It has been shown that Bm86-based vaccines affect the fitness of R. microplus by decreasing the number, weight and fecundity of engorging females [7, 9]. It has also been reported that anti-Bm86 sera affect the endocytotic activity of R. microplus gut cells . In the present study we took one step further and showed that the silencing of the Bm86 gene decreases the fitness of R. microplus ticks fed on B. bovis-infected cattle. Evidence from two independent experiments demonstrated that Bm86 plays a role during the feeding period and blood digestion in R. microplus females fed on cattle acutely infected with B. bovis. Gene silencing significantly decreased the number and percentage of survival of engorged females, and decreased the weight of egg masses. We also show that Bm86 is expressed in larvae, nymphs and adults of R. microplus and its expression was significantly down-regulated in ovaries of ticks fed on cattle during acute B. bovis infection.
It was recently shown that silencing of Bm86 had no statistical effect on the fitness of R. microplus females fed on uninfected cattle, despite the tendency of lower tick weight and egg mass weight in the silenced group than in the control group . In contrast, it has been demonstrated that the silencing of a Bm86 homologue of H. longicornis ticks significantly decreased the weight of engorged females . Our data show that the silencing of the R. microplus Bm86 gene significantly decreased tick fitness in the presence of acute B. bovis infection. The B. bovis-infected calves used in this study showed evidence of acute infection during the tick feeding period. Clinical indications of acute infection included a drop in PCV, fever and detection of parasites in peripheral blood by PCR. As shown in Figure 4B, there was a profound drop in PCV of the calf used in experiment two and the animal had to be euthanized 15 days after the B. bovis infection (7 days of tick feeding). As a result, clinical signs and parasitemia may have affected the tick fitness; however the overall biological impact cannot be solely attributed to B. bovis infection, considering the significant biological differences between the Bm86 silenced group and control group.
Two independent experiments were performed to investigate the effect of Bm86 silencing on R. microplus fitness in context with B. bovis infection. Although comparisons between experiments were not intended, some aspects of both experiments should be addressed en bloc. In experiment one, the weight of egg mass was significantly decreased by gene silencing; however, it was not significantly affected in experiment two. Additionally, there was a pronounced difference in survival of the replete females between experiment one and experiment two. These discrepancies may be explained by the different number of female ticks evaluated in each experiment, and by the fact that in experiment two the effect of gene silencing was investigated only in the most rapidly engorging cohort of females fed on a calf experiencing severe acute B. bovis infection. Notably, the silencing of Bm86 significantly decreased the survival of engorged R. microplus females and this effect was pronounced in experiment two where the PCV dropped from 25 to 11% during the tick feeding period. Therefore, it is reasonable to hypothesize that the effect of gene silencing on the mortality of engorged females may have been exacerbated by low PCV values. However, caution should be exercised when interpreting data from experiment two considering that it was untimely terminated due to the severity of the B. bovis infection and consequently only a small number of engorged R. microplus females were evaluated. Experiment one showed no significant differences in the infection rate of B. bovis in larval progeny from the Bm86-silenced group and the control group, demonstrating that gene silencing did not affect transovarial transmission of the parasite. It has been demonstrated that B. bovis-infected R. microplus larval progeny are very efficient in transmitting the parasite [4, 5]. The effect of Bm86 silencing on the ability of larval progeny to transmit B. bovis was not tested in this study; however, considering the published data and the infection rates of the larval progeny in the present study, there was no rationale to expect that larval progeny would not transmit the parasite in subsequent feedings.
Amino acid analyses have revealed that Bm86 contains several EGF-like domains . Proteins containing EGF-like regions fall into two general categories: those involved in blood coagulation and complement cascade, and those associated with the regulation of cell growth . It has been proposed that Bm86 resembles the latter group, which is characterized by multiple EGF repeats, transmembrane regions and location on the extracellular surface . It has been also argued that Bm86 could act as a cell membrane-bound ligand transmitting positional or cell-type information to adjacent cells in a manner similar to the Drosophila Notch protein . It has been reported that anti-Bm86 sera affect the endocytotic activity of tick gut cells, suggesting that this protein is somehow involved in the intracellular process of blood digestion . Here we show that silencing of the Bm86 gene in context with B. bovis infection decreased the number of engorging females and caused an increase in mortality of replete females. Additionally, histological analyses revealed the presence of structures resembling undigested red blood cells in gut cells of Bm86 silenced ticks. Despite the low number of ticks evaluated in the histological analyses, the data suggest that silencing of Bm86 caused some level of impairment in digesting the blood meal. It was beyond the scope of this study to perform a full functional analysis of the Bm86 gene, but the data demonstrate that Bm86 plays a role in blood digestion during engorgement and survival after repletion. Considering the histological results and the significantly higher mortality of replete females in the silenced group than in the control group, we hypothesize that silencing of Bm86 compromised the ability of the guts to maintain and digest a blood meal from cattle acutely infected with B. bovis.
It has been demonstrated that Bm86 protein is present mainly on the surface of the digestive tract of R. microplus females [10, 11]. However, information about gene expression and/or presence of the protein in other tick tissues and in different tick stages is scarce. This study shows that the Bm86 gene is expressed at different levels in larvae, nymphs and adult R. microplus, confirming previous observation . Although gene expression is not necessarily an indication of protein expression, it is reasonable to expect the presence of the Bm86 protein in other tick stages than adult females and in other tick tissues than gut, and this could be an important factor for the efficacy of Bm86 vaccines as a direct binding site for anti-Bm86 antibodies. The relevance of this result needs to be investigated in context with the efficiency of Bm86-based vaccines to control one-host and multiple-host tick species.
It has been shown that Babesia spp infection can affect tick fitness and the severity of these effects is related to the degree of parasitemia . In fact, it was recently demonstrated that B. bovis infection changes the protein expression profile of R. microplus females [20, 21]. Our results reinforce the published data and show that the expression of Bm86 decreased significantly in ovaries of R. microplus fed on cattle during B. bovis infection. The biological relevance of this data needs to be further addressed in context with protozoa infection and the use of Bm86-based vaccines.
Off-target effects caused by dsRNA have been described in numerous species [22–24] and cannot be entirely ruled out as the cause of the results observed in this study. The R. microplus genome sequence is not currently available , consequently alignment analyses are restricted to DNA and cDNA tick sequences listed in the GenBank and Gene Index Project databases http://compbio.dfci.harvard.edu. The 403-bp sequence used to synthesize the dsRNA does not have significant identity to any known sequence other than with the R. microplus Bm86 gene. Additionally, we demonstrate that the R. microplus Bm86 gene was significantly silenced by the injection of the 403-bp dsRNA. Collectively, these aspects support the phenotype data present in this study and represent the best possible strategy to make solid scientific observation regarding the biological effect of the silencing of Bm86 in R. microplus in context with B. bovis infection.
In conclusion, the results show that Bm86 plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. The data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation. This strategy may be particularly relevant for controlling ticks in B. bovis endemic areas, considering the assumption that Bm86 plays a more critical role in the fitness of R. microplus fed on B. bovis-infected cattle than in ticks fed on uninfected animals . Interestingly, the efficiency of transovarial transmission of B. bovis from surviving tick females to their larval progeny was not affected by silencing Bm86, despite the lower number of females that fed to repletion in the silenced group than in the control group. This study also demonstrated that Bm86 is expressed in larvae, nymphs, males, and gut and ovaries of female R. microplus ticks and its expression was down-regulated in ovaries by B. bovis infection. Additional investigations are required to elucidate the function of Bm86, and Bm86 homologues and orthologues, and its interaction with protozoa infection. Efforts should also be concentrated in discovering novel tick antigens and chemical acaricides that, in combination with Bm86-based vaccine, could be used in the development of an efficient program to control tick infestation and protozoa transmission.