997) or in
652 crickets (Sinclair et al., 2011) once the effects of fuel-use have been
653 accounted for (since the amount of CO
2
produced varies whether
654 the insect is burning lipids or carbohydrates). However, another
655 study showed that starvation slightly decreased an insects meta-
656 bolic rate but then resulted in an extreme increase once feeding
657 did occur (Bennett et al., 1999). Additionally, one recent study on
658 diapausing plant bugs (Lygus hesperus) did not nd any difference
659 in metabolic rate between winter-diapausing and non-diapausing
660 individuals, because brief periods of activity and feeding occurred
661 (Brent et al., 2013). In conclusion, we predict that mosquitoes hid-
662 den in cool, underground shelters may exhibit a reduced metabolic
663 rate (Clarke and Fraser, 2004) while in a dormant, energy-conserv-
664 ing state, but that those mosquitoes which are seeking bloodmeals
665 aboveground no longer show this reduction. It is also unknown if
666 they revert to a reduced metabolism once they presumably return
667 to these shelters after feeding (Huestis et al., 2012); however,
668 pulses of increased metabolic activity followed by a return to lower
669 levels are known to occur in insect winter diapause (Hahn and
670 Denlinger, 2011). Clearly, if mosquitoes could be found within their
671 as-yet-unknown dry-season shelters, it would open up a new
672 avenue of investigation regarding their physiology.
673 4.2. Nutritional reserves during diapause
674 To survive a period of inactivity without feeding, insects
675 accumulate nutritional reserves in preparation for diapause
676 (Denlinger, 2002; Hahn and Denlinger, 2011) and reduce their
677 metabolic rate to conserve these acquired reserves as described
678 above. In insect winter diapause, triacylglycerides are the most
679 common form of lipid storage, although insects also continue to
680 use other lipids, carbohydrates, and amino acids (Hahn and
681 Denlinger, 2011). For example, adult Cx. pipiens mosquitoes accu-
682 mulate lipids prior to winter diapause, and it was found that any
683 remaining lipid resources not used during diapause can be used
684 for egg-production by females after breaking diapause (Zhou and
685 Miesfeld, 2009).
686 Only a few studies on insect aestivation have measured nutri-
687 tional reserves accumulated before and fuel usage during aestiva-
688 tion. For example, pupae of the cotton bollworm Helicoverpa
689 armigera undergo summer aestivation and increase their energy
690 storage of lipids and glycogen prior to aestivation (Liu et al.,
691 2006). Similarly, the tiger moth C. pudica also undergoes aestiva-
692 tion in the pupal stage and was found to increase triacylglyceride
693 levels and have altered ratios of saturated to unsaturated lipids
694 (Kostal and Simek, 1998). Adults of the beetle Stenotartus rotundus
695 increase their glycerol and glucose content during dry-season dia-
696 pause (Pullin and Wolda, 1993). Prior to aestivation, another cole-
697 opteran, the weevil H. postica, increases its fat and protein levels
698 while reducing water content, and is hypothesized to use fat as
699 its primary fuel source during aestivation, due to its linear decrease
700 during the 3-month period (Tombes, 1964). In summer-aestivating
701 Cs. inornata, female mosquitoes increased their lipid content nearly
702 fourfold prior to aestivation; lipid levels returned to normal after
703 the 3-month aestivation period, indicating usage of this fuel during
704 this time (Barnard and Mulla, 1978). Similar results were obtained
705 by rearing female Cs. inornata under short- and long-day photope-
706 riods (Barnard and Mulla, 1977). Although nutritional reserves
707 have not yet been explicitly measured in An. coluzzii during the
708 dry season, this work is currently underway and we hypothesize
709 that increased lipid reserves may be found, given the 70% reduction
710 in egg-laying by blood-fed females during the dry season (see
711 above; Yaro et al., 2012). Furthermore, a study of non-aestivating
712 female anophelines showed that, during ight trials, blood-fed
713 females had lower carbohydrate usage than sugar-fed females,
714 indicating that blood-fed females may be able to use some of the
715 nutrients from the bloodmeal for their own activity (Kaufmann
716 and Briegel, 2004). However, as with metabolic rate above, it will
717 be difcult to draw conclusions about the nutritional physiology
718 of aestivating An. coluzzii in the Sahel without being able to identify
719 their shelters and measuring mosquitoes in their dormant state.
720 4.3. Activity level during diapause
721 Coupled with increased nutritional reserves and a decreased
722 metabolic rate to make these reserves last longer is a decrease in
723 the activity level of the insect (ight, foraging, and/or reproduc-
724 tion). While this is given for overwintering under low temperature,
725 during aestivation of the tropical beetle S. rotundus, the size of the
726 ight muscles was greatly reduced and ight activity ceased (Pullin
727 and Wolda, 1993). Similarly, during adult winter diapause of the
728 mosquito Cx. pipiens, ight muscle size (measured as amount of
729 beta-tubulin) decreased while ight activity was much reduced
730 (Kim and Denlinger, 2009). For insects which are not actively for-
731 aging during dormancy, reducing the amount of energy required
18 June 2014
732 to maintain large ight muscles is very benecial (Denlinger,
733 1986). In our study comparing anophelines at a Sahelian site with
734 those from a riparian site, we found signicantly reduced ight
735 activity prior to and during the dry season in the Sahelian popula-
736 tion but no signicant seasonal variation in ight activity in the
737 riparian population after accounting for temperature variation
738 (Huestis et al., 2012; Table 1). This study analyzed sound record-
739 ings from eld-caught mosquitoes placed in individual chambers,
740 generated over a 2-h timeframe in the eld (Huestis et al., 2011).
741 Thus, these results indicate that although we found a signicant
742 increase in metabolic rate during the late dry season in the Sahel,
743 mosquitoes may still modify their behavior to conserve energy
744 during the dry season.
745 Based on these patterns, we presume that, while in shelters, An.
746 coluzzii minimizes its activity and reduces its resting metabolic
747 rate to conserve resources. However once its nutritional resources
748 are depleted, it becomes active and forages for sugars and blood
749 sources. This strategy minimizes the number of foraging trips
750 and bloodmeals a single female will take during the dry season,
751 similar to the strategy of the winter-diapausing plant bug L.
752 hesperus, which reduces its feeding rate during diapause to one-
753 fth of that during normal activity (Brent et al., 2013). This strategy
754 may explain why malaria transmission is very low throughout the
755 dry season, even if the mosquitoes are very old.
756 5. Stress tolerance during diapause
757 During the diapause period, organisms are often inactive (and
758 sometimes incapable of movement), and must cope in situ with
759 the harsh environmental conditions that occur; therefore,
760 increased stress resistance is another hallmark of diapause
761 (Box 1). Desiccation, temperature extremes, and starvation repre-
762 sent stress factors that insects often withstand during inhospitable
763 seasons, especially while in dormancy (Denlinger, 1986; Denlinger
764 and Armbruster, 2014; Masaki, 1980; Tauber et al., 1986).
765 5.1. Desiccation tolerance
766 Desiccation tolerance has long been considered a key adapta-
767 tion of insects living in arid environments, whether they undergo
768 dormancy or not (Benoit et al., 2010a; Chown and Nicolson,
769 2004; Kostal et al., 1998; Tauber et al., 1986). However, during dia-
770 pause, water sources may be scarce, the air is dry, and often the
771 insects mobility is limited; thus desiccation resistance is essential.
772 Higher body water-content, lower rate of water loss, and lower
773 threshold for critical body water-content at death are all compo-
774 nents of desiccation tolerance (Chown, 2002; Gibbs et al., 2003).
775 The physiological mechanisms most commonly studied reduce
776 the rate of water loss by i) reducing the amount of time spiracles
777 are open to minimize water vapor loss during gas exchanges, dis-
778 continuous gas exchange (Gibbs and Johnson, 2004; Lighton,
779 1996), or ii) increased waterproong via a higher amount of wax
780 layer in the epicuticle and/or changes in the cuticular hydrocarbon
781 composition (Benoit and Denlinger, 2007; Gibbs et al., 1997).
782 Reduced surface-to-volume ratio associated with increased body
783 size also increases desiccation resistance (Hadley, 1994).
784 Desiccation resistance mediated by an increased cuticular
785 hydrocarbon layer is considered key to egg diapause in Ae. albopic-
786 tus (Lounibos et al., 2011; Urbanski et al., 2012). During winter dia-
787 pause, female Cx. pipiens suppress water loss by doubling the total
788 amount of cuticular hydrocarbons in the epicuticle, lowering met-
789 abolic rate (reduces gas exchange), and by increased body size
790 (Benoit and Denlinger, 2007). Whether they drink water or
791 sugar-feed during diapause under natural conditions is unclear.
792 Dehydration stress uses up lipids and glycogen (Benoit et al.,
793 2010b), suggesting that desiccation resistance, nutritional reserves,
794 and starvation resistance may be selected together in populations
795 where extreme winters select for diapause. Unless provided with a
796 sugar source during diapause, depleted nutritional reserves due to
797 desiccation stress reduced female reproductive success after win-
798 ter diapause (Benoit et al., 2010b).
799 Rather than comparing their desiccation tolerance under aesti-
800 vating vs. normal conditions, previous studies compared species,
801 populations, and genotypes (within a population) inhabiting dry
802 vs. humid environments. Early studies found clines in frequencies
803 of the inversions 2La and 2Rb to be strongly correlated with aridity
804 on spatial and seasonal scales (Bayoh et al., 2001; Coluzzi et al.,
805 1985, 1979; Toure et al., 1994). These inversions are found in
806 markedly different frequencies between species of the complex
807 (2La is xed in An. arabiensis and is especially variable between
808 populations of An. coluzzii; Bayoh et al., 2001; Coluzzi et al.,
809 1979, 1985; Toure et al., 1994). It has been hypothesized that the
810 2La and 2Rb inversions confer higher desiccation tolerance and
811 possibly higher temperature tolerance to their carriers (Bayoh
812 et al., 2001; Coluzzi et al., 1979, 1985; Toure et al., 1994). Using
813 laboratory colonies established from Kenya, female An. arabiensis
814 exhibited a higher desiccation resistance than that of An. gambiae
815 s.s. (Gray and Bradley, 2005). Notably, desiccation resistance of
816 teneral mosquitoes (<24 h after adult emergence) of both species
817 was higher than that of 4- and 8-day-old mosquitoes. Additional
818 evidence suggested that the higher water content of An. arabiensis
819 enhances its desiccation resistance and may also explain the
820 advantage of teneral mosquitoes (Gray and Bradley, 2005; Table 1).
821 As in other parts of East Africa, An. arabiensis predominates during
822 the drier season in accordance with this observation. It is also not
823 clear if the larger body size of An. arabiensis (Huestis et al., 2011;
824 Lehmann and Diabate, 2008) has contributed to its higher survival.
825 Comparison of larval tolerance to desiccation between colonies of
826 these species revealed lower tolerance of An. arabiensis from Zim-
827 babwe compared to that of An. gambiae from the Gambia (G3 col-
828 ony), and that the latter was very similar to the tolerance of An.
829 arabiensis from Sudan (Benedict et al., 2010). However, since the
830 mean difference in survival measured only a few minutes, its
831 importance to existence in dry environments remains unclear.
832 Using F1s of eld-collected females from Mali, An. coluzzii exhib-
833 ited higher desiccation tolerance than An. gambiae s.s. (22.2 vs.
834 17.6; 22%), although variation in body size or age were not con-
835 sidered (Lee et al., 2009; Table 1). Notably, the variation between
836 females and males within a species was larger than differences
837 between species (22.4 vs. 16.8; 27%), and the effect of 2La inver-
838 sion was not signicant, although a small sample size reduced the
839 power of this test (Lee et al., 2009).
840 Within-species variation in desiccation resistance between
841 lines of mosquitoes selected as homozygotes for either the 2La
842 inversion or the standard karyotype showed that the former were
843 more resistant early in life than the latter at 1 and 4 days post-
844 eclosion (14.7 vs. 11.6 h and 14.7 vs. 12.6 h, respectively; Gray
845 et al., 2009; Table 1). At 8 days of age, the difference was reversed
846 (13.3 vs. 13.9 h) or disappeared because it was not statistically sig-
847 nicant. Higher water content contributed to higher resistance of
848 2La homozygotes at day 4 and slower water loss of this karyotype
849 contributed to the resistance of the teneral mosquitoes. In a subse-
850 quent study, using 1-day-old mosquitoes representing different
851 karyotypes within An. gambiae s.s. (previously the S molecular
852 form), 2La-homozygotes exhibited higher desiccation tolerance
853 than the standard-homozygotes (612 vs. 537 min or 13%), although
854 the heterozygotes exhibited apparently lower resistance than both
855 (529 min; Fouet et al., 2012; Table 1). Notably, body size of the
856 2La-homozygotes was larger than that those carrying the standard
857 karyotype, but the heterozygotes were apparently the largest
858 (Fouet et al., 2012). The difference was only found in teneral
18 June 2014
859 mosquitoes and no difference was detected after 1 day post-eclo-
860 sion (Fouet et al., 2012).
861 Environmental factors such as larval nutrition and adult access
862 to water signicantly affected survival of An. coluzzii females under
863 desiccation stress (Aboagye-Antwi and Tripet, 2010). In addition to
864 the effect of higher body-water content (prior to desiccation
865 stress), higher glycogen reserves (reecting access to better larval
866 food) also increased desiccation resistance without affecting body
867 size. In populations of Anopheles stephensi from dry vs. humid envi-
868 ronments, smaller spiracle size relative to body size was proposed
869 as a morphological mechanism conferring desiccation tolerance
870 (Nagpal et al., 2003), although desiccation tolerance was not
871 directly assessed.
872 During the dry season, female An. coluzzii from Sahelian popula-
873 tions exhibited higher desiccation tolerance than that during the
874 wet season (14.6 h vs. 10.112.1, P < 0.001), consistent with
875 aestivation (Dao et al., unpublished; Table 1). However, whether
876 a difference of 25% (2035%) in aestivation tolerance explains
877 the long survival of these mosquitoes throughout the long dry sea-
878 son remains unclear.
879 5.2. Tolerance to temperature extremes
880 Fewer studies have measured tolerance to temperature
881 extremes in mosquitoes. Using laboratory colonies, An. arabiensis
882 exhibited a higher tolerance to high temperatures than An. gam-
883 biae, surviving 112 vs. 67 min at 40 C (Kirby and Lindsay, 2004;
884 Table 1). The effect of larger body size of An. arabiensis was not
885 evaluated. A colony of An. gambiae was used to compare homozy-
886 gotes of the 2La inversion vs. the standard arrangement in toler-
887 ance to high temperature of 4th instar larvae and pupae (Rocca
888 et al., 2009). No difference was found among larvae, but, after accli-
889 mation, homozygotes of the 2La inversion exhibited signicantly
890 higher tolerance than carriers of the standard arrangement
891 (Table 1). No difference was detected in between pupae regardless
892 of acclimation (Rocca et al., 2009).
893 In summary, multiple studies have compared desiccation toler-
894 ance between species and populations of An. gambiae s.l. selected
895 to represent the extreme opposites in this trait, but the differences
896 measured were modest (<40% although statistically signicant, and
897 often restricted to narrow age group), whilst the overlap between
898 these populations was large. The difference required to explain
899 aridity tolerance has not been dened, yet we question whether
900 the available estimates (above) would approach this value.
901 6. Molecular underpinnings of diapause
902 The molecular regulation of diapause has been previously
903 reviewed thoroughly by Denlinger (2002), MacRae (2010), and
904 Storey and Storey (2012), and was recently summarized in mosqui-
905 toes by Denlinger and Armbruster (2014). Additionally, metabolo-
906 mic and ecdysteroid variation in An. gambiae s.l. under desiccating
907 conditions was recently reviewed (Mamai et al., 2014). Here we
908 will describe common gene-expression changes during winter dia-
909 pause and focus mainly on those few studies examining molecular
910 changes during aestivation.
911 6.1. Gene expression during winter diapause
912 Gene-expression changes associated with winter diapause
913 reveal genes and processes which many organisms have in com-
914 mon, leading to the notion of a genetic toolkit for diapause
915 (Poelchau et al., 2013a). For example, in winter-diapausing
916 Drosophila melanogaster, regulation of the insulin pathway shifts
917 so that nutrients are depleted slowly and juvenile hormone
918 increases so that ovarian development ceases (Emerson et al.,
919 2009). Furthermore, genes associated with photoperiod often have
920 widespread downstream effects, as shown in the bean bug Riptor-
921 tus pedestris, where RNAi knockdown of two photoperiod genes
922 affected both cuticle deposition and ovarian development, impor-
923 tant traits for winter diapause of this species (Ikeno et al., 2010).
924 Additionally, defensive proteins such as antifungal and antibacte-
925 rial peptides are often up-regulated, such as the gene drosomycin
926 in Drosophila triauraria (Daibo et al., 2001).
927 The Asian tiger mosquito, Ae. albopictus, which undergoes dia-
928 pause in the pharate rst instar stage (i.e., within the egg), has been
929 used as a model for egg diapause. Expression of a gene involved in
930 the cell-cycle arrest process and another gene associated with
931 sugar metabolism were both found to increase in the pre-diapause
932 stage (Poelchau et al., 2013a). These same genes have been identi-
933 ed in other taxa and t into the diapause toolkit concept
934 (above). In a related study, it was found that several genes associ-
935 ated with carbohydrate metabolism and lipid metabolism were
936 over-expressed in the early stages of diapause, while expression
937 of amino-acid metabolism genes were generally reduced
938 (Poelchau et al., 2013b), indicating that, in the early phase of dia-
939 pause, the dormant embryos are engaged in utilization of stored
940 resources. Later in diapause, however, only lipid metabolism genes
941 were found to be signicantly differentially expressed (Poelchau
942 et al., 2013b). Surprisingly, this study did not nd evidence for
943 up-regulation of heat-shock proteins throughout diapause
944 (Poelchau et al., 2013b), in contrast to many other studies of insect
945 diapause (Denlinger, 2002).
946 The expression of structural proteins in the northern house
947 mosquito, Cx. pipiens, such as actin, was shown to be up-regulated
948 in early diapause and further increased after exposure to freezing
949 temperatures (Kim et al., 2006). Actin is presumed to strengthen
950 the cytoskeleton and protect against cellular damage due to freez-
951 ing temperatures (Kim et al., 2006). The expression level of another
952 structural protein, beta-tubulin, was also studied in midgut and
953 ight muscles of diapausing female Cx. pipiens (Kim and
954 Denlinger, 2009). A signicant decrease in microtubule abundance
955 in the ight muscles of diapausing females was found, correspond-
956 ing to the reduction in ight activity due to low temperature and
957 diapause; in contrast, there was no signicant change in the mid-
958 gut muscles (Kim and Denlinger, 2009). Expression levels of genes
959 associated with feeding and reproduction have also been studied,
960 as it has been shown that female mosquitoes increase sugar intake
961 and decrease blood feeding prior to entering diapause (Robich and
962 Denlinger, 2005). In accordance with this shift in meal intake, the
963 enzymes needed to digest a bloodmeal (trypsin and a chymotryp-
964 sin-like protease) were down-regulated in the pre-diapause period,
965 while a gene associated with increasing the size of the fat reserve
966 (fatty acid synthase) was up-regulated (Robich and Denlinger,
967 2005). This preparation happens before diapause begins, consistent
968 with the observation that female Cx. pipiens usually will not take a
969 bloodmeal before or during diapause (and even if they do so, will
970 often expel the blood; see Robich and Denlinger, 2005 and refer-
971 ences therein); however, it is possible that they could continue
972 sugar-feeding if the opportunity was available. As the end of dia-
973 pause nears, the trypsin and chymotrypsin-like expression gradu-
974 ally increases, in preparation for breaking diapause and
975 beginning the gonotrophic cycle (Robich and Denlinger, 2005).
976 Lastly, insulin-signaling genes have been found to be instrumental
977 in initiating the diapause cascade in Cx. pipiens (Sim and Denlinger,
978 2008), and insulin-like peptides are crucial for stopping ovarian
979 development during overwintering (Sim and Denlinger, 2009).
980 However, ovarian development can be rescued by applying juve-
981 nile hormone (Sim and Denlinger, 2009; Spielman, 1974). These
18 June 2014
982 studies on adult-diapausing Cx. pipiens can predict some of the
983 gene-expression differences that could be utilized by aestivating
984 An. gambiae s.l. during the Sahelian dry season (see below).
985 6.2. Gene expression during aestivation
986 In a review covering gene expression during aestivation in
987 toads, snails, and nematodes, several key classes of genes were
988 found across these taxa, including nutrient-regulation, growth sig-
989 naling, dehydration responses, and antioxidant defenses (Storey
990 and Storey, 2012). Similarly, a review of gene-expression changes
991 during dormancy across taxa including arthropods, fungi, and
992 mammals revealed that small heat-shock proteins (namely the
993 Hsp70 and Hsp90 families) are up-regulated in different forms of
994 dormancy, including aestivation (Denlinger, 2002), and likely act
995 as protective chaperone proteins. However, few studies have
996 examined changes in gene expression during insect aestivation.
997 For example, pupae of the onion maggot, Delia antiqua, can enter
998 both summer and winter diapause, and suppressive subtractive
999 hybridization identied genes related to stress response (heat-
1000 shock proteins), antimicrobial defense, metabolism, and food stor-
1001 age that were expressed signicantly more in summer diapause
1002 than in winter diapause (Hao et al., 2012). The authors of that
1003 study hypothesized that (i) heat-shock proteins are needed more
1004 in the summer than in winter, due to the extreme heat, and (ii)
1005 more defense proteins are needed because there are more patho-
1006 gens active in the soil in summer than in winter, given increased
1007 moisture and warmth in the summer. Similarly, a recent study of
1008 protein expression in the aestivating snail Pomacea canaliculata
1009 also revealed an upregulation of immune-response proteins, anti-
1010 oxidative proteins, and a suite of proteins involved lipid fuel-usage
1011 (Sun et al., 2013). Lastly, prior to migration, monarch butteries
1012 (Danaus plexippus) enter a state of reproductive diapause/arrest
1013 and increased longevity, and changes in expression of genes asso-
1014 ciated with circadian rhythm and feeding, along with juvenile hor-
1015 mone, were found in these butteries, relative to reproductively
1016 active summer butteries (Zhu et al., 2008).
1017 Although no studies to date have measured gene expression
1018 during mosquito aestivation, two studies have examined gene-
1019 expression changes due to relevant phenotypes: aging and desicca-
1020 tion stress. Using the laboratory G3 colony of An. gambiae (a mix of
1021 M and S molecular forms), age-related changes in gene expression,
1022 separate from gonotrophic-cycle effects, were measured across
1023 ages up to 28 days (Wang et al., 2010). Genes which increased in
1024 expression throughout the aging process included two detoxica-
1025 tion genes (cytochrome P450 and glutathione S-transferases) and
1026 Hsp70, a key stress-response gene (Wang et al., 2010); impor-
1027 tantly, these classes of genes have been found in previous studies
1028 on aestivation, as mentioned above, and may implicate these genes
1029 as part of the aestivation toolkit. In a similar study using the G3
1030 colony, changes in gene expression due to desiccation stress were
1031 measured, and overall, more genes showed decreased expression
1032 during desiccation than those which increased (Wang et al.,
1033 2011). Genes which were down-regulated during desiccation
1034 stress included chitin metabolism genes, oxidative-stress response
1035 genes, and binding function genes, while those which were up-reg-
1036 ulated during desiccation stress included amino-acid biosynthesis
1037 and Hsp40 (Wang et al., 2011).
1038 Combining these studies on An. gambiae with previous studies
1039 during aestivation of other insects, we hypothesize that changes
1040 in gene expression will likely occur in key classes of genes. Specif-
1041 ically, similar to previous studies, we predict increased levels of
1042 lipid metabolism genes, detoxication and immune-related pro-
1043 teins, genes related to building cuticular hydrocarbons and thicken-
1044 ing the cuticle, and heat-shock proteins. Simultaneously, we predict
1045 decreased levels of vitellogenesis-related genes and other genes
1046 associated with reproduction. Conversely, we predict that expres-
1047 sion of some other classes, including ight- and feeding-related
1048 genes, will not change dramatically, as mosquitoes are found (albeit
1049 in low numbers) throughout the dry season. How gene expression
1050 varies during the dry season between active and inactive phases
1051 (Adamou et al., 2011; Huestis et al., 2012; Lehmann et al., 2014),
1052 as well as throughout the progression of the dry season, might be
1053 unique for this strategy and thus render predictions based on stud-
1054 ies of overwintering diapause less powerful.
1055 7. Discussion and conclusions
1056 Here, we have focused on the physiology of mosquitoes during
1057 the dry season and mainly on those mechanisms that would poten-
1058 tially allow their persistence in environments without surface
1059 water across vast areas for at least three months (Adamou et al.,
1060 2011; Lehmann et al., 2010; Omer and Cloudsley-Thompson,
1061 1968, 1970; Simard et al., 2000). Insects, including mosquitoes,
1062 employ different forms of dormancy to survive harsh seasons
1063 (Box 1). Until recently, aestivation in African anophelines has been
1064 discounted by most entomologists, although winter diapause in
1065 temperate malaria vectors has been well-documented (Jetten and
1066 Takken, 1994), and a few studies provided evidence consistent
1067 with it in Sudan (Omer and Cloudsley-Thompson, 1968, 1970). In
1068 part, this stemmed from studies conducted in areas that experi-
1069 ence a mild dry season, during which some larval sites remain
1070 available within 510 km radius (Charlwood et al., 2000; Jawara
1071 et al., 2008; Koenraadt et al., 2003; Minakawa et al., 2001;
1072 Ramsdale and Fontaine, 1970a,b; Sogoba et al., 2007). Although
1073 less abundant, constantly available larval sites during the dry sea-
1074 son probably act as a strong selection force against aestivation.
1075 Future studies on this topic must recognize this key criterion in
1076 the selection of eld sites and source populations of mosquitoes
1077 for future laboratory studies on aestivation.
1078 The big picture of the physiological mechanisms that allow
1079 mosquitoes to cope with seasonal extremes is well understood
1080 with respect to winter diapause (e.g., Cx. pipiens, Cx. tarsalis, Wy.
1081 smithii, and Ae. albopictus), but is poorly understood with respect
1082 to aestivation (e.g., Cs. inornata) and even less so with respect to
1083 African anophelines in seasonally dry habitats such as the Sahel
1084 (Lehmann et al., 2010). Little attention has been paid to low-trans-
1085 mission periods in the study of disease vectors in sub-Saharan
1086 Africa, when it is often difcult to collect enough mosquitoes for
1087 experiments, and except for one study (Omer and Cloudsley-
1088 Thompson, 1968), induction of aestivation under laboratory condi-
1089 tions have failed. Here, we appraised the current knowledge on the
1090 dry-season physiology of African anophelines in reference to char-
1091 acteristics of the diapause syndrome, which includes aestivation,
1092 seeking to address the question, Is aestivation the strategy used
1093 by An. gambiae s.l.?
1094 A dramatic extension of lifespan during the dry season was
1095 reported for two members of the complex: An. arabiensis (Omer
1096 and Cloudsley-Thompson, 1968) and An. coluzzii (Lehmann et al.,
1097 2010), adding to an earlier observation of an unknown member(s)
1098 of An. gambiae s.l. that survived for over three months after collec-
1099 tion from the vicinity of Bobo Dioulasso, Burkina Faso (Holstein,
1100 1954). The physiological processes that facilitate such a dramatic
1101 change may well be aestivation or quiescence. Consistent with
1102 the dormancy framework, female An. arabiensis were reproduc-
1103 tively suppressed in a state of gonotrophic dissociation (Omer
1104 and Cloudsley-Thompson, 1970); similarly a 70% reduction in
1105 reproduction (Yaro et al., 2012) and an 80% reduction in ight
1106 activity (Huestis et al., 2012) was measured in An. coluzzii during
1107 the dry season as opposed to the wet season. On the other hand,
1108 metabolic rate was actually highest during the late dry season
18 June 2014
1109 (Huestis et al., 2012). Although elevated metabolic rate ies in the
1110 face of traditional expectations for dormancy (but see Brent et al.,
1111 2013), it is important to qualify studies that were conducted on
1112 host-seeking mosquitoes (most of them bloodfed) that were col-
1113 lected indoors, which may not represent the physiological state
1114 of the same mosquitoes if found in shelters. These mosquitoes
1115 may have interrupted their dormancy to replenish nutritional
1116 reserves and therefore may suspend at least certain aspects of their
1117 dormancy during that time (Huestis et al., 2012).
1118 Consistent with aestivation rather than with quiescence, the
1119 switch from reproduction to long-term survival was not a direct
1120 result of the absence of surface water for oviposition (Artis et al.,
1121 2014; Dieter et al., 2012), indicating that a token environmental
1122 stimulus is involved. Accordingly, An. coluzzii has been observed
1123 to nearly disappear from villages approximately one month before
1124 the larval sites dry up, presumably in anticipation of the coming
1125 dry season, as would be expected according to aestivation and con-
1126 trary to the quiescence hypothesis (Adamou et al., 2011; Huestis
1127 et al., 2012; Lehmann et al., 2010; Yaro et al., 2012). Likewise,
1128 induction of diapause by short photoperiod produced only a mod-
1129 est extension of An. coluzzii lifespan in either a eld insectary or the
1130 laboratory and a similarly small increase in the total cuticular
1131 hydrocarbons (Table 1). These results revealed a response to pho-
1132 toperiod in the expected direction, but its limited intensity indi-
1133 cates that the insectary conditions were unsuitable to attain full
1134 dormancy, possibly because of inadequate conditions to maintain
1135 dormant adults. Accordingly, more eld studies (64%, n = 11) have
1136 revealed evidence consistent with aestivation than laboratory
1137 studies (33%, n = 6; Table 1).
1138 A number of studies (some unpublished, Table 1) measured a
1139 slight increase in desiccation tolerance of An. coluzzii, either in
1140 comparison with An. gambiae s.s. (Lee et al., 2009), or in mosquitoes
1141 homozygous for the 2La inversion compared with homozygotes to
1142 the standard karyotype (Fouet et al., 2012; Gray et al., 2009). How-
1143 ever, most of these studies were not designed to test whether mos-
1144 quitoes were dormant, and they measured minimal differences
1145 (although statistically signicant) during the wet season (Lee
1146 et al., 2009) or between lines that were not induced to undergo
1147 dormancy; thus our inferences from their results are limited.
1148 Nonetheless, they have demonstrated that the tolerance to desicca-
1149 tion of An. coluzzii during the wet season cannot explain, by itself,
1150 this species persistence throughout the dry season in the face of
1151 the virtual disappearance of the An. gambiae s.s.
1152 Recognizing the diversity in the manifestation of diapause (aka
1153 diapause syndrome) between and within species and popula-
1154 tions (above), it is difcult to interpret the physiological changes
1155 over the season (Table 1 and above) outside of the dormancy spec-
1156 trum. The specic requirements for induction and latency implied
1157 by aestivation (Box 1) are not evident based on classical photope-
1158 riod experiments (Table 1). However, the early disappearance of
1159 An. coluzzii a month before larval sites dried up (above), the modest
1160 extension of lifespan in response to shortened photoperiod
1161 (Lehmann et al., 2010, 2014; Table 1), and the suppression of
1162 reproduction in the presence of water for oviposition (Yaro et al.,
1163 2012; Table 1), as well as the failure to utilize articial oviposition
1164 sites during the dry season by anophelines but not culicines
1165 (Lehmann et al., 2010), all agree with an induction and latency.
1166 We therefore propose that the existing ndings support a form of
1167 aestivation as the basis of the strategy of An. coluzzii in the West
1168 African Sahel and An. arabiensis in the East African Sahel and pos-
1169 sibly in Senegal (Lemasson et al., 1997; Simard et al., 2000). This
1170 form of aestivation differs from the hibernal diapause typied by
1171 Cx. pipiens and other temperate mosquito species that undergo
1172 winter diapause as adults, as explained below.
1173 The winter in temperate latitudes implies freezing tempera-
1174 tures, limited insect activity (ight), a lack of sugar sources (ow-
1175 ers and plant juices), and desiccating conditions; combined, these
1176 factors result in greatly reduced mobility or complete immobility
1177 of the insect and hence total reliance on physiological solutions
1178 to cope with the harsh environment. For Sahelian anophelines,
1179 on the other hand, the primary restrictive force of the dry season
1180 is the absence of surface waters for larval development (Fig. 2). Diel
1181 uctuations in temperature (Fig. 1) and the corresponding changes
1182 in relative humidity likely conne ight to certain parts of the
1183 night (Fig. 1), but they promote foraging for blood and available
1184 sugar sources and even male swarming (above; Fig. 2). We hypoth-
1185 esize that behavioral changes in selecting suitable microhabitats in
1186 shelters and suitable periods of activity and rest may have a large
1187 role in complementing physiological changes, rather than relying
1188 on them completely, as is the case for winter diapause. A greater
1189 reliance on behavioral elements probably requires a different set
1190 of physiological changes (Fig. 2), as may be reected by the bouts
1191 of elevated metabolism (Huestis et al., 2012) and local movement
1192 (Lehmann et al., 2014) in the late dry season. Specialized behaviors
1193 such as digging into ground and/or cocoon formation exhibited by
1194 caterpillars and beetles (Tauber et al., 1986) and the switch into
1195 sugar feeding and accumulating lipid reserves in mosquitoes
1196 (Robich and Denlinger, 2005) are of paramount importance to
1197 overwintering diapause, although they are expressed in the dia-
1198 pause-initiation phase rather than the diapause-maintenance
1199 phase, which we presume would be the case for African anophe-
1200 lines. Clearly this hypothesis requires empirical evidence, which
1201 presents attractive challenges for future research. Finding the shel-
1202 ters used by mosquitoes during the dry season would allow us to
1203 measure the metabolic rate, activity patterns, and blood-feeding
1204 responses of mosquitoes in this active dormant state. More
1205 research is needed to dissect the environmental cues which induce
1206 aestivation, the conditions required to maintain it, and those that
1207 signal its end. Lastly, gene-expression studies on the molecular
1208 basis of the physiological changes which occur during aestivation
1209 will benet from starting with those essential genes noted by stud-
1210 ies on hibernal diapause and aestivation in other organisms (e.g.,
1211 heat-shock, storage-utilization, and reproduction-related proteins,
1212 see above).
1213 A comprehensive picture of the strategy used by mosquitoes to
1214 persist throughout the dry season could yield novel vector control
1215 strategies and make positive impacts on malaria eradication in the
1216 future. Specically, if the shelters used by these mosquitoes could
1217 be identied, predicted, and subjected to vector control, aestivat-
1218 ing mosquitoes could be targeted during the dry season, markedly
1219 reducing the overall disease burden during the transmission sea-
1220 son. Likewise, if the genes underlying this unique physiological
1221 state are known, they may be targeted to prevent mosquitoes from
1222 completing their aestivation. Thus, the continued studies on the
1223 physiological and behavioral strategies used by aestivating anoph-
1224 elines are ongoing and remain a frontier in medical entomology.
1225 Acknowledgements
1226 We thank Dia Elnaiem, Peter Armbruster, Phil Lounibos, and 1
1227 anonymous reviewer for their comments on earlier versions of this
1228 manuscript. Research conducted by the authors mentioned
1229 throughout was supported by the Division of Intramural Research,
1230 NIAID, NIH.
1231 References
1232 Aboagye-Antwi, F., Tripet, F., 2010. Effects of larval growth condition and water
1233
availability on desiccation resistance and its physiological basis in adult
1234
Anopheles gambiae sensu stricto. Malar. J. 9, 225.
1235 Adamou, A., Dao, A., Timbine, S., Kassogue, Y., Yaro, A.S., Diallo, M., Traore, S.F.,
1236
Huestis, D.L., Lehmann, T., 2011. The contribution of aestivating mosquitoes to
1237 the persistence of Anopheles gambiae in the Sahel. Malar. J. 10, 151.
18 June 2014
1238 Andrewartha, H.G., 1952. Diapause in relation to the ecology of insects. Biol. Rev.
1239
Camb. Philos. Soc. 27, 50107.
1240 Barnard, D.R., Mulla, M.S., 1977. Effects of photoperiod and temperature on blood
1241
feeding, oogenesis, and fat body development in the mosquito, Culiseta inornata.
1242 J. Insect Physiol. 23, 12611266.
1243
Barnard, D.R., Mulla, M.S., 1978. The ecology of Culiseta inornata in the Colorado
1244
Desert of California: seasonal abundance, gonotrophic status, and oviparity of
1245 adult mosquitoes. Ann. Entomol. Soc. Am. 71, 397401.
1246
Bates, M., 1949. The Natural History of Mosquitoes. MacMillan, New York.
1247 Bayoh, M.N., Thomas, C.J., Lindsay, S.W., 2001. Mapping distributions of
1248
chromosomal forms of Anopheles gambiae in West Africa using climate data.
1249
Med. Vet. Entomol. 15, 267274.
1250 Beach, R., 1978. Required day number and timely induction of diapause in
1251
geographic strains of mosquito Aedes atropalpus. J. Insect Physiol. 24, 449455.
1252 Beier, J.C., Copeland, R.S., Oyaro, C., Masinya, A., Odago, W.O., Odour, S., Koech, D.K.,
1253
Roberts, C.R., 1990. Anopheles gambiae complex egg stage survival in dry soil
1254 from larval development sites in western Kenya. J. Am. Mosq. Control Assoc. 6,
1255
105109.
1256
Bennett, V.A., Kukal, O., Lee, R.E., 1999. Metabolic opportunists: feeding and
1257 temperature inuence the rate and pattern of respiration in the high arctic
1258
woollybear caterpillar Gynaephora groenlandica (Lymantriidae). J. Exp. Biol. 202,
1259 4753.
1260
Benoit, J.B., Denlinger, D.L., 2007. Suppression of water loss during adult diapause in
1261
the northern house mosquito, Culex pipiens. J. Exp. Biol. 210, 217226.
1262 Benoit, J.B., Lopez-Martinez, G., Phillips, Z.P., Patrick, K.R., Denlinger, D.L., 2010a.
1263
Heat shock proteins contribute to mosquito dehydration tolerance. J. Insect
1264 Physiol. 56, 151156.
1265
Benoit, J.B., Patrick, K.R., Desai, K., Hardesty, J.J., Krause, T.B., Denlinger, D.L., 2010b.
1266
Repeated bouts of dehydration deplete nutrient reserves and reduce egg
1267 production in the mosquito Culex pipiens. J. Exp. Biol. 213, 27632769.
1268
Bradshaw, W.E., Lounibos, L.P., 1977. Evolution of dormancy and its photoperiodic
1269 control in pitcher-plant mosquitoes. Evolution 31, 546567.
1270
Brakeeld, P.M., Pijpe, J., Zwaan, B.J., 2007. Developmental plasticity and
1271 acclimation both contribute to adaptive responses to alternating seasons of
1272
plenty and of stress in Bicyclus butteries. J. Biosci. 32, 465475.
1273
Brent, C.S., Klok, C.J., Naranjo, S.E., 2013. Effect of diapause status and gender on
1274 activity, metabolism, and starvation resistance in the plant bug Lygus hesperus.
1275
Entomol. Exp. Appl. 148, 152160.
1276 Buth, J.L., Brust, R.A., Ellis, R.A., 1990. Development time, oviposition activity and
1277
onset of diapause in Culex tarsalis, Culex restuans, and Culiseta inornata in
1278
southern Manitoba. J. Am. Mosq. Control Assoc. 6, 5563.
1279 Canzano, A.A., Krockenberger, A.A., Jones, R.E., Seymour, J.E., 2006. Rates of
1280
metabolism in diapausing and reproductively active tropical butteries,
1281 Euploea core and Euploea sylvester (Lepidoptera: Nymphalidae). Physiol.
1282
Entomol. 31, 184189.
1283 Chambers, R.J., 1982. Maternal experience of crowding and duration of aestivation
1284
in the sycamore aphid. Oikos 39, 100102.
1285
Charlwood, J.D., Vij, R., Billingsley, P.F., 2000. Dry season refugia of malaria-
1286 transmitting mosquitoes in a dry savannah zone of east Africa. Am. J. Trop. Med.
1287
Hyg. 62, 726732.
1288 Charlwood, J.D., Cuamba, N., Tomas, E.V., Briet, O.J., 2013. Living on the edge: a
1289
longitudinal study of Anopheles funestus in an isolated area of Mozambique.
1290
Malar. J. 12, 208.
1291 Chown, S.L., 2002. Respiratory water loss in insects. Comparative biochemistry and
1292
physiology A-molecular and integrative physiology 133, 791804.
1293 Chown, S.L., Nicolson, S.W., 2004. Insect Physiological Ecology: Mechanisms and
1294
Patterns. Oxford University Press, Oxford.
1295 Clarke, A., Fraser, K.P.P., 2004. Why does metabolism scale with temperature?.
1296
Funct. Ecol. 18, 243251.
1297
Clements, A.N., 1963. The Physiology of Mosquitoes. Pergamon Press, Oxford.
1298 Clements, A.N., 1992. The Biology of Mosquitoes. Chapman & Hall, London.
1299
Coetzee, M., Hunt, R.H., Wilkerson, R., Della Torre, A., Coulibaly, M.B., Besansky, N.J.,
1300 2013. Anopheles coluzzii and Anopheles amharicus, new members of the
1301
Anopheles gambiae complex. Zootaxa 3619, 246274.
1302
Coluzzi, M., 1964. Morphological divergences in the Anopheles gambiae complex.
1303 Riv. Malariol. 43, 197232.
1304
Coluzzi, M., Sabatini, A., Petrarca, V., Di Deco, M.A., 1979. Chromosomal
1305 differentiation and adaptation to human environments in the Anopheles
1306
gambiae complex. Trans. R. Soc. Trop. Med. Hyg. 73, 483497.
1307 Coluzzi, M., Petrarca, V., Di Deco, M.A., 1985. Chromosomal inversion intergradation
1308
and incipient speciation in Anopheles gambiae. Boll. Zool. 52, 4563.
1309
Daibo, S., Kimura, M.T., Goto, S.G., 2001. Upregulation of genes belonging to the
1310 drosomycin family in diapausing adults of Drosophila triauraria. Gene 278, 177
1311
184.
1312 Danilevskii, A.S., 1965. Photoperiodism and Seasonal Development of Insects. Oliver
1313
and Boyd, Edinburgh and London.
1314
Davidson, G., 1964. Anopheles gambiae, a complex of species. Bull. World Health
1315 Organ. 31, 625634.
1316
Denlinger, D.L., 1986. Dormancy in tropical insects. Annu. Rev. Entomol. 31, 239264.
1317 Denlinger, D.L., 2002. Regulation of diapause. Annu. Rev. Entomol. 47, 93122.
1318
Denlinger, D.L., Armbruster, P.A., 2014. Mosquito diapause. Annu. Rev. Entomol. 59,
1319
7393.
1320 Depner, K.R., Harwood, R.F., 1966. Photoperiodic responses of 2 latitudinally diverse
1321
groups of Anopheles freeborni (Diptera Culicidae). Ann. Entomol. Soc. Am. 59,
1322 711.
1323 Dieter, K.L., Huestis, D.L., Lehmann, T., 2012. The effects of oviposition-site
1324
deprivation on Anopheles gambiae reproduction. Parasit. Vectors 5, 235.
1325 Dingle, H., 1996. Migration: The Biology of Life on the Move. Oxford University
1326
Press, New York.
1327 Dixon, A.F.G., 1966. The effect of population density and nutritive status of the host
1328
on the summer reproductive activity of the sycamore aphid, Drepanosiphum
1329
platanoides (Schr.). J. Anim. Ecol. 35, 105112.
1330 Dixon, A.F.G., Wellings, P.W., Carter, C., Nichols, J.F.A., 1993. The role of food quality
1331
and competition in shaping the seasonal cycle in the reproductive activity of the
1332 sycamore aphid. Oecologia 95, 8992.
1333
Djawdan, M., Rose, M.R., Bradley, T.J., 1997. Does selection for stress resistance
1334
lower metabolic rate?. Ecology 78, 828837.
1335 Donnelly, M.J., Simard, F., Lehmann, T., 2002. Evolutionary studies of malaria
1336
vectors. Trends Parasitol. 18, 7580.
1337 Douglas, A.E., 2000. Reproductive diapause and the bacterial symbiosis in
1338
the sycamore aphid Drepanosiphum platanoidis (Schr.). Ecol. Entomol. 25,
1339 256261.
1340
Eldridge, B.F., 1987. Diapause and Related Phenomena in Culex Mosquitoes: Their
1341
Relation to Arbovirus Disease Ecology, Current Topics in Vector Research.
1342 Springer, New York, pp. 128.
1343
Emerson, K.J., Bradshaw, W.E., Holzapfel, C.M., 2009. Complications of complexity:
1344 integrating environmental, genetic and hormonal control of insect diapause.
1345
Trends Genet. 25, 217225.
1346
Focks, D.A., Linda, S.B., Craig, G.B., Hawley, W.H., Pumpuni, C.B., 1994. Aedes
1347 albopictus (Diptera: Culicidae): a statistical model of the role of temperature,
1348
photoperiod, and geography in the induction of egg diapause. J. Med. Entomol.
1349 31, 276286.
1350
Fouet, C., Gray, E., Besansky, N.J., Costantini, C., 2012. Adaptation to aridity in the
1351
malaria mosquito Anopheles gambiae: chromosomal inversion, polymorphism,
1352 and body size inuence resistance to desiccation. PLoS ONE 7, e34841.
1353
Gibbs, A.G., Johnson, R.A., 2004. The role of discontinuous gas exchange in insects:
1354 the chthonic hypothesis does not hold water. J. Exp. Biol. 207, 34773482.
1355
Gibbs, A.G., Chippindale, A.K., Rose, M.R., 1997. Physiological mechanisms of
1356 evolved desiccation resistance in Drosophila melanogaster. J. Exp. Biol. 200,
1357
18211832.
1358
Gibbs, A.G., Fukuzato, F., Matzkin, L.M., 2003. Evolution of water conservation
1359 mechanisms in Drosophila. J. Exp. Biol. 206, 11831192.
1360
Gillies, M.T., De Meillon, B., 1968. The Anophelinae of Africa south of the Sahara,
1361 second ed. South African Institute for Medical Research, Johannesburg, South
1362
Africa.
1363
Gray, E.M., Bradley, T.J., 2005. Physiology of desiccation resistance in Anopheles
1364 gambiae and Anopheles arabiensis. Am. J. Trop. Med. Hyg. 73, 553559.
1365
Gray, E.M., Rocca, K.A., Costantini, C., Besansky, N.J., 2009. Inversion 2La is
1366 associated with enhanced desiccation resistance in Anopheles gambiae. Malar.
1367
J. 8, 215.
1368 Guppy, M., 2004. The biochemistry of metabolic depression: a history of
1369
perceptions. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 139, 435442.
1370
Guppy, M., Withers, P., 1999. Metabolic depression in animals: physiological
1371 perspectives and biochemical generalizations. Biol. Rev. Camb. Philos. Soc. 74,
1372
140.
1373 Hadley, N.F., 1994. Ventilatory patterns and respiratory transpiration in adult
1374
terrestrial insects. Physiol. Zool. 67, 175189.
1375
Hahn, D.A., Denlinger, D.L., 2011. Energetics of insect diapause. In: Berenbaum, M.R.,
1376 Carde, R.T., Robinson, G.E. (Eds.), Annual Review of Entomology, vol. 56. Annual
1377
Reviews, Palo Alto, pp. 103121.
1378 Hairston, N.G., Kearns, C.M., 1995. The interaction of photoperiod and temperature
1379
in diapause timing a copepod example. Biol. Bull. 189, 4248.
1380 Hao, Y.J., Li, W.S., He, Z.B., Si, F.L., Ishikawa, Y., Chen, B., 2012. Differential gene
1381
expression between summer and winter diapause pupae of the onion maggot
1382
Delia antiqua, detected by suppressive subtractive hybridization. J. Insect
1383 Physiol. 58, 14441449.
1384
Harwood, R.F., Halfhill, E., 1964. The effect of photoperiod on fat body and ovarian
1385 development of Culex tarsalis (Diptera: Culicidae). Ann. Entomol. Soc. Am. 57,
1386
596600.
1387
Held, C., Spieth, H.R., 1999. First evidence of pupal summer diapause in Pieris
1388 brassicae L.: the evolution of local adaptedness. J. Insect Physiol. 45, 587598.
1389
Holstein, M.H., 1954. Biology of Anopheles gambiae. Research in French West Africa,
1390 World Health Organization, Geneva.
1391
Hudson, J.E., 1977. Induction of diapause in female mosquitoes, Culiseta inornata, by
1392 a decrease in daylength. J. Insect Physiol. 23, 13771382.
1393
Huestis, D.L., Yaro, A.S., Traore, A.I., Adamou, A., Kassogue, Y., Diallo, M., Timbine, S.,
1394
Dao, A., Lehmann, T., 2011. Variation in metabolic rate of Anopheles gambiae and
1395 A. arabiensis in a Sahelian village. J. Exp. Biol. 214, 23452353.
1396
Huestis, D.L., Yaro, A.S., Traore, A.I., Dieter, K.L., Nwagbara, J.I., Bowie, A.C., Adamou,
1397 A., Kassogue, Y., Diallo, M., Timbine, S., Dao, A., Lehmann, T., 2012. Seasonal
1398
variation in metabolic rate, ight activity and body size of Anopheles gambiae in
1399
the Sahel. J. Exp. Biol. 215, 20132021.
1400 Ikeno, T., Tanaka, S.I., Numata, H., Goto, S.G., 2010. Photoperiodic diapause under
1401
the control of circadian clock genes in an insect. BMC Biol. 8.
1402 Jaenson, T.G.T., Ameneshewa, B., 1991. Prehibernation diet and reproductive
1403
condition of female Anopheles messeae in Sweden. Med. Vet. Entomol. 5, 243
1404
252.
1405 Jawara, M., Pinder, M., Drakeley, C.J., Nwakanma, D.C., Jallow, E., Bogh, C., Lindsay,
1406
S.W., Conway, D.J., 2008. Dry season ecology of Anopheles gambiae complex
1407 mosquitoes in The Gambia. Malar. J. 7, 156.
18 June 2014
1408 Jetten, T.H., Takken, W., 1994. Anophelism without malaria in Europe: a review of
1409
the ecology and distribution of the genus Anopheles in Europe. Wageningen
1410 Agricultural University Papers 94-5, pp. 169..
1411
Kaufmann, C., Briegel, H., 2004. Flight performance of the malaria vectors Anopheles
1412 gambiae and Anopheles atroparvus. J. Vector Ecol. 29, 140153.
1413
Keilin, D., 1959. The problem of anabiosis or latent life: history and current concept.
1414
Proc. R. Soc. Lond. B Biol. Sci. 150, 149191.
1415 Kim, M., Denlinger, D.L., 2009. Decrease in expression of beta-tubulin and
1416
microtubule abundance in ight muscles during diapause in adults of Culex
1417 pipiens. Insect Mol. Biol. 18, 295302.
1418
Kim, M., Robich, R.M., Rinehart, J.P., Denlinger, D.L., 2006. Upregulation of two actin
1419
genes and redistribution of actin during diapause and cold stress in the
1420 northern house mosquito, Culex pipiens. J. Insect Physiol. 52, 12261233.
1421
Kirby, M.J., Lindsay, S.W., 2004. Responses of adult mosquitoes of two sibling
1422 species, Anopheles arabiensis and A. gambiae s.s. (Diptera: Culicidae), to high
1423
temperatures. Bull. Entomol. Res. 94, 441448.
1424 Koenraadt, C.J., Paaijmans, K.P., Githeko, A.K., Knols, B.G., Takken, W., 2003. Egg
1425
hatching, larval movement and larval survival of the malaria vector Anopheles
1426
gambiae in desiccating habitats. Malar. J. 2, 20.
1427 Kostal, V., 2006. Eco-physiological phases of insect diapause. J. Insect Physiol. 52,
1428
113127.
1429 Kostal, V., Hodek, I., 1997. Photoperiodism and control of summer diapause in the
1430
Mediterranean tiger moth Cymbalophora pudica. J. Insect Physiol. 43, 767777.
1431
Kostal, V., Simek, P., 1998. Changes in fatty acid composition of phospholipids and
1432 triacylglycerols after cold-acclimation of an aestivating insect prepupa. J. Comp.
1433
Physiol. B, Biochem. Syst. Environ. Physiol. 168, 453460.
1434 Kostal, V., Sula, J., Simek, P., 1998. Physiology of drought tolerance and cold
1435
hardiness of the Mediterranean tiger moth Cymbalophora pudica during
1436
summer diapause. J. Insect Physiol. 44, 165173.
1437 Lanciani, C.A., 1993. Photoperiod and longevity in Anopheles crucians. J. Am. Mosq.
1438
Control Assoc. 9, 308312.
1439 Lanciani, C.A., Anderson, J.F., 1993. Effect of photoperiod on longevity and metabolic
1440
rate in Anopheles quadrimaculatus. J. Am. Mosq. Control Assoc. 9, 158163.
1441 Lee, Y., Meneses, C.R., Fofana, A., Lanzaro, G.C., 2009. Desiccation resistance among
1442
subpopulations of Anopheles gambiae s.s. From Selinkenyi, Mali. J. Med. Entomol.
1443
46, 316320.
1444 Lehmann, T., Diabate, A., 2008. The molecular forms of Anopheles gambiae: a
1445
phenotypic perspective. Infect. Genet. Evol. 8, 737746.
1446 Lehmann, T., Dao, A., Yaro, A.S., Adamou, A., Kassogue, Y., Diallo, M., Sekou, T.,
1447
Coscaron-Arias, C., 2010. Aestivation of the African malaria mosquito, Anopheles
1448
gambiae, in the Sahel. Am. J. Trop. Med. Hyg. 83, 601606.
1449 Lehmann, T., Dao, A., Yaro, A.S., Diallo, M., Timbine, S., Huestis, D.L., Adamou, A.,
1450
Kassogue, Y., Traore, A.I., 2014. Seasonal variation in spatial distributions of
1451 Anopheles gambiae in a Sahelian village: evidence for aestivation. J. Med.
1452
Entomol. 51, 2738.
1453 Lemasson, J.J., Fontenille, D., Lochouarn, L., Dia, I., Simard, F., Ba, K., Diop, A., Diatta,
1454
M., Molez, J.F., 1997. Comparison of behavior and vector efciency of Anopheles
1455
gambiae and An. arabiensis (Diptera: Culicidae) in Barkedji, a Sahelian area of
1456 Senegal. J. Med. Entomol. 34, 396403.
1457
Lighton, J.R.B., 1996. Discontinuous gas exchange in insects. Annu. Rev. Entomol. 41,
1458 309324.
1459
Liu, Z.D., Gong, P.Y., Wu, K.J., Sun, J.H., Li, D.M., 2006. A true summer diapause
1460
induced by high temperatures in the cotton bollworm, Helicoverpa armigera
1461 (Lepidoptera: Noctuidae). J. Insect Physiol. 52, 10121020.
1462
Lounibos, L.P., Escher, R.L., Lourenco-de-Oliveria, R., 2003. Asymmetric evolution of
1463 photoperiodic diapause in temperate and tropical invasive populations of Aedes
1464
albopictus (Diptera: Culicidae). Ann. Entomol. Soc. Am. 96, 512518.
1465 Lounibos, L.P., Escher, R.L., Nishimura, N., 2011. Retention and adaptiveness of
1466
photoperiodic egg diapause in Florida populations of invasive Aedes albopictus. J.
1467
Am. Mosq. Control Assoc. 27, 433436.
1468 Luker, L.A., Hatle, J.D., Juliano, S.A., 2002. Reproductive responses to photoperiod by
1469
a south Florida population of the grasshopper Romalea microptera (Orthoptera:
1470 Romaleidae). Environ. Entomol. 31, 702707.
1471
MacRae, T.H., 2010. Gene expression, metabolic regulation and stress tolerance
1472
during diapause. Cell. Mol. Life Sci. 67, 24052424.
1473 Mamai, W., Mouline, K., Blais, C., Larvor, V., Dabire, K.R., Ouedraogo, G.A., Simard, F.,
1474
Renault, D., 2014. Metabolomic and ecdysteroid variations in Anopheles gambiae
1475 s.l. mosquitoes exposed to the stressful conditions of the dry season in Burkina
1476
Faso, West Africa. Physiol. Biochem. Zool. 87, 486497.
1477 Masaki, S., 1980. Summer diapause. Annu. Rev. Entomol. 25, 125.
1478
Minakawa, N., Githure, J.I., Beier, J.C., Yan, G.Y., 2001. Anopheline mosquito survival
1479
strategies during the dry period in western Kenya. J. Med. Entomol. 38, 388
1480 392.
1481
Nagpal, B.N., Srivastava, A., Kalra, N.L., Subbarao, S.K., 2003. Spiracular indices in
1482 Anopheles stephensi: a taxonomic tool to identify ecological variants. J. Med.
1483
Entomol. 40, 747749.
1484
Omer, S.M., Cloudsley-Thompson, J.L., 1968. Dry season biology of Anopheles
1485 gambiae Giles in the Sudan. Nature 217, 879880.
1486
Omer, S.M., Cloudsley-Thompson, J.L., 1970. Survival of female Anopheles gambiae
1487 Giles through a 9-month dry season in Sudan. Bull. World Health Organ. 42,
1488
319330.
1489
Poelchau, M.F., Reynolds, J.A., Elsik, C.G., Denlinger, D.L., Armbruster, P.A., 2013a.
1490 Deep sequencing reveals complex mechanisms of diapause preparation in the
1491
invasive mosquito, Aedes albopictus. Proc. R. Soc. B Biol. Sci., 280.
1492 Poelchau, M.F., Reynolds, J.A., Elsik, C.G., Denlinger, D.L., Armbruster, P.A., 2013b.
1493
RNA-Seq reveals early distinctions and late convergence of gene expression
1494 between diapause and quiescence in the Asian tiger mosquito, Aedes albopictus.
1495
J. Exp. Biol. 216, 40824090.
1496 Pullin, A.S., Wolda, H., 1993. Glycerol and glucose accumulation during diapause in
1497
a tropical beetle. Physiol. Entomol. 18, 7578.
1498 Ragland, G.J., Fuller, J., Feder, J.L., Hahn, D.A., 2009. Biphasic metabolic rate
1499
trajectory of pupal diapause termination and post-diapause development in a
1500
tephritid y. J. Insect Physiol. 55, 344350.
1501 Ramsdale, C.D., Fontaine, R.E., 1970a. Ecological investigations of Anopheles gambiae
1502
and Anopheles funestus II. Dry season studies with colony-reared A. gambiae
1503 species B, Kaduna Nigeria. WHO/VBC/70.249, pp. 18..
1504
Ramsdale, C.D., Fontaine, R.E., 1970b. Ecological investigations of Anopheles gambiae
1505
and Anopheles funestus I. Dry season studies in villages near Kaduna Nigeria.
1506 WHO/VBC/70.248..
1507
Rao, V.V., 1947. On gonotrophic discordance among certain Indian Anopheles. Indian
1508 J. Malariol. 1, 4350.
1509
Reisen, W.K., Meyer, R.P., Milby, M.M., 1989. Studies on the seasonality of
1510 Culiseta inornata in Kern County, California. J. Am. Mosq. Control Assoc. 5,
1511
183195.
1512
Reisen, W.K., Thiemann, T., Barker, C.M., Lu, H.L., Carroll, B., Fang, Y., Lothrop, H.D.,
1513 2010. Effects of warm winter temperature on the abundance and gonotrophic
1514
activity of Culex (Diptera: Culicidae) in California. J. Med. Entomol. 47,
1515 230237.
1516
Robich, R.M., Denlinger, D.L., 2005. Diapause in the mosquito Culex pipiens evokes a
1517
metabolic switch from blood feeding to sugar gluttony. Proc. Natl. Acad. Sci.
1518 U.S.A. 102, 1591215917.
1519
Rocca, K.A.C., Gray, E.M., Costantini, C., Besansky, N.J., 2009. 2La chromosomal
1520 inversion enhances thermal tolerance of Anopheles gambiae larvae. Malar. J. 8,
1521
147.
1522
Sim, C., Denlinger, D.L., 2008. Insulin signaling and FOXO regulate the overwintering
1523 diapause of the mosquito Culex pipiens. Proc. Natl. Acad. Sci. U.S.A. 105, 6777
1524
6781.
1525 Sim, C., Denlinger, D.L., 2009. A shut-down in expression of an insulin-like peptide,
1526
ILP-1, halts ovarian maturation during the overwintering diapause of the
1527 mosquito Culex pipiens. Insect Mol. Biol. 18, 325332.
1528
Simard, F., Lehmann, T., Lemasson, J.J., Diatta, M., Fontenille, D., 2000. Persistence of
1529
Anopheles arabiensis during the severe dry season conditions in Senegal: an
1530 indirect approach using microsatellite loci. Insect Mol. Biol. 9, 467479.
1531
Sinclair, B.J., Bretman, A., Tregenza, T., Tomkins, J.L., Hosken, D.J., 2011. Metabolic
1532 rate does not decrease with starvation in Gryllus bimaculatus when changing
1533
fuel use is taken into account. Physiol. Entomol. 36, 8489.
1534
Sogoba, N., Doumbia, S., Vounatsou, P., Baber, I., Keita, M., Maiga, M., Traore, S.F.,
1535 Toure, A., Dolo, G., Smith, T., Ribeiro, J.M.C., 2007. Monitoring of larval habitats
1536
and mosquito densities in the Sudan savanna of Mali: implications for malaria
1537 vector control. Am. J. Trop. Med. Hyg. 77, 8288.
1538
Spielman, A., 1974. Effect of synthetic juvenile hormone on ovarian diapause of
1539 Culex pipiens mosquitoes. J. Med. Entomol. 11, 223225.
1540
Spielman, A., 2001. Structure and seasonality of nearctic Culex pipiens populations.
1541
In: White, D.J., Morse, D.L. (Eds.), West Nile Virus: Detection, Surveillance, and
1542 Control. New York Acad Sciences, New York, pp. 220234.
1543
Spielman, A., Wong, J., 1973. Environmental control of ovarian diapause in Culex
1544 pipiens (Diptera: Culicidae). Ann. Entomol. Soc. Am. 66, 905907.
1545
Storey, K.B., Storey, J.M., 1990. Metabolic rate depression and biochemical
1546
adaptation in anaerobiosis, hibernation, and estivation. Q. Rev. Biol. 65, 145
1547 174.
1548
Storey, K.B., Storey, J.M., 2004. Metabolic rate depression in animals: transcriptional
1549 and translational controls. Biol. Rev. 79, 207233.
1550
Storey, K.B., Storey, J.M., 2012. Aestivation: signaling and hypometabolism. J. Exp.
1551 Biol. 215, 14251433.
1552
Sun, J., Mu, H.W., Zhang, H.M., Chandramouli, K.H., Qian, P.Y., Wong, C.K.C., Qiu, J.W.,
1553
2013. Understanding the regulation of estivation in a freshwater snail through
1554 iTRAQ-based comparative proteomics. J. Proteome Res. 12, 52715280.
1555
Swellengrebel, N.H., 1929. La dissociation des fonctions sexuelles de nutritives
1556 (dissociation gonotrophique) dAnopheles maculipennis comme cause du
1557
paludisme dans les Pays-Bas et ses rapports ave linfection domiciliare. Ann.
1558
Inst. Pasteur 43, 13701389.
1559 Tauber, M.J., Tauber, C.A., Masaki, S., 1986. Seasonal Adaptations of Insects. Oxford
1560
University Press, New York.
1561 Tauber, M.J., Tauber, C.A., Nyrop, J.P., Villani, M.G., 1998. Moisture, a vital but
1562
neglected factor in the seasonal ecology of insects: hypotheses and tests of
1563 mechanisms. Environ. Entomol. 27, 523530.
1564
Tombes, A.S., 1964. Respiratory and compositional study on the aestivating insect,
1565
Hypera postica (Gyll) (Curculionidae). J. Insect Physiol. 10, 9971003.
1566 Toure, Y.T., Petrarca, V., Traore, S.F., Coulibaly, A., Maiga, H.M., Sankare, O., Sow, M., Di
1567
Deco, M.A., Coluzzi, M., 1994. Ecological genetic studies inthe chromosomal form
1568 Mopti of Anopheles gambiae s.str. in Mali, West Africa. Genetica 94, 213223.
1569
Tsuda, Y., Kim, K.S., 2008. Sudden autumnal appearance of adult Culex
1570
tritaeniorhynchus (Diptera: Culicidae) at a park in urban Tokyo: rst eld
1571 evidence for prediapause migration. J. Med. Entomol. 45, 610616.
1572
Urbanski, J., Mogi, M., ODonnell, D., DeCotiis, M., Toma, T., Armbruster, P., 2012.
1573 Rapid adaptive evolution of photoperiodic response during invasion and range
1574
expansion across a climatic gradient. Am. Nat. 179, 490500.
1575
Vinogradova, 1960. An experimental investigation of the ecological factors inducing
1576 imaginal diapause in blood sucking mosquitoes. Entomol. Rev. 39,
1577
210219.
1578 Vinogradova, E.B., Pantyuchov, G.A., 1995. Adult diapause and its physiological
1579
characteristic in the ragweed leaf beetle, Zygogramma suturalis F (Coleoptera,
18 June 2014
1580 Chrysomelidae), introduced to Russia from north America. Folia Biol. (Krakow)
1581
43, 137141.
1582 Vinogradova, E.B., De Statsio, B., Gilbert, J.J., 2007. Diapause in aquatic insects, with
1583
emphasis on mosquitoes. In: Alekseev, V.R. (Ed.), Diapause in Aquatic
1584 Invertebrates. Springer, pp. 83113.
1585
Wachira, S.W., Ndungu, M., Njagi, P.G.N., Hassanali, A., 2010. Comparative
1586
responses of ovipositing Anopheles gambiae and Culex quinquefasciatus females
1587 to the presence of Culex egg rafts and larvae. Med. Vet. Entomol. 24, 369374.
1588
Wagoner, K.M., Lehmann, T., Huestis, D.L., Cech, N.B., Ehrmann, B.M., Wasserberg,
1589 G., 2014. Identication of morphologic and chemical markers of dry- and wet-
1590
season conditions in female Anopheles gambiae mosquitoes. Parasit. Vectors (in
1591
press).
Q4
.
1592 Wang, M.H., Marinotti, O., James, A.A., Walker, E., Githure, J., Yan, G.Y., 2010.
1593
Genome-wide patterns of gene expression during aging in the African malaria
1594 vector Anopheles gambiae. PLoS ONE 5, e13359.
1595
Wang, M.H., Marinotti, O., Vardo-Zalik, A., Boparai, R., Yan, G.Y., 2011. Genome-wide
1596 transcriptional analysis of genes associated with acute desiccation stress in
1597
Anopheles gambiae. PLoS ONE 6, e26011.
1598 Washino, R.K., 1970. Physiological condition of overwintering female Anopheles
1599
freeborni in California (Diptera: Culicidae). Ann. Entomol. Soc. Am. 63, 210.
1600 Washino, R.K., 1977. The physiological ecology of gonotrophic dissociation and
1601
related phenomena in mosquitoes. J. Med. Entomol. 13, 381388.
1602 Washino, R.K., Bailey, S.F., 1970. Overwintering of Anopheles punctipennis (Diptera:
1603
Culicidae) in California. J. Med. Entomol. 7, 95.
1604
WHO, 2013. World Malaria Report 2013. World Health Organization, Geneva,
1605 Switzerland.
1606
Yaro, A.S., Traore, A.I., Huestis, D.L., Adamou, A., Timbine, S., Kassogue, Y., Diallo, M.,
1607 Dao, A., Traore, S.F., Lehmann, T., 2012. Dry season reproductive depression of
1608
Anopheles gambiae in the Sahel. J. Insect Physiol. 58, 10501059.
1609
Zhou, G.L., Miesfeld, R.L., 2009. Energy metabolism during diapause in Culex pipiens
1610 mosquitoes. J. Insect Physiol. 55, 4046.
1611
Zhu, H.S., Casselman, A., Reppert, S.M., 2008. Chasing migration genes: a brain
1612 expressed sequence tag resource for summer and migratory monarch
1613
butteries (Danaus plexippus). PLoS ONE 3, e1345.
1614
18 June 2014

Infection, Genetics and Evolution: Diana L. Huestis, Tovi Lehmann

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