A spatiotemporal wave of turnover and functional maturation of olfactory receptor neurons in the spiny lobster Panulirus argus

Abstract

Olfactory receptor neurons (ORNs) of crustaceans are housed in aesthetasc sensilla that are located on the lateral flagellum of the antennule. We used young adult spiny lobsters to examine turnover of aesthetascs and functional maturation of their ORNs after molting. The proliferation zone for new aesthetascs is located in the proximal part of the aesthetasc-bearing region and progressively moves along a distoproximal axis. Older aesthetascs are lost in the distal part of the aesthetasc-bearing region. As a result, an aesthetasc may be shed three to six molts after it differentiates. Taurine-like immunoreactivity is elevated in ORNs of aesthetascs that have yet to emerge on the cuticular surface and thereafter decreases gradually and asynchronously. ORNs from the distalmost-developing aesthetascs lose taurine-like immunoreactivity immediately before sensillar emergence, whereas ORNs from the most proximal and lateral new aesthetascs retain taurine-like immunoreactivity throughout the intermolt stage after sensillar emergence. Furthermore, taurine-like immunoreactivity is inversely correlated with odor responsiveness. These results suggest that taurine-like immunoreactivity reveals immature ORNs and that their functional maturation is not synchronized with molting and may not be completed until many weeks after sensillar emergence. Our data suggest successive spatiotemporal waves of birth, differentiation and functional maturation, and death of ORNs.

Fig. 1.

Lateral flagellum of the spiny lobster P. argus. Drawing,Top, The aesthetasc-bearing region that is located in the distal half of the lateral flagellum. LettersA–F indicate the relative position of the flagellar regions that are shown in each respective micrograph. A, Scanning electron micrograph showing the distal part of the flagellum. Note the absence of aesthetascs (a), normal-sized guard hairs (g), asymmetric hairs (as), and companion hairs (c) on the four most distal annuli. B, Scanning electron micrograph showing a mesial view of the flagellum with the aesthetascs (a) and their accompanying sensilla, including mesial companion hairs (mc) and mesial guard hairs (mg). Note the presence of one to two mesial companion hairs per annulus.C, Scanning electron micrograph showing a high-magnification view of rows of aesthetascs (a), asymmetric hairs (as), mesial and lateral guard hairs (mg and lg, respectively), and lateral companion hairs (lc).D, Scanning electron micrograph showing a lateral view of the flagellum with the aesthetascs (a) and their accompanying sensilla, including lateral companion hairs (lc), lateral guard hairs (lg), a long lateral seta (ls), and a plumose seta (ps). E, Scanning electron micrograph showing a high-magnification view of the plumose seta (ps) that usually replaces the lateral companion hair on an annulus that possesses a long lateral seta.F, Scanning electron micrograph of the proximal end of the aesthetasc region. Note the small number of aesthetascs (a) on the most proximal row and the presence of guard hairs (g) on the two annuli just proximal to the aesthetasc-bearing annuli. Scale bars: A, B, D, 200 μm; C, 100 μm; E, 20 μm;F, 400 μm.

Fig. 2.

Example of the distribution of sensilla in the aesthetasc region on the premolt flagellum (exuvium;left) and its corresponding postmolt flagellum (right). Most of the proximal nonaesthetasc region is not shown. Each row corresponds to an annulus of the flagellum. Premolt and postmolt flagella are aligned so that patterns of distribution of the mesial companion hairs (column mc) and long lateral setae (column ls) are matching. Note the good matching of the pattern of the mesial companion hairs (column mc) in both premolt and postmolt flagella, with a few exceptions indicated in the postmolt flagellum asgray boxes for loss of sensilla and black boxes for gain of sensilla. Note that the distribution of the long lateral setae (column ls) is identical in the aesthetasc region of the premolt flagellum and its corresponding region on the postmolt flagellum, with the exception of the three most distal long lateral setae and associated plumose setae that disappeared after the molt (indicated as gray boxes). Note that proximal to the aesthetasc region of the premolt flagella, the position of the long lateral setae does not match with the position of the long lateral setae on the postmolt flagellum. This is caused by the addition of annuli between two consecutive setae. * indicates that the lateral companion hair was replaced by a plumose seta (Fig. 1D,E, ps).a, Aesthetascs; as, asymmetric hairs;lc, lateral companion hairs; lg, lateral guard hairs; mg, mesial guard hairs.

Fig. 3.

Net changes in the number of annuli in different parts of the lateral flagellum after molting. Values are means ± SEM, for seven flagella from four animals. Data are based on changes in the number of annuli between consecutive long lateral setae in different parts of the flagellum. These parts are the following:Distal tip, region at the tip with no or few aesthetasc-bearing annuli; “Old” aesthetascs, region of annuli with existing rows of aesthetascs; All new aesthetascs, region of annuli with newly emerged aesthetascs; and No aesthetascs, region of annuli without aesthetascs. For the nonaesthetasc-bearing region, the net change is based on the change in the number of annuli between the most distal long lateral setae along this region and the flagellar base. To quantify and compare changes in annuli after the molt on many different flagella of variable lengths, we standardized the length of each postmolt flagellum by using as fixed reference points the tip of the flagellum (point 100) and the most proximal annuli with 1-molt-cycle-old aesthetascs (point0). Negativenumbers along the standardized flagella indicate regions with annuli that did not bear aesthetascs before the last molt. To illustrate these regions and their standardized positions, drawings (top) of an exuvium (i.e., premolt flagellum) and its corresponding postmolt flagellum are shown such that an annulus on the exuvium is aligned with its corresponding annulus on the postmolt flagellum.Breaks in the drawings of the premolt and postmolt flagella and on the x-axis indicate that not all of the proximal nonaesthetasc-bearing annuli are shown in this figure.

Fig. 4.

The number of aesthetascs per row (filled circles), the aesthetasc length (opentriangles), and net changes in the number of aesthetascs per row (open circles) along different parts of the lateral flagellum after molting. Values are means ± SEM, for 10 flagella from six animals. To analyze and compare flagella of variable lengths, we standardized the length of each postmolt flagellum as described in Figure 3.Drawings (top) of a premolt and postmolt flagellum are shown such that an annulus on the premolt flagellum (i.e., exuvium) is aligned with its corresponding annulus on the postmolt flagellum.

Fig. 5.

Drawings of a flagellum showing the turnover of aesthetasc-bearing annuli and the relative position of representative sets of annuli over a period of six molts. A few sets of annuli along the flagellum are colored to illustrate the relative movement of annuli toward the distal part of the flagellum as new rows of aesthetascs are developing and emerging on the cuticular surface of the proximal region and as the distal aesthetasc-bearing annuli are gradually losing their aesthetascs and are finally shed. Regions numbered from Ito VII represent zones of the flagellum where distinct turnover-related events occur: zone I, addition of annuli; zone II, addition of annuli (indicated as an increase in the number of green annuli) and new rows of aesthetascs; zone III, addition of a few aesthetascs on existing rows of aesthetascs; zone IV, no net change in aesthetasc numbers; zone V, net loss of aesthetascs;zone VI, loss of all aesthetascs; and zone VII, no aesthetascs, with shedding of annuli between molts, presumably by damage, and at molting. This figure is based on the extrapolation over six molts of the data presented in the Results and Figures 3 and 4. It assumes that a given zone occupies the same proportion of the flagellar length over a period of six molts.

Fig. 6.

Taurine-like immunoreactivity in ORNs of an intermolt lobster (stage C). Drawing, Bottom, The aesthetasc-bearing region with the positions in the developmental zones of the transverse sections shown in A–C.A, Transverse section through clusters of ORN cell bodies (cl) associated with existing and mature aesthetascs in the medial part of the aesthetasc region (zone IV; see drawing). Note a lack of taurine-like IR in ORNs but a presence of taurine-like IR in the supporting cells (*).B, Transverse section through clusters of ORN cell bodies (cl) associated with newly emerged aesthetascs in a new row of sensilla located six annuli distal to the proximal end of the aesthetasc-bearing region (medial part ofzone III; see drawing). C, Transverse section through clusters of ORN cell bodies (cl) associated with newly emerged aesthetascs in a new row of sensilla located one annulus distal to the proximal end of the aesthetasc-bearing region (proximal part of zone III; see drawing). In B andC, note on the lateralside the presence of cell body clusters (cld) and inner dendrites (arrow) of developing ORNs associated with aesthetascs that have not yet emerged at the cuticular surface but will at the next molt. Note the very high and uniform level of taurine-like IR in ORNs of these developing clusters, compared with the weaker and more variable taurine-like IR in ORNs of the newly emerged aesthetascs. Note also the greater degree of taurine-like IR in ORN clusters of emerged aesthetascs (cl) in C than inB. id, Inner dendrites of ORNs associated with a single aesthetasc; *, supporting cells. Scale bar, 100 μm.

Fig. 7.

Taurine-like immunoreactivity in ORNs of a premolt lobster (stage D₀–D₁ or ∼3 weeks before molting). Drawing, Bottom, The positions along the flagellum of the horizontal sections shown in A andB. A, Horizontal section through clusters of ORN cell bodies associated with aesthetascs in the most proximal annulus with emerged aesthetascs (proximal part of zone III; see drawing). Clusters of ORNs without taurine-like IR (cl) are associated with aesthetascs that emerged at the cuticular surface at the previous molt. Clusters of ORNs with taurine-like IR (cld) are associated with developing aesthetascs that will emerge at the cuticular surface at the next molt and are located on the lateral side of rows of emerged aesthetascs. Supporting cells (*) of the aesthetascs also show elevated taurine-like IR. B, Horizontal section through clusters of ORN cell bodies (cld) associated with developing aesthetascs in the third most distal annuli with no emerged aesthetascs (distal part of zone II; seedrawing). This figure shows that the ORNs of aesthetascs ready to emerge (cld) on the lateral side of existing rows (in zone III; A) and in the nonaesthetasc-bearing annuli (zone II; B) had relatively lower levels of taurine-like IR than ORNs of developing aesthetascs during the intermolt stage (Fig.6B,C, cld). By the end of the premolt stage (1–3 d before molting; stage D₃–D₄), most of the ORNs from the most distal developing aesthetascs (in zone III) that are ready to emerge did not have taurine-like IR, whereas ORNs from more proximal developing aesthetascs (in zone II) still showed taurine-like IR (data not shown). Scale bar, 100 μm.

Fig. 8.

Taurine-like immunoreactivity in ORNs of a postmolt lobster (stage AB). Drawing, Bottom, The aesthetasc-bearing region with the positions in the developmental zones of the transverse sections shown in A–C.A, Transverse section through clusters of ORN cell bodies (cl) associated with existing and mature aesthetascs in the medial part of the aesthetasc region (zone IV; see drawing). Note a lack of taurine-like IR in ORNs. B, Transverse section through clusters of ORN cell bodies (cl) associated with newly emerged aesthetascs in a new row of sensilla located 11 annuli distal to the proximal end of the aesthetasc-bearing region (distal part ofzone III; see drawing). Note that clusters located on the lateralside have more ORNs with high taurine-like IR than do ORNs on themesialside. C, Transverse section through clusters of ORN cell bodies (cl) associated with newly emerged aesthetascs in a new row of sensilla located four annuli distal to the proximal end of the aesthetasc-bearing region (proximal part of zone III; see drawing). By contrast to A, note inB and C the high taurine-like IR in many ORNs. Scale bar, 100 μm.

Fig. 9.

Taurine-like immunoreactivity in ORNs shows a spatiotemporal gradient in rows of newly emerged aesthetascs (zone III). Taurine-like IR in new ORNs decreases slowly but asynchronously over time after a molt, with ORNs of proximal and lateral aesthetascs retaining high levels of taurine-like IR for longer time periods than ORNs of distal and mesial aesthetascs.A, Distal rows of new aesthetascs that are located 9–15 annuli distal from the proximal end of the aesthetasc region (Fig. 4, region ∼0 to −10; see drawing, bottom right). At the postmolt stage, aesthetascs have ORNs with variable levels of taurine-like IR. A small proportion of mesial aesthetascs contains ORNs without taurine-like IR, whereas a large proportion of lateral aesthetascs contains only ORNs with taurine-like IR. At the intermolt stage, a large proportion of aesthetascs has only ORNs without taurine-like IR, especially in the mesial aesthetascs.B, Proximal rows of new aesthetascs that are located on the six most proximal annuli of the aesthetasc region (Fig. 4, region approximately −10 to −20; see drawing, bottom right). At the postmolt stage, all aesthetascs have all or most of their ORNs with taurine-like IR. At the intermolt stage, an increasing proportion of aesthetascs has ORNs without taurine-like IR, especially in the mesial aesthetascs. Lateral and mesial aesthetascs are the two most lateral and mesial aesthetascs within a row, respectively.

Fig. 10.

Schematic model of the spatiotemporal wave of differentiation and maturation of ORNs in clusters of developing aesthetascs (circles with red line) and newly emerged aesthetascs (circles with black line) throughout a molt cycle. Levels of grayswithin the circles indicate different levels of taurine-like IR in ORNs of a cluster; an empty circlesymbolizes a cluster for which no ORNs have taurine-like IR. ORNs from newly emerged aesthetascs, particularly those with high taurine-like IR, are also less likely to be odor responsive (see Fig. 11). Developmental zones (Fig. 5, II,III) are given on the rightside of the figure.

Fig. 11.

Odor-dependent activity labeling of ORNs in different stages of development. In lateral flagella of intermolt (stage C) and very early premolt (beginning stage D₀) lobsters, newly emerged aesthetascs contain fewer odor-dependent activity-labeled ORNs than do old aesthetascs (>1 molt cycle old), and new aesthetascs with many ORNs with elevated levels of taurine-like IR contain fewer odor-dependent activity-labeled ORNs than do new aesthetascs with no or a few ORNs with taurine-like IR. A, Flagella (n = 9) stimulated with a single odorant compound (either NH₄, AMP, glycine, proline, or cysteine) and flagella (n = 2) not odor stimulated [only agmatine (AGBcontrol)]. All flagella stimulated with any of the single odorants showed the same trend and were pooled together. The percentage of ORNs with taurine-like IR is inversely correlated with the percentage of ORNs with odor-dependent activity labeling in flagella stimulated with single odorants (p< 0.05, nonparametric gamma correlation test, Statistica; StatSoft, Tulsa, OK). B, Flagella (n = 3) stimulated with the oyster mixture (a 33-component odor mixture) and flagella (n = 2) not odor stimulated [agmatine only (AGB control)]. There was a nonsignificant trend toward an inverse correlation between the percentage of ORNs with odor-dependent activity labeling and the percentage of taurine-like IR ORNs in flagella stimulated with either the oyster mixture or agmatine alone (p > 0.05, nonparametric gamma correlation test). *Significant difference in odor activity labeling (p < 0.05, Kruskal–Wallis nonparametric ANOVA).

Fig. 12.

Schematic summary of the turnover of aesthetascs in the lateral flagellum of the spiny lobster (see Discussion).Open circles represent emerged aesthetascs; dark circles indicate developing aesthetascs that will emerge at the cuticular surface at the following molt. The developmentalzonesI–VII are described in Results and Figure 5.