A team of Brazilian researchers this month reported for the first time on the discovery of long-term persistence and parental transmission of RNAi in Rhodnius prolixus, a species of bloodfeeding bugs called triatomines.
As vectors of Trypanosoma cruzi, a parasite that causes trypanosomosis, triatomines are a medically important insect. R. prolixus is also a classical model in insect biology, and a number of recent studies have demonstrated that RNAi occurs in the bugs.
"However, there is no information about the persistence of the RNAi effect and if the signal is transmitted to the progeny," the investigators wrote in a paper appearing in Insect Biochemistry and Molecular Biology.
To fill in this gap, the team examined RNAi effects in R. prolixus in vivo by targeting nitrophorins, which are abundant multifunctional molecules expressed in the bugs' salivary glands, and rhodniin, an intestinal inhibitor of coagulation.
Double-stranded RNA against the molecules were injected into the midguts of third and fifth instar nymphs, which were later dissected and evaluated.
Suppression of nitrophorin in third instar nymphs was apparent 48 hours after dsRNA administration, with levels falling 98 percent, according to the paper. "However, the RNAi-induced phenotype was only apparent after the insect had blood fed and molted to the next instar, when a significant reduction in the salivary hemoprotein content appeared."
The reduction in nitrophorin persisted over all of the insects' life stages — more than seven months of knockdown after dsRNA injection, the researchers noted.
Parental RNAi was not detected in the first instar generation derived from the third instar-injected nymphs, even when the amount of dsRNA was doubled. However, the transmission of RNAi effects to this next generation of bugs could be "clearly observed" with the injection of nitrophorin-targeting dsRNA to fifth instar nymphs.
Meanwhile, a greater than 60 percent knockdown in rhodniin mRNA was observed in the anterior midguts in all R. prolixus groups two days after injection, the scientists wrote. After molting to the fourth instar, mRNA levels fell 93 percent, reaching 97.3 percent in the 60th day after dsRNA administration.
The phenotypic effects of rhodniin inhibition in the midgut were apparent earlier than those observed with RNAi in the salivary glands, with anticoagulate activity in the midgut content of the bugs approaching 70 percent 15 days after dsRNA treatment. Target knockdown subsided somewhat after feeding and molting, but persisted at least five months in third instar nymphs.
As in the salivary glands, parental RNAi was not detected, either by qPCR or anticoagulate assay, in the midguts of first instars derived from the dsRNA-treated third instar nymphs. However, the transmission of RNAi to the next generation occurred when the knockdown was performed in the fifth instar.
Overall, the study represents the first report of the "prolonged persistence of RNAi effects and their transmission to the next generation beyond the embryos stage in insect vectors of diseases," the researchers stated. Notably, the persistence of RNAi in R. prolixus is the longest among any insect species studied thus far, they added.
As for the reasons why parental RNAi occurred when dsRNA was administered to fifth istar nymphs but not third instar ones, the team suggested that this peculiarity may be due to the RNAi molecules reaching the germline cells of the bugs only when it is introduced during the later developmental stage.
"Once the oocytes had already started to develop in the fifth stage, but not in the third, it is possible that RNAi effects are transmitted to the next generations after the oviposition by females," they wrote.
Perhaps "the dsRNA injected in third instar nymphs can enter in any cell which has some dsRNA uptake mechanism, but the RNAi amplification step would occur only in the target cells, where the endogenous target mRNA act as a template for the production of secondary siRNAs through a still unknown mechanism," they speculated. "When this insect reaches the fifth instar and the oocytes are developed, there is no remaining dsRNA in the hemolymph to enter in the germ cells and the RNAi effects cannot pass to the next generation."
In line with this reasoning, it is possible that the dsRNA injected into the fifth instar is able to reach both the target cells and the oocytes, they added. "The target mRNA is not expressed in oocytes and so the dsRNA/siRNA will not be processed, but can be transmitted to the next generation."
Additional research is required to better understand the spread of dsRNA between cells and the persistence of an RNAi effect without the additional dsRNA, the investigators concluded.
"Unveiling the factors involved in a possible [RNAi] amplification step and spread are important to understand the mechanisms and apply this tool more effectively in insect functional genomic studies and can also provide new insights into the evolution of these mechanisms in insects and among other animals."