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Aestivation of the African Malaria Mosquito, Anopheles gambiae in the Sahel

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Aestivation of the African Malaria Mosquito, Anopheles gambiae in the Sahel
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  601  Am. J. Trop. Med. Hyg.,  83(3), 2010, pp. 601–606doi:10.4269/ajtmh.2010.09-0779Copyright © 2010 by The American Society of Tropical Medicine and Hygiene  INTRODUCTION Malaria causes over 1 million deaths every year, most of which are African children. One important reason that this burden is so heavy in Africa is that the malaria-transmission machinery consists of an exceptionally robust vectorial sys-tem including  Anopheles gambiae  s.s. (representing two incipi-ent species known as the M and S molecular forms), a sibling species  An. arabiensis  , and  An. funestus  . These vectors exploit diverse environments including expansive dry savannas and semi-desert areas, where the surface waters required for lar-val development disappear for 4–8 months each year. Extreme seasonal fluctuation is a hallmark of the population dynamics of these malaria vectors, especially in arid environments where these anophelines apparently disappear during the dry season but population sizes rapidly increase after the onset of rains. 1–   3  Eggs, larvae, and pupae of  An. gambiae  cannot withstand des-iccation over a few days, 4–   7  and adults seldom survive beyond 2 months. 8,   9  Therefore, a mosquito that survives over 3 months during the dry season when no larval sites are available can be defined as aestivating. 10,   11  Aestivation is a recurring state of summer dormancy, typically characterized by suppressed reproduction and/or growth that facilitate extended survival during harsh conditions. Temperate mosquito species com-monly undergo winter diapause, 12–   16  but summer diapause is virtually unknown in mosquitoes. Many insects, including those inhabiting tropical regions, undergo seasonal dormancy, 17,   18  but whether  An. gambiae  aestivates is one of the longest-lasting mysteries of malariology. Several studies reported finding a few mosquitoes during the late dry season when no larval sites could be found, 10,   19–   21  but resolving if these mosquitoes sur-vived throughout the long dry season (aestivation) or whether they migrated from areas with ongoing permanent breed-ing has not been possible. To date, only two studies provided evidence for aestivation. Omer and Cloudsley-Thompson 22  found low density of  An. arabiensis  adults 20 km away from the Nile in Sudan when no larval sites were available. During the early dry season, adult females (mostly collected in houses and wells) did not develop eggs despite feeding on blood. This physiological condition, known as gonotrophic dissociation, is consistent with aestivation. In the laboratory,  An. arabien- sis  survived during the dry season for 206 days, 11  whereas  An.  gambiae  s.l. from Bobo Dioulasso, Burkina Faso survived in the laboratory up to 150 days. 10  However, similar studies could not replicate these results 5,   19,   23–   25  and thus, cast doubt about the generality of these findings. Furthermore, estimates of survival in nature have never matched these records. Population genet-ics studies aimed at detecting dry-season bottlenecks in vector populations have found evidence for the contrary. Surprisingly large effective population size (N e  ~ 2,000) of  An. arabiensis  was estimated in the Sahel and dry savannas, 21,   26  as was the case (N e  ~ 6,500) for  An. gambiae  s.s. in wet savannas, 27  sug-gesting a large aestivating population or mass migration. 28  To test the hypothesis that  An. gambiae  aestivates in the Sahel, we undertook a mosquito demographic study including a mark release–recapture (MRR) study conducted from one rainy season (September to November 2008) to the beginning of the next (April to June 2009) in the village Thierola, located in the Sahelian belt of Mali. In the course of this experiment, a total of 6,931  An. gambiae  s.l. were captured, marked, and released, each with a unique identification code based on a combination of up to four dots of nine colors placed on four positions on the body. MATERIALS AND METHODS The study was performed in Thierola (13.40° N, 7.13° W) ( Table 1 ), a small village (276 inhabitants living in a total of 120 houses) 3 km away from its nearest neighboring village, Zanga ( Table 1 ), and 6 km from the next closest neighboring village, Bako ( Table 1 ). The rectangular mud-brick, mud-roof houses of the Bambara ethnic group (80% of the population) are clustered together in adjacent compounds. The circular, Aestivation of the African Malaria Mosquito,  Anopheles gambiae  in the Sahel Tovi Lehmann ,* Adama Dao , Alpha Seydou Yaro , Abdoulaye Adamou , Yaya Kassogue , Moussa Diallo , Traoré Sékou , and Cecilia Coscaron-Arias Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases  , National Institutes of Health, Rockville, Maryland; Malaria Research and Training Center, University of Bamako, Bamako, Mali  Abstract.  The African malaria mosquito,  Anopheles gambiae  , inhabits diverse environments including dry savannas, where surface waters required for larval development are absent for 4–8 months per year. Under such conditions,  An.  gambiae  virtually disappears. Whether populations survive the long dry season by aestivation (a dormant state promoting extended longevity during the summer) or are reestablished by migrants from distant locations where larval sites persist has remained an enigma for over 60 years. Resolving this question is important, because fragile dry season populations may be more susceptible to control. Here, we show unequivocally that  An. gambiae  aestivates based on a demographic study and a mark release–recapture experiment spanning the period from the end of one wet season to the beginning of the next. During the dry season,  An. gambiae  was barely detectable in Sahelian villages of Mali. Five days after the first rain, before a new generation of adults could be produced, mosquito abundance surged 10-fold, implying that most mos-quitoes were concealed locally until the rain. Four days after the first rain, a marked female  An. gambiae  s.s. was recap-tured. Initially captured, marked, and released at the end of the previous wet season, she has survived the 7-month-long dry season. These results provide evidence that  An. gambiae  persists throughout the dry season by aestivation and open new questions for mosquito and parasite research. Improved malaria control by targeting aestivating mosquitoes using existing or novel strategies may be possible. * Address correspondence to Tovi Lehmann, National Institutes of Health, National Institute of Allergy and Infectious Diseases , MS 8132, 12735 Twinbrook Parkway, Rockville, MD 20852. E-mail: tlehmann@niaid.nih.gov  602 LEHMANN AND OTHERS mud-brick, thatch-roof houses of the Fulani ethnic group (20% of the population) are organized in five compounds sep-arated by 200 m from each other, along an arc 500 m south of the main village. The community grows primarily millet, sor-ghum, maize (corn), and peanuts during the rainy season (June to September). Cattle, sheep, goats, and chickens are raised by most families. The rains fill two large ponds and numerous small puddles, but usually, all surface waters dry by November. From November until May, rainfall is altogether absent or negligible (total precipitation <  30 mm). After the harvest (October to November), the fields surrounding the village lay bare. Water is only available in four deep wells (~30 m deep). Annual precipitation is approximately 500 mm (513 mm in Segou, which lies 30 km south and 100 km east of Thierola and 380 mm in Nara, which lies 170 km north of Thierola). The nat-ural vegetation consists of grasses, low shrubs, and scattered trees. Only few refugia, such as holes in baobab trees, can be found in a radius of 2 km around the village. Table 1 House density and composition of  An. gambiae  s.l. in villages around Thierola before (upper rows) and after (lower rows) the first rain VillageDistance (km) to Thierola/Niger (geoposition coordinates)Date (2009)   An. gambiae  s.l. density (houses sprayed)   An. gambiae  s.l. composition M/S/A (%) ‡ Before rain Thierola * 0/45 (13.40°N, 7.13°W)May 80.11 y   (120)100/0/0 Zanga * † 3/48 (13.41°N, 7.13°W)May 40.04 y   (102)100/0/0 Bako † 6/49 (13.39°N, 7.16°W)May 60.06 y   (50)100/0/0 Filanibougou16/52 (13.47°N, 7.08°W)May 50.16 y   (50)75/0/25After rain Thierola * 0/45 (13.40°N, 7.13°W)June 40.31 y   (120)95/0/5 Zanga * † 3/48 (13.41°N, 7.13°W)June 30.12 y   (111)100/0/0 Bako † 6/49 (13.39°N, 7.16°W)June 30.42 y   (48)100/0/0 Kondo14/59 (13.47°N, 7.15°W)June 90.09 y   (67)75/0/25 Serimana16/31 (13.32°N, 7.09°W)June 90.23 y   (65)100/0/0 Kolimana46/0 (13.21°N, 6.56°W)June 81.29 z   (55)100/0/0  Significantly different density values are marked by different letters ( P    <  0.05; Dunnett T   test comparing all values with that of Thierola after analysis of variance). Note that the spray collection in Thierola was performed after the surge in density, when numbers were in decline ( Figure 1 ). * Spray catch sampling (presented in this table) is known to provide higher estimates than aspiration of individual live mosquitoes (presented in Figures 1 – 3 ). † Zanga and Bako are the two nearest villages to Thierola. ‡ The relative percentage of the M and S molecular forms of  An. gambiae  (M/S) and  An. arabiensis  (A) are ordered accordingly. Figure  1. Abundance ( Bottom  ) and composition ( Top  ) of  An. gambiae  from the late wet season (2008) to the early subsequent wet season (2009). Density is measured by daily live indoor density per house ( Bottom  ). The left axis (black) corresponds to the data from the wet season, and the right axis (red) corresponds to the data from the dry season (note difference in scale). Vertical gray lines depict one standard error of the mean density per house. Composition of  An. gambiae  is shown in bars based on pooled collections representing 1- to 18-day intervals centered on the date shown above the bars. Sample size used in calculation of composition is shown beneath each bar, and percentage of each population is given inside the bars. Stars indicate day of desiccation of the last larval site in the village (November 8) and 1.5 km away (November 25), which was the last larval site in a radius of 6 km or more from Thierola. Blue ellipse indicates day of scattered rains (March 22) during the dry season.  603 AESTIVATION OF  AN. GAMBIAE  The first phase of the study was a multiple capture, mark, release, and recapture experiment performed during the end of the wet season (mid-September to mid-November 2008). The second and third phases consisted of identical surveys con-ducted at the end of the subsequent dry season before the first rains and immediately after the first rains, respectively; how-ever, mosquitoes captured in these phases were not released. Live collection by aspiration from all houses ( N   = 120, includ-ing abandoned houses and those used for kitchens, storage, and animals), outdoor clay-pot traps, and emergence traps (set over natural larval sites) was conducted every other day dur-ing phase I. Live collection in every house was carried out by two trained collectors, each searching for mosquitoes for 10–15 minutes (and until no mosquitoes were being collected for 3–5 minutes). Mosquitoes were released ~12 hours after capture within 5 m from their point of capture. A total of 2,397 male and 4,534 female  An. gambiae  s.l. were captured, marked, and released—each with a unique identification code based on a combination of two to four dots of nine possible colors placed on four positions on the body (three on the ventral side of the abdomen and one on the mesothorax) following methods pre-viously described. 29  Each mosquito was anesthetized in diethyl ether, examined under dissecting scope, and unless found to be marked, was marked before being placed in a cup and provided with 5% sugar water until release (9:00–11:00 pm  ). During phase I, a total of 248 mosquitoes (58 males and 190 females) were recaptured, some of which were recaptured two or three times. During phases II and III, extensive surveys were conducted in Thierola, including daily live adult collections by aspiration from all houses ( N   = 120) as described above. Collections were also performed outdoors in clay pots ( N   = 28) and Centers for Disease Control and Prevention (CDC) traps ( N   = 20) baited with an open fruit (mango/melon) set for 6 nights. Fruit-baited large traps ( N   = 8, set for 6 nights), well traps ( N   = 8, set for 10 nights), and toilet traps ( N   = 8, set for 6 nights) were checked every 3 hours from 6:00 pm  to 6:00 am  . These traps consisted of standard bed nets (without insecticide) hung above the well or toilet pit, leaving no space for mosquitoes to enter or exit, whereas an approximately 30-cm gap above ground was left for mosquitoes to enter to the fruit-baited traps. Human land-ing catch was performed by 16 collection sites (8 indoors and 8 outdoors) from 6:00 pm  to 6:00 am  for 6 nights. Additionally, large (50 cm diameter) and small (15 cm diameter) artificial oviposition sites ( N   = 8 and N   = 20, respectively) were main-tained and surveyed for over 3 weeks in Thierola and Zanga. Finally, extensive surveys of possible larval sites in an area of 20 km diameter around Thierola were conducted over 5 days. Pyrethrum spray collection was conducted after 12 days of live collection in Thierola and Zanga. Similar pyrethrum spray catches were performed in neighboring villages. RESULTS Extreme seasonal variation in abundance and composition of  An. gambiae  s.l. is one of its hallmarks, especially in drier environments. 2,   5,   8,   23,   30,   31  Rapid population growth over a few generations, however, is distinct from a rapid surge of den-sity within one generation. Therefore, we measured popula-tion density on a daily basis during the end of the wet season (October to November 2008), the end of the dry season (April to May 2009), and the early part of the subsequent wet sea-son (May to June 2009), providing a comprehensive description of the population dynamics. In early November, mosquitoes almost vanished from Thierola ( Figure 1 ). Although scattered rains fell on March 22, 2009 larval sites only had water for up to 6 days, which is insufficient time for development of an egg into an adult (9–11 days); moreover, no larvae were found in those larval sites. Adult density dropped 100-fold from its wet season levels. However, a few  An. gambiae  and  An. arabiensis  could be collected until the end of the dry season (May) ( Figure 1 and Table 1 ). At that time, females comprised the majority of  An.  gambiae  s.l. specimens (44 females versus 8 males). All females were collected shortly after taking a blood meal (32% fed, 29% semigravid, and 39% gravid), and most were inseminated (86%). All mosquitoes were collected indoors, except a single female that was collected from human landing catch and one male that was collected in a CDC light trap baited with fruit. Despite hav-ing no available larval sites, egg developed normally with little evidence for gonotrophic dissociation (mature or maturing ova-ries were observed in all but 5 of 28 gravid females dissected). By the end of the dry season, the S form apparently dis-appeared, and the M form predominated (90%) ( Figure 1 ), whereas the rest consisted of  An. arabiensis  ( Figure 1 ). The mosquitoes found during the dry season in neighboring vil-lages in a radius of 20 km from Thierola had similarly low den-sities and composition ( Table 1 ). Extensive surveys revealed a single small larval site in a dry streambed in the village Filanibougou, 16 km from Thierola, which dried out 2 days later. Adult mosquito density in that village was comparable with that of Thierola ( Table 1 ), suggesting that it too could not serve as a source of migrants. The first rain fell on May 24, 2009 and filled many larval sites, some of which remained with water for over 3 weeks without additional rain. Five days after the rain,  An. gambiae  density shot up and peaked on the seventh day at a level (0.36/house) 10-fold higher than that during the weeks preceding the rain (mean/house = 0.031) ( Figures 1 and 2 ). Because embryonic and larval development takes at least 9 days, these adults must have emerged before the rain. The surge in density 5–7 days after the rain consisted almost exclusively of M form of  An. gambiae  (237 of a total of 240 collected in Thierola and Zanga after the rain) ( Figure 1 ). Importantly, a surge was not observed in  An. funestus  or  An. rufipes  that coexisted during the dry season ( Figure 2 ), indicating that the surge could not be attributed to collection efficiency. Every mosquito collected from April to June was inspected for markings painted during the previous wet season (September to November). On the fourth day after the rain (May 27, 2009), a marked female mosquito was recaptured indoors ( Figure 3 ). Originally captured, marked, and released on October 29, 2008 (570 m away from her recapture point), she had survived from the end of the wet season until the beginning of the next wet season, which provides definite evi-dence for aestivation as a mechanism that allows  An. gambiae  to persist during the dry season in the Sahel. DISCUSSION The long-lasting enigma of whether  An. gambiae  aesti-vates in arid environments or migrates from neighboring locations where breeding continues has remained unresolved for over 60 years, because the key findings in support of aes-tivation, namely extended longevity (over 3 months) 10,   11  and gonotrophic dissociation, 11  have not been reproduced despite  604 LEHMANN AND OTHERS repeated attempts. 5,   19,   23–   25  Moreover, extended longevity under laboratory settings 10,   11  may not be relevant to natural condi-tions. The current results, however, resolve this problem by providing definitive evidence for aestivation, confirming the early studies 10,   11  and conjectures made on the basis of ecologi-cal 28,   32–   35  and genetic data. 21,   26,   27  The recapture of a marked (M form) female at the begin-ning of the wet season after her release 7 months earlier at the end of the previous wet season is unequivocal evidence for aestivation. Placed in the context of mosquito MRR experi-ments, it is rare to recapture more than two to three mosqui-toes 1 week after the release of thousands. 34,   36,   37  Therefore, a realistic expectation for the number to be recaptured after 7 months cannot be much larger than one. The surge of M-form adults 5–7 days after the first rain, before a new generation of adults could be produced, also attests to the fact that these adults were hidden in the vicinity of the village. A surge was not observed in  An. funestus  or  An. rufipes  (or other mem-ber of the  An. gambiae  complex) that coexisted during the dry season ( Figure 2 ), attesting to the fact that the surge could not be attributed to higher collection efficiency. Consistently, the mosquitoes found during the dry season were mostly M form. They probably were residents rather than migrants, because neighboring villages could not serve as a source of migrants given the low densities of  An. gambiae  throughout the area ( Table 1 ) and the isolation of these Sahelian villages. Notably, dispersal over 2–3 km is very rare, even during the wet sea-son. 31,   34,   36,   37  The concordance in molecular form between the population that persisted throughout the dry season, the surge of adults 5 days after the rain, and the identity of the marked 7-month-old female together provides strong evidence that the M form of  An. gambiae  undergoes aestivation. Despite finding  An. arabiensis  at the end of the dry season, it remains unclear if it too aestivates. The S form apparently van-ished from Thierola during the dry season (between November and May) ( Figure 1 ), and no resurgence of the S form was detected up to 10 days after the first rain. Nonetheless, one S form female appeared in Thierola 9 days after the rain ( Figure 1 ), suggesting that aestivation in this form is rare rather than absent (yet, being unmarked, migration from a distant source cannot be ruled out). Likewise, it is unknown how  An. funestus  (and the zoophilic  An. rufipes  ) persists throughout the long dry sea-son, because all known semi-permanent water with submerged vegetation disappears by December. Possibly, the capacity for aestivation is widespread in these anophelines, despite being so elusive for induction in laboratory conditions. Accordingly, in a typical Sahelian village, hundreds of mos-quitoes may aestivate throughout the dry season, hidden in as of yet unknown sites. Aestivating females take blood meals at a lower frequency than non-aestivating females, which is the case for other mosquitoes where diapause is known to occur, 12,   13,   38,   39  accounting for the low density indoors over the dry season. These findings prompt new investigations on such topics as aging in adult insects, summer diapause in mosquitoes, the molecular basis, physiology, ecology, and population variation in aestivation, and the effect of aestivation on Plasmodium  and malaria transmission as well as on novel approaches for malaria control by targeting aestivating mosquitoes. Aestivation may not be the only strategy malaria mosqui-toes use to survive the dry season in arid environments. The specific contribution of aestivation to the persistence of popu-lations inhabiting dry areas throughout the dry season should be evaluated in future studies. That larvae were collected from Filanibougou (M form of  An. gambiae  and  An. arabien- sis  ) in April (above) indicates that some females break their aestivation more readily than others. Gonotrophic dissocia-tion was not observed in our study. This seems to be contra-dictory to aestivation, but it was also observed at the end of the dry season in Sudan in the same population that exhibited gonotrophic dissociation earlier in the dry season 11,   22  as well as by  An. gambiae  s.l. maintained in the laboratory for over 4 months in Burkina Faso. 10  Notably, no anopheline in Thierola and Zanga laid eggs in the artificial larval sites constructed there (nor in the larval sites formed by the scattered rain in Figure  2. House density of  An. gambiae  ,  An. Funestus  , and  An. rufipes  before and after the first rain (May 24, 2009) measured by the total daily live collections indoors in all 120 houses of Thierola. In a box-whisker plot, the box extends between the 25th and 75th percentiles [i.e., across one interquartile range (IQR)], and the whiskers extend up to the most extreme value but not beyond 1.5 times the IQR. Values located over 2.5 IQR from the median are shown. Non-overlapping notched belts indicate significant difference between means ( P    <  0.05). This figure appears in color at www.ajtmh.org .  605 AESTIVATION OF  AN. GAMBIAE March), although larvae of Culex quinquefasciatus  inhabited most of these sites. Furthermore, after the first rain, larvae were scarce for 10 days in larval sites that held water for over 3 weeks, suggesting that females reappeared from their refugia but awaited additional signals before breaking aestivation. Finding that aestivation is key to the persistence of vector populations in arid environments may hold promise for mil-lions of people. For example, long-lasting insecticides applied indoors during the dry season could greatly diminish the num-bers of aestivating mosquitoes that would survive to seed the next wet-season generation, thus delaying the build-up of populations after the rains and cutting malaria transmis-sion. During the dry season, mosquitoes were found in all compounds of Thierola, without apparent clustering (data not shown), suggesting that their hiding places are numer-ous. Insecticide applications during the dry season, therefore, should include all houses. Because aestivating mosquitoes are probably found in every village where larval sites are unavail-able for over 3 months, interruption of aestivation must cover large clusters of villages to prevent migration from untreated villages nearby. It is unclear if such a strategy will also reduce malaria transmission where the dry season is shorter. Received December 21, 2009. Accepted for publication May 7, 2010. Acknowledgments: We are grateful to the residents of Thierola and Zanga, who went out of their way to accommodate our studies and for their hospitality. We thank Drs. Diana Huestis, Dia Elnaiem, Jen Hume, Nick Manoukis, Jose Ribeiro, Michael Service, Martin Donnelly, Peter Armbruster, Frederic Tripet, Robert Gwadz, Jennifer Anderson, and Thomas Wellems for helpful discussions and comments. The authors thank Drs. Robert Gwadz and Cheick Traore for supporting and facili-tating our project and especially, Dr. Jennifer Anderson for her dedi-cated assistance with IRB-related issues. Financial support: This study was supported by the Intramural Research Program in National Institutes of Health , National Institute of Allergy and Infectious Diseases. Authors’ addresses: Tovi Lehmann and Cecilia Coscaron-Arias, Lab-oratory of Malaria and Vector Research, National Institutes of Health, National Institute of Allergy and Infectious Diseases , Rockville, MD, E-mails: tlehmann@niaid.nih.gov , and coscaron@verizon.net . Adama Dao, Alpha Seydou Yaro, Abdoulaye Adamou, Yaya Kassogue, Moussa Diallo, and Traoré Sékou, Malaria Research and Training Center, University of Bamako, Bamako, Mali, E-mails: adama@MRTCBKO.org , yaro@icermali.org , Adamou@MRTCBKO.org , ykassogue@ MRTCBKO.org , moussad@icermali.org , and cheick@mrtcbko.org . REFERENCES 1. Toure YT , Petrarca V , Traore SF , Coulibaly A , Maiga HM , Sankare O , Sow M , Di Deco MA , Coluzzi M , 1998 . The distribu- tion and inversion polymorphism of chromosomally recognized taxa of the  Anopheles gambiae  complex in Mali, West Africa. Parassi tologia   40:  477 – 511 . 2. Toure YT , Petrarca V , Traore SF , Coulibaly A , Maiga HM , Sankare O , Sow M , Di Deco MA , Coluzzi M , 1994 . Ecological genetic studies in the chromosomal form Mopti of  Anopheles  gambiae  s.str. in Mali, West Africa. Genetica   94:  213 – 223 . 3. Fontenille D , Lochouarn L , Diagne N , Sokhna C , Lemasson JJ , Diatta M , Konate L , Faye F , Rogier C , Trape JF , 1997 . High annual and seasonal variations in malaria transmission by anophelines and vector species composition in Dielmo, a holoendemic area in Senegal .  Am J Trop Med Hyg    56:  247 – 253 . 4. Beier JC , Copeland RS , Oyaro C , Masinya A , Odago WO , Odour S , Koech DK , Roberts CR , 1990 .  Anopheles gambiae  complex egg stage survival in dry soil from larval development sites in western Kenya .  J Am Mosq Control Assoc   6:  105 – 109 . 5. Minakawa N , Githure JI , Beier JC , Yan G , 2001 . Anopheline mosquito survival strategies during the dry period in western Kenya .  J Med Entomol     38:  388 – 392 . 6. Koenraadt CJ , Paaijmans KP , Githeko AK , Knols BG , Takken W , 2003 . Egg hatching, larval movement and larval survival of the malaria vector  Anopheles gambiae  in desiccating habitats . Malar J     2:  20 . 7. Yaro AS , Dao A , Adamou A , Crawford JE , Ribeiro JM , Gwadz R , Traore SF , Lehmann T , 2006 . The distribution of hatching time in  Anopheles gambiae  . Malar J     5:  19 . 8. Gillies MT , De Meillon B , 1968 . The Anophelinae of Africa South of the Sahara  , 2nd ed . Johannesburg : South African Institute for Medical Research . 9. Lehmann T , Diabate A , 2008 . The molecular forms of  Anophe les  gambiae  : a phenotypic perspective .  Infect Genet Evol    8:  737 – 746 . 10. Holstein MH , 1954 . Biology of Anopheles gambiae. Research in French West Africa  . Geneva : World Health Organization . 11. Omer SM , Cloudsley-Thomson JL , 1968 . Dry season biology of  Anopheles gambiae  Giles in the Sudan . Nature    217:  879 – 880 . 12. Benoit JB , Denlinger DL , 2007 . Suppression of water loss during adult diapause in the northern house mosquito, Culex pipiens  .  J Exp Biol     210:  217 – 226 . 13. Robich RM , Denlinger DL , 2005 . Diapause in the mosquito Culex  pipiens  evokes a metabolic switch from blood feeding to sugar gluttony . Proc Natl Acad Sci USA   102:  15912 – 15917 . Figure  3. Photographs of the aestivating female recaptured after the first rains nearly 7 months (212 days) after being marked. The painted dot on the dorsal side of the thorax ( Top  ) and the two dots on the ventral side of the abdomen ( Bottom  ) are visible. Thick arrows point to light-blue paint dots that are clearly visible on the image. The narrow arrow points to the white paint dot that is faintly visible on the image (contrast and brightness of the images were optimized using GIMP2 by Dr. Nick Manoukis).
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