The Passive Transfer And Immune Function Of Maternal Complement Components And The Ontogeny Of Complement System In Zebrafish (Danio Rerio)

Fish larvae are exposed to pathogens long before their lymphoid organs mature and immunocompetence is attained. It appears that fish embryo and larvae mainly rely on the maternal immune factors transferred from parents. The presence of humoral non-specific factors like lysozymes, lectins and complement components has been reported in the eggs of different teleost species. However, little information is available on the maternal immunity in fish. The main objectives of this study were to examine the transfer of maternal complement components in zebrafish (Danio rerio), their roles in early development, and the ontogeny and maturation of complement system in D. rerio.The paper first dealt with the bacteriolytic activity of maternal complement in the fertilized eggs of zebrafish and its mode of action. We showed for the first time that the Gram-negative bacterium E. coli was sensitive to the lysis by the cytosol prepared from the fertilized eggs of zebrafish D. rerio, and all the findings point to complement system being one of the most important factors involved in the bacteriolytic activity. First, the bacteriolytic activity was abolished by pre-incubation of anti-C3 antibody with the egg cytosol, a process that would cause the precipitation of the central component of all known complement pathways, C3. Second, the lytic activity was depleted by heating at 45℃, a temperature known to inactivate fish complement.To determine which pathway of complement activation might be involved in the bacteriolytic activity of the egg cytosol, the antibodies against C1q (a key component of CP), C4 (a key component of both CP and LP) and Bf (a key component of AP) were utilized to block the CP, LP or AP, respectively. It is found that precipitation of C1q and C4 causes little loss of the bacteriolytic activity of the egg cytosol, whereas precipitation of Bf results in a significant reduction of the lytic activity. Furthermore, addition of EGTA to remove Ca2+ from the egg cytosol, which can inhibit both CP and LP, induces little decrease in the bacteriolytic activity. In contrast, pre-incubation of EDTA with the egg cytosol leads to a substantial reduction of the bacteriolytic activity, and saturation of the chelator with Mg2+ is capable of restoring the lytic activity, but not by addition of Ca2+. Moreover, selective inhibition of the AP by zymosan A induces marked loss of bacteriolytic activity, while addition of L-lysine, an inactivator of the CP, is not inhibitory. Taken together, all these undoubtedly indicate that activation of the AP is responsible for the bacteriolytic activity of the egg cytosol, but the CP and LP have little contribution to the lytic activity.Second, this paper addressed the maternal transfer of complement components in zebrafish and the role of the components in offspring. The in vivo experiments showed that the maternal complement components can be trasfer from mother to offspring and they play key roles in protecting the embryos against Aeromonas hydrophila challenge. The immunization of female zebrafish with formalin-killed A. hydrophila resulted in the elevation of the alternative complement activity (ACH50) and the content of complement C3 and Bf in the vaccinated mother fish and their eggs, and both ACH50 and the complement content increased more significantly when the mother fish were vaccinated a second time, which indicated the transfer of maternal complement components and their immunocompetence in the eggs of D. rerio. Moreover, the eggs released by the vaccinated fish were more resistant to the infection by A. hydrophila than the eggs released by the non-vaccinated fish, and this proved that the maternally transferred immune factors can enhance the immunity of zebrafish offspring. Furthmore, microinjection of anti-C3 and anti-Bf antibodies into the zebrafish eggs led to a significant increase in the motality of the eggs challenged with H. hydrophila, providing additional evidence for the participation of complement components in the eggs of D. rerio.Finally, this paper examined the ontogenic and expression of complement key component genes (C3, C1r/s, C4, C6, Bf, MBL and MASP) of the CP, AP, LP and lytic pathway, and their responses to challenge with LPS during development. The findings showed that all components, except C6, show a similar trend of low expression level before hatch and increasing expression after hatching; Expression of C6 was detected at very high level just after fertilization, then it significantly droped in the early time-point samples and steady increase followed. Moreover, Bf transcript representing the AP is expressed at much higher levels than C1r/s and C4 involved in the CP. Thus it is likely that the AP may be more significant than the CP during embryonic and post-hatching development of D. rerio.When the embyos/larvae of zebrafish was challenged with LPS, it was found that: The contents of Bf transcript representing the AP and C3 transcript important in all the activation pathways are both markedly increased by challenge with LPS soon after hatching, while the expression levels of C1r/s and MASP transcripts representing the CP and LP, respectively, are not elevated, or even decreased by the same challenge throughout the study period; The expression of C4 mRNA involved in both the CP and LP is first down-regulated by LPS challenge and then fluctuates; The expression of MBL mRNA associated with the LP is up-regulated by LPS challenge only after 18 dpf; and C6 transcript representing the lytic pathway is peculiarly expressed in response to LPS challenge, which is significant decreased before hatching, followed by a significant drop post hatching. Taken together, these results hints at the clue that the complement operating via the AP is already competent by responding to challenge with LPS in the hatched larvae of D. rerio. Moreover, C6 expression is relatively high in early embryos, but it is significantly suppressed following LPS challenge. It is probable that there exists a large amount of maternal C6 mRNA stored in the eggs, which is rapidly consumed by challenge with LPS. On the other hand, none of C1r/s and C4 expression is fully responsive to challenge with LPS in the 3-week old larvae, implicating that the CP is still developing. However, the developmental status of the LP remains to be further determined at present.