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A Hemocyte-like Cell Line Established from the Malaria VectorAnopheles gambiae Expresses Six Prophenoloxidase Genes

1999; Elsevier BV; Volume: 274; Issue: 17 Linguagem: Inglês

10.1074/jbc.274.17.11727

1083-351X

Hans‐Michael Müller, George Dimopoulos, Claudia Blass, Fotis C. Kafatos,

Insect Utilization and Effects

Cell lines from the malaria vectorAnopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, andPPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae. Cell lines from the malaria vectorAnopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro. Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO). This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, andPPO6). The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner. Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation. Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage. The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts. This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A. gambiae. prophenoloxidase 20-hydroxyecdysone bacterial artificial chromosome fetal calf serum phosphate-buffered saline phenoloxidase polymerase chain reaction reverse transcription-PCR Anopheles gambiae mosquitoes are the principle vectors of Plasmodium falciparum, the parasite causing the most severe form of human malaria. However, within the A. gambiaecomplex mosquitoes may differ in their efficiency of transmitting malaria (1Coluzzi M. Parasitol. Today. 1992; 8: 113-118Abstract Full Text PDF PubMed Scopus (87) Google Scholar). Transmission requires completion of a complex sporogonic cycle, which takes place over 2 weeks within the mosquito and can be aborted by innate immune responses, such as the encapsulation of early oocysts soon after invasion of the midgut (2Collins F.H. Sakai R.K. Vernick K.D. Paskewitz S. Seeley D.C. Miller L.H. Collins W.E. Campbell C.C. Gwadz R.W. Science. 1986; 234: 607-610Crossref PubMed Scopus (421) Google Scholar). Genes responsible for such refractory phenotypes would be candidates for inclusion in a vector control strategy based on genetically manipulated mosquitoes (3Collins F.H. Besansky N.J. Science. 1994; 264: 1874-1875Crossref PubMed Scopus (73) Google Scholar,4Zheng L. Arch. Insect Biochem. Physiol. 1997; 34: 1-18Crossref Scopus (14) Google Scholar). Vertebrate immune defense has been attributed to two general systems, innate and adaptive immunity, which are interconnected (5Medzhitov R. Janeway C.A. Curr. Opin. Immunol. 1998; 10: 12-15Crossref PubMed Scopus (279) Google Scholar). In contrast, insect immune defense lacks the adaptive component and therefore an antibody-mediated immune response. Instead, it fully relies on innate immune mechanisms, such as the inducible synthesis of antimicrobial peptides, and the coagulation and melanization cascades (6Hoffmann J.A. Reichhart J.-M. Trends Cell Biol. 1997; 7: 309-316Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 7Richman A. Kafatos F.C. Curr. Opin. Immunol. 1995; 8: 14-19Crossref Scopus (63) Google Scholar). The ongoing exploration of immune mechanisms in A. gambiae is hampered by the small size of this insect, limited knowledge of its genetics, and the absence of an efficient method for germline transformation. Insect cell lines were previously shown to exhibit immune properties, including the inducible synthesis of antibacterial peptides (8Komano H. Kasama E. Nakanishi Y. Matsuyama K. Ando K. Nagasawa Y. Natori S. Kuroda Y. Kurstak E. Maramorosch K. Invertebrate and Fish Tissue Culture. Springer Verlag, New York1988: 75-78Crossref Google Scholar, 9Samakovlis C. Asling B. Boman H.G. Gateff E. Hultmark D. Biochem. Biophys. Res. Commun. 1992; 188: 1169-1175Crossref PubMed Scopus (132) Google Scholar, 10Hernandez V.P. Gerenday A. Fallon A.M. Am. J. Trop. Med. Hyg. 1994; 50: 440-447Crossref PubMed Scopus (18) Google Scholar). Therefore, we have taken the approach of establishing A. gambiae cell lines as an in vitro system to analyze the immune mechanisms of the mosquito. We have reported that one such line expresses in an inducible manner a panel of immune marker genes (11Dimopoulos G. Richman A. Müller H.-M. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11508-11513Crossref PubMed Scopus (325) Google Scholar). In the course of establishing a larger variety of immune-responsiveA. gambiae cell lines, we identified some that secrete prophenoloxidase (PPO)1 in the culture medium and adopted one such line, 4a-3B, as a standard. No continuous cell lines of this type have been reported previously in any insect. Phenoloxidases (POs) are enzymes that serve multiple tasks in insects, including cuticle pigmentation and sclerotization, wound healing and the melanotic encapsulation of protozoan (plasmodia) and metazoan (filaria) pathogens (12Ashida M. Yamazaki H.I. Ohnishi E. Ishizaki H. Molting and Metamorphosis. Japan Scientific Societies Press, Tokyo1990: 239-265Google Scholar, 13Vernick K.D. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 261-309Google Scholar). The PPO zymogens are known to be synthesized in hemocytes, the cellular component of the insect immune defense, and are activated by trypsin-like serine protease components of the PPO activation system, also called the phenoloxidase cascade (see under "Discussion"). The PO cascade is inducible in vitro by microbial cell wall constituents and is considered part of the insect non-self recognition system (14Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, London1998: 135-172Google Scholar, 15Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1108) Google Scholar). PO catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine and the oxidation of dihydroxyphenylalanine and dopamine to their respective quinones, mediators of protein cross-linking, and precursors of the melanin polymer that is a central component of the melanotic capsule (16Nappi A.J. Sugumaran M. Pathak J.P.N. Insect Immunity. Kluwer Academic Publishers, Dordrecht, The Netherlands1993: 131-148Crossref Google Scholar, 17Zhao X. Ferdig M.T. Jianyong L. Christensen B.M. Dev. Comp. Immunol. 1995; 19: 205-215Crossref PubMed Scopus (55) Google Scholar). In addition, melanin synthesis leads to local increase of toxic quinones and free radicals (16Nappi A.J. Sugumaran M. Pathak J.P.N. Insect Immunity. Kluwer Academic Publishers, Dordrecht, The Netherlands1993: 131-148Crossref Google Scholar). Molecular cloning has yielded 12 PPO sequences to date, 1 crustacean, 6 lepidopteran, and 5 from diptera. All the sequences are closely related and show homology to hemocyanin, the arthropod oxygen transporter, which is functionally replaced in insects by the tracheal system. Using degenerate oligonucleotides and cDNA from the PPO producingA. gambiae cell lines as template, we amplified a PCR product consisting of a mixture of six distinct PO sequences. Three of these corresponded to PPO genes found in studies then in press (18Jiang H. Wang Y. Korochkina S.E. Benes H. Kanost M.R. Insect Biochem. Mol. Biol. 1997; 27: 693-699Crossref PubMed Scopus (49) Google Scholar,19Lee W.-J. Ahmed A. della Torre A. Kobayashi A. Ashida M. Brey P.T. Insect Mol. Biol. 1998; 7: 41-50Crossref PubMed Scopus (47) Google Scholar); the other three were novel and were designated PPO4, PPO5, and PPO6. Corresponding full-length cDNA clones were isolated and their sequences were determined. Reverse transcription PCR (RT-PCR) expression analysis using specific primer pairs confirmed that all six PPO genes are expressed constitutively in the same cell line but show distinct expression profiles during mosquito development. Transcription of the genes that are larva-specific is transiently up-regulated in the adult female mosquito following a blood meal. The same genes are up-regulated when our standard cell line is exposed to 20-HOE. Interestingly, the only adult-specific PPO gene, PPO5, shows a converse behavior: it is repressed upon a blood meal in the mosquito and upon 20-HOE treatment of the cell line. Thus, this cell line is a useful in vitro model for the study of hormone-regulated as well as immune-regulated gene expression, phenomena that are frequently connected in insects. The A. gambiae strains 4a r/r, L3–5, and Suakoko 2La were maintained at 27 °C and 75% relative humidity on a 12-h light-dark cycle. Larvae were kept in deionized water supplemented with 0.1% marine salt and fed with dry cat food. Adult mosquitoes had access to 20% sucrose solution ad libidum and were blood-fed on a volunteer's arm (H.-M. M.). Schneider medium (Sigma) supplemented with 10% FCS, 20 mm EGTA, and 10 mm thiourea was used to perfuse 1-day-old female 4a r/r mosquitoes. No further protease inhibitors were added, as FCS alone was found to be an efficient inhibitor of the PO cascade otherwise triggered by exposure of the hemolymph to air (blackening). Mosquitoes were chilled on ice and transferred on a 8-well glass slide containing 5 μl of perfusion medium per well, and a cut was made at the second-to-last abdominal segment. A fine glass needle containing 5 μl was inserted in the neck, and the perfusion medium, injected by air pressure, was collected in the well. Cells were allowed to settle for 2 min, and then 10 μl of 4% formaldehyde in PBS (130 mm NaCl, 7 mmNa2HPO4, 3 mm NaH2PO4, pH 7.2) were added. After 20 min of fixation at ambient temperature, samples were processed as described below for immunofluorescence. Pupae or decapitated female mosquitoes of the 4a r/r strain were placed in a drop of 5-μl PBS containing 2 mg/ml of the serine protease inhibitor Pefabloc (Boehringer Mannheim) and 20 mm EGTA. After cutting apart thorax and abdomen, the body parts and the fluid were transferred in a vial containing 100 μl of the same buffer, prechilled on ice. After the dissection of 10 animals, thoraces and abdomens were gently vortexed. The fluid phase was recovered and mixed with an equal volume of 2-fold concentrated reducing SDS-polyacrylamide gel electrophoresis sample buffer. Samples were boiled for 5 min and stored at −20 °C. Cell lines were established according to the method of Pudney et al. (20Pudney M. Varma M.G.R. Leake C.J. Tissue Culture Association Manual. 1979; 5: 997-1003Crossref Scopus (16) Google Scholar) using neonate A. gambiae larvae as starting material. Repeated agitation starting 36 h after egg deposition led to synchronous hatching 6–7 h later; only larvae hatched within 1 h were used for primary cultures. Large scale primary cultures were set up in 50-ml tissue culture flasks (Falcon 3014) in a volume of 5 ml using about 1000–3000 larvae, each cut, on average, in 3–4 pieces, followed by a trypsin treatment (20Pudney M. Varma M.G.R. Leake C.J. Tissue Culture Association Manual. 1979; 5: 997-1003Crossref Scopus (16) Google Scholar). Two alternative media were used, Schneider medium (Sigma) or MK/VP-medium (20Pudney M. Varma M.G.R. Leake C.J. Tissue Culture Association Manual. 1979; 5: 997-1003Crossref Scopus (16) Google Scholar), supplemented with 20% heat-inactivated FCS, penicillin-streptomycin (300 units/ml-300 μg/ml), gentamycin (100 μg/ml), and Fungizone (2.5 μg/ml); the cultures were kept at 27 °C. Once per week, half of the culture medium was replaced by fresh medium containing 20% FCS and penicillin-streptomycin (100 units/ml-100 μg/ml). After subculture 10, the FCS content in the medium was gradually decreased to 10%. All the lines are kept continuously in Schneider medium. Cells were seeded in 75 cm2 tissue culture flasks (Greiner) containing 25 ml of Schneider medium/10% FCS. After having reached half confluency, cell layers were rinsed several times with protein-free medium (Insect-Xpress, BioWhittaker), 15 ml of protein-free medium was added, and cultures were grown for 2 more weeks at 27 °C. Cell growth was retarded in Insect-Xpress medium, although without any sign of deterioration of the cells. The conditioned medium was transferred in a dialysis bag and 5-fold concentrated on polyethylene glycol-35,000 powder. After dialysis against 10 mm phosphate buffer, pH 6.0, preparations were stored at −20 °C. Cell cultures were immune-stimulated by adding to a confluent culture for 4 h a mixture of heat-inactivatedEscherichia coli 1106 and Micrococcus luteus A270, 1000 bacteria per mosquito cell. 20-Hydroxyecdysone (Sigma) in ethanol was added yielding a final concentration of 0.5 μg/ml (1 μm), with the ethanol concentration below 0.1%. For the wash-out experiment, 20-HOE-containing medium was aspirated, and cell layers were rinsed several times with Schneider medium before conditioned 20-HOE-free medium, taken from a culture of identical age, was added. Unless otherwise indicated, all molecular techniques were performed as described (21Perbal B. A Practical Guide to Molecular Cloning. Wiley-Interscience, New York1988Google Scholar). Total RNA from cell lines and the various mosquito developmental stages was purified using the RNaid PLUS kit (bio101) according to the manufacturer's instructions. First strand cDNA was synthesized using Moloney murine leukemia virus-reverse transcriptase and supplied solutions (Life Technologies, Inc.) with 2 μg total RNA in a reaction volume of 15 μl. cDNA synthesis was primed using the equivalent of 3 μl of oligo(dT)25 magnetic beads (Dynal), washed with distilled water before use. After incubation at 37 °C for 1 h with occasional suspension, reactions were terminated by heating to 95 °C for 5 min. Beads were rinsed several times and resuspended in 200 μl of distilled water. 1.5 μl of bead suspension was used in 20-μl PCRs containing 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 8.3, 200 μm dNTP (Amersham Pharmacia Biotech), 0.05 μl of each [α-32P]dATP and [α-32P]dCTP (10 μCi/μl, 3000 Ci/mmol), 0.5 units Taq polymerase (Perkin-Elmer), and 20 pmol of each primer. PCRs were performed in a Perkin-Elmer thermal cycler (GeneAmp 9600), 1 min at 63 °C, 1 min at 72 °C, and 1 min at 94 °C per cycle. Cycle numbers are indicated in the figure legends. 4 μl of each PCR were subjected to electrophoresis on 6% nondenaturing polyacrylamide gels. Dried gels were exposed on Kodak X-Omat AR films with Ilford Fast Tungstate screens. The ribosomal gene S7 (22Salazar C.E. Mills-Hamm D. Kumar V. Collins F.H. Nucleic Acids Res. 1993; 21: 4147Crossref PubMed Scopus (77) Google Scholar) served as internal control and was used for normalization as follows: the S7 signals resulting from the initial PCRs of a given set of cDNA samples were quantified with a PhosphorImager (Molecular Dynamics), the S7 values were equalized, and the template amounts were adjusted accordingly. The S7 signals obtained from the second round of PCRs were again quantified by phosphorimaging and normalized against S7 in order to fine-tune the amount of sample loaded on the final gel, resulting in deviations between any S7 signal in a given experiment of no more than ±5%. The sequences of the primers used in RT-PCR experiments were as follows: S7-A, 5′-GGCGATCATCATCTACGT-3′; S7-B, 5′-GTAGCTGCTGCAAACTTCGG-3′. Def-A, 5′-CTGTGCCTTCCTAGAGCAT-3′; Def-B, 5′-CACACCCTCTTCCCAGGAT-3′. GNBP-A, 5′-GCAACGAGAATCTGTACC-3′; GNBP-B, 5′-TAACCACCAGCAACGAGG-3′. PO1-A, 5′-TTCGATGCCTCTAACCGGGCGA-3′; PO1-B, 5′-GCGGGATGCGGTTACCGGATTCA-3′. PO2-A, 5′-CGGTTCTGCGCCAAGCTGAAGAA-3′; PO2-B, 5′-CTGCCATACAGCTGGGCATTCGG-3′. PO3-C, 5′-GGGTCCCGACCGTGTCGTCAAC-3′; PO3-D, 5′-ACGATTACCACCGGGGCCCACG-3′. PO4-C, 5′-CGCGGGCCGGATCGTATCGTGC-3′; PO4-E, 5′-CTCAACTCGTTTAAGATCACTCAA-3′. PO5-C, 5′-ACTGGTCCGGATCGGGTTGTGC-3′; PO5-D, 5′-GAACACGATCGCCATTCGTCGC-3′. PO6-A, 5′-GGCGAGGGTCCGAATAACGTA-3′; PO6-B, 5′-TCCGATTTCCTCCGGGGGCAACA-3′. The resulting lengths of the corresponding PCR products were as follows: S7, 460 bp; defensin, 404 bp; GN BP, 511 bp; PPO1, 358 bp; PPO2, 344 bp; PPO3, 343 bp; PPO4, 246 bp; PPO5, 346 bp; and PPO6, 349 bp. Except for PPO2, the primer combinations denoted above do not work on genomic DNA, e.g. on bacterial artificial chromosomes (BACs), as the polymorphic sequence block chosen for the design of the antisense primers turned out to span an intron/exon border that occurs in PPO1, 2P. Brey, personal communication. which is obviously conserved in all PPO genes except PPO2. Two lambda ZAP cDNA libraries were screened, one was constructed using mRNA isolated from fourth instar larvae of A. gambiae G3 (23Barillas-Mury C. Charlesworth A. Gross I. Richman A. Hoffmann J.A. Kafatos F.C. EMBO J. 1996; 15: 4961Crossref Scopus (104) Google Scholar) and one using mRNA isolated from abdomens of adult female A. gambiae Suakoko 2La mosquitoes (24Dimopoulos G. Seeley D. Wolf A. Kafatos F.C. EMBO J. 1998; 17: 6115-6123Crossref PubMed Scopus (259) Google Scholar). Both libraries were constructed using the ZAP Express system (Stratagene). As probe, the gel-purified PCR product amplified from cDNA of cell line 4a-3B using the primers PO-CuA and PO-CuB was enriched with gel-purified PPO4, PPO5, and PPO6 fragments and 32P-labeled via PCR. From a total of 700,000 plaques screened, 200,000 larval and 500,000 abdominal, approximately 60 positive signals were obtained in each library. Before starting the plaque purification procedure, the supernatants of the positive phage plaque zones of the primary screen were PCR-typed using the set of six specific PPO primer pairs. The identified PPO4, PPO5, and PPO6 samples were plaque purified via hybridization, followed by PCR typing. The 0.6-kb PCR product amplified from cell line cDNA using the degenerate primers PO-CuA (5′-CAICA(TC)TGGCA(TC)TGGCA(TC)CTIGT(GATC)TA(TC)CC-3′) and PO-CuB (5′-CITGCCAICG(AG)TA(AG)AAIA(ACT)(CG)GG(AG)TCIC(TG)CAT-3′) (1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, 25 cycles) was gel-purified and cloned using the TA Cloning Kit (Invitrogen). Plasmid clones containing PPO cDNA inserts were obtained from the purified lambda ZAP phage clones using the in vivo excision method according to the manufacturer's instructions (Stratagene). The sequences of the cloned insertions were determined by the EMBL sequencing facility. The RGD sequence in PPO6 was confirmed by sequencing the corresponding region of four independent cDNA clones; in PPO4 and PPO5, the corresponding region of a second cDNA clone was sequenced. Hybridizations were essentially performed as described (25Kumar V. Collins F.H. Insect Mol. Biol. 1994; 3: 41-47Crossref PubMed Scopus (76) Google Scholar), using semi-gravid female mosquitoes of the Suakoko 2La strain, during the second gonotrophic cycle. DNA of whole BACs that were shown by PCR typing to contain PPO3 and PPO6(BAC clone 27E19) or PPO4 and PPO5 (BAC clone 28C11) were used for in situ hybridization. In addition,in situ hybridizations were carried out using gel-purified insertions of cDNA clones corresponding to PPO4,PPO5, and PPO6. The TA-plasmid vectors (Invitrogen) containing the PCR fragments amplified by the degenerate oligonucleotides PO-CuA and PO-CuB wereEcoRI-digested, and the PO inserts were subcloned in theEcoRI site of the polylinker of the modified expression vector pDS56/RBSII,6xHis/E−(-2) (26Müller H.-M. Catteruccia F. Vizioli J. della Torre A. Crisanti A. Exp. Parasitol. 1995; 81: 371-385Crossref PubMed Scopus (98) Google Scholar), resulting in a fusion of six histidine residues at the amino termini. Expression of recombinant proteins in E. coli and purification by nickel-chelate affinity chromatography was performed as described previously (27Stüber D. Matile H. Garotta G. Lefkovits I. Pernis B. Immunology Methods. 4. Academic Press Inc., Orlando, FL1990: 121-152Google Scholar). The purified PO proteins were insoluble and were stored at −20 °C in 8 m urea, 0.1 m sodium phosphate, 0.01 m Tris-HCl, pH 8.0, at a concentration of 5 mg/ml. Two male rats were immunized with each construct using 100 μg of purified protein and the Ribi Adjuvant System following the manufacturer's instructions (RIBI ImmunoChem Research). Rats were bled 1 week after the fourth booster injection. Polypeptides contained in mosquito hemolymph or in protein-free medium (Insect-Xpress, BioWhittaker) conditioned by the growth of cell lines 4a-3A and 4a-3B were separated on 7% SDS-polyacrylamide gel electrophoresis under reducing conditions. Immunoblots on nitrocellulose filters (Protran, Schleicher & Schuell) were performed as described previously (26Müller H.-M. Catteruccia F. Vizioli J. della Torre A. Crisanti A. Exp. Parasitol. 1995; 81: 371-385Crossref PubMed Scopus (98) Google Scholar). The anti-PPO6 serum dilution used was 1:1000; bound antibodies were detected by goat anti-rat IgG (H+L) conjugated to alkaline phosphatase (Promega). 4a-3A and 4a-3B cells were seeded on round glass coverslips placed in 24-well tissue culture plates (Nunc) yielding half confluent cell layers. After 3 days of growth at 27 °C the Schneider medium was removed, and the coverslips were washed once with PBS and then fixed in PBS/4% formaldehyde for 20 min. After fixative removal followed by one PBS wash, cells were exposed for 2 min to 0.2% Triton X-100 in PBS and then washed twice with PBS. Coverslips were blocked for 2 h with 1% bovine serum albumin in PBS and then incubated overnight at 4 °C with anti-PPO4 serum or preimmune serum diluted 1:500 in 1% bovine serum albumin/PBS. After three PBS washes, samples were incubated for 1 h with a fluorescein isothiocyanate-conjugated anti-rat IgG antibody (The Jackson Laboratory) diluted 1:300 in 1% bovine serum albumin/PBS. After the first PBS wash, nuclei were counterstained with DAPI (Boehringer Mannheim) diluted 1:15,000 in PBS for 5 min, and three further PBS washes followed before mounting. In a previous report, we described the cell line Sua1B, which has immune-responsive properties, such as inducible expression of the antibacterial peptide defensin (11Dimopoulos G. Richman A. Müller H.-M. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11508-11513Crossref PubMed Scopus (325) Google Scholar). In a further effort to establish A. gambiae cell lines with novel properties, primary cultures were set up starting from neonate larvae of threeA. gambiae strains, Suakoko 2La, 4a r/r, and L3–5. Further variation was brought in by differing culture conditions,e.g. initial seeding density, medium, and splitting strategy. Some primary cultures gave rise to cell lines that developed relatively slowly and were finally identified serendipitously as PPO producers, when we observed spontaneous blackening of stored culture supernatants. Such lines were most frequently derived from the 4a r/r strain. The presence of PPO in the culture supernatant could be monitored directly by artificial activation on nitrocellulose (28Brey P.T. Stoltz D.B. Cook D.I. Ashida M. Anal. Biochem. 1991; 194: 359-364Crossref PubMed Scopus (7) Google Scholar). This rather simple test enabled us to follow PPO expression in all the lines during increasing subculture number. Whereas some primary cultures gradually lost the ability to produce PPO during establishment of the line, probably due to overgrowth by non-PPO producing fast growing cell types, others manifested the PPO phenotype stable, over 2 years (subculture > 60), until submission of this study for publication. We have chosen cell line 4a-3B as an example for our stable PPO producing cell lines and cell line 4a-3A as a PPO-negative control. These lines were derived from two primary cultures initiated from a single batch of minced neonate larvae of the A. gambiae strain 4a r/r. Both lines show inducible expression of certain immune markers (see below). To verify the expression of PPO on the molecular level, we PCR-amplified RNA sequences from several PPO-positive lines using degenerate oligonucleotides designed according to two conserved sequence blocks within the PO copper binding sites CuA (HHWHWHLVYP) and CuB (MRDP(F/V/I)FYRWH), respectively (Fig. 1) (29Hall M. Scott T. Sugumaran M. Söderhäll K. Law J.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7764-7768Crossref PubMed Scopus (185) Google Scholar). A PCR product of the expected size (approximately 600 bp) was obtained and cloned. Among 13 sequenced clones, we identified six distinct PPO sequences, which are different enough to represent distinct genes rather than polymorphism (see below). Subsequently, we designed primer pairs specific for each of the six PPO sequences (see under "Experimental Procedures"); analysis of 20 additional PCR-derived PPO clones with these specific primer pairs did not yield any evidence of an additional PPO gene: for every clone tested, one (and only one) of the six PPO primer pairs amplified a fragment of the expected size, thus confirming that the gene-specific primer pairs are diagnostic. All six PPOs were expressed in each of the six independent PPO-positive cell lines analyzed. 3H.-M. Müller, unpublished data. Three of the sequences matched the PPO1 gene published by Lee et al. (19Lee W.-J. Ahmed A. della Torre A. Kobayashi A. Ashida M. Brey P.T. Insect Mol. Biol. 1998; 7: 41-50Crossref PubMed Scopus (47) Google Scholar) and the PPO2 (formerly PPO-p1) andPPO3 (formerly PPO-p2) sequences published by Jiang et al. (18Jiang H. Wang Y. Korochkina S.E. Benes H. Kanost M.R. Insect Biochem. Mol. Biol. 1997; 27: 693-699Crossref PubMed Scopus (49) Google Scholar). However, the three other PPO genes were novel and were named PPO4, PPO5, and PPO6. The mixed PCR products generated by the degenerate primers were enriched with these novel sequences, and were used as probe to screen two A. gambiae cDNA phage libraries: one constructed with mRNA of whole fourth instar larvae (23Barillas-Mury C. Charlesworth A. Gross I. Richman A. Hoffmann J.A. Kafatos F.C. EMBO J. 1996; 15: 4961Crossref Scopus (104) Google Scholar), and one with mRNA purified from abdomens of female mosquitoes (24Dimopoulos G. Seeley D. Wolf A. Kafatos F.C. EMBO J. 1998; 17: 6115-6123Crossref PubMed Scopus (259) Google Scholar). The screening yielded approximately 60 positive signals from each of the two libraries, and rescreening of a subset of the positives, using the six PPO gene-specific primer pairs, identified PPO clones that were evidently differentially represented: from the adult library 10 PPO5, 5 PPO6, and 1 PPO2 clone were identified, whereas from the larval library, 5 PPO2 and 4 PPO3 clones were identified. The original 120 positives were rescreened with the PPO4-specific primers, leading to the identification of four PPO4 clones from the larval and one from the adult library. At least three full-length cDNA clones were characterized for each of the novel PPO genes PPO4, PPO5, and PPO6. Fig. 1 shows the amino acid sequences of PPO4,PPO5, and PPO6 as deduced from their cDNA sequences, aligned with those of the previously identified A. gambiae PPO genes PPO1, PPO2, andPPO3. Like all the arthropod PPO sequences published so far, the mosquito PPOs do not encode a potential signal peptide sequence. An arginine-phenylalanine zymogen cleavage site (Fig. 1, vertical arrow) that has been experimentally determined in Bombyx mori (30Kawabata T. Yasuhara Y. Ochiai M. Matsuura S. Ashida M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7774-7778Crossref PubMed Scopus (170) Google Scholar) and Holotrichia diomphalia (31Lee S.Y. Kwon T.H. Hyun J.H. Choi J.S. Kawabata S.-I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 254: 50-57Crossref PubMed Scopus (119) Google Scholar) is conserved in both sequence and position relative to the initiator methionine. A second cleavage site (Fig. 1, arrowhead) was found inMusca domestica PPO (32Hara T. Miyoshi T. Tsukamoto T. Comp. Biochem. Physiol. 1993; 106B: 287-292Google Scholar), and the sequence encompassing that site (REE) is present in all six A. gambiae PPO sequences; furthermore, the amino-terminal sequence of the resulting activated PO (EEATVVPDG) is conserved with only one substitution in A. gambiae PPO1. Cleavage sites at similar positions were determined experimentally in H. diomphalia PPO (31Lee S.Y. Kwon T.H. Hyun J.H. Choi J.S. Kawabata S.-I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 254: 50-57Crossref PubMed Scopus (119) Google Scholar) andPacifastacus leniusculus PPO (33Aspán A. Huang T.-S. Cerenius L. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (230) Google Scholar), located th