Left-right positioning of the adult rudiment in sea urchin larvae is directed by the right side.

Indirect-developing sea urchins eventually form an adult rudiment on the left side through differential left-right development in the late larval stages. Components of the adult rudiment, such as the hydropore canal, the hydrocoel and the primary vestibule, all develop on the left side alone, and are the initial morphological traits that exhibit left-right differences. Although it has previously been shown that partial embryos dissected in cleavage stages correctly determine the normal left-right placement of the adult rudiment, the timing and the mechanism that determine left-right polarity during normal development remain unknown. In order to determine these, we have carried out a series of regional operations in two indirect-developing sea urchin species. We excised all or a part of tissue on the left or right side of the embryos during the early gastrula stage and the two-armed pluteus stage, and examined the left-right position of the adult rudiment, and of its components. Excisions of tissues on the left side of the embryos, regardless of stage, resulted in formation of a left adult rudiment, as in normal development. By contrast, excisions on the right side of the embryos resulted in three different types of impairment in the left-right placement of the adult rudiment in a stage-dependent manner. Generally, when the adult rudiment was definitively formed only on the right side of the larvae, no trace of basic development of the components of the adult rudiment was found on the left side, indicating that a right adult rudiment results from reversal of the initial left-right polarity but not from a later inhibitory effect on the development of an adult rudiment. Thus, we suggest that determination of the left-right placement of the adult rudiment depends on a process, which is directed by the right side, of polarity establishment during the gastrula and the prism stages; however, but commitment of the cell fate to initiate formation of the adult rudiment occurs later than the two-armed pluteus stage.     

Identification of adult midgut precursors in Drosophila

The adult Drosophila midgut is thought to arise from an endodermal rudiment specified during embryogenesis. Previous studies have reported the presence of individual cells termed adult midgut precursors (AMPs) as well as “midgut islands” or “islets” in embryonic and larval midgut tissue. Yet the precise relationship between progenitor cell populations and the cells of the adult midgut has not been characterized. Using a combination of molecular markers and directed cell lineage tracing, we provide evidence that the adult midgut arises from a molecularly distinct population of single cells present by the embryonic/larval transition. AMPs reside in a distinct basal position in the larval midgut where they remain through all subsequent larval and pupal stages and into adulthood. At least five phases of AMP activity are associated with the stepwise process of midgut formation. Our data shows that during larval stages AMPs give rise to the presumptive adult epithelium; during pupal stages AMPs contribute to the final size, cell number and form. Finally, a genetic screen has led to the identification of the Ecdysone receptor as a regulator of AMP expansion.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Larval and post-larval development of the branchiopod clam shrimp Cyclestheria hislopi (Baird, 1859) (Crustacea, Branchiopoda, Conchostraca, Spinicaudata)

The larval and post-larval development of Cyclestheria hislopi is examined by SEM. There are at least nine stages (excluding the adult) – six larval and three post-larval stages. The first four stages are passed within the egg-membrane. The larval and the post-larval phase are separated by a profound change in morphology that takes place between stages VI and VII. The larva shifts from a dorso-ventrally flattened 'larval' appearance up to stage VI to a laterally flattened, more 'adult' appearance from stage VII. New morphological data have been revealed by this study, including (1) a large and globular larval dorsal organ; (2) the carapace starts its development from the segments of the first and second maxillae; (3) the anterior ramus of the second antenna in adult Cyclestheria hislopi is the endopod, and the posterior ramus the exopod. Direct development of the brood in Cyclestheria hislopi – unique among conchostracans – is compared with that of the Cladocera. If Cyclestheria is the sister group to the Cladocera, as favoured in this work, the classical neoteny theory of the Cladocera must be reconsidered, as there is no particular similarity between any adults of the Cladocera and any of the larval stages of Cyclestheria . It is suggested that Cyclestheria displays the type of development present in a cladoceran ancestor. A comparison between Cyclestheria and the Upper Cambrian 'Orsten' fossil Rehbachiella kinnekullensis reveals a remarkable similarity in the endite morphology of the trunk limbs.     

Baboon/dSmad2 TGF-beta signaling is required during late larval stage for development of adult-specific neurons

The intermingling of larval functional neurons with adult-specific neurons during metamorphosis contributes to the development of the adult Drosophila brain. To better understand this process, we characterized the development of a dorsal cluster (DC) of Atonal-positive neurons that are born at early larval stages but do not undergo extensive morphogenesis until pupal formation. We found that Baboon(Babo)/dSmad2-mediated TGF-beta signaling, known to be essential for remodeling of larval functional neurons, is also indispensable for proper morphogenesis of these adult-specific neurons. Mosaic analysis reveals slowed development of mutant DC neurons, as evidenced by delays in both neuronal morphogenesis and atonal expression. We observe similar phenomena in other adult-specific neurons. We further demonstrate that Babo/dSmad2 operates autonomously in individual neurons and specifically during the late larval stage. Our results suggest that Babo/dSmad2 signaling prior to metamorphosis may be widely required to prepare neurons for the dynamic environment present during metamorphosis.     

SEM studies on the early larval development of Triops cancriformis (Bosc)(Crustacea: Branchiopoda, Notostraca)

We investigated early larval development in the notostracan Triops cancriformis (Bosc, 1801–1802) raised from dried cysts under laboratory conditions. We document the five earliest stages using scanning electron microscopy. The stage I larva is a typical nauplius, lecithotropic and without trunk limbs. The stage II larva is feeding and has trunk limb precursors and a larger carapace. Stage III larvae have larger trunk limbs and a more adult shape. Stage IV larvae have well developed trunk limbs, and stage V larvae show atrophy of the antennae. We describe the ontogeny of selected features such as trunk limbs and carapace, discuss ontogeny and homologization of head appendages, follow the development of the feeding mechanism, and discuss trunk limb ontogeny.     

Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis

The origin of germ cells in the ascidian is still unknown. Previously, we cloned a vasa homologue (CiVH) of Ciona intestinalis from the cDNA library of ovarian tissue by polymerase chain reaction and showed that its expression was specific to germ cells in adult and juvenile gonads. In the present study, we prepared a monoclonal antibody against CiVH protein and traced the staining for this antibody from the middle tailbud stage to young adulthood. Results showed that positive cells are present in the endodermal strand in middle tailbud embryos and larvae. When the larval tail was absorbed into the trunk during metamorphosis, the CiVH-positive cells migrated from the debris of the tail into the developing gonad rudiment, and appeared to give rise to a primordial germ cell (PGC) in the young juvenile. The testis rudiment separated from the gonad rudiment, the remainder of which differentiated into the ovary. PGCs of the testis rudiment and the ovary rudiment differentiated into spermatogenic and oogenic cells, respectively. When the larval tail containing the antibody-positive cells was removed, the juveniles did not contain any CiVH-positive cells after metamorphosis, indicating that the PGCs in the juvenile originated from part of the larval tail. However, even in such juveniles, positive cells newly appeared in the gonad rudiment at a later stage. This observation suggests that a compensatory mechanism regulates germline formation in C. intestinalis.     

Studies on induction of polydactyly in rats with cytosine arabinoside.

The development of the retino-tectal projection in Rana pipiens has been studied by the intraocular injection of small amounts of [³H]proline at late embryonic and at several larval stages. After survival periods varying from 1–24 hr the distribution of the radioactively labeled proteins in the axons of the retinal ganglion cells was studied autoradiographically. It is evident from the appearance of labeled proteins in the optic nerve and chiasm at late embryonic and early larval stages that there is a rapid phase of axonal transport at these stages and that some fraction of the materials transported in this phase are distributed to the tips of the growing axons.The first retinal fibers reach the contralateral optic tectum at embryonic Stage 22; at this stage they are confined to the rostrolateral portion of the tectum where the first tectal neurons are generated. At successively later stages the fibers appear to grow across the surface of the tectum in a general rostrolateral to caudomedial direction, reaching the dorsal part of the mid-tectum at larval Stage II and the lateral part of its caudal third by Stage V. However, it is not until relatively late larval stages (XVIII) that the fibers reach the caudomedial region of the tectum, and it is only at the time of metamorphosis (Stage XXV) that the retinal projection appears to cover the entire tectum.     

孤雌生殖长角血蜱的哈氏器超微结构与发育

为阐明长角血蜱Haemaphysalis longicornis孤雌生殖种群的哈氏器结构及发育特征, 用扫描电镜对其各虫期哈氏器进行了观察, 分析了血餐对哈氏器发育的影响。结果表明： 该种群幼蜱、 若蜱和成蜱哈氏器形态结构基本相同, 均由前窝和后囊构成。幼蜱前窝感毛6根, 位于同一基盘; 若蜱和成蜱哈氏器相似, 前窝感毛7根, 其中1根孔毛位于外侧基盘, 另6根感毛位于内侧基盘。各虫期饱血后哈氏器大小均比饥饿状态下显著增大（P<0.05）。幼蜱前窝与后囊面积比值与若蜱相比无显著差异（P>0.05）, 若蜱前窝与后囊面积比值与成蜱相比差异显著（P<0.05）。各虫期哈氏器均在发育, 且血餐对哈氏器发育有重要影响。幼蜱至若蜱期哈氏器前窝与后囊的发育速度相似, 若蜱至成蜱期哈氏器前窝发育快于后囊。本研究结果在一定程度上揭示了孤雌生殖长角血蜱的哈氏器发育规律。     

A Novel Technique for Identifying the Instar of Field-Collected Insect Larvae

Many field studies of insects have focused on the adult stage alone, likely because immature stages are unknown in most insect species. Molecular species identification (e.g., DNA barcoding) has helped ascertain the immature stages of many insects, but larval developmental stages (instars) cannot be identified. The identification of the growth stages of collected individuals is indispensable from both ecological and taxonomic perspectives. Using a larval–adult body size relationship across species, I present a novel technique for identifying the instar of field-collected insect larvae that are identified by molecular species identification technique. This method is based on the assumption that classification functions derived from discriminant analyses, performed with larval instar as a response variable and adult and larval body sizes as explanatory variables, can be used to determine the instar of a given larval specimen that was not included in the original data set, even at the species level. This size relationship has been demonstrated in larval instars for many insects (Dyar’s rule), but no attempt has been made to include the adult stage. Analysis of a test data set derived from the beetle family Carabidae (Coleoptera) showed that classification functions obtained from data sets derived from related species had a correct classification rate of 81–100%. Given that no reliable method has been established to identify the instar of field-collected insect larvae, these values may have sufficient accuracy as an analytical method for field-collected samples. The chief advantage of this technique is that the instar can be identified even when only one specimen is available per species if classification functions are determined for groups to which the focal species belongs. Similar classification functions should be created for other insect groups. By using those functions together with molecular species identification, future studies could include larval stages as well as adults.     

Evolutionary and experimental change in egg volume, heterochrony of larval body and juvenile rudiment, and evolutionary reversibility in pluteus form

SUMMARY Heterochronic developmental plasticity of the juvenile rudiment and larval body of sea urchin larvae occurs in response to supply of food. Evolutionary increase in egg size can also be associated with earlier development of the juvenile rudiment. We examined effects of egg volume of feeding larvae on this heterochrony and other changes in larval form. (1) Evolutionary and experimental enlargements of egg volume did not accelerate formation of the rudiment relative to the larval body. Development of the larval body and juvenile rudiment was compared for the echinoids Strongylocentrotus purpuratus (with an egg of 78–82 μm) and Strongylocentrotus droebachiensis (with an egg of 150–160 μm diameter). Development of both larval body and rudiment were accelerated in S. droebachiensis relative to S. purpuratus but with greater acceleration of the larval body, so that the rudiment of S. droebachiensi s was initiated at a later larval stage even though at an earlier age. Also, experimentally doubling the egg volume of S. purpuratus did not accelerate development of the juvenile rudiment relative to the larval body. (2) Both species exhibited similar plasticity in timing of rudiment development in response to food supplies. (3) Doubling egg volume of S. purpuratus produced a larval form more similar to that of S. droebachiensis . This result mirrors previous experiments in which larvae from half embryos of S. droebachiensis were more similar to larvae of S. purpuratus . Many of the effects of egg volume on larval form are similar against either species' genetic background and are thus evolutionarily reversible effects on larval form.     

Reproductive cycle and releasing time for increase of resource of adult sea cucumber Apostichopus japonicus released to seed breeding grounds

The reproductive cycle and releasing time for effective increase of resource of adult sea cucumber (Apostichopus japonicus) have been studied in the west coast of Korea. Adult sea cucumbers collected in Seocheon-gun April 2013 for first release and in the uncontaminated Taean-gun area in the west coast of Korea. Divers monthly collected the specimens in the released area from December 2013 to November 2014 in order to investigate the reproductive cycle of A. japonicas in Taean-gun. Random specimens were dissected to examine the gonadal developmental stages and discharge rates of the guts and gonads. The reproductive cycle of A. japonicus in both sexes are classified into the following six successive stages in the Taean-gun: (1) Stage I (recovery stage from December to February), (2) Stage II (early growing stage from February to March), (3) Stage III (late growing stage from March to April), (4) Stage IV (mature stage from April to July), (5) Stage V (partly spawned stage in July), and (6) Stage VI (spent stage from August to November). The estivation period of this species is from July to October in the Taean-gun region while surface water temperature is approximately 20–25.4°C. Thus, the optimum period to easily harvest them is from October to November in the uncontaminated Taean-gun area. It is also the best releasing time because the recovery stage starts from December to February in the Taean-gun region.     

The larval development of an Australian limnadiid clam shrimp (Crustacea, Branchiopoda, Spinicaudata), and a comparison with other Limnadiidae

The adult morphology of the Australian Limnadopsis shows some remarkable differences to that of other Limnadiidae. These differences are not reflected in its larval development. In Limnadopsis parvispinus, larval development comprises six stages. In stages I and II only the three naupliar appendages are present: the antennule as a small bud, the biramous antenna as the main swimming organ, and the mandible. The antennal protopod bears two endites, the proximal naupliar process and a more distal endite. In stage III a bifid naupliar process (in earlier stages not bifid) and the first signs of the carapace and trunk limb anlagen (undifferentiated rudiments) appear. In stage IV the carapace anlagen become more pronounced. The number of trunk limb anlagens increases to five, and differentiation has commenced. In stage V the first five pairs of trunk limbs are differentiated to varying degrees. The anterior-most four pairs of trunk limbs are subdivided into five endites, a small endopod, an exopod and an epipod. The bivalved carapace covers the anterior-most limbs. In larval stage VI the carapace is larger and the trunk limbs are further differentiated. A general pattern in the sequence of larval stages is the increasing number of sensilla on the antennules. From the last larval to the first postlarval stage, a significant change in morphology takes place. The trunk limbs are now used for swimming. Typical larval organs are much smaller than in the last larval stage. A comparison with other representatives of the Limnadiidae shows a high degree of correspondence, with most differences explained by the heterochronous appearance of characters during development. Five to seven stages are described for all studied Limnadiidae, including one particular stage in which four fully developed setae, a bifid naupliar process and the first signs of carapace anlagen are present. These characters are found in stage III in L. parvispinus, Limnadia stanleyana, Eulimnadia texana, and Imnadia yeyetta but in stage IV in E. braueriana and L. lenticularis. Based on a comparison of the larval stages of six limnadiid and one cyzicid species, we conclude that at least six naupliar stages belong to the limnadiid ground pattern.     

Embryonic Appearance of α, β, and γ Crystallins in the Periodic Albinism (ap) Mutant of Xenopus laevis

The appearance of the crystallins during lens development in the periodic albinism (a^p/a^p) mutant of Xenopus laevis has been studied. Using antibodies specific for total crystallins, α+β crystallins, and γ crystallins in the immunofluorescence technique, the first positive reaction for all could be demonstrated in the Nieuwkoop-Faber Stage 31 lens rudiment. The antibody to α+β crystallins exhibited differences in intensity from cell to cell in the early rudiment, while the reaction to the other antibodies was uniform throughout the rudiment. As lens differentiation progressed, immunofluorescence was restricted in all cases to the lens fiber area, up to and including Nieuwkoop-Faber Stage 45. The lens epithelium of the one-year-old adult a^p/a^p was positive, however, for total lens crystallins.
These results are at variance with earlier studies on lens development and the crystallins in wild-type (+/+) X. laevis , where a positive reaction for y and total crystallins could be detected earlier, and in the lens epithelium of Nieuwkoop-Faber Stage 41 embryos for total lens crystallins. That this divergence in the mutant is due to a pleiotropic effect or directly to the inductive failure of the endomesoderm to initiate melanogenesis, is discussed.     

Early events in sea urchin metamorphosis,description and analysis

The larval epithelium of the sea urchin, Lytechinus pictus, consists of squamous cells and bands of columnar epithelial cells bearing cilia. During metamorphosis this tissue undergoes a series of rapid, complex changes. Through the scanning and transmission electron microscope, we describe and analyse these changes. The changes can be divided into three steps. (1) The larval arms bend away from the left side of the larva, exposing the urchin rudiment. Cells which are identical to smooth muscle cells are in a position to bring about this bending. (2) The squamous epithelial cells assume a cuboidal shape. This change in shape results in the collapse of the larval epithelium onto the presumptive aboral surface. These cells possess a subapical band of microfilaments. The cellular shape change but not the bending of the arms is reversibly inhibited by Cytochalasin B. These observations suggest a mechanism for this change. (3) The former lining of the vestibule of the urchin rudiment comes to lie over the collapsed larval tissue and forms the adult epithelium. At this point, after only one hour, the larva has assumed the external shape of an adult sea urchin.     

Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development

Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat α-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II–III tadpoles and in successive larval stages. Soon after hatching, stages II–III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI–XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone. Received: 28 July 1997 / Accepted: 8 December 1997