Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses

Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga “Oophila amblystomatis” (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the ‘Oophila’ clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae.     

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae,Chlamydomonadales)

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.     

Symbiotic green algae in eggs of Hynobius nigrescens,an amphibian endemic to Japan

The egg capsules of some amphibians' eggs are known to become green colored before hatching. This is due to the increase of green symbionts in the egg capsule surrounding the embryo. The green symbionts in North American amphibian eggs were reported to be unicellular green algae in the Oophila‐clade of Volvocales, Chlorophyceae. However, it remains unclear whether this is also the case in other parts of the world. In this study, we analyzed the green symbionts in green‐colored eggs of Hynobius nigrescens, an amphibian endemic to Japan, obtained from five distinct locations. Microscopic observations revealed that the green symbionts were similar in appearance to Oophila amblystomatis, which was reported in some amphibian eggs in North America, in which non‐motile cells of the algae had thick cell walls with reticulate protuberances. PCR‐DGGE followed by phylogenetic analyses of partial 18S rRNA sequences revealed that the symbionts from the five locations were identical and most likely unialgal in each egg capsule. They formed an independent subclade within the Oophila‐clade, indicating that H. nigrescens has a unique symbiont. Our data are consistent with the previous report on North American amphibian eggs and support the specific symbiotic relationships between Oophila‐clade symbionts and the eggs of amphibians. This is the first report on the specific symbiont‐and‐host association between an Oophila‐clade symbiont and an amphibian outside of North America. We also discuss several possibilities regarding the origin of green symbionts (vertical transmission or invasion) on the basis of the discovery and detailed observation of H. nigrescens eggs without any green symbionts.     

Capsule walls as barriers to oxygen availability: Implications for the development of brooded embryos by the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

Encapsulation of developing embryos imposes potential restrictions, because the capsule wall must allow for adequate inward diffusion of oxygen and for increased diffusion of oxygen as metabolic demand increases with continued development. Samples of egg capsules from the gastropod Crepipatella dilatata were used to document surface characteristics, composition of the different capsule wall layers, and alterations in wall thickness during development. The diffusion coefficient and capsule wall permeability were determined experimentally for capsules containing embryos at different developmental stages. We also determined oxygen consumption rates for various embryonic stages and for nurse eggs, which provide food for embryos during development. The capsule wall of C. dilatata possesses 2 differentiated layers: the external capsular wall (ECW) and the internal capsular wall (ICW). The ECW is compact and fibrous, features that remain invariable during development, and lacks surface features that might make some portions of the capsule wall more permeable to oxygen than others. On the other hand, the ICW is initially spongy and thick, but significantly decreases in thickness over time, particularly before the embryos begin feeding on nurse eggs. Although the capsule wall is a serious barrier to diffusion, permeability to oxygen increases over time by 112% due to the dramatic thinning of the inner capsule wall layer. Nurse eggs consume oxygen but at very low rates, supporting the idea that they correspond to living embryonic cells that have stopped their development. Respiration measurements indicated that embryos are initially supplied with enough oxygen within the egg capsules to carry out the activities characteristic of embryogenesis, even though the capsular walls show their maximum thickness and lowest permeability at this time. However, as the embryo develops its velum and becomes more active, capsule wall thickness decreases and capsule permeability to oxygen increases. Correspondingly, the oxygen demands of metamorphosed but still encapsulated specimens are approximately 135% higher than those of pre-metamorphosed sibling embryos.     

Development of the Embryonic Envelopes of Mesocestoides lineatus (Cestoda: Cyclophyllidea)

Summary

Transmission electron microscopy revealed that the eggs of Mesocestoides lineatus consisted of an oncosphere larva surrounded by various coverings. The outermost of these was the embryonic capsule, which appeared as a thin electron-dense membranous sac. The capsule enclosed inner and outer embryonic envelopes, each of which was syncytial and apparently formed from embryonic blastomeres. The envelopes became increasingly vesiculated during embryogenesis, and were attached to each other by desmosomes by the time the larva was fully formed. An electron-dense intracellular embryophore was produced by the inner envelope; it first appeared under the distal plasma membrane as a series of blocks, which grew and fused to form a thick unbroken layer. Early in development, the proximal plasma membrane of the inner envelope was connected to the larval epithelium by a multilaminate membrane complex that was ultrastructurally similar to a continuous junction. At the end of embryogenesis, this appeared to detach from its formative cells on both sides to form the distinctive oncospheral membrane. Several eggs were bound together in clusters by a cluster capsule that was ultrastructurally identical to the individual embryonic capsules. This type of egg packaging has not been described previously for any cestode. Both the cluster and individual capsules broke down by the end of embryogenesis.     

The surface ultrastructure of the egg capsule of Transversotrema patialense (Transversotrematidae: Digenea)

Bundy D. A. P. 1981. The surface ultrastructure of the egg capsule of Transversotrema patialense (Transversotrematidae : Digenea). International Journal for Parasitology11: 19–22. Scanning and transmission electron micrographs show that the egg capsule of the digenean Transversotrema patialense bears thread-like extensions 3 μm long and 0.12 μm in diameter at a density of one per μm^?2. These extensions trap bacteria and detritus against the egg capsule surface. The ultrastructural topography of the egg capsules of this species differs from the forms previously described for other parasitic platyhelminths. It is suggested that the capsular sculpturing arises as an incidental consequence of moulding effects at the egg capsule-reproductive tract interface during ovogenesis.     

Population growth rate of the parasitic rotifer Proales gigantea,and susceptibility to parasitization in the snail Lymnaea acuminata at different stages of embryonic development

Developing eggs of the host snail Lymnaea acuminata were experimentally parasitized with the parasitic rotifer Proales gigantea to study the population growth rate of the parasite within the snail egg capsule and the susceptibility of the host eggs at different stages of embryonic development. The population growth rate of P. gigantea was 0.46 ± 0.07 individual^–1 day^–1 at the ambient temperature of 18–22 °C. Snail eggs were most susceptible to rotifer attack during the initial stages of development, becoming progressively more resistant after the hippo stage. Yet, regardless of the stage of development, the host embryo was doomed to die without hatching even if one individual rotifer gained entry inside the egg capsule. The presence of P. gigantea within the parasitized egg capsules or in the mucilage had no effect on the developmental rates and hatching success of non-parasitized eggs within the same egg mass.     

Über die Entwicklung der Eikapselvolumina bei verschiedenen Opisthobranchier-Arten (Mollusca,Gastropoda)

An attempt has been made to estimate the volumes of eggs and egg-capsules employing the formula for the rotation-ellipsoid, first after oviposition, second for the capsules during different stages of embryonic development until hatching. The results of the calculations permitted analysis of: (a) the relationship between egg-case volume and egg volume immediately after oviposition; (b) the size of capsules during development until hatching; (c) the relationship of the capsule volume at hatching in comparison to that after oviposition (enlargement-factor).Placida dendritica shows no increase in capsule volume during development.Gonidoris nodosa, Rostanga rufescens, Trinchesia viridis, Acanthadoris pilosa, Archidoris pseudoargus, Tritonia hombergi andAeolidia papillosa show a distinct increase in capsule volume at the time of velarcilia appearance. The degree of egg-case enlargement and the time of its beginning reveal interspecific differences; within some of the species, both also depend on the number of larvae present in the egg-case. The reasons for such behaviour are not yet known; some possible explanations are discussed.     

Parental size and environmental conditions affect egg capsule production by Nassarius reticulatus (Linnaeus 1758) (Gastropoda: Nassariidae)

In shallow coastal habitats scavenging netted whelks Nassarius reticulatus attached egg capsules to the stipes of red algae Chondrus crispus and occasionally on Furcellaria lumbricalis and Plumaria plumose. In the laboratory egg capsules were laid on aquaria sides and lids by individuals ≥ 21 mm shell length. Larger size classes produced more egg capsules and spawned over a longer period and in doing so partitioned less energy into shell growth. Large netted whelks (25-28.9 mm) produced larger capsules which contained significantly more and larger eggs than those produced by smaller individuals (21-24.9 mm). Egg capsule production continued throughout the year by regularly fed N. reticulatus held at ambient seawater temperatures. Egg production increased in the spring and summer with peak production during June (15 °C), decreased between August and October and resumed again during the winter (November to February at ∼ 7 °C). During the summer (15-16 °C) egg capsules were smaller and contained smaller eggs than those deposited during the winter (7-10 °C), although the number of eggs · capsule⁻¹ was similar. Enforced food limitation reduced the number and size of the egg capsules, the number and size of eggs produced · female⁻¹ and the duration of the breeding period. Hatching success of N. reticulatus egg capsules was high (95%) even at winter seawater temperatures (11-8.5 °C) and the duration of embryonic development was fastest between 15 and 17.5 °C.     

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

Summary

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.     

Embryonic development in the Patagonian red snail Odontocymbiola magellanica (Neogastropoda: Volutidae): Morphology and biochemistry

Embryo morphology, feeding mechanism and changes in composition of the egg capsule content during development (intracapsular fluids and embryos) were studied in Odontocymbiola magellanica from newly spawned egg capsules to the pre-hatching juvenile stage. Changes in embryo morphology and behavior are presented, based on observations and micrographs of living specimens and scanning electron microscopy. The arrangement of velar cilia and athrocytes and shell gland location and development differed markedly from other studied caenogastropods. Embryo ingestion of intracapsular fluid was promoted by velar ciliary currents at least from the early veliger stage, while feeding by grazing on the inner membranous layer of the egg capsule was rarely observed until juveniles were about to hatch. The main growth of embryos occurred during the veliger stages. A significant nutritional investment in egg capsules, as compared with other South American volutids was observed. Nutrition from proteins seemed to predominate at the expense of a high molecular weight fraction (>220 kDa). Calcium concentration in the intracapsular fluid remained constant during development, but notably, the total intracapsular content (i.e., the amount contained in both fluid and embryos) increased 3-fold, which may be explained by extraction from the egg capsule magnesium-rich calcite cover, or alternatively, by uptake of calcium from the surrounding sea water. Ammonia, a major end-product of nitrogen metabolism in marine invertebrates, was present in both embryos and intracapsular fluid, from which it may easily diffuse to the surrounding sea water through the egg capsule wall. Our results on embryo morphology, development and biochemical changes provide useful comparative data for evolutionary and developmental studies in the Volutidae as well as in other caenogastropods.     

Effects of oxidation and reduction on the spectral properties of the egg capsules of Raja erinacea Mitchill

At oviposition, egg capsules of Raja erinacea Mitchill were a deep greenish-brown. The dorsal wall was translucent while the ventral wall appeared transparent. Spectral analysis of capsulai material was performed by placing capsular specimens in microcuvettes in a scanning spectrophotometer and measuring absorbance at wavelengths in the visible spectrum between 350 and 900 nm. The capsule wall completely absorbed light between 350- and 460-nm wavelengths. At higher wavelengths, absorbance decreased while the amount of transmitted light increased. The dorsal wall absorbed more light > 460 nm than did the ventral wall because the dorsal wall was thicker and contained higher catechol concentrations. Reduction of capsule specimens with NaBH₄ decreased absorbance of light but did not alter the wall thickness or catechol concentration. Reduced specimens appeared transparent light green. Oxidation of capsule with FeCl₃ effectively increased absorbance of light > 460 nm so that little light of any wavelength passed through the specimen. In reflected light, FeCl₃-treated specimens appeared dark brownish-black, mimicking the natural darkening of capsules which occurs during incubation. These results indicate that R. erinacea egg capsules remain chemically reactive following oviposition. Capsules possess inherent redox potential at oviposition and contain catechols which can be oxidized to quinones forming dark pigments. These observations suggest that catechol oxidation plays a role in the change in color and transparency of R. erinacea egg capsules during incubation.     

Development and the influence of nurse egg allotment on hatching size in Searlesia dira (Reeve, 1846) (Prosobranchia : Buccinidae)

The reproductive biology of the intertidal prosobranch Searlesia dira (Reeve, 1846) was examined with special attention given to variability in the nurse egg to embryo ratio among capsules, among clutches and among geographically isolated populations. Embryos and nurse eggs were distributed among the capsules in a manner consistent with the hypothesis that nurse eggs were genetically predetermined, that each female had a genetically defined nurse egg to embryo ratio, and that each capsule represented a random sample of that ratio. The binomial distribution of embryos and nurse eggs among the capsules resulted in some capsules receiving many more embryos per nurse egg than others. The number of nurse eggs an embryo succeeded in eating was proportional to the number of capsule-mates sharing a capsule. Embryos eating more nurse eggs hatched out at a larger size. Differences in the nurse egg to embryo ratios among capsules in the same clutch were much larger than that of the mean ratios among clutches. Among-site differences in the mean nurse egg to embryo ratios suggest that selection pressure for different mean hatching sizes may have acted on the mean nurse egg to embryo ratios.In contrast to the predictions of optimal hatching size theory, hatching size varied widely within clutches as a consequence of differences in nurse egg to embryo ratios among capsules. This variance may be adaptive for species that lay their eggs months before juveniles emerge into an unpredictable environment, or simply be a consequence of an imperfect mechanism for increasing hatching size.     

The Combined Effects of Bacterial Symbionts and Aging on Life History Traits in the Pea Aphid,Acyrthosiphon pisum

While many endosymbionts have beneficial effects on hosts under specific ecological conditions, there can also be associated costs. In order to maximize their own fitness, hosts must facilitate symbiont persistence while preventing symbiont exploitation of resources, which may require tight regulation of symbiont populations. As a host ages, the ability to invest in such mechanisms may lessen or be traded off with demands of other life history traits, such as survival and reproduction. Using the pea aphid, Acyrthosiphon pisum, we measured survival, lifetime fecundity, and immune cell counts (hemocytes, a measure of immune capacity) in the presence of facultative secondary symbionts. Additionally, we quantified the densities of the obligate primary bacterial symbiont, Buchnera aphidicola, and secondary symbionts across the host''s lifetime. We found life history costs to harboring some secondary symbiont species. Secondary symbiont populations were found to increase with host age, while Buchnera populations exhibited a more complicated pattern. Immune cell counts peaked at the midreproductive stage before declining in the oldest aphids. The combined effects of immunosenescence and symbiont population growth may have important consequences for symbiont transmission and maintenance within a host population.     

Egg capsules of the dusky catshark Bythaelurus canescens (Carcharhiniformes,Scyliorhinidae) from the south‐eastern Pacific Ocean

The external morphology of the egg capsule of Bythaelurus canescens and its fixation to the substratum are described. Bythaelurus canescens egg capsules are typically vase‐shaped, dorso‐ventrally flattened, pale yellow in colour when fresh and covered by 12–15 longitudinal ridges. The anterior border of the capsule is straight, whereas the posterior border is semicircular. Two horns bearing long, coiled tendrils arise from the anterior and posterior ends of the capsule. The presence of longitudinal ridges and long coiled tendrils at both anterior and posterior ends of the capsule readily distinguish these egg capsules from those of other chondrichthyans occurring in the south‐east Pacific Ocean.     

Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida

The lumbricid earthworms (annelid family Lumbricidae) harbor gram-negative bacteria in their excretory organs, the nephridia. Comparative 16S rRNA gene sequencing of bacteria associated with the nephridia of several earthworm species has shown that each species of worm harbors a distinct bacterial species and that the bacteria from different species form a monophyletic cluster within the genus Acidovorax, suggesting that there is a specific association resulting from radiation from a common bacterial ancestor. Previous microscopy and culture studies revealed the presence of bacteria within the egg capsules and on the surface of embryos but did not demonstrate that the bacteria within the egg capsule were the same bacteria that colonized the nephridia. We present evidence, based on curing experiments, in situ hybridizations with Acidovorax-specific probes, and 16S rRNA gene sequence analysis, that the egg capsules contain high numbers of the bacterial symbiont and that juveniles are colonized during development within the egg capsule. Studies exposing aposymbiotic hatchlings to colonized adults and their bedding material suggested that juvenile earthworms do not readily acquire bacteria from the soil after hatching but must be colonized during development by bacteria deposited in the egg capsule. Whether this is due to the developmental stage of the host or the physiological state of the symbiont remains to be investigated.     

Morphological changes during diapause stages in the embryonic cortex of the annual killifish <Emphasis Type="Italic">Millerichthys robustus</Emphasis> (Cyprinodontiformes: Cynolebiidae) under natural conditions

The annual killifish inhabits in extreme locations with unpredictable rainy season where survives through the massive generation of embryos resistant of drought, capable to remain in a state of metabolic dormancy (three moments of diapause during embryonic development) protected by embryonic cortical structures: perivitelline space, egg envelope and its ornamented structures (trapeze-shaped projections and filaments in Millerichthys robustus). This research describes, for the first time, changes in cortical structures during three diapause stages in embryos of annual fish M. robustus during an annual life cycle. Embryos were collected in three periods through the year in a temporal water body: flood, drought and wet. During flood period all embryos were found in diapause I (during epiboly, dispersion of the blastomeres stage) with maximum thickness in all cortical structures and presence of egg envelope filaments. During drought period all embryos were in diapause II (development during somitogenesis, before the organogenesis) and its structures reduced its thickness significantly and lost the egg envelope filaments. Interestingly, embryos in diapause II and III (embryonic development completed in a pre hatching stage) were found during wet period (an example of bet-hedging strategy) in which all structures presented a recovery tending to its original condition observed during flood period. This research demonstrates that annual fish embryos respond to their exposure to seasonal environmental variations with dynamic structural changes that are fundamental for their survival.     

Response of Tetraselmis suecica to nutrient and grazer manipulation

Three methods of algal quantification (direct cell counts, chlorophyll a extraction, in vivo fluorescence) were used to evaluate the response of the unicellular green flagellate Tetraselmis suecica to nutrients and grazers. Nutrient enrichment enhanced total cell counts, chlorophyll a concentration and in vivo and DCMU-fluorescence. Photosynthetic efficiency was reduced in the complete F2 medium as indicated by the high level of in vivo fluorescence, whereas photosynthetic efficiency was increased by the introduction of mussels to the F2 medium. The addition of mussels significantly increased the proportion of non-motile cells, but did not reduce the total cell count. The effect of mussel grazing on algae could be underestimated if only total cells were counted or only the chlorophyll a concentration was measured. The results indicate that these three methods measure different properties of an algal culture and are complementary to each other in assessing the quality and quantity of an algal population. Direct algal counting offers a reliable numerical assessment for cell population abundance. Chlorophyll a concentration was closely correlated to the total cell count. In the presence of mussels, in vivo fluorescence did not correlate with either algal cell counts or chlorophyll a concentration, indicating that the measurement of in vivo fluorescence may be misleading for estimating algal abundance under different culture conditions. This revised version was published online in August 2006 with corrections to the Cover Date.