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16S rRNA Gene Sequences from Bacteria Associated withAdult Anopheles darlingi (Diptera Culicidae) MosquitoesAuthor(s) Olle Terenius Caroline Dantas de Oliveira Waleria Dasso PinheiroWanderli Pedro Tadei Anthony Amade James and Osvaldo MarinottiSource Journal of Medical Entomology 45(1)172-175 2008Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-2585(2008)45[172SRGSFB]20CO2URL httpwwwbiooneorgdoifull1016030022-258528200829455B1723ASRGSFB5D20CO3B2

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SHORT COMMUNICATIONS

16S rRNA Gene Sequences from Bacteria Associated with AdultAnopheles darlingi (Diptera Culicidae) Mosquitoes

OLLE TERENIUS1 CAROLINE DANTAS DE OLIVEIRA2 WALERIA DASSO PINHEIRO2

WANDERLI PEDRO TADEI2 ANTHONY AMADE JAMES13 AND OSVALDO MARINOTTI14

J Med Entomol 45(1) 172ETH175 (2008)

ABSTRACT The microbial szligora associated with Anopheles darlingi Root (Diptera Culicidae) amajor Neotropical malaria vector was investigated for the development of a paratransgenesis-basedapproach to control malaria transmission in Brazil Female mosquitoes were collected using humanland catches and captured insects provided a bloodmeal The controlled blood feeding resulted inincreased detection of mosquito bacterial population because it was possible to retrieve bacterial DNAfrom all blood-fed mosquitoes The 16S sequences of bacteria recovered include some closely relatedto those found in other vector mosquitoes including Aeromonas Pantoea and Pseudomonas species

KEY WORDS Anopheles darlingi paratransgenesis malaria

The malaria burden in Brazil is estimated to 600 000cases per year (Coura et al 2006) Both Plasmodiumfalciparum and Plasmodium vivax infections are prev-alent and the principal malaria vector in the Amazonregion isAnopheles darlingiRoot (Diptera Culicidae)(Deane 1986 Tadei and Dutary-Thatcher 2000) Re-lease of genetically engineered adult mosquitoes re-sistant to parasite infections has been proposed as away to control malaria transmission (James 2003)However the use of transgenic An darlingi to controlmalaria transmission in the Amazon region has beenhampered by the failure to rear this species in cap-tivity To circumvent this problem an alternativewould be to use paratransgenesis where bacteria aremodiTHORNed genetically to produce antiparasitic factorsand then introduced into the insect midgut (Beard etal 2002) To search for bacteria suitable for paratrans-genesis investigations of cultured and uncultured bac-teria have been performed in Culex quinquefasciatusSay (Pidiyar et al 2004) and in African Anophelesspecies (Lindh et al 2005) Both studies used capturedblood-fed mosquitoes for gene ampliTHORNcation of bac-terial 16S rRNA Despite the sensitivity of this methodrather few culture-independent bacteria were de-tected In an attempt to increase the proportion ofmosquitoes from which 16S rRNA sequences can beretrieved we explored the outcome of controlledblood feeding of host-seeking An darlingi In thisarticle we also present a THORNrst survey of the uncultured

bacteria associated with An darlingi and show thatthey mainly belong to the Enterobacteriaceae family

Materials and Methods

Host-seeking An darlingi females were capturedwhen attempting to feed on humans during August2006 6ETH9 pm in Manaus (3 08 S 60 01 W) Ama-zonas Brazil They were identiTHORNed using morpholog-ical characters (Faran and Linthicum 1981 Forattini1962) and conTHORNned individually in plastic cups Themosquitoes were allowed to feed on domestic ducks(Cairinamoschata) within 2 h of capture or they weregiven access to a 10 sucrose solution Twenty-sixhours after capture mosquitoes were immersed inisopropanol and stored at room temperature DNAwas extracted individually from nine whole mosqui-toes (Wizard Genomic DNA PuriTHORNcation kit Pro-mega Madison WI) To avoid contamination THORNlteredtips were used throughout the process and the DNAand the primers were dissolved in DNA-free water(Ambion Austin TX) The 16S rRNA gene ampliTHORN-cations were performed in 25-l reactions (SupermixInvitrogen Carlsbad CA) containing 50 ng of DNAand the primers 8f (5-AGAGTTTGATIITGGCT-CAG-3) and 1501r (5-CGGITACCTTGTTACGAC-3) (Lindh et al 2005) The DNA ampliTHORNcation pro-gram was as follows 94C for 3 min followed by 30cycles of 94C for 30 s 58ETH48C for 30 s (the temper-ature was decreased by 1C every cycle for 10 cyclesand thenheldat 48Cfor20cycles) 72Cfor1min30 sfollowed by a THORNnal extension step at 72C for 25 min(Lindh et al 2005) AmpliTHORNcation products (15 kb)were cloned into TOPO 21 (Invitrogen) and 10 16SrRNA clones from each mosquito were sequenced(Laguna ScientiTHORNc Laguna Beach CA) by using M13forward and reverse primers The 16S rRNA gene

1 Department of Molecular Biology and Biochemistry 3205 Mc-Gaugh Hall University of California Irvine CA 92697

2 Instituto Nacional de Pesquisas da Amazonia Manaus AM 69011-970 Brazil

3 Department of Microbiology and Molecular Genetics Universityof California Irvine CA 92697

4 Corresponding author e-mail omarinotuciedu

0022-2585080172ETH0175$04000 2008 Entomological Society of America

sequences were analyzed in BLASTn (httpwwwncbinlmnihgovBLAST) by using GenBank as thereference library Chimerical sequences were elimi-nated after analysis with the Ribosomal DatabaseProject II Chimera Check program (Cole et al 2005)The percentage of coverage of the sequence analysiswas calculated by GoodOtildes method with the formula[1 (nN)] 100 where n is the number of mo-lecular species represented by one clone (single-cloneoperational taxonomic units [OTUs]) and N is thetotal number of sequences (Good 1953) An OTU wasdeTHORNned as consisting of all sequences with 2 di-vergence from the aligned homologous nucleotides(Pidiyar et al 2004)

Results and Discussion

The bacteria colonizing midguts of insect diseasevectors have drawn special attention for their in-teractions with both the insect hosts and the patho-genic organisms (for review see Azambuja et al2005) Based on these interactions paratransgenesishas been developed as a strategy to control Chagasdisease transmission by triatomine bugs (Beard et al2002) It is anticipated that paratransgenesis also canbe used to deliver anti-Plasmodium effector mole-cules to wild mosquito populations resulting in thecontrol of malaria transmission So far no true mos-quito symbionts have been found but recent re-search has provided the basis for mosquito para-transgenesis (Riehle and Jacobs-Lorena 2005) andit has been shown that it is possible to enhance thecapability of a gut bacterium to remain in the mos-quito gut (Riehle et al 2007) Many of the bacteriashown to inhabit the guts of mosquitoes are envi-ronmental and isolated commonly from soil orplants For example a species of the genus Asaiaisolated mainly from tropical szligowers in Asia canstably inhabit the gut of the Asian malaria vectorAnopheles stephensi Liston (Favia et al 2007) TheidentiTHORNcation of 16S rRNA sequences from bacterialpopulations found in the gut of vector mosquitoeswas performed previously on mosquitoes from Asia(Pidiyar et al 2004) and from Africa (Lindh et al2005) Here we present results of a polymerasechain reaction (PCR)-based approach to identifybacteria associated with An darlingi a major ma-laria vector in Latin America

From the nine mosquitoes analyzed we retrieved 56bacterial 16S sequences from six mosquitoes corre-sponding to a coverage of 93 by using the criteria set inPidiyar et al (2004) The coverage is the probability ofthe next cloned sequence falling in a novel (not yetobserved)OTUormolecular speciesThis valuegives anestimation of how well the clones analyzed account forthebiodiversitywithintheoriginal samplebythepresentmethodology (ie with the used PCR conditions andprimer set)Acoverageof93 indicates that sequencingmorecloneswouldresult inonly7of thesamplesbeingnovel

The majority of 16S sequences detected are frombacteria belonging to the Enterobacteriaceae family

(Fig 1) The Enterobacter hormaechei-cluster and theant lion gut bacterium 7s5-cluster contain bacteriaobtained from most of the specimens analyzed Thisdiffers from the results from other malaria vectorswhere only a single bacterial species was found to bepresent in more than one specimen of 91 Anophelesgambiae and 25 Anopheles funestus Giles mosquitoesexamined (Lindh et al 2005) Our result is encourag-ing because the use of paratransgenesis requires abacterium that could be introduced to the majority ofthe mosquitoes in a vector population Many of the 16SrRNA sequences retrieved from An darlingi are sim-ilar to those of gut bacteria found in other mosquitoesbut some are related to bacteria from insects as evo-lutionary distanced as thrips (Thysanoptera) and antlions (Neuroptera) The THORNrstAeromonas speciesAero-monas culicicola isolated from a mosquito was foundin Cx quinquefasciatus (Pidiyar et al 2002 Huys et al2005)Aeromonas species were found subsequently inAfrican anophelines (Lindh et al 2005) and now inthis study Representatives from the previous studies(Fig 1) reveal that one of the sequences from Andarlingi (313) is closely related to the Aeromonas spclone H22629 (AY837743) from An gambiae (Lindhet al 2005) whereas the other sequence is closer toalthough different from Aeromonas culicicola (Aero-monas veronii) Similarly the Pseudomonas sequencesretrieved from An darlingi are related closely to aPseudomonas putida relative isolated fromAn gambiaess from Kenya (Lindh et al 2005)

Three clades of sequences belong to potentiallynew species of bacteria based on their sequencesimilarities being below 98 to other sequences inGenBank The 45ETH23 clade is 96ETH97 similar to un-cultured Enterobacter relatives the 32ETH42 clade is97ETH98 similar toEnterobacter species and the 51ETH52clade is only 96 similar to P putida None of thesesequences were found in the DNA from the nonblood-fed mosquito therefore they could have their originfrom the ducks that were used for blood feeding

Gene ampliTHORNcation of all (THORNve) blood-fed mosquitoesgenerated a 16S rDNA ampliTHORNcation product whereasonly one of four nonblood-fed mosquitoes resulted inany product These data indicate that any bacteria pos-sibly existing on the cuticle were in too low numbers toyield any PCR product and that the bacterial DNA orig-inated from either bacterial populations increased by abloodmeal or the bloodmeal itself The results are inagreement with previous work onAnophelesmosquitoesthat found 11ETH40-fold increase of bacteria 24 h after abloodmeal (Pumpuni et al 1996) That this increase issufTHORNcient for genetically modiTHORNed bacteria to affect ma-lariaparasitedevelopmenthasbeenshownintwostudieswhere Escherichia coli were fed to An stephensi In theTHORNrst article on successful paratransgenesis in mosquitoesYoshida et al (2001) used a single-chain antibody tar-geting Plasmodium berghei ookinete Pbs21 linked to thelytic peptide Shiva-1 and obtained 956 transmissionblockage Recently Riehle et al (2007) showed that Ecoli expressing the anti-Plasmodium effector moleculesphospholipase-A(2)andSM1inhibitedoocyst formationby 23 and 41 respectively That we obtained 16S rRNA

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 173

N42 (2) EF179819

41 (3) EF179824

33 (3) EF179815

AJ853890 Enterobacter h steigerwaltii

N49 EF179837

210 EF179812

N48 EF179836

AJ853889 Enterobacter hormaechei oharae

44 EF179827

NC 000913 Escherichia coli

48 EF179828

43 EF179826

N45 EF179835

AJ871858 Klebsiella oxytoca

14 EF179809

512 EF179833

510 EF179832

AF364847 Pantoea ananatis

AJ233423 Pantoea agglomerans

Z96080 Pantoea stewartii

EF189920 Pantoea stewartii Ae aegypti

EF189919 Pantoea stewartii An gambiae

DQ068811 Ant lion gut bacterium f5s7

AF025369 Citrobacter murliniae

N43 (2) EF179834

56 EF179830

DQ068848 Ant lion gut bacterium 5s9

59 EF179831

45 EF179816

23 EF179822

N46 EF179820

21 (5) EF179821

DQ068853 Ant lion gut bacterium 6s25

AJ233404 Buttiauxella izardii

DQ068923 Ant lion bacterium s4w5a

32 EF179814

42 EF179825

DQ068862 Ant lion gut bacterium 7s5

53 EF179817

410 EF179829

12 (3) EF179807

24 (2) EF179823

31 EF179813

111 (2) EF179811

N41 (2) EF179818

ECU80197 Erwinia c carotovora

NC 004547 Erwinia carotovora atroseptica

Z96094 Erwinia cypripedii

U80205 Erwinia persicinus

15 EF179810

AF024609 Thrip gut bacterium

13 EF179808

11 (2) EF179843

NC 006155 Yersinia pseudotuberculosis

AF170914 Aeromonas culicicola

310 EF179838

NC 008570 Aeromonas h hydrophila

AY837743 Aeromonas sp An gambiae

313 EF179844

51 EF179845

52 EF179841

314 EF179839

55 (2) EF179842

NC 002947 Pseudomonas putida

AY837753 Pseudomonas sp An gambiae

37 EF179840

001

Fig 1 Phylogenetic tree of all 16S rRNAclones in the study (shaded) and selected da-tabase sequences The tree is based on Clust-alW alignment (Thompson et al 1994) of se-quences 1500 bp in length with Kimura-2distance parameters and was generated usingneighbor-joining with 2000 bootstrap repli-cates in MEGA version 31 (Kumar et al 2004)Branch points supported by bootstrap values of90 are represented by black circles 70are represented by gray circles and 50 arerepresented by white circles Branches with-out circles are unresolved (bootstrap values of50) Sequence ID consists of mosquitonumber followed by clone number (N non-blood fed) EF179808 to EF179845 refer to theGenBank accession numbers If two or moresequences from one specimen are 995 sim-ilar the total number of clones represented iswritten in parentheses The bar indicates 1difference

174 JOURNAL OF MEDICAL ENTOMOLOGY Vol 45 no 1

sequencesfromallblood-fedmosquitoeswhereasLindhet al (2005) only detected uncultured bacteria in 9 ofthemosquitoesanalyzedusing thesamePCRconditionssuggests that the controlled blood feeding increases thelikelihood to retrieve bacterial DNA However becausethe number of specimens analyzed is small the gener-ality of our THORNnding remains to be conTHORNrmed

From this pilot investigation we can conclude thefollowing

1 Human land catches without the mosquitoes bitingor touching the skin of the human volunteer (toavoid contamination) select for the subpopulationof female mosquitoes most attracted to humansTherefore bacteria obtained from these mosqui-toes are especially suitable for paratransgenesis

2 Controlled blood feeding 24 h before DNA extrac-tion enhances the recovery of 16S rRNA sequencesfrom mosquitoes The mosquitoes should prefera-bly be fed sterile blood to avoid contamination ofthe blood with bacteria As a control the sterility ofthe blood should be assessed by using the samemethods of DNA extraction and gene ampliTHORNcationas for the mosquitoes

3 Nonblood-fed mosquitoes in general did not gen-erate any product indicating that bacteria on theexoskeleton of the mosquitoes were too few toaffect the results

4 There is a great diversity of bacteria inAn darlingiincluding potentially new species This justiTHORNes fur-ther search and isolation of bacteria useful for para-transgenesis

Acknowledgment

We thank J M Lindh (Stockholm University StockholmSweden) for valuable comments on the manuscript This studywas supported by funds from the National Institutes of Health(NIH) (to AAJ) and CIPETRO (Plano Nacional de Cienciae Tecnologia do setor de petroleo e gas natural) PIATAM(Programa Potenciais Impactos e Riscos Ambientais da Indu-stria do Petroleo e Gas no Amazonas and FAPEAM (Fundacaode Amparo a Pesquisa do Estado do Amazonas) (to WPT)

References Cited

Azambuja P E S Garcia and N A Ratcliffe 2005 Gutmicrobiota and parasite transmission by insect vectorsTrends Parasitol 21 568ETH572

Beard C B C Cordon-Rosales and R V Durvasula 2002Bacterial symbionts of the triatominae and their potentialuse in control of Chagas disease transmission Annu RevEntomol 47 123ETH141

Cole J R B Chai R J Farris Q Wang S A Kulam D MMcGarrell G M Garrity and J M Tiedje 2005 TheRibosomal Database Project (RDP-II) sequences andtools for high-throughput rRNA analysis Nucleic AcidsRes 33 D294ETHD296

Coura J R M Suarez-Mutis and S Ladeia-Andrade 2006 Anew challenge for malaria control in Brazil asymptomaticPlasmodium infection a review Mem Inst Oswaldo Cruz101 229ETH237

Deane L M 1986 Malaria vectors in Brazil Mem InstOswaldo Cruz 81 5ETH14

Faran M E and K J Linthicum 1981 A handbook ofthe Amazonian species of Anopheles (Nyssorhynchus)(Diptera Culicidae) Mosq Syst 13 1ETH81

Favia G I Ricci C Damiani N Raddadi E Crotti MMarzorati A Rizzi R Urso L Brusetti S Borin et al2007 Bacteria of the genus Asaia stably associate withAnopheles stephensi an Asian malarial mosquito vectorProc Natl Acad Sci USA 104 9047ETH9051

Forattini O P 1962 Entomologia Medica vol 1 Facul-dade de Higiene e Saude Publica USP Sao Paulo Brasil

Good I J 1953 The population frequencies of species andthe estimation of population parameters Biometrica 40237ETH264

Huys G M Cnockaert and J Swings 2005 Aeromonasculicicola Pidiyar et al 2002 is a later subjective synonymof Aeromonas veronii Hickman-Brenner et al 1987 SystAppl Microbiol 28 604ETH609

JamesAA 2003 Blocking malaria parasite invasion of mos-quito salivary glands J Exp Biol 206 3817ETH3821

Kumar SKTamura andMNei 2004 MEGA3 integratedsoftware for molecular evolutionary genetics analysis andsequence alignment Brief Bioinform 5 150ETH163

Lindh J M O Terenius and I Faye 2005 16S rRNAgene-based identiTHORNcation of midgut bacteria from THORNeld-caughtAnopheles gambiae sensu lato andA funestusmos-quitoes reveals new species related to known insect sym-bionts Appl Environ Microbiol 71 7217ETH7223

Pidiyar V A Kaznowski N B Narayan M Patole and Y SShouche 2002 Aeromonas culicicola sp nov from themidgut of Culex quinquefasciatus Int J Syst Evol Mi-crobiol 52 1723ETH1728

Pidiyar V J K Jangid M S Patole and Y S Shouche 2004StudiesonculturedandunculturedmicrobiotaofwildCulexquinquefasciatus mosquito midgut based on 16S ribosomalRNA gene analysis Am J Trop Med Hyg 70 597ETH603

Pumpuni C B J Demaio M Kent J R Davis and J CBeier 1996 Bacterial population dynamics in threeanopheline species the impact on Plasmodium sporo-gonic development Am J Trop Med Hyg 54 214ETH218

RiehleM A andM Jacobs-Lorena 2005 Using bacteria toexpress and display anti-parasite molecules in mosqui-toes current and future strategies Insect Biochem MolBiol 35 699ETH707

Riehle M A C K Moreira D Lampe C Lauzon and MJacobs-Lorena 2007 Using bacteria to express and dis-play anti-Plasmodiummolecules in the mosquito midgutInt J Parasitol 37 595ETH603

Tadei W P and B Dutary-Thatcher 2000 Malaria vec-tors in the Brazilian Amazon Anopheles of the subgenusNyssorhynchus Rev Inst Med Trop Sao Paulo 42 87ETH94

Thompson J D D G Higgins and T J Gibson 1994CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weight-ing position-speciTHORNc gap penalties and weight matrixchoice Nucleic Acids Res 22 4673ETH4680

Yoshida SD IokaHMatsuokaHEndo andA Ishii 2001Bacteria expressing single-chain immunotoxin inhibit ma-laria parasite development in mosquitoes Mol BiochemParasitol 113 89ETH96

Received 24 April 2007 accepted 11 September 2007

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 175

sequences were analyzed in BLASTn (httpwwwncbinlmnihgovBLAST) by using GenBank as thereference library Chimerical sequences were elimi-nated after analysis with the Ribosomal DatabaseProject II Chimera Check program (Cole et al 2005)The percentage of coverage of the sequence analysiswas calculated by GoodOtildes method with the formula[1 (nN)] 100 where n is the number of mo-lecular species represented by one clone (single-cloneoperational taxonomic units [OTUs]) and N is thetotal number of sequences (Good 1953) An OTU wasdeTHORNned as consisting of all sequences with 2 di-vergence from the aligned homologous nucleotides(Pidiyar et al 2004)

Results and Discussion

The bacteria colonizing midguts of insect diseasevectors have drawn special attention for their in-teractions with both the insect hosts and the patho-genic organisms (for review see Azambuja et al2005) Based on these interactions paratransgenesishas been developed as a strategy to control Chagasdisease transmission by triatomine bugs (Beard et al2002) It is anticipated that paratransgenesis also canbe used to deliver anti-Plasmodium effector mole-cules to wild mosquito populations resulting in thecontrol of malaria transmission So far no true mos-quito symbionts have been found but recent re-search has provided the basis for mosquito para-transgenesis (Riehle and Jacobs-Lorena 2005) andit has been shown that it is possible to enhance thecapability of a gut bacterium to remain in the mos-quito gut (Riehle et al 2007) Many of the bacteriashown to inhabit the guts of mosquitoes are envi-ronmental and isolated commonly from soil orplants For example a species of the genus Asaiaisolated mainly from tropical szligowers in Asia canstably inhabit the gut of the Asian malaria vectorAnopheles stephensi Liston (Favia et al 2007) TheidentiTHORNcation of 16S rRNA sequences from bacterialpopulations found in the gut of vector mosquitoeswas performed previously on mosquitoes from Asia(Pidiyar et al 2004) and from Africa (Lindh et al2005) Here we present results of a polymerasechain reaction (PCR)-based approach to identifybacteria associated with An darlingi a major ma-laria vector in Latin America

From the nine mosquitoes analyzed we retrieved 56bacterial 16S sequences from six mosquitoes corre-sponding to a coverage of 93 by using the criteria set inPidiyar et al (2004) The coverage is the probability ofthe next cloned sequence falling in a novel (not yetobserved)OTUormolecular speciesThis valuegives anestimation of how well the clones analyzed account forthebiodiversitywithintheoriginal samplebythepresentmethodology (ie with the used PCR conditions andprimer set)Acoverageof93 indicates that sequencingmorecloneswouldresult inonly7of thesamplesbeingnovel

The majority of 16S sequences detected are frombacteria belonging to the Enterobacteriaceae family

(Fig 1) The Enterobacter hormaechei-cluster and theant lion gut bacterium 7s5-cluster contain bacteriaobtained from most of the specimens analyzed Thisdiffers from the results from other malaria vectorswhere only a single bacterial species was found to bepresent in more than one specimen of 91 Anophelesgambiae and 25 Anopheles funestus Giles mosquitoesexamined (Lindh et al 2005) Our result is encourag-ing because the use of paratransgenesis requires abacterium that could be introduced to the majority ofthe mosquitoes in a vector population Many of the 16SrRNA sequences retrieved from An darlingi are sim-ilar to those of gut bacteria found in other mosquitoesbut some are related to bacteria from insects as evo-lutionary distanced as thrips (Thysanoptera) and antlions (Neuroptera) The THORNrstAeromonas speciesAero-monas culicicola isolated from a mosquito was foundin Cx quinquefasciatus (Pidiyar et al 2002 Huys et al2005)Aeromonas species were found subsequently inAfrican anophelines (Lindh et al 2005) and now inthis study Representatives from the previous studies(Fig 1) reveal that one of the sequences from Andarlingi (313) is closely related to the Aeromonas spclone H22629 (AY837743) from An gambiae (Lindhet al 2005) whereas the other sequence is closer toalthough different from Aeromonas culicicola (Aero-monas veronii) Similarly the Pseudomonas sequencesretrieved from An darlingi are related closely to aPseudomonas putida relative isolated fromAn gambiaess from Kenya (Lindh et al 2005)

Three clades of sequences belong to potentiallynew species of bacteria based on their sequencesimilarities being below 98 to other sequences inGenBank The 45ETH23 clade is 96ETH97 similar to un-cultured Enterobacter relatives the 32ETH42 clade is97ETH98 similar toEnterobacter species and the 51ETH52clade is only 96 similar to P putida None of thesesequences were found in the DNA from the nonblood-fed mosquito therefore they could have their originfrom the ducks that were used for blood feeding

Gene ampliTHORNcation of all (THORNve) blood-fed mosquitoesgenerated a 16S rDNA ampliTHORNcation product whereasonly one of four nonblood-fed mosquitoes resulted inany product These data indicate that any bacteria pos-sibly existing on the cuticle were in too low numbers toyield any PCR product and that the bacterial DNA orig-inated from either bacterial populations increased by abloodmeal or the bloodmeal itself The results are inagreement with previous work onAnophelesmosquitoesthat found 11ETH40-fold increase of bacteria 24 h after abloodmeal (Pumpuni et al 1996) That this increase issufTHORNcient for genetically modiTHORNed bacteria to affect ma-lariaparasitedevelopmenthasbeenshownintwostudieswhere Escherichia coli were fed to An stephensi In theTHORNrst article on successful paratransgenesis in mosquitoesYoshida et al (2001) used a single-chain antibody tar-geting Plasmodium berghei ookinete Pbs21 linked to thelytic peptide Shiva-1 and obtained 956 transmissionblockage Recently Riehle et al (2007) showed that Ecoli expressing the anti-Plasmodium effector moleculesphospholipase-A(2)andSM1inhibitedoocyst formationby 23 and 41 respectively That we obtained 16S rRNA

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 173

N42 (2) EF179819

41 (3) EF179824

33 (3) EF179815

AJ853890 Enterobacter h steigerwaltii

N49 EF179837

210 EF179812

N48 EF179836

AJ853889 Enterobacter hormaechei oharae

44 EF179827

NC 000913 Escherichia coli

48 EF179828

43 EF179826

N45 EF179835

AJ871858 Klebsiella oxytoca

14 EF179809

512 EF179833

510 EF179832

AF364847 Pantoea ananatis

AJ233423 Pantoea agglomerans

Z96080 Pantoea stewartii

EF189920 Pantoea stewartii Ae aegypti

EF189919 Pantoea stewartii An gambiae

DQ068811 Ant lion gut bacterium f5s7

AF025369 Citrobacter murliniae

N43 (2) EF179834

56 EF179830

DQ068848 Ant lion gut bacterium 5s9

59 EF179831

45 EF179816

23 EF179822

N46 EF179820

21 (5) EF179821

DQ068853 Ant lion gut bacterium 6s25

AJ233404 Buttiauxella izardii

DQ068923 Ant lion bacterium s4w5a

32 EF179814

42 EF179825

DQ068862 Ant lion gut bacterium 7s5

53 EF179817

410 EF179829

12 (3) EF179807

24 (2) EF179823

31 EF179813

111 (2) EF179811

N41 (2) EF179818

ECU80197 Erwinia c carotovora

NC 004547 Erwinia carotovora atroseptica

Z96094 Erwinia cypripedii

U80205 Erwinia persicinus

15 EF179810

AF024609 Thrip gut bacterium

13 EF179808

11 (2) EF179843

NC 006155 Yersinia pseudotuberculosis

AF170914 Aeromonas culicicola

310 EF179838

NC 008570 Aeromonas h hydrophila

AY837743 Aeromonas sp An gambiae

313 EF179844

51 EF179845

52 EF179841

314 EF179839

55 (2) EF179842

NC 002947 Pseudomonas putida

AY837753 Pseudomonas sp An gambiae

37 EF179840

001

Fig 1 Phylogenetic tree of all 16S rRNAclones in the study (shaded) and selected da-tabase sequences The tree is based on Clust-alW alignment (Thompson et al 1994) of se-quences 1500 bp in length with Kimura-2distance parameters and was generated usingneighbor-joining with 2000 bootstrap repli-cates in MEGA version 31 (Kumar et al 2004)Branch points supported by bootstrap values of90 are represented by black circles 70are represented by gray circles and 50 arerepresented by white circles Branches with-out circles are unresolved (bootstrap values of50) Sequence ID consists of mosquitonumber followed by clone number (N non-blood fed) EF179808 to EF179845 refer to theGenBank accession numbers If two or moresequences from one specimen are 995 sim-ilar the total number of clones represented iswritten in parentheses The bar indicates 1difference

174 JOURNAL OF MEDICAL ENTOMOLOGY Vol 45 no 1

sequencesfromallblood-fedmosquitoeswhereasLindhet al (2005) only detected uncultured bacteria in 9 ofthemosquitoesanalyzedusing thesamePCRconditionssuggests that the controlled blood feeding increases thelikelihood to retrieve bacterial DNA However becausethe number of specimens analyzed is small the gener-ality of our THORNnding remains to be conTHORNrmed

From this pilot investigation we can conclude thefollowing

1 Human land catches without the mosquitoes bitingor touching the skin of the human volunteer (toavoid contamination) select for the subpopulationof female mosquitoes most attracted to humansTherefore bacteria obtained from these mosqui-toes are especially suitable for paratransgenesis

2 Controlled blood feeding 24 h before DNA extrac-tion enhances the recovery of 16S rRNA sequencesfrom mosquitoes The mosquitoes should prefera-bly be fed sterile blood to avoid contamination ofthe blood with bacteria As a control the sterility ofthe blood should be assessed by using the samemethods of DNA extraction and gene ampliTHORNcationas for the mosquitoes

3 Nonblood-fed mosquitoes in general did not gen-erate any product indicating that bacteria on theexoskeleton of the mosquitoes were too few toaffect the results

4 There is a great diversity of bacteria inAn darlingiincluding potentially new species This justiTHORNes fur-ther search and isolation of bacteria useful for para-transgenesis

Acknowledgment

We thank J M Lindh (Stockholm University StockholmSweden) for valuable comments on the manuscript This studywas supported by funds from the National Institutes of Health(NIH) (to AAJ) and CIPETRO (Plano Nacional de Cienciae Tecnologia do setor de petroleo e gas natural) PIATAM(Programa Potenciais Impactos e Riscos Ambientais da Indu-stria do Petroleo e Gas no Amazonas and FAPEAM (Fundacaode Amparo a Pesquisa do Estado do Amazonas) (to WPT)

References Cited

Azambuja P E S Garcia and N A Ratcliffe 2005 Gutmicrobiota and parasite transmission by insect vectorsTrends Parasitol 21 568ETH572

Beard C B C Cordon-Rosales and R V Durvasula 2002Bacterial symbionts of the triatominae and their potentialuse in control of Chagas disease transmission Annu RevEntomol 47 123ETH141

Cole J R B Chai R J Farris Q Wang S A Kulam D MMcGarrell G M Garrity and J M Tiedje 2005 TheRibosomal Database Project (RDP-II) sequences andtools for high-throughput rRNA analysis Nucleic AcidsRes 33 D294ETHD296

Coura J R M Suarez-Mutis and S Ladeia-Andrade 2006 Anew challenge for malaria control in Brazil asymptomaticPlasmodium infection a review Mem Inst Oswaldo Cruz101 229ETH237

Deane L M 1986 Malaria vectors in Brazil Mem InstOswaldo Cruz 81 5ETH14

Faran M E and K J Linthicum 1981 A handbook ofthe Amazonian species of Anopheles (Nyssorhynchus)(Diptera Culicidae) Mosq Syst 13 1ETH81

Favia G I Ricci C Damiani N Raddadi E Crotti MMarzorati A Rizzi R Urso L Brusetti S Borin et al2007 Bacteria of the genus Asaia stably associate withAnopheles stephensi an Asian malarial mosquito vectorProc Natl Acad Sci USA 104 9047ETH9051

Forattini O P 1962 Entomologia Medica vol 1 Facul-dade de Higiene e Saude Publica USP Sao Paulo Brasil

Good I J 1953 The population frequencies of species andthe estimation of population parameters Biometrica 40237ETH264

Huys G M Cnockaert and J Swings 2005 Aeromonasculicicola Pidiyar et al 2002 is a later subjective synonymof Aeromonas veronii Hickman-Brenner et al 1987 SystAppl Microbiol 28 604ETH609

JamesAA 2003 Blocking malaria parasite invasion of mos-quito salivary glands J Exp Biol 206 3817ETH3821

Kumar SKTamura andMNei 2004 MEGA3 integratedsoftware for molecular evolutionary genetics analysis andsequence alignment Brief Bioinform 5 150ETH163

Lindh J M O Terenius and I Faye 2005 16S rRNAgene-based identiTHORNcation of midgut bacteria from THORNeld-caughtAnopheles gambiae sensu lato andA funestusmos-quitoes reveals new species related to known insect sym-bionts Appl Environ Microbiol 71 7217ETH7223

Pidiyar V A Kaznowski N B Narayan M Patole and Y SShouche 2002 Aeromonas culicicola sp nov from themidgut of Culex quinquefasciatus Int J Syst Evol Mi-crobiol 52 1723ETH1728

Pidiyar V J K Jangid M S Patole and Y S Shouche 2004StudiesonculturedandunculturedmicrobiotaofwildCulexquinquefasciatus mosquito midgut based on 16S ribosomalRNA gene analysis Am J Trop Med Hyg 70 597ETH603

Pumpuni C B J Demaio M Kent J R Davis and J CBeier 1996 Bacterial population dynamics in threeanopheline species the impact on Plasmodium sporo-gonic development Am J Trop Med Hyg 54 214ETH218

RiehleM A andM Jacobs-Lorena 2005 Using bacteria toexpress and display anti-parasite molecules in mosqui-toes current and future strategies Insect Biochem MolBiol 35 699ETH707

Riehle M A C K Moreira D Lampe C Lauzon and MJacobs-Lorena 2007 Using bacteria to express and dis-play anti-Plasmodiummolecules in the mosquito midgutInt J Parasitol 37 595ETH603

Tadei W P and B Dutary-Thatcher 2000 Malaria vec-tors in the Brazilian Amazon Anopheles of the subgenusNyssorhynchus Rev Inst Med Trop Sao Paulo 42 87ETH94

Thompson J D D G Higgins and T J Gibson 1994CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weight-ing position-speciTHORNc gap penalties and weight matrixchoice Nucleic Acids Res 22 4673ETH4680

Yoshida SD IokaHMatsuokaHEndo andA Ishii 2001Bacteria expressing single-chain immunotoxin inhibit ma-laria parasite development in mosquitoes Mol BiochemParasitol 113 89ETH96

Received 24 April 2007 accepted 11 September 2007

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 175

N42 (2) EF179819

41 (3) EF179824

33 (3) EF179815

AJ853890 Enterobacter h steigerwaltii

N49 EF179837

210 EF179812

N48 EF179836

AJ853889 Enterobacter hormaechei oharae

44 EF179827

NC 000913 Escherichia coli

48 EF179828

43 EF179826

N45 EF179835

AJ871858 Klebsiella oxytoca

14 EF179809

512 EF179833

510 EF179832

AF364847 Pantoea ananatis

AJ233423 Pantoea agglomerans

Z96080 Pantoea stewartii

EF189920 Pantoea stewartii Ae aegypti

EF189919 Pantoea stewartii An gambiae

DQ068811 Ant lion gut bacterium f5s7

AF025369 Citrobacter murliniae

N43 (2) EF179834

56 EF179830

DQ068848 Ant lion gut bacterium 5s9

59 EF179831

45 EF179816

23 EF179822

N46 EF179820

21 (5) EF179821

DQ068853 Ant lion gut bacterium 6s25

AJ233404 Buttiauxella izardii

DQ068923 Ant lion bacterium s4w5a

32 EF179814

42 EF179825

DQ068862 Ant lion gut bacterium 7s5

53 EF179817

410 EF179829

12 (3) EF179807

24 (2) EF179823

31 EF179813

111 (2) EF179811

N41 (2) EF179818

ECU80197 Erwinia c carotovora

NC 004547 Erwinia carotovora atroseptica

Z96094 Erwinia cypripedii

U80205 Erwinia persicinus

15 EF179810

AF024609 Thrip gut bacterium

13 EF179808

11 (2) EF179843

NC 006155 Yersinia pseudotuberculosis

AF170914 Aeromonas culicicola

310 EF179838

NC 008570 Aeromonas h hydrophila

AY837743 Aeromonas sp An gambiae

313 EF179844

51 EF179845

52 EF179841

314 EF179839

55 (2) EF179842

NC 002947 Pseudomonas putida

AY837753 Pseudomonas sp An gambiae

37 EF179840

001

Fig 1 Phylogenetic tree of all 16S rRNAclones in the study (shaded) and selected da-tabase sequences The tree is based on Clust-alW alignment (Thompson et al 1994) of se-quences 1500 bp in length with Kimura-2distance parameters and was generated usingneighbor-joining with 2000 bootstrap repli-cates in MEGA version 31 (Kumar et al 2004)Branch points supported by bootstrap values of90 are represented by black circles 70are represented by gray circles and 50 arerepresented by white circles Branches with-out circles are unresolved (bootstrap values of50) Sequence ID consists of mosquitonumber followed by clone number (N non-blood fed) EF179808 to EF179845 refer to theGenBank accession numbers If two or moresequences from one specimen are 995 sim-ilar the total number of clones represented iswritten in parentheses The bar indicates 1difference

174 JOURNAL OF MEDICAL ENTOMOLOGY Vol 45 no 1

sequencesfromallblood-fedmosquitoeswhereasLindhet al (2005) only detected uncultured bacteria in 9 ofthemosquitoesanalyzedusing thesamePCRconditionssuggests that the controlled blood feeding increases thelikelihood to retrieve bacterial DNA However becausethe number of specimens analyzed is small the gener-ality of our THORNnding remains to be conTHORNrmed

From this pilot investigation we can conclude thefollowing

1 Human land catches without the mosquitoes bitingor touching the skin of the human volunteer (toavoid contamination) select for the subpopulationof female mosquitoes most attracted to humansTherefore bacteria obtained from these mosqui-toes are especially suitable for paratransgenesis

2 Controlled blood feeding 24 h before DNA extrac-tion enhances the recovery of 16S rRNA sequencesfrom mosquitoes The mosquitoes should prefera-bly be fed sterile blood to avoid contamination ofthe blood with bacteria As a control the sterility ofthe blood should be assessed by using the samemethods of DNA extraction and gene ampliTHORNcationas for the mosquitoes

3 Nonblood-fed mosquitoes in general did not gen-erate any product indicating that bacteria on theexoskeleton of the mosquitoes were too few toaffect the results

4 There is a great diversity of bacteria inAn darlingiincluding potentially new species This justiTHORNes fur-ther search and isolation of bacteria useful for para-transgenesis

Acknowledgment

We thank J M Lindh (Stockholm University StockholmSweden) for valuable comments on the manuscript This studywas supported by funds from the National Institutes of Health(NIH) (to AAJ) and CIPETRO (Plano Nacional de Cienciae Tecnologia do setor de petroleo e gas natural) PIATAM(Programa Potenciais Impactos e Riscos Ambientais da Indu-stria do Petroleo e Gas no Amazonas and FAPEAM (Fundacaode Amparo a Pesquisa do Estado do Amazonas) (to WPT)

References Cited

Azambuja P E S Garcia and N A Ratcliffe 2005 Gutmicrobiota and parasite transmission by insect vectorsTrends Parasitol 21 568ETH572

Beard C B C Cordon-Rosales and R V Durvasula 2002Bacterial symbionts of the triatominae and their potentialuse in control of Chagas disease transmission Annu RevEntomol 47 123ETH141

Cole J R B Chai R J Farris Q Wang S A Kulam D MMcGarrell G M Garrity and J M Tiedje 2005 TheRibosomal Database Project (RDP-II) sequences andtools for high-throughput rRNA analysis Nucleic AcidsRes 33 D294ETHD296

Coura J R M Suarez-Mutis and S Ladeia-Andrade 2006 Anew challenge for malaria control in Brazil asymptomaticPlasmodium infection a review Mem Inst Oswaldo Cruz101 229ETH237

Deane L M 1986 Malaria vectors in Brazil Mem InstOswaldo Cruz 81 5ETH14

Faran M E and K J Linthicum 1981 A handbook ofthe Amazonian species of Anopheles (Nyssorhynchus)(Diptera Culicidae) Mosq Syst 13 1ETH81

Favia G I Ricci C Damiani N Raddadi E Crotti MMarzorati A Rizzi R Urso L Brusetti S Borin et al2007 Bacteria of the genus Asaia stably associate withAnopheles stephensi an Asian malarial mosquito vectorProc Natl Acad Sci USA 104 9047ETH9051

Forattini O P 1962 Entomologia Medica vol 1 Facul-dade de Higiene e Saude Publica USP Sao Paulo Brasil

Good I J 1953 The population frequencies of species andthe estimation of population parameters Biometrica 40237ETH264

Huys G M Cnockaert and J Swings 2005 Aeromonasculicicola Pidiyar et al 2002 is a later subjective synonymof Aeromonas veronii Hickman-Brenner et al 1987 SystAppl Microbiol 28 604ETH609

JamesAA 2003 Blocking malaria parasite invasion of mos-quito salivary glands J Exp Biol 206 3817ETH3821

Kumar SKTamura andMNei 2004 MEGA3 integratedsoftware for molecular evolutionary genetics analysis andsequence alignment Brief Bioinform 5 150ETH163

Lindh J M O Terenius and I Faye 2005 16S rRNAgene-based identiTHORNcation of midgut bacteria from THORNeld-caughtAnopheles gambiae sensu lato andA funestusmos-quitoes reveals new species related to known insect sym-bionts Appl Environ Microbiol 71 7217ETH7223

Pidiyar V A Kaznowski N B Narayan M Patole and Y SShouche 2002 Aeromonas culicicola sp nov from themidgut of Culex quinquefasciatus Int J Syst Evol Mi-crobiol 52 1723ETH1728

Pidiyar V J K Jangid M S Patole and Y S Shouche 2004StudiesonculturedandunculturedmicrobiotaofwildCulexquinquefasciatus mosquito midgut based on 16S ribosomalRNA gene analysis Am J Trop Med Hyg 70 597ETH603

Pumpuni C B J Demaio M Kent J R Davis and J CBeier 1996 Bacterial population dynamics in threeanopheline species the impact on Plasmodium sporo-gonic development Am J Trop Med Hyg 54 214ETH218

RiehleM A andM Jacobs-Lorena 2005 Using bacteria toexpress and display anti-parasite molecules in mosqui-toes current and future strategies Insect Biochem MolBiol 35 699ETH707

Riehle M A C K Moreira D Lampe C Lauzon and MJacobs-Lorena 2007 Using bacteria to express and dis-play anti-Plasmodiummolecules in the mosquito midgutInt J Parasitol 37 595ETH603

Tadei W P and B Dutary-Thatcher 2000 Malaria vec-tors in the Brazilian Amazon Anopheles of the subgenusNyssorhynchus Rev Inst Med Trop Sao Paulo 42 87ETH94

Thompson J D D G Higgins and T J Gibson 1994CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weight-ing position-speciTHORNc gap penalties and weight matrixchoice Nucleic Acids Res 22 4673ETH4680

Yoshida SD IokaHMatsuokaHEndo andA Ishii 2001Bacteria expressing single-chain immunotoxin inhibit ma-laria parasite development in mosquitoes Mol BiochemParasitol 113 89ETH96

Received 24 April 2007 accepted 11 September 2007

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 175

sequencesfromallblood-fedmosquitoeswhereasLindhet al (2005) only detected uncultured bacteria in 9 ofthemosquitoesanalyzedusing thesamePCRconditionssuggests that the controlled blood feeding increases thelikelihood to retrieve bacterial DNA However becausethe number of specimens analyzed is small the gener-ality of our THORNnding remains to be conTHORNrmed

From this pilot investigation we can conclude thefollowing

1 Human land catches without the mosquitoes bitingor touching the skin of the human volunteer (toavoid contamination) select for the subpopulationof female mosquitoes most attracted to humansTherefore bacteria obtained from these mosqui-toes are especially suitable for paratransgenesis

2 Controlled blood feeding 24 h before DNA extrac-tion enhances the recovery of 16S rRNA sequencesfrom mosquitoes The mosquitoes should prefera-bly be fed sterile blood to avoid contamination ofthe blood with bacteria As a control the sterility ofthe blood should be assessed by using the samemethods of DNA extraction and gene ampliTHORNcationas for the mosquitoes

3 Nonblood-fed mosquitoes in general did not gen-erate any product indicating that bacteria on theexoskeleton of the mosquitoes were too few toaffect the results

4 There is a great diversity of bacteria inAn darlingiincluding potentially new species This justiTHORNes fur-ther search and isolation of bacteria useful for para-transgenesis

Acknowledgment

We thank J M Lindh (Stockholm University StockholmSweden) for valuable comments on the manuscript This studywas supported by funds from the National Institutes of Health(NIH) (to AAJ) and CIPETRO (Plano Nacional de Cienciae Tecnologia do setor de petroleo e gas natural) PIATAM(Programa Potenciais Impactos e Riscos Ambientais da Indu-stria do Petroleo e Gas no Amazonas and FAPEAM (Fundacaode Amparo a Pesquisa do Estado do Amazonas) (to WPT)

References Cited

Azambuja P E S Garcia and N A Ratcliffe 2005 Gutmicrobiota and parasite transmission by insect vectorsTrends Parasitol 21 568ETH572

Beard C B C Cordon-Rosales and R V Durvasula 2002Bacterial symbionts of the triatominae and their potentialuse in control of Chagas disease transmission Annu RevEntomol 47 123ETH141

Cole J R B Chai R J Farris Q Wang S A Kulam D MMcGarrell G M Garrity and J M Tiedje 2005 TheRibosomal Database Project (RDP-II) sequences andtools for high-throughput rRNA analysis Nucleic AcidsRes 33 D294ETHD296

Coura J R M Suarez-Mutis and S Ladeia-Andrade 2006 Anew challenge for malaria control in Brazil asymptomaticPlasmodium infection a review Mem Inst Oswaldo Cruz101 229ETH237

Deane L M 1986 Malaria vectors in Brazil Mem InstOswaldo Cruz 81 5ETH14

Faran M E and K J Linthicum 1981 A handbook ofthe Amazonian species of Anopheles (Nyssorhynchus)(Diptera Culicidae) Mosq Syst 13 1ETH81

Favia G I Ricci C Damiani N Raddadi E Crotti MMarzorati A Rizzi R Urso L Brusetti S Borin et al2007 Bacteria of the genus Asaia stably associate withAnopheles stephensi an Asian malarial mosquito vectorProc Natl Acad Sci USA 104 9047ETH9051

Forattini O P 1962 Entomologia Medica vol 1 Facul-dade de Higiene e Saude Publica USP Sao Paulo Brasil

Good I J 1953 The population frequencies of species andthe estimation of population parameters Biometrica 40237ETH264

Huys G M Cnockaert and J Swings 2005 Aeromonasculicicola Pidiyar et al 2002 is a later subjective synonymof Aeromonas veronii Hickman-Brenner et al 1987 SystAppl Microbiol 28 604ETH609

JamesAA 2003 Blocking malaria parasite invasion of mos-quito salivary glands J Exp Biol 206 3817ETH3821

Kumar SKTamura andMNei 2004 MEGA3 integratedsoftware for molecular evolutionary genetics analysis andsequence alignment Brief Bioinform 5 150ETH163

Lindh J M O Terenius and I Faye 2005 16S rRNAgene-based identiTHORNcation of midgut bacteria from THORNeld-caughtAnopheles gambiae sensu lato andA funestusmos-quitoes reveals new species related to known insect sym-bionts Appl Environ Microbiol 71 7217ETH7223

Pidiyar V A Kaznowski N B Narayan M Patole and Y SShouche 2002 Aeromonas culicicola sp nov from themidgut of Culex quinquefasciatus Int J Syst Evol Mi-crobiol 52 1723ETH1728

Pidiyar V J K Jangid M S Patole and Y S Shouche 2004StudiesonculturedandunculturedmicrobiotaofwildCulexquinquefasciatus mosquito midgut based on 16S ribosomalRNA gene analysis Am J Trop Med Hyg 70 597ETH603

Pumpuni C B J Demaio M Kent J R Davis and J CBeier 1996 Bacterial population dynamics in threeanopheline species the impact on Plasmodium sporo-gonic development Am J Trop Med Hyg 54 214ETH218

RiehleM A andM Jacobs-Lorena 2005 Using bacteria toexpress and display anti-parasite molecules in mosqui-toes current and future strategies Insect Biochem MolBiol 35 699ETH707

Riehle M A C K Moreira D Lampe C Lauzon and MJacobs-Lorena 2007 Using bacteria to express and dis-play anti-Plasmodiummolecules in the mosquito midgutInt J Parasitol 37 595ETH603

Tadei W P and B Dutary-Thatcher 2000 Malaria vec-tors in the Brazilian Amazon Anopheles of the subgenusNyssorhynchus Rev Inst Med Trop Sao Paulo 42 87ETH94

Thompson J D D G Higgins and T J Gibson 1994CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weight-ing position-speciTHORNc gap penalties and weight matrixchoice Nucleic Acids Res 22 4673ETH4680

Yoshida SD IokaHMatsuokaHEndo andA Ishii 2001Bacteria expressing single-chain immunotoxin inhibit ma-laria parasite development in mosquitoes Mol BiochemParasitol 113 89ETH96

Received 24 April 2007 accepted 11 September 2007

January 2008 TERENIUS ET AL BACTERIAL DNA FROM An darlingi 175