Fig 1.
Overview of spiny lobster chemosensory systems.
Modified from Derby et al. (2016) [9]. (a) Location of aesthetascs mediating olfaction (blue dots) and bimodal chemo- and mechanosensory sensilla mediating distributed chemoreception (yellow dots) on different body parts and appendages of P. argus (1—lateral flagellum of antennule, 2—medial flagellum of antennule, 3—second antenna, 4—mouthpart appendages, 5—walking legs, 6—gill chamber, 7—tail fan, 8—pleopods). Location of pieces of appendages used for immunocytochemistry and PCR indicated by gray boxes: dactyl, 2nd antenna (A2). (b) Location of aesthetascs and bimodal chemo- and mechanosensory sensilla on the antennules. Aesthetascs (blue) are restricted to a tuft of sensilla on the distal third of the lateral flagellum. Bimodal chemo- and mechanosensory sensilla (yellow) among them, guard setae (GS) are associated with the aesthetascs but also occur on the proximal part of the lateral flagellum and on the entire medial flagellum. Location of pieces of appendages used for immunocytochemistry and PCR indicated by gray boxes: lateral flagellum of antennule proximal (LFP), lateral flagellum of antennule distal (LFD). (c) Schematic drawing of the cellular organization of olfactory sensilla. Olfactory sensilla, called aesthetascs, are exclusively innervated by olfactory receptor neurons (ORN, blue). The outer dendritic segments of the ORNs (modified cilia) are highly branched and covered by extremely thin and permeable cuticle (pC). (d) Schematic drawing of the cellular organization of distributed chemosensilla. Distributed chemosensilla are bimodal chemo- and mechanosensory sensilla innervated by a few mechanoreceptor neurons (MRN) and several chemoreceptor neurons (CRN). Dendrites of CRNs are unbranched and extend to a terminal pore (tP) at the tip of the sensillum. (e) Schematic drawing of the molecular structure of chemoreceptor proteins (iGluRs and Co-IRs, IRs, GRs, and TRP channels). Transmembrane domains of iGluRs and IRs (M1 –M3), pore loop (P), ligand binding domains (S1, S2), amino terminal domain (ATD), coiled-coil domain (CC), ankyrin repeats (A), TRP domain (TRP).
Fig 2.
Maximum likelihood phylogenetic tree of iGluRs and IRs.
Colored areas of the tree represent iGluRs (shades of grey) and co-receptor IRs (shades of green). ■ indicates conserved IR sequences of the IR40a family. Sequence colored in black indicates the Parg divergent IR that has an amino acid substitution in the conserved glutamate binding site in the S1 region of the LBD. The tree was built under LG+F+G4 model of substitution with 1000 ultrafast bootstrap (UFBoot) replications and visualized on FigTree v1.4.2. Bootstrap values on some internal branches of divergent IRs are low due to incomplete sequences and high sequence divergence across the different species. The tree was unrooted but drawn with the NMDA clade as root. The scale bar represents expected number of substitutions per site.
Table 1.
Number of unique iGluRs and IRs in P. argus, D. pulex, and D. melanogaster.
Fig 3.
Multiple sequence alignment of iGluRs and IRs of Parg, Dmel, and Dpul.
This analysis shows that the LBD is highly divergent within the IRs and in comparison to iGluRs. Parg (red), Dmel (mint), and Dpul (blue) sequences are organized based on sequence homology in Fig 2. Divergent IR, PargIR1059, an exception to ‘R’ conservation in S1 lobe, is indicated in black. Glutamate binding sites are indicated with arrows. (a) S1 lobe of LBD. (b) S2 lobe of LBD. The sequences were aligned with MAFFT and visualized on Jalview. The residues were colored according to the Clustal X color scheme on Jalview. Criteria for the color scheme: http://www.jalview.org/help/html/colourSchemes/clustal.html.
Fig 4.
Maximum likelihood phylogenetic tree of homologous sequences of conserved IRs.
Only conserved IR groups with Parg homologues are colored. Pink branches represent homologous sequences of IR68a with the Ccly sequence, the putative PargIR68a-put, and DpulIR298 as crustacean representatives. Sequences were aligned with MAFFT and visualized on Jalview for editing. The tree was built on IQ-Tree under LG+F+G4 model of substitution with 1000 UFBoot replications and visualized on FigTree v1.4.2. Bootstrap values in some internal nodes are low due to incomplete sequences. The tree was unrooted but drawn with the NMDA clade as the root. The scale bar represents the expected number of substitutions per site.
Fig 5.
Immunolabeling with anti-HaIR25a in the lateral flagellum of the antennule.
(a–s) Aesthetascs-bearing tuft region of the lateral flagellum. (a) Outer morphology—stereomicroscopical image. Each annulus (horizontal bar) bears two rows of transparent aesthetascs (A) accompanied by guard setae (GS). Dashed line shows direction of cross section through aesthetascs in (g–k). (b) Sagittal section labeled with Hoechst 33258 showing full length of aesthetasc setae (A). Each annulus (horizontal bar) bears two rows of aesthetascs. ORN clusters (ORN) are labeled by Hoechst whereas cuticle (C) is autofluorescent. Only the proximal 20% of the cuticle of the aesthetasc setae containing the inner dendritic segments of the ORNs is autofluorescent (arrow). (c) Sagittal view of the lateral flagellum (modified from Schmidt et al. (2006) [87]). Each aesthetasc (A) is innervated by a cluster of ORNs (blue) whose inner dendritic segments traverse the cuticle (C) in a wide canal. ORN clusters are associated with subcuticular aesthetasc tegumental glands (ATG) whose thin drainage ducts terminate in pores at the base of the aesthetascs. Guard setae (GS) are located at the lateral margins of the aesthetasc rows. (d) Reconstruction of aesthetasc ultrastructure based on transmission electron microscopy; inset: region of transition from inner to outer dendritic segments within the base of aesthetasc setae at higher magnification (modified from Grünert and Ache (1988) [94]). Each aesthetasc (A) is innervated by about 320 ORNs whose somata form a cluster (ORN) below the cuticle (C). Inner dendritic segments arising apically from the ORN somata are wrapped by auxiliary cells (AC). Inset: each inner dendritic segment (iD) gives rise to two highly-branched outer dendritic segments (oD). (e, f) Sagittal section through medial plane of the tuft region of a lateral flagellum labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at low magnification (confocal images). Scale bar in (e) also applies to (f). (e) Overlay of all three fluorescence channels. (f) anti-HaIR25a channel. Two rows of aesthetascs setae (A) arise from the intensely autofluorescent (blue) cuticle of an annulus (horizontal bar in e). Each aesthetasc seta is associated with a clearly delineated cluster of ORN somata (ORN). The somata and inner dendritic segments (arrows) are intensely labeled by anti-HaIR25a and anti-tubulin. (g–k) Cross sections through aesthetasc setae (direction of section indicated by dashed line in (a)). (g, h) Low magnification epifluorescence images; scale bar in (h) also applies to (g). (g) Overlay of all three fluorescence channels. (h) anti-HaIR25a channel. Aesthetasc setae (A) located on different annuli (horizontal bars with numbers) are cut at different levels systematically progressing from the tips (1) to the base emerging from the annulus cuticle (5). Note that the lumen of the aesthetasc setae containing outer dendritic segments (1, 2, 3, 4—left aesthetasc row) is more intensely labeled by anti-HaIR25a than the lumen of aesthetasc setae containing only inner dendritic segments (4 –right aesthetasc row, 5). (i–k) High magnification epifluorescence images of sections through aesthetasc setae on annulus 3; insets: very high magnification (confocal images); scale bars in (j) (top: 50 μm; bottom: 10 μm) also apply to (i) and (k). (i) Overlay of anti-HaIR25a and anti-tubulin fluorescence channels. (j) anti- HaIR25a channel. (k) anti-tubulin channel. Note that entire lumen of aesthetasc setae is filled by outer dendritic segments of ORNs intensely labeled by anti-HaIR25a and anti-tubulin. (l–o) Sagittal section through ORN clusters labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (confocal images–maximum intensity projection of entire stack of optical sections spanning about 60 μm). Scale bar in (m) also applies to (l), (n), and (o). (l) Overlay of all 3 fluorescence channels. (m) anti-HaIR25a channel. (n) anti-tubulin channel. (o) Hoechst channel. Insets in (m): Apical region of ORN cluster at high magnification; left, overlay of all 3 fluorescence channels; right, anti-HaIR25a channel (confocal images of a single optical section). The somata (ORN), axons (Ax), and inner dendritic segments (iD) of all ORNs are intensely labeled by anti-HaIR25a and anti-tubulin. Somata of auxiliary cells (nuclei indicated by arrows) are not labeled by either of the antibodies and epithelial cells (nuclei indicated by arrowheads) and aesthetasc tegumental glands (asterisks) are labeled by anti-tubulin but not anti-HaIR25a. Autofluorescent (blue) cuticle (C). (p–s) Sagittal section through lateral plane of tuft region of lateral flagellum labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue). (p, q) Section at low magnification (confocal images); scale bar in (q) also applies to (p). (p) Overlay of all three fluorescence channels. (q) anti-HaIR25a channel. In addition to clusters of ORN somata, clusters of sensory neurons innervating bimodal sensilla accompanying the aesthetascs (one cluster outlined by white dots) are also labeled by anti-HaIR25a and anti-tubulin. The intensity of labeling with anti-HaIR25a is higher in ORN somata and axons (Ax) than in somata and axons of the other sensory neurons. (r, s) Section at high magnification (confocal images; scale bar in (r) also applies to (s). (r) anti-HaIR25a and anti-tubulin channel. (s) anti-HaIR25a channel. All sensory neurons of the clusters are labeled by anti-tubulin, but three of them (asterisks) mostly located in the distal aspect of the cluster are not double-labeled by anti-HaIR25a. (t–v) Sagittal section through cluster of sensory neurons innervating a bimodal sensillum in the proximal part of the lateral flagellum labeled with anti-HaIR25a (red), anti-tubulin (green) and Hoechst 33258 (blue). (t) Overlay of all three fluorescence channels. (u) anti-HaIR25a channel. (v) anti-tubulin channel. All sensory neurons of the clusters are labeled by anti-tubulin but one of them (asterisk) located in the distal aspect of the cluster is not double-labeled by anti-HaIR25a. The intensity of labeling with anti-HaIR25a and anti-tubulin differs substantially but independent of each other between labeled somata.
Fig 6.
Immunolabeling with anti-HaIR25a in the walking leg dactyl and in the flagellum of the 2nd antenna.
(a–l) Walking leg dactyl. (a) Outer morphology of the dactyl of a third pereiopod of a late juvenile animal, shown in a stereomicroscopical image. The dactyl bears rows of evenly spaced bundles of smooth setae (asterisks), except on the epicuticular cap (Cap) at the tip. Propodus (P). (b, c) Outer morphology of the distal part of the dactyl of a second pereiopod of a late juvenile animal, shown in scanning electron micrographs (SEM). The main body of the dactyl bears rows of dense bundles of about 20 smooth setae (asterisks). Single smooth spines (S) are located between rows of bundled setae. The epicuticular cap (Cap) does not bear setae but instead holds numerous small depressions (arrows) that likely represent the outer structures of bimodal sensilla called funnel-canal organs. (d–f) Sagittal section through the distal aspect of a third pereiopod dactyl (proximal to epicuticular cap) labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at low magnification (confocal images); scale bar in (e) also applies to (d) and (f). (d) Overlay of all three fluorescence channels. (e) anti-HaIR25a channel. (f) anti-tubulin channel. Numerous spindle-shaped clusters of sensory neurons (one outlined by white dots), each innervating one of the smooth bundled setae are 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). Overlay of all three channels reveals that in the clusters of sensory neurons, neurons that are labeled by anti-tubulin but not by anti-HaIR25a (and therefore appear green) are located at the distal pole of the clusters. Single bipolar sensory neurons labeled by anti-tubulin but not anti-HaIR25a (arrows) are interspersed between clusters of sensory neurons. (g–l) Clusters of sensory neurons labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (confocal image); scale bar in (h) also applies to (g), (i); scale bar in (k) also applies to (j) and (l). (g, j) Overlay of all three fluorescence channels. (h, k) anti-HaIR25a channel. (i, l) anti-tubulin channel. Each cluster contains about 15 bipolar sensory neurons, all strongly labeled by anti-tubulin. Neurons in the proximal part of the cluster are also intensely labeled by anti-HaIR25a, but two or three neurons located at the distal pole of the cluster are not labeled by anti-HaIR25a and two other neurons in the distal region are only weakly labeled by anti-HaIR25a (arrowheads). Axons (Ax), inner dendritic segments (iD). (m–o) Flagellum of 2nd antenna. Cluster of sensory neurons labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (confocal images); scale bar in (n) also applies to (m) and (o). (m) Overlay of all three fluorescence channels. (n) anti-HaIR25a channel. (o) anti-tubulin channel. All sensory neurons of the cluster are labeled by anti-tubulin, although to different degrees. All but 3 sensory neurons are also labeled by anti-HaIR25a. Two of the HaIR25a-negative neurons (asterisks) are the largest neurons of the clusters suggesting that they are MRNs.
Fig 7.
Immunolabeling with anti-HaIR25a in the brain.
(a) Schematic drawing of the olfactory pathway (light blue overlay) in the brain of P. argus (modified from Schmidt and Ache (1996) [8]). Afferent axons of ORNs (1, blue) enter the brain via the antennular nerve (A1Nv) and project to the ipsilateral olfactory lobe (OL) where they terminate in one of its cone-shaped glomeruli. The OL is closely linked to another glomerular neuropil, the accessory lobe (AL). OL and AL are innervated by local interneurons (green) whose somata form the medial soma clusters (MC) and ascending projection neurons (red) whose somata form the lateral soma clusters (LC). Axons of projections neurons form the olfactory glomerular tracts (OGT) that run within the protocerebral tracts (PT) connecting the brain with the eyestalk ganglia. Median protocerebral neuropils (MPN). (b) Horizontal section through brain stained with ethyl gallate (EG). Large cells (arrows in insets) are located in the axon sorting zone of the A1Nv before it reaches the OL. Location of insets is shown by black squares; scale bar in left inset is 100 μm and also applies to right inset. Antenna 2 nerve (A2Nv). (c–e) Cross section through the antennular nerve where it enters the brain, stained with methylene blue (MB). (c) Low magnification. The axon fascicles in the antennular nerve form three large divisions. The lateral division is formed by axon fascicles from the lateral flagellum that are more intensely stained than other axon fascicles (because they contain numerous extremely thin ORN axons). Large, intensely stained cells selectively occur in the lateral division (white rectangle). (d, e) Large, intensely stained cells (arrows) in the lateral division of the antennular nerve at higher magnification. (e) Region highlighted in (c). The large cells have voluminous cytosol and a spherical nucleus containing at least one dense nucleolus. (f, g) Confocal image of a sagittal section through brain labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at low magnification); scale bar in (f) also applies to (g). (f) Overlay of all three fluorescence channels. (g) anti-HaIR25a channel. Anti-HaIR25a intensely labels a loose assembly of about 100 large cells located in the axon sorting zone of the A1Nv before it reaches the OL. Axons within the antennular nerve are intensely labeled by anti-tubulin but not by anti-HaIR25a, and HaIR25a-positive cells are not labeled by anti-tubulin. (h) Confocal image of a sagittal section through brain of different animal labeled with anti-HaIR25a at low magnification () shows a similar assembly of about 100 intensely labeled large cells in the axon sorting zone of the antennular nerve. (i–n) Morphology of single large cells in the axon sorting zone of the antennular nerve labeled with anti-HaIR25a (red), anti-tubulin (green), and Hoechst 33258 (blue) at high magnification (confocal images). Scale bar in (l) applies to all images. (i, k, m) Overlay of all three fluorescence channels. (j, l, n) anti-HaIR25a channel. (i, j) Unipolar cells. Most of the HaIR25a-positive cells have one process (arrows) projecting from the cell body. Generally, this process projects toward the OL. (k, l) Bipolar cell. Some of the HaIR25a-positive cells have two processes (arrows) projecting from both poles of the cell body. (m, n) Pseudo-unipolar cell. Rarely HaIR25a-positive cells have two processes (arrows) projecting from the same region of the cell body. (o–t) Double-labeling with anti-HaIR25a (red) and WGA-AF488 (Hoechst 33258—blue) at high magnification (confocal images). Scale bar in (q) applies to all images. (o, r) Overlay of all three fluorescence channels. (p, s) WGA-AF488 channel. (q, t) Hoechst 33258 channel. (o–q) Large cells in the axon sorting zone of the OL. (r–t) Neuronal somata in the medial soma cluster. Large cells in axon sorting zone are intensely labeled by anti-HaIR25a but not by WGA whereas somata in the MC are not labeled by anti-HaIR25a but are intensely labeled by WGA. Nuclei of large cells and neurons (arrows) are similar in shape (spherical) and in having very loose heterochromatin (Hoechst labeling of low intensity).
Fig 8.
(a–e) Gel images of PCR products amplified from all target tissues, antenna 2 (A2), central brain (Brain), dactyl of second pereiopod (Dactyl), green gland (GG), aesthetasc-bearing tuft region of the distal lateral flagellum of antennule (LFD), and proximal region of the lateral flagellum of antennule (LFP), using specific primer pairs for GAPDH (a), NMDA-R1 (b), IR25a (c), IR8a (d), IR93a (e), PargIR1028 (f) found only in LF; PargIR1074 (g) found only in dactyl. The predicted amplicon length of the PCR product for each primer pair is given in parentheses. The left side of each gel shows a 100 bp DNA ladder.
Table 2.
PCR results on expression of iGluRs and IRs in different tissues of P. argus.
Fig 9.
PargGR1 fragment sequence alignment.
The multiple sequence alignment of PargGR1 (red) with GRs from arthropods, Eaff (green), Dpul (pink), and Dmel (grey) shows the TM7 region of the 7tm_7 superfamily. Sequences were aligned using MAFFT and visualized on Jalview. Conservation of amino acids across GRs of various species is highest at the ‘TYxxxxxQF’ motif (grey bar) as shown in the consensus histogram. The residues were colored according to the Clustal X color scheme on Jalview.
Fig 10.
Maximum likelihood phylogenetic tree of TRP channels.
Various subfamilies of TRP channels are indicated by different colors where shades of each color indicates a class of TRP channels within a subfamily: TRPA subfamily (grey), TRPM (green), TRPV (yellow), TRPC (pink), TRPN (orange), TRPP (light blue), and TRPML (dark blue). Sequences were aligned with MAFFT and visualized on Jalview. The tree was built on IQ-Tree under LG+G4 model of substitution with 1000 UFBoot replications and visualized on FigTree v1.4.2. Tree was unrooted but drawn with Pkd2 and TRPML clades as the root. The scale bar represents expected number of substitutions per site.