Azadirachtin-A from Azadirachta indica impacts multiple biological targets in cotton bollworm Helicoverpa armigera

Azadirachtin-A (AzaA) from the Indian neem tree (Azadirachta indica) has insecticidal properties; however, its molecular mechanism remains elusive. The “targeted and nontargeted proteomic profiling”, metabolomics, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) imaging, gene expression, and in silico analysis provided clues about its action on Helicoverpa armigera. Fourth instar H. armigera larvae fed on AzaA-based diet (AzaD) suffered from significant mortality, growth retardation, reduced larval mass, complications in molting, and prolonged development. Furthermore, death of AzaD-fed larvae was observed with various phenotypes like bursting, blackening, and half-molting. Liquid chromatography–mass spectrometry (LC–MS) data showed limited catabolic processing of ingested AzaA and dramatic alternations of primary metabolism in H. armigera. MALDI-TOF imaging indicated the presence of AzaA in midgut of H. armigera. In the gut, out of 79 proteins identified, 34 were upregulated, which were related to digestion, immunity, energy production, and apoptosis mechanism. On the other hand, 45 proteins were downregulated, including those from carbohydrate metabolism, lipid metabolism, and energy transfer. In the hemolymph, 21 upregulated proteins were reported to be involved in immunity, RNA processing, and mRNA-directed protein synthesis, while 7 downregulated proteins were implicated in energy transfer, hydrolysis, lipid metabolism, defense mechanisms, and amino acid storage-related functions. Subsequently, six target proteins were identified using labeled AzaA that interacted with whole insect proteins. In silico analysis suggests that AzaA could be efficiently accommodated in the hydrophobic pocket of juvenile hormone esterase and showed strong interaction with active site residues, indicating plausible targets of AzaA in H. armigera. Quantitative polymerase chain reaction analysis suggested differential gene expression patterns and partly corroborated the proteomic results. Overall, data suggest that AzaA generally targets more than one protein in H. armigera and hence could be a potent biopesticide

Insecticidal Potential of Defense Metabolites from <i>Ocimum kilimandscharicum</i> against <i>Helicoverpa armigera</i>

<div><p>Genus <i>Ocimum</i> contains a reservoir of diverse secondary metabolites, which are known for their defense and medicinal value. However, the defense-related metabolites from this genus have not been studied in depth. To gain deeper insight into inducible defense metabolites, we examined the overall biochemical and metabolic changes in <i>Ocimum kilimandscharicum</i> that occurred in response to the feeding of <i>Helicoverpa armigera</i> larvae. Metabolic analysis revealed that the primary and secondary metabolism of local and systemic tissues in <i>O. kilimandscharicum</i> was severely affected following larval infestation. Moreover, levels of specific secondary metabolites like camphor, limonene and β-caryophyllene (known to be involved in defense) significantly increased in leaves upon insect attack. Choice assays conducted by exposing <i>H. armigera</i> larvae on <i>O. kilimandscharicum</i> and tomato leaves, demonstrated that <i>O. kilimandscharicum</i> significantly deters larval feeding. Further, when larvae were fed on <i>O. kilimandscharicum</i> leaves, average body weight decreased and mortality of the larvae increased. Larvae fed on artificial diet supplemented with <i>O. kilimandscharicum</i> leaf extract, camphor, limonene and β-caryophyllene showed growth retardation, increased mortality rates and pupal deformities. Digestive enzymes of <i>H. armigera -</i> namely, amylase, protease and lipase- showed variable patterns after feeding on <i>O. kilimandscharicum,</i> which implies striving of the larvae to attain required nutrition for growth, development and metamorphosis. Evidently, selected metabolites from <i>O. kilimandscharicum</i> possess significant insecticidal activity.</p></div

Two way analysis of variance for growth inhibition and percentage mortality of <i>H. armigera</i> upon exposure to <i>O. kilimandscharicum</i> leaf extract and selected metabolites on various days.

<p>DF = Degrees of freedom, SS = Sum of squares, MS = Mean square, n = numerator, d = denominator, p = probability of significance, α = 0.05.</p

Metabolic changes in leaves of <i>O. kilimandscharicum</i> following <i>H. armigera</i> infestation.

<p>Heat map representing relative expression of a sub-set of volatiles elicited in leaf tissue during <i>O. kilimandscharicum</i>-<i>H. armigera</i> interaction; comparison between metabolite profiles of local (L) and systemic (S) leaf tissue in <i>O. kilimandscharicum</i>, 12 h and 24 h after feeding by <i>H. armigera</i>, and also on days 3 (D3) and 6 (D6), compared to control (C) plants.</p

Digestive enzymes of <i>H. armigera</i> larvae fed on <i>O. kilimandscharicum</i> leaves.

<p>Changes in the levels of <b>A.</b> protease <b>B.</b> amylase <b>C.</b> lipase activity of <i>H. armigera</i> second-instar larvae fed on <i>O. kilimandscharicum</i> plants at 12 h, 24 h, day 3 and day 6. One way ANOVA followed by Tukey's multiple comparisons test suggested significant difference between the data at. <i>p</i><0.001 (indicated as ‘***’), <i>p</i><0.01 (indicated as ‘**’), <i>p</i><0.05 (indicated as ‘*’). Results are an average of three independent experiments conducted in duplicate. Error bars represent Mean ± SD.</p

Fig. 2 in Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis

Fig. 2. Heat map for the RPKM values of transcripts which involved in isopreniod biosynthesis. Triterpenoid biosynthesis related genes were highly expressed in kernel and pericarp, which is in line with total triterpenoid profiling.Published as part of &lt;i&gt;Pandreka, Avinash, Chaya, Patil S., Kumar, Ashish, Aarthy, Thiagarayaselvam, Mulani, Fayaj A., Bhagyashree, Date D., B, Shilpashree H., Jennifer, Cheruvathur, Ponnusamy, Sudha, Nagegowda, Dinesh &amp; Thulasiram, Hirekodathakallu V., 2021, Limonoid biosynthesis 3: Functional characterization of crucial genes involved in neem limonoid biosynthesis, pp. 1-12 in Phytochemistry (112669) 184&lt;/i&gt; on page 4, DOI: 10.1016/j.phytochem.2021.112669, &lt;a href="http://zenodo.org/record/10126978"&gt;http://zenodo.org/record/10126978&lt;/a&gt

Metabolic changes in stems and roots of <i>O. kilimandscharicum</i> following <i>H. armigera</i> infestation.

<p>Heat map representing relative expression of a sub-set of volatiles elicited in <b>A.</b> stems and <b>B.</b> roots during <i>O. kilimandscharicum</i>- <i>H. armigera</i> interaction at 12 h, 24 h, and on days 3 (D3) and 6 (D6) as compared to control (C) plants.</p