Darker eggs resist more to desiccation: the case of melanin in Aedes, Anopheles and Culex mosquito vectors

Mosquito vectors lay their eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secretes the serosal cuticle, localized right below the endochorion, which greatly reduces water flow and allows the egg to survive outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if melanization enhances serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control.

can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult 48 insects, darkerbody individuals show greater resistance to desiccation than lighter ones. 49 We asked if melanization enhances serosal cuticledependent ERD. Species with higher 50 ERD at late embryogenesis exhibit more melanized eggshells. The melanizationERD 51 hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type 52 and the mutant GORO, with a darkbrown and a golden eggshell, respectively. In all 53 cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD 54 but egg viability outside the water is much higher in mosquitoes with darker eggshells than 55 in those with lighter ones. The finding that pigmentation influences egg water balance is 56 relevant to understand the evolutionary history of insect coloration. Since eggshell and 57 adult cuticle pigmentation ensure insect survivorship in some cases, they should be 58 considered regarding species fitness and novel approaches for vector or pest insects 59 control. interval between blood meal and egg laying induction, as well as the procedure adopted 147 for obtaining eggs, varied according to the species. 148 Aedes aegypti and all anopheline females were anaesthetized in ice three to four days 149 after blood feeding. Groups of five to ten sleeping females were then rapidly transferred to 150 upside down 8.5 cm diameter Petri dishes, where the lid became the base. This base was 151 internally covered with Whatman No. 1 filter paper. After the females were awaken, a 152 process that took 310 minutes, the filter paper was soaked with the same water employed 153 to rearing each species, thus stimulating the laying of the eggs that were deposited 154 individually or in small disorganized groups. 155 Groups of five to ten Culex quinquefasciatus females were anaesthetized in ice five to six 156 days after the blood meal and then transferred to 8.5 cm diameter Petri dishes in the 157 normal position (not upside down) without filter paper. After insect recovery, dechlorinated 158 water was added with the aid of a micropipette through a small hole in the lid until the 159 females were pressed against it, which stimulated egg laying. A second small hole was 160 present in the lid to allow air outlet while water was being introduced. Eggs were deposited 161 in organized rafts containing from few dozens to hundreds of eggs.

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In all cases egg laying lasted one hour in the dark, inside an incubator at 25 ± 1 °C. Petri 163 dishes were then opened inside a large cage where the females were released. Eggs were 164 allowed to develop at 25 °C until being employed in the experiments. For Ae. aegypti and 165 anopheline eggs the sides of the Petri dishes were sealed with parafilm, in order to avoid 166 water evaporation. For Cx. quinquefaciatus eggs, rafts were kept intact prior to the first 167 experimental point, when they were transferred to Petri dishes whose base was covered 168 with Whatman No. 1 filter paper soaked with dechlorinated water. Rafts were carefully 169 disrupted and the eggs were spread with the aid of a painting brush. The procedure and use of live chicken followed the UFIACUC Protocol no. 201003892.

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The procedure and use of anaesthetized guinea pigs was reviewed and approved by the Eggs at approximately 80% of embryogenesis completion had their exochorion removed 178 with bleach (NaOCl, 6% active chlorine) treatment for one minute followed by three 179 washes with dechlorinated water. These exochoriondepleted eggs were then kept in moist 180 filter paper until hatching. Eggshells were then transferred into a microscopy slide and 181 brightfield images were obtained with a digital imaging acquisition system coupled to a    spite of this, overall, Aedes aegypti exhibits the greater eggshell pigmentation, followed by 274 An. aquasalis and Cx. quinquefasciatus. 275 Although establishing a direct relationship between eggshell pigmentation and ERD is 276 tempting, other eggshell related factors, such as differences in thickness or components of 277 the endochorion or the serosal cuticle, might account for this distinctness (Harwood, 1959; 278 Christophers, 1960; Clements, 1992; Monnerat et al., 1999;Farnesi et al., 2015). 279 Moreover, since we are studying mosquitoes of different genus, whose common ancestor  In order to directly evaluate the relationship between melanization and ERD without any 283 other confounding factor, we took advantage of a mutant strain of the species Anopheles 284 quadrimaculatus, which shows a significant melanization deficit: the GORO strain.  Likewise, the period necessary for entire embryogenesis, approximately 56 hours after egg 303 laying, is similar in both strains ( Figure 3F). Therefore, the lack of melanization in the An.   Hatching rates were assessed at the end of embryogenesis ( Figure 5 and Table 1). In all 319 cases serosal cuticle formation significantly increases egg viability outside the water before, but increases considerably after serosal cuticle synthesis, as previously described 327 for An. quadrimaculatus (Darrow, 1949) and An. gambiae (Goltsev et al., 2009 for two hours before serosal cuticle synthesis survive but similar aged eggs exposed to a 331 dry environment for longer periods do not resist. Moreover, egg viability after serosal 332 cuticle formation is inversely proportional to the exposure period outside the water.   Insect eggs occur in a myriad of colors, ranging from white to black with tones of yellow, 363 orange, red, pink, green and brown, among others. Egg color may occur uniformly or in 364 patches throughout the eggshell, or can appear in restricted areas (Hinton, 1981). These    In any organism, an increase in resistance to desiccation is related with three aspects: a 429 higher initial body water store, a reduction in the rate of water loss and an increase in the

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The moderate level of ERD before serosal cuticle formation, shown in Ae. aegypti and Cx. 451 quinquefasciatus, but not in Anopheles spp., cannot be related to the presence of melanin.

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This viability might be due to an increased tolerance to water loss or a higher initial egg 453 water content. Percentage of eggshell weight in relation to total egg weight indeed suggest 454 that total body water content is lower in An. aquasalis (Farnesi et al., 2015).

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Aedes aegypti shows an outstanding success in keeping its eggs viable outside the water, 480 up to 8 months in the dry (Christophers, 1960;Clements, 1992). There is even a report 481 15 that shows hatching of Ae. aegypti eggs after 15 months, when kept at 9 ºC (Bacot, 1918). 482 Indeed, more detailed analysis reveals this hatching success is directly related to higher 483 relative humidity (Kliewer, 1961). The present results show that the increased Ae. aegypti 484 eggshell melanization is one of the traits responsible for the extremely efficient ERD seen 485 in this species (Figure 6).   Clements, 1992). Beyond being darker than other eggshell regions (Figure 2), the 505 posterior tip is the only endochorion region whose surface is rough and irregular, similar to 506 the whole endochorion of Ae. aegypti eggshells (Farnesi et al., 2015). Given that Culex 507 eggs at raft edges were found dead after exposure to strong dry winds (Clements, 1992), it 508 seems that the raft per se can act as a protection against dehydration, according to the 509 egg clusterdesiccation hypothesis (Clark and Faeth, 1998). This could relax the selection

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In summary, we believe that the differential egg resistance to desiccation observed in 516 distinct mosquito species is a trait with multifactorial origins. Eggshell melanization and 517 serosal cuticle formation increases this protection ( Figure 6). However, other factors might 518 also contribute such as the thickness and texture of the distinct eggshell layers and the 519 parental investment, observed in Culex species.