The monophyletic origin of a remarkable sexual system in akentrogonid rhizocephalan parasites: A molecular and larval structural study

We use sequences from the nuclear ribosomal genes, 18S and 28S to analyze the phylogeny of the Rhizocephala Akentrogonida including two species, Clistosaccus paguri and Chthamalophilus delagei, that are critical for understanding rhizocephalan evolution but have not previously been part of a molecularly based study. In addition we use light and scanning electron microscopy to compare the cypris larvae of C. paguri, Sylon hippolytes and two species of the family Thompsoniidae, since this larval stage offers a suite of characters for analyzing the evolution of these otherwise highly reduced parasites. The Rhizocephala Akentrogonida form a monophyletic group nested within a paraphyletic “Kentrogonida”. C. paguri and S. hippolytes are sistergroups confirming the monophyly of the Clistosaccidae that was originally based on similarities in the cypris larvae. We find numerous LM and SEM level similarities between the two species, many of which appear to be correlated with their specialized sexual system, where male cyprids use an antennule to implant cells into the virgin female parasite. Some of these traits are also found in cyprids of the thompsoniid species. We conclude that the special cypris morphology and the implantation of males by antennular penetration was present in the stem species to the Thompsoniidae and the Clistosaccidae and emphasize the power of larval characters in rhizocephalan systematics. C. delagei is a sister group to Boschmaella balani and the two are nested deep within the Akentrogonida. This confirms the monophyly of the Chthamalophilidae and falsifies the theory that C. delagei should represent the most primitive extant rhizocephalan. Instead, chthamalophilid rhizocephalans represent some of the most highly advanced members of the parasitic barnacles.     

The anatomy and development of the rhizocephalan barnacle Clistosaccus paguri Lilljeborg and relation to its host pagurus bernhardus (L.)

The anatomy of all developmental stages of Clistosaccus paguri Lilljeborg including internal primordia only 200 μm across has been studied by conventional and ultrastructural techniques. The earliest primordia lack any roots and organs. A receptacle and ovary arise as separate structures. The colleteric gland develops by infolding of the mantle cavity epithelium. The primordium becomes external during a host intermoult. The externa is apparently female. It is proposed that male cyprids, through the integument, have implanted cells found in the mantle of the externa. These male cells travel through the mantle into the single receptacle, where they proceed with spermatogenesis. Externae lacking male cells will not grow. The larvae are released as cyprids, in which no preformed kentrogon is present. About half of the infected hosts carry more than one externa. Multiple externae are separate and may represent different invading female cyprids. C. paguri infects all sizes of its host, and castrates it. The effect of the parasite on the host is discussed. The life span of the parasite seems to be one year and the parasite population shows an annual cycle.     

Metamorphosis in the Cirripedia Rhizocephala and the homology of the kentrogon and trichogon

The Rhizocephala are considered to be monophyletic due to several synapomorphies in the ontogeny of the cndoparasitic phase. The various types of metamorphosis described in the Rhizocephala are discussed and compared to metamorphosis in the Cirripedia Thoracica and Acrothoracica. In males and females of the suborder Kentrogonida. the cyprid settles and metamorphoses into a new instar, in males the trichogen and in females the infective kentrogon. The kentrogon goes through yet another. incomplete moult associated with the development of the stylet. Within the three kentrogonidan families. the Iernaeodiscid-peltogastrid type of kentrogon differs from the sacculinid type in the mode of attachment to the host. in the complexity of internal anatomy. in the position and penetration of the stylet, and in whether or not the cyprid carapace must be shed prior to penetration of the stylet. In the Akentrogonida metamorphosis never results in a new instar. Where observed (Clistosaccidae and Thompsoniidae). both male and female cyprids settle and penetrate into their substrate (female parasite or new host) with one of the antennules. Using the antennule as a syringe. male cyprids inject spermatogonia while female cyprids injects embryonic cells developing into an endoparasite. By comparison with metamorphosis in the Cirripedia Thoracica and Acrothoracica it is concluded that the presence of a metamorphic moult leading to a post-cyprid instar is plesiomorphic and that the trichogon and kentrogon are homologous with the first metamorphosed juvenile in these outgroups. The abbreviated ontogeny in the Akentrogonida without metamorphic moult and post-cyprid larval instars is considered apomorphic. This contradicts the long-held supposition that the Akentrogonida are the most‘primitive’Rhizocephala and dovetails with new information that this suborder contains many advanced traits. Within the Kentrogonida. the lernacodiseid-peltogastrid type of kentrogon is considered more plesiomorphic than the sacculinid type, which resembles the clistosaccidthompsoniid type in having the antennules involved in the penetration process. The homologization of the kentrogon with a juvenile barnacle indicates that presence of a kentrogon is plesiomorphic within the Rhizocephala and that the Kentrogonida is paraphyletic.     

Preliminary investigation of the digestive enzymes in Pagellus erythrinus (Linneo 1758) larvae

To gain a better understanding of the role of the olfactory system in sexual selection, we conducted a series of behavioral assays to correlate the number of aesthetascs with pre-copulatory behavior and male mating success in a protandrous simultaneous hermaphroditic shrimp, Lysmata wurdemanni. We demonstrated that male pre-copulatory behavior and mating success were closely associated with the number of aesthetascs. Individuals with a higher numbers of aesthetascs started approaching/searching females earlier and had higher mating success than ones with lower numbers. Sexual selection theory predicts that small males in a pure searching mating system have advantages to mate with females because they are cryptic and agile. However, our results show that aesthetasc number, rather than shrimp size, may be the major factor in determining male mating success.     

Rosette-type tegumental glands associated with aesthetasc sensilla in the olfactory organ of the Caribbean spiny lobster, Panulirus argus

The lateral antennular flagellum of decapod crustaceans bears unique olfactory sensilla, namely the aesthetascs, and other sensilla types. In this study, we identify a new major tissue in the lateral flagellum of the Caribbean spiny lobster, Panulirus argus, namely “aesthetasc tegumental glands” (ATGs), based on immunostaining with antibodies against CUB serine protease (Csp), in situ hybridization with csp-specific probes, labeling with the F-actin marker phalloidin, labeling with the nuclear marker Hoechst 33258, and staining with methylene blue. Each ATG has 12–20 secretory cells arranged in a rosette. Each secretory cell has a Csp-immunoreactive basal portion and an apical portion containing granular material (metachromatic staining indicative of acid mucopolysaccharides). At the center of each secretory rosette is a phalloidin-positive common locus that gives rise to a main drainage duct projecting toward the cuticle. Scanning electron and light microscopy show that thin ducts traverse the cuticle and connect to “peg pores” proximal to the bases of the aesthetascs, with 3.4 peg pores per aesthetasc. Since the number of common loci is correlated with the number of peg pores, we conclude that each pore represents the outlet of one ATG, and that the secretions are released from them. We conclude further that ATGs and aesthetascs are functionally linked. We hypothesize that ATG secretions have antifouling and/or friction-reducing properties, and that they are spread over the surface of the aesthetascs by antennular grooming. A review of the literature suggests that ATGs are common in decapod crustacean antennules, and that rosette glands and grooming might be functionally coupled in other body areas.This study was supported by NSF IBN 0077474 and NIH DC00312.     

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.     

Role of the olfactory pathway in agonistic behavior of crayfish, <Emphasis Type="Italic">Procambarus clarkii</Emphasis>

Crayfish establish social dominance hierarchies through agonistic interactions, and these hierarchies are maintained through assessment of social status. Chemical signals influence several aspects of fighting behavior, but the specific chemosensory sensilla involved in detecting these signals in crayfish are unknown. The goal of our study was to examine the importance of aesthetasc sensilla—olfactory sensors on the antennules of decapod crustaceans—in regulating changes in fighting behavior in crayfish, Procambarus clarkii, over the course of repeated pairings. We selectively ablated aesthetascs from pairs of crayfish after the first day of trials and compared the behavior of these ablated animals to that of pairs of intact controls. Results show that unablated crayfish significantly decreased the number and duration of fights over repeated pairings, whereas crayfish lacking aesthetascs continued to engage in similar amounts of fighting across all three trial days. This difference shows that aesthetascs regulate fighting behavior in P. clarkii.     

Fine structure of the aesthetasc hairs ofPagurus hirsutiusculus dana

Summary The thin-walled aesthetasc pegs on the antennules of a small hermit crab,Pagurus hirsutiusculus, were studied by light and electron microscopy. The lumen of each aesthetasc was found to be filled with the dendrites of 300–500 bipolar neurons whose cell bodies lie beneath the base of the aesthetasc. These dendrites are ciliary in nature, having well developed basal bodies and rootlets.Each basal body gives rise to a cilium which divides to form a cluster of slender branches, each of which contains a microtubule running lengthwise. These structures occupy most of the length of the hair.The cuticle of the aesthetasc wall is thin and tenuous. Except for the pore canals in the basal region, we have found no pores at either light or electron microscope level, but as the hair is extremely permeable, we conclude that the cuticle itself may permit the passage of solutions. This permeability of the cuticle and the large numbers of dendrites within support the hypothesis that the aesthetascs are chemoreceptors.     

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     

Morphology and Histochemistry of the Aesthetasc-Associated Epidermal Glands in Terrestrial Hermit Crabs of the Genus Coenobita (Decapoda: Paguroidea)

Crustaceans have successfully adapted to a variety of environments including fresh- and saltwater as well as land. Transition from an aquatic to a terrestrial lifestyle required adaptations of the sensory equipment of an animal, particularly in olfaction, where the stimulus itself changes from hydrophilic to mainly hydrophobic, air-borne molecules. Hermit crabs Coenobita spp. (Anomura, Coenobitidae) have adapted to a fully terrestrial lifestyle as adults and have been shown to rely on olfaction in order to detect distant food items. We observed that the specialized olfactory sensilla in Coenobita, named aesthetascs, are immersed in a layer of mucous-like substance. We hypothesized that the mucous is produced by antennal glands and affects functioning of the aesthetascs.Using various microscopic and histochemical techniques we proved that the mucous is produced by aesthetasc-associated epidermal glands, which we consider to be modified rosette-type aesthetasc tegumental glands known from aquatic decapods. These epidermal glands in Coenobita are multicellular exocrine organs of the recto-canal type with tubulo-acinar arrangement of the secretory cells. Two distinct populations of secretory cells were clearly distinguishable with light and electron microscopy. At least part of the secretory cells contains specific enzymes, CUB-serine proteases, which are likely to be secreted on the surface of the aesthetasc pad and take part in antimicrobial defense. Proteomic analysis of the glandular tissue corroborates the idea that the secretions of the aesthetasc-associated epidermal glands are involved in immune responses.We propose that the mucous covering the aesthetascs in Coenobita takes part in antimicrobial defense and at the same time provides the moisture essential for odor perception in terrestrial hermit crabs. We conclude that the morphological modifications of the aesthetasc-associated epidermal glands as well as the functional characteristics of their secretions are important adaptations to a terrestrial lifestyle.     

Ultrastructure of the peduncular aesthetascs of Speleonectes tanumekes (Crustacea, Remipedia) in comparison with crustaceans from stressful environments

Abstract. Osmotic stress associated with the freshwater environment and desiccation stress associated with the terrestrial environment may have a shortening effect on the length of the innervation of crustacean aesthetascs. Physical stress of the littoral environment may have a similar effect on the length of the cuticular portion of aesthetascs. The aesthetascs of crustaceans that inhabit these environments share a similar ultrastructural feature, which may help animals cope with these environmental stresses. This ultrastructural feature, the position of the basal bodies proximal to the lumen of the aesthetasc, is absent from the aesthetascs of crustaceans that occur in the typical marine environment. Interestingly, the ultrastructural feature associated with these stressful habitats is present in the peduncular aesthetascs of the remipede Speleonectes tanumekes , even though the environmental stresses that may invoke the reduction of aesthetascs are absent in the marine-cave environment where this animal occurs. The importance of the sensitivity of aesthetascs for survival in this lightless environment may result in a selective pressure that favors basal bodies to be positioned proximal to the lumen of the aesthetasc.     

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     

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.     

Comparison of turnover in the olfactory organ of early juvenile stage and adult Caribbean spiny lobsters

Proliferation and turnover of neurons occurs in the olfactory systems of many animals. In this study, we examined developmental changes in turnover in the olfactory organ of the Caribbean spiny lobster Panulirus argus by examining two life-history stages—early juveniles and young adults. Turnover was compared using external morphology of the olfactory organ before and after molting to determine addition and loss of aesthetascs and other chemosensilla, and BrdU labeling to identify newly proliferated cells. The number of olfactory receptor neurons (ORNs) innervating each aesthetasc increased only slightly over development, but there was a net increase of olfactory sensory units (i.e. aesthetascs and their ORNs) at each molt. This increase was similar in early juveniles and young adults when expressed as absolute number of ORNs neurons but greater in early juveniles when expressed as a proportion of existing ORNs. The net increase in olfactory sensory units in early juveniles is due solely to addition, since virtually no aesthetascs are lost. In contrast, the net increase in olfactory sensory units in adults reflects addition of new units accompanied by considerable loss of old units. These developmental changes result in expansive enlargement of the olfactory organ without turnover in early juveniles, and a more modest growth combined with continuous turnover and replenishment of ORNs in adults.     

Fine structure of the aesthetasc hairs of Coenobita compressus Edwards

The aesthetascs, short thin-walled pegs on the antennule flagella of Coenobita clypeatus, a terrestrial hermit crab, are similar to those of other decapod crustacea in containing the dendrites of many bipolar neurons whose cell bodies are grouped in spindle-shaped masses beneath the bases of each hair. The dendrites contain rootlets, basal bodies, and cilia, which divide dichotomously before entering the aesthetasc, so that within the hair, each cilium becomes represented by a group of slender branches. The aesthetascs themselves are short, blunt, and partially recumbent so that each has an exposed and an unexposed side. The cuticle on the exposed side is thinner and more tenuous than that on the protected side, and the dendrite branches are concentrated just underneath. The protected side, on the other hand, is lined with nondendritic supporting cells, and the cuticle is thicker, more lamellar, and probably less permeable. All dendritic elements proximal to the dendrite branches are enclosed within the main body of the antennular flagellum, and the initial segments of the cilia lie within a vacuole. In these respects, the aesthetascs of Coenobita resemble the thin-walled pegs on insect antennae more than they do those of the marine decapods thus far examined. This convergence in the terrestrial forms may be in response to the need to conserve water.     

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.     

The olfactory pathway mediates sheltering behavior of Caribbean spiny lobsters, <Emphasis Type="Italic">Panulirus </Emphasis><Emphasis Type="Italic">argus</Emphasis>, to conspecific urine signals

The “noses” of diverse taxa are organized into different subsystems whose functions are often not well understood. The “nose” of decapod crustaceans is organized into two parallel pathways that originate in different populations of antennular sensilla and project to specific neuropils in the brain—the aesthetasc/olfactory lobe pathway and the non-aesthetasc/lateral antennular neuropil pathway. In this study, we investigated the role of these pathways in mediating shelter selection of Caribbean spiny lobsters, Panulirus argus, in response to conspecific urine signals. We compared the behavior of ablated animals and intact controls. Our results show that control and non-aesthetasc ablated lobsters have a significant overall preference for shelters emanating urine over control shelters. Thus the non-aesthetasc pathway does not play a critical role in shelter selection. In contrast, spiny lobsters with aesthetascs ablated did not show a preference for either shelter, suggesting that the aesthetasc/olfactory pathway is important for processing social odors. Our results show a difference in the function of these dual chemosensory pathways in responding to social cues, with the aesthetasc/olfactory lobe pathway playing a major role. We discuss our results in the context of why the noses of many animals contain multiple parallel chemosensory systems.     

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.     

The cytological basis for reproductive variability in the Anoetidae (Sarcoptiformes: Acari)

Meiosis is described in a thelytokous strain of the anoetid mite Histiostoma feroniarum (Dufour) and in both sexes of the arrhenotokous strain of this species. Oogenesis in the thelytokous strain is accomplished by ameiotic mitosis with only one pseudo-maturation division. During this division one or more chromosomes may move to the poles precociously and while in this position can be mistaken for centrioles. Fourteen chromosomes are found at metaphase of the pseudo-maturation division and in cleaving eggs of this strain. In the arrhenotokous strain, male meiosis consists of a single mitotic division. Oogenesis is regular and 7 bivalents are observed at the first maturation division. Metaphases of the first cleavage division in fertilized eggs show 14 chromosomes and 7 chromosomes in unfertilized eggs.It is postulated that the thelytokous strain has arisen from the arrhenotokous strain. This assumption is in agreement with that suggested for several insect species previously reported. The evolution in the Acari and the variability in the modes of reproduction in this suborder are discussed in light of the findings in this paper on the Anoetidae.