Egg viability of mosquito species and strains under dry conditions during embryogenesis, before and after serosal cuticle (SC) formation.

<p>Egg viability of mosquito species and strains under dry conditions during embryogenesis, before and after serosal cuticle (SC) formation.</p

Mosquito eggshell melanization varies among species.

<p>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 <i>Ae</i>. <i>aegypti</i> 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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.g001" target="_blank">Fig 1</a>). 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).</p

Mosquito vectors egglaying behavior and water flux through the eggshell before and after serosal cuticle formation.

<p>From top to bottom, leftmost panel: while <i>Ae</i>. <i>aegypti</i> and <i>An</i>. <i>aquasalis</i> females lay their eggs individually, the females of <i>Cx</i>. <i>quinquefasciatus</i> 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 <i>Ae</i>. <i>aegypti</i>, with a black endochorion, most of the water is retained inside the egg, in <i>An</i>. <i>aquasalis</i>, with a dark-brown endochorion, some of the water is retained inside the egg, but not all. Finally, in <i>Cx</i>. <i>quinquefasciatus</i>, 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 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.ref019" target="_blank">19</a>] and egg sizes among species are depicted in their natural proportion [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.ref020" target="_blank">20</a>]. 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 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.ref020" target="_blank">20</a>].</p

Embryogenesis of the weakly pigmented <i>Anopheles quadrimaculatus</i> GORO strain proceeds similarly to the WT.

<p>GORO means ‘GOlden cuticle and ROse eyes’. (<b>A</b>) eggs, (<b>B</b>) larvae, (<b>C</b>) pupae and (<b>D</b>) adults. (<b>E</b>) 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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.g004" target="_blank">Fig 4</a>). Each lozenge represents mean ± s.e. of three independent experiments, each one with 30 eggs per time point (total of 630 eggs per strain) (<b>F</b>) 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). (<b>G</b>, <b>H</b>) The lack of proper melanization can be phenocopied in the mosquito <i>An</i>. <i>gambiae</i>: while eggs laid in water become dark-brown (<b>G</b>), those laid on a benserazide solution, a melanization inhibitor (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.g001" target="_blank">Fig 1B</a>) develop a golden color (<b>H</b>).</p

Mosquitoes with darker eggshells resist more to desiccation.

<p>Mosquito eggs were laid on water. Values in the <i>x</i>-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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.g001" target="_blank">1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.g003" target="_blank">3</a>). 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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006063#pntd.0006063.t001" target="_blank">Table 1</a>); the exception being <i>Cx</i>. <i>quinquefasciatus</i> at 10 hours in dry conditions. After serosal cuticle formation, <i>An</i>. <i>quadrimaculatus</i> 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% (<i>An</i>. <i>quadrimaculatus</i>) or 20–55% (other species).</p

Period duration of <i>Ae. aegypti</i> larval ecdysis.

<p>Symbols represent the cumulative percentage of specimens at different immature stages. Bars represent the standard deviation of three independent experiments. Arrows indicate the experimental points, defined in hours, as early (e), intermediate (int) and late (l) moments for each instar.</p

Novaluron alters cuticular chitin presence in late L4 larvae.

<p>WGA-FITC labeling was used to detect chitin by fluorescence microscopy. (<b>A</b>, <b>B</b>) Control larvae exhibiting continuous cuticle labeling. (<b>C–F</b>) Novaluron EI₉₉ larvae show uneven cuticle labeling. Chitin labeling of cuticle in distinct regions of the larva is rather uniform (arrow in <b>C</b>; panel <b>D</b>), absent (asterisk in <b>C</b>; panel <b>E</b>) or irregular (arrowhead in <b>C</b>; panel <b>F</b>). Peritrophic matrix chitin labeling was not altered (<b>A</b>, <b>C</b>). Bar = 100 µm in <b>A</b>, <b>C</b> and 10 µm in <b>B</b>, <b>D–F</b>. All images were also recorded in bright field, in order to assure that images were in focus and that a cuticle was present (data not shown).</p

Novaluron inhibits <i>Ae. aegypti</i> adult emergence.

<p>(<b>A</b>) Dose-dependent effect of novaluron over emergence inhibition. EI₅₀ and EI₉₀ indicate novaluron concentrations resulting in emergence inhibition of 50 and 99% of adults, respectively. Black and white bars indicate death at larval and pupal stages, respectively (<b>B</b>) Percentage of surviving adults (males and females) after novaluron treatment (EI₅₀). Bars indicate mean and standard deviation of three experiments. Asterisks indicate significant differences (ANOVA, P<0.05).</p

Novaluron modifies cuticle and epidermis aspect of <i>Ae. aegypti</i> larvae.

<p>DIC microscopy was performed on histological sections of late L4 larvae stained with HE. (<b>A</b>) Control. Note the close association among cuticle, epidermis and the subjacent fat body layer. (<b>B–D</b>) Novaluron EI₉₉. Cuticle presents a semitransparent and discontinuous aspect being detached from the epidermis (<b>B</b>); epidermis is thinner (<b>C</b>) or degenerated, with a rope-like cuticle (<b>D</b>). ct: cuticle, did: disorganized imaginal disc, ep: epidermis, fb: fat body.</p

Novaluron induces delay in the development of <i>Ae. aegypti</i> immatures.

<p>Symbols represent the cumulative percentage of specimens in relation to eliminated exuviae of the preceding instar: squares, triangles and lozenges indicate newly emerged L4, pupae and adults, respectively. (<b>A</b>) control; (<b>B</b>) EI₅₀ and (<b>C</b>) EI₉₉. Bars represent the standard deviation of three independent experiments. Arrow indicates the moment of novaluron administration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030363#s2" target="_blank">Methods</a>).</p