Michael G. Hadfield

Animals in a bacterial world, a new imperative for the life sciences

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal–bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other’s genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal–bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

The apical sensory organ of a gastropod veliger is a receptor for settlement cues.

On the basis of anatomy and larval behavior, the apical sensory organ (ASO) of gastropod veliger larvae has been implicated as the site of perception of cues for settlement and metamorphosis. Until now, there have been no experimental data to support this hypothesis. In this study, cells in the ASO of veliger larvae of the tropical nudibranch Phestilla sibogae were stained with the styryl vital dye DASPEI and then irradiated with a narrow excitatory light beam on a fluorescence microscope. When its ASO cells were bleached by irradiation for 20 min or longer, an otherwise healthy larva was no longer able to respond to the usual metamorphic cue, a soluble metabolite from a coral prey of the adult nudibranch. The irradiated cells absorbed the dye acridine orange, suggesting that they were dying. When larvae not stained with DASPEI were similarly irradiated, or when stained larvae were irradiated with the light beam focused on other parts of the body, there was no loss of ability to metamorphose. Together these data provide strong support for the hypothesis. Potassium and cesium ions, known to induce metamorphosis in larvae of many marine-invertebrate phyla, continue to induce metamorphosis in larvae that have lost the ability to respond to the coral inducer due to staining and irradiation. These results demonstrate that (1) the ASO-ablated larvae have not lost the ability to metamorphose and (2) the ions do not act only on the metamorphic-signal receptor cells, but at other sites downstream in the metamorphic signal transduction pathway.

EXCESS POTASSIUM INDUCES LARVAL METAMORPHOSIS IN FOUR MARINE INVERTEBRATE SPECIES

An increase in the concentration of K+ in defined seawater medium induces settlement and metamorphosis in larvae of the marine molluscs Phestilla sibogae, Haliotis rufescens, and Astraea undosa, and in larvae of the marine annelid Phragmatopoma californica. The effect is dose-dependent, optimal at approximately double the normal concentration of K+ in seawater, and specific for the K+ ion. The ability of K+ to directly influence cell membrane potential is proposed as an explanation for its broad effectiveness as a metamorphic inducer for larvae that recruit to different habitats. Depolarization of externally accessible, excitable cells thus is suggested to be a mechanism common to the induction of settlement and metamorphosis of a number of species. For Phestilla and Haliotis, the inductive effect of excess K+ is additive with that of the substratum-derived inducers or analogs. The sensitivity of induction by K+ to external tetraethylammonium (TEA, a K+-channel blocker) reported previously for Haliotis (B...

Settlement requirements of molluscan larvae: New data on chemical and genetic roles

Abstract Recent studies in the authors laboratory and elsewhere indicate that a high degree of substratum chemical specificity is necessary to induce settlement and metamorphosis in a number of molluscan species. The chemical nature of natural and artificial inducers, as well as the nature of inducer-larval interactions, provides insights into both stimulus and response aspects of the metamorphic process. Larvae of Phestilla sibogae undergo rapid and complete metamorphosis in response to a soluble, coral-produced substance. These larvae undergo slow but complete metamorphosis in response to choline, GABA, and some related compounds; they exhibit partial metamorphosis in the presence of certain catecholamines. Reversible inhibition of metamorphosis (habituation) occurs when larvae are exposed to an inducer prematurely. Analysis of these observations allows the formulation of new models for molluscan metamorphic induction. It may be that a single model will not be appropriate for all marine molluscs. Both age at metamorphic competence and concentration of natural inducer necessary to elicit metamorphosis in competent larvae of Phestilla are known to show wide variance among larvae. Long-term selective inbreeding experiments have failed to indicate obvious genetic components of the observed variances. High variance in age at competence occurs even among the egg masses of a single, highly inbred individual with a single mate.

Metamorphosis of the marine gastropod Phestilla Sibogae Bergh (nudibranchia: aeolidacea). I. Light and electron microscopic analysis of larval and metamorphic stages

Abstract The structure and fate of transitory larval organs (velum, shell, operculum, retractor muscles, part of the epidermis) of Phestilla sibogae Bergh were studied before, during, and after metamorphosis with both light and electron microscopy to elucidate the morphology of these organs and the mechanisms by which they are lost. Loss of the velar lobes is the first morphological sign of metamorphosis, and involves selective dissociation and subsequent ingestion of the ciliated velar cells; the remaining aggregate of supportive cells is apparently incorporated into cephalic epidermis. Attachment of the larval body to shell and operculum is primarily at sites of retractor muscle insertions; once the velum is gone, the attachment between shell and larval body is lost and the shell is cast off as the visceral organs exit through the shell aperture. Merger of visceral and cephalopedal elements results in flattening of the postlarval body and reorientation of internal organs. Simultaneously, a rapid spreading of epipodial epidermis over the lateral, dorsal, and posterior sides of the body produces the definitive integument. The squamous cells which comprise the larval perivisceral epidermis are pushed ahead of the definitive epidermis and are seen shortly after the shell is cast as a constricted aggregate of cells on the posterior end of the body. Autolysis of the left and right retractor muscles begins during metamorphosis and no trace of them is left after 24 to 48 h. The metapodial mucous glands which hypertrophy before metamorphosis are also lost within 48 h following exit of the post larva from the shell. Metamorphosis produces a detorsion caused in part by muscular action and in part by continuing growth and development.

Observations on development, larval growth and metamorphosis of four species of aplysiidae (gastropoda: Opisthobranchia) in laboratory culture

The development of simple, reliable techniques for the laboratory culture of aplysiid gastropods through their complete life cycle, has enabled us to study the larval biology, metamorphosis, and early juvenile development of these animals. Egg masses, duration of the embryonic phase, veligers, and larval growth and development are described for four species of Hawaiian Aplysiidae, namely, Aplysia dactylomela Rang, Aplysia Juliana Quoy and Gaimard, Dolabella auricularia (Lightfoot) and Stylocheilus longicauda (Quoy and Gaimard). Metamorphosis and early juvenile development of A. Juliana are described in detail with additional comments on these processes in the other three species. Length of the embryonic phase and size of the veliger at hatching are a function of the size of the uncleaved egg. All four species develop planktotrophically and have ≈ 30-day larval phases. In each species the larval phase includes a period of rapid shell growth to a species-specific size followed by a non-growth period during which other morphological developments occur to culminate in metamorphic competence. The larvae of each species metamorphose preferentially on a particular species of benthic algae. The events of metamorphosis require 2 to 4 days for completion and transform the planktonic filter-feeding larva into a benthic, radular-feeding juvenile. Postlarval development includes growth of the shell, parapodia, oral tentacles, rhinophores, anal siphon, and structures of the mantle cavity.

Microbial Biofilms Facilitate Adhesion in Biofouling Invertebrates

Much interest has focused on the role of microbial layers—biofilms—in stimulating attachment of invertebrates and algae to submerged marine surfaces. We investigated the influence of biofilms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test surfaces that were either clean or coated with a natural biofilm. Measuring larval removal under precisely controlled flow forces, we found that biofilms significantly increased adhesion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attachment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the presence of a biofilm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research.

Chemical Interactions in Larval Settling of a Marine Gastropod

Prior to the 1930s, it was a generally accepted notion that larvae of benthic marine invertebrates were, in the timing of their metamosphosis, at the mercy of chance. If the ocean’s currents carried them over substrata suitable for adult life when the time for metamorphosis arrived, they survived; if the substrate were not suitable, the larvae perished. Beginning with observations of Mortensen1 and Day and Wilson2, students of marine ecology began to see that the situation relative to larval settling was more controlled, that larvae could execute a “choice” of substratum. It was additionally recorded that larvae could actually delay metamorphosis until suitable substrata were found3.

Rapid Behavioral Responses of an Invertebrate Larva to Dissolved Settlement Cue

Larvae of the nudibranch Phestilla sibogae were used to study whether a natural dissolved settlement cue (from their prey, Porites compressa, an abundant coral on Hawaiian reefs) induces behavioral responses that can affect larval transport to suitable settlement sites. As cue and larvae are mixed in the turbulent flow over a reef, cue is distributed in fine-scale filaments that the larva experiences as rapid (seconds) on/off encounters. To examine larval responses in this setting, individual larvae were tethered in a small flume with flow simulating water velocity relative to a freely swimming larva, and their responses to realistic temporal patterns of cue encounter were videotaped. Competent larvae quickly ceased swimming in cue filaments and resumed swimming after exiting filaments. The threshold cue concentration eliciting a response was 3%–17% of concentrations within heads of P. compressa in nature. When moving freely in filtered seawater, competent larvae swam along straight paths in all directions at ∼0.2 cm s−1, whereas in water conditioned by P. compressa, most ceased swimming and sank at ∼0.1 cm s−1. The ability of larvae to rapidly respond (by sinking) to brief encounters with dissolved settlement cues can enhance their rapid transport to the substratum, even in wave-driven turbulent flow.

The vermetidae (Mollusca: Gastropoda) of the Hawaiian Islands

The Hawaiian vermetid fauna comprises 8 species, 7 of which are here described as new. The generic distribution includes 5 species of Dendropoma and 1 each of Petaloconchus, Vermetus and Serpulorbis. The species descriptions rely little on conchology, stressing instead descriptions of animals, habitats and reproductive and developmental characteristics. Feeding is accomplished in all species by a combination of mucous nets and detrital collection by ctenidial cilia. Only in the single species of Vermetus, an inhabitant of quiet waters, does ciliary feeding predominate. Four small species of Dendropoma inhabit shallow, coralline algal-encrusted, wave-washed reef areas, while Serpulorbis and Dendropoma platypus are found not only in intertidal areas subjected to heavy surf, but subtidally to depths of 10 m or more. The single species of Petaloconchus is a characteristic associate of the 4 Dendropoma spp. of shallow waters but, being strongly associated with the coral Porites, Petaloconchus also extends subtidally. The Hawaiian vermetids are very abundant in some localities, with densities ranging up to 60,000/m3 in one species of Dendropoma. Reproduction is continuous in all Hawaiian vermetids, most of which produce small hatching juveniles rather than swimming veligers. Only Serpulorbis and Vermetus have obligatory planktonic stages. Petaloconchus and Dendropoma species may produce a mixture of hatching juveniles and short-term planktonic veligers. Larval or juvenile size is correlated with available nurse yolk, not with egg size.