Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali

Development of a reliable method for RNA interference (RNAi) by orally-delivered double-stranded RNA (dsRNA) is potentially promising for crop protection. Considering that RNAi efficiency considerably varies among different insect species, it is important to seek for the practical conditions under which dsRNA-mediated RNAi effectively works against each pest insect. Here we investigated RNAi efficiency in the brown-winged green stinkbug Plautia stali, which is notorious for infesting various fruits and crop plants. Microinjection of dsRNA into P. stali revealed high RNAi efficiency–injection of only 30 ng dsRNA into last-instar nymphs was sufficient to knockdown target genes as manifested by their phenotypes, and injection of 300 ng dsRNA suppressed the gene expression levels by 80% to 99.9%. Knockdown experiments by dsRNA injection showed that multicopper oxidase 2 (MCO2), vacuolar ATPase (vATPase), inhibitor of apoptosis (IAP), and vacuolar-sorting protein Snf7 are essential for survival of P. stali, as has been demonstrated in other insects. By contrast, P. stali exhibited very low RNAi efficiency when dsRNA was orally administered. When 1000 ng/μL of dsRNA solution was orally provided to first-instar nymphs, no obvious phenotypes were observed. Consistent with this, RT-qPCR showed that the gene expression levels were not affected. A higher concentration of dsRNA (5000 ng/μL) induced mortality in some cohorts, and the gene expression levels were reduced to nearly 50%. Simultaneous oral administration of dsRNA against potential RNAi blocker genes did not improve the RNAi efficiency of the target genes. In conclusion, P. stali shows high sensitivity to RNAi with injected dsRNA but, unlike the allied pest stinkbugs Halyomorpha halys and Nezara viridula, very low sensitivity to RNAi with orally-delivered dsRNA, which highlights the varied sensitivity to RNAi across different species and limits the applicability of the molecular tool for controlling this specific insect pest.

Attractiveness of Pheromone Components With and Without the Synergist, Methyl (2E,4E,6Z)-2,4,6-Decatrienoate, to Brown Marmorated Stink Bug (Hemiptera: Pentatomidae)

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) is attracted to its male-produced aggregation pheromone, a ~3.5:1 mixture of (3S,6S,7R,10S)- and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (SSRS and RSRS respectively), and also to the pheromone of its Asian sympatric species Plautia stali Scott (Hemiptera: Pentatomidae), methyl (2E,4E,6Z)-2,4,6-decatrienoate (MDT). A stereoisomeric mixture of (7R) 10,11-epoxy-1-bisabolen-3-ols (= mixed murgantiols) is used together in commercialized products with MDT because the latter is a synergist for H. halys attraction to mixed murgantiols. However, the optimal ratio for MDT combination with mixed murgantiols, and the sensitivity of bug captures to variation in ratio of the two pheromone components, have not been tested to date. Using black pyramid traps at two sites (in Maryland and West Virginia, United States), different ratios of mixed murgantiols to MDT were tested over two entire seasons. Also, captures using various ratios of the two active pheromone stereoisomers were undertaken in month-long trials with and without MDT. Results showed that H. halys adult and nymphal captures were relatively insensitive to the ratio of synthetic pheromone (mixed murgantiols) to MDT, as long as each was present in the trap. Captures of adults and nymphs were responsive to the lure loading of the SSRS isomer, but relatively insensitive to levels of the minor component, RSRS. The relative insensitivity of H. halys to these attractant ratios gives flexibility to development of more cost-efficient synthesis and trapping as well as other semiochemical-based management tactics.