ABSTRACT
In insects, lipid digestion is controversial because insects have no bile salts to solubilize dietary lipids. One hypothesis is that a secretory type of phospholipase A2 (sPLA2 ) provides lysophospholipid (LPL) from dietary phospholipids (PLs). We identified a sPLA2 , Se-sPLA2 , in beet armyworm, Spodoptera exigua, that hydrolyses PLs at sn-2. Our goal was to investigate its role in lipid digestion. Se-sPLA2 was expressed in the entire alimentary canal. Incubating the isolated midgut in a cell culture medium led to secretion of Se-sPLA2 and other proteins. Ex vivo RNA interference (RNAi) of Se-sPLA2 expression in isolated midgut culture led to significantly decreased Se-sPLA2 secretion into the medium. Feeding double-stranded RNA specific to Se-sPLA2 to larvae suppressed sPLA2 activity in gut contents. A recombinant Se-sPLA2 was susceptible to benzylideneacetone (BZA), a specific PLA2 inhibitor. After feeding BZA to larvae, we recorded significant decreases in gut content sPLA2 activity, body growth and total haemolymph lipid contents. RNAi against Se-sPLA2 resulted in reduced digestibility. Addition of a specific LPL, 1-palmitoyl-sn-glycero-3-phosphocholine, to BZA-treated larvae rescued digestibility and larval growth. These results strongly bolster our hypothesis that Se-sPLA2 secreted from the midgut acts in lipid digestion by providing necessary LPL to solubilize dietary neutral lipids.
Subject(s)
Digestion , Insect Proteins/metabolism , Lipid Metabolism , Phospholipases A2, Secretory/metabolism , Spodoptera/physiology , Amino Acid Sequence , Animals , Insect Proteins/chemistry , Intestines/physiology , Larva/enzymology , Larva/growth & development , Larva/physiology , Phospholipases A2, Secretory/chemistry , Spodoptera/enzymology , Spodoptera/growth & developmentABSTRACT
Helicoverpa armigera is a significant agricultural pest and particularly notorious for its resistance to many types of common insecticides. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing and trigged by double-strand RNA (dsRNA), has become a widely used reverse genetics and potent tool for insect pest control. In this study, the effect of ingestion and injection delivery methods of dsRNA related two important enzyme genes, α-amylase (HaAMY48, Ha-AMY49) and juvenile hormone esterase (Ha-JHE), were examined on growth and development of H. armigera. After 24, 48, 72 and 96 h of feeding bioassay, significant down regulation was observed about; 56, 68, 78, 80.75% for HaAMY48, 60, 70, 86.5 and 96.75%, for Ha-AMY49 and 14, 27.5, 23 and 31.7% for Ha-JHE, respectively. The results for injection assay was 61.5, 71.5, 74 and 95.8% for Ha-AMY48; 70, 88, 91.5 and 97.7% for Ha-AMY49 and 22, 61, 75 and 74% for Ha-JHE after 24, 48 and 72 h of last injecting, respectively. Larvae that treated with dsRNA, fed or injected, lost more than half of their weight. 50% mortality in treated larvae was observed in the case injection bioassay with dsHa-JHE and 59% of larvae that fed of dsRNA-treated cubes survived. DsHa-AMY48 and 49 have significant mortality, but mixing of them is more effective in both bioassays. Injection bioassay has a potent inhibitory effect on α-amylase-specific activity about more than 87% in treated larvae with mix of dsHa-AMY48 and 49. Adult malformation percent was evaluated for feeding (28, 35.5 and 43% for Ha-AMY48, 49 and Ha-JHE, respectively) and injection bioassay (23, 42 and 29% for Ha-AMY48, 49 and Ha-JHE, respectively). All these finding suggest that Ha-AMY48, Ha-AMY49 and Ha-JHE can be new candidates to scheming effective dsRNAs pesticide for H. armigera control.
