Crustacean Gills Research Articles

Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein.

To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

Differential effects of cobalt ions in vitro on gill (Na+, K+)-ATPase kinetics in the Blue crab Callinectes danae (Decapoda, Brachyura)

We used the gill (Na+, K+)-ATPase as a molecular marker to provide a comprehensive kinetic analysis of the effects of Co2+in vitro on the modulation of K+-phosphatase activity in the Blue crab Callinectes danae. Co2+ can stimulate or inhibit K+-phosphatase activity. With Mg2+, K+-phosphatase activity is almost completely inhibited by Co2+. Co2+ stimulates K+-phosphatase activity similarly to Mg2+ although with a ≈4.5-fold greater affinity. At saturating Mg2+ concentrations, Mg2+ displaces bound Co2+ from the Mg2+-binding site in a concentration dependent manner, but Co2+ cannot displace Mg2+ from its binding site even at millimolar concentrations. Saturation by Co2+ of the Mg2+ binding site does not affect pNPP recognition by the enzyme. Substitution of Mg2+ by Co2+ slightly increases enzyme affinity for K+ and NH4+. Independently of Mg2+, inhibition by ouabain or sodium ions is unaffected by Co2+. Investigation of gill (Na+, K+)-ATPase K+-phosphatase activity provides a reliable tool to examine the kinetic effects of Co2+ with and without Na+ and ATP. Given that the toxic effects of Co2+ at the molecular level are poorly understood, these findings advance our knowledge of the mechanism of action of Co2+ on the crustacean gill (Na+, K+)-ATPase.

Effect of Cothurnia variabilis and Epistylis gammari (Ciliophora: Peritrichia) on Metabolic Rate of the Crayfish Cambarellus (Cambarellus) montezumae

Peritrichs usually settle on the external surface and gills of crustaceans. However, their physiological effect on the host has not been studied. Respiratory rate is a susceptible index for some factors that affect an organism. To test if ciliates attached to the crustacean gills have a physiological effect, we measured crustacean oxygen consumption in a closed system. Crayfish Cambarellus (Cambarellus) montezumae were collected in Lago Xochimilco in Mexico City and were isolated in chambers. Two peritrich species, Cothurnia variabilis (32.5%) and Epistylis gammari (67.5%), were observed on the gills. The metabolic rate was evaluated by oxygen consumption with a polarimetric oximeter. Statistical analyses demonstrated that a density of ciliates attached to the gills of the crayfish above ~50 individuals increased oxygen consumption with a positive correlation to epibiont density. We conclude that C. variabilis and E. gammari ciliate epibionts attached to the gill of the crayfish Cambarellus (Cambarellus) montezumae cause an increase in metabolic rate.

Exploring the versatility of the perfused crustacean gill as a model for transbranchial transport processes.

The study of transbranchial ion and gas transport of water-breathing animals has long been a useful means of modeling transport processes of higher vertebrate organs through comparative physiology. The molecular era of biological research has brought forward valuable information detailing shifts in gene expression related to environmental stress and the sub-cellular localization of transporters; however, purely molecular studies can cause hypothetical transport mechanisms and hypotheses to be accepted without any direct physiological proof. Isolated perfused gill experiments are useful for testing most of these hypotheses and can sometimes be used outright to develop a well-supported working model for transport processes relating to an animal's osmoregulation, acid-base balance, nitrogen excretion, and respiratory gas exchange as well as their sensitivity to pollutants and environmental stress. The technique allows full control of internal hemolymph-like saline as well as the ambient environmental fluid compositions and can measure the electrophysiological properties of the gill as well as the transport rates of ions and gases as they traverse the gill epithelium. Additives such as pharmaceuticals or peptides as well as the exclusion of ions from the media are commonly used to identify the importance of specific transporters to transport mechanisms. The technique can also be used to identify the penetrance, retention, and localization of pollutants within the gill epithelium or to explore the uptake and metabolism of nutrients directly from the ambient environment. While this technique can be applied to virtually any isolatable organ, the anatomy and rigidity of the decapod crustacean gill make it an ideal candidate for most experimental designs.

Dopamine binding directly up-regulates (Na+, K+)-ATPase activity in the gills of the freshwater shrimp Macrobrachium amazonicum

We examined the effects of exogenous dopamine on gill (Na+, K+)-ATPase activity in vitro in microsomal preparations from juvenile or adult freshwater shrimp, Macrobrachium amazonicum. Dopamine had no effect on enzyme activity in juveniles but stimulated activity in adult shrimp gills by ≈35%. Stimulation of the gill (Na+, K+)-ATPase in adult shrimps by 100 mmol L−1 dopamine was characterized kinetically by varying ATP, MgATP, and Na+ and K+ concentrations, together with inhibition by ouabain. Dopamine stimulated ATP hydrolysis by ≈40% obeying Michaelis-Menten kinetics, reaching VM = 190.5 ± 15.7 nmol Pi min−1 mg−1 protein, KM remaining unaltered. Stimulation by Na+ (≈50%) and K+ (≈25%) revealed distinct kinetic profiles: although KM values were similar, Na+ stimulation followed cooperative kinetics, contrasting with the Michaelian kinetics seen for K+. Stimulation by MgATP increased activity by ≈30% with little change in KM. Similar saturation profiles were seen for ouabain inhibition with very similar calculated KI values. Our findings suggest that dopamine may be involved in hemolymph sodium homeostasis by directly binding to the gill (Na+, K+)-ATPase at a site different from ouabain, thus stimulating enzyme activity in an ontogenetic stage-specific manner. However, dopamine binding does not affect enzyme affinity for cations and ouabain. This is the first report of the direct action of dopamine in stimulating the crustacean gill (Na+, K+)-ATPase.

Pathology, Effects, and Transmission of Black Gill in Commercial Penaeid Shrimp from the South Atlantic Bight

Severe outbreaks of black gill (BG), heavily melanized gills of crustaceans, have been reported in white (Litopenaeus setiferus) and brown shrimp (Farfantepenaeus aztecus) from coastal Georgia and South Carolina during late summer and fall since the mid-1990s. The cause of this condition is an apostome ciliate that elicits the innate immune response of the shrimp, resulting in the formation of melanized nodules in shrimp gill tissue. In the absence of a definitive identification, the causative ciliate is referred to as the shrimp black gill (sBG) ciliate. During outbreaks, necrosis of gill tissue was often seen in microscopic sections; in some cases, there appeared to be penetration of the ciliate into gill tissue. Shrimp with BG exhibited reduced physical endurance and escape responses compared with shrimp without symptomatic BG. Physical impairment due to BG may contribute to higher predation rates and increased vulnerability to environmental conditions. The infection transmission rate of the sBG ciliate appears to be atypically low for apostome ciliates, raising the question of how epidemic levels of BG reoccur annually. Asymptomatic shrimp placed in direct contact with carcasses (heads) from BG symptomatic shrimp exhibited a significant increase in the development of BG symptoms after 7 days (P = 0.028), but waterborne transmission was not detected. A preliminary survey of sympatric crustacean species, including syntopic species of grass shrimp (Palaemonetes spp.), indicated the possible presence of the sBG ciliate, suggesting that other crustacean species may serve as infection reservoirs. These studies support the conclusion that BG is negatively impacting the penaeid shrimp fishery and highlight the challenges that remain in understanding and managing the ongoing sBG epidemic in the southeastern United States.

Polyamines regulate phosphorylation-dephosphorylation kinetics in a crustacean gill (Na+, K+)-ATPase.

Aiming to clarify the mechanism of inhibition of (Na+, K+)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na+, K+)-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006s-1) in adults than in juveniles (0.053 ± 0.003s-1) for spermidine, but similar to juveniles (0.059 ± 0.004s-1) for putrescine. Maximum phosphointermediate formation for the (Na+, K+)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na+, K+)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.

Effects of salinity on the accumulation of hemocyte aggregates and bacteria in the gills of Callinectes sapidus, the Atlantic blue crab, injected with Vibrio campbellii

In addition to respiration and ion regulation, crustacean gills accumulate and eliminate injected particles, along with hemocyte aggregates that form in response to those particles. Here we report that the dose of Vibrio campbellii previously shown to induce a decrease in respiration and hemolymph flow across the gill in the Atlantic blue crab, Callinectes sapidus, also triggered the formation of aggregates containing four or more hemocytes in the gills, compared with saline-injected controls. More bacteria were trapped and rendered non-culturable per unit weight by anterior respiratory gills than posterior gills specialized for ion regulation. Further, more bacteria accumulated in the anterior gills of animals held at 30ppt than those at 10ppt. Thus, the role of the gills in immune defense comes at an energetic cost to this and likely to other crustaceans; this cost is influenced by acclimation salinity and the position and specialized function of individual gills.

Synergistic stimulation by potassium and ammonium of K+-phosphatase activity in gill microsomes from the crab Callinectes ornatus acclimated to low salinity: Novel property of a primordial pump

We provide an extensive characterization of the modulation by p-nitrophenylphosphate, Mg2+, Na+, K+, Rb+, NH4+ and pH of gill microsomal K+-phosphatase activity in the posterior gills of Callinectes ornatus acclimated to low salinity (21‰). The synergistic stimulation by K+ and NH4+ of the K+-phosphatase activity is a novel finding, and may constitute a species-specific feature of K+/NH4+ interplay that regulates crustacean gill (Na+, K+)-ATPase activity. p-Nitrophenylphosphate was hydrolyzed at a maximum rate (V) of 69.2±2.8nmolPimin−1mg−1 with K0.5=2.3±0.1mmolL−1, obeying cooperative kinetics (nH=1.7). Stimulation by Mg2+ (V=70.1±3.0nmolPimin−1mg−1, K0.5=0.88±0.04mmolL−1), K+ (V=69.6±2.7nmolPimin−1mg−1, K0.5=1.60±0.07mmolL−1) and NH4+ (V=90.8±4.0nmolPimin−1mg−1, K0.5=9.2±0.3mmol L−1) all displayed site-site interaction kinetics. In the presence of NH4+, enzyme affinity for K+ unexpectedly increased by 7-fold, while affinity for NH4+ was 28-fold greater in the presence than absence of K+. Ouabain partially inhibited K+-phosphatase activity (KI=320±14.0μmolL−1), more effectively when NH4+ was present (KI=240±12.0μmolL−1). We propose a model for the synergistic stimulation by K+ and NH4+ of the K+-phosphatase activity of the (Na+, K+)-ATPase from C. ornatus posterior gill tissue.

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals.

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail.

Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10days to salinities up to 21‰. Specific activity was highest in fresh water (26.5±2.1U mg(-1)), decreasing in 5‰ to 21‰, attaining 3-fold less at 15‰. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10‰, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21‰, maximum specific activity decreased 2.5- to 4-fold within 1 to 24h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24h and 2.4-fold after 1h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1h, remaining constant up to 120h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water.

Microarray-detected changes in gene expression in gills of green crabs ( Carcinus maenas) upon dilution of environmental salinity

The interaction between environmental salinity and gene expression was studied in gills of the euryhaline green shore crab Carcinus maenas. A 4462-feature oligonucleotide microarray was used to analyze changes in transcript abundance in posterior ion-transporting gills at 8 time periods following transfer of animals from 32 to 10 or 15 ppt salinity. Transcripts encoding Na +/K +-ATPase α-subunit and cytoplasmic carbonic anhydrase were upregulated with significant changes between 6 and 24 h post-transfer. Other transport proteins showing similar transcriptional upregulation were an organic cation transporter, a sodium/glucose cotransporter, an endomembrane protein associated with regulating plasma membrane protein composition, and a voltage-gated calcium channel. Transport proteins showing little transcriptional response included Na +/H + exchanger, Na +/K +/2Cl − cotransporter, and V-type H +-ATPase B subunit, all of which have been implicated in osmoregulatory ion transport across crustacean gill. Interestingly, there was little affect of salinity dilution on transcriptional expression of stress proteins, suggesting that salinity acclimation is part of normal physiology for C. maenas. Expression of transcripts encoding a variety of mitochondrial proteins was significantly upregulated between 4 days and 7 days post-transfer, consistent with the proliferation of mitochondria-rich cells previously observed at this time.

Ammonia and urea transporters in gills of fish and aquatic crustaceans

Ammonia and urea transporters in gills of fish and aquatic crustaceans

Ammonia and urea transporters in gills of fish and aquatic crustaceans

The diversity of mechanisms of ammonia and urea excretion by the gills and other epithelia of aquatic organisms, especially fish and crustaceans, has been studied for decades. Although the decades-old dogma of ;aquatic species excrete ammonia' still explains nitrogenous waste excretion for many species, it is clear that there are many mechanistic variations on this theme. Even within species that are ammonoteles, the process is not purely ;passive', often relying on the energizing effects of proton and sodium-potassium ATPases. Within the ammonoteles, Rh (Rhesus) proteins are beginning to emerge as vital ammonia conduits. Many fishes are also known to be capable of substantial synthesis and excretion of urea as a nitrogenous waste. In such species, members of the UT family of urea transporters have been identified as important players in urea transport across the gills. This review attempts to draw together recent information to update the mechanisms of ammonia and urea transport by the gills of aquatic species. Furthermore, we point out several potentially fruitful avenues for further research.

The crustacean gill (Na +, K +)-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na +, K +)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na + and K + of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na +, K +)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na + and K +. However, in contrast to Na +, a threshold K + concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na +, K +)-ATPase compared to the vertebrate enzyme.

Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crabCallinectes sapidus

Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill, CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill, and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the alpha-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative ;housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity, but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.

Immune Defense Reduces Respiratory Fitness inCallinectes sapidus,the Atlantic Blue Crab

Crustacean gills function in gas exchange, ion transport, and immune defense against microbial pathogens. Hemocyte aggregates that form in response to microbial pathogens become trapped in the fine vasculature of the gill, leading to the suggestion by others that respiration and ion regulation might by impaired during the course of an immune response. In the present study, injection of the pathogenic bacterium Vibrio campbellii into Callinectes sapidus, the Atlantic blue crab, caused a dramatic decline in oxygen uptake from 4.53 to 2.56 micromol g-1 h-1. This decline in oxygen uptake is associated with a large decrease in postbranchial PO2, from 16.2 (+/-0.46 SEM, n=7) to 13.1 kPa (+/-0.77 SEM, n=9), while prebranchial PO2 remains unchanged. In addition, injection of Vibrio results in the disappearance of a pH change across the gills, an indication of reduced CO2 excretion. The hemolymph hydrostatic pressure change across the gill circulation increases nearly 2-fold in Vibrio-injected crabs compared with a negligible change in pressure across the gill circulation in saline-injected, control crabs. This change, in combination with stability of heart rate and branchial chamber pressure, is indicative of a significant increase in vascular resistance across the gills that is induced by hemocyte nodule formation. A healthy, active blue crab can eliminate most invading bacteria, but the respiratory function of the gills is impaired. Thus, when blue crabs are engaged in the immune response, they are less equipped to engage in oxygen-fueled activities such as predator avoidance, prey capture, and migration. Furthermore, crabs are less fit to invade environments that are hypoxic.

Association of tracheal placodes with leg primordia inDrosophilaand implications for the origin of insect tracheal systems

Adaptation to diverse habitats has prompted the development of distinct organs in different animals to better exploit their living conditions. This is the case for the respiratory organs of arthropods, ranging from tracheae in terrestrial insects to gills in aquatic crustaceans. Although Drosophila tracheal development has been studied extensively, the origin of the tracheal system has been a long-standing mystery. Here, we show that tracheal placodes and leg primordia arise from a common pool of cells in Drosophila, with differences in their fate controlled by the activation state of the wingless signalling pathway. We have also been able to elucidate early events that trigger leg specification and to show that cryptic appendage primordia are associated with the tracheal placodes even in abdominal segments. The association between tracheal and appendage primordia in Drosophila is reminiscent of the association between gills and appendages in crustaceans. This similarity is strengthened by the finding that homologues of tracheal inducer genes are specifically expressed in the gills of crustaceans. We conclude that crustacean gills and insect tracheae share a number of features that raise the possibility of an evolutionary relationship between these structures. We propose an evolutionary scenario that accommodates the available data.

Gill microsomal (Na +,K +)-ATPase from the blue crab Callinectes danae: Interactions at cationic sites

Euryhaline crustaceans tolerate exposure to a wide range of dilute media, using compensatory, ion regulatory mechanisms. However, data on molecular interactions occurring at cationic sites on the crustacean gill (Na +,K +)-ATPase, a key enzyme in this hyperosmoregulatory process, are unavailable. We report that Na + binding at the activating site leads to cooperative, heterotropic interactions that are insensitive to K +. The binding of K + ions to their high affinity sites displaces Na + ions from their sites. The increase in Na + ion concentrations increases heterotropic interactions with the K + ions, with no changes in K 0.5 for K + ion activation at the extracellular sites. Differently from mammalian (Na +,K +)-ATPases, that from C. danae exhibits additional NH 4 + ion binding sites that synergistically activate the enzyme at saturating concentrations of Na + and K + ions. NH 4 + binding is cooperative, and heterotropic NH 4 + ion interactions are insensitive to Na + ions, but Na + ions displace NH 4 + ions from their sites. NH 4 + ions also displace Na + ions from their sites. Mg 2+ ions modulate enzyme stimulation by NH 4 + ions, displacing NH 4 + ion from its sites. These interactions may modulate NH 4 + ion excretion and Na + ion uptake by the gill epithelium in euryhaline crustaceans that confront hyposmotic media.

The evolution of arthropod limbs

Limb morphology across the arthropods is reviewed using external morphological and internal anatomical data from both recent and fossil arthropods. Evolutionary trends in limb structure are identified primarily by reference to the more rigorous of the many existing phylogenetic schemes, but no major new phylogenetic inferences are presented. Tagmosis patterns are not considered, although the origins and patterns of heteronomy within the postantennulary limb series are analysed. The phenomenon of annulation is examined and two basic types of annuli are recognised: terminal and intercalary. The annulation of the apical segment of a limb results in the formation of terminal flagella, and is typical of primarily sensory appendages such as insect and malacostracan antennules and maxillary palps of some hexapods. Intercalary annulation, arising by subdivision of existing subterminal segments, is common, particularly in the tarsal region of arthropodan walking limbs. Differentiating between segments and annuli is discussed and is recognised as a limiting factor in the interpretation of fossils, which usually lack information on intrinsic musculature, and in the construction of groundplans. Rare examples of secondary segmentation, where the criteria for distinguishing between segments and annuli fail, are also highlighted. The basic crown-group arthropodan limb is identified as tripartite, comprising protopodite, telopodite and exopodite, and the basic segmentation patterns of each of these parts are hypothesised. Possible criteria are discussed that can be used for establishing the boundary between protopodite and telopodite in limbs that are uniramous through loss of the exopodite. The subdivision of the protopodite, which is typical of the postantennulary limbs of mandibulates, is examined. The difficulties resulting from the partial or complete failure of expression of articulations within the mandibulate protopodite and subsequent incorporation of partial protopodal segments into the body wall, are also discussed. The development and homology between the various exites, including gills, on the postantennulary limbs of arthropods are considered in some detail, and the question of the possible homology between crustacean gills and insect wings is critically addressed. The hypothesis that there are only two basic limb types in arthropods, antennules and postantennulary limbs, is proposed and its apparent contradiction by the transformation of antennules into walking limbs by homeotic mutation is discussed with respect to the appropriate level of serial homology between these limbs.