Fig 1.
Schematic drawing of the molecular structure of putative chemoreceptor proteins co-receptor IRs and iGluRs, tuning IRs, GRs, and TRP channels.
IRs, co-receptor IRs, and iGluRs contain the following domains: extracellular amino terminal domain (ATD); ion channel domain (ICD) that forms the ion channel, consisting of three transmembrane domains (M1, M2, M3) and a pore loop (P); ligand binding domain (LBD) consisting of two half-domains (S1, S2). TRP channels contain the following domains: coiled-coil domain (CC), ankyrin repeats (A), TRP domain (TRP). Adapted from [11].
Fig 2.
Panel A shows all arthropod groups. Panel B shows an expanded view of the Malacostraca. Names of species used in our analysis are included. Based on [39–43].
Fig 3.
Pre-absorption control for anti-HaIR25a.
Cross sections through aesthetascs of P. argus; single optical sections of 0.5 μm thickness. a–c. Sections labeled with control anti-HaIR25a (red) and Hoechst 33258 (blue). d–f. Sections labeled with preabsorbed (with P1Pa and P2Pa) anti-HaIR25a (red) and Hoechst 33258 (blue). Scale bar in e applies to all images. Arrows in a, b, d, and e point to cross-sections of aesthetascs in which anti-HaIR25a labeling is captured at the very surface of the section (highest intensity). Images a, b, d, and e were collected with the same intensity setting of the red fluorescence channel of the confocal microscope; images c and f were collected at a higher (but between c and f consistent) intensity setting to compensate for the fact that labeling intensity of anti-HaIR25a is much higher in the inner dendritic segments (iDS) and outer dendritic segments (oDS) of OSNs than in the somata as was previously reported [11]. Labeling intensity is high in (a) oDS, (b) iDS, and (c) somata of OSNs with the control anti-HaIR25a, but below detectability in (d) oDS), (e) iDS, and (f) somata of OSNs with preabsorbed anti-HaIR25a, demonstrating the specificity of anti-HaIR25a for IR25a in P. argus in addition to that demonstrated by Stepanyan et al. [32] for IR25a in H. americanus.
Table 1.
Number of predicted IRs and iGluRs in transcriptomes of four decapod crustacean species, based on either or both PF domains.
Fig 4.
Phylogenetic tree of IRs in four decapod species and tissue expression.
a. Maximum likelihood phylogenetic tree of IRs and iGluRs from four decapod crustaceans. Clades with co-receptor IRs (IR25a, IR8a, IR76b, and IR93a) are colored in shades of green; clades with tuning IRs that are conserved across crustaceans and insects (IR21a, IR40a, IR68a, and IR75-family) are colored dark grey; clades with tuning IRs that are conserved across all four decapod crustaceans are colored light blue; clades with tuning IRs that are conserved in at least three decapod crustaceans are colored dark blue; clades with iGluRs are colored light grey. * with underline indicates higher expression in dactyl than LF. The tree was built using IQ-Tree with 1000 UFBoot replications under the WAG+F+G4 model of substitution according to BIC as selected by ModelFinder. The tree was visualized on FigTree v.1.4.4. The tree is unrooted but the root is drawn at the iGluR/IR25a/IR8a clade. Scale bar represents expected number of substitutions per site. b. Venn diagrams showing tissue specific differential expression (LF–lateral flagella of antennules, Da–dactyls of walking legs) of IRs and iGluRs in each decapod crustacean as calculated by DESeq2, where a ~ 2.8 fold difference or greater in expression (i.e. log2[fold change] ≥ 1.5 or log2[fold change] ≤ -1.5) between tissue types is considered higher expression in one tissue.
Fig 5.
Phylogenetic tree of conserved IRs across arthropods.
Maximum likelihood phylogenetic tree shows the different IRs that are conserved across major groups of arthropods: chelicerates, myriapods, crustaceans, and insects. IR25a sequences from two gastropods are also included. Among crustaceans, species are colored by their subclass as follows: thecostracan–brown; copepods–pink; isopods–navy blue; amphipods–green; decapods–red; branchiopod–fluorescent blue. The tree was built using IQ-Tree with 1000 UFBoot replications under the LG+F+G4 model of substitution according to BIC as selected by ModelFinder. The tree was visualized on FigTree v.1.4.4. The tree is unrooted but the root is drawn at the iGluR/IR25a/IR8a clade. Scale bar represents expected number of substitutions per site.
Table 2.
Number of predicted IRs and iGluRs in transcriptomes from four species of decapod crustaceans.
Fig 6.
Immunolabeling with anti-HaIR25a in the aesthetasc-bearing tuft region of the lateral flagellum of the antennule.
a–d. Panulirus argus. e–j. Homarus americanus. a, b, e, f. Sagittal sections through the medial plane of the tuft region of lateral flagellum labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at low magnification (maximum intensity projections of confocal image stacks). Scale bar in a also applies to b, scale bar in e also applies to f. a and b adapted from Kozma et al. (2018) [11]. a, e. Overlay of all 3 confocal channels. b, f. anti-HaIR25a channel. Overall organization of aesthetascs is similar in both species: two rows of aesthetasc setae (A) arise from the autofluorescent (blue in P. argus, blue and green in H. americanus) cuticle (C) of an annulus (An + horizontal bar). Each aesthetasc seta is associated with a large cluster of olfactory sensory neuron (OSN) somata which are distinctly labeled by anti-HaIR25a and labeled with moderate intensity by anti-tubulin. Bundles of inner dendritic segments (iD) arising at the apical pole of the OSN clusters are labeled with moderate intensity by both antibodies. Bundles of axons (Ax) arising at the basal pole of OSN clusters are intensely labeled by anti-tubulin labeled with moderate intensity by anti-HaIR25a. Tegumental glands (asterisks) are located between bundles of inner dendritic segments. OSN clusters in H. americanus are more elongated than in P. argus and contain fewer OSNs. c, d, g, h. Sagittal section through OSN clusters labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (confocal images–maximum intensity projection of confocal image stacks with a total thickness of about 1 μm). Scale bar in c also applies to d, scale bar in g also applies to h. c, g. Overlay of all 3 confocal channels. d, h. anti-HaIR25a channel. The somata (OSN) of all OSNs identified by having almost spherical nuclei are distinctly labeled by anti-HaIR25a. Note that the overall shape of OSNs is close to spherical in P. argus but more elliptical in H. americanus. Somata of auxiliary cells (identified by having flat, elongated nuclei—arrows) are not labeled by anti-HaIR25a. In H. americanus, auxiliary cells are not only present at the apical pole of the OSN cluster (white arrows) but also in its center (black arrows). i, j. Horizontal section through an aesthetasc of H. americanus labeled with anti-HaIR25a (red) and anti-tubulin (green) (confocal images of one optical section of 1 μm thickness). Scale bar in j (100 μm) also applies to i. Note that anti-tubulin non-specifically labeled cuticle (C) in addition to dendrites enclosed in the thin cuticular tube of the aesthetasc seta. The bulge at the bottom of the seta (arrowhead) indicates the transition region between inner dendritic segments (iD) and outer dendritic segments (oD). Note that labeling intensity of anti-HaIR25a is considerably higher in oD compared to iD.
Fig 7.
Immunolabeling with anti-HaIR25a in the walking leg dactyl.
a–f. Panulirus argus. g–k. Homarus americanus. a, b, g, h. Sagittal sections through distal part (excluding the epicuticular cap) of the dactyl of the 3rd pereiopod labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at low magnification (maximum intensity projections of confocal image stacks that are 5–10 μm thick). Scale bar in a also applies to b, scale bar in g also applies to h. a, g. Overlay of all 3 confocal channels. b, h. anti-HaIR25a channel. In both species, the numerically dominant sensilla are smooth setae (SS) organized into large, distinct groups. Each smooth seta is innervated by an elongated cluster of sensory neurons that is more than 200 μm long and about 50 μm in diameter and intensely labeled by anti-HaIR25a and anti-tubulin. Both antibodies label the somata of sensory neurons as well as their axons (Ax) and inner dendritic segments (iD). Note that in P. argus, but not in H. americanus, single bipolar sensory neurons labeled by anti-tubulin but not anti-HaIR25a (arrows) are interspersed between the double-labeled clusters of sensory neurons. c–f, i—k. Two examples of clusters of sensory neurons labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (maximum intensity projections of two adjacent optical sections of 0.4 μm thickness); scale bar in f also applies to c–e; scale bar in k also applies to i and j. c, i. Overlay of all three channels. d, j. anti-HaIR25a channel. e. anti-tubulin channel. f. Hoechst channel. k. Overlay of anti-HaIR25a and Hoechst channel.
Fig 8.
Phylogenetic tree of TRP channels across animals.
The maximum likelihood phylogenetic tree shows the conservation of TRP channel sequences from the transcriptomes of four decapod crustaceans with TRP channels from insects, nematodes, and mammals. Among crustaceans, decapods are in red and branchiopod in light blue. All four decapod crustaceans have several homologues to each subfamily of TRP channels. The tree was constructed on IQ-Tree with 1000 UFBoot replications under the LG+F+G4 model of substitution according to BIC, as determined by ModelFinder. Tree was visualized on FigTree v.1.4.4. Tree was unrooted but is drawn with the Group 2 subfamilies, TRPML and TRPP, clades as the root. Support for some inner nodes is low due to incomplete sequences and high divergence. Scale bar represents expected number of substitutions per site.
Fig 9.
Multiple sequence alignment of GRL fragments in decapod crustaceans and GRs in arthropods.
Multiple sequence alignment shows the TM7 region of the sequences that have the highly conserved “TYxxxxxQF” motif (red bar). Sequences were aligned using MAFFT and visualized on Jalview. Decapod crustaceans are in red. Green–Eaff–E. affinis; Blue–Dpul–D. pulex; Grey–Dmel–D. melanogaster. Conservation of amino acids and the consensus histogram were annotated on Jalview. Clustal X color scheme was used to color residues.