Comparative morphology,morphometry and distribution pattern of the trigeminal nerve branches supplying the bill tip in the ostrich (Struthio camelus) and emu (Dromaius novaehollandiae)

The subdivisions of the trigeminal nerve (N. ophthalmicus R. medialis and N. intramandibularis) innervating the upper and lower bill tip, respectively, were well developed in both the ostrich and emu and displayed extensive branching. Transmission electron microscopy revealed that both nerves displayed features typical of a mixed, peripheral nerve. Nerve fibre size in the ostrich and emu was larger than that reported in domestic poultry. There were a significantly higher number of myelinated nerve fibres in the N. ophthalmicus R. medialis in the emu by comparison with the same nerve in the ostrich, or by comparison with the N. intramandibularis of either species. As myelinated nerve fibres supply Herbst corpuscles, and these structures have been demonstrated in this region in these two species, this may indicate that the upper bill of the emu is more sensitive to vibrational stimuli than the upper bill of the ostrich or the lower bill of both species. The large size of the nerve fibres, the high nerve fibre count and the particular distribution of the nerves in the bill tip support the existence of a well‐developed sensory area in this region of the ostrich and emu.     

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos¹. Different structures such as DNA plasmids encoding genes^2-4, small interfering RNA (siRNA) plasmids⁵, small synthetic RNA oligos⁶, and morpholino antisense oligonucleotides⁷ can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)⁸ and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes⁹ and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos^10-12.     

Early life traits of farm and wild Atlantic salmon Salmo salar and first generation hybrids in the south coast of Newfoundland

This study examined fertilization rates, survival and early life‐trait differences of pure farm, wild and first generation (F1) hybrid origin embryos after crossing farm and wild Atlantic salmon Salmo salar. Results show that despite a trend towards higher in vitro fertilization success for wild females, differences in fertilization success in river water are not significantly different among crosses. In a hatchery environment, wild females' progeny (pure wild and hybrids with wild maternal parent) hatched 7–11 days earlier than pure farm crosses and hybrids with farm maternal parents. In addition, pure wild progeny had higher total lengths (L_T) at hatch than pure farm crosses and hybrids. Directions in trait differences need to be tested in a river environment, but results clearly show the maternal influence on early stages beyond egg‐size differences. Differences in L_T were no longer significant at 70 days post hatch (shortly after the onset of exogenous feeding) showing the need to investigate later developmental stages to better assess somatic growth disparities due to genetic differences. Higher mortality rates of the most likely hybrids (farm female × wild male hybrids) at egg and fry stages and their delayed hatch suggest that these F1 hybrids might be less likely to survive the early larval stages than wild stocks.     

Size matters: predation risk and juvenile growth in North Island brown kiwi (

This study investigated how predation risk in North Island brown kiwi changes as the birds grow and develop. Over a 10-year period, 53 adult and 126 young kiwi were radio-tagged at Lake Waikaremoana and studied to determine survival rates, causes of death, and rates of growth. Predation loss amongst adults was low (2.49% year^-1) and caused mainly by ferrets. Young kiwi suffered intense predation from stoats during their first four months of life, but thereafter became too large (> 800 g) for stoats to kill. Juveniles took at least 880 days to attain adult size, about four times longer than expected for a 2–3 kg bird. Growth rates peaked at about the point of hatch, rather than later on in development as in other birds. We suggest that a long evolutionary history dominated by resource limitation rather than predation may account for slow rates of development in kiwi, and that differences among kiwi species in their ability to persist in the presence of stoats are explained by differences in the time that they take to reach safe-size.     

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage,diapause III

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage, diapause III. The embryo, surrounded by a transparent vitelline envelope, is in the pre‐hatching stage. A prominent eye and part of the pigmented body and tail are apparent. Why annual fishes? Annual fishes (Order Cyprinodontiformes) are a special kind of teleost, found in Africa and South America, with developmental strategies closely related to their life cycle. These fishes inhabit temporary pools that undergo drying during summer, when all adults die. The embryos remain buried in the bottom mud and are resistant to desiccation. In the subsequent rainy season they hatch a few hours after the pool is flooded and a new reproductive cycle begins. This developmental pattern is characterized by the presence of a unique stage between cleavage and embryogenesis, dispersion‐aggregation of blastomeres and because the embryos show reversible developmental arrests (diapauses) at different stages. Annual fish embryos are transparent, large, hardy and easy to maintain in the laboratory. Adults show continuous production of eggs and juveniles reach sexual maturity a few weeks after hatching (an unusual condition in fishes). Their particular developmental features confer unique opportunities for research on cell behavior during early development, the effect of environmental factors on development, the regulation of diapauses and the mechanisms involved in sex determination, among others topics. Image provided by Nibia Berois, Universidad de la República, Montevideo, Uruguay.     

Primordial germ cell‐specific expression of eGFP in transgenic chickens

PR domain zinc finger protein 14 (PRDM14) plays an essential role in the development of primordial germ cells (PGCs) in mice. However, its functions in avian species remain unclear. In the present study, we used CRISPR/Cas9 to edit the PRDM14 locus in chickens in order to demonstrate its importance in development. The eGFP gene was introduced into the PRDM14 locus of cultured chicken PGCs to knockout PRDM14 and label PGCs. Chimeric chickens were established by a direct injection of eGFP knocked‐in (gene‐trapped) PGCs into the blood vessels of Hamburger–Hamilton stages (HH‐stages) 13–16 chicken embryos. Gene‐trapped chickens were established by crossing a chimeric chicken with a wild‐type hen with very high efficiency. Heterozygous gene‐trapped chickens grew normally and SSEA‐1‐positive cells expressed eGFP during HH‐stages 13–30. These results indicated the specific expression of eGFP within circulating PGCs and gonadal PGCs. At the blastodermal stage, the ratio of homozygous gene‐trapped embryos obtained by crossing heterozygous gene‐trapped roosters and hens was almost normal; however, all embryos died soon afterward, suggesting the important roles of PRDM14 in chicken early development.     

Developmental biology of medaka fish (Oryzias latipes) exposed to alkalinity stress

Alkalinity stress is common in cultured aquatic animals and considered to be one of the major stress factors for fishes when they are transferred to saline‐alkali waters. To evaluate potential effects of alkalinity on the developmental biology of Oryzias latipes, fertilized eggs, larvae and breeding fish were exposed to different carbonate alkalinity concentrations of 1.5–64.5 meq l^?1, for 9, 120, and 60 days, respectively. The mortality of embryos significantly increased when exposed to the high concentrations (16.5–64.5 meq l^?1). Although more than 50% of survived embryos hatched in 16.5 and 31.4 meq l^?1 concentrations of carbonate alkalinity, most were not able to swim up after hatching. Morphological abnormalities such as coagulated embryos, halted embryo development, and hatching failure were observed at stages 15, 29–33 and 38 in high concentrations (31.4, 64.5 meq l^?1). Almost all larvae in 16.5 and 31.4 meq l^?1 treatments died 70 d post‐hatch. Growth of juveniles exposed to carbonate alkalinity of 5.3 and 8.8 meq l^?1 was not significantly different at 70 d and 120 d post‐hatch. The number of eggs released by breeders, the fertilization rate and the hatching rate of eggs were significantly lower in the 31.4 meq l^?1 treatment than in other treatments. Although medaka are capable of surviving in high alkalinities (31.4, 64.5 meq l^?1) for an extended period of time, these conditions are stressful to the fish, especially at the embryonic and reproductive stages.     

Embryonic development of adriatic sturgeon,Acipenser naccarii (Bonaparte 1836), in farming conditions: a guide

The sequence and timing of the embryonic development of Adriatic sturgeon (Acipenser naccarii) are described at a constant temperature (17 ± 1 °C), from fertilisation to hatch in a hatchery. Fertilised eggs, obtained from one female and two males, were held in a flow‐through system. On the first day, embryos were sampled every 20 min during the first 12 h, and every 40 min in the following 12 h. Embryos were sampled hourly on the second day. On the third and fourth days, embryos were sampled every 2 h, and every 3 h on the fifth and sixth days. The first cleavage furrow appeared at 1 h 40 min postfertilisation. Blastulation concluded at 12 HPF (hours postfertilisation), when the dorsal blastopore lip formed. The completion of gastrulation was at 18 HPF, closing of the neural tube at 23 HPF and formation of the s‐shape heart at 31 HPF. The mass hatch occurred at 120 HPF. A. naccarii is a relatively common sturgeon species in aquaculture and possesses similar developmental patterns to those of other sturgeons; hence, we can use this species as a model for further studies about sturgeon species which are more difficult to breed artificially. Information about the embryonic development of the Adriatic sturgeon could assist restocking programmes.     

Sublethal Effects of Nitrite on Eastern Tiger Salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) and Wood Frog (<Emphasis Type="Italic">Rana sylvatica</Emphasis>) Embryos and Larvae: Implications for Field Populations

Ephemeral pools, which can have high animal biomass and low dissolved oxygen, may be prone to nitrite accumulation. As such, it is important to understand how exposure to nitrite might affect development and growth of amphibians that breed in these ephemeral pools. Wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and tadpoles and young larvae were exposed to elevated concentrations of nitrite derived from sodium nitrite: 0, 0.3, 0.6, 1.2, 2.1, 4.6, and 6.1 mg l⁻¹ NO₂–N. Increasing nitrite exposure slowed embryonic and larval development in both the eastern tiger salamander and the wood frog, reduced growth in tiger salamander embryos and larvae, and delayed metamorphosis in the wood frog. At concentrations less than 2 mg l⁻¹ NO₂–N nitrite delayed hatching, and at concentrations above 2 mg l⁻¹ time to hatching decreased causing more individuals to hatch at less developed stages. Nitrite also increased asynchrony in tiger salamander hatching. The sublethal effects of nitrite on amphibian development, growth and hatching could have serious repercussions on amphibian fitness in ephemeral environments. Potential increases in mortality on field populations caused by sublethal effects of nitrite are discussed.     

Embryonic development of the Caridean prawn Macrobrachium mammillodactylus (Crustacea: Decapoda: Palaemonidae)

The freshwater knob-tooth prawn Macrobrachium mammillodactylus is a commercially exploited species in the Philippines. To study the biology of this species, broodstock from the wild was collected, transported to the laboratory and kept in pairs in indoor polyethylene tanks for breeding. Eggs from berried females were sampled to follow the stages of embryonic development until hatching to zoea larva. It took 18.0?±?2.1?days for the eggs to hatch at ambient water temperature between 25 and 28?°C. The morphological landmarks of development at the different stages (pre-cleavage, cleavage, blastula, pre-nauplius, post-nauplius and pre-hatching) of the live embryos are described. Incremental percentage staging was adopted from 0% at fertilization to 100% at hatching and were matched with corresponding morphological development. Egg volume increased significantly toward the mid-to-later stages of development. The eye index also showed a significant increase as the egg developed. The colour of the egg mass changed from light olive green to grey as the eggs progressed in development. The general pattern of development was comparable to other members of the genus Macrobrachium.     

Waiting for a sign: extended incubation postpones larval stage in the beach spawning California Grunion <Emphasis Type="Italic">Leuresthes tenuis</Emphasis> (Ayres)

Unlike most embryos that hatch on a predetermined timetable, California Grunion Leuresthes tenuis can prolong the embryonic period up to three times longer than the time required for hatching readiness. L. tenuis are teleosts that spawn tidally around the highest spring tides of spring and summer, incubating eggs above the water line. Embryos are competent to hatch in 10 days, however they do not hatch until triggered by an environmental cue, agitation in seawater, as the next spring tides rise. This study examined the growth and survival of L. tenuis embryos and larvae that were all fertilized on the same day, then triggered to hatch after different durations of incubation, up to 35 days post fertilization. L. tenuis embryos that survive extended incubation had decreased yolk reserves and did not advance appreciably in morphological development, even when incubation time was extended to its upper limit. After extended incubation, length of hatchlings was significantly longer than hatchlings from the primary incubation time. Regardless of the duration of incubation, larvae provided food ad libitum grew rapidly and were not significantly different in length at three weeks post hatch. Dry mass increased over time and was not significantly different between larval groups within any post-hatch age. Larval growth and survival after one additional tidal cycle of incubation are not adversely affected, but longer incubation significantly decreases embryonic and larval survival. Large reproductive output, environmentally cued hatching, and plasticity in incubation duration enable L. tenuis to reproduce successfully in the unpredictable sandy intertidal ecosystem.     

Studies on avian erythrocyte metabolism: IX. Relationship of changing organic phosphate composition to whole blood oxygen affinity during development of the ostrich (Struthio camelus camelus)

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in erythrocytes (RBC) of the 37-day ostrich embryo at a concentration of 3.7 μmole/ml RBC, representing 23.5% of the cell phosphate. (2) Inositol tetraphosphate (inositol-P₄) is absent in red cells of the 37-day embryo, appears in the RBC about 63 days after hatch, and thereafter gradually accumulates to a level of 2.8 μmole/ml RBC in the adult bird, representing 30–35% of the cell phosphate. (3) Inositol pentaphosphate (inositol-P₅) is present in the erythrocytes of the 37-day embryo at a concentration of 0.8 μmole/ml RBC, reaches a peak level of 2.9 μmole/ml RBC by Day 63 after hatch, and thereafter declines to a level of 1.1 μmole/ml RBC in the adult bird. (4) The crossover time where the molar concentrations of inositol-P₅ and inositol-P₄ are equal in the erythrocyte appears to be about 150 days after hatch. (5) The p₅₀ of whole blood from the 37-day ostrich embryo, 5-day ostrich chick, and adult ostrich was 15.4, 31.8, and 24.9 Torr. The p₅₀ of whole blood immediately after hatch correlates best with an abrupt rise in ATP concentration but after 54 days posthatch correlates best with the appearance of increasing concentrations of inositol-P₄ and the decrease in concentrations of ATP and inositol-P₅. (6) The appearance of inositol-P₄ in the cells could be an adaptive mechanism for regulating oxygen supply by switching to a modulator which maintains a higher oxygen affinity than would a predominance of inositol-P₅.     

Phenotypically plastic responses of green frog embryos to conflicting predation risk

Predators have been shown to alter the timing of switch points between life history stages, but few studies have addressed switch point plasticity in prey exposed simultaneously to conflicting predation pressure. We tested hatching responses of green frog (Rana clamitans) embryos subject to perceived predation risk from chemical cues released by two stage-specific predators, predicting that these predators would elicit: (1) directional hatching responses when presented independently, and (2) intermediate phenotypic responses when presented simultaneously. R. clamitans embryos in outdoor exclosures were exposed to cues from an egg predator (freshwater leeches; Nephelopsis obscura), a larval predator (dragonfly nymphs, Aeschna canadensis), and both predators in a 2 × 2 factorial experiment, and changes in hatchling size, hatchling developmental stage, and hatching time were compared to those for control embryos. Leeches alone induced embryos to hatch at a smaller size and an earlier developmental stage than controls, while dragonfly nymphs elicited a delay in egg hatching time that was associated with larger size and later developmental stage at hatching. Embryos failed to respond to simultaneous exposure to both predators, implying that responses to each occurred concurrently and were therefore dampened. Our results indicate that prey under threat from conflicting predators may manifest intermediate defensive phenotypes. Such intermediate responses may result in elevated rates of prey mortality with possible consequences at the population level.     

Remote touch prey‐detection by Madagascar crested ibises Lophotibis cristata urschi

Birds that forage by probing must often rely on sensory systems other than vision to detect their buried prey. Such senses may include hearing (e.g. Australian magpies (Atramidae), American robins (Turdidae)) or chemical senses/olfaction (e.g. kiwi (Apterygidae) and some shorebirds (Scolopacidae)). Probe foraging kiwi and shorebirds are also able to use vibrotactile cues to locate prey buried in the substrate at some distance from their bill‐tips (‘remote touch’). These birds possess an organ consisting of a honey‐comb of sensory pits in bone of the bill‐tips, packed with mechanoreceptive nerve ending (Herbst corpuscles). Such a bill‐tip organ has recently also been described in ibises (Threskiornithinae), but its function not elucidated. We designed a foraging experiment presenting mealworm prey to three captive Madagascar crested ibises Lophotibis cristata urschi under a variety of trial conditions to discover whether they were using remote touch, mediated by their bill‐tip organ; chemosense/olfaction; or hearing to locate buried prey. The ibises were reliant on remote touch for prey detection – the first time this sensory system has been demonstrated for this group of birds. They did not appear to use hearing or chemical senses/olfaction to aid in prey detection.     

Desiccation tolerance in embryonic stages of the tardigrade

Desiccation tolerance commonly found among tardigrades allows them to cope with temporal variation of available water. Although the long-term survival of adults has been demonstrated in several species, desiccation tolerance of eggs and embryos is less well studied, however it is an important aspect from an ecological and evolutionary point of view. For the first time we evaluated the desiccation tolerance and subsequent hatching success of five different developmental stages of the tardigrade species Milnesium tardigradum , when rehydrated following drying at eight different humidity levels (10, 20, 31, 40, 54, 59, 72, 81%). Humidity level and developmental stage are significant factors in determining successful hatch rates. The results showed that the less developed stages were quite sensitive to desiccation. Low humidity levels during the first 3 days of development lead to a decrease in hatch rates following rehydration. Later developmental stages showed higher hatch rates than embryos dried at earlier stages. However, fast drying at low humidity levels resulted in delayed development and lower hatch rates following rehydration. In general, further developed embryos exhibit a better survival capacity compared with younger stages.     

Embryology and development of a freshwater prosobranch,Melania scabra

Embryology and development of the snail, Melania scabra was studied. The embryos complete their development in a special incubatory pouch called the broodpouch and hatch out in a form completely resembling that of the adult. The embryonic development of the snail represents specialized features like precocious development of the statocyst, presence of a well developed velar retractor muscle and torsion.     

Effects of environmental calcium deprivation on the egg masses of Physa marmorata Guilding (Gastropoda: Physidae) and Biomphalaria glabrata Say (Gastropoda: Planorbidae)

Eggs of the basommatophoran snails Physa marmorata and Biomphalaria glabrata were cultured in low concentrations of calcium to determine effects on growth and development. In both species there was some development in media with 0.12 mg/l Ca²⁺ but embryos were unable to hatch. 61.04% of embryos of P. marmorata could develop to hatching in 0.22 mg/l Ca²⁺ but those of B. glabrata required a level of 0.42 mg/l Ca²⁺, to attain even a 31.07% hatch. Marked effects on growth rate, embryo size and on time taken to achieve hatching were noted in both species at very low calcium levels. The possibility of cation-controlling mechanisms in the egg membrane is discussed.