> Abstract The intensive rearing of various fish species in aquaculture has revealed intimate relationships between fish and bacteria that eventually may affect establishment of a "normal" mucosal microflora or result in disease epizootics. Interactions between bacteria and mucosal surfaces play important roles both at the egg and larval stages of marine fish. Bacterial adhesion and colonization of the egg surface occur within hours after fertilization. The diverse flora which eventually develops on the egg appears to reflect the bacterial composition and load of the ambient water, but species-specific adhesion at the egg surface may also play a role in development of the egg epiflora. Proteolytic enzymes produced by members of the adherent epiflora may cause serious damage to the developing egg and may also affect further adhesion of the epiflora. Ingestion of bacteria at the yolk sac stage results in establishment of a primary intestinal microflora which seems to persist beyond first feeding. Establishment of a gut microflora is likely to undergo several stages, resulting in an "adult" microflora weeks to months after first feeding. Ingested bacteria may serve as an exogenous supply of nutrients or essential factors at an early life stage. Early exposure to high bacterial densities is probably important for immune tolerance, and thus for the establishment of a protective intestinal microflora. Successful rearing of early life stages of several marine fish species depends on knowledge of the complex interactions among the cultured organisms and the bacterial communities which develop at the mucosal surfaces and in the ambient water and rearing systems. The routine use of antibiotics during rearing of fish larvae is not advisable, since it may increase the risk of promoting antibiotic resistance and adversely affect the indigenous microflora of the larvae. The use of probiotics has proven advantageous in domestic animal production, and the search for effective probiotics may have a great potential in aquaculture of marine organisms. Bacteria with antagonistic effects against fish pathogens have been successfully administered to several fish species, resulting in decreased mortality or increased growth rate.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p1.html

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibriofischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.

A psychrotrophic Ffexiibacter sp., Flexibacter ovolyticus sp. nov., was isolated from the adherent bacterial epiflora of Atlantic halibut (Hippoglossus hippoglossus L.) eggs and was shown to be an opportunistic pathogen for halibut eggs and larvae. The strains which we isolated had the enzymatic capacity to dissolve both the chorion and the zona radiata of the egg shells. A total of 35 isolates were characterized by using morphological and biochemical tests. These strains were rod shaped, gram negative, Kovacs oxidase positive, and pale yellow and exhibited gliding motility, They did not produce acid from any of the wide range of carbohydrates tested. Our isolates had the ability to degrade gelatin, tyrosine, DNA, and Tween 80. Starch, cellulose, and chitin were not degraded. The strains were catalase and nitrate reductase positive, did not produce H,S, and did not grow under anaerobic conditions. F. ovofyticus resembles Ffexiibacter maritimus, but differs from the latter species in several biochemical and physiological characteristics. DNAs from F. ovofyticus strains had guanine-pluscytosine contents which ranged from 30.3 to 32.0 mol% (strains EKC001, EKD002T [T = type strain], and VKB004), and DNA-DNA hybridization studies revealed levels of relatedness between F. ovoZyticw EKD002T and F. maritimus NCMB 2154T and NCMB 2153 of 42.7 and 30.0%, respectively. Compared with previously described CytophQga and FZexibacter spp. with low guanine-plus-cytosine contents, F. ovofyticus constitutes a new species. Strain EKD002 (= NCIMB 13127) is the type strain of the new species.

Scanning electron micrographs of halibut eggs with an epiflora dominated by Flexibacter sp., showed ulcerations colonized by large numbers of bacteria. The chorion was dissolved in most ulcerations and the zona radiata was severely damaged. Infection experiments showed that exposure to these bacteria caused high mortalities at the late egg stage, hatching and the early yolk sac stage. Eggs exposed to three different Vibrio spp. showed different mortality patterns, with low mortality at hatching, followed by a eontinuous high mortality throughout the yolk sac stage. Mortality in the uninfected control group was low throughout the experiment. Transmission electron microscopy of the l''/7>no-infccted larvae showed baeteria present in the gill, heart and frontal yolk sae regions.

The aerobic intestinal microflora of 2-week-old herring (Clupea harengus) larvae was characterized by using conventional microbiological methods and electron microscopy. Larvae were hatched and kept in filtered seawater or in seawater with peniciUlin and streptomycin. The gastrointestinal tract of herring larvae is essentially a straight tube divided into two compartments. Light microscopy revealed bacteria present in a progressively increasing amount throughout the length of the gastrointestinal tract from esophagus to anus. The posterior region of the intestinal lumen appeared completely occluded with bacteria. The intestinal microflora consisted mainly of members of the genera Pseudomonas and Alteromonas in the larvae incubated in filtered seawater, whereas Flavobacterium spp. dominated in larvae exposed to antibiotics. The intestinal microflora of untreated fish larvae was sensitive to all tested antibiotics, whereas multiple resistance was found in the intestinal microflora of the group given antibiotics. Thus, a dramatic change in the microfiora resulted from incubation with antibiotics. Nonpigmented yeasts were detected in both larval groups. Ciliated epithelial cells were observed in the midgut, probably propeling bacteria towards the hindgut, where endocytosis of bacteria has been demonstrated. These findings suggest that transport and sequestering mechanisms resembling those of invertebrates may be found in the gut of fish larvae. The possible significance for larval health and nutrition is discussed.