Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly,Calliphora vicina,with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Metamorphosis of cyclorrhaphous flies takes place inside a barrel‐like puparium, formed by the shrinking, hardening and darkening of the third‐instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro‐computed tomography (micro‐CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau‐Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra‐puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X‐ray images and micro‐CT virtual sections. The origin of this gas bubble has been largely unknown, but micro‐CT virtual sections show that it is connected to one of the main tracheal trunks. Micro‐CT virtual sections also provided enough resolution for determining the completion of the larval‐pupal and pupal‐adult apolyses, thus enabling an accurate timing of the different intra‐puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra‐puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre‐helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.     

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the “hour‐glass” model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well‐defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within‐group variance and the largest disparity level (between‐group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.     

Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second

Non‐invasive biological imaging is crucial for understanding in vivo structure and function. Optical coherence tomography (OCT) and reflectance confocal microscopy are two of the most widely used optical modalities for exogenous contrast‐free, high‐resolution, three‐dimensional imaging in non‐fluorescent scattering tissues. However, sample motion remains a critical barrier to raster‐scanned acquisition and reconstruction of wide‐field anatomically accurate volumetric datasets. We introduce spectrally encoded coherence tomography and reflectometry (SECTR), a high‐speed, multimodality system for simultaneous OCT and spectrally encoded reflectance (SER) imaging. SECTR utilizes a robust system design consisting of shared optical relays, scanning mirrors, swept laser and digitizer to achieve the fastest reported in vivo multimodal imaging rate of 2 gigapixels per second. Our optical design and acquisition scheme enable spatiotemporally co‐registered acquisition of OCT cross‐sections simultaneously with en face SER images for multivolumetric mosaicking. Complementary axial and lateral translation and rotation are extracted from OCT and SER data, respectively, for full volumetric estimation of sample motion with micron spatial and millisecond temporal resolution.      

Three‐dimensional rendering of otolith growth using phase contrast synchrotron tomography

A three‐dimensional computer reconstruction of a plaice Pleuronectes platessa otolith is presented from data acquired by the Diamond Light synchrotron, beamline I12, X‐ray source, a high energy (53–150 keV) source particularly well suited to the study of dense objects. The data allowed non‐destructive rendering of otolith structure, and for the first time allows otolith annuli (internal ring structures) to be analysed in X‐ray tomographic images.     

Comparison of pulsed photothermal radiometry,optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non‐invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three‐dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 μm–1.7 mm and 120–910 μm respectively) but poor axial resolution (75 and 120 μm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14–450 μm) but highest axial resolution (14 μm). The Monte Carlo simulation and three‐dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (<$>><$>400 μm), while OCT is better to measure thin melanoma lesions (<$><<$>400 μm). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Challenges of metamorphosis in invertebrate hosts: maintaining parasite resistance across life‐history stages

1. Insects lack the acquired immune system of vertebrates, but there is some evidence that insect immunity can be primed against an encountered pathogen to mitigate the intensity of future infections within a life stage. 2. Many invertebrates have multiple life‐history stages separated by complete metamorphosis, but different life stages can often be infected by the same pathogens, and the potential loss of immune priming during metamorphosis could therefore have detrimental effects on the host. Evidence that invertebrate immune priming can persist through metamorphosis is still missing, and consequently it is unclear how host–parasite interactions change across different life‐history stages in the context of infection history. 3. By experimentally manipulating the infection history of the flour beetle Tribolium confusum, we show that intestinal gregarine parasite infections during the larval stage reduced parasite load in adults, demonstrating that a host‐controlled mechanism for parasite resistance can persist through complete metamorphosis in insects. 4. Infections reduced larval developmental rates and increased host mortality but only during the crucial metamorphic stage, indicating that parasites impact multiple life stages. In general, our results demonstrate that invertebrates can show surprisingly robust immune priming despite dramatic physiological changes and protect hosts across completely different life‐history stages.     

Short‐term larval food stress and associated compensatory growth reduce adult immune function in a damselfly

Abstract 1. In animals with a complex life cycle, larval stressors may carry over to the adult stage. Carry‐over effects not mediated through age and size at metamorphosis have rarely been studied. The present study focuses on the poorly documented immune costs of short‐term food stress both in the larval stage and after metamorphosis in the adult stage. 2. The present study quantified immune function [number of haemocytes, activity of prophenoloxidase (proPO) and phenoloxidase (PO)] in an experiment where larvae of the damselfly Lestes viridis were exposed to a transient starvation period. 3. Directly after starvation, immune variables were reduced in starved larvae. Levels of proPO and PO remained low after starvation, even after metamorphosis. In contrast, haemocyte numbers were fully compensated by the end of the larval stage, yet were lower in previously starved animals after metamorphosis. This can be explained as a cost of the observed compensatory growth after starvation. Focusing only on potential costs of larval stressors within the larval stage may therefore be misleading. 4. The here‐identified immunological cost in the adult stage of larval short‐term food stress and associated compensatory growth strongly indicates that physiological costs may explain hidden carry‐over effects bridging metamorphosis. This adds to the increasing awareness that the larval and adult stages in animals with a complex life cycle should be jointly studied, as trade‐offs may span metamorphosis.     

Development of the olfactory and vomeronasal organs in Discoglossus pictus (Discoglossidae,Anura)

Using histological techniques and computer‐aided three‐dimensional reconstructions of histological serial sections, we studied the development of the olfactory and vomeronasal organs in the discoglossid frog Discoglossus pictus. The olfactory epithelium in larval D. pictus represents one continuous unit of tissue not divided into two separate portions. However, a small pouch of olfactory epithelium (the “ventromedial diverticulum”) is embedded into the roof of the buccal cavity, anteromedial to the internal naris. The lateral appendix is present in D. pictus through the entire larval period and disappears during the onset of metamorphosis. The disappearance of the lateral appendix at this time suggests that it is a typical larval organ related to aquatic life. The vomeronasal organ develops during hindlimb development, which is comparatively late for anurans. The development of the vomeronasal organ in D. pictus follows the same general developmental pattern recognized for neobatrachians. As with most anurans, the vomeronasal glands appear later than the vomeronasal organ. After metamorphosis, the olfactory organ of adult D. pictus is composed of a series of three interconnected chambers: the cavum principale, cavum medium, and cavum inferius. We suggest that the ventromedial diverticulum at the anterior border of the internal naris of larval D. pictus might be homologous with the ventral olfactory epithelium of bufonids and with the similar diverticulum of Alytes. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

The differential impact of a native and a non‐native ragwort species (Senecioneae) on the first and second trophic level of the rhizosphere food web

Whereas the impact of exotic plant species on above‐ground biota is relatively well‐documented, far less is known about the effects of non‐indigenous plants on the first and second trophic level of the rhizosphere food web. Here, rhizosphere communities of the invasive narrow‐leaved ragwort Senecio inaequidens and the native tansy ragwort Jacobaea vulgaris, co‐occurring in three semi‐natural habitats are compared. For both species, two life stages were taken into consideration. Quantitative PCR assays for the analyses of bacterial and fungal communities at a high taxonomic level were optimized, and it was investigated whether changes in the primary decomposer community were translated in alterations in bacterivorous and fungivorous nematode communities. In contrast to J. vulgaris, small but significant reductions were observed for Actinobacteria and Bacteroidetes (both p < 0.05) in case of the invasive S. inaequidens. More pronounced changes were detected for the overall nematode community density, and, more specifically, for the bacterivorous genus Anaplectus and the family Monhysteridae (both p < 0.05), as well as the necromenic Pristionchus (p < 0.001). At high taxonomic level, no differences were observed in fungal rhizosphere communities between native and non‐native ragwort species. The impact of plant developmental stages on rhizosphere biota was prominent. The overall bacterial and fungal biomasses, as well as a remarkably consistent set of constituents (Actinobacteria, α‐ and β‐Proteobacteria and Bacteroidetes) were negatively affected by plant stage for both ragwort species. Although later developmental stages of plants generally coincided with lower levels for individual nematode taxa, densities of the fungivorous genera Diphtherophora and Tylolaimophorus remain unaltered. Hence, even at a high taxonomic level, differential effects of native and non‐native ragwort could be pinpointed. However, plant developmental stage has a more prominent impact and this impact was similar in nature for both native and non‐native ragwort species.     

Estimation of gonad volume, fecundity, and reproductive stage of shovelnose sturgeon using sonography and endoscopy with application to the endangered pallid sturgeon

Most species of sturgeon are declining in the Mississippi River Basin of North America including pallid (Scaphirhynchus albus F. and R.) and shovelnose sturgeons (S. platorynchus R.). Understanding the reproductive cycle of sturgeon in the Mississippi River Basin is important in evaluating the status and viability of sturgeon populations. We used non‐invasive, non‐lethal methods for examining internal reproductive organs of shovelnose and pallid sturgeon. We used an ultrasound to measure egg diameter, fecundity, and gonad volume; endoscope was used to visually examine the gonad. We found the ultrasound to accurately measure the gonad volume, but it underestimated egg diameter by 52%. After correcting for the measurement error, the ultrasound accurately measured the gonad volume but it was higher than the true gonad volume for stages I and II. The ultrasound underestimated the fecundity of shovelnose sturgeon by 5%. The ultrasound fecundity was lower than the true fecundity for stage III and during August. Using the endoscope, we viewed seven different egg color categories. Using a model selection procedure, the presence of four egg categories correctly predicted the reproductive stage ± one reproductive stage of shovelnose sturgeon 95% of the time. For pallid sturgeon, the ultrasound overestimated the density of eggs by 49% and the endoscope was able to view eggs in 50% of the pallid sturgeon. Individually, the ultrasound and endoscope can be used to assess certain reproductive characteristics in sturgeon. The use of both methods at the same time can be complementary depending on the parameter measured. These methods can be used to track gonad characteristics, including measuring Gonadosomatic Index in individuals and/or populations through time, which can be very useful when associating gonad characteristics with environmental spawning triggers or with repeated examinations of individual fish throughout the reproductive cycle.     

Life‐history traits of Tubastraea coccinea: Reproduction,development, and larval competence

The sun coral Tubastraea coccinea Lesson, 1829 (Dendrophylliidae) is a widely distributed shallow‐water scleractinian that has extended its range to non‐native habitats in recent decades. With its rapid spread, this coral is now one of the main invasive species in Brazil. Its high invasive capability is related to opportunistic characteristics, including several reproductive strategies that have allowed it to disperse rapidly and widely. To better understand the reproductive biology of T. coccinea and aid in developing management strategies for invaded areas, we investigated aspects of its reproductive performance and life cycle, including the effects of colony size, seawater temperature and salinity, and lunar periodicity on offspring production and larval metamorphosis competence. A total of 18,139 offspring were released in different developmental stages, mainly from the larger colonies, which also produced larvae with longer competence periods. The main reproductive peak occurred during the First Quarter and New Moon phases and was highest in water temperatures around 26°C. Together, these results help to explain the rapid expansion of T. coccinea into non‐native habitats such as the Caribbean and southwestern Atlantic, and will inform actions of the recent Brazilian National Plan for the prevention, eradication, control, and monitoring of sun corals.     

Plasticity in newt metamorphosis: the effect of predation at embryonic and larval stages

1. Some organisms under variable predator pressure show induced antipredator defences, whose development incurs costs and may be associated with changes to later performance. This may be of especial relevance to animals with complex life histories involving metamorphosis. 2. This study examines the effect of predation environment, experienced both during embryonic and larval stages, on palmate newt (Triturus helveticus) metamorphosis. Newt eggs were raised until hatching with or without exposure to chemical cues from brown trout (Salmo trutta), and larval development was monitored in the presence or absence of the cues. 3. Exposure to predator cues during the embryonic stage resulted in higher growth rates at the larval stage, reduced time to metamorphosis and size at metamorphosis. Metamorphs also had narrower heads and shorter forelimbs than those from predator‐free treatments. In contrast, exposure to predator cues during the larval stage did not affect metamorph characteristics. 4. These results indicate that developing embryos are sensitive to predator chemical cues and that the responses can extend to later stages. Reversion of induced defences when predation risk ceased was not detected. We discuss the possible adaptive significance of these responses.     

Stage‐specific regulation of Gremlin1 on the differentiation and expansion of human urinary induced pluripotent stem cells into endothelial progenitors

Human urinary induced pluripotent stem cells (hUiPSCs) produced from exfoliated renal epithelial cells present in urine may provide a non‐invasive source of endothelial progenitors for the treatment of ischaemic diseases. However, their differentiation efficiency is unsatisfactory and the underlying mechanism of differentiation is still unknown. Gremlin1 (GREM1) is an important gene involved in cell differentiation. Therefore, we tried to elucidate the roles of GREM1 during the differentiation and expansion of endothelial progenitors. HUiPSCs were induced into endothelial progenitors by three stages. After differentiation, GREM1 was obviously increased in hUiPSC‐induced endothelial progenitors (hUiPSC‐EPs). RNA interference (RNAi) was used to silence GREM1 expression in three stages, respectively. We demonstrated a stage‐specific effect of GREM1 in decreasing hUiPSC‐EP differentiation in the mesoderm induction stage (Stage 1), while increasing differentiation in the endothelial progenitors' induction stage (Stage 2) and expansion stage (Stage 3). Exogenous addition of GREM1 recombinant protein in the endothelial progenitors' expansion stage (Stage 3) promoted the expansion of hUiPSC‐EPs although the activation of VEGFR2/Akt or VEGFR2/p42/44MAPK pathway. Our study provided a new non‐invasive source for endothelial progenitors, demonstrated critical roles of GREM1 in hUiPSC‐EP and afforded a novel strategy to improve stem cell‐based therapy for the ischaemic diseases.     

钩藤钩器官形态发育的解剖学研究

为了解钩藤钩的微观形态发育过程,该文利用石蜡切片的方法,对钩藤营养器官分枝茎钩的微观形态特征进行解剖和比较分析。结果表明:钩与茎之间在解剖学上既有相关性,又各有其自身的特征; 其相关性表现在茎的内部结构大体上决定着其钩的内部结构,充分体现钩是由着生在茎上的侧枝变态发育形成的; 在钩发育中期逐渐形成与茎类似的内部结构; 茎的内部结构组成在数量上比钩更丰富,钩只有初生结构; 钩藤茎与钩的药用成分积累没有本质的区别,均主要分布在韧皮部和木质部及皮层薄壁细胞。由此可知,钩藤钩器官是由茎变态发育形成,但只有初生结构。该研究结果可辅助解决某些仅靠形态学性状难以解决的分类鉴定问题,为判断钩状茎或卷须茎的变态来源提供理论参考依据。     

Does detritus quality predict the effect of native and non‐native plants on the performance of larval amphibians?

1. The lack of consistent differences between the traits of native and non‐native plant species makes it difficult to make general predictions about the ecological impact of invasive plants; however, the increasing number of non‐native plants in many habitats makes the assessment of the impact of each individual species impracticable. General knowledge about how specific plant traits are linked to their effects on communities or ecosystems may be more useful for predicting the effect of plant invasions. Specifically, we hypothesised that higher carbon‐to‐nitrogen ratio (C:N) and percent lignin in plant detritus would reduce the rate of development and total mass at metamorphosis of tadpoles, resulting in lower metamorph production (total fresh biomass) and amphibian species richness. 2. To test these hypotheses, we raised five species of tadpoles in mesocosms containing senescent leaves of three common native and three common non‐native wetland plants that varied in C:N ratio and % lignin. 3. Leaf mass loss, total metamorph production and the number of species that metamorphosed declined as a function of increasing C:N ratio of plant leaves. Plant lignin content was not related to the production of metamorphs or the number of species that metamorphosed. The percentage of wood frog (Lithobates sylvaticus) and American toad (Anaxyrus americanus) tadpoles reaching metamorphosis declined as a function of increasing plant C:N ratio. Mean time to metamorphosis increased and mean mass at metamorphosis declined as a function of increasing plant C:N ratio. Tadpole performance and metamorph diversity and production (biomass) were similar between native and non‐native plant species with similar C:N ratio in leaves. Percent lignin was not a significant predictor of tadpole performance. 4. Our results show that the impact of a plant invasion on tadpole performance could depend on differences between the quality of the detritus produced by the invading species and that of the native species it replaces. We suggest that plant community changes that lead to dominance by more recalcitrant plant species (those with higher leaf C:N ratio) may negatively affect amphibian populations.     

The moult cycle of the terrestrial isopod Armadillo officinalis Duméril, 1816 (Crustacea: Isopoda: Oniscidea)

The present work focuses on the moult cycle of Armadillo officinalis. For a 100‐day period, 134 animals were observed and routinely examined with the aim of detecting distinctive morphological characters in the several stages and substages of the moult cycle and of disclosing their duration. Statistical tests and Poisson regression models with robust standard errors were used to investigate differences and relationships between moult and the size and gender of the animals. The appearance of the calcium carbonate deposits on the pereon sternites during the premoult stage was documented in detail, and three main substages were identified. The average duration of the premoult and of the biphasic ecdysis was about 12 and 1.5 days, respectively. This observation period, however, did not allow to establish a determined average duration of the intermoult stage, which was extremely variable. This stage lasted for 2 months or more in most of the cases observed, but about 1‐month‐long intermoult stages were also recorded. No statistically significant association was found between the number of moults and gender and size of the animals.     

An invasive species imposes selection on life‐history traits of a native frog

As well as their direct ecological impacts on native taxa, invasive species can impose selection on phenotypic attributes (morphology, physiology, behaviour, etc.) of the native fauna. In anurans, body size at metamorphosis is a critical life‐history trait: for most challenges faced by post‐metamorphic anurans, larger size at metamorphosis probably enhances survival. However, our studies on Australian frogs (Limnodynastes convexiusculus) show that this pattern can be reversed by the arrival of an invasive species. When metamorph frogs first encounter invasive cane toads (Bufo marinus), they try to eat the toxic invader and, if they are able to do so, are likely to die from poisoning. Because frogs are gape‐limited predators, small metamorphs cannot ingest a toad and thus survive long enough to disperse away from the natal pond (and thus from potentially deadly toads). These data show that larger size at metamorphosis can reduce rather than increase anuran survival rates, because larger metamorphs are more easily able to ingest (and thus be poisoned by) metamorph cane toads. Our results suggest that patterns of selection on life‐history traits of native taxa (such as size and age at metamorphosis, seasonal timing of breeding and duration of pondside aggregation prior to dispersal) can be modified by the arrival of an invasive species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 329–336.     

Ultrastructure and protein composition of the oocyte envelope in the whitemouth croaker (Micropogonias furnieri, Desmarest, 1823, Sciaenidae, Perciformes)

The organization, time‐course deposition and protein composition of the oocyte envelope in the whitemouth croaker, Micropogonias furnieri, were analyzed at different stages of oocyte maturation. Adult females were sampled in the Uruguayan coast of the Río de la Plata during three annual periods. Morphological organization and temporal deposition were assessed by histology and electron microscopy. Protein composition was analyzed using gel electrophoresis, followed by MALDI‐TOF‐MS. Oocyte envelope deposition starts in lipid‐yolk oocytes, reaching maximum width in fully grown oocytes when it shows a three‐layer organization. In mature oocytes, the envelope becomes narrower than in the previous stage and loses its trilaminar structure. In envelopes from fully grown oocytes, one‐dimensional gel electrophoresis revealed five bands; mature oocytes showed only three bands. Following two‐dimensional gel electrophoresis, 14 major polypeptides were detected in envelopes from fully grown oocytes. Considering that morphological and biochemical results obtained from samples of the three annual periods were remarkably similar, data reported here might provide a useful baseline to assess the future impact of pollutants on the oocyte envelope and reproductive success of whitemouth croakers inhabiting the geographic area.