| The study was conducted to investigate the toxic effects of microcystin (MC) on fish from the perspectives of the immune and reproductive system, respectively. A batch of immune related parameters and histological changes of immune organs were determined in crucian carp which were fed with diets added with cyanobacterial containing MC. On the other side, the influence of microcystin-LR (MC-LR) on reproductive capacity, histological changes of gonad and liver, sex hormone levels, vitellogenin (VTG) expression level were explored in adult zebrafish. Furthermore, the influence of MC-LR on gonad apoptosis and apoptosis related factors in zebrafish was also involved in the study.1. The changes in immune related parameters and histological changes of immune organs in crucian carp(Carassius auratus) which were fed with diets added with cyanobacterial containing MC. Two diets containing20%and40%of cyanobacterial for20%and40%exposed group respectively and the diet without cyanobacterial for control group were prepared. Crucian carp (mean individual initial weight=85.0±8.2g) were fed for30d. The sampling for determination of immune organs indexes, blood NBT positive cell activity, plasma lysozyme activity, macrophage bactericidal activity and histological observation was carried out at the end of feeding trials. Results show that head kidney and spleen index in40%group were significantly higher than that in control group (P<0.01); By optical and electron microscopic observation, we found hyperemia and hemorrhage in head kidney and spleen in40%group. In addition, edematous mitochondria, deformation of nucleus and compaction of chromatin occurred in lymphocytes of head kidney and spleen in both exposed groups; In the two exposed groups, blood NBT positive cell activity were significantly higher than that in control group (P<0.01), and the value in40%group was nearly twice as much as that in control group; fish in20%group had a higher lysozyme activity than the control value (P<0.01), but in40%group it was significantly lower than that in control group (P<0.05); In20%group, significant increase of macrophage bactericidal activity was found in20%group (P<0.01), but there was no significant difference between the value in40%group and the control value (P>0.05). Results show that different cyanobacteria proportions added in the diets caused some tissue damage in immune organs in crucian carp and it tends to proceed toward the direction of immunostimulative response at a lower MC concentration but toward the trend of immunosuppressive answer at a higher MC concentration.2. Influence of MC-LR on zebrafish reproductive ability, histological changes of gonad and liver, sex hormone levels, vitellogenin (VTG) expression level. Adult female and male zebrafish are fed in water containing0,1,5,20μg/L MC-LR for30d. The sampling for determination of spawning, hatching rate, gonad index, histological changes of gonad and liver, β-estradiol (E2) and testosterone levels, VTG level and transcription level was carried out at10,20,30days of feeding trials. Results show that there was no significant different spawn number between the exposed zebrafish and the control ones at every sampling (P>0.05); Hatching rates in all exposed zebrafish were lower than that of control group at every sampling, especially in1,20μg/L group at20d and20μg/L group at30d there were significant differences (P<0.05); ovarian index in exposed groups at30d were all lower than that in control group, but there was no significant difference (P>0.05). At each sampling there was no significant difference in the testis index between the exposed zebrafish and the control ones (P>0.05); No obvious tissue changes were found in liver, ovary and testis in exposed fish at10and20d by optical microscopic observation, however the obvious histopathological damage occurred at30d. In the liver of exposed fish, a considerable disruption of the spatial architecture and intercellular spaces were observed. Some cytoplasmic inclusions were no longer detectable and some nuclei appeared pycnotic and deformative. In the testis of exposed fish, a few cellular deteriorations and optically empty intercellular spaces were detectable. The reduced proportion of mature sperm was visible. In the ovary of exposed fish, increased number of early-stage follicles, swelling oocyte membrane, an increase in oocyte atresia and degenerating vitellogenic oocytes were found; In20μg/L group at30d, gonad E2levels were significant lower than that in control group in both females and males (P<0.05) However there were no significant difference in testosterone level between all the exposed groups and the control ones (P>0.05); At each sampling in nearly all exposed zebrafish, liver VTG1gene transcription level were lower than the control value (P<0.05), but at30d the whole body VTG level in5,20μg/L exposed female fish were higher than that in control group (P<0.05). In male fish, the VTG levels in5,20μg/L at30d were lower than control value (P<0.01), but it was higher than control value in1,5μg/L at20d (P<0.05). The results suggested that chronic MC-LR exposure to adult zebrafish affected its reproductive ability, sex hormone levels and caused histological damage in gonad. The adverse impact almost only happened at30d. On the other hand, the results also proved that the MC-LR did not cause any estrogen effect in adult zebrafish.3. Influence of MC-LR on zebrafish gonadal apoptosis and apoptosis related factors. Gonadal apoptosis rate and apoptosis related factors (Bcl-2, Bax and caspase-3) transcription levels were obtained by flow cytometry and real-time fluorescence quantitative PCR, caspase-3activity in gonad was also measured at30d. The results showed that apoptosis rates in exposed group were all significantly higher than that in control fish in females (P<0.05), but not in males (P>0.05). In all exposed females and males, Bcl-2transcription levels in gonads were significantly lower than control level (P <0.05), but only in20μg/L female group, Bax transcription level was significantly higher than that of control fish (P<0.05), while in males it was significantly lower than control value in20μg/L group (P<0.01). Caspase-3transcription level in all exposed groups were lower than that of control group, but only in female5,20μg/L groups the difference reached significant levels (P<0.01). By further testing caspase-3activity in gonads, we found that gonadal caspase-3activity were significantly higher than control level (P<0.05) except for male1μg/L group (P>0.05). The results suggested that MC-LR would cause zebrafish gonadal apoptosis, and this effect is more obvious in female zebrafish. By detection of apoptosis related factors, it could be speculated that the mechanism of gonadal apoptosis caused by MC-LR is closely related to mitochondrial pathway. |
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