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. 2016 Aug 17;11(8):e0160928.
doi: 10.1371/journal.pone.0160928. eCollection 2016.

Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae

Marilza da Silva Costa  1 , Sérgio Oliveira de Paula  2 , Gustavo Ferreira Martins  2 , José Cola Zanuncio  1 , Antônio Euzébio Goulart Santana  3 , José Eduardo Serrão  2
Affiliations

Multiple Modes of Action of the Squamocin in the Midgut Cells of Aedes aegypti Larvae

Marilza da Silva Costa et al. PLoS One. .

Abstract

Annonaceous acetogenins are botanical compounds with good potential for use as insecticides. In the vector, Aedes aegypti (L.) (Diptera: Culicidae), squamocin (acetogenin) has been reported to be a larvicide and cytotoxic, but the modes of action of this molecule are still poorly understood. This study evaluated the changes in the cell morphology, and in the expression of genes, for autophagy (Atg1 and Atg8), for membrane ion transporter V-ATPase, and for water channel aquaporin-4 (Aqp4) in the midgut of A. aegypti larvae exposed to squamocin from Annona mucosa Jacq. (Annonaceae). Squamocin showed cytotoxic action with changes in the midgut epithelium and digestive cells of A. aegypti larvae, increase in the expression for autophagy gene Atg1 and Atg8, decrease in the expression of V-ATPase, decrease in the expression of Aqp4 gene in LC20 and inhibition of Apq4 genes in the midgut of this vector in LC50. These multiple modes of action for squamocin are described for the first time in insects, and they are important because different sites of action of squamocin from A. mucosa may reduce the possibility of resistance of A. aegypti to this molecule.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Photomicrographs of the midgut of Aedes aegypti third instar larvae (control).
(A) Epithelium with a single layer of columnar digestive cells (DC), with spherical nucleus (N) containing nucleolus (arrow). (B) Epithelium showing well-developed apical brush border (arrowhead). L = lumen. Bar = 5μm.
Fig 2
Fig 2. Photomicrographs of the midgut in Aedes aegypti third instar larvae exposed to sublethal doses LC20 and LC50 of squamocin.
(A) Disorganized midgut epithelium (EP) (LC20). (B) Digestive cells (DC) with vacuoles (*) in the apical cytoplasm and disorganized brush border (arrowhead) (LC50). (C) Digestive cells with vacuoles (v) in the basal cytoplasm (LC20). (D) Digestive cells with damaged striated border (arrowhead) and vacuoles (v) (LC20). N = nucleus, L = lumen. Bars = 5μm.
Fig 3
Fig 3. Transmission electron micrographs of the digestive cells in the midgut of Aedes aegypti third instar larvae.
(A) Midgut cell with long microvilli (arrows) associated with mitochondria (M) in the control larvae. (B) Midgut cell showing disorganized microvilli (arrows) in larvae exposed to LC50 acetogenin. (C) Middle-basal cell region showing mitochondria (M) and large vacuoles (V) in the larvae exposed to LC20 acetogenin. (D) Middle-basal cell region showing presence of large vacuoles (V) with lamellar content (arrowhead) in larvae exposed to LC20 acetogenin. bl–basal lamina.
Fig 4
Fig 4. Relative mRNA levels of Atg1 in the midgut of Aedes aegypti third instar larvae exposed to sublethal doses of squamocin and at different times.
(A) Lethal concentration of 20% of population (LC20). (B) Lethal concentration of 50% of population (LC50). The y-axis indicate the relative gene expression, corresponding to the Atg1 mRNA levels relative to ribosomal protein rp7S (reference) gene mRNA level (mean ± se). * p < 0.05.
Fig 5
Fig 5. Relative mRNA levels of Atg8 in the midgut of Aedes aegypti third instar larvae exposed to sublethal doses (LC20 and LC50) of squamocin and control at different times.
(A) Lethal concentration of 20% of population (LC20). (B) Lethal concentration of 50% of population (LC50). The y axis indicates the relative gene expression corresponding to the Atg8 mRNA levels relative to ribosomal protein rp7S (reference) gene mRNA level (mean ± se). * p < 0.05.
Fig 6
Fig 6. Relative mRNA levels of V-ATPase in the midgut of Aedes aegypti third instar larvae exposed to sublethal doses (LC20 and LC50) of squamocin and control at different times.
(A) Lethal concentration of 20% of population (LC20). (B) Lethal concentration of 50% of population (LC50). The y axis indicates the relative gene expression, corresponding to the V-ATPase mRNA levels relative to ribosomal protein rp7S (reference) gene mRNA level (mean ± se). * p < 0.05.
Fig 7
Fig 7. Relative mRNA levels of Aqp4 in the midgut of Aedes aegypti third instar larvae exposed to sublethal dose (LC20) of squamocin and control at different times.
The y-axis indicates the relative gene expression corresponding to the Aqp4 mRNA levels relative to ribosomal protein rp7S (reference) gene mRNA level (mean ± se). * p < 0.05.
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