Ultrastructure of the developing thymus of the leopard frog (Rana pipiens)

Summary A study of the ultrastructure of the developing thymus of the leopard frog (Rana pipiens) revealed that the thymus had undergone all of the major changes which would persist through larval life and metamorphosis by the time that the animals had reached larval stage IV of Taylor and Kollros (1946). These changes included development of an outer, lymphoid cortical region and an inner, essentially nonlymphoid medulla; mitotic activity among lymphoid cell precursors and the formation of the first small lymphocytes; development of complex cysts containing PAS-positive material and the appearance of other signs of secretory activity among epithelial cells of the medulla; and differentiation of large myoid cells containing bundles of striated muscle fibrils. The changes are particularly noteworthy because they first appear during a period in which the animals are known to be developing the capacity to respond immunologically to allografts.Supported by a grant from the National Institutes of Health number GM-11782 to E.P.V.     

Maturation and degeneration of the fat body in the Drosophila larva and pupa as revealed by morphometric analysis

Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.     

Changes in growth performance and proximate composition in Japanese flounder during metamorphosis

Morphological changes are described in Japanese flounder Paralichthys olivaceus larvae and juveniles with emphasis on growth during the period of metamorphosis. Ontogenetic changes in condition factor and lipid, glycogen and protein levels were also analysed to determine the utilization of stored energy. Fish grew from 6·6 to 20·3 mm L _T(1·15 to 84·4 mg in mass, M ) during the period from 11 to 40 days after hatching (DAH) at 19·3° C. Per cent specific growth rate per day (% G day⁻¹) for wet mass was lowest during the metamorphic phase (21–30 DAH) compared to pre‐(11–20 DAH) and post‐metamorphic (31–40 DAH) phases. When L _T and M were expressed as     , the b value was highest during the pre‐metamorphic phase and lowest during the metamorphic phase. These findings indicate that the developmental changes that occur during metamorphosis of Japanese flounder are closely related to the growth pattern. Moisture, lipid and glycogen contents were also at the lowest level during metamorphosis compared to pre‐ and post‐metamorphosis, which suggest that Japanese flounder use their energy reserves to accomplish metamorphosis due to an apparent decline in feeding during this period.     

Intraspecific developmental variation in the life cycle of the Andean Treefrog (Boana riojana): A temporal analysis

Intraspecific variation during the anuran larval period has been analyzed mainly in relation to the timing of metamorphosis and body size at metamorphosis. However, other traits may vary as well. We examined two developmental series of Boana riojana from the same population in two consecutive years and describe intraspecific variation in larvae of this species. We discuss how variation, if present, may influence its life cycle. We found that both larval series differed in the larval period length, one twice as long as the other. This variation primarily depended on when breeding occurred, metamorphosis was achieved during late spring in both generations and at similar sizes, and only the rate of larval development during premetamorphosis varied extensively between years. This is consistent with thyroid gland activity because when it became active the developmental trajectory became more canalized. No variation of staging sequence occurred in relation to the different durations of the larval period. However, in the long-lasting series we found two different morphs. Also, integument, thyroid gland, skeleton, and testis differentiation events occurred at the same developing stages. In contrast, ovarian differentiation proceeded at the same absolute age in both series. Sexual dimorphism becomes evident within the year after metamorphosis. The intraspecific heterochrony that we describe for the larval development of B. riojana does not lead to phenotypic variation at the end of metamorphosis. We discuss the importance of analyzing growth and development independently. Each proceeds differently in time, but with an interdependence at some point, because size and shape do not vary at the end of metamorphosis.     

Models of metamorphic timing: an experimental evaluation with the pond-dwelling salamander Hemidactylium scutatum (Caudata: Plethodontidae)

Amphibian larvae vary tremendously in size at metamorphosis and length of larval period. We raised pond-dwelling four-toed salamander (Hemidactylium scutatum) larvae to test two models that predict a larva’s age and size at metamorphosis. The Wilbur-Collins model proposes that the developmental rate of a larva responds to changes in growth rate in an adaptive manner throughout the larval period, and that metamorphosis can be initiated after a minimum size has been reached. The Leips-Travis or fixed-rate model states that developmental rate is set early in the larval period, perhaps by early growth rate or food availability and their positive correlation with developmental rate, and that changes in growth rate during the larval period affect size at metamorphosis, but have no effect on the age of an individual at metamorphosis. A modified version of the Wilbur-Collins model suggests that a larva’s developmental rate becomes fixed about two-thirds of the way through the larval period, with changes in growth rate after that point only affecting size at metamorphosis. Larvae were raised on eight different feeding regimes which created two constant and six variable growth histories. Growth history did significantly affect size at metamorphosis. However, an a posteriori statistical test revealed a group of seven and an overlapping group of six treatments with indistinguishable lengths of larval period, indicating a general picture of a fixed developmental rate regardless of growth history. This result is unique among similar studies on invertebrates, fish, and frogs. There was no association between early growth or food level and development rates. Neither the Wilbur-Collins nor the Leips-Travis fixed-rate models were supported. The invariable developmental rate of Hemidactylium and recent osteological evidence from the literature suggest that larvae begin the process of metamorphosis as soon as they hatch, probably a trait selected for by strong predation pressure in the aquatic environment. A variety of different approaches (ecological, developmental, phylogenetic) are necessary to fully evaluate the adaptive nature of the timing of transitions between life cycle stages. Received: 3 June 1999 / Accepted: 18 March 2000     

Effect of changes in resource level on age and size at metamorphosis in Hyla squirella

Recent experiments suggest that timing of metamorphosis is fixed during development in some anurans, insects, and freshwater invertebrates. Yet, these experiments do not exclude a growth rate optimization model for the timing of metamorphosis. I manipulated food resources available to larvae of squirrel treefrogs (Hyla squirella) to determine if there is a loss of plasticity in duration of larval period during development and to critically test growth rate models for the timing of metamorphosis. Size-specific resource levels for individual tadpoles were switched from low to high or high to low at three developmental stages spaced throughout larval development. The effects of changes in resource availability on larval period and mass at metamorphosis were measured. Switching food levels after late limb bud development did not significantly affect larval period in comparison to constant food level treatments. Therefore, developmental rate in H. squirella is better described by a fixed developmental rate model, rather than a growth rate optimization model. The timing of fixation of developmental rate in H. squirella is similar to that found in other anuran species, suggesting a taxonomically widespread developmental constraint on the plasticity of larval period duration. Mass at metamorphosis was not significantly affected by the timing of changes in food levels; the amount of food available later in development determined the size at metamorphosis. Larval period and mass at metamorphosis were negatively correlated in only one of two experiments, which contrasts with the common assumption of a phenotypic trade-off between decreased larval period and increased mass at metamorphosis. Received: 19 August 1996 / Accepted: 20 June 1997     

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.     

家蚕蛹变态期丝腺组织的退化与细胞凋亡特征

利用形态学观察方法、分子生物学检测方法以及20-羟基蜕皮酮(20-hydroxyecdysone)和放线菌酮(cycloheximide)体外培养方法, 研究了家蚕Bombyx mori 蛹变态期丝腺组织的退化与细胞凋亡特征。显微镜的观察显示家蚕丝腺的逐渐退化发生在吐丝期间。DNA梯度电泳的分析表明程序性细胞死亡(programmed cell death)可能伴随发生在丝腺的退化过程中。在离体培养条件下, 用20-羟基蜕皮酮处理5龄第6天幼虫的丝腺, 导致的细胞凋亡提前于对照, 提示在进入蛹变态期前, 20-羟基蜕皮酮提早激发了介导家蚕丝腺细胞凋亡与水解机制的遗传调控级联系统。上述结果表明, 20-羟基蜕皮酮能够诱导家蚕丝腺组织在蛹变态期发生程序性细胞死亡。     

Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus

The developing thymus in Xenopus was examined at four different levels: 1) precursor immigration of cytogenetically distinct embryonic stem cells; 2) waves of colonization during tadpole life and metamorphosis; 3) inter-thymic exchange of cells between separate lobes; and 4) development of cortical and medullary thymocytes. Based on the flow cytometric analysis of cytogenetically distinct thymocytes, there were at least two periods of stem cell immigration into the thymus, one during early larval life and the second before or during metamorphosis. Within the thymus, cohorts of cells derived from the first wave of immigration expanded at different times. The initial expansion occurred before 35 days of development. Cells involved in the second period of expansion were also derived from the initial immigrants, expanded after 35 days, and resulted in a turnover of thymocytes during the larval period. Precursor cells entering the thymus during metamorphosis expanded and resulted in an additional replacement of thymocytes. Cortical and medullary thymocytes were isolated from animals that received embryonic stem cell grafts. No differences in the presence or absence, or in the percentages, of donor thymocytes in these different fractions were observed. When limiting numbers of stem cells were transplanted, several cases of asymmetrical thymic lobe colonization were observed. These data suggested that an inter-thymic exchange of cells did not occur during larval life.     

Differentiations of 5-HT and GAS cells in the digestive canals of Rana chensinensis tadpoles

In the current study, 5-nydroxytryptamine(5-HT) and gastrin(GAS) cells in the digestive canals of Rana chensinensis tadpoles at different developmental stages were investigated by immunohistochemistry. Results showed that the 5-HT cells were only detected in the duodenum before metamorphosis began, and were extensively distributed in the stomach, duodenum, small intestine, and rectum thereafter, with the highest counts found in the duodenum and rectum when metamorphosis was completed. The GAS cells were only distributed in the stomach and duodenum, and only rarely detected in the duodenum before metamorphosis began, but increased in the stomach during metamorphosis and showed zonal distribution in the gastric mucosa when metamorphosis was completed. Metamorphosis is a critical period for amphibians, during which structural and functional physiological adaptations are required to transition from aquatic to terrestrial environments. During metamorphosis, the differentiations of 5-HT cells in the gastrointestinal canals of tadpoles could facilitate mucus secretion regulation, improve digestive canal lubrication, and help watershortage food digestion in terrestrial environments. Conversely, GAS cell differentiations during metamorphosis might contribute to the digestive and absorptive function transition from herbivore to omnivore.     

Expression,subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval?pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real‐time quantitative polymerase chain reaction in different tissues during the larval–pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein?protein interaction were explored by transient expression and far‐western blotting, respectively. All the four genes were significantly up‐regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20‐hydroxyecdysone (20E) induction except for USP2, and USP1 could be up‐regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein?protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval?pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.     

Nutritional condition of metamorphosing sole: spatial and temporal analyses

Sole Solea solea larvae were collected during five cruises in the Bay of Biscay in 1992 and 1993. The larval growth rate, triacylgycerol (TAG) and sterol (ST) content, and the width of the marginal otolith increments were determined on the same individual larva as indicators of nutritional condition. The TAG content increased during the ontogenetic larval development up to an elevated value corresponding to the end of metamorphosis. During metamorphosis, sole larvae continued to accumulate TAG and to increase in length. Seasonal and yearly variations of growth and nutritional condition of sole larvae at the onset of metamorphosis were more important than spatial variations. Analysis of larval growth showed that, during each cruise, the faster growing larvae were generally those with the highest TAG content (and TAG : ST ratio). In 1993, larvae began to metamorphose earlier and their growth (both larval growth and recent growth) were significantly higher than in 1992. Their relatively low nutritional condition, however, seemed to be the result of a low accumulation of TAG. Sole metamorphosis occurs without growth arrest and the majority of the larvae during this phase are judged to be in good nutritional condition (TAG : ST ratio > 1). In the Bay of Biscay, metamorphosis does not seem to be a critical period in the life cycle of the sole. It is suggested that larvae originating from different cohorts probably have the same chance of survival during or just after metamorphosis when they enter the benthic environment.     

Metamorphosis of the mountain brook lamprey Ichthyomyzon greeleyi

Synopsis Metamorphosis of the nonparasitic mountain brook lamprey, I. greeleyi, is described within 7 phases based on external morphological changes. The commencement of metamorphosis in early August is consistent with the pattern for northern hemisphere lampreys which display an inverse relationship with latitude. The sequence of external changes including the development of eyes, oral disc, teeth, tongue, oral fimbriae and lateral line its consistent among individuals of I. greeleyi and lampreys in general. Some of the changes realized during metamorphosis of I. greeleyi are important to the adult period while others reflect their presumed parasitic ancestry. Energy reserves by nonfeeding I. greeleyi during the 104–140 days required to complete metamorphosis are replaced by water so that total length and body weight do not change significantly during this time.     

How does fish metamorphosis affect aromatic amino acid metabolism?

Aromatic amino acids (AAs, phenylalanine and tyrosine) may be specifically required during fish metamorphosis, since they are the precursors of thyroid hormones which regulate this process. This project attempted to evaluate aromatic AA metabolism during the ontogenesis of fish species with a marked (Senegalese sole; Solea senegalensis) and a less accentuated metamorphosis (gilthead seabream; Sparus aurata). Fish were tube-fed with three l-[U-¹⁴C] AA solutions at pre-metamorphic, metamorphic and post-metamorphic stages of development: controlled AA mixture (Mix), phenylalanine (Phe) and tyrosine (Tyr). Results showed a preferential aromatic AA retention during the metamorphosis of Senegalese sole, rather than in gilthead seabream. Senegalese sole’s highly accentuated metamorphosis seems to increase aromatic AA physiological requirements, possibly for thyroid hormone production. Thus, Senegalese sole seems to be especially susceptible to dietary aromatic AA deficiencies during the metamorphosis period, and these findings may be important for physiologists, fish nutritionists and the flatfish aquaculture industry.     

Developmental profile of annexin IX and its possible role in programmed cell death of the Bombyx mori anterior silk gland

During pupal metamorphosis, the anterior silk gland (ASG) of the silkworm, Bombyx mori, undergoes programmed cell death (PCD), which is triggered by 20-hydroxyecdysone (20E). Annexin IX (ANX IX) has been identified as a 20E-inducible gene in dying ASGs, and we show here that its expression is down-regulated in tissues destined to die but not in tissues that survive pupal metamorphosis. ANX IX expression was high in the ASGs during the feeding period, when the ecdysteroid titer was low, and decreased in response to the rising ecdysteroid titer that triggered pupal metamorphosis. Before gut purge, in vitro exposure of the ASGs to 20E levels corresponding to the ecdysteroid concentration present at the time of gut purge caused a decrease in ANX IX messenger RNA levels. Expression profiles of EcR and USP, and the 20E concentration-responses of these genes, indicate the importance of the relative abundance of EcR-A and EcR-B1 isoforms in ANX IX regulation. These results suggest an involvement of ANX IX in the determination of PCD timing by delaying or suppressing the response to the increase in hemolymph ecdysteroid concentration during the prepupal period.     

Preventing ascidian fouling in aquaculture: screening selected allelochemicals for anti-metamorphic properties in ascidian larvae

Fouling by ascidians causes major stock losses and disrupts production in marine aquaculture, especially bivalve aquaculture. Currently, no cost effective solution exists despite the testing of many prospective control techniques. This study examined a range of allelochemicals suspected to inhibit metamorphosis in marine larvae. Five allelochemicals were screened in a larval metamorphosis bioassay using Ciona savignyi Herdman to determine their potential as a remedy for ascidian fouling in bivalve aquaculture. Three of the compounds tested inhibited ascidian larval metamorphosis and increased mortality at low concentrations. These were radicicol (99% inhibition of metamorphosis [IC??], 0.8 μg ml?1; 99% lethal concentration [LC??], 2.5 μg ml?1; 99% lethal time [LT??], 7.0 days), polygodial (IC??, 0.003 μg ml?1; LC??, 0.9 μg ml?1; LT??, 6.4 days), and ubiquinone-10 (IC??, 3.2 μg cm?2; LC??, 14.5 μg cm?2; LT??, 5.6 days; expressed as μg cm?2 due to insolubility in water and ethanol). While spermidine significantly affected metamorphosis and mortality of C. savignyi, the effect was insufficient to achieve inhibition in 99% of larvae over the 7-day timeframe of the assay. Muscimol did not affect metamorphosis or mortality at the concentrations tested. The present study demonstrates that radicicol, polygodial and ubiquinone-10 have potential for future development in antifoulant formulations targeted towards the inhibition of metamorphosis in ascidian larvae, while spermidine and muscimol appear unsuitable.     

The developing visual system and metamorphosis in the lamprey

Metamorphosis of the sea lamprey, Petromyzon marinus, is a true metamorphosis. The larval lamprey is a filter-feeder who dwells in the silt of freshwater streams and the adult is an active predator found in large lakes or the sea. The transformation usually occurs in the fifth or sixth year of life. Enlargement of the eye has been long accepted as a distinctive indication of metamorphosis in the sea lamprey, but it had been thought that this was because eye development in the larva was arrested after the formation of only the small central region. Recent studies indicate that all of the retina begins its development in the larva and that ganglion, amacrine, and horizontal cells differentiate in the peripheral retina of the larva. Retinal development is arrested during the premetamorphic period, to be resumed during metamorphosis. Metamorphic contributions include the differentiation of photoreceptor and bipolar cells. With the early appearance of ganglion cells, retinal pathways to the thalamus and tectum are established in larvae, as is a centripetal pathway. Tectal development spans the larval period but a spurt in tectal growth and differentiation is correlated with the completion of the retinal circuitry late in metamorphosis. The metamorphic changes in retina and tectum complete the functional development of the visual system and provide for the adult lamprey's predatory and reproductive behavior.     

Gene expression during metamorphosis: An ideal model for post-embryonic development

The precocious induction in vivo and in culture of insect and amphibian metamorphosis by exogenous ecdysteroids and thyroid hormones, and its retardation or inhibition by juvenile hormone and prolactin, respectively, has allowed the analysis of such diverse processes of post-embryonic development as morphogenesis, tissue remodelling, functional reorganization, and programmed cell death. Metamorphosis in vertebrates also shares many similarities with mammalian development in the late foetal and perinatal period. This review describes the regulation of expression of some of the ‘adult’ gene products during metamorphosis in invertebrates and vertebrates. Recent studies on metamorphosis have revealed the important role played by auto-induction of hormone receptor genes, based on which a model will be presented to explain the activation of ‘downstream’ genes which give rise to the adult phenotype. It will also be argued that metamorphosis is an ideal model for analyzing some of the major mechanisms governing post-embryonic development.     

Immunological studies of insect metamorphosis. II. The role of a sex-limited blood protein in egg formation by the Cecropia silkworm

1. In the pupal stage of the cecropia silkworm, antigen 7, a protein with the solubility characteristics of an albumin, is present in female blood in approximately a thousand times higher concentration than in the blood of males. Antigen 7 is undetectable in the blood of larvae of either sex. It first appears in the blood after the larva has spun its cocoon, and is present throughout all subsequent stages of metamorphosis. Late in the pupal-adult transformation, when the eggs are produced, the concentration of antigen 7 in female blood decreases significantly. 2. An antigen which could not be distinguished from antigen 7 immunologically is present in solution in the yolk of unfertilized eggs. 3. In females which, by ovariectomy, were prevented from forming eggs, the concentration of antigen 7 in the blood increased during the usual period of egg formation rather than undergoing the normal decrease. Ovaries transferred to the hemocoel of males produced eggs but were unable to incorporate antigen 7 in the yolk unless a detectable amount of the protein was present in the blood. The ovaries of polyphemus females which had been transfused with cecropia blood incorporated cecropia antigen 7 into the eggs they produced. These lines of evidence indicate that antigen 7 is secreted into the blood by some tissue other than the ovaries, and that it is subsequently drawn from the blood and deposited in the yolk. 4. The concentration of antigen 7 in the clear, liquid fraction of the yolk is four times higher than the maximum concentration attained in the blood during metamorphosis, and twenty times higher than that of the blood at the conclusion of egg formation. The protein thus appears to be transferred from blood to yolk against a concentration gradient.     

Survival, development, and gonadal differentiation in Rana dalmatina chronically exposed to chlorpyrifos

Chlorpyrifos is an organophosphate pesticide among the most widely used in the world, which is suspected to be an endocrine-disrupting compound. To determine the capacity of chlorpyrifos to affect gonadal differentiation in Rana dalmatina, tadpoles were exposed to this pesticide during larval development until completion of metamorphosis at ecologically relevant concentrations (0.025 and 0.05 mg/L). No effects of chlorpyrifos exposure on survival, development, or metamorphosis were observed. After a 1 month metamorphosis, the gonadal phenotype was determined by gross morphology and histological examination. Morphological and histological analysis revealed normal ovaries or testes in froglets belonging to control group, whereas testes from several froglets exposed to chlorpyrifos were interspersed with testicular oocytes in histological sections. Chlorpyrifos exposure during the entire larval period did not affect sex ratio, but reduced the percentage of males with histologically normal testes. The findings suggest that chlorpyrifos exposure has significant effects on gonadal differentiation in some animals by inducing an intersex condition and alterations to testicular morphology, and that R. dalmatina is sensitive to endocrine disruption. Thereby, this study provides evidence that the ecologically relevant concentrations of chlorpyrifos, although not adversely affect the survival, development, or metamorphosis, may interfere with sex differentiation and reproductive development of R. dalmatina via endocrine-disrupting mechanisms.