Morphology and distribution of setae on the antennules of the Caribbean spiny lobster Panulirus argus reveal new types of bimodal chemo-mechanosensilla.

This study describes the morphology and distribution of setae on the lateral and medial flagella of the antennules of the spiny lobster Panulirus argus in an effort to identify antennular chemoreceptors in addition to the well-studied aesthetasc chemosensilla. Setae were examined using light and electron microscopy, and their distribution on flagellar annuli was analyzed. We identified ten setal types based on external morphology: hooded, plumose, short setuled, long simple, medium simple, short simple, aesthetasc, guard, companion, and asymmetric setae, with the last four types being unique to the "tuft" located on the distal half of the lateral flagellum. The three setal types whose ultrastructure was examined--hooded, long simple, and medium simple setae--had characteristics of bimodal (chemo-mechanoreceptive) sensilla. The antennules have four distinct annular types based on their setal complement, as shown by cluster analysis. This basic distribution of non-tuft setal types is similar for both lateral and medial flagella. Annuli in the tuft region have tuft setal types superimposed on a basic organization of non-tuft setal types. These results show that the antennules possess a diverse set of setae, that these setae have a highly ordered arrangement on the antennules, that at least four (and probably many more) of these setal types are chemosensilla, and suggest that most antennular chemosensilla are bimodally sensitive.

Postembryonic proliferation in the spiny lobster antennular epithelium: rate of genesis of olfactory receptor neurons is dependent on molt stage.

Olfactory systems undergo continuous growth and turnover in many animals. Many decapod crustaceans, such as lobsters and crayfish, have indeterminate growth, and in these animals, turnover of both peripheral and central components of the olfactory system occurs continuously throughout life. In this study, we examine the dynamics of olfactory receptor neuron (ORN) proliferation in the antennule of the Caribbean spiny lobster, Panulirus argus, using in vivo incorporation of the cell proliferation marker BrdU. We show that addition of ORNs occurs in a "proximal proliferation zone" (PPZ), which exists on the proximo-lateral margin of the existing ORN population. The PPZ is spatially and temporally dynamic in that it travels as a wave in the proximal and lateral directions in the antennule. This wave results in continuous addition of ORNs throughout the molt cycle. The rate of proliferation, as measured by the size and shape of the PPZ, changes depending on the animal's molt stage. The rate is highest during premolt and lowest during intermolt. ORNs are the most prominent cell-type produced in the PPZ, but other cell types, including glia, are also produced. Patches of proliferating epithelial cells occur immediately proximal to the PPZ, suggesting that neuronal and glial precursors reside in this region. Possible mechanisms for peripheral and central modulation of ORN development are discussed.

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

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.

Functional units of a compound nose: aesthetasc sensilla house similar populations of olfactory receptor neurons on the crustacean antennule.

The lateral flagellum of the antennule of the spiny lobster Panulirus argus houses more than 1,000 morphologically similar olfactory sensilla, called aesthetascs. By using a high-resolution activity labeling technique that depends on entry of agmatine into olfactory receptor neurons (ORNs) through cation channels during odor stimulation, we examined the distribution of different functional types of ORNs within and across mature aesthetascs. A significant number of ORNs in mature aesthetascs are labeled with agmatine during stimulation by single odorants, including adenosine-5'-monophosphate, ammonium chloride, cysteine, glycine, proline, and taurine. The percentage of ORNs per aesthetasc that was agmatine labeled during odor stimulation averaged 0.5-1.6% for single compounds and 4.6% for a 33-component mimic of oyster tissue. For most antennules and antennular regions studied, the percentage of agmatine-labeled ORNs by stimulation with single or complex odorants was statistically homogeneous across most or all aesthetascs. The extent of heterogeneity among mature aesthetascs was correlated with their age: extensive heterogeneity was observed only in the distal part of the flagellum containing the oldest aesthetascs and their ORNs. Thus, it appears that over most of the length of the aesthetasc-bearing region of the lateral flagellum, different and distinct functional types of aesthetascs do not exist. Rather, aesthetascs appear to be repetitive morphological and functional units in olfactory coding. However, because odor sensitivity of ORNs can change with the age of an aesthetasc, some development-related functional heterogeneity exists among aesthetascs.

High-resolution functional labeling of vertebrate and invertebrate olfactory receptor neurons using agmatine, a channel-permeant cation.

Methods are described for odor-stimulated labeling of olfactory receptor neurons (ORNs) of the freshwater zebrafish Danio rerio and the marine spiny lobster Panulirus argus. Permeation of a cationic molecule, 1-amino-4-guanidobutane ( = agmatine, AGB), through ion channels following odor stimulation, and its detection by an anti-AGB antibody, allow labeling of odor-stimulated ORNs. Parameters adjusted to optimize activity-dependent labeling included labeling medium ionic composition, stimulation times, and AGB concentration. For lobsters, 7% of ORNs were labeled by a complex odor, oyster mixture, under optimal conditions, which was stimulation for 5 s per min for 60 min with 20 mM AGB in artificial seawater with reduced sodium and calcium concentrations. AGB was a weak odorant for lobsters; it elicited only a small electrophysiological response from ORNs and labeled < 1% of the ORNs during stimulation with AGB in the absence of odors. For the zebrafish, stimulation for 10 s per min for 10 min with 5 mM AGB plus odorant (L-glutamine) in fish Ringer's solution was the optimal labeling condition, resulting in labeling of 17% of the olfactory epithelial area. Approximately 6% of the olfactory epithelium was labeled during stimulation with a control stimulus, AGB alone. This labeling by AGB alone suggests it is an olfactory stimulus for zebrafish; a conclusion supported by electrophysiological recordings. We used electrophysiological assays and channel blockers to examine, for each species, potential ion channels for entry of AGB into ORNs. These results show that AGB can be used as an activity-dependent label for chemoreceptor neurons of diverse phyla living in a range of environmental conditions.

Functional significance of the co-localization of taste buds and teeth in the pharyngeal jaws of the largemouth bass, Micropterus salmoides.

Studies of feeding behavior in the largemouth bass, Micropterus salmoides, revealed that live goldfish or artificial food balls are ingested in three discrete steps: inhalation of the food into the oral cavity, passage through the pharyngeal cavity, and swallowing. Food balls with or without a feeding stimulant were inhaled with equal frequency; thus, vision was clearly the major sense affecting inhalation. However, food balls with defined concentrations of a feeding stimulant were swallowed in a dose-dependent manner, whereas food balls without a feeding stimulant were promptly expelled. Thus, gustation played a major role in stimulating swallowing. Videotaped observations of feeding behavior suggested that both food processing and gustation occur in the pharynx and take place before the swallowing of either goldfish or food balls. The well-developed pharyngeal jaws of largemouth bass consist of six major pads of caniform teeth in the upper pharynx and two pads in the lower pharynx. Scanning electron microscopy showed that taste buds were abundant around most of these pharyngeal teeth. Histological sections prepared from all pharyngeal pads revealed that both elevated and flattened taste buds occur with the teeth. The morphology of these taste buds was typical of that described in other teleosts. Neuronal profiles, visualized with an HNK-1 monoclonal antibody, were observed entering each taste bud. The antibody also selectively stained a group of one to four putative sensory cells in each taste bud and the distal processes of these cells in the receptor area. The co-localization of teeth and taste buds on the pharyngeal jaws indicates that food processing and gustation both occur there, and that together these processes determine whether a potential food item is swallowed.

Perireception in olfaction: molecular mass sieving by aesthetasc sensillar cuticle determines odorant access to receptor sites in the Caribbean spiny lobster Panulirus argus.

The responsiveness of chemoreceptor neurons depends on a combination of perireceptor and receptor events. Olfactory neurons of crustaceans are packaged into distinctive cuticular sensilla called aesthetascs. The cuticle of aesthetascs is thin and permeable, even though it does not contain any obvious surface pores or pore tubules. This suggests that this 'spongy' aesthetasc cuticle may act as a molecular sieve that restricts large odorant molecules from entering the sensilla and binding to the olfactory neurons. We examined whether this is so for the aesthetasc cuticle of the Caribbean spiny lobster Panulirus argus. We used a chromatographic column packed with aesthetasc cuticle and connected to a flow-through ultraviolet spectrophotometer to measure the elution times of ultraviolet-absorbent molecular mass markers between 165 and 2 x 10(6) Da. Molecules larger than approximately 8.5 kDa had similar elution times, indicating that they did not penetrate the cuticle. Molecules smaller than 8.5 kDa had longer elution times that were directly and inversely proportional to their molecular mass. These results suggest that aesthetasc cuticle excludes molecules larger than 8.5 kDa from having access to the olfactory receptor neurons. We conclude that the molecular sieving capacity of the aesthetasc cuticle of P. argus is a perireceptor mechanism that is a critical determinant of the types of molecules capable of stimulating its olfactory receptors.