Egg envelopes and cuticle renewal in Porcellio embryos and marsupial mancas

An important adaptation to land habitats in terrestrial isopod crustaceans is development of embryos in a fluid-filled female brood pouch, marsupium. The study brings insight into the structure and protective role of egg envelopes and cuticle renewal during ontogenetic development of Porcellio embryos and marsupial mancas. Egg envelopes cover embryos, the outer chorion until late-stage embryo and the inner vitelline membrane throughout the whole embryonic development. Egg envelopes of Porcellio have relatively simple ultrastuctural architecture compared to Drosophila egg envelopes. Exoskeletal cuticle is produced in late embryonic development by hypodermal cells of the embryo and is renewed in further development in relation to growth of developing embryos and mancas. Cuticle structure and renewal in prehatching late-stage embryos and marsupial mancas exhibit main features of cuticle in adults. Epicuticle is thin and homogenous. The characteristic arrangement of chitin-protein fibers and the dense distal layer in exocuticle are hardly discernible in prehatching embryo and distinct in marsupial mancas. Endocuticle consists of alternating electron dense and electron lucent sublayers and is perforated by pore canals in both stages. Differences from adult cuticle are evident in cuticle thickness, ultrastructure and mineralization. Signs of cuticle renewal in prehatching embryo and marsupial mancas such as detachment of cuticle from hypodermis, partial disintegration of endocuticle and assembly of new cuticle are described.     

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.     

Formation of the hindgut cuticular lining during embryonic development of Porcellio scaber (Crustacea,Isopoda)

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellio scaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage – stage 18 – an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.     

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.     

Molting and cuticle deposition in the subterranean trichoniscid Titanethes albus (Crustacea, Isopoda)

Terrestrial isopods are a suitable group for the study of cuticle synthesis and calcium dynamics because they molt frequently and have evolved means to store calcium during molt. Little data is currently available on molting in Synocheta and subterranean isopods. We studied the molting dynamics in the subterranean trichoniscid Titanethes albus under laboratory conditions and performed a microscopic investigation of sternal CaCO(3) deposits and the tergal epithelium during molt in this species. In accordance with its lower metabolic rate, molting in the laboratory is roughly 2-3 times less frequent in Titanethes albus than would be expected for an epigean isopod under similar conditions. Animals assumed characteristic postures following the molt of each body half and did not consume the posterior exuviae after posterior molt. The structure of sternal calcium deposits and the ultrastructural characteristics of the epidermis during cuticle formation in Titanethes albus are similar to those described in representatives of Ligiidae. During the deposition of the exocuticle, the apical plasma membrane of epidermal cells forms finger-like extensions and numerous invaginations. In the ecdysial space of individuals in late premolt we observed cellular extensions surrounded by bundles of tubules.     

The Caenorhabditis elegans Patched domain protein PTR-4 is required for proper organization of the precuticular apical extracellular matrix

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.     

Orchestin, a calcium-binding phosphoprotein, is a matrix component of two successive transitory calcified biomineralizations cyclically elaborated by a terrestrial crustacean

Orchestia cavimana is a crustacean that cyclically replaces its calcified cuticle during molting cycles in order to grow. Its terrestrial way of life requires storage of calcium during each premolt period, as calcareous concretions, in tubular diverticula of the midgut. During the postmolt period the stored calcium is reabsorbed and is translocated through the storage organ epithelium as calcified small spherules. In a previous study, we sequenced and characterized a remarkable component of the organic matrix of the premolt storage structures, Orchestin, which is a calcium-binding phosphoprotein. In this paper, we analyzed the spatiotemporal expression of the orchestin gene by Northern blotting and in situ hybridization, and its translated product by immunocytochemistry. We found evidence that the gene and the protein are expressed specifically during premolt in the storage organs. More interestingly, we demonstrated that the protein is synthesized also during the postmolt period, as a component of the organic matrix of the calcium resorption spherules. Thus, Orchestin is a matrix component that is synthesized by the same cells to contribute alternately to the elaboration of two different calcifications. These results, in addition to the physical and chemical features of the protein, suggest that Orchestin is probably a key molecule in the calcium carbonate precipitation process leading to the cyclic elaboration of two transitory calcified mineralizations by the crustacean Orchestia.     

New Functions of Arthropod Bursicon: Inducing Deposition and Thickening of New Cuticle and Hemocyte Granulation in the Blue Crab,Callinectes sapidus

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.     

Fine structure and function of the digestive tract of Cyathura carinata (Krøyer) (Crustacea,Isopoda)

Summary The entire gut of Cyathura carinata is lined by a cuticle indicating its completely ectodermal origin. By flattening of the epithelial folds and possibly also of reserve-folds of the plasma membrane the intestine is highly dilatable, an adaptation towards a rapid uptake of the food which is sucked in by means of specialized mouthparts, which pierce the body wall of its main prey, the polychaete Nereis diversicolor. Bundles of microtubules within the intestinal cells presumably represent cytoskeletal structures providing protection against mechanical stress. Spirally arranged muscle fibres, which form peculiar contact areas with the gut, can easily follow any dilatation. A few indications of the metabolic functions of the anterior gut epithelium have been found: Basally and apically located labyrinthine structures of the plasma membrane, apically located clear vesicles, positive reactions for lysosomal, mitochondrial and membraneous enzymes, a strikingly thin and loosely arranged cuticle through which food substances of low molecular weight may diffuse. The cells of the gut and also of the digestive caeca are interconnected by desmosomes, extensive pleated septate junctions, and gap junctions. In the pleon the gut is less dilatable and devoid of plasma membrane specializations. In this area tendon cells, particularly rich in microtubules, serve as attachment sites for the dilating muscles of the rectum. The digestive caeca synthetize and secrete digestive enzymes, mix food and enzymes in their lumen, resorb food molecules, store lipids and glycogen. In the glandular epithelium small cells, rich in rough ER, and a majority of large cells, rich in lipid droplets, occur which, however, are interconnected by a series of morphologically intermediate cells. All cells bear an apical brush border, form a basal labyrinth and contain high to medium activities of acid phosphatase, nonspecific esterases, ATPase, and succinic dehydrogenase. The ER-rich cells are far less frequent than in the omnivorous or herbivorous isopods (Sphaeroma, Idothea, Asellidae, Oniscoidea).     

中国米虾bursicon基因的功能及作用机制

为探讨米虾鞣化激素在其蜕皮周期及表皮角质层形成过程中的作用, 采用PCR技术克隆得到了米虾鞣化激素两个亚基基因的开放阅读框(ORF)序列。bursicon-α ORF全长441 bp, 共编码146个氨基酸; bursicon-β ORF全长411 bp, 共编码136个氨基酸。利用实时荧光定量PCR分析米虾整个蜕皮周期中鞣化激素2个亚基基因的表达特征, 结果发现, 鞣化激素bursicon-α和bursicon-β在米虾蜕皮周期的各个阶段的相对表达量存在差异, 在蜕皮前期(D期)相对表达量开始上升, 到D₃期时相对表达量最高, 蜕皮期E期相对表达量最低。RNA干扰(RNA interference, RNAi)介导bursicon-α和bursicon-β基因沉默后, 发现米虾的蜕皮周期延长, 表皮角质层明显变薄。结果提示, 鞣化激素(Bursicon)与新形成的外骨骼中角质层的加厚与硬化密切相关, 进而影响蜕皮时间。     

Activation of old cuticle chitin as a substrate for chitinase in the molt of Manduca

During the molt, chitin in the old cuticle of $\text{Manduca}$ is digested by chitinase taken up from molting fluid, but the chitin in intact (= premolt) cuticle is not accessible to chitinase. As a prerequisite of digestion, old cuticle chitin is rendered competent to serve as chitinase substrate in a reaction attributable to trypsin-like proteolytic activity of molting fluid.     

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus)

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.     

An ultrastructural investigation of muscle attachment in the opercular filament of a polychaete annelid

The ultrastructure of peduncle muscle attachment to the cuticular flange in the opercular filament of the serpulid Pomatoceros lamarckii Quatrefages is described. The cuticular flange is composed of layers of orthogonally arranged fibres. Specialized epithelial cells (tendon cells) and a collagenous matrix intervene between the peduncle muscles and the cuticular flange. The tendon cells are characterized by hemidesmosomes at both apical and basal ends, connected by thick bundles of tonofilaments. Apically long specialized microvilli from the tendon cells penetrate the cavities in the orthogonally arranged layers of fibres of the cuticular flange. The basal surfaces of the tendon cells and the terminal ends of the peduncle muscles anchor independently of one another in the collagenous matrix. The peduncle muscles appear to be smooth muscles which contain thin filaments, 5 nm in diameter, and thick filaments, 40-100 nm in diameter, with a faint axial periodicity 12-14 nm. The method of peduncle muscle attachment in the opercular filament is compared with those of other invertebrates.     

Prominent actin fiber arrays in Drosophila tendon cells represent architectural elements different from stress fibers

Tendon cells are specialized cells of the insect epidermis that connect basally attached muscle tips to the cuticle on their apical surface via prominent arrays of microtubules. Tendon cells of Drosophila have become a useful genetic model system to address questions with relevance to cell and developmental biology. Here, we use light, confocal, and electron microscopy to present a refined model of the subcellular organization of tendon cells. We show that prominent arrays of F-actin exist in tendon cells that fully overlap with the microtubule arrays, and that type II myosin accumulates in the same area. The F-actin arrays in tendon cells seem to represent a new kind of actin structure, clearly distinct from stress fibers. They are highly resistant to F-actin–destabilizing drugs, to the application of myosin blockers, and to loss of integrin, Rho1, or mechanical force. They seem to represent an important architectural element of tendon cells, because they maintain a connection between apical and basal surfaces even when microtubule arrays of tendon cells are dysfunctional. Features reported here and elsewhere for tendon cells are reminiscent of the structural and molecular features of support cells in the inner ear of vertebrates, and they might have potential translational value.     

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+-ATPase

Periodical changes in Ca2+-ATPase and Mg2+-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca2+-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt , when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg2+-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca2+-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg2+-ATPase activity. There results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.     

Formation of the inner branchiostegal cuticle of the blue crab,Callinectes sapidus

The noncalcified inner branchiostegal cuticle, which lines the branchial chamber, was examined histologically and ultrastructurally over the molt cycle in the blue crab, Callinectes sapidus. In intermolt crabs (stage C₄) the epithelium underlying the inner cuticle is cuboidal and has abundant intercellular spaces and a prominent basement membrane. Apolysis occurs at stage D₀ and dissolution of the cuticle is accompanied by the formation of numerous lysosomes in the epithelium. During stage D₁, cells increase in height, apical mitochondria become more abundant, and the cuticle continues to be resorbed. An epicuticle is formed in early D₂, arising from a fusion of small subunits apparently attached to short apical microvilli. Cuticle deposition continues through D₂ and is complete by stage D₃. By the time cuticle deposition is complete, the epithelium has become extremely columnar and cells are filled with bundles of microtubules. In stage D₄, an amorphous electron‐dense core appears in the microtubule‐filled cells, which are attached to the cuticle at their apical end and anchored to their basement membrane at the basal surface. These microtubule‐filled cells persist through ecdysis, stage E, but during stage A₁ the cores disappear and some organelles begin to reappear in the cytoplasm. By stage A₂, the cells return to the cuboidal morphology seen in intermolt and remain so throughout the remainder of the molt cycle. This new pattern of cuticle deposition resembles that observed in the gills of crustaceans in that the cuticle is uncalcified and there is no postecdysial cuticle formation. However, instead of apolysis being delayed until just before ecdysis, the inner cuticle is formed during the first half of premolt, allowing the epithelial cells time to differentiate into a morphology that provides tensile strength for the stress of ecdysis. These new observations demonstrate that cuticle formation can follow very diverse structural and temporal patterns. In order to integrate and coordinate these diverse patterns, it is suggested that a suite of feedback mechanisms must be present. J. Morphol. 240:267–281, 1999. © 1999 Wiley‐Liss, Inc.     

Ultrastructural investigation of the mechanism of muscle attachment to the gastropod shell

The ultrastructure of the muscle-shell attachment was investigated in the land pulmonate snails Helix aspersa, Anguispira altemata, in the freshwater pulmonate Laevipex sp., and in the freshwater prosobranch Pomacea paludosa. In all cases, a collagenous intercellular matrix and a specialized epithelium (tendon cells) intervene between the columellar muscle and the shell. These tendon cells are characterized by hemidesmosomes at both apical and basal ends, connected by thick bundles of microfilaments. The tendon cells do not insert into the shell directly by microvilli, as formerly thought, but by an extensive network of extracellular organic fibers.     

Formation of the arthrodial membrane in the blue crab,Callinectes sapidus

In this study the pattern of arthrodial membrane deposition in Callinectes sapidus was determined by histological and ultrastructural examination of tissues from the carpus joint of the cheliped collected during premolt, ecdysis, postmolt, and intermolt. Apolysis in the arthrodial membrane occurs at stage D(0) and is synchronous with apolysis of the calcified cuticle. Epicuticle formation begins at early stage D(1) and is completed in late stage D(1). Procuticle deposition starts at D(2) and continues until ecdysis. Numerous cytoplasmic extensions occur throughout the lamellae. Component fibers of the arthrodial membrane are intimately associated with dense plaques on the apical membrane of the underlying hypodermal cells, suggesting a site for fiber polymerization. Deposition of the arthrodial membrane continues after ecdysis, with most of the cuticle thickening occurring during stage C. When stained with PAS and counterstained with hematoxylin, a difference can be discerned between preecdysial and postecdysial procuticle of the arthrodial membrane, a distinction not made in previous studies. The boundary between the arthrodial membrane and calcified cuticle is thicker than either of the two layers and the layers overlap rather than butting up against one another. This pattern suggests that underlying hypodermal cells have to produce multiple types of cuticle over the molt cycle. A summary of the various molting patterns in C. sapidus suggests that the control of these diverse events may prove to be complex.     

Ovoviviparous reproduction in Australian specimens of the intertidal isopod Cirolana harfordi

Cirolana harfordi is an intertidal isopod found in the SW Pacific (Eastern Australia: New South Wales and Victoria), NW Pacific (Japan and Russia) and the Pacific coast of North America. C. harfordi from North America has been reported to reproduce by depositing eggs into a marsupial pouch formed by oostegite plates that grow from the base of the 3rd to 6th pereopods (walking legs), so as to incubate embryos and mancas (young that resemble the adult except the seventh pair of pereopods are absent) external to the ventral cuticle of the mother. In this study, however, C. harfordi individuals from New South Wales, Australia were found to incubate embryos and mancas inside the pereon (thorax) cavity. C. harfordi in Australia is, therefore, displaying ovoviviparous reproduction and not incubation in a marsupial pouch external to the pereon as previously thought. Embryos have a direct development pathway to the internal manca stage that is held within the mother’s body cavity until the time of live birth. Additionally, internal mancas show some morphological differences to adults, such as a lack of cusps on the mandibles.