Immunogenetics (2006) 58: 168–179DOI 10.1007/s00251-006-0096-3

ORIGINAL PAPER

Marie Løvoll . Terje Kilvik . Hani Boshra .Jarl Bøgwald . J. Oriol Sunyer . Roy A. Dalmo

Maternal transfer of complement components C3-1, C3-3, C3-4,C4, C5, C7, Bf, and Df to offspring in rainbow trout(Oncorhynchus mykiss)

Received: 15 November 2005 / Accepted: 19 January 2006 / Published online: 21 March 2006# Springer-Verlag 2006

Abstract Defense mechanisms in developing fish arepoorly known but before maturation of lymphoid organsand immunocompetence, innate mechanisms are essential.The complement system represents a major part of innateimmunity. Our main objective was to map the presence ofcomplement components early in fish development. Rain-bow trout eggs, embryos, and hatchlings were assayed forthe onset and duration of C3-1, C3-3, C3-4, C4, C5, C7,factor B, and factor D transcription using real-time reversetranscription-polymerase chain reaction. In general, com-plement transcript levels increased steadily from day 28post-fertilization to hatch, followed by a decrease duringyolk–sac resorption. All the complement proteins studiedwere found in unfertilized eggs. There was no correlationbetween the transcript and protein levels throughout thestudy period. Complement proteins appeared in the liver,kidney, and intestine between day 7 and 35 but not untilday 77 in the heart. This study is the first to address theontogeny of several complement components and repre-sents the first evidence that maternal transfer of comple-ment components, other than C3, occurs in teleost fish.

Keywords Complement . Ontogeny . Rainbow trout

Introduction

The complement system is a major component of the innateimmune system and it plays a role in adaptive immunity.(Morgan et al. 2005). Bacteria, viruses, and parasites all

trigger the cascade involving about 30 soluble andmembrane proteins. Activated complement componentsdisplay a wide range of functions in inflammatory reactionssuch as chemotaxis, opsonization, and destruction of targetmicroorganisms. In addition to being a crucial first line ofdefense, the complement system serves to protect thenewborn before the maturation of the immune system(Ogundele 2001). Fish embryos and hatchlings are exposedto aquatic pathogens before their lymphoid organs matureand immunocompetence is attained, but few studies wereconducted to elucidate defense mechanisms at these earlydevelopmental stages (Zapata et al. 1997). Leukocytic cellsmay be present in zebrafish (Danio rerio) and carp(Cyprinus carpio) embryos (Rombout et al. 2005). In-formation about vertical transfer of innate defense factors ismeager, but lectin-like agglutinins were observed in theova of brown trout (Salmo trutta), Atlantic herring (Clupeaharengus), roach (Rutilus rutilus) (Anstee et al. 1973), andin the serum and eggs of plaice (Pleuronectes platessa)(Bly et al. 1985). Likewise, maternal transfer of IgM tooffspring may occur in carp (C. carpio), tilapia (Oreochro-mis mossambicus), gilthead seabream (Sparus aurata),Indian major carp (Labeo rohita) (Avtalion and Mor 1992;Picchietti et al. 2001; Scapigliati et al. 1999; Swainet al. 2006; van Loon et al. 1981), and rainbow trout(Oncorhynchus mykiss) (Castillo et al. 1993). The func-tional role of complement components in the fish embryowas not studied (Ellingsen et al. 2005; Lange et al.2004a,b; Magnadottir et al. 2004), but it is likely thatthese components contribute to protection againstpathogens.

The complement cascade can be activated through threedistinct, but partially overlapping pathways: the alternative,the lectin, and the classical pathway (Holland andLambris 2002). Factor B (Bf) is essential as a catalyticsubunit in the alternative pathway, which is activated bymicrobial pathogens. This equals the role of C2 in theclassical pathway. Factor D (Df) is unique to the alternativepathway generating the convertase C3bBb by cleavage ofBf complexed with activated C3. Mammalian Df issynthesized by macrophages and is identical to adipsin

M. Løvoll . T. Kilvik . J. Bøgwald . R. A. Dalmo (*)Department of Marine Biotechnology,Norwegian College of Fishery Science,University of Tromsø,Tromsø 9037, Norwaye-mail: Roy.Dalmo@nfh.uit.no

H. Boshra . J. O. Sunyer (*)Department of Pathobiology,School of Veterinary Medicine, University of Pennsylvania,Philadelphia, PA 19104, USAe-mail: sunyer@vet.upenn.edu

produced by adipocytes (Hajnik et al. 1998). C4 (Boshra etal. 2004) is a key component of the classical pathway,being activated by antigen-antibody complexes. Activationinduces cleavage of C3 (Sahu and Lambris 2001), whichleads to the assembly of the membrane-attack-complex(MAC) with the end result that pores form in the cellmembrane of pathogens, e.g., bacteria resulting in celldeath by lysis (Janeway et al. 2001). Being integralcomponents of the MAC, C5 and C7 play central roles inthe terminal complement cascade (Nonaka et al. 1981;Zarkadis et al. 2004).

In diploid and tetraploid fish, such as teleosts, severalcomplement components are encoded by multiple genesgiving rise to subtypes displaying structural and functionaldiversity. The rainbow trout is in the process ofdiploidization, which possibly explains the findings ofthree C3 proteins: C3-1, C3-3, and C3-4 (Zarkadis etal. 2001). Two rainbow trout Bf molecules, Bf-1 and Bf-2,were characterized (Sunyer et al. 1998). Bf-2 is involved inthe alternative and the classical pathway, displaying bothBf- and C2-like functions. The potential role of Bf-1 in theclassical pathway remains unknown. Complement tran-scription may be modulated by proinflammatory cytokines,which are under complex hormonal control (growth factorsand glucocorticoids) (Falus 1990; Volanakis 1995). In-tricate promoter regions (Carney et al. 1991; Vik etal. 1991) and extrahepatic synthesis, however, indicate thatcomplement components may exert additional roles totheir well-known inflammatory functions (Mastellos andLambris 2002). A main objective of our work is to studywhether the complement system may be involved indefense at early developmental stages in the rainbow trout.As a first step, the ontogenic and differential appearance ofcomplement component transcripts and proteins of thealternative and classical pathways (C3-1, C3-3, C3-4, C4,C5, C7, Bf, and Df) was examined.

Materials and methods

Sample collection

Unfertilized rainbow trout eggs and sperm were obtainedfrom AquaGen (Trondheim, Norway) mixed and disin-fected with buffodine. Incubation of fertilized eggs andmaintenance of hatchlings was performed in upwellingincubators and aquaria supplied with aerated, runningwater at 6°C for 15 weeks (Kårvika Aquaculture ResearchStation, Tromsø, Norway). Hatchlings were maintainedunder a photoperiod of 12 h light/12 h dark. The eyed stagewas reached approximately 35 days after fertilization, eggshatched at day 58–60, and yolk–sac resorption wascompleted ∼70 days post-fertilization. Samples werecollected weekly. Hatchlings were overanesthetized using0.01% benzocaine. Samples to be processed for immuno-blotting were frozen at −20°C while samples for RNAextraction were submerged in RNAlater (Ambion, Austin,TX, USA), kept at room temperature overnight, and storedat −20°C. Samples for immunohistochemistry were fixed

in 4% paraformaldehyde in phosphate-buffered saline(PBS) for 2 days, transferred to 70% ethanol, and storedat room temperature. Total RNA was extracted using theTRIzol method (Chomczynski and Sacchi 1987), theRNeasy MiniKit (Qiagen, Hilden, Germany), or acombination of both. Three eggs, embryos, or hatchlingsat each time-point were pooled and homogenized in 3 mlTRIzol reagent using a rotor–stator homogenizer (UltraTurrax; IKA Werke, Staufen, Germany). For additionalremoval of DNA and proteins, the water phase of the initialTRIzol/chloroform separation was added to a secondvolume of TRIzol reagent. To remove any contaminatinggenomic DNA, samples were treated with DNase (TURBODNA-free, Ambion). Purified RNA was confirmed to beintact by gel electrophoresis. Samples with A260/A280 ratiosfrom 1.8 to 2.2 were considered pure and contaminatedsamples were cleaned-up using the RNeasy MiniKit.Proteins were extracted from ten pooled eggs, embryos,or five hatchlings at each time-point. Samples werehomogenized in 2.5 ml 0.1 M Tris, pH 7.6 containingprotease inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich, Steinberg, Germany) using the rotor–statorhomogenizer (UltraTurrax). The homogenates were cen-trifuged at 4.500×g for 15 min at 4°C and the supernatantswere removed and centrifuged three times at 14.000×g for5 min at 4°C. Protein extracts were stored at −20°C. RNAand protein concentrations were measured using either theRiboGreen RNA Quantitation Kit (Molecular Probes,Eugene, OR, USA) (Jones et al. 1998) or the BioRadProtein Assay (BioRad, Hercules, CA, USA).

Quantitative real-time reversetranscription-polymerase chain reaction

RNA was reversely transcribed using random hexamers(TaqMan RT-reagents, Applied Biosystems, CA, USA).Reaction volumes of 50 μl contained 100 ng of total RNA.Polymerase chain reactions (PCRs) were performed induplicates with an ABI PRISM 7000 Sequence DetectionSystem (Applied Biosystems) using default parameters(50°C for 2 min, 95°C for 15 min, 40 cycles of 95°C/15 s,and 58°C/60 s). Every PCR contained 2× SYBR GreenPCR Master Mix, Applied Biosystems [AmpliTaq GoldDNA Polymerase, dNTPs, passive reference (ROX)and optimized buffer components]; specific forward andreverse primers (300 nM final concentration each); and2.5 μl of cDNA template and MilliQ water to a finalvolume of 25 μl. Templates to be used with the 18S primerswere diluted 1:100. Primers were designed from partialcDNA sequences deposited in the GeneBank or TheInstitute for Genomic Research (TIGR) database (acces-sion numbers are listed in Table 1) using the PrimerExpress software (version 2.0, Applied Biosystems) andsynthesized by MedProbe (Oslo, Norway). Primers weretargeted at exon–exon boundaries that were located byaligning the cDNA sequences with their respectivegenomic DNA sequences from pufferfish (Fugu rubripes)and zebrafish (D. rerio) (Ensembl Genome Browser). The

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amplification efficiency of each primer set was assessedfrom twofold liver cDNA dilutions (Fig. 1, Table 2).

To verify specific amplification, melting curves weregenerated for all amplicons. A single peak matching theamplicon’s melting temperature (Tm) (Ririe et al. 1997)verified specific priming and absence of potential dimers.In addition, the PCR products were analyzed on a 1.5%agarose gel. Amplicons representing C3-1, C3-3, and C3-4were cloned (TOPO TA Cloning Kit for Sequencing;Invitrogen, Carlsbad, CA, USA) and sequenced using the

ABI PRISM BigDye Terminator Cycle Sequencing ReadyReaction Kit (Applied Biosystems) in compliance with themanufacturer’s protocol. To verify that there was nogenomic DNA contamination of the RNA, several sampleswithout reverse transcription (RT) were subjected to real-time PCR under analogous conditions to those used in thereal-time RT-PCR. Differences in slope values betweenreference and target genes of the standard curves were<0.1, allowing quantitative translation of the Ct values incompliance with the manufacturer’s instructions (AppliedBiosystems); target Ct values were normalized to thecorresponding 18S RNA Ct values and compared to acalibrator sample. All data were captured using Sequence

Table 1 Primers used in quantitative real-time RT-PCR for RNA analysis

Function Gene Oligonucleotides Amplicon (bp) GeneBank/TIGR acc. no.

Central complement component C3-1 Forward 5′-ggccagtccctggtggtta-3′ 157 L24433Reverse 5′-ggtggactgtgtggatccgta-3′

C3-3 Forward 5′-ttctactctggaggccacagctt-3′ 150 U61753Reverse 5′-tggaacaccatgatagtggactg-3′

C3-4 Forward 5′-tgccatactgtaggctaaaagtgaa-3′ 184 AF271080Reverse 5′-atcatgtcccagatcttagtctgagtaa-3′

Alternative/classical pathway Bf-1/2 Forward 5′-tgtgctgaccctgggattc-3′ 181 AF089861/AF089860Reverse 5′-catacgtgtgcttgtagtaacatttagg-3′

Df Forward 5′-gctgctctgctggctgtctt-3′ 151 TC79023Reverse 5′-actgatcggccaccagaaac-3′

Classical pathway C4 Forward 5′-tctacaaccctacacagcaagtgag-3′ 105 AJ544262Reverse 5′-tgcccgcagcattaaaaatag-3′

Lytic pathway C5 Forward 5′-aaccctggatacctgtgctca -3′ 151 AF349001Reverse 5′-ctcaacacgtgccaagacatg -3′

C7 Forward 5′-gagggccactacatcactgga-3′ 102 AJ566190Reverse 5′-cggaacatcacatgttgcact-3′

Reference gene 18S Forward 5′-tgtgccgctagaggtgaaatt-3′ 101 AF308735Reverse 5′-cgaacctccgactttcgttct-3′

Due to few and scattered nucleotide differences, specific primers separating Bf-1 and Bf-2 were difficult to design for PCR usingSYBR Green as the detection reagent. Mutual primers annealing to both subtypes were madeC3-1 and C3-3 primers annealed to the regions encoding the α-chains of the molecules. C3-4, C4, and C5 primers annealed to theregions encoding the β-chains

0

5

10

15

20

25

30

35

40

-2,5-2-1,5-1-0,500,5

Log Quantity

Ct

Val

ues

18S

C3-1

C3-3

C3-4

C4

C5

C7

Bf

Df

Fig. 1 Standard curves for calculation of PCR efficiencies. Logrelative input amount liver cDNA (abscissa axis) vs Ct cycles(ordinate axis) were plotted and analyzed using linear regression.The slope (m) was defined from the equation Ct=m (log Q)+c whereCt is the threshold cycle, Q is the initial amount cDNA and c is theintercept on the ordinate axis

Table 2 Primer amplification efficiencies (% E) were calculatedfrom the slope values of the standard curves according to theequation: E=10(1/−slope)−1

Gene Slope r2 % E

C3-1 −3.370 0.999 98.0C3-3 −3.312 0.998 100.4C3-4 −3.286 0.993 101.5Bf-1/2 −3.311 0.976 100.5Df −3.357 0.997 98.5C4 −3.359 0.998 98.5C5 −3.386 0.972 97.4C7 −3.355 0.990 98.618S −3.383 0.999 97.5

One hundred percent efficiency corresponds to a slope of −3.32 as3.32 cycles are required to generate a tenfold increase of product(Bogerd et al. 2001)

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Detection Software (SDS version 1.1; Applied Biosystems)and exported to Microsoft Excel worksheets for furtheranalysis.

Immunization and antibody purification

Complement component proteins were purified fromrainbow trout serum and antibodies prepared as previouslydescribed (Sunyer et al. 1996). Antibody reactivity with thedifferent C3 subtypes is shown in Fig. 2. Anti C3-1α, antiC3-1β, anti C3-4α, and anti C3-4β specifically bound tothe correct components. Some cross-reactivity was de-tected for anti C3-3α with C3-4 and to a lesser extent foranti C3-3β with C3-4.

Immunoblotting

All reagents were purchased from Invitrogen and used incompliance with the manufacturer’s protocol. Fortymicrograms of proteins from each sampled time-pointwere mixed with 4× lithium dodecyl sulfate sample bufferand 10× sample reducing agent, denatured for 10 min at70°C, and fractionated on NuPage 4–12% Bis–Tris gels.The proteins were transferred to a polyvinylidene difluo-ride (PVDF) membrane at 30 V for 1 h and nonspecificbinding was prevented by incubation in blocking solution[Hammersten caseine solution diluted in tris-bufferedsaline (TBS)] for 30 min. The membranes were incubated

with primary antibody (polyclonal rabbit anti-rainbow troutcomplement component sera, for dilutions, see Table 3) for1 h. After rinsing, the membranes were incubated withalkaline phosphatase-conjugated secondary antibody (West-ernBreeze Chemiluminescent Western Blot Immunodetec-tion Kit) for 30 min. For detection, the membranes werecovered with chemiluminescent detection phosphatase-starchemiluminescent substrate solution for alkaline phospha-tase for 5 min, wiped off, and exposed to an X-ray-sensitivefilm for 20 min (Lumi-Film Chemiluminescent DetectionFilm, Roche Diagnostics, Basel, Switzerland). Normalrabbit serum (Sigma) served as the negative control.

Immunohistochemistry

The immunostaining procedure was performed as de-scribed earlier (Abelseth et al. 2003). Eggs, embryos, andhatchlings were dehydrated, embedded in paraffin wax,and sectioned. The sections were mounted on poly-L-lysinecoated glass microscope slides, melted at 60°C, dewaxed inxylene, rehydrated via ethanol to water, and demasked in0.1 M citric acid using a pressure cooker at 1 bar and 100°Cfor 9 min. Antigen retrieval was accomplished by treatingthe slides with 1% sodium borohydride in TBS. A solutionof 30% skimmed milk, 3% bovine serum albumin, 0.3%Tween 20, and heparin (500 IU ml−1) in TBS, pH 7.5 wasused to prevent nonspecific binding. The sections wereincubated with the different polyclonal rabbit anti-rainbowtrout complement component sera (for dilutions, seeTable 3), washed in TBS, then incubated with affinitypurified biotinylated goat anti-rabbit Ig serum (Vectastain,Vector Laboratories, Burlingame, USA). The sections werewashed in TBS, incubated with avidin:biotinylated enzymecomplex-alkaline phosphatase reagent (Vectastain ABC-AP), washed in TBS, and incubated with FastRed TR/Naphtol AS-MX (Sigma). The sectionswere counterstainedwith Harris’ hematoxylin solution (Merck, Darmstadt,Germany).

Fig. 2 Antibodies were tested for their reactivity with trout C3-1,C3-3, C3-4, and C5. One microgram samples of purified trout C3-1,C3-3, C3-4, and C5 proteins were run in separate lanes on 7.5%SDS-PAGE under nonreducing conditions and blotted to PVDFmembranes. Each protein was incubated with polyclonal antibodies(1:1,000) against the α- and β-chains of trout C3-1, C3-3, and C3-4.The upper part of the figure represents the region of the gel (180–200 kDa) showing the Coomassie staining of the various troutcomplement proteins. The lower part of the figure shows the sameregion represented in the upper part after immunoblotting, using theantibodies indicated by the arrows

Table 3 Primary antibody dilutions for immunoblotting (IB) andimmunohistochemical (IHC) analysis

Antibody Dilutions

IB IHC

C3-1 α 1:1,000 1:500C3-1 β 1:2,000 1:2,000C3-3 α 1:500 1:500C3-3 β 1:1,000 1:1,000C3-4 α 1:250 1:500C3-4 β 1:500 1:1,000Bf-1/2 1:1,000 –Df 1:200 1:200C4 1:100 1:100C5 1:500 1:500N. r. s. 1:200 1:200

N. r. s. Normal rabbit serum

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Results

For the real-time RT-PCR, all transcripts were compared tothe levels of C3-1 transcripts in homogenates from day 7. Ifdifferences in Ct values between duplicates exceeded 0.4

cycles, the data were not retained. No signals wereobserved in control reactions performed without addingreverse transcriptase to the reverse transcription reaction(data not shown). Hatchlings were not sampled for RNApurification on day 77. In immunoblotting, negativecontrols using a 1:200 dilution of normal rabbit serum(Fig. 3) revealed weak signals from a protein of ∼15 kDa,but the signals did not interfere with signals representingthe complement components analyzed. In addition, back-ground signals from proteins of approximately 70 and80 kDa were detected. The intensities of these bands were,however, very weak, which made the identification of theC3 and C4 β-chains (∼75 kDa) relatively easy. Nonreducedsamples from all time-points were tested using mixed antiC3-1α and anti C3-1β sera. The blot revealed the presenceof a ∼182 kDa protein (intact C3-1) at all time-points,though at very low intensities from day 0 to 28 (results notshown).

Central complement component subtypes C3-1, C3-3,and C3-4

Results are shown in Fig. 4. From the first sampled time-point post-fertilization (day 7) and onward, C3-1 tran-scripts were detected at increasing levels with a significant27-fold increase by post-hatch (day 63). During yolk–sac

Fig. 3 Negative control using normal rabbit serum. Forty micro-grams of proteins from each sampled time-point (day 0 to 105) werefractionated by gel electrophoresis under reducing conditions. Theproteins were transferred to a PVDF membrane and incubated withnormal rabbit serum (1:200) followed by alkaline phosphatase-conjugated secondary antibody. Proteins were visualized by chemi-luminescence and exposure to an X-ray-sensitive film for 20 min

Fig. 4 Expression levels of complement component C3 transcripts(real-time RT-PCR) and proteins (immunoblotting). Eggs, embryos,and hatchlings were sampled weekly before hatching throughoutcomplete yolk–sac resorption (no sampling for RNA purification onday 77). Transcript expression levels were relatively compared to

C3-1 transcript levels, day 7. Ct values for the first time-pointswhere significant signals could be detected were as follows: C3-1:26.9; C3-3: 32.5; and C3-4: 35.1. Proteins were visualized bychemiluminescence and exposure to an X-ray-sensitive film for20 min. Life history events are included

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resorption, the C3-1 transcripts decreased significantly. Incontrast to the C3-1 and C3-3 primers, which weredesigned to anneal to the regions encoding the α-chains,the C3-4 primers were directed at the β-chain. Despite this,the overall transcription patterns were quite similar for allthree subtypes. Transcription of C3-3 and C3-4 started atday 28 and was significantly lower compared to C3-1.Immunoblotting revealed signals assumed to correspond tothe β-chains of the C3 subtypes (C3-1 and C3-3: 70 kDa,

C3-4: 73 kDa) in homogenates of eggs, embryos, andhatchlings at seemingly decreasing intensities at all time-points throughout the study period. The α-chains of C3-1(112 kDa), C3-3 (112 kDa), and C3-4 (107 kDa) wereobserved from the hatch throughout yolk–sac resorption(day 56 and onward). The intensities of the bandscorresponding to C3-3 and C3-4 α-chains were, however,very low (data not shown) compared to the bandcorresponding to the C3-1 α-chain.

Fig. 5 Expression levels of complement component transcripts(real-time RT-PCR) and proteins (immunoblotting). Eggs, embryos,and hatchlings were sampled weekly before hatching throughoutcomplete yolk–sac resorption (no sampling for RNA purification onday 77). Transcript expression levels were relatively compared tothe C3-1 transcript level at day 7 (Fig. 4). Ct values for the first time-

points where significant signals could be detected were as follows:C4: 35.9; Bf: 26.2; Df: 33.2; C5: 29.9; and C7: 35.6. Proteins werevisualized by chemiluminescence and exposure to an X-ray-sensitive film for 20 min. No antiserum was raised against C7.Life history events are included

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Classical pathway represented by C4

Results are shown in Fig. 5 (classical pathway). No C4transcripts were detected from day 0 to 56. From day 63and onward, very low levels were detected and day 91samples showed no transcription. From the immunoblot-ting, the C4 β-chain was present in homogenates at alltime-points including in the unfertilized egg. The contentsof C4 proteins in late time-point homogenates (day 98 and105, in particular) appeared to be lower than in the earlytime-point samples. Before hatch, the rabbit anti-rainbowtrout C4 serum recognized one distinct ∼75-kDa protein,while additional signals from ∼29, ∼43, and ∼90 kDaproteins were detected after hatch (data not shown).Presumably, the ∼90 and ∼75 kDa proteins representedthe C4 α- and β-chains, respectively. The low molecularproteins (∼29 and ∼43 kDa) may have represented the C4γ-chain (C4-1γ: 39 kDa, C4-2γ: 34 kDa) (Boshra etal. 2004) or degradation products of activated C4.

Alternative pathway represented by Bf and Df

Results are shown in Fig. 5 (alternative/classical pathway).Bf transcripts were detected from day 7, increased, andpeaked after hatch. During yolk–sac resorption, a signif-icant drop in the contents of Bf transcripts was seen exceptin homogenates from day 84. The contents of Bf transcriptswere, in general, higher at all time-points than all the othercomplement components except for C3-1. Immunoblottingrevealed Bf (81 kDa) proteins in homogenates of theunfertilized egg. The signal then disappeared from day 7 to28 to reappear with increasing intensity from day 35 to 105.A background signal (∼84 kDa) identical to the signaldetected using normal rabbit serum was seen from day 0 to63. Transcription of Df was generally low starting at day 28and peaking at day 84 and 98. Probing with antibodiesspecific to Df (29 kDa) revealed signals in homogenatesfrom all sampled time-points including day 0.

Lytic pathway represented by C5 and C7

Results are shown in Fig. 5 (lytic pathway). Detection ofC5 transcripts revealed a pattern that differed from theother components studied. Transcription was at its highestat day 28 followed by a decrease. Immunoblotting revealedsignals from a ∼133-kDa protein from day 0 to day 105.

This high-molecular weight protein was assumed to be theα-chain of C5 and signals seemed to increase throughoutthe period. Transcription of C7 started at day 28 and peakedat day 42 and 63 (hatching completed). After hatch thecontents of C7 transcripts decreased. A significant drop intranscript levels was detected immediately after hatch(day 70).

Localization of complement component proteinsby immunohistochemistry

Localization of complement components C3-1, C3-3, C3-4,C4, C5, and Df in different tissues of embryos, yolk–sachatchlings, and first feeding hatchlings was studied byimmunohistochemistry. Apart from the heart where all thecomplement components appeared approximately atday 77, the other tissues and organs analyzed containedall complement proteins at developmental stages betweenday 7 and 35 and onward (Fig. 6). Cells with resemblance toyolk periblast cells were clearly stained using the anti-C3-1β serum. Epithelial cells of the esophagus, embryonalskeletal muscle cells, myocardial cells of the heart,parenchymal cells of the liver, mucosal columnar epithelialcells of the intestine, and kidney tubular epithelial cells werestained for all components. All complement components(one micrograph each) studied are shown in Figs. 7 and 8.

Discussion

Fish immunocompetence is determined by the functionalcapability of lymphocytes rather than the morphology ofthe primary (thymus) and secondary, peripheral lymphoidorgans (kidney, spleen, and gut-associated lymphoidtissue) and the presence of lymphoid cells in them (Zapataet al. 1997). At the time of hatching, the lymphoid systemwas still developing and neither the organization nor thefunctional capabilities displayed by adult fish was achieved(Ellis 1988). Whether the fish embryo is capable ofinitiating a complete array of immune responses is not yetclear. One-day-old carp (C. carpio) embryos respond tomicroinjection of bacterial lipopolysaccharide by increas-ing levels of IL-1β transcripts (Rombout et al. 2005) andtranscripts of MHC class Ia, TCRβ, and CD8a were foundin rainbow trout (O. mykiss) at day 7 to 14 (Fischer et al.2005).

Fig. 6 Schematic view of theimmunohistological detection ofcomplement component C3subtypes, C4, C5, and Df indifferent organs of the rainbowtrout embryo from 7 to 105 dayspost-fertilization. The unfertil-ized egg was not studied

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In the current study, no complement componenttranscripts were present before egg fertilization, whichexcludes the possibility of maternal transfer of transcripts.All components, except C5, followed a similar expressionpattern. There was a general increase in the contents oftranscripts after fertilization with a peak post-hatch(day 63). After hatch, transcription seemed to decrease,but the profiles were no longer reciprocal. Homogenatesfrom day 70 appeared to have low transcript levels of allcomplement components studied. No C4 transcripts weredetected at day 91, but transcription of this component wasgenerally very low. A high variation in individual expres-sion is a well-known problem when working with fish andas analyses were based on pooled samples, this possiblycould explain the inconsistency. Immunoblotting, as asemiquantitative method, indicated increased levels of Bfand C5 proteins post-hatch, which was opposed to thedecreasing transcript levels detected by real-time RT-PCR.In general, there was little correlation between thetranscript and protein levels throughout the study periodand this was particularly evident for C4 and C5. ThemRNA levels may, however, not reflect the levels ofprotein expressed by the cell (Gygi et al. 1999). For many

proteins, regulation occurs at the post-transcriptional ortranslational stage, giving delayed turnover, and there mayalso be variable mRNA half lives between the differentcomplement components.

Hatching is complex (Yamagami 1988) and may inducestress. Analyses of hatching in medaka (Oryzias latipes)revealed that corticosteroids stimulated secretion of hatch-ing enzymes (Cloud 1981; Schoots et al. 1982). Corticoidresponse elements are located in the 5′-flanking regions ofhuman and mouse C3 and C5 genes and this may provide abasis for the elevated complement component transcriptlevels found at hatch in our study. On the other hand,studies on carp (C. carpio) and gilthead seabream (S.aurata) have indicated reduced serum complement activityafter induction of stress (crowding, handling, transporta-tion, and anesthesia treatment) (Ortuno et al. 2001, 2002;Yin et al. 1995). Regulatory mechanisms may, however,differ between the natural stress that occurs during adevelopmental event, such as hatching, and duringenvironmentally induced stress.

Bf and Df transcripts representing the alternativepathway were expressed at much higher levels than C4that is central to the classical pathway. Thus, it is likely that

Fig. 7 A selection of micro-graphs of tissue sections fromrainbow trout embryos and lar-vae at different stages of devel-opment. Sections to the left wereimmunohistochemically stainedcomplement C3 subtype pro-teins and counterstained withHarris’ hematoxylin solution(blue). Sections to the rightrepresent the correspondingnormal rabbit serum controls.C3-1β Periblast cells werestrongly stained (day 7). C3-3βEmbryonal kidney tubuli epi-thelial cells were stained. Inaddition, some interstitial cellswere weakly stained (day 35).C3-4α Epithelial cells of theesophagus were clearly stained(day 35)

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the alternative pathway may be more significant than theclassical pathway during embryonic and post-hatch devel-opment. Low levels of C4 transcripts may, however, becompensated by maternally derived proteins. In the spottedwolffish (Anarhichas minor), the ontogenic appearance ofC3 transcripts was reported to coincide with the major celldivision phase during liver development (Ellingsen etal. 2005). In cod (Gadus morhua), C3 transcripts weredetected in various organs from 11 days post-fertilization(Lange et al. 2005). These findings support the notion thattranscription of most complement components starts at anearly developmental stage in fish. Caution, however, mustbe exercised because there is high diversity among teleostfish species with respect to the time of appearance ofimmune mechanisms (Zapata et al. 1997). At the earlieststages of development, the finding of reference genes forquantitative real-time RT-PCR is a challenge. As theactivation of the embryonic genome occurs at the two-cellstage (Picton et al. 1998), transcripts for housekeepinggenes must also be maternally transferred. Thus, adjust-ment to any housekeeping gene at the earliest stages willnot be fully correct. However, as close to undetectableamounts of complement component transcripts were

detected at the earliest time-points, this did not representa problem in this study. From day 14, the embryo’s 18Stranscription level had reached a steady state.

As revealed by the immunoblotting experiments, allcomplement component proteins examined in this studywere detected in the unfertilized egg. In particular, Bfshowed an unambiguous pattern of maternal transfer by itspresence in the unfertilized egg followed by its absence insubsequent samples (day 7 to 28). Maternal transfer of C3also occurs in the spotted wolffish (Ellingsen et al. 2005).Low levels of maternal IgM were found in eggs of rainbowtrout (O. mykiss) (Castillo et al. 1993), gilthead sea bream(S. aurata) (Picchietti et al. 2001), and sea bass(Dicentrarchus labrax) (Scapigliati et al. 1999). Thematernal supplies of IgM, however, were low and did notpersist, questioning their efficacy as protection, but mater-nal IgM might be involved in protection against verticaltransfer of certain pathogens. Castillo et al. (1993) showedthat in the rainbow trout, the amount of IgM g-1 eggsincreased from fertilization until hatch. From this stageonward, expression decreased until 2 months after hatchwhen a recovery was observed. This resembles the generalexpression pattern of complement component transcripts in

Fig. 8 A selection of micro-graphs of tissue sections fromrainbow trout embryos and lar-vae at different stages of devel-opment. Sections to the left wereimmunohistochemically stainedcomplement proteins andcounterstained with Harris’hematoxylin solution (blue).Sections to the right representthe corresponding normal rabbitserum controls. C4 Myocardialcells of the heart were clearlystained (day 77). Df Parenchy-mal cells of the liver werestained (day 42). C5 Embryonalkidney tubuli epithelial cellswere stained (day 35)

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our study. The elevated levels of IgM and complement foundat the time of hatch may be of importance because the newlyhatched fish will be exposed to waterborne pathogens.Antibodies may opsonize bacteria, e.g., for final destructionand eradication by phagocytic cells (Magnadottir 2006;Magnadottir et al. 2005; Rombout et al. 2005). The presenceof antibodies in eggsmay also act in concert with complementcomponents and thus facilitate binding of opsonized bacteriato complement receptors on phagocytes (Holland andLambris 2002). As yet, no such functional studies werecarried out, so this must be considered speculative.

For a functional immunoresponse intact C3 would berequired. The immunoblotting experiments revealed intactC3 under nonreducing conditions. Reducing conditionsmade the detection of the α-chain of the C3 subtypes andC4 difficult. The absence of an intact α-chain was oftenexplained by the chains’ relative susceptibility to enzy-matic cleavage compared to the β-chains (Lange etal. 2004c). Proteolytic degradation or activation of thecomplement cascade induces α-chain cleavage by C3convertase, factor I, and cofactors. This generates a ∼68-kDa fragment of the α-chain, which would be hard toseparate from the 70/73 kDa β-chain under gel electro-phoretic conditions. It is likely that endogenous proteolyticevents in eggs cause degradation of the α-chain of C3,explaining the low amounts of this chain. In addition,intracellular bacteria horizontally transferred to the eggsmay contribute to α-chain depletion by the action ofbacterial enzymes. Sample preparation procedures mayalso induce proteolytic activation and α-chain degradation.Vertical transfer of single β-chains was not likely from animmunological point of view because the α-chain is crucialfor covalent binding to pathogens. Normal rabbit serumshowed some cross-reactivity with ∼80 and ∼15 kDaproteins in the rainbow trout eggs, embryos, and hatchlingsfrom day 0 to 63. This could be derivatives of vitellogeninB, a storage protein for egg yolk, which represents a majormaternal protein, e.g., in cod (G. morhua) (Magnadottir etal. 2004).

Immunohistochemical studies revealed the presence ofC3 proteins at day 7. Different cell types are difficult todistinguish at this developmental stage but periblast cells inthe yolk near the embryo were shown to be involved in con-version of yolk into embryo nutrition (Timmermans 1987).The appearance of complement component transcriptsfrom the real-time RT-PCR correlated with the firstimmunochistochemical detection of proteins in tissuessuch as the liver, skeletal muscle, and intestine. The levelsof complement component transcripts, however, were low,so the embryo’s own protein synthesis could hardlyaccount for the entire immunohistochemical signalsdetected. If complement component proteins were mater-nally transferred, it would be interesting to study theembryo’s transfer and uptake mechanisms and how theproteins are directed to their correct cell types and tissues(Picchietti et al. 2004). Complement component C3 wasfound to be transported into developing chicken oocytesthrough receptor-mediated endocytosis (Recheis etal. 2005). Whether such a process occurs in fish is

unknown. Our findings with regard to the presence ofcomplement components in several tissues may support ahypothesis that complement components have nonimmu-nological functions (Mastellos and Lambris 2002).Individual complement gene promoters have uniquestructures and contain a large array of regulatorysequences (Volanakis 1995). From the multiplicity ofsubtypes and complexity of regulation, it is reasonable toassume that complement components can be linked tononimmunological processes ranging from fertilization(Anderson et al. 1993; Llanos et al. 2000) and regeneration(Del Rio-Tsonis et al. 1998; Kimura et al. 2003) to signaltransduction (Bohana-Kashtan et al. 2004) and energymetabolism in the peripheral nerve (Chrast et al. 2004).From several descriptive studies, a role of complement inorganogenesis is suggested (Lange et al. 2004b), but nofunctional studies on fish were conducted.

This study is the first to address the ontogeny of severalcomplement components. Our findings represent the firstevidence that maternal transfer of complement componentsother than C3 occurs in fish. Whether the presence ofcomplement components in developing animals has im-munological significance is not yet known. Furthermore,our immunohistochemical results support previous reportson complement that suggest putative nonimmunologicalfunctions during organogenesis.

Acknowledgements We wish to thank Dr. Frederick W. Goetz(Marine Biological Laboratory, Marine Resources Center, USA) andDr. Ivar Hordvik (Department of Biology, University of Bergen,Norway) for helpful suggestions. We are grateful to ProfessorMalcolm Jobling (Department of Aquatic BioSciences, University ofTromsø, Norway) for carefully reading this manuscript. The financialsupport of the European Commission grant IMAQUANIM (contractno. 007103) and the University of Tromsø is acknowledged.

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