Figure 1.
Structure of the isobutylamide alkaloid pellitorine.
Figure 2.
Light micrographs of midgut, thorax, and anal gill parts of third instar Ae. aegypti larvae without (A) and with treatment with 5 mg/l of natural pellitorine (B) ×35 magnification.
Pellotorine treatment induced toxic effects on many different regions of the body including thorax, abdomen, and anal gills. All experiments were conducted in triplicate in which 20 mosquito larvae were used in each replicate. More than 10 live larvae from control and treated groups were randomly collected and used for histological analysis. All histological observations showed similar results throughout experiments same in other results.
Figure 3.
Histology of thorax and midgut regions of third instar Ae. aegypti larvae.
(A) Control mosquito. A1. anterior midgut region of control larvae has well-developed gastric caeca (GC) and single-layered midgut epithelium. A2. Central midgut region of control larvae consisted of well-developed lumen content (LC), peritrophic space (PS), and peritrophic membrane (PM). A3. Posterior midgut region of control larvae consisted of distinguished midgut epithelial layer, lumen contents, and peritrophic membrane. (B) Treated mosquito with 5 mg/l natural pellitorine showed undistinguished enlarged portion of gastric caeca and damaged single-layered epithelial cells. B1. Pellitorine-treated larvae had an undistinguished enlarged portion of gastric caeca and damaged single-layered epithelial cells (asterisks). B2. Central midgut region of pellitorine-treated larvae showed demolished epithelial layer residues mixed with a few LC (asterisks). B3. Complete damaged residue of epithelial and peritrophic membranes was observed in pellitorine-treated larvae (asterisk).
Figure 4.
Histology of anal gill region of third instar Ae. aegypti larvae.
(A) Control larvae showed that the anal gill has inner lined epithelium layer (EP) with well-organized anal gill cells (AGC) surrounded by thick cuticle layer. (B) Undistinguished damaged cuticle leading to completely destroyed anal gill cells and shrinked anal gill of the treated larvae with 5 mg/l natural pellitorine compared to the anal gill of the control larvae.
Figure 5.
Confocal laser scanning micrograph histology observations by immunostaining.
(A1–3) Anterior midgut region of the control larvae showed well-developed cardial nerves and subpopulation of neuron ends in rich neuropils. (A4–6) Cardial nerves in thorax and anterior midgut regions of larvae treated with 5 mg/l alkaloid pellitorine showed disappearance of neural processes. (B1–3) In posterior midgut region, neuronal cells and processes near the epithelial layer of the posterior midgut region were present. (B4–6) Posterior midgut region in the pellitorine-treated larvae also showed the lack of cells in the epithelial layers. (C1–3) Anal gill region of the control larval mosquito showed well-developed neurons elongated throughout large swelling anal gill cells. (C4–6) Neurons were damaged and disappeared in treated larvae. Arrowheads shown in (A) represent cardial nerves, gastric caeca, and neural processes. Arrowheads in (B) represent neuronal cells in epithelial layers of the gut. Arrowheads in (C) indicate neuronal elongation and branches to anal gills. Scale bars depict 40 µm.
Figure 6.
Transmission electronic micrographs of anterior midgut regions of third instar Ae. aegypti larvae without (A) and with treatment with 5 mg/l of natural pellitorine (B).
A1. A control larva showed well-developed peritrophic membrane (PM) enclosing midgut lumen contents. The midgut lumen cells consist of prominent nucleus and other cellular content present in the cytoplasm. A2. Organization of anterior midgut epithelial cells with large nucleus in a control larva. A3. Anterior midgut lumen content possesses numerous fat body (FB) tissue. B1-2. In the anterior midgut region of larvae treated with 5 mg/l natural pellitorine, all cellular contents including the nucleus were destroyed and cell masses in the cytoplasm were extruded. B3. Fat body tissues of lumen content were demolished by pellitorine treatment. N, nucleus;
Figure 7.
Transmission electronic micrographs of posterior midgut regions of third instar Ae. aegypti larvae without (A) and with treatment with 5 mg/l of natural pellitorine (B).
A1–2. The posterior midgut region of the control larva was characterized by few epithelial cells with electron-dense cytoplasm. Also, posterior midgut lumen has shown numerous dark cells with nucleus and polysomes. B1. Posterior midgut region of a larva treated with 5 mg/l natural pellitorine showed the compound destroyed all posterior midgut epithelial cells layer and also caused degeneration of dark cells and polysomes. B2. Residual tissues inside the gut lumen were observed in the posterior midgut in a pellitorine-treated larva. PM, peritrophic membrane; LC, lumen contents; PS, polysomes; DC, dark cell
Figure 8.
Transmission electronic micrographs of anal gill regions of third instar Ae. aegypti larvae.
(A) The anal gill of the control larva was surrounded by thick cuticle (CU) and inner surface covered with epithelial layer (EP) having large swelling anal gill cells (AGC) filled with hemolymph. (B) The anal gill of a larva treated with 5 mg/l natural pellitorine showed damaged outer membrane surrounded by a thick cuticle, which led to internal lumen content destruction, particularly degeneration of all anal gill cells.
Figure 9.
Gene expression patterns by pellitorine treatment.
(A) RT-PCR of AaAQP4, AaV-H+-ATPase and Aarps7 genes in control and treatment groups. (B) Relative gene expression level of AaV-type H+-ATPase and aquaporin 4 (AaAQP4), compared to the expression level of Aarps7 gene. The gene expression of AaV-type H+-ATPase and aquaporin 4 (AaAQP4) in the anal gills was inhibited in a larva treated with 5 mg/l natural pellitorine as compared to the control larvae. * indicates significant difference in gene expression levels in control and pellitorine treated groups (Student's t-test, N = 3, P<0.01).