Reproductive Biology and Embryonic Diapause as a Survival Strategy for the East Asian Endemic Eagle Ray Aetobatus narutobiei

Batoids comprise five of the seven most threatened families of sharks and rays. The East Asian endemic Naru eagle ray Aetobatus narutobiei is a large bodied ray whose estuarine habitat overlaps with an economically valuable bivalve fishery. In response to decreased bivalve yields, the government initiated a predator control program and as a result, Naru eagle rays have faced intense and targeted fishing pressure during the last two decades. The long-term impacts of the predator control program on the population of rays and bivalves and their balance in the ecosystem are unknown because the life history of the Naru eagle ray has not been characterized. To begin to fill these critical knowledge gaps, the reproductive life history of the Naru eagle was described. Females mature at a larger size than males and require nearly twice as many years to reach maturity (DW50, 952.0 mm vs. 764.2 mm; Age50, 6.0 years vs. 3.5 years). Both males and females reproduce annually and their reproductive cycles are synchronized and seasonal. Females have a single ovary and paired uteri, are viviparous, and reproduce via matrotrophic histotrophy. Mating occurs in August and September and gestation lasts approximately 12 months including a 9.5-month diapause that begins soon after mating and ends in June of the following year, leaving 2.5 months for embryos to complete development. Fecundity ranged from 1 to 7 embryos per brood (n = 158, mean ± SD = 3.36 ± 1.26) and was positively correlated with female disc width (linear regression; F = 105.73, d.f. = 151, P < 0.05). Naru eagle rays are vulnerable to overfishing because of their low fecundity, long reproductive cycle and long time to reach sexual maturity. Obligate embryonic diapause during overwintering and seasonal migrations is a survival strategy that benefits the adults and neonates. This research is a valuable resource to help guide science-based management, conservation and protection of the endemic Asian A. narutobiei and its nursery areas.

Phenological Asynchrony Is Associated With Diapause Program and Heat Shock Protein Expression in Three Grasshopper Species in the Inner Mongolian Steppe

Phenological asynchrony is a common and important natural phenomenon that affects interspecific interaction, resource allocation, species survival, and range shift in sympatric species. However, the underpinnings for regulating phenological asynchrony at physiological and molecular levels remains less explored. We investigated the seasonal pattern of emergence period and abundance in three dominant grasshopper species, namely, Dasyhipus barbipes, Oedalus asiaticus, and Chorthippus dubius, which occur sympatrically in the Inner Mongolian steppe. The three grasshopper species decoupled their population occurrence phenology that occurred in a growing season between May and September and diverged into early, middle, and late seasonal species. We also examined the association of embryonic diapause and heat shock protein (Hsp) expression with phenological asynchrony in the three species. The species developed different embryonic diapause programs, i.e., obligate diapause, facultative diapause, and non-diapause, to control the timing of egg hatching and seasonality of population occurrence. The diapausing eggs exhibited significantly enhanced supercooling capacity compared with pre- and post-diapausing eggs. Gene expression analysis in the developmental process revealed that three Hsps, e.g., Hsp20.6, Hsp40, and Hsp90, were significantly upregulated in diapause state relative to that in pre- and post-diapause states; expression of these genes seems to be associated with the diapause program regulation. This study provides a possible mechanistic explanation for phenological differentiation among sympatric species in a typical steppe habitat and establishes a potential linkage among phenological asynchrony, diapause, and Hsp gene expression. The findings will facilitate our prediction of population dynamics and pest management.

Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm

Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

Molecular Regulation of Paused Pluripotency in Early Mammalian Embryos and Stem Cells

The energetically costly mammalian investment in gestation and lactation requires plentiful nutritional sources and thus links the environmental conditions to reproductive success. Flexibility in adjusting developmental timing enhances chances of survival in adverse conditions. Over 130 mammalian species can reversibly pause early embryonic development by switching to a near dormant state that can be sustained for months, a phenomenon called embryonic diapause. Lineage-specific cells are retained during diapause, and they proliferate and differentiate upon activation. Studying diapause thus reveals principles of pluripotency and dormancy and is not only relevant for development, but also for regeneration and cancer. In this review, we focus on the molecular regulation of diapause in early mammalian embryos and relate it to maintenance of potency in stem cells in vitro. Diapause is established and maintained by active rewiring of the embryonic metabolome, epigenome, and gene expression in communication with maternal tissues. Herein, we particularly discuss factors required at distinct stages of diapause to induce, maintain, and terminate dormancy.

The formation of a hatching line in the serosal cuticle confers multifaceted adaptive functions on the eggshell of a cicada

Insect eggshells must meet various demands of developing embryos. These demands sometimes conflict with each other; therefore, there are tradeoffs between eggshell properties, such as robustness and permeability. To meet these conflicting demands, particular eggshell structures have evolved in diverse insect species. Here, we report a rare eggshell structure found in the eggshell of a cicada, Cryptotympana facialis. This species has a prolonged egg period with embryonic diapause and a trait of humidity-inducible hatching, which would impose severe demands on the eggshell. We found that in eggs of this species, unlike many other insect eggs, a dedicated cleavage site, known as a hatching line, was formed not in the chorion but in the serosal cuticle. The hatching line was composed of a fine furrow accompanied by ridges on both sides. This furrow-ridge structure formed in the terminal phase of embryogenesis through the partial degradation of an initially thick and nearly flat cuticle layer. We showed that the permeability of the eggshell was low in the diapause stage, when the cuticle was thick, and increased with degradation of the serosal cuticle. We also demonstrated that the force required to cleave the eggshell was reduced after the formation of the hatching line. These results suggest that the establishment of the hatching line on the serosal cuticle enables flexible modification of eggshell properties during embryogenesis, and we predict that it is an adaptation to maximize the protective role of the shell during the long egg period while reducing the barrier to emerging nymphs at the time of hatching.

Lipid droplets in mammalian eggs are utilized during embryonic diapause

Embryonic diapause (ED) is a temporary arrest of an embryo at the blastocyst stage when it waits for the uterine receptivity signal to implant. ED used by over 100 species may also occur in normally “nondiapausing” mammals when the uterine receptivity signal is blocked or delayed. A large number of lipid droplets (LDs) are stored throughout the preimplantation embryo development, but the amount of lipids varies greatly across different mammalian species. Yet, the role of LDs in the mammalian egg and embryo remains unknown. Here, using a mouse model, we provide evidence that LDs play a crucial role in maintaining ED. By mechanical removal of LDs from zygotes, we demonstrated that delipidated embryos are unable to survive during ED. LDs are not essential for normal prompt implantation, without ED. We further demonstrated that with the progression of ED, the amount of intracellular lipid reduces, and composition changes. This decrease in lipid is caused by a switch from carbohydrate metabolism to lipid catabolism in diapausing blastocysts, which also exhibit increased release of exosomes reflecting elevated embryonic signaling to the mother. We have also shown that presence of LDs in the oocytes of various mammals positively corelates with their species-specific length of diapause. Our results reveal the functional role of LDs in embryonic development. These results can help to develop diagnostic techniques and treatment of recurrent implantation failure and will likely ignite further studies in developmental biology and reproductive medicine fields.