Biology

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The sub-cellular and humoral concentrations of a collectin-like protein from the solitary tunicate, Styela plicata, were measured after in vivo challenge with the inflammatory agent, zymosan. Tunicates were injected with zymosan before hemocytes and serum were harvested, subjected to western blotting and immunostained with an anti-S. plicata collectin antibody to determine the relative titers of collectin-like proteins. Concentrations of the predominant 43 kDa collectin polypeptide were found to decrease in hemocytes immediately after zymosan injection, before rising to levels that were six times higher than controls within 96 h. Similarly, immunohistochemistry showed that the frequency of collectin-positive hemocytes in the circulating hemolymph increased significantly within 96 h of injection. Levels of the 43 kDa polypeptide in serum mirrored those of hemocytes. Humoral collectin concentrations decreased immediately after zymosan injection before rising, within 96 h post-injection, to levels three times higher than controls. This response to an inflammatory stimulus resembles that of mammalian collectins like mannose-binding lectin. The data suggest that, like its mammalian counterparts, the tunicate collectin acts as an acute phase antigen recognition protein.

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

Proteins with obvious similarities to mammalian complement are widely distributed in the animal kingdom. In the vertebrate lineage, deuterostomes like sea urchins and tunicates express proteins that are homologues of C3, the central component of the vertebrate complement cascade. Their genomes also encode molecules resembling factor B from the "alternative" complement activation pathway; and tunicates have collagenous lectins of the type that can activate complement in the absence of antibodies. This suggests that the core components of the complement system evolved before antibodies, which first appear in jawed fish.

Toxic metals, such as tributyltin (TBT), contribute substantially to anthropogenic pollution in many estuarine environments. Animals that live in those environments, particularly invertebrate filter feeders like tunicates, are likely to be exposed to substantial metal contamination. This study investigates the effects of TBT and other metals on the phenoloxidase activity of the estuarine tunicate, Styela plicata, in an effort to identify a biochemical marker of metal pollution. Hemocytes harvested from S. plicata that were exposed to tributyltin or copper in aquaria had significantly enhanced phenoloxidase activities relative to non-exposed controls. This enhanced phenoloxidase activity could be explained by an increased frequency of morula cells, which contain high levels of phenoloxidase's proenzyme, prophenoloxidase. Unlike those from tunicates exposed to metals in aquaria, the phenoloxidase activities of hemocytes incubated with tributyltin in vitro were significantly reduced when compared with hemocytes cultured without tributyltin. The ability of tributyltin to decrease phenoloxidase activity in tissue culture may reflect its known inhibitory effects on calcium-dependent signaling systems such as those involved in the exocytosis of prophenoloxidase from morula cells.

This study identi®es a complement component C3-like protein in the solitary tunicate, Styela plicata. Three different polyclonal antibodies raised against C3 molecules from two species (humans and the tunicate, Halocynthia roretzi) were used to identify the C3-like protein in S. plicata hemolymph. The C3 cross-reactive protein is a 170 kDa heterodimer composed of polypeptides (116 and 80 kDa) that have molecular weights comparable to those of C3a and C3b from other species. Amino acid sequencing and amino acid composition analysis con®rmed that the C3-like protein from S. plicata is closely related to C3 from H. roretzi.

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an 'inducer' bacterium stimulated the production of antimicrobial molecules by 'producer' bacteria that were active against the 'inducer' bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.

The Sydney rock oyster, Saccostrea glomerata, is susceptible to infection by the protozoan parasite, Marteilia sydneyi, the causative agent of QX disease. M. sydneyi infection peaks during summer when QX disease can cause up to 95% mortality. The current study takes a proteomic approach using 2-dimensional electrophoresis and mass spectrometry to identify markers of QX disease resistance among Sydney rock oysters. Proteome maps were developed for QX disease-resistant and-susceptible oysters. Six proteins in those maps were clearly associated with resistance and so were characterized by mass spectrometry. Two of the proteins (p9 and p11) were homologous to superoxide dismutase-like molecules from the Pacific oyster, Crassostrea gigas, and the Eastern oyster, Crassostrea virginica. The remaining S. glomerata proteins had no obvious similarities to known molecules in sequence databases. p9 and p11 are currently being investigated as potential markers for the selective breeding of QX disease-resistant oysters.

This study investigates the exocytic responses of invertebrate hemocytes to pathogen-associated antigens. It demonstrates that a homologue of complement component C3, a key defensive protein of the innate immune system, is expressed by phagocytic hemocytes (non-refractile vacuolated cells) of the tunicate, Styela plicata. C3-like molecules are localized in subcellular vesicles and are rapidly exocytosed after stimulation with bacterial, fungal or algal cell surface molecules. Signal transduction analysis indicated that the induced secretion of C3-like molecules is mediated by a G-protein dependent signaling pathway, which modulates tubulin microtubules. All of this evidence indicates that hemocytes can contribute to host defense responses by rapidly exocytosing C3-like proteins at sites of infection.

Genome sequences and high diversity cDNA arrays have provided a detailed molecular understanding of immune responses in a number of invertebrates, including sea urchins. However, complementary analyses have not been undertaken at the level of proteins. Here, we use shotgun proteomics to describe changes in the abundance of proteins from coelomocytes of sea urchins after immunological challenge and wounding. The relative abundance of 345 reproducibly identified proteins were measured 6, 24 and 48 h after injection. Significant changes in the relative abundance of 188 proteins were detected. These included pathogen-binding proteins, such as the complement component C3 and scavenger receptor cysteine rich proteins, as well as proteins responsible for cytoskeletal remodeling, endocytosis and intracellular signaling. An initial systemic reaction to wounding was followed by a more specific response to immunological challenge involving proteins such as apolipophorin, dual oxidase, fibrocystin L, aminopeptidase N and a-2-macroglobulin.

In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring.

Ostreid herpes virus causes serious disease in the Pacific oyster (Crassostrea gigas), but not in the Sydney Rock Oyster (Saccostrea glomerata). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas, and that the gill tissue is an important site of dsRNA processing and response.

Many social birds produce food-associated calls. In galliforms, these vocalizations are typically accompanied by a distinctive visual display, creating a multimodal signal known as tidbitting. This system is ideal for experimental analysis of the way in which signal components interact to determine overall efficacy. We used high-definition video playback to explore perception of male tidbitting by female fowl, Gallus gallus. Hens experienced four treatments consisting of multimodal tidbitting, visual tidbitting without sound, audible tidbitting without a male present, and a silent empty cage control. Hens took longer to begin food search when the display was silent, but the overall rate of this response did not differ among the multimodal, visual only or audio only playback treatments. These results suggest that the visual and vocal components of tidbitting are redundant, but they also highlight the importance of a temporal dimension for any categorization scheme. Visual displays also evoked inspection behavior, characterized by close binocular fixation on the head of the playback male, which is known to facilitate individual recognition. This may also allow hens to assess male quality. Such social responses reveal that tidbitting probably has multiple functions and provide a new insight into the selective factors responsible for the evolution of this complex multimodal signal.

With the notable exception of bee dances, there are no established examples of multimodal referential signals. The food calls of male fowl, Gallus gallus, are functionally referential and the acoustic component of a multimodal display. However, the specificity of the receiver's response to the visual component (tidbitting) has never been tested. Here we provide the first detailed analysis of tidbitting, and test the hypothesis that these characteristic movements are functionally referential. We conducted a playback experiment with five high-definition video stimuli: Silent tidbit, Matched-frequency motion in the opposite direction, Silent crows, Inactive male and Empty cage. Females searched for food more during Silent tidbitting than under any other condition, suggesting that this visual display specifically predicts the presence of food and hence has similar functional properties to food calls. Silent tidbitting was also singularly effective at evoking approach and close inspection, which may enhance signal memorability. These social responses suggest that the visual component of the display has the unique function of triggering assessment of signaler identity and quality as a potential mate. The acoustic and visual components are hence redundant as a food signal, but synergistic when additional functions are considered. These findings emphasize the perceptual complexity of multimodal displays and provide the first demonstration of multimodal referential signaling in a vertebrate.

The presence of eavesdroppers within a communication network can increase the costs associated with signalling. Hence, selection should favour the ability to vary signal structure with social context. One possible mechanism is the flexible combination of the components that form a multimodal signal. This phenomenon clearly occurs in social mammals, particularly primates, and has been identified as one of the foundation elements for the evolution of complex communication. However, this flexibility in signal component composition in relation to social context has not previously been demonstrated in other taxa. Here we show that subordinate male fowl, Gallus gallus, show facultative variation in the structure of their multimodal signals. Intriguingly, signallers were not sensitive to the behaviour of the intended receivers (hens) but rather to the attentional state of eavesdropping rival males. Subordinates switched from multimodal displays (movements and calls) to unimodal (silent) displays when the alpha male was attentive. Unimodal and multimodal displays had equivalent efficacy in attracting hens, but multimodal signals were associated with more rapid approach by the alpha male and increased probability of severe attack. Variation in signal type is hence driven by social costs. This is the first demonstration of facultative multimodal signalling in birds. Ó

Many animal signals have been produced by selection operating upon behavioural precursors that lack signal function, a process known as 'ritualization'. When the precursor remains in the repertoire, comparisons of its structure with that of the derived signal permit inference of the forces responsible. Selection for signal efficacy and recognition produce characteristic changes (e.g., increases in intensity and duration, and repetition). The tidbitting display of male fowl, Gallus gallus, has long been considered an example of ritualization. However, the changes associated with this evolutionary process have never been characterized, nor has there been any test of whether the signal is perceived as distinct from its putative precursor (self-feeding). To address this, we first analysed video recordings of males feeding and tidbitting. We found a significant increase in the intensity, duration and repetition of specific movements during the tidbitting display compared to feeding. These results suggest that tidbitting is a ritualized form of self-feeding behaviour, but the critical test requires assessment of receiver response. If females recognized the tidbitting signal as distinct from male feeding behaviour, we predicted that females would food search significantly more in response to tidbitting. The results of high-definition video playbacks support this hypothesis. This is the first empirical demonstration of the perceptual and functional predictions made by a classic ethological hypothesis.

Alarm calling is a classic problem in evolutionary biology. Although a signaller may increase the likelihood of survival for group members, which typically include kin and mates, there are inherent risks associated with any behaviour that increases conspicuousness to predators. Callers can increase their indirect benefits by calling only in the presence of an appropriate audience and manage concomitant costs by judicious investment. Possible tactics for controlling costs include facultative variation in call structure and timing, as well as sensitivity to the environmental and social factors that predict personal vulnerability. We examined individual variation in the alarm-calling behaviour of male fowl in naturalistic social groups. Previous studies of cost management have focused on variation at the level of alarm call rate. We took advantage of recent advances in wireless sound recording and remote video monitoring to test for more subtle variation in signal structure and timing. These were then mapped onto individual mating success and moment-to-moment changes in environmental and social context. Results replicate the previous finding that alarm calling is sensitive to both social rank and recent mating success. In addition, we detected systematic variation in call structure as a function of personal vulnerability and proximity to a rival male. The frequency bandwidth of alarms was reliably influenced by degree of vigilance prior to calling, suggesting that this acoustic dimension reflects motivational state. Taken together, these results reveal several novel tactics for risk management, complementing those previously described at the level of gross variation in alarm-calling behaviour.

Alarm calls and food-associated calls from a diverse range of species are said to be functionally refer- 68 ential, in that receivers can use these sounds to predict environmental events in the absence of other 69 contextual cues. The evolutionary driver for referential alarm calls has been hypothesized to be the 70 mutually incompatible escape behaviours required to avoid different predators. However, some species 71 produce acoustically distinctive and referential alarm calls but do not show highly referential abilities in 72 other domains. We examined whether food-associated calls in many species are likely to be functionally 73 referential and whether they specifically communicate about characteristic features of food. Food- 74 associated calls are given in both feeding and nonfeeding contexts, and the types of information con- 75 tained vary greatly. Most species do not produce unique calls for different foods; more common is 76 variation in the call rate, which suggests that call structure reflects the callers’ internal state rather than 77 the food type. We also examined the ultimate function of food-associated calls to evaluate whether there
is a unifying explanation for the evolution of functionally referential food calls. Based on the literature, 78 there does not appear to be a unifying function. In conclusion, while functionally referential food- 79 associated calls have been convincingly demonstrated in a few species, it is more common for these 80 vocalizations to reflect arousal rather than additionally providing specific referential information about 81 the feeding event. At this point, there is no compelling hypothesis to explain the evolution of functionally 82 referential food-associated calls. Given the multiple functions of food-associated signals, we should not 83 expect a unitary explanation.