Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.     

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue‐specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better‐known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10–15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.     

Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata

In marine benthic invertebrates with complex life cycles, recruitment success, juvenile survival, and growth may be affected by variation in both maternal factors and environmental conditions prevailing during preceding embryonic or larval development. In an estuarine crab, Chasmagnathus granulata, previous investigations have shown that initial larval biomass is positively correlated with the biomass of recently extruded eggs, and it depends also on the salinity experienced during embryogenesis. Biomass at hatching has consequences for the subsequent larval development which, in this species, comprises two alternative developmental pathways with four or five zoeal instars (short or long pathway) and a megalopa. Larvae hatching with a lower than average biomass tend to develop through the long pathway and metamorphose to megalopae with higher biomass. In the present study, we show experimentally that the long pathway produces also significantly larger juveniles (crab size measured as carapace width, biomass as dry mass, carbon and nitrogen contents). Compared with juveniles originating from the short pathway, those from the long pathway showed in successive instars longer moulting cycles and larger carapace width, but lower size increments at ecdysis. In consequence, differences in size or biomass of long pathway vs short pathway crabs tended to disappear in later instars (after stage V). Furthermore, we tested in juveniles the tolerance of starvation at three salinities (5‰, 15‰, 32‰). Tolerance of starvation was significantly higher in juveniles originating from the long pathway, indicating higher energy reserves. While salinity played only a minor role for survival, it exerted significant effects on the time of moulting to the second juvenile instar, regardless of the preceding developmental pathway. The biomass of first juveniles obtained from the short pathway showed a significant positive correlation with the biomass of the freshly hatched zoea I, but not in those from the long pathway. In conclusion, the fitness of juvenile C. granulata is linked with previous developmental processes and environmental conditions during the embryonic and larval phase. Hence, a better understanding and prediction of the recruitment success of marine benthic invertebrates with a complex life cycle may require more comprehensive life‐history investigations.     

Early development of marine catfishes (Ariidae): from mouth brooding to the release of juveniles in nursery habitats

The development and allometric growth patterns of the ariid catfishes Cathorops spixii and Cathorops agassizii were studied from neurula embryos to juveniles. The ontogenetic sequence revealed that prior to hatching, embryos of both species are well developed, and their axial and appendicular skeletons are well ossified. Embryos of both species grow slowly longitudinally, but positively allometric growth (growth coefficient, β₁ > 1) was observed in head width and eye diameter. It is hypothesized that these growth patterns might be related to functional priorities for the development of sensory organs, such as the inner ears (otoliths), the Weberian apparatus, eyes and nostrils, during the embryonic period. The first appearance of vertebrae and otoliths, as well as the ossification of otoliths and the Weberian apparatus, occur earlier in embryos of C. agassizii than in embryos of C. spixii. After hatching, mouth‐brooded free embryos of both species grow isometrically. Negatively allometric growth was observed in head width and eye diameter during the yolk‐sac period, which is expected given that the sensory organs are already formed. Free embryos of C. agassizii are distinguishable from those of C. spixii by their larger eyes, longer snouts, longer heads and heavier yolk sacs. The end of the yolk‐sac period is characterized by a direct change from free embryo to juvenile, without a true larval period. The juveniles of the two species can also be distinguished from each other by the larger eyes of C. agassizii compared with C. spixii, as in adult fishes.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

Quantitative staging of embryonic development of the tobacco hawkmoth,Manduca sexta

Summary A complete timetable of embryonic development of the tobacco hawkmoth,Manduca sexta (Lepidoptera: Sphingidae), is presented. Using living embryos, 20 developmental stages from oviposition to hatching are described with respect to their morphological and physiological maturation. This staging series provides a simple method to identify the stage ofManduca development during all phases of embryogenesis.     

Prothoracicotropic hormone activity in the embryonic brain of the tobacco hornworm,Manduca sexta

Summary Head segments and brains were extirpated from embryos of the tobacco hornworm,Manduca sexta, extracted and the resulting extracts assayed for prothoracicotropic hormone (PTTH) activity on prothoracic glands from day 3 fifth instar larvae and day 0 pupae. Dose-response curves were generated and indicated the presence of PTTH activity in embryonic brains and head segments, suggesting a role(s) for this neurohormone during embryogenesis. Maximal PTTH activity was found in brains from embryos 117 h post-oviposition, just prior to hatching, but activity was also noted in head segments as early as 24 h postoviposition. These data on PTTH and those on ecdysteroids and juvenile hormones in embryos suggest that these 3 classes of hormones which control insect post-embryonic development, may also be involved in the regulation of developmental processes in the embryo.     

Responses of embryonic stages of the estuarine mud crab,Macrophthalmus hirtipes (Jacquinot), to salinity

Females of the estuarine mud crab, Macrophthalmus hirtipes (Jacquinot, 1853) (Ocypodidae) carrying newly-deposited eggs were maintained in salinities of 11‰, 18‰, and 36‰ at 10°C. In 11‰ salinity eggs swelled more than in the other salinities; embryonic development was retarded; and eggs did not hatch. In 18‰ and 36‰, rates of development were generally similar and successful hatching occurred after approximately 75 days. However, late-stage eggs in 18‰ were significantly (P<0.001) larger than those in 36‰, even though initial egg volumes were the same. Zoeae were morphologically similar, except that the dorsal spine was significantly (P<0.001) longer in 36‰ compared with 18‰.     

Adriamycin inhibits embryonic development in zebrafish through downregulation of Kruppel‐like factor4

Adriamycin is an effective anticancer drug used in a wide range of cancers. Anticancer drugs modulate oncogenes and nodal regulatory molecules that affect cell differentiation and organismal development. In this study, we explore the effect of adriamycin on Kruppel‐like factor4 (Klf4), an essential pluripotent factor by choosing zebrafish embryos as a model system. Klf4 is involved in the regulation of cellular growth, proliferation, and differentiation. In zebrafish embryogenesis, Klf4 is a major regulator of differentiation of polster in the anterior mesendoderm region of cells into hatching gland cells. The importance of this study is to check the effect of adriamycin on embryonic development. We found, adriamycin dose dependently altered the gene expression level of Klf4 that occurs in parallel to its detrimental effect on hatching. Supportively, cathepsin L and cyclase‐associated protein1 are the other two markers of hatching that are altered along with Klf4.     

Hatching ofCoregonus albula andC. lavaretus embryos at different stages of development

Synopsis Hatching in Coregoninae embryos is not closely connected with a determined stage of embryonic development. Electrical stimulation (AC) induced precocious hatching, and incubation at low temperature caused delay of hatching compared to controls. Embryos hatched precociously had shorter bodies and larger yolksacs than those whose hatching was normal or delayed. This was accompanied by respective changes in duration of the eleutheroembryonic phase (DEP) — inCoregonus lavaretus DEP decreased from 7 days in embryos hatched precociously to 0 days in embryos from delayed hatching.     

The apple sawfly, Hoplocampa testudinea: egg development and forecasting of egg hatch

The effect of temperature on egg development of the apple sawfly Hoplocampa testudinea Klug was studied under controlled conditions. Based on a linear model describing the relationship between temperature and developmental rates a thermal threshold of 6.9 °C and a thermal constant of 85 day-degrees were established. The experimental results were used to expand and parameterise an existing phenology model to include egg development and hatching of first instar larvae. The model thus became a valuable forecasting tool not only for adult emergence and the optimal monitoring period but also for egg hatch and the best timing of control measures against first instar larvae. The model was validated by comparing predictions with independent observations on egg-laying and subsequent hatching of first instar larvae under semi-field conditions.     

Acute toxic effects of 8‐epidiosbulbin E,a 19‐norclerodane diterpene from yam Dioscorea antaly,on medaka Oryzias latipes embryos

The fractionation of an aqueous extract of yam Dioscorea antaly from Madagascar led to the isolation of terpenoids and flavonoids. Compounds were identified on the basis of modern mass spectrometry and two‐dimensional nuclear magnetic resonance (2D‐NMR). Toxicological effects of the most abundant isolated compound, 8‐epidiosbulbin E were studied on medaka Oryzias latipes embryo‐larval development. The lethal concentration (killing 50%; LC₅₀) to embryos treated 24 h before hatching and for 3 days after hatching was estimated to be 0·56 mg ml^?1 (P< 0·05). No mortality was observed with O. latipes larvae exposed after hatching until day 4. Anatomo‐pathological studies of embryos exposed to 0·56 mg ml^?1 showed development anomalies of the central nervous system, liver, muscle and intestine. The present data thus extend the model of O. latipes embryos as a useful animal model to analyse the effects of food toxins.     

Latitudinal differences in temperature effects on the embryonic development and hatchling phenotypes of the Asian yellow pond turtle,Mauremys mutica

The effect of incubation temperature on embryonic development and offspring traits has been widely reported for many species. However, knowledge remains limited about how such effects vary across populations. Here, we investigated whether incubation temperature (26, 28, and 30 °C) differentially affects the embryonic development of Asian yellow pond turtle (Mauremys mutica) eggs originating from low‐latitude (Guangzhou, 23°06′N) and high‐latitude (Haining, 30°19′N) populations in China. At 26 °C, the duration of incubation was shorter in the high‐latitude population than in the low‐latitude population. However, this pattern was reversed at 30 °C. As the incubation temperature increased, hatching success increased in the low‐latitude population but slightly decreased in the high‐latitude population. Hatchlings incubated at 30 °C were larger and righted themselves more rapidly than those incubated at 26 °C in the low‐latitude population. In contrast, hatchling traits were not influenced by incubation temperature in the high‐latitude population. Overall, 30 °C was a suitable developmental temperature for embryos from the low‐latitude population, whereas 26 and 28 °C were suitable for those from the high‐latitude population. This interpopulation difference in suitable developmental temperatures is consistent with the difference in the thermal environment of the two localities. Therefore, similarly to posthatching individuals, reptile embryos from different populations might have evolved diverse physiological strategies to benefit from the thermal environment in which they develop. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 35–43.     

Cardiac development in crayfish: ontogeny of cardiac physiology and aerobic metabolism in the red swamp crayfish Procambarus Clarkii

The cardiovascular system performs key physiological functions even as it develops and grows. The ontogeny of cardiac physiology was studied throughout embryonic and larval development in the red swamp crayfish Procambarus clarkii using videomicroscopic dimensional analysis. The heart begins to contract by day 13 of development (at 25°C, 20 kPa O₂). Prior to eclosion, heart rate (ƒH) decreases significantly. Previous data suggests that the decrease in cardiac parameters prior to hatching may be due to an oxygen limitation of the embryo. Throughout development, metabolizing mass and embryonic oxygen consumption primarily increased while egg surface area remains constant. The limited area for gas exchange of the egg membrane, in combination with the increasing oxygen demand of the embryo could result in an inadequate diffusive supply of oxygen to developing tissues. To determine if the decrease in cardiac function was the result of an internal hypoxia experienced during late embryonic development, early and late stage embryos were exposed to hyperoxic water (PO₂ =40 kPa O₂). The ƒH in late stage embryos increased significantly over control values when exposed to hyperoxic water suggesting that the suppression in cardiac function observed in late stage embryos is likely due to a limited oxygen supply.     

Juvenile Hormone Stimulation of Oocyte Development and Embryogenesis in the Parthenogenetic Ovaries of an Aphid,Aphis fabae

Summary

The parthenogenetic ovaries of the black bean aphid, Aphis fabae, contain developing embryos. When reared at 15°C in long days (LD 16:8) oocyte development begins within the ovaries of the largest embryos of a fourth instar mother 24–48 hr after her ecdysis from the third instar. Starvation, decapitation and precocene III treatment inhibit embryonic oocyte development; juvenile hormone treatment reverses this inhibition. A method for the in vitro culture of embryos is described and under these conditions juvenile hormone again stimulates oogenesis. Embryogénie growth in vivo, as measured by the increase in length of the oldest daughter embryos, is also stimulated by juvenile hormone treatment. The results are discussed in relation to other roles proposed for juvenile hormone in aphid development.     

Egg environments have large effects on embryonic development,but have minimal consequences for hatchling phenotypes in an invasive lizard

Plastic responses of embryos to developmental environments can shape phenotypes in ways that impact fitness. The mechanisms by which developmental conditions affect offspring phenotypes vary substantially among taxa and are poorly understood in most systems. In this study, we evaluate the effects of thermal and hydric conditions on patterns of egg water uptake, embryonic development and yolk metabolism in embryos of the lizard Anolis sagrei to gain insights into how these factors shape morphological variation in hatchlings. Our 3 × 2 experimental design (3 thermal and 2 hydric conditions) revealed that developmental temperature has strong effects on rates of development and yolk metabolism, but the impacts of moisture were minimal. Increased water uptake by eggs under relatively wet conditions resulted in larger hatchlings with less internalized residual yolk than hatchlings from dry‐incubated eggs. However, the relatively small phenotypic differences among treatments may have small fitness consequences. These results demonstrate that embryos of A. sagrei can tolerate a broad range of environmental conditions without substantial impacts on critical morphological traits. Such embryonic tolerances may facilitate colonization and establishment in novel environments. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 25–41.     

Gold climates and the evolution of viviparity in reptiles: cold incubation temperatures produce poor-quality offspring in the lizard, Sceloporus virgatus

Evolutionary origins of viviparity among the squamate reptiles are strongly associated with cold climates, and cold environmental temperatures are thought to be an important selective force behind the transition from egg-laying to live-bearing. In particular, the low nest temperatures associated with cold climate habitats are thought to be detrimental to the developing embryos or hatchlings of oviparous squamates, providing a selective advantage for the retention of developing eggs in utero, where the mother can provide warmer incubation temperatures for her eggs (by actively thermoregulating) than they would experience in a nest. However, it is not entirely clear what detrimental effects cold incubation temperatures may have on eggs and hatchlings, and what role these effects may play in favouring the evolution of viviparity. Previous workers have suggested that viviparity may be favoured in cold climates because cold incubation temperatures slow cmbryogenesis and delay hatching of the eggs, or because cold nest temperatures are lethal to developing eggs and reduce hatching success. However, incubation temperature has also been shown to have other, potentially long-term, effects on hatchling phcnotypcs, suggesting that cold climates may favour viviparity because cold incubation temperatures produce offspring of poor quality or low fitness. We experimentally incubated eggs of the oviparous phrynosomatid lizard, Sceloporus virgatus, at temperatures simulating nests in a warm (low elevation) habitat, as is typical for this species, and nests in a colder (high elevation) habitat, to determine the effects of cold incubation temperatures on embryonic development and hatchling phenotypes. Incubation at cold nest temperatures slowed embryonic development and reduced hatching success, but also affected many aspects of the hatchlings' phenotypes. Overall, the directions of these plastic responses indicated that cold-incubated hatchlings did indeed exhibit poorer quality phenotypes; they were smaller at hatching (in body length) and at 20 days of age (in length and mass), grew more slowly (in length and mass), had lower survival rates, and showed greater fluctuating asymmetry than their conspecifics that were incubated at warmer temperatures. Our findings suggest that cold nest temperatures are detrimental to S. virgatus, by delaying hatching of their eggs, reducing their hatching success, and by producing poorer quality offspring. These negative effects would likely provide a selective advantage for any mechanism through which these lizards could maintain warmer incubation temperatures in cold climates, including the evolution of prolonged egg retention and viviparity.     

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.