Many organisms naturally defend themselves against microbial attachment and biofouling in the marine environment. In this study, we investigated microbial fouling on 2 molluscan egg capsules using scanning electron microscopy (SEM), two-photon laser scanning microscopy (TPLSM) with bacterial viability staining and bacterial attachment experiments with the biofilm-forming Pseudoalteromonas sp. S91 in flow chambers. Results indicated that early stage egg capsules of Dicathais orbita (Neogastropoda) are relatively free of surface microorganisms. Egg capsules during the trocophore stage had a regularly ridged microtexture, but as capsules matured, shedding of the outer wall was observed, followed by the extrusion of unidentified droplets, which then accumulated on the capsule surface in association with bacteria. By comparison, the egg capsules of Sepioteuthis australis (Cephalopoda) were found to have an irregular surface with many hills and valleys that accommodate colonization by a variety of microorganisms. At the later stages of development these squid egg capsules become heavily colonized by algal spores. Cross sections of egg capsules revealed that S. australis capsule walls were about 12 times thicker than D. orbita egg capsules. Staining the egg capsules with BacLight™ also revealed a significantly thicker biofilm, with more live and dead bacteria on S. australis capsules than on those of D. orbita (p < 0.05). Flow chamber experiments indicated that the surface of S. australis capsules provided a suitable substrate for colonization by Pseudoalteromonas sp. S91, whereas colonization was significantly less on D. orbita egg capsules after 24 and 72 h (p < 0.01). These experiments indicated that D. orbita egg capsules are better defended against fouling microbes than are the eggs of S. australis. D. orbita appears to use a combination of physical, mechanical and possibly chemical defense mechanisms to reduce fouling on their egg capsules.KEY WORDS: Biofilm · Egg capsules · Mollusc · Scanning electron microscopy · Two-photon laser scanning microscopy · Bacterial attachment Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 47: [275][276][277][278][279][280][281][282][283][284][285][286][287] 2007 an estimated density of 5 × 10 5 prokaryotic cells ml -1seawater (Whitman et al. 1998), sessile invertebrates and algae are exposed to a constant onslaught of potentially detrimental microbes. These include biofilm-forming bacteria along with single-cell diatoms that rapidly settle, attach and form colonies on any surface placed in the marine environment (Davis et al. 1989). The formation of a microbial biofilm promotes the attachment of algal spores, protozoa, barnacle cyprids and marine fungi, followed by the settlement of other marine invertebrate larvae and macroalgae (Maki 2000, Callow & Callow 2002. Heavy surface fouling could lead to the accumulation of toxic wastes, a reduction in oxygen and nutrient availability and increased drag, which can cause sessil...

Pseudoalteromonas sp. S91 is a marine bacterium known to secrete chitinases and proteases, hydrolytic enzymes responsible for the degradation of chitin and protein, respectively, which enable access to nutrients contained in chitinous materials such as squid pen. In a dual species biofilm grown on squid pen, Pseudoalteromonas sp. S91 was able to support the accumulation of Vibrio sp. S141, which is unable to degrade squid pen but able to metabolise the chitin subunit N '-acetylglucosamine (GlcNAc), a product of squid pen hydrolysis. When grown on a glass substratum in the presence of a soluble carbon source that only Pseudoalteromonas sp. S91 could use, its biofilm provided no support to Vibrio sp. S141. KEY WORDS: Chitin · Biofilm · Marine bacteria · CommensalismResale or republication not permitted without written consent of the publisher

Green fluorescent protein (gfp) tagging has enabled the spatial analysis of different bacterial species within biofilms [1]. The use of GFP in conjunction with a Two-Photon Laser Scanning Microscope (TPLSM) facilitates the real time analysis of bacterial cells within a fully hydrated, living biofilm grown in a flow chamber [2]. Biofilms consist of vertically complex aggregates of microbial cells and secreted polymer attached to, and growing on, a surface or at an interface exposed to an aqueous phase [3].This study investigated whether the chitinolytic marine bacterium Pseudoalteromonas sp. S91, which is able to completely hydrolyse squid pen can support Vibrio sp. S141 or Psychrobacter sp. SW5 in mixed species biofilms growing on squid pen in once through continuous flow chambers. Patterns of attachment on squid pen for gfp tagged Vibrio or gfp tagged Psychrobacter grown in monoculture were determined. Neither strain can grow on squid pen as the sole carbon source. Numbers of gfp tagged cells in mixed species biofilms containing S91 were determined.Images of the bacterial biofilms were collected using a Bio-Rad Radiance 2000MP visualising system in conjunction with a Nikon Eclipse TE300 inverted microscope. The microscope was equipped with a 60x water immersion lens with a numerical aperture of 1.2 and a Coherent Mira900-F titanium:sapphire ultrafast laser which has an excitation spectrum of 700 -980 nm. An excitation wavelength (λ) at 800 nm was used with an emission λ of 515 -530 nm, detected as green, for GFP visualisation and an emission λ 450 -480 nm, detected as blue, for squid pen autofluorescence. Image analysis was performed using ImageJ 1.32j [4] computer image analysis software. Numbers of GFP + bacteria in TPLSM images were quantified, essentially as done by Fitch et al. [5]; GFP + SW5, S91 and S141 were grown in liquid monoculture and separately viewed as wet mounts using the TPLSM.This study showed that the motile marine bacterium S141, unable to efficiently metabolise squid pen, was supported by S91. After 72 h, following various time periods of colonisation and growth within flow chambers (Fig. 1a), S141 cell numbers within dual species biofilms exhibited on average a 5-fold increase in the presence of S91. GFP + S141 cells were localised on the pen surface in monoculture. In the presence of S91, S141 cells were displaced or prompted to migrate away from the pen surface and attach to the vertically complex S91 biofilm (Fig. 1d).In contrast, the marine bacterium SW5 was unable to be supported by S91 when grown in a dual species biofilm on squid pen. After 72 hours exposed to squid pen in monoculture, SW5 cells occurred singly and in small clusters in patches over the pen surface (Fig. 1b). In the presence of S91, SW5 cells were either absent or exhibited a significant decrease in cell numbers.[1] A.