Download L`utilizzo di probiotici ed immunostimolanti in acquacoltura può

ABSTRACT
Probiotics are beneficial bacteria able to improve the host welfare and can be supplied to fish
through diet or water. They are able to positively act on fish growth and development by
modulating innate immune system and digestive system, and perhaps by improving water quality.
Probiotics may represent in some cases a valid alternative to the misuse of medicinal drugs widely
used in animal husbandry and larviculture. In particular, finfish aquaculture industry broadly
developed during last decades and unfortunately antibiotics and chemotherapics have been widely
used in order to increase aquaculture productivity. In fact the high mortality that usually occurs
during the fish larval stage, leads to high economical losses for aquaculture industry. Thus studies
on fish welfare improvement upon probiotic administration are necessary in order to clarify the
mechanisms involved in the improvements and at last, to increase aquaculture industry productivity.
In this Doctorate thesis we sought to determine the effects of the application of several probiotic
species on larval fish survivorship, growth, development and stress resistance. Several fish models
(Solea solea, Sparus aurata, Amphiprion ocellaris, Danio rerio GnRH3- EGFP transgenic line)
have been used in order to better clarify the effects and the molecular pathways involved in the
improvements observed upon probiotic administration.
A Bacillus mixture containing B. subtilis, B. pumilus and B. licheniformis was supplied to
gilthead sea bream larvae until day 75 post hatch. The control group was fed with a standard diet
based on rotifers and Artemia nauplia, in the group 1 the Bacillus mixture was supplied via live
prey, while in the group 2, probiotics were delivered through live prey and with addition to rearing
water. The Bacillus mixture significantly increased growth in terms of standard length and body
weight in both experimental groups, at larval and juvenile stages, 47 and 75 days after hatching,
respectively. In addition, the expression of genes involved in growth metabolism (Insulin-like
Growth Factors I and Myostatin) and animal welfare (70KDa-Heat shock protein and
Glucocorticoid receptor) were analysed. The morphometric analysis was supported by molecular
results which clearly evidenced higher expression of IGFI and lower levels of myostatin in groups
fed on probiotics. In addition, a better tolerance to farming conditions was also found as evidenced
by the lower expression of HSP70 and glucocorticoid receptor, suggesting beneficial effects of the
supplied Bacillus mixture on fish welfare.
Regarding the application of lactic acid bacteria, the autochthonous probiotic species
Enterococcus faecium IMC 511 was supplied at different concentrations (103 and 105 CFU · L-1)
and from the mouth opening day or from the Artemia phase to common sole larvae. The effects on
larval microbial community has been investigated. Beside, at days 10, 30 and 50 post hatch, the
effects on the fish welfare, survival and growth have been evaluated by morphometric approach and
by molecular approach through the relative gene expression of 70KDa Heat Shock Protein and
Myostatin. Moreover, cortisol levels have been estimated. The results showed that the Vibrio
populations of the intestine of S. solea larvae were thwarted in a significant way by the probiotic
Ent. faecium IMC 511 which seemed to be predominant by day 50 ph, probably due to a
stabilization of the intestinal microflora; this could in turn lead to an improvement of animal
wellbeing and growth as also supported by the lower levels of HSP70 and myostatin in the
probiotic treated groups. The microbiological data together with the molecular and morphometric
results here achieved testimony the importance of administering Enterococcus faecium to common
sole larvae ever since the mouth opening day.
Another lactic acid bacteria species, Lactobacillus rhamnosus (IMC501TM) was challenged
as probiotic in order to improve larval development in the false percula clownfish Amphiprion
ocellaris and to determine what molecular responses were observed in the larvae upon probiotic
exposure. We supplied the probiotic strain to clownfish larvae from the first day post hatch. We
supplied probiotic via live prey and with addition to rearing water (group 2) or exclusively via live
prey (group 3). We observed two times higher body weight in both clownfish larvae and juveniles
in group 2. In addition, in both groups 2 and 3 development was accelerated with metamorphosis
occurring 3 days before than in the control group. Alteration in molecular biomarkers supported the
faster growth observation; probiotic treatment significantly increased the gene expression of factors
involved in growth and development (Insulin-like Growth Factors I and II, Myostatin, Peroxisome
Proliferators Activated Receptor α and β, Vitamin D Receptor α, Retinoic Acid Receptor γ) when
probiotics were delivered via live prey and addition to the rearing water. Moreover probiotic
treatment lessened the severity of the stress response as exhibited by the lower levels of the
Glucocorticoid receptor and 70 kDa Heat shock protein gene expression. Furthermore, positive
effects on skeletal head development was observed, with a 10 to 20% decrease in deformities of
juveniles treated with probiotic. All data suggest a potent effect through administration of lactic
acid bacteria on early larval development of clownfish. These data provide a preliminary molecular
entry path into the investigation of mechanisms responsible for probiotic enhancement in fish
development.
The same probiotic species, L. rhamnosus was supplied to GnRH3-EGFP transgenic
zebrafish larvae from the opening mouth day until puberty. Here we sought to determine if 9 weeks
L. rhamnosus IMC 501TM treatment could affect Danio rerio early development and gonadal
maturation and differentiation. From 6 to 10 dpf we analyzed the effects of probiotics on GnRH3
neurons soma size. Moreover at 3, 6 and 9 weeks post fertilization we observed gonad development
by histological sections. By quantitative PCR we analyzed the expression of an array of genes
involved in growth and development and stress response, at 6, 10 and 20 days post fertilization. At
day 12 post fertilization we checked the ongoing calcification of backbone vertebra.
A significant increase of Insuline like Growth Factors I and II, Peroxisome Proliferator Activated
Receptor  and , Vitamin D Receptor and Retinoic Acid Receptor  was found in the probiotic
treated larvae with respect to the control ones. The ongoing calcification of vertebral bodies seemed
to occur faster in probiotic treated larvae .Moreover probiotic seemed to less the severity of
physiological and cellular stress as the lower levels of GR and HSP70 registered in probiotic treated
group. Moreover, L. rhamnosus treatment seemed to affect also GnRH3 neurons soma size. More
incredibly, 3 weeks old zebrafish treated with probiotic already showed 20% of males. In the
control group male appeared only at 9 weeks post fertilization. At the end of the experiment the
control group showed a sex ratio of 94% females and 6% males vs. the 55% females and 45% males
ratio found in the probiotic group. We hypothesized that probiotic treatment can act on early larval
development by delivering organic molecules and increasing the calcium absorption as indicated by
the higher calcification of the axial skeleton. The early gonadal development, may be related with
the activation of GnRH3 neurons as indicated by the larger soma of treated larvae.
These results evidenced that Lactobacillus rhamnosus may improve several cornerstones of fish
development. Further studies are necessary in order to deepen these outstanding findings.
Finally, for the first time we challenged the potentiality of Magnetic Resonance Imaging
(MRI) to depict, with sufficient resolution and satisfactory contrast, the anatomy of clownfish
during metamorphosis, without dissecting the larvae. Once the method was optimized, MRI was
used to identify internal anatomical changes during metamorphosis climax and by Real time PCR
the gene expression trend of TRα and β
associated with the metamorphosis process first occur in the inner body, while outer changes, such
as the appearance of the typical adult pigmentation, occur soon after. The morphological changes at
brain, eyes, swim bladder and digestive tract were associated with higher TRα and β
expression levels. Our findings represent the starting point for future applications of MRI to marine
fish developmental studies.
The present doctoral thesis tried to deep investigate into the signalling pathways involved
upon probiotic administration in a number of marine and freshwater fish species. The molecular
insights, together with the morphological observations, allowed us to detect some of the molecular
mechanisms involved in the host physiological response to beneficial bacteria supply. The results
here achieved could provide new insights to the emerging blue biotechnology, for a further increase
of productivity and competitiveness of the aquaculture industry and to the field of developmental
biology. Next step will be the study of the effects of those beneficial bacteria on the ageing
mechanisms. We will seek to determine whether the anticipation of some developmental milestones
by probiotic treatment lead in turn to a faster ageing. Moreover, studies on probiotics are important
in order to develop a environmental friendly and sustainable aquaculture, together with an increase
of productivity.