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

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 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

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

Assessing the Effects of <i>Aedes aegypti kdr</i> Mutations on Pyrethroid Resistance and Its Fitness Cost

<div><p>Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (Na_V), inducing the knockdown effect. In <i>Aedes aegypti</i>, the main dengue vector, the AaNa_V substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (<i>kdr</i>) mutations. We evaluated the fitness cost of these <i>kdr</i> mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both <i>kdr</i> mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the <i>kdr</i> mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the <i>Ae. aegypti kdr</i> mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the <i>kdr</i> mutation is found before new adaptive alleles can be selected for diminishing the <i>kdr</i> deleterious effects.</p> </div

Activity of enzymes related to insecticide metabolic resistance in <i>Aedes aegypti</i> strains.

<p>The cut-offs are (dashed lines) determined by the Rockefeller 99 percentile value of each enzyme (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060878#pone.0060878-Montella1" target="_blank">[11]</a>). Rockefeller is a reference strain of insecticide susceptibility and vigor. Distributions with less than 15% of individuals beyond the cut-off are considered unaltered. Between 15 and 50% are altered and above 50% are highly altered. CIT-32 is the original <i>kdr</i> strain, derived from a pyrethroid resistant Brazilian <i>Aedes aegypti</i> population. Rock-kdr is the <i>kdr</i> strain, backcrossed for eight generations with Rockefeller in order to reduce the contribution of detoxification enzymes to pyrethroid resistance.</p

Developmental timing of <i>Ae. aegypti</i> Rock and Rock-kdr male adult emergence competing under a stringent condition.

<p>A – Cumulative rate of male emergence up to the 8th day after the beginning of adult emergence when the controls Rock and Rock-kdr were reared separately (‘intra-strain’ conditions). B – Cumulative proportion of Rock or Rock-kdr male emergence from the inter-strain competition. Male strain was daily determined by randomly genotyping 30% of emerging individuals.</p

Comparison of larval development time between Rockefeller and Rock-kdr <i>Ae. aegypti</i> strains.

<p>Numbers represent the cumulative daily proportion of Rock and Rock-kdr pupae formation after larvae eclosion under standard laboratory conditions. SEM is indicated. Gray dotted line indicates equal proportion (rate = 1) between strains.</p