Proteomic analysis of castor bean tick Ixodes ricinus: a focus on chemosensory organs

Highlights

• In insects soluble olfactory proteins are widely studied, while proteins with similar functions are unknown in Acari.

• We hypothesized that Niemann Pick C2 proteins (NPC2) act as odorant carriers in tick chemosensilla.

• We performed a proteomic study of chemosensory organs of the tick Ixodes ricinus to search for soluble olfactory proteins.

• A member of NPC2 proteins was detected in chemosensilla by immunocytochemistry; its affinity to some odorants was measured.

Abstract

In arthropods, the large majority of studies on olfaction have been focused on insects, where most of the proteins involved have been identified. In particular, chemosensing in insects relies on two families of membrane receptors, olfactory/gustatory receptors (ORs/GRs) and ionotropic receptors (IRs), and two classes of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). In other arthropods, such as ticks and mites, only IRs have been identified, while genes encoding for OBPs and CSPs are absent. A third class of soluble proteins, called Niemann-Pick C2 (NPC2) has been suggested as potential carrier for semiochemicals both in insects and other arthropods.

Here we report the results of a proteomic analysis on olfactory organs (Haller's organ and palps) and control tissues of the tick Ixodes ricinus, and of immunostaining experiments targeting NPC2s. Adopting different extraction and proteomic approaches, we identified a large number of proteins, and highlighted those differentially expressed. None of the 13 NPC2s known for this species was found. On the other hand, using immunocytochemistry, we detected reaction against one NPC2 in the Haller's organ and palp sensilla. We hypothesized that the low concentration of such proteins in the tick's tissues could possibly explain the discrepant results. In ligand-binding assays the corresponding recombinant NPC2 showed good affinity to the fluorescent probe N-phenylnaphthylamine and to few organic compounds, supporting a putative role of NPC2s as odorant carriers.

Introduction

The study of chemical communication in arthropods has been almost exclusively focused on insects, while very little is known in other arthropods. This fact is likely related to the great economical impact of insect on agriculture and health, as well as to wide information available on insect pheromones.

However, many species of ticks and mites are parasites or vectors of serious diseases to mammals and insects, such as Ixodes, Amblyomma and Varroa (Rosenkranz et al., 2010), or agricultural pests, as the spider mite Tetranychus urticae (Zhu et al., 2016a, Zhu et al., 2016b). Therefore, a study of olfactory mechanisms at the molecular level in acari olfaction is of great medical and economical interest. At the same time, kairomones and pheromones have been discovered in Acari mediating several behaviours such as aggregation, and search for sexual mates and hosts (Carr and Roe, 2016).

In insects, chemoreception utilises membrane-bound receptors (olfactory receptors: ORs, gustatory receptors: GRs and ionotropic receptors: IRs) as well as two classes of soluble proteins, OBPs (odorant-binding proteins) and CSPs (chemosensory proteins) (Clyne et al., 1999, Vosshall et al., 1999, Pelosi et al., 2006, Vogt and Riddiford, 1981, Vogt et al., 1991, Angeli et al., 1999, Wanner et al., 2004, Vieira and Rozas, 2011, Pelosi et al., 2014, Leal, 2013, Wicher, 2015, Carraher et al., 2015). Both OBPs and CSPs are small compact proteins, mainly made of α-helical domains (Sandler et al., 2000, Tegoni et al., 2004, Campanacci et al., 2003) present in milli-molar concentrations in the sensillar lymph surrounding olfactory neurons, where they bind volatile odorants.

The number of OBPs and CSPs is higly variable between species, ranging from a dozen to about one hundred for OBPs and from four to 70 for CSPs (Pelosi et al., 2014) and include members involved in pheromone release as well as in functions unrelated to chemical communication, from development to nutrition and resistance to insecticides (Kitabayashi et al., 1998, Maleszka et al., 2007, Marinotti et al., 2014, Liu et al., 2014a, Xuan et al., 2014, Ishida et al., 2013, Liu et al., 2014b).

From a phylogenetical perspective it is interesting to observe that OBPs are only found in Hexapoda. They are present in all insects including Zygentoma (Tysanura) and also in Ectognatha such as springtails (Collembola), but are absent in all other arthropods, as well as in any other metazoan (Pelosi et al., 2014).

A very recent study investigating proteins expressed in chemosensory organs in the tick Amblyomma americanum identified two proteins named by the authors OBP-like based on the presence of four cysteines with a pattern similar to that found in insect OBPs (Renthal et al., 2016). However, these polypeptides show poor amino acid sequence identity with OBPs and are not part of a multigene family, as expected for proteins binding the large variety of semiochemicals.

Contrary to OBPs, CSPs have been also found in crustacea and myriapoda (Pelosi et al., 2014), but only one or two sequences are annotated in each species, suggesting that these proteins are not likely involved in chemoreception.

Given the anatomical similarities of chemoreception structures (sensilla) between insects and other arthropods, we can reasonably hypothesize that soluble binding proteins could be present in crustacea and chelicerata, performing roles analogous to those of insect OBPs and CSPs.

In acari, the foretarsal sensory organ (Carr and Roe, 2016), known as the Haller's organ in ticks, is reported as the main chemoreception structure. Haller's organ presents four parts: capsule, anterior pit, posterior group of sensilla and distal knoll, and exhibits both gustatory and olfactory hairs (Foelix and Axtell, 1971, Foelix and Axtetl, 1972, Leonovich, 1977).

Olfactory sensilla contain up to 35 receptor cells (Hess and Vlimant, 1986) several of them responding to pheromones and other volatiles in Amblyomma variegatum (Steullet and Guerin, 2014a, Steullet and Guerin, 2014b). In I. ricinus the distan knoll bears two pair of setae reacting to steer wool volatiles and to 2,6-dichlorophenol, a sex pheromone component of several tick species (Leonovich, 2004). Moreover, contact chemosensilla on the foreleg tarsi of I. ricinus respond to faeces and faecal breakdown products implicated in aggregation (Grenacher et al., 2001). Other chemoreception hairs are present on the terminal segments of palpi. Some of these sensilla are also endowed with mechanoreceptive functions (Ronghang and Roy, 2014).

Based on their relatively large number and diversity, we have suggested that in Chelicerata Niemann-Pick type C2 (NPC2) proteins might perform a carrier function, similar to that of OBPs and CSPs in insects (Pelosi et al., 2014, Zhu et al., 2016a, Zhu et al., 2016b). Transcriptome and proteome studies have found genes encoding NPC2 proteins and their protein products in the salivary glands of three species of ticks, Ixodes scapularis, I. ricinus and Rhipicephalus appendiculatus (Cotté et al., 2014, de Castro et al., 2016, Kotsyfakis et al., 2015, Schwarz et al., 2014). One of these proteins, the allergen of the common house mite, Der-p2, has been crystallised and its three-dimensional structure (PDB ID: 1KTJ) shown to enclose a binding cavity for a hydrophobic ligand (Derewenda et al., 2002). Moreover, a recent proteomic study identified two NPC2 proteins in the chemosensory organs of the lone star tick Amblyomma americaum (Renthal et al., 2016).

To search for NPC2s and other soluble proteins possibly involved in chemoreception in ticks, we have performed a proteomic analysis on chemosensory organs and other parts of the body of the blacklegged tick Ixodes ricinus. The choice of the species was mainly dictated by the easier availability of the biological material compared to I. scapularis, whose genome had been sequenced at the time we started this research. The two species are very similar in terms of amino acid sequences, as documented now by the available information on I. ricinus. An antiserum against an I. scapularis NPC2 protein (IscaNPC2-1, acc. no. EEC00381), chosen because of its similarity with Camponotus japonicus NPC2, which was found to be expressed in antennal chemosensilla (Ishida et al., 2014), was also prepared to study the localization of this protein through Western blot and immunocytochemistry.