Trochophore Larvae Research Articles

The expression of dopa decarboxylase and dopamine beta hydroxylase and their responding to bacterial challenge during the ontogenesis of scallop Chlamys farreri

Dopa decarboxylase (DDC) and dopamine beta hydroxylase (DBH) is responsible for the synthesis of dopamine and norepinephrine, respectively. In the present study, dopa decarboxylase (CfDDC) and dopamine beta hydroxylase (CfDBH) were selected as indicator to investigate the development of catecholaminergic nervous system in the larvae of scallop Chlamys farreri. The CfDDC and CfDBH transcripts were all detectable during the whole ontogenesis expect for the CfDDC transcripts in 2-cell embryos stage. The expression level of CfDDC and CfDBH mRNA increased significantly in the veliger stage, and reached the peak in late (35.64-fold, P < 0.05) and mid-veliger (400.21-fold, P < 0.05) larvae, respectively. By immunofluorescence, two CfDDC immunoreactive areas were observed in the trochophore and D-hinged larvae, and then three CfDDC immunoreactive areas and two immunopositive fibres formed in early and late veliger larvae, respectively. Two CfDBH immunopositive fibers appeared initially in the early D-hinged stage, and another two similar fibers developed in the late D-hinged stage. The bacteria Vibrio anguillarum challenge could induce the mRNA expression of CfDDC and CfDBH in different developmental stage. The significantly increase of CfDDC mRNA was observed in the trochophore larvae at 12 h (8.61-fold, P < 0.05) and in late D-hinged larvae at 24 h (1.56-fold, P < 0.05) post challenge. The expression level of CfDBH mRNA decreased significantly in late D-hinged larvae at 6 h (0.45-fold, P < 0.05), whereas it increased significantly in late veliger larvae at 12 h after bacterial challenge (14.52-fold, P < 0.05). These results concluded that the scallop catecholaminergic nervous system appeared firstly as the form of dopaminergic neurons in the trochophore larvae, and the developing catecholaminergic nervous system in the trochophore, D-hinged and veliger larvae of scallop could respond to the immune stimulation in different patterns.

Giant Clam Aquaculture: a Review on Induced Spawning and Larval Rearing

Giant clams are the largest extant bivalves and widespread inhabitants of the Indo-Pacific coral reefs. These organisms are particularly sought after by the food industry because of their large mantle and adductor muscle, while also are relevant to the aquarium trade market. The aquaculture of giant clams is dependent of a land-based hatchery divided in several tanks with different purposes for larval rearing. Prior to reproductive events, broodstock are conditioned and gametogensis is stimulated by frequent food additions, increased photoperiod and intense physical-chemical stability at optimum conditions. Several different methods can be employed to induce spawning, most notoriously the intragonadal injection of serotonin, thermal stress and addition of macerated gonadal tissue of a conspecific. The success of the method is based on spawning occurrence, time for response and events of broodstock mortality. After spawning, gametes are fertilized and a succession of planktonic larval stages is cultured in hatching tanks and raceways. Embryonic development lasts for approximately 12 hours until the hatch of free-swimming and non-feeding trochophore larvae. After 24 hours post-fertilization larvae morph into veliger stage, made evident by the presence of calcium carbonate shells and velum. Veliger larva are fed with live or preserved phytoplankton and must also acquire symbiotic zooxanthellae. The last stage is the pediveliger stage at approximately one week post-fertilization, when settlement takes place and metamorphosis is soon attained.

The golden age of comparative morphology: Laser scanning microscopy and neurogenesis in trochophore animals

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.

Swimming Performance in Early Development and the "Other" Consequences of Egg Size for Ciliated Planktonic Larvae

The evolutionary significance of egg size in marine invertebrates is commonly perceived in energetic terms. Embryonic size should also have direct effects upon the forces that govern swimming, a behavior common to early larval development in the plankton. If swimming is ecologically important, early larvae may need to perform to a certain "standard", or threshold of speed and/or stability. The existence of performance standards in early development could therefore act to constrain the evolution of egg size and the evolution of development. Here we present the key parameters that characterize the upward swimming speed of ciliated spheroidal larvae moving at very low Reynolds numbers. The dependence of maximum supported mass upon larval size, and the independence of neutral-weight swimming speed from size, lead to hypotheses about scaling of swimming speed with size. Experimental studies with thirteen broadcast-spawning planktotrophs demonstrate that free-living embryonic swimmers in all of these species conform to a strong negative scaling of density with size that offsets increases in mass with increasing size. This trend suggests that swimming ability is broadly under selection in early development. In experimental studies and in a hydrodynamic model of larval swimming, the performance of trochophore larvae provides support for our hypothesized scaling relationships, and also for the concept of a standard in swimming speed. Echinoid blastulae, however, show relationships between speed and size that are not predicted by our scaling arguments. Results for echinoids suggest that differences in ciliary tip speed, or possibly in spatial density of cilia over the blastula's surface, result in significant differences in species' performance. Strong phyletic differences in the initial patterning and growth of structures used for swimming thus appear to cause significant differences in the relationship of swimming ability with embryo size.

Effects of pH on growth development of larva Coelomactra antiquata and survival rate of fertilized eggs.

The effects of pH on the hatching of Coelomactra antiquata fertilized eggs and on the development as well as attachment metamorphosis of velige larva were studied.The results showed that the optimal pH for fertilized eggs to be hatched to trochophore larva was 7.0~8.5,while the optimal pH for trochophore larva to grow to straight line hinge larva was 7.5~8.5.Velige grew faster when pH was 8.0~8.2 and the average daily growth gain of shell length was 15.0~16.5 μm on average.When water temperature was 26.4±0.45 ℃,salinity 25.50 and pH 8.0~8.2,C.antiquata veliger with an average shell length of 196 μm,after 72 hours of cultivation,reached attachment metamorphosis at a rate of 51.66%~53.00%.

Molecular markers comparing the extremely simple body plan of dicyemids to that of lophotrochozoans: insight from the expression patterns of Hox, Otx, and brachyury

Because of their extremely simple body plan, dicyemids have long been the subject of phylogenetic controversy, regarding whether their body plan reflects their primitiveness or a degeneration from complex metazoans. Several studies have argued that the simple body plan of dicyemids are likely secondarily derived from higher lophotrochozoan animals, as a result of their endoparasitic, or endosymbiotic, lifestyle in the cephalopod kidney. To clarify the evolution of their simple body plan, we investigated the developmental expression patterns of three important regulatory genes, the central type Hox gene (DoxC), otx, and brachyury homologs in the dicyemid mesozoa, Dicyema orientale. DoxC was expressed in the trunk and tail of the asexually developing vermiform embryo, with clear anterior boundaries. Do-otx was expressed in the vegetal pole cells of the developing infusoriform embryos, suggesting that the invagination in infusoriform embryo is homologous to the gastrulation of other metazoans. Do-bra is expressed in the presumptive ventral cells, which are ventral to the opening of the urn cavity. The expression of Do-bra suggests that the urn cavity opening of the infusoriform embryo is comparable to the stomodium of trochophore larvae. These gene expression patterns provide molecular clues to trace the evolutionary history of degeneration in the dicyemid embryogenesis and life cycle from those of ancestral lophotrochozoan animals.

The Larval Development, Metamorphosis and Juvenile Growth of the Turban Snail Lithopoma (Astraea) undosa (Wood, 1828) (Gastropoda:Turbinidae)

This work presents the embryonic, larval, and postlarval development of Lithopoma undosa (L. undosa) (20 ± 2 C and the effect of natural inducers on the larval metamorphosis. Embryonic development until hatching of the trochophore larva took 15 h. Early veliger larvae with well‐developed shell were after 24 h. At 48 h, the operculum was completely formed. Cephalic tentacles were visible after 72 h. Larvae reached competence 6 to 7 d after fertilization, and was characterized by the presence of branched cephalic tentacles. The effect of natural inducers was evaluated. The percentage of metamorphosis with diatoms and adult mucus was 94% and 58% respectively, differing from the 25% without inducer. When was repeated, the metamorphosis was 100%, and 16% with benthic diatoms. The initial mean size of the postlarvae was 0.316 ± 0.02 mm. After 264 d the juveniles had attained a mean size of 5.98 ± 0.16 mm. These results will not only be of use in the evaluation of the culture of L. undosa, but also in studies on the characterization of inducers.

Developmental stages, larval and post-larval growth of angelwing clamPholas orientalis

Angelwing clam were induced to spawn by thermal stimulation. Mature eggs measured 50 μm in diameter. Cell division occurred within 36 min after fertilization. Mobile trochophore larvae were seen after 12 h and larvae developed within 18 h. Reared on a diet of Isochrysis galbana the larvae reached the umbo stage in 6–7 days. On day 10 the foot could be seen and settlement occurred if a suitable substrate was present. The larvae completed metamorphosis into juveniles within 20 days after settling.

Associations between the White Sea invertebrates and oxygen-evolving phototrophic microorganisms

Eleven species of White Sea invertebrates (sponges, actinians, hydroids, polychaetes, and nudibranch mollusks) were tested for the presence of associated oxygen-evolving phototrophic microorganisms (OPM) (microalgae and cyanobacteria). Two main approaches were applied: (a) light and electron microscopy of intact samples and fixed preparations of invertebrates, and (b) isolation of microorganisms from samples of invertebrates after mild surface sterilization. The obtained results lead to conclusions on the formation of multicomponent associations by White Sea invertebrates and OPM based on the following data: (1) isolation of 27 cultures of OPM from eight species of invertebrates (sponges, hydroids, polychaete trochophore larva), (2) specificity of association between epibiontic communities of microorganisms and macroorganisms within the same biotope, and (3) spatial integration of micro- and macropartners resulting in the formation of morphological structures within the interorganismic contact zones.

Spawning and development in Osedax boneworms (Siboglinidae, Annelida)

We report observations on spawning and early development in bone-eating worms of the genus Osedax. Individual females of Osedax rubiplumus were observed at 1820 m depth freely spawning hundreds of oocytes, and females of an undescribed species, Osedax “orange collar”, were observed spawning in laboratory aquaria. Cytological and molecular analysis of the spawned oocytes of two Osedax species revealed no evidence for the bacterial endosymbionts that the female worms require for their nutrition, suggesting that the bacteria must be acquired later from the environment, as they are in other siboglinids. Individual O. “orange collar” females released an average of 335 (±130) eggs per day, but the number of oocytes spawned per day varied greatly, suggesting that not all the females spawned daily. Fertilization rates of the spawned oocytes varied from 0 to 100%, though most females showed nearly 100% fertilization rates. Oocytes spawned in the laboratory at 4–6°C were negatively buoyant. If fertilized, these oocytes extruded polar bodies and then after at least four hours cleaved unequally. Subsequent cleavages occurred in a spiral pattern at roughly 2-h intervals, resulting in free-swimming trochophore larvae after 24 h. These lecithotrophic trochophores swam for 9–16 days before settling with several hooked chaetae, similar to those of dwarf Osedax males. The larval life span of the Osedax species studied in the laboratory appears to be shorter than in closely related Vestimentifera. Osedax rubiplumus, on the other hand, has much larger oocytes and so may have greater dispersal potential than these other Osedax species. The high fecundity and apparently continuous reproduction of Osedax boneworms permits the opportunistic exploitation of sunken vertebrate bones.

Karyotypes of Diploid and Triploid Mercenaria mercenaria (Linnaeus)

Northern quahogs (=hard clam; Mercenaria mercenaria) is a commercially important bivalve naturally distributed along the American Atlantic coast. Because the hard clam was first introduced into China as a candidate for aquaculture in 1997, research has been conducted on its ecologic habits, ecological physiology, metabolism, growth, reproduction, artificial propagation, and seed culture. Little information about their chromosomes and karyotype is available. The present study is designed to study the karyotype of the diploids and triploids to provide a reference to genetic research on introduced M. mercenaria. Triploids were produced by inhibiting the release of second polar body (PBII) with a treatment of 450 μmol/mL 6-deimethylaminopurine (6-DMAP), with untreated fertilized eggs developing as normal diploid controls. Chromosome preparations were made from trochophore larvae with drop-splash method. Chromosomes were measured and classified according to Leven criteria. Statistical analysis of relative length and arm ratio revealed that, this species had a normal diploid chromosome number of 2n = 38, and the karyotype consists of 15 pairs of metacentric (M) and 4 pairs of submetacentric (SM) chromosomes, NF = 76. The triploid karyotype formula was 45 M 12 SM, NF = 114. The heterochromosomes and satellite chromosomes were not observed in all chromosomes in the complement.

Cyclic AMP concentration and protein kinase A (PKA) gene expression at different developmental stages of the polychaete Hydroides elegans

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed inductive effect on larval settlement of the polychaete Hydroides elegans. It has been suggested that IBMX triggers larval settlement by elevating the cellular adenosine 3',5'-cyclic monophosphate (cAMP) level in this species. To test this hypothesis, we first examined cAMP-level changes in both the competent (CL) and attached larvae (AL) and then characterized the cAMP-dependent protein kinase in H. elegans, which is the major mediator of cAMP action. Tissue extracts of the larvae were assayed for cAMP by enzyme immunoassay; the results showed that IBMX increased cAMP production up to approximately two-folds in the CL. However, there was no significant difference in the cAMP concentration between the CL and AL that were not treated with IBMX. The catalytic subunit of protein kinase A gene from H. elegans (designated HePKAc) was cloned, and its expression in different developmental stages of H. elegans was examined using quantitative real-time polymerase chain reaction. The gene expression level in the pre-competent trochophore larvae was the lowest, increased in the CL, reached the highest in the larvae undergoing normal and IBMX-induced metamorphosis, and then decreased in the adult stage. In situ hybridization results showed that HePKAc expressed mainly around eye regions and along body fragments of the CL and AL. Our results indicated that the IBMX-induced cAMP changes and the cAMP-dependent protein kinase gene may mediate larval development and settlement of H. elegans.

Mesoderm in spiralians: the organizer and the 4d cell

The Spiralia is a clade of protostome invertebrate phyla that share a highly conserved mode of early development. Spiralian development is characterized by regularities in the arrangement of early cleavages, the fates of the cells that are produced by these divisions, and the development of the distinctive trochophore larva. Because of the strong conservation in early development, homologies can be identified between cells in divergent taxa. Some of the most striking examples of conservation in the spiralian embryo are in the cells that generate the mesoderm. The specification of the mesodermal precursors has been well characterized by embryological approaches, and recently the molecular mechanisms of mesoderm specification are starting to be elucidated. This review examines the development of mesoderm in spiralians in a comparative context, with particular focus on the relationship between the mesendodermal cell 4d and the embryonic organizer.

Extension of the breeding season and its effects on fertilization and development in two species of lugworm ( Arenicola marina and A. defodiens)

In the autumn/winter breeding polychaetes, Arenicola marina and A. defodiens, spawning can be advanced or delayed by a number of months through temperature manipulation of the adults. However, this manipulation may have significant consequences for fertilization rates and embryo developmental success and so in vitro fertilizations were performed to assess the impact of manipulation. Firstly, we used oocytes and sperm obtained from advanced or delayed individuals. For both species, using gametes from 4 weeks advanced individuals did not result in a significant reduction in development, however, gametes from individuals advanced ( A. marina only) or delayed by 8 weeks resulted in significantly fewer embryos developing normally. Reciprocal crosses of temperature-manipulated A. marina gametes (from 4 weeks advanced and 4 weeks delayed individuals) with those at the natural spawning time confirmed that the reduction in developmental success in both was attributable to the oocytes. After 5 h post-fertilization, the majority of oocytes from advanced individuals had fertilized, but by 24 h most were abnormal. For fertilizations with gametes from delayed individuals, nearly 100% of the embryos were developing normally after 24 h, but after 144 h significantly more were abnormal in crosses involving oocytes from delayed females. Although both species have reproductive plasticity to extend their breeding season, the significant reduction in the numbers of competent larvae produced as the spawning is delayed or advanced further may be a significant bottleneck in aquaculture and it may also have considerable implications for the long-term reproductive success of a population in response to environmental change. Sympatric populations of the species exist in many locations and the inherent variability in the breeding seasons could allow spawning times to overlap. Artificially delaying A. marina individuals enabled fertilizations to be performed with A. defodiens at the natural spawning time in the laboratory. Both conspecific fertilizations produced 100% trochophore larvae after 120 h, but A. defodiens oocytes failed to fertilize after incubation with A. marina sperm, in comparison to the A. marina oocytes incubated with A. defodiens sperm where 40% developed to the trochophore stage. This asymmetric gamete incompatibility may be one of a suite of mechanisms to minimise hybridisation.

Subtype Variation Among Bacterial Endosymbionts of Tubeworms (Annelida: Siboglinidae) from the Gulf of California

Symbiosis involving chemoautotrophic bacteria allows vestimentiferan tubeworms to thrive in sulfidic marine environments. This study examined genetic variation among endosymbionts associated with three vestimentiferan species from the Gulf of California. Small subunit (16S) rRNA sequences identified two evolutionary lineages of -Proteobacteria in these worms. Phylotype-II bacteria associated with the hydrothermal vent species Riftia pachyptila exhibited no subtype variation upon examination of form II (cbbM) RuBisCO, whereas the phylotype-I bacteria associated with two cold-seep species, Escarpia spicata and Lamellibrachia barhami, were polymorphic. Bacterial subtypes distinguished by three RuBisCO alleles occurred at similar frequencies in both host species when sampled together from tubeworm clusters, offering, therefore, no evidence for host-specificity. Instead, the frequencies of these subtypes varied significantly among patchily distributed tubeworm clusters. Subtype variation on small spatial scales is consistent with prior evidence that vestimentiferans acquire their symbionts locally from the environment in which they settle as larvae. Adult vestimentiferans are nourished by endosymbiotic bacteria that oxidize inorganic sulfides and fix carbon via the Calvin-Benson cycle (1). These essential bacteria infect vestimentiferans de novo in each generation by penetrating the epidermis of trochophore larvae that settle on benthic substrates (2). Previous studies (summarized in reference 3) revealed two related phylotypes (i.e., a clade defined by 16S rRNA sequences) of -Proteobacteria associated with vestimentiferans worldwide. The two phylotypes segregate geographically and according to the kind of chemosynthetic habitat in which the hosts settle. Phylotype-I is found in cold-seep vestimentiferans worldwide and in hydrothermal vent vestimentiferans from the western Pacific. Phylotype-II has been found only in vestimentiferans from eastern Pacific hydrothermal vents. When distinct vestimentiferan species co-occur in a hab

Homology of ciliary bands in Spiralian Trochophores

A number of hypotheses have been presented regarding the origins of the metazoans and, more specifically, the Bilateria. Using various phylogenetic analyses, characteristics have been mapped on phylogenetic trees to infer ancestral body plans and life history strategies of those ancestors. Many arguments on the evolution of the Bilateria are based on the presumed homology of certain characteristics of extant larva and adults, including various ciliated bands involved in feeding and locomotion. This article considers a recent study indicating that the second, downstream-collecting, ciliated band in the veliger larva of the gastropod mollusc, Crepidula fornicata, is actually derived from secondary trochoblasts (derived from second quartet micromeres), that normally form part of the prototrochal band found in other spiralian phyla (Hejnol et al. 2007). Despite previous arguments, these new findings suggest that the second ciliated band in the veliger larva is not homologous to the metatroch found in the trochophore larva of some other spiralians, such as the annelid, Polygordius lacteus. In the latter case, the metatroch was reported to be formed by a different set of lineage precursors (derived from third quartet micromeres) (Woltereck 1904). These findings have important implications for the interpretation of various hypotheses related to the evolution of metazoan phyla.

Sinistral equal‐size spiral cleavage of the indirectly developing polychaete Hydroides elegans

Two major variants of the stereotypic spiral cleavage correlate with distinct developmental modes in polychaetes. Indirect development through a feeding trochophore larva correlates with development from four equal-sized blastomeres, whereas direct development correlates with unequal cleavage characterized by a large dorsal blastomere precursor maternally predetermined. The equal-size spiral cleavage of the indirectly developing serpulid Hydroides elegans has been reconstructed from serial sections of nuclei-stained embryos. The order of cell divisions has been determined from the 2-cell stage to the 80-cell stage, when gastrulation cell movements start to overlap with late spiral-cleavage divisions. In contrast to related species, the third cleavage in Hydroides elegans is invariably sinistral. The four quadrants remain indistinct until the 60-cell stage, when the small 2d22 and large 2d21 cells are generated. The developmental significance of the invariant spiral cleavage relates to the spatial distribution of gene functions that it partitions and their relation to blastomere fate commitments. The conservation and divergence of the cleavage pattern among spiralians is well suited to study the developmental control of the cell-cleavage machinery and its evolution.

Early development of the aplacophoran mollusc Chaetoderma

AbstractThe early development of the trochophore larva of the aplacophoran Chaetoderma nitidulum (Mollusca: Caudofoveata = Chaetodermomorpha) is described using scanning and transmission electron microscopy and using fluorescence staining and confocal laser scanning microscopy of the muscle system. The most important features include the presence of a prototroch and a telotroch, serially arranged glandular cells (spiculoblasts) at the dorsal mantle, the occurrence of a ventral suture as a possible remnant of a former foot sole, and the presence of a pair of protonephridia. The development of the early anlagen of the circular body wall muscles does not show the anterior–posterior mode of formation that is typical for annelids, thus strengthening the hypothesis of a non‐segmented ancestry of Mollusca.

HeOtx expression in an indirectly developing polychaete correlates with gastrulation by invagination

The expression of an Otx homolog in the indirectly developing polychaete Hydroides elegans was characterized during embryo, trochophore, and feeding-larva stages. In the animal hemisphere, HeOtx is first expressed in 1q(12) blastomeres and their immediate descendants. Such discrete embryonic animal hemisphere Otx expression perhaps relates to cell-type specification functions of the larva. During feeding stages, transcripts are detected in adult cerebral ganglia precursors and putative adult eye precursors, where it may have adult brain regionalization functions. HeOtx is not expressed in primary trochoblast precursors, but it is expressed in cells adjacent to the ciliary band. HeOtx is also expressed in a group of cells in the dorsal midline of the early trochophore larva in putative posterior sensory organ precursors. The vegetal hemisphere expression starts in oral and lateral sides of the blastopore and later expands to central blastomeres that lead the gastrulation movements. During late gastrulation stages, the expression declines in foregut precursors, but it is maintained in midgut precursors, suggesting its involvement in tripartite gut subdivision functions. HeOtx broader and earlier endoderm expression correlates with gastrulation by invagination associated with the formation of the feeding trochophore, in contrast with a later and orally restricted Otx expression found in a polychaete that gastrulates by epiboly and forms a non-feeding trochophore. The endoderm expression and functional roles in other bilaterians suggest an ancestral role of Otx related to gastrulation by invagination.

Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete

The trochophore larvae of indirectly developing spiralians have ciliary bands with motor and feeding functions. The preoral prototroch ciliary band is the first differentiating organ in annelid and mollusk embryos. Here we report the expression of several ciliary band markers during embryogenesis and early larval stages of the indirectly developing polychaete Hydroides elegans. Genes with similarity to caveolin, β- tubulin, α- tubulin, and tektin are expressed in the eight primary prototroch precursors, 1q 221 and 1q 212. Blastomeres 1q 221 and 1q 212 locate at the same equatorial latitude after the complementary asymmetric division of their 1q 22 and 1q 21 precursors. In addition, caveolin and α- tubulin are expressed in the metatroch and adoral ciliary zone. Caveolin is expressed in foregut ciliated cells, and α- tubulin is expressed in apical tuft ciliated cells. The expression of a β- thymosin homolog is restricted to 1q 122 and 1q 121 blastomeres, which locate just above and in close association with the eight primary prototroch cells 1q 221 and 1q 212. In addition, the β- thymosin homolog has a transient expression in the hindgut and apical zone. The expression of all these genes provides a landmark for the early specification of ciliary bands and other ciliated organs.