Apoptosi e HSP70 come marcatori di adattamento ambientale nei pesci telostei
Author(s)
Ceccarelli, Giuseppina
Date Issued
January 8, 2009
Type
Doctoral Thesis
Abstract
The worldwide decline of fisheries stocks has provided a rapid growth in fish farming and a
growing economical interest in intensive aquaculture, thus gained great importance to plan a proper management in order to limit stressful stocking conditions and environmental impact.
This management should improve fish welfare and counteract adverse water characteristics.
In aquaculture fish are exposed to stressful conditions that can be due to routine procedures
(handling, selection, weighing), management factors (high stocking density, feeding,
temperature and water quality or disease), transport conditions and slaughter procedures.
Direct parameters such as haematocrit, total haemoglobin, blood glucose and hydrocortisone,
frequently reported as descriptors of the fish conditions, may be not sufficiently reliable when
chronic stresses are applied. It is therefore necessary to search for further parameters, such as alternative markers, capable to describe fish welfare to keep under control fishes in
aquaculture. In this thesis work it was examined two different parameters: apoptotic process
and the expression of one of the Heat Shock Protein (HSP70), to the aim of use these as
markers of stress in fish farming and possibly in environmental monitoring.
With this aim, the study in the beginning analysing the physiological apoptosis presence in
developmental thymus and in the late-juveniles/adult of fresh/marine temperate water species (Dicentrarchus labrax, Epinephelus marginatus, Cyprinus carpio), and after that in species adapted to polar water (- 1°C) such as the Trematomus species. The fish thymus plays an
important role in the development of a fully functional immune system in maturing
lymphocytes (T) where the selection process of autoreactive/damaged cells plays a central
role. The developmental results showed that probably in Cypriniformes and Percyformes this
selection process can be occur with similar features in thymus, and that it is mainly restricted to cortical/medullary border and cortex. Particularly, in carp, it was confirm and amplified previous studies (Romano et al., 1999b). The cells appeared scattered in the parenchyma in the first week post hatch (ph). From the first to the 4th week ph, the number of apoptotic cells increased in the outer-cortical region of the thymus. From the week 4 until 30-40 weeks post hatch (ph) the mean diameter of non-apoptotic lymphoid cells varied following the formation of cortex and medulla. At 22 untill 48-50 weeks ph apoptotic cells were more numerous in the cortex and cortical/medullary border than in the medulla. From 22 months ph onwards the
apoptotic cells remains confined minly in the cortex. In Perciformes Teleosts the distribution
was evidenced like in carp: i) In sea bass, it was confirmed the observations done in previous
studies (Abelli et al., 1998) and further analysis revealed a cortical and cortical/medullary border distribution of apopotic cells from 70 days ph until 165 days ph. ii) in Epinephelus
marginatus the first anlagen of lymphoid thymus appears at day 15, while apoptotic cells
were evidenced at day 48 ph. During thymus development the number of apoptotic cell
increased in cortex and, contemporary decreased the average of diameter of the counted
elements. In late juveniles/adult (9 months ph onwards), the apoptotic cells were mainly
restricted in the cortex similarly to other species. iii) In adult Antarctic Perciformes the
thymus is extremely adapted to low temperature (high vascularisation and strongly compact
stroma) and. the apoptosis was 4-7 fold more intense than in temperate water fish and mainly
distributed in the cortex.
Although gradually temperature changing may be physiologically balanced, rapid changes, as
occur in land aquaculture, can disturb homeostasis acting as a stressor. Subsequently it was analysed the effects of the thermic stress on the European sea bass (Dicentrarchus labrax), a
largely reared fish species in offshore cages and in land aquaculture. Although gradually
temperature changing may be physiologically balanced, rapid changes disturb homeostasis
acting as a stressor. With this aim it was studied the effect of temperature enhance (+ 4°C) on most superficial lymphoid organs (thymus, head kidney and gills) of larva (47-70 days p.h.)
by using as markers of stress: the apoptotic process (by in situ end-labelling of fragmented
DNA) and the expression of HSP70. The temperature of experimental group of larvae have
been raised at 50th days for two hours, and the stressed individuals are sampled after 40
minutes, 2 hours, 24 hours and 10 days and 20 days post-treatment. In control, the gills and
thymus the number of apoptotic cells progressively growth following the develop of the
organs. In the stressed larvae, 40 minutes after stress, the apoptotic processes peaked
significantly in thymus (P<0,001) and head kidney (P< 0,05), while in gills (P<0,05) the peak
was delayed at 2 hours after stress. At 10 and 20 days post-treatment the apoptosis number
decreased and was comparable to control levels. The expression of HSP70 was detectable
with the ELISA assay and immuno-histochemistry techniques. In non-treated larvae, HSP70
was constitutively expressed whereas in stressed larvae, significantly enhance of the
expression was revealed at 24 hours after stress, in thymus (P<0,01) and head kidney
(P<0,01). In gills, HSP70 was more expressed at 10 days after stress (P<0,05). In all organs
the expression level of HSP70 remained high as compare to control samples. Taken together,
this study suggest that apoptosis and HSP70 as potential candidates to describe the fish
welfare.
In respect with the new European directives (2006), new vaccine therapies are requested by
fish farming to prevent various infectious disease and contemporary maintaining the welfare
of fish and the integrity of environment. In this work we evaluate the efficiency of different
vaccination trials, against Vibrio anguillarum, on the specific and non-specific immune
system of juveniles sea bass. In parallel it was inquired the eventual induction of apoptosis
and expression of HSP70 in thymus, gills and head kidney. From the 95th days p.h. the larvae
were submitted to two trials: a) two vaccinations for immersion (95 and 165 day ph) or b) two
vaccinations for immersion (95/165 days ph) followed to a intraperitoneal injection (233 day
ph). The specific immunity activation was demonstrated in vaccinated group by the
significative enhance of B lymphocytes in the head kidney and spleen, contemporary to
intensification of specific anti-Vibrio Ig serum content, and the agglutination capacity of the
bacteria. Non-specific immunity activation was contemporary demonstrated by the
enhancement of the respiratory burst in leucocytes of head kidney and spleen. The
Respiratory Burst test (by ROS test) on phagocytes (macrophages and granulocytes) of nontreated and experimental groups, evidenced in the latter groups a enhance of phagocytic
capability already after two vaccination for immersion. A decrease of the apoptotic processes
was revealed in thymus, head kidney and gills during the trials in both experimrntal and nontreated groups. Morover, also the expression of HSP70 in organs showed a similar trend.
Probably, it was due to a normal differentiation mechanism of the organ. Interestingly, in
thymus of the experimental groups the HSP70 had a significant enhance of expression in
cortical and cortical/medullary border cells as compare with control group (P<0,01). These
data demonstrates that the agglutination test, ROS test and HSP70 expression are possible
markers for the efficiency of the vaccination therapy. Morover, vaccinations early done by
immersion (90 days followed by an other one 60-70 days later) can be sufficient to protect sea
bass juveniles by V. anguillarum infection.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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