NPs regulate the levels of NO which, when released in the feeding wound, induces vasodilatation and reduced platelet aggregation (Ribeiro et al., 1993
; Champagne et al., 1995
). Anticoagulant activity has been associated only with NP2 (Moreira et al., 2003
). The above results support these findings, showing that introduction of NP2 dsRNA significantly reduces the anticoagulant activity of R. prolixus
saliva and that this reduction can be achieved by injection or ingestion of the dsRNA. Although RNAi has been shown in many species, including hemipteran insects (Hughes and Kaufman, 2000
), this is the first time it has been used for triatomine bugs.
Hematophagous insects must overcome host hemostasis, which they do by producing a sophisticated cocktail of potent biological compounds in their saliva. Recent advances in transcriptome and proteome research allow an unprecedented insight into the complexity of these compounds, indicating that their molecular diversity as well as the diversity of their targets is even larger than previously thought (Ribeiro et al., 2004
). Several tools have emerged for post-genomic studies and among them there is great interest in exploring RNAi via the use of dsRNA as a means of assessing specific function(s) of genes (Hannon, 2002
). The main hallmarks of RNAi are its simplicity and the ease with which the method can be applied to an organism in vitro or in vivo (Fjose et al., 2001
NPs are hemeproteins corresponding to a considerable proportion of the proteins in the saliva of R. prolixus
(Champagne et al., 1995
). The NPs start being produced in the salivary glands in different stages and once started, they continue being produced until they reach the adult stage. Moreira et al. (2003)
found that NP2 proteins are the sole NPs in N1 salivary glands, with NP4 also found in N2 saliva and NP1 starts to appear in N3. NP3 protein is the last to be identified and is only found in N5 saliva. Using northern blot analysis, Sun et al. (1998)
did not see NP3 transcripts in N4 instars. In this work, RT-PCR carried out with salivary glands identified that RNA encoding NP3 is not found in unfed first instar, while all the other NPs are transcribed in all instars.
The results obtained with injections of dsRNA resuspended in water or saline induced about the same level of inhibition in triatomine bugs. This is an important point because the volume of the injections (2 μl) corresponds to around 10% of the weight of the fourth instar, suggesting that water could cause any kind of osmotic damage to the nymphs. For these reasons, we opted for using saline instead of water.
In salivary gland genes from hematophagous arthropods, RNAi was previously demonstrated in ticks (Karim et al., 2004
; Narasimhan et al., 2004
). But different from ticks, triatomines have the saliva stored in a reservoir comprised of the epithelial and muscular cells of the salivary gland (Meirelles et al., 2003
). During the blood meal, only half of the salivary proteins are lost and a single blood meal allows the bug to molt to the next stage, when the next feed occurs usually after the 3rd–5th day (Nussenzveig et al., 1995
). For this reason, reduction in protein levels are only observed after molt to the next instar while reduction in RNA expression occurred as early as 48 h after dsRNA introduction. Loss of salivary activity is only seen after new saliva is resynthesized, without or with low levels of the knockdown protein, to refill the saliva reservoir (Nussenzveig et al., 1995
). In addition, there is a considerable expansion of the glands after molt, as observed for other triatomine species, whose reservoir more than doubles its size and the amount of saliva as well (Guarneri et al., 2003
The introduction of NP2 dsRNA in R. prolixus
nymphs reduced NP2 expression, but also reduced levels of other NPs. This low specificity could be due to the considerable homology observed between the NPs (Andersen and Montfort, 2000
). It has already been observed for other insect species that RNAi can also trigger the destruction of mRNAs that contain significant stretches of sequence identity (Jackson et al., 2003
The activity assays performed and the protein content suggest that introduction of unspecific dsRNA induce slight alterations in the gland machinery, but do not compromise the functioning of the glands, which enforces the idea of specificity of the RNAi. It is important to highlight the variation observed among specimens in each of the experiments and the constraints that this will place on future experimental design.
An important finding of this work was the verification that RNAi can be achieved by ingestion of dsRNA in triatomine bugs. Ingestion inducing RNAi was previously achieved in nematodes (Hunter, 1999
; Timmons and Fire, 1998
) and in ticks (Soares et al., 2005
). In C. elegans
, RNA can be absorbed through the gut and distributed to somatic tissues and germ line (Kamath et al., 2001
; Timmons and Fire, 1998
). However, the same methodology has not been shown in insects. Curiously, host intact immunoglobulin G has been reported to occur in the hemolymph of insects with marked differences in digestive tract anatomy and physiology such as ticks (Ackerman et al., 1981
; Ben-Yakir, 1989
; Tracey-Patte et al., 1987
), some mosquitos (Hatfield, 1988
; Lackie and Gavin, 1989
; Ramasamy et al., 1988
), fleshflies (Sarcophaga falculata
) (Schlein et al., 1976
) and the blood-fed fly Haematobia irritans
(Allingham et al., 1992
), showing that molecules as big as immunoglobulin G can cross the gut epithelium with no damage even in flies that have the peritrophic membrane.
As observed in C. elegans
), ingestion of dsRNA is less effective in inducing RNAi in triatomines than injection. While two injections of 15 μg of dsRNA for NP2 in fourth-instar R. prolixus
was shown to reduce gene expression in 75%, an experiment in which two ingestions of 80 μg of NP2 dsRNA were offered to the same instar showed no reduction at all (data not shown). But delivering dsRNA by ingestion is less traumatic to the nymphs than injections. After ingestion, nymphs remain healthier and the mortality is considerable lower. Ingestion has another great advantage, it is easier to perform in first and second-instar nymphs in which injection would need special equipment and result in a high mortality.
In the past years, our laboratory has been working in identifying and characterizing novel genes expressed in the salivary glands of triatomines (Sant Anna et al., 2002
). The optimization of the RNAi technique and the possibility of introduction by ingestion in early nymph stages will greatly help at the functional characterization of new genes and better analyze the functions of the NPs in the feeding process. Similar methodology will be used in future experiments with R. prolixus
and other triatomine bugs. After silencing, bugs saliva will be used in in vitro assays for specific activity and knockdown bugs used for in vivo feeding behavior experiments, such as electric monitoring of cibarial pumps and intravital microscopy, in order to precisely analyze the lack of the protein in the feeding process.