The complement system present in circulating blood is an effective mechanism of host defense, responsible for the killing of pathogens and the production of potent anaphylatoxins. Inhibitors of the complement system have been described in the saliva of hematophagous arthropods that are involved in the protection of digestive tissues against complement system–mediated damage. In this study, we describe albicin, a novel inhibitor of the alternative pathway of complement from the salivary glands of the malaria vector, Anopheles albimanus. The inhibitor was purified from salivary gland homogenates by reverse-phase HPLC and identified by mass spectrometry as a small (13.4-kDa) protein related to the gSG7 protein of Anopheles gambiae and Anopheles stephensi. Recombinant albicin was produced in Escherichia coli and found to potently inhibit lysis of rabbit erythrocytes in assays of the alternative pathway while having no inhibitory effect on the classical or lectin pathways. Albicin also inhibited the deposition of complement components on agarose-coated plates, although it could not remove previously bound components. Antisera produced against recombinant albicin recognized both the native and recombinant inhibitors and also blocked their activities in in vitro assays. Using surface plasmon resonance and enzymatic assays, we found that albicin binds and stabilizes the C3-convertase complex (C3bBb) formed on a properdin surface and inhibits the convertase activity of a reconstituted C3bBb complex in solution. The data indicate that albicin specifically recognizes the activated form of the complex, allowing more efficient inhibition by an inhibitor whose quantity is limited.

Rhipicephalus (Boophilus) microplus is the main ectoparasite affecting livestock worldwide. For a successful parasitism, ticks need to evade several immune responses of their hosts, including the activation of the complement system. In spite of the importance of R. microplus, previous work only identified one salivary molecule that blocks the complement system. The current study describes complement inhibitory activities induced by R. microplus salivary components and mechanisms elicited by putative salivary proteins on both classical and alternative complement pathways. We found that R. microplus saliva from fully- and partially engorged females was able to inhibit both pathways. Saliva acts strongly at the initial steps of both complement activation pathways. In the classical pathway, the saliva blocked C4 cleavage, and hence, deposition of C4b on the activation surface, suggesting that the inhibition occurs at some point between C1q and C4. In the alternative pathway, saliva acts by binding to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and reducing C3b production and deposition as well as cleavage of factor B. Saliva has no effect on formation or decay of the C6 to C8 components of the membrane attack complex. The saliva of R. microplus is able to inhibit the early steps of classical and alternative pathways of the complement system. Saliva acts by blocking C4 cleavage and deposition of C4b on the classical pathway activation surface and, in the alternative pathway, saliva bind to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and the production and deposition of additional C3b.

Rhipicephalus microplus is a cattle ectoparasite responsible for economic losses in livestock systems. The present study aimed to evaluate the essential oil (EO) of Piper hemmendorffii and its major constituent, limonene, for their activity against R. microplus larvae. The EO of P. hemmendorffii and its major compound, limonene, were evaluated against pyrethroid-resistant R. microplus larvae using larval immersion bioassays, biochemical analyses of detoxification enzymes and acetylcholinesterase, and scanning electron microscopy (SEM). The EO showed higher larvicidal potency (LC₅₀ = 10.46 mg/mL; 95% CI: 9.86–11.09) compared to limonene (LC₅₀ = 31.62 mg/mL; 95% CI: 30.86–32.40). Piper hemmendorffii EO affected the following classes of enzymes investigated: α-Esterase (α-EST) activity increased at LC₂₅ and LC₅₀, reaching its highest level at LC₅₀, whereas β-esterase (β-EST) activity increased only at LC₂₅. Cytochrome P450 (CYP450)-associated heme content activity showed a significant increase exclusively at LC₅₀, suggesting a response of the surviving larvae to EO exposure, although this increase does not by itself demonstrate the activation of detoxification pathways. In contrast, limonene did not significantly affect the activity of the three evaluated enzymes. The inhibition of AChE by the EO suggests a possible effect on the cholinergic system, which may be associated with neurotoxic activity, whereas limonene had no significant effect. The absence of detectable ultrastructural changes by SEM suggests that treatment with either EO or limonene did not induce evident external morphological damage to the larval cuticle under the evaluated conditions. EO toxicity seems to be associated with changes in detoxification enzyme and AChE activities, suggesting the involvement of these biochemical pathways in larval response.