Triatoma infestans is the main vector of Chagas disease in the Americas, currently transmitting it in Argentina, Paraguay, and Bolivia. Many T. infestans populations present insecticide resistance, reducing the efficiency of control campaigns. Alternative vector control methods are needed, and molecular targets mediating fundamental physiological processes can be a promising option to manipulate kissing bug behavior. Therefore, it is necessary to characterize the main sensory targets, as well as to determine whether they are modulated by physiological factors. In order to identify gene candidates potentially mediating host cue detection, the antennal transcripts of T. infestans fifth instar larvae were sequenced and assembled. Besides, we evaluated whether a blood meal had an effect on transcriptional profiles, as responsiveness to host-emitted sensory cues depends on bug starvation.
We propose that the set of genes presenting nutritionally-triggered modulation on their expression represent candidates to mediate triatomine host-seeking behavior. Besides, the triatomine-specific gene lineages found represent molecular adaptations to their risky natural history that involves stealing blood from an enormously diverse set of vertebrates. Heteropteran gene orthogroups identified may represent unknown features of the sensory specificities of this largest group of hemipteroids. Our work is the first molecular characterization of the peripheral modulation of sensory processes in a non-dipteran vector of human disease.
Olfaction is critical for insect survival , as it mediates the detection of food [5, 6], sexual partners [7,8,9], danger [10, 11] and pathogens [12, 13], among other resources and threats. Insects have developed a diverse array of receptors to detect both host cues and communication signals . The main groups of insect proteins mediating the detection of volatile molecules are odorant receptors (ORs), ionotropic receptors (IRs), odorant-binding proteins (OBPs), chemosensory proteins (CSPs), odorant-degrading enzymes (ODEs), and sensory neuron membrane proteins (SNMPs) . The genes coding for membrane receptors mediating odor recognition (ORs and IRs) are expressed by olfactory sensory neurons (OSNs), which are housed in the olfactory sensilla mostly located on insect antennae. This confers these neurons the ability to detect minute quantities of volatile compounds circulating in the environment, granting that brain centers dealing with olfactory information are updated on their presence, abundance, and fluctuations. Nevertheless, this ability is not a static feature, but one that varies with the season, time of the day, development, nutritional and mating status . The modulation of sensory neuron responsiveness seems to be mediated by a variety of correlated changes in gene expression as an underlying molecular substrate [17,18,19,20,21,22]. Characterizing such molecular changes seems critical to understanding sensory processes and uncovering key components that may become targets for controlling insect pests more rationally. Other genes coding for sensory components that detect mechanical stimuli, heat, humidity, ammonia or salinity are also fundamental to grant insect survival. Gene families like those of transient receptor potential (TRP) channels, pickpockets (PPKs) and ammonium transporters (AmTs) represent the molecular substrate underlying these sensory abilities. Detecting heat and humidity seems to play a key sensory role in host recognition by triatomines and mosquitoes [23,24,25,26].
In recent years, the advent of next-generation sequencing techniques has allowed the characterization of the sensory gene repertoires of many insects through genomic and transcriptomic studies . The resulting datasets have improved our knowledge about the molecular processes that underlie the behavioral and sensory plasticity of these animals. For example, the characterization of antennal gene expression changes triggered by blood ingestion allowed a better understanding of the molecular bases of host-seeking behavior at the peripheral level in several mosquitoes [17, 20,21,22]. Even though T. infestans is the main vector species transmitting Chagas disease, its genome sequence annotation is not available, hindering homology-based searches of molecular targets. Lacking such a massive source of gene sequences limits our characterization of gene families that have suffered expansions or contractions through the evolution of the species. Besides, it limits our capacity to uncover key genetic components deserving functional studies. Furthermore, this impedes evaluating whether the recent evolutionary pressures caused by bug domiciliation have had any impacts on its genetic sensory machinery.
Few transcriptomic studies have been published for T. infestans [28,29,30,31], however, none was performed on sensory tissues. In this work, we sequenced the antennal transcripts of T. infestans fifth instar larvae, generated a de novo assembly, and studied the effect of blood ingestion on transcriptional profiles in this tissue. The sensory gene repertoire of T. infestans was annotated and compared to that of Rhodnius prolixus and other hemipterans. This process revealed gene lineages unique to triatomines. Besides, we identified a set of sensory-related genes that had antennal expression levels significantly altered after feeding, which suggests that they are candidates to mediate host-seeking behavior in triatomines. This work represents the first characterization of peripheral modulation of host-seeking in a non-dipteran human disease vector.
A total of 127 ORs were identified in the antennal transcriptome, with an average length of 346 amino acids (ranging from 203 to 474) (Additional file 5: Supplementary Table S3). Fifty-six (44%) had their sequences complete, while 71 ORs (66%) presented partial sequences, mainly due to the absence of the initial methionine. All sequences had the PFAM domain PF02949.23 characteristic of the OR family, except for 3 candidates. Besides, 89 sequences (70%) mapped against an insect OR from the UniProtKB/Swiss-Prot database. A total of 95 (75%) T. infestans ORs had between 4 and 7 transmembrane domains. The size of the OR repertoire of T. infestans (127) is similar to that of O. fasciatus (120) and larger than the repertoires found for R. prolixus (110) and other hemipterans, such as Cimex lectularius (47), Diaphorina citri (46) Tessaratoma papillosa (59) and Acyrthosiphon pisum (79) (Table 1). On the other hand, the OR sets of Apolygus lucorum and Sogatella furcifera (135), Halyomorpha halys (138) and Nilaparvata lugens (141) slightly exceed the size of that of T. infestans (Table 1).
Except for most sequences belonging to the Ir41 subfamily and a few sequences of the Ir75 subfamily, T. infestans IRs (30) were annotated based on their relationships to R. prolixus IRs. This was the case of the three IR co-receptors Ir25a, Ir8a, and Ir76b, as well as the orthologues of several antennal IRs, such as Ir21a, Ir40a, Ir41, Ir68a and Ir93a (Fig. 1). As observed for R. prolixus , orthologues of the other highly conserved IRs, including Ir31a, Ir92a, Ir60a, Ir76a, Ir64a and Ir84a, were not detected for T. infestans. Most of the IRs presented a 1:1 orthology relation between T. infestans and R. prolixus, except for Ir40a and Ir21a lineages that seem to have expanded in T. infestans, with 5 and 3 transcripts each (Fig. 1). Among the divergent IRs, only orthologues of Ir105 and Ir106 were identified for both triatomines, while the remaining were only detected for R. prolixus. As previously reported for R. prolixus (13 Ir75 members; ), a large expansion of the Ir75 subfamily was identified for T. infestans, with 19 paralogues and some duplicated members for Ir75e, Ir75f, Ir75i and Ir75j (Fig. 1, highlighted in green). The expansion of the Ir75 lineage is an evolutionary process that seems to include C. lectularius and O. fasciatus, with 12 and 10 Ir75 genes, respectively. For non-heteropteran hemipterans, this lineage seems to have contracted or had no Ir75 orthologues, such as for N. lugens, S. furcifera, and D. citri (Table 1).
A total of 41 OBPs were identified in the de novo assembled transcriptome, with an average length of 146 amino acids, and only eight of them represented incomplete transcripts (Additional file 5: Supplementary Table S3). The analysis of the OBP sequences of T. infestans revealed the six conserved cysteines in 28 transcripts; the characteristic conserved alpha helices (between 6 and 7) in 36 sequences; and the presence of the signal peptide in 28 of them. Except for one OBP candidate, the rest showed positive hits against the PFAM domain PF01395.25, typical of this gene family.
The takeout (TO) repertoire of T. infestans was composed of 36 candidates (Additional file 5: Supplementary Table S3), all of them having the PF06585.14 domain that is characteristic of this family. Only 5 TOs presented partial sequences and all of them presented very similar lengths (247 amino acids on average). Seventeen new TO genes were identified in the R. prolixus genome (annotated from RproTo17 to RproTo32), resulting in a total of 32 (Additional file 5: Supplementary Table S3).
Our work establishes the basis of molecular research on the sensory processes of T. infestans, as we describe the sensory gene repertoire of this relevant Chagas disease vector species. As part of this characterization, we report a set of sensory genes whose antennal expression is modulated by the ingestion of a blood meal. We suggest that they mediate triatomine host-seeking and propose functional studies to determine their specific roles in the detection of multimodal host cues. Therefore, these genes represent potential targets for bug behavioral manipulation for more sustainable control of Chagas disease in the future. 781b155fdc