Fig 1.
Events related to mosquito embryogenesis.
(A) Periods of egg darkening and serosal cuticle formation. Data are shown as percentages of the total embryonic development for each species, which is 77.4, 51.3 and 34.2 hours after egg laying for Ae. aegypti, An. aquasalis and Cx. quinquefasciatus, respectively. (B) Melanization pathway. Chromes are formed non-enzymatically (dashed arrows). DHICA: 5,6-dihydroxyindole-2-carboxylic acid, DHI: 5,6-dihydroxyindole. NADA (N-acetyldopamine) and NBAD (N-β-alanyldopamine) are also substrates for Laccase 2, originating quinones that participate in the sclerotization pathway. Grey background: Dopa contribution for melanin formation is minor since it is a poor substrate for Laccase2 (see main text). Enzyme names are shown in blue and Drosophila melanogaster mutants, in italic. PO: phenoloxidase, TH: tyrosine hydroxylase, DCE: dopachrome conversion enzyme, DDC: dopa decarboxylase, NAT: N-acetyltransferase, tan: N-β-alanyldopamine hydrolase, ebony: N-β-alanyldopamine synthase. Red inhibition symbol: the drug benserazide inhibits DDC activity. (C) Egg resistance to desiccation at the end of embryogenesis. At 80% of total embryogenesis, eggs were transferred from water to dry conditions (20–55% relative humidity), and their viability monitored at regular intervals. *Ae. aegypti eggs are viable outside water for even longer periods, up to several months [1,17,21]. All data in A and C were recovered from Vargas et al. [19], except darkening period obtained from Christophers [1] and Clements [2].
Fig 2.
Mosquito eggshell melanization varies among species.
Melanization degree was quantified in empty eggshell images obtained with bright field microscopy employing the ImageJ software (lower right graphic). The maximum melanization level was arbitrarily attributed to Ae. aegypti eggshells. The measured region, always near the hatching line, is indicated by dashed white circles. A direct correlation between melanization and ERD degree occurs (compare with Fig 1). Values represents the mean ± s.d. of two experiments, each consisting of at least 9 eggshells. All observed differences are statistically significant (Kruskal-Wallis, P < 0.0001).
Fig 3.
Embryogenesis of the weakly pigmented Anopheles quadrimaculatus GORO strain proceeds similarly to the WT.
GORO means ‘GOlden cuticle and ROse eyes’. (A) eggs, (B) larvae, (C) pupae and (D) adults. (E) Eggs at different embryonic ages developing at 25°C were air-dried for 15 minutes and the percentage of eggs that did not shrink (i.e. intact eggs) was then registered. Relative humidity ranged between 65 and 75%. The abrupt alteration in egg permeability is coupled with serosal cuticle formation, highlighted by a blue stripe (see Fig 4). Each lozenge represents mean ± s.e. of three independent experiments, each one with 30 eggs per time point (total of 630 eggs per strain) (F) Cumulative larval hatching at 25°C; data were normalized by total eclosion, obtained 24 hours after the expected embryogenesis completion. Each curve represents mean and standard error of three independent experiments consisting of 120 eggs each (total of 360 eggs per strain). (G, H) The lack of proper melanization can be phenocopied in the mosquito An. gambiae: while eggs laid in water become dark-brown (G), those laid on a benserazide solution, a melanization inhibitor (Fig 1B) develop a golden color (H).
Fig 4.
Resistance to air-drying is related to serosal cuticle formation in both An. quadrimaculatus strains.
Serosal cuticle presence was determined by chorion digestion driven by bleach (6% active chlorine). (A, D) Intact eggs. (B, E) Eggs treated with bleach before acquisition of air-drying resistance are totally digested while (C, F) eggs exposed to the same procedure after acquisition of air-drying resistance remain intact due to the presence of the serosal cuticle (see Fig 3E). Arrow: endochorion remnants not yet digested; arrowheads: serosal cuticle boundaries; asterisk: posteriormost end of the germ band. All images are in the same magnification.
Fig 5.
Mosquitoes with darker eggshells resist more to desiccation.
Mosquito eggs were laid on water. Values in the x-axis indicate the moment that eggs were transferred to dry conditions, staying outside the water for 2, 5 or 10 hours. Eggs were then returned to moist filter paper until completion of embryo development, when hatching rates were evaluated. Data were normalized regarding to control samples, kept on moist conditions throughout development. Blue stripes indicate the serosal cuticle formation period (as shown in Figs 1 and 3). Each point represents mean ± s.e. of three independent experiments consisting of at least 120 eggs each. A total of at least 3,240 eggs were employed for each species or strain. In all cases viability was significantly different between the two first experimental points (i.e. before and after serosal cuticle formation) (ANOVA followed by Tukey’s test, P < 0.05, see Table 1); the exception being Cx. quinquefasciatus at 10 hours in dry conditions. After serosal cuticle formation, An. quadrimaculatus GORO eggs were less viable than WT ones under equivalent conditions, in all cases (Student’s t-test, P < 0.001). All experiments were conducted at 25°C and relative humidity of 60–80% (An. quadrimaculatus) or 20–55% (other species).
Table 1.
Egg viability of mosquito species and strains under dry conditions during embryogenesis, before and after serosal cuticle (SC) formation.
Fig 6.
Mosquito vectors egglaying behavior and water flux through the eggshell before and after serosal cuticle formation.
From top to bottom, leftmost panel: while Ae. aegypti and An. aquasalis females lay their eggs individually, the females of Cx. quinquefasciatus lay their eggs as an organized raft that floats on the water surface. In all species, before serosal cuticle formation water passes freely through the eggshell. Serosal cuticle formation diminished water passage through the eggshell in a color-dependent manner: while in Ae. aegypti, with a black endochorion, most of the water is retained inside the egg, in An. aquasalis, with a dark-brown endochorion, some of the water is retained inside the egg, but not all. Finally, in Cx. quinquefasciatus, with a light-brown/light-tanned endochorion, most of the water escapes and only a small portion of it is retained inside the egg. The depicted embryonic morphology are representative for each stage and species [19] and egg sizes among species are depicted in their natural proportion [20]. For the sake of simplicity, the outermost eggshell layer (the exochorion) and the other extraembryonic membrane (the amnion) are not depicted here. The exochorion does not participate in the ERD [20].