Physiological and Morphological Aspects of Aedes aegypti Developing Larvae: Effects of the Chitin Synthesis Inhibitor Novaluron

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes

Darker eggs of mosquitoes resist more to dry conditions: Melanin enhances serosal cuticle contribution in egg resistance to desiccation in Aedes, Anopheles and Culex vectors

Submitted by Sandra Infurna ([email protected]) on 2018-02-12T16:40:58Z No. of bitstreams: 1 denise_valle_etal_IOC_2017.pdf: 10391762 bytes, checksum: 1c8618f8236c8dd686ae9a03c18c9761 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-02-12T16:59:28Z (GMT) No. of bitstreams: 1 denise_valle_etal_IOC_2017.pdf: 10391762 bytes, checksum: 1c8618f8236c8dd686ae9a03c18c9761 (MD5)Made available in DSpace on 2018-02-12T16:59:28Z (GMT). No. of bitstreams: 1 denise_valle_etal_IOC_2017.pdf: 10391762 bytes, checksum: 1c8618f8236c8dd686ae9a03c18c9761 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Insetos. Rio de Janeiro, RJ, Brasil.Universidade Estadual do Norte Fluminense Darcy Ribeiro. Campos dos Goytacazes, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivurs. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Estadual do Norte Fluminense Darcy Ribeiro. Campos dos Goytacazes, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular. Rio de Janeiro, RJ, Brasil.Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is 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 secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival 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 egg melanization enhances mosquito 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 egg 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

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

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

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

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

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