Studies on the embryogenesis of Aulocara elliotti (Thomas) (Orthoptera, Acrididae). II. Developmental variability and the effects of maternal age and environment

The stages of diapause embryos of A. elliotti reared in the laboratory were described, and the lengths of embryos of stage 19 were compared with a second group of diapause embryos of advanced ages. Collections of diapause embryos of the same species were made from wild populations during the autumn and winter of two consecutive years. The stages of embryos present in these samples were found to vary widely. The mean lengths of embryos in stage 19 from different populations were compared and found to differ with statistical significance. The numbers of retarded embryos in different populations were found to vary significantly between populations of diapause embryos. In two populations, mean lengths of embryos of stage 19 were found to differ from one year to the next. Post-diapause embryos were collected from wild populations in the spring of two consecutive years and their stages described. No significant difference was found in the incidence of retardation in post-diapause samples. Results of these studies and those of other authors indicate that there may be wide developmental variation between populations of acridid embryos as well as in one population in two different years. Rearing adult A. elliotti in varying numbers under two different environmental conditions was found to produce significant effects upon the rate of development and variability of their embryonic progeny. Embryos laid by young females were found to be more variable, in general, and to develop more slowly than those laid by older females.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

ORIGIN AND ONTOGENESIS OF SOMATIC EMBRYOS IN HEYEA BRASILIENSIS (EUPHORBIACEAE)

In Hevea, currently described culture conditions allow the induction of two modes of embryogenesis in callus formed on excised portions of the internal integument of immature seeds. One mode is of unicellular origin and is transitory, only resulting in the production of globular proembryos. The other is of multicellular origin and produces embryos. Specific culture conditions appear to favor one or the other mode of development in a given genotype. The ontogenesis of embryos of multicellular origin bypasses the classical stages of zygotic embryogenesis. The structural abnormalities observed in most somatic embryos are probably responsible for the low germination rates obtained.     

Differences in embryological development in two closely related species: Ihlea racovitzai and Ihlea magalhanica (Tunicata,Thaliacea)

Differences in the early stages of embryological development confirm the validity of the closely related salp species Ihlea racovitzai and Ihlea magalhanica. In the latter 8–12 oocytes and thus 8–12 follicles and uterine sacs are observed in the ovary; however most of them undergo a progressive degradation and only two embryos complete their development. In Ihlea racovitzai two embryos also develop but from the very beginning only the two successful follicles are found.     

Development of the Osteocranium in Natrix natrix (Serpentes,Colubridae) Embryogenesis II: Development of the jaws,palatal complex and associated bones

Snakes differ from the other vertebrates with their hyperkinetic skull. To establish the developmental features of the skull bones, involved in prey capture and ingestion, the Grass snake Natrix natrix (Serpentes, Colubridae) embryos are studied at all the successive stages of embryogenesis. Thirty-five N. natrix embryos are examined. Twenty embryos are studied with histological methods; fifteen embryos are cleared and double-stained with alizarin red and alcian blue. The sequence of appearance and formation of the upper and lower jaw bones, palatal complex and associated bones is described in accordance with the table of developmental stages. New features in the ossification mode of some bones are revealed: each bone, namely, the vomer, septomaxilla and maxilla, is formed from three separate ossification centres. Three ossification centres in the maxilla, two ossification centres in the bodies of the septomaxilla and vomer, as well as the unknown additional ossification centre in the vomer had not been previously described in snake embryos. The new data can be used in further comparative research on the reptile skull development and vertebrate phylogeny.     

Fine structure of dicyemid mesozoans,with special reference to cell junctions

The fine structure of the dicyemid mesozoan, Dicyema acuticephalum, from Octopus vulgaris, was studied with special attention to intercellular junctional complexes between various kinds of cells. Two types of intercellular junction, namely, adherens junctions and gap junctions, were found in both vermiform stages and in infusoriform embryos. Adherens junctions were classified into two types. Zonulae adherentes-like junctions were observed between adjacent peripheral cells at vermiform stages, between adjacent external cells of infusoriform embryos, and between members of groups of internal cells that covered the urn in infusoriform embryos. Maculae adherentes-like junctions were seen between a peripheral cell and an axial cell at vermiform stages. In infusoriform embryos, these junctions were observed between various types of cells, excluding urn cells. Gap junctions were found between adjacent peripheral cells at vermiform stages, whereas in infusoriform embryos these junctions were located between various types of cells excluding urn cells. Dicyemids might be the most primitive multicellular animals to possess these basic types of cell junctions. Ciliary rootlet systems at vermiform stages and in infusoriform embryos were unique in structure compared with those of other primitive multicellular animals. J Morphol 231:297–305, 1997. © 1997 Wiley-Liss, Inc.     

A preliminary method for estimating the age of brown kiwi (Apteryx mantelli) embryos

Morphological features of a collection of unknown-age wild kiwi (Apteryx mantelli) embryos from early development to point of hatch are described. Using these features, we assign developmental stages to each embryo and compare the progress of development to similar-staged ostrich (Struthio camelus) and chicken (Gallus gallus) embryos. Two ageing schemes for the kiwi embryos are developed by comparing measurements of their hindlimb segments, bills and crown–rump lengths with those of ostrich and chicken embryos at various stages of development. One of the 20 kiwi embryos was of known age. Both the ostrich model and the chicken model gave identical predictions for the marker and four other embryos. Developmental timing of some features differed between all three species, most markedly in the bill, with growth in the kiwi bill being relatively faster to achieve its larger relative and absolute size at hatch.     

GA3 stimulates the formation and germination of somatic embryos and the expression of a KNOTTED-like homeobox gene of Cocos nucifera (L.)

The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 μM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem.     

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.     

Comparative study of reserve lipid accumulation during somatic and zygotic <Emphasis Type="Italic">Acca sellowiana</Emphasis> (O. Berg.) Burret embryogenesis

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.     

Report on fetal mummification in the scalloped hammerhead shark Sphyrna lewini

This study describes the fetal mummification process in two embryos of a 310 cm total length scalloped hammerhead shark Sphyrna lewini caught in southeastern Brazil, in December 2017. Fourteen embryos were observed in total, in which two males in the left uterus presented different stages of mummification. Both mummified embryos were covered by an exudate (i.e., a mucous substance), indicating a hematic mummification process. All embryos were at the placentotrophic stage of development, indicating that they were close to parturition. An intrinsic characteristic is suggested as possible etiology for this condition, such as umbilical torsion, because both embryos were at different sizes and, therefore, at different development stages. In addition, the sample size did not allow the authors to presume any pollution effect once only one female was observed. Finally, fetal mummification and other embryonic development disorders might have populational impacts due to reduction in embryo survival and, consequently, recruitment. For this reason and considering that S. lewini is categorized as a “critically endangered species,” this study's results have conservational relevance.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Reproductive biology of the bigeye thresher shark,Alopias superciliosus (Lowe, 1839) (Chondrichthyes: Alopiidae), in the northwestern Pacific

Reproductive aspects ofAlopias superciliosus in the northwestern Pacific were described in detail, on the basis of 629 specimens (429 females and 200 males) collected from January 1984 to October 1984 and from October 1992 to March 1994.Alopias superciliosus embryos are oophagous. Six developmental stages (3 encapsulative and 3 posthatching) based on embryonic morphology and source of nutrition were recognized. The species bears 2 embryos per litter, their size at birth being between 135 and 140 cm TL. The sex ratio of embryos was 1∶1. Total length of females at maturity was 332–341. 1. cm; of males 270.1–287.6 cm. The gestation period could not be determined because most adult females were pregnant throughout the year. The typical reproductive strategy ofA. superciliosus is the production of a few large embryos per litter, with no fixed mating or birthing season.     

Development of the salivary glands in embryos of Ixodes ricinus (Acari: Ixodidae)

We examined Ixodes ricinus embryos between 18 and 28 days of development with light, scanning and transmission electron microscopy. The differences in inner structure attested to establish three successive developmental stages: days 18–20, day 23, and days 26–28. Between 18 and 20 days the embryos are at early stages of organogenesis. Salivary glands cannot be identified at that stage. In 23-day-old embryos salivary glands are already outlined but the structure of alveoles is still different from that in larvae in which the embryonic development has been completed. Gland cells start to form alveoles and become active between 26 and 28 days of the development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Developmental stages of zebrafish (Danio rerio) embryos and toxicological studies using foldscope microscope

Zebrafish (Danio rerio), is a well‐established vertebrate animal model widely used in developmental biology and toxicological research. In the present study, foldscope is used as an innovative tool to study the developmental stages and toxicological analysis of the zebrafish embryos. Briefly, the developmental stages, such as zygote, cleavage, blastula, gastrula, segmentation, and pharyngula formation are observed and documented using simple foldscope. Toxicological parameters upon exposure to different concentration of ethanol extract of Curcuma longa and its lead compound, ar‐turmerone along with rhodamine B (bio‐coupler) on zebrafish embryos are analyzed upto 72 hr using foldscopes in live condition. The lethal endpoints, such as coagulation, lack of somite formation, non‐detachment of tail, and lack of heartbeat are clearly monitored and documented using foldscope. Bio‐evaluation of test compounds with the aid of foldscope confirms that the toxicity is directly proportional to the concentration. Our results conclude that, ethanol extract of C. longa, ar‐turmerone and rhodamine B exposed embryos remains healthy up to 96, 48, and 24 µg concentrations, respectively. Embryos exposed to higher concentrations become coagulated, however normal physiological active movement of tail lashing and heartbeat are evident in lower concentration exposed embryos. Except coagulation, no other abnormalities are observed and interestingly, the hatching ability is not delayed, when compared with the control embryos. It is confirmed that the test compounds are not highly toxic to zebrafish embryos. Hence it can be used for further analysis, especially for studying the neural‐regeneration and its neuronal development in zebrafish embryos.     

Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra- and inter-species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four-cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l^?1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.