Pyriproxyfen, a juvenile hormone analog, damages midgut cells and interferes with behaviors of Aedes aegypti larvae

Abstract

Juvenile hormone analogs (JHA) are known to interfere with growth and biosynthesis of insects with potential for insecticide action. However, there has been comparatively few data on morphological effects of JHA on insect organs. To determine pyriproxyfen effects on Aedes aegypti larvae, we conducted toxicity, behavioral bioassays and assessed ultrastructural effects of pyriproxyfen on midgut cells. A. aegypti larvae were exposed in aqueous solution of pyriproxyfen LC₅₀ concentrations and evaluated for 24 h. This study fulfilled the toxic prevalence of pyriproxyfen to A. aegypti larvae (LC₅₀ = 8.2 mg L^-1). Behavioral observations confirmed that pyriproxyfen treatment significantly changes swimming behavior of larvae, limiting its displacement and speed. The pyriproxyfen causes remarkable histopathological and cytotoxic alterations in the midgut of larvae. Histopathological study reveals presence of cytoplasmic vacuolization and damage to brush border of the digestive cells. The main salient lesions of cytotoxic effects are occurrence of cell debris released into the midgut lumen, cytoplasm rich in lipid droplets, autophagosomes, disorganized microvilli and deformed mitochondria. Data suggest that pyriproxyfen can be used to help to control and eradicate this insect vector.

Keywords: Aedes aegypti; Autophagy; Juvenile Hormone; Locomotory behavior; Ultrastructure.

Figure 1. Larval mortality of Aedes aegypti caused by aqueous solution of pyriproxyfen.

Lethal concentrations were estimated based on concentration-mortality assays using Probit analysis (χ² = 20.20; df = 5; P < 0.001). Lines denote 95% confidence intervals. Black point represents LC₂₅, LC₅₀, LC₇₅ and LC₉₀ concentrations, while LC₅₀ was selected to evaluate histological, ultrastructural changes and immunofluorescence.

Figure 2. Displacement trails of Aedes aegypti larvae from control (A) and exposure to LC₅₀ concentrations of pyriproxyfen (B).

Red tracks indicate high swimming speed; green tracks indicate low (initial) velocity.

Figure 3. Means ± SD of swimmed area (A) and resting time (B) of third instar Aedes aegypti larvae exposed in aqueous solution for 24 h to LC₅₀ pyriproxyfen concentrations.

Bars followed by different letters differ at P < 0.05 (Tukey’s mean separation test). The bars represent mean values and the error bars are standard errors of the mean, asterisks (*) indicate significant differences between treatments.

Figure 4. Light micrographs of the midgut of third instar Aedes aegypti larva.

(A) Single layered epithelium with columnar digestive cells (DC) from control larvae, with spherical nucleus (N), well-developed brush border (arrows) and basal membrane (arrowheads). Note a regenerative cell (RC) in differentiation. (B) Midgut epithelium of larvae exposed to aqueous solution of LC₅₀ pyriproxyfen showing digestive cells (DC) with cytoplasmic vacuoles (asterisks) and disorganized brush border (arrows). Regenerative cells (RC) from the larvae exposed in concentrations. Note many regenerative cells (RC) in differentiation with large nucleus (black arrow head). L – lumen.

Figure 5. Transmission electron micrographs of the digestive cells from midgut of control third instar Aedes aegypti larvae.

(A) General view of digestive cell, showing microvilli (MV) and rough endoplasmic reticulum (ER). (B) Basal region of digestive cell showing rough endoplasmic reticulum (ER), glycogen island (GL), regular basal labyrinth and muscle (M). (C) Perinuclear cytoplasm showing lipid droplets (asterisks), rough endoplasmic reticulum (ER), glycogen (GL), and mitochondria (arrowhead). (D) Detail of rough endoplasmic reticulum (ER), mitochondria (arrowhead) and glycogen (GL).

Figure 6. Transmission electron micrographs of the digestive cells from midgut of third instar Aedes aegypti larvae exposed to LC₅₀ pyriproxyfen aqueous solution.

(A) General view showing damaged microvilli (MV), Nucleus (N), mitochondria (arrowhead) and enlarged basal labyrinth (BL) and muscle (M). (B) Midgut lumen showing cell debris similar to mitochondria (arrows) and rough endoplasmic reticulum (arrowheads). (C) Basal cell region showing big lipid droplets (asterisks) and enlarged basal labyrinth (BL). (D) Perinuclear cytoplasm with autophagic vacuoles (arrows), lipid droplet (asterisk) and damaged mitochondria (arrowheads). (E) Details of damaged mitochondria (arrowheads) and empty glycogen deposit (GL).

Figure 7. Transmission electron micrograph of the digestive cell form midgut of third instar Aedes aegypti larvae exposed to LC₅₀ pyriproxyfen aqueous solution.

Apical region showing damaged mitochondria (M) and vesicular rough endoplasmic reticulum (arrowheads). MV – microvilli.

Figure 8. Micrographs of midgut epithelium of third instar Aedes aegypti larvae in the control and exposed to LC₅₀ pyriproxyfen.

Micrographs of midgut epithelium of third instar Aedes aegypti larvae in the control (A) and exposed to LC₅₀ pyriproxyfen (B) showing negative staining for phosphorylate histone-H3, but with increase in the number of cell nucleus (blue) in treated ones.