Micro-scale fluid and odorant transport to antennules of the crayfish, Procambarus clarkii

A numerical model was developed to determine advective-diffusive transport of odorant molecules to olfactory appendages of the crayfish, Procambarus clarkii. We tested the extent of molecule transport to the surfaces of aesthetasc sensilla during an antennule flick and the degree of odorant exchange during subsequent flicks. During the rapid downstroke of a flick, odorant molecules are advected between adjacent aesthetascs, while during the slower return stroke, these odorants are trapped between the sensilla and molecular diffusion occurs over a sufficient time period to transport odorants to aesthetasc surfaces. During subsequent flicks, up to 97.6?% of these odorants are replaced with new odorant molecules. The concentration of molecules captured along aesthetasc surfaces was found to increase with increased gap spacing between aesthetascs, flick speed, and distance from the proximal end of the aesthetasc, but these changes in morphology and flicking kinematics reduce the animal's ability to take discrete samples of the odorant-laden fluid environment with each flick. Results suggest that antennule flicking allows discrete sampling of the time- and space-varying odorant signal, and high concentration odorant filaments can be distinguished from more diffuse, low concentration filaments through changes in both the timing and the encounter rate of odorant molecules to aesthetasc surfaces.     

Identification of chemosensory sensilla mediating antennular flicking behavior in Panulirus argus, the Caribbean spiny lobster

Crustaceans sample odorants by a rapid series of flicks of the two flagella composing the distal segments of each of the paired antennules. The lateral flagella contain aesthetasc sensilla that house unimodal chemosensory neurons. Nine types of nonaesthetasc setae with putative chemosensory and mechanosensory functions are distributed on the lateral and medial flagella. Sensory neurons in aesthetascs and nonaesthetasc sensilla terminate in separate regions of the brain, the olfactory lobe, and the lateral antennular neuropil, resulting in two odorant-processing pathways. Distilled water ablation of flagella and excision of specific setae were used to identify chemosensory sensilla mediating antennular flick behavior in Panulirus argus. The flick rates of sham-ablated and ablated or excised lobsters toward squid extract were compared. Complete attenuation of flick response to squid extract occurred as a result of (1) distilled water ablation of lateral flagella, (2) excision of aesthetascs and asymmetric sensilla, and (3) excision of aesthetascs. Distilled water ablation of medial flagella resulted in a mean flick rate 52% of that observed for sham-ablated lobsters toward squid extract. Flicking was unaffected by excision of asymmetric, guard, or companion sensilla. We propose that odorant mediation of flicking behavior requires both the aesthetasc and nonaesthetasc pathways.     

Molecule capture by olfactory antennules: Mantis shrimp

 A critical step in the process of olfaction is the movement of odorant molecules from the environment to the surface of a chemosensory structure. Many marine crustaceans capture odorant molecules with arrays of chemosensory sensilla (aesthetascs) on antennules that they flick through the water. We developed a model to calculate molecule flux to the surfaces of aesthetascs in order to study how the size, aesthetasc spacing, and flick kinematics of olfactory antennules affect their performance in capturing molecules from the surrounding water. Since the three-dimensional geometry of an aesthetasc-bearing antennule is complex, dynamically-scaled physical models can often provide an efficient method of determining the fluid velocity field through the array. Here we present a method to optimize the incorporation of such measured velocity vector fields into a numerical simulation of the advection and diffusion of odorants to aesthetasc surfaces. Furthermore, unlike earlier models of odorant interception by antennae, our model incorporates odorant concentration distributions that have been measured in turbulent ambient flows. By applying our model to the example of the olfactory antennules of mantis shrimp, we learned that flicking velocity can have profound effects on odorant flux to the aesthetascs if they operate in the speed range in which the leakiness of the gaps between the aesthetascs to fluid movement is sensitive to velocity. This sensitivity creates an asymmetry in molecule fluxes between outstroke and return stroke, which results in an antennule taking discrete samples in space and time, i.e. “sniffing”. As stomatopods grow and their aesthetasc Reynolds number increases, the aesthetasc arrangement on the antennule changes in a way that maintains these asymmetries in leakiness and molecule flux between the outstroke and return stroke, allowing the individual to continue to take discrete samples as it develops. Received: 24 May 2000 / Revised version: 8 May 2001 / Published online: 7 December 2001     

The innervation and chemical sensitivity of single aesthetasc hairs

Summary Each aesthetasc hair of the lateral antennule of the California spiny lobsterPanulirus interruptus (Randall) is shown by light and scanning electron microscopy to be innervated by a basally situated cluster of sensory neurons encased in a glial sheath which isolates each cluster from those of other hairs (Figs. 1, 3, 4). The dendrites of these neurons penetrate the aesthetasc hairs and their axons extend to the central nervous system. Extracellular recordings with suction electrodes from the axons of single neuronal clusters were used to determine the responsiveness of individual hairs to a spectrum of amino acids, amines, amides, carbohydrates, carboxylic acids, nucleotides, and a tripeptide (Tables 1, 2, Figs. 6, 8). Randomly selected hairs from the antennules of juvenile, and male and female adult lobsters were shown to be broadly sensitive to a variety of stimuli and are homogeneous in their breadth of responsiveness (Figs. 5, 7). Cluster analysis does not reveal distinct chemoreceptive hair types based on their response spectra, suggesting that the receptor populations of single hairs are uniformly competent to respond to diverse chemical stimuli (Figs. 6, 8). Further, the sensitivity profile of aesthetascs to these stimuli correlates well with behavioral responses ofPanulirus interruptus to these same stimuli (Tables 1, 2).Abbreviation ² Chi-squared     

The fluid mechanics of arthropod sniffing in turbulent odor plumes

Many arthropods capture odorant molecules from the environment using antennae or antennules bearing arrays of chemosensory hairs. The penetration of odorant-carrying water or air into the spaces between these chemosensory hairs depends on the speed at which they are moved through the surrounding fluid. Therefore, antennule flicking by crustaceans and wing fanning by insects can have a profound impact on the odorant encounter rates of the chemosensory sensilla they bear; flicking and fanning are examples of sniffing. Odors are dispersed in the environment by turbulent wind or water currents. On the scale of an antenna or antennule, an odor plume is not a diffuse cloud but rather is a series of fine filaments of scent swirling in odor-free water. The spatiotemporal pattern of these filaments depends on distance from the odor source. The physical interaction of a hair-bearing arthropod antennule with the surrounding fluid affects the temporal patterns of odor concentration an animal intercepts when it sniffs in a turbulent odor plume.     

Numerical simulations of odorant detection by biologically inspired sensor arrays

The antennules of many marine crustaceans enable them to rapidly locate sources of odorant in turbulent environmental flows and may provide biological inspiration for engineered plume sampling systems. A substantial gap in knowledge concerns how the physical interaction between a sensing device and the chemical filaments forming a turbulent plume affects odorant detection and filters the information content of the plume. We modeled biological arrays of chemosensory hairs as infinite arrays of odorant flux-detecting cylinders and simulated the fluid flow around and odorant flux into the hair-like sensors as they intercepted a single odorant filament. As array geometry and sampling kinematics were varied, we quantified distortion of the flux time series relative to the spatial shape of the original odorant filament as well as flux metrics that may be important to both organisms and engineered systems attempting to measure plume structure and/or identify chemical composition. The most important predictor of signal distortion is the ratio of sensor diameter to odorant filament width. Achieving high peak properties (e.g. sharpness) of the flux time series and maximizing the total number of odorant molecules detected appear to be mutually exclusive design goals. Sensor arrays inspired specifically by the spiny lobster Panulirus argus and mantis shrimp Gonodactylaceus falcatus introduce little signal distortion but these species' neural systems may not be able to resolve plume structure at the level of individual filaments via temporal properties of the odorant flux. Current chemical sensors are similarly constrained. Our results suggest either that the spatial distribution of flux across the aesthetasc array is utilized by P. argus and G. falcatus, or that such high spatiotemporal resolution is unnecessary for effective plume tracking.     

Identification of chemosensory sensilla activating antennular grooming behavior in the Caribbean spiny lobster,Panulirus argus

Crustaceans such as crabs and lobsters clean or 'groom' their olfactory organ, the antennule, by wiping it through a pair of mouthpart appendages, the third maxillipeds. In the lobster, only a few chemicals found in prey extracts, especially glutamate, elicit grooming. Chemosensory input driving grooming is likely to be mediated via sensilla located on antennules and third maxillipeds. Chemosensory antennular sensilla are innervated by neurons with central projections either to the glomerular olfactory lobe (aesthetasc sensilla) or to non-glomerular antennular neuropils (nonaesthetasc sensilla). By selectively ablating the chemosensory sensilla on the antennules and the third maxillipeds we have determined that the aesthetascs are necessary and sufficient to drive grooming behavior. Chemosensory activation of antennular grooming behavior likely follows a 'labeled-line' model in that aesthetasc neurons tuned to glutamate provide adequate input via the olfactory lobe to motor centers in the brain controlling antennular movements.     

Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.     

Structural and functional changes in regenerating antennules in the crayfish Orconectes sanborni

Crayfish rely on the chemosensory neurons in their antennules to help them find food and habitat and to mediate social interactions. These structures often sustain damage from aggressive interactions or from the environment, but they have the ability to regenerate. In this study, we examine whether the effects of antennule ablation and regeneration on odor-tracking ability correlate with structural changes in the antennule that occur during regeneration. We initiated the regeneration process by removing the right antennules from 55 individuals of Orconectes sanborni. We developed a method to nondestructively sample the regenerating antennules so that we could follow the growth of new antennular tissue in the same animals over time. We used dental epoxy to make molds of the regenerating antennule after each molt. We then made resin positives, which were visualized using scanning electron microscopy. Structural parameters including aesthetasc length, diameter, segment length, and number per row were measured from scanning electron micrographs using Image J software. Crayfish were tested in a tabletop water Y-maze before and after surgery and after each molt to assess their ability to track food odors. The structural and the behavioral data indicate that the antennules possessed many aspects of their original structure by the end of the second molt. Flicking of antennules, investigation of substrate, success rate at finding the odor-containing Y-maze branch, and time to completion of Y-maze regained pre-antennulectomy values by the end of the third molt.     

Development of functional adaptation to clasping behaviour in harpacticoid copepods (Copepoda,Harpacticoida)

The copepodite antennule development of several harpacticoid families is studied and male antennule development of 5 species is schematically illustrated. The origin of newly formed segments can be determined by seta numbers of segments, relative segment length, furrows indicating previous articulations and by the position of the segment which bears the proximal aesthetasc. At least in some species sexual dimorphism of antennules is present from C II onwards. Females practically reach the adult antennular state at C V whereas males undergo drastic changes from C V to C VI with proximal addition of segments, and often with distal fusions and formation of a unique armature due to functional adaptations in clasping behaviour. No correlation has been found between clasping mode, male antennule type and taxonomic level. In closely related species of the same genus both chirocer and subchirocer antennules are found. The postnaupliar development of the chirocer type can proceed along different ways. Consequently it is suggested that at least chirocer antennules have originated several times independently during harpacticoid evolution.     

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.     

Integumental organs of the phyllosoma larva of the rock lobster Jasus edwardsii (Hutton)

The external morphology and distribution of the integumentalorgans of the final-stage phyllosoma of the rock lobster Jasusedwardsii were examined. Seven types of organs were recognizedon the integument of the body trunk, antennules and antennae:plumose setae, simple setae, porous setae, aesthetasc setae,simple pores, dorsal cuticular organs and dome-shaped structures.The plumose setae and simple pores were abundant on the dorsalsurface of the cephalosome, abdomen and telson. The dorsal cuticularorgans were present only on the dorsal surface of the cephalosome,and the aesthetasc setae were restricted to the antennule. A comparison of the morphology of these organs with the senseorgans of other decapods with known function suggests that thedorsal surface of the body trunk is one of the major sites ofreception of near-field water movement in the Jasus phyllosoma.     

Ultrastructure of aesthetasc innervation and external morphology of the lateral antennule setae of the spiny lobster Panulirus interruptus (Randall)

Summary Six types of setae and one type of cuticular depression were examined on the lateral antennule of the spiny lobster Panulirus interruptus using scanning electron microscopy. The organization and ultrastructure of the innervation of the most numerous setal type, the aesthetasc, were investigated using light-and transmission electron microscopy.Each aesthetasc is innervated by approximately 300 bipolar neurons whose sensory dendrites penetrate the hair and extend toward the tip, and whose axons project towards the central nervous system. The neuronal somata and two types of glia form a cluster within the antennular lumen. The inner sheath-cell somata encircle the dendritic tract distal to the sensory somata. These cells appear to extend distal processes which wrap the dendritic tract to the base of the aesthetasc. Elongate outer sheath cells are interposed between the glia-wrapped dendritic tract and the hypodermis which underlies the antennule cuticle. A continuous investment of neural lamella separates the hypodermis, the entire cluster of somata, and sensillar nerve from the antennule lumen. The organization of the neuronal somata and their association with outer and inner sheath cells in this marine species appear similar to those of crustaceans from freshwater and terrestrial habitats.     

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.     

Morphological characteristics facilitating stimulus access and removal in the olfactory organ of the spiny lobster, Panulirus argus: insight from the design

The olfactory organ (antennule) of the spiny lobster, Panulirusargus, has from 1000–2000 olfactory sensilla (aesthetascs)which are grouped in a dense tuft along the distal portion ofthe lateral filament. This assemblage of aesthetascs, togetherwith other associated sensilla, forms a substantial boundarylayer through which odor stimuli must diffuse in moving to andfrom the aesthetascs. Periodic flicking of the antennule, abehavior analogous to sniffing in certain vertebrate species,is considered to be a means of reducing the thickness of thisboundary layer. In this report we describe the structure ofthe aesthetasc tuft and examine certain of its dynamic properties.We propose that the unique configuration of the aesthetasces,together with their orientation, serves to channel water flowbetween these sensilla during a flick, thereby reducing diffusiondistances and consequently facilitating the access and removalof odor stimuli in a rapid, synchronized manner. The functionalsignificance of this and other design features of the aesthetasctuft is considered in light of the current understanding offundamental olfactory process.     

A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus.

csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full-length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1). RT-PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains.     

Morphology of stomatopod chemosensory sensilla facilitates fluid sampling

Abstract. Stomatopods, like many marine crustaceans, rely on their sense of smell to detect prey and to find mates (Ache 1982; Zimmer-Faust 1989; Atema & Voigt 1995). In lobsters, crabs, crayfishes, prawns, leptostracans, anaspidans, mysids, amphipods, tanaids, isopods, ostracodes, phyllopods, and cumaceans (Heimann 1984; Hallberg et al. 1992), this detection of odors from distant sources involves specialized chemosensory setae called aesthetascs located on the antennules. The external structure of stomatopod sensilla appears to follow the typical crustacean aesthetasc pattern, but their internal structure has not been previously examined. In this study, we use serial reconstruction from transmission electron microscopy to show that the stomatopod sensilla are aesthetascs. For chemoreception to occur, chemical-containing fluid must be very close to the surface of the aesthetascs, such that odor molecules can diffuse to chemoreceptors on the olfactory receptor neurons inside the aesthetasc. Flicking of stomatopod antennules maximizes fluid penetration near the parts of the sensilla where the cuticle is thinnest, and where the outer dendritic segments are most fully branched with the greatest surface area. Thus, the external and the internal structure of the stomatopod aesthetasc are "matched" to maximize the efficiency of odor arrival at the surface of the olfactory receptor neurons.     

Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue.     

Molecular Cloning of a Lobster Gαq Protein Expressed in Neurons of Olfactory Organ and Brain

Abstract: We have isolated from an American lobster ( Homarus americanus ) olfactory organ cDNA library a clone, hGα_q, with >80% identity to mammalian and arthropod Gα_q sequences. In brain and olfactory organ, hGα_q mRNA was expressed predominantly in neurons, including virtually all the neuronal cell body clusters of the brain. Gα_q protein was also expressed broadly, appearing on western blots as a single band of 46 kDa in brain, eyestalk, pereiopod, dactyl, tail muscle, olfactory organ, and aesthetasc hairs. These results suggest that hGα_q plays a role in a wide variety of signal transduction events. Its presence in the olfactory aesthetasc hairs, which are almost pure preparations of the outer dendrites of the olfactory receptor neurons, the expression of a single hGα_q mRNA species (6 kb) in the olfactory organ, and the localization of hGα_q mRNA predominantly in the olfactory receptor neurons of the olfactory organ strongly suggest that one function of hGα_q is to mediate olfactory transduction.     

Morphology and distribution of antennular setae of scyllarid lobsters (Scyllarides aequinoctialis, S. latus, and S. nodifer) with comments on their possible function

Abstract. Lateral flagella of the antennules of scyllarid lobsters were examined for setal morphology and distribution via scanning electron microscopy. Setal distribution patterns were mapped directly for 3 regions of the antennule ( base, tuft , and tip ) and analyzed for differences: (1) between left and right antennules, (2) between males and females within a species, and (3) among species by comparing counts of setae per annulus in the ventral tuft region only. Six types of antennular setae were identified based on their external morphology: aesthetases, simple, modified simple, asymmetric, hemi-plumose, and toothbrush setae. These different types were organized in a clear pattern over the ventral and dorsal surfaces of the lateral flagella of the antennule. Aesthetase, asymmetric, modified simple, and hemi-plumose setae were found only on annuli in the tuft region between the distal and proximal ends of the flagellum. Simple setae were found on all annuli of all regions of the antennule, and toothbrush setae were mainly concentrated on all annuli of the base region and on proximal annuli of the tuft region . All species of scyllarids examined had the same general pattern of setal distribution and no differences were found between left and right, or male and female antennules. Similar setae located on the lateral antennules of species from the families Nephrophidae and Palinuridae (clawed and spiny lobsters) have been previously described as chemo- and/or mechanoreceptive for use in distance chemoreception (i.e., detection and orientation to olfactory stimuli). Based on work on clawed and spiny lobsters, we predict that the aesthetases on slipper lobsters have a chemoreceptive function and that simple and toothbrush setae may have a bimodal chemo- and mechanoreceptive function.