Comparison of surface microfouling and bacterial attachment on the egg capsules of two molluscan species representing Cephalopoda and Neogastropoda

Abstract

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...

Histochemical correlations between egg capsule laminae and the female gonoduct reveal the process of capsule formation in the Muricidae (Neogastropoda: Mollusca)

“…Undulations in the surface of L1 (Fig. 4a) correlate with ridges observed on the exterior of D. orbita capsules by SEM (Lim et al, 2007). Surface corrugations have also been detected in Muricinae capsules and are thought to result from pedal molding (D'Asaro, 1988), which may explain the mixture of orientated and unorientated fibers observed in L1 of D. orbita capsules.…”

“…4b) capsules. Degradation and finally delamination of this outer fibrous layer has also been observed in SEM analyses of veliger stage D. orbita capsules (Lim et al, 2007). Shedding of this outer fibrous lamina was thought to occur in response to excessive fouling (Lim et al, 2007).…”

“…A thin layer (L0) of proteinaceous material was observed on surface of fresh egg capsules. Previous research into the ultrastructure of D. orbita egg capsules (Lim et al, 2007), revealed a "crust" on the surface of #1-week-old capsules. The age-dependent correlation between changes observed in SEM micrographs by Lim et al (2007) and those observed in this histochemical study strongly suggest that the previously described "crust" is the outer lamina layer L0 detected in this investigation (Fig.…”

“…Muricid capsules are typically composed of 3-4 proteinaceous and carbohydrate-rich laminae. The outermost fibrous laminae (L1-2) impart protection (Sullivan and Maugel, 1984;D'Asaro, 1988;Garrido & Gallardo, 1993;Rawlings, 1995;Ram et al, 2000;Lim et al, 2007) and structural support (D'Asaro, 1988) to the capsule. The third lamina (L3) is homogeneous in composition and often considered an extension of the apical plug, which is part of the hatching mechanism in many species (D'Asaro, 1988).…”

Cooperative interactions within a marine bacterial dual species biofilm growing on a natural biodegradable substratum

“…Following completion of flow chamber experiments, images of the bacterial biofilms within the chambers 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 60× water immersion lens with a numerical aperture of 1.2 and a Coherent Mira900-F titanium:sapphire ultra fast laser, which has an excitation spectrum of pulsed 800 nm light equivalent to 1 photon of 400 nm light, suitable for exciting GFP without damaging cells (Lim et al 2007). An excitation wavelength (λ) of 2 photons of 800 nm and an emission λ of 515 to 530 nm was used to visualise GFP.…”