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Passage of Ingested Mansonella ozzardi (SpiruridaOnchocercidae) Microfilariae Through the Midgut of Aedesaegypti (Diptera Culicidae)Author(s) Jefferson A Vaughan Jeffrey A Bell Michael J Turell and DaveD ChadeeSource Journal of Medical Entomology 44(1)111-116 2007Published By Entomological Society of AmericaDOI httpdxdoiorg1016030022-2585(2007)44[111POIMOS]20CO2URL httpwwwbiooneorgdoifull1016030022-258528200729445B1113APOIMOS5D20CO3B2

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VECTORPATHOGENHOST INTERACTION TRANSMISSION

Passage of Ingested Mansonella ozzardi (Spirurida Onchocercidae)Microfilariae Through the Midgut of Aedes aegypti

(Diptera Culicidae)

JEFFERSON A VAUGHAN12 JEFFREY A BELL1 MICHAEL J TURELL3 AND DAVE D CHADEE4

J Med Entomol 44(1) 111ETH116 (2007)

ABSTRACT When virus and microTHORNlariae are ingested concurrently by a mosquito microTHORNlariae(mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoelallowing mosquitoes to become infectious much sooner than normal and enhancing transmission ofviruses by mosquitoesMansonella ozzardi (Manson) is a benign THORNlarial nematode parasite of humansin Latin America and is transmitted by black szligies (Diptera Simuliidae) and biting midges (DipteraCeratopogonidae) Because M ozzardi and dengue are sympatric we wanted to know whether Mozzardimf had the ability to penetrate the midgut ofAedes aegypti (L) (Diptera Culicidae) and thusplay a potential role in the enhancement of dengue transmission To test this the F1 progeny fromlocally collected Ae aegypti were fed on M ozzardi-infected human males in an endemic village innorthern Trinidad Mosquitoes were dissected at various times after feeding and examined for mf inthe midguts and thoraces MicroTHORNlariae penetrated the midguts of 43 of 63 mosquitoes that ingestedmf Overall 11 of mf penetrated the midgut by 17 h after being ingested The intensity of midgutpenetration was positively correlated to the numbers of mf ingested Because midgut penetration isa key requirement for mf enhancement to occur the potential exists thatM ozzardi could be involvedin the enhancement of dengue virus transmission

KEY WORDS Mansonella ozzardi Aedes aegypti dengue microTHORNlaria transmission

Concurrent ingestion of microTHORNlariae (mf) and arbo-viruses by vector arthropods has been shown to resultin signiTHORNcantly more efTHORNcient transmission of the virusthan when the same dose of virus was ingested alone(Mellor and Boorman 1980 Turell et al 1984 1987Zytoon et al 1993 Vaughan and Turell 1996 Vaughanet al 1999) This has been referred to as microTHORNlarialenhancement of arboviral transmission As part oftheir normal developmental cycle in a vector most mfspecies penetrate the vector midgut after being in-gested and migrate to a preferred site of development(eg szligight muscle and fat body) If a microTHORNlaremichost is also viremic when fed upon by a vector thenthere is the possibility that penetration of the midgutby mf will allow some of the ingested virus to enterdirectly into the vector hemocoel The facilitatedmovement of virus directly into the hemocoel canhave two important epidemiological consequences

First it can increase vector competence by allowingvirus to bypass both midgut infection and midgut es-cape barriers (Chamberlain and Sudia 1961 Kramer etal 1981) transforming otherwise incompetent vectorspecies into competent vector species and thus in-creasing the number of vector species involved in anarbovirus transmission cycle Second mf enhance-ment can accelerate arboviral development within avector and shorten the time required for a virus-ex-posed mosquito to become infectious (ie shorten theextrinsic incubation period [EIP]) Because the EIPaffects transmission exponentially (MacDonald 1952)small reductions in EIP can lead to large increases invectorial capacity even within natural arbovirusETHvec-tor systems

Most laboratory studies of mf enhancement haveused various arboviruses with Brugia spp mf andAedesOchlerotatus spp mosquitoes as experimentalmodel systems (Turell et al 1984 1987 Vaughan andTurell 1996 Vaughan et al 1999) But the concept ofmf enhancement could apply in theory to almost anyTHORNlarialETHvector system (Mellor and Boorman 1980)where THORNlarial and arboviral infections are sympatricAn important consideration is whether the mf canpenetrate the midgut and enter the hemocoel and thusallow the virus to bypass the midgut Further devel-opment of the parasite is irrelevant ie a mosquitospecies does not have to be a competent vector of

The opinions interpretations conclusions and recommendationsare those of the authors and are not necessarily endorsed by the USArmy

1 Department of Biology University of North Dakota Grand ForksND 58202ETH9019

2 Corresponding author e-mail jefferson_vaughanundnodakedu

3 Virology Division US Army Medical Research Institute of In-fectious Diseases Fort Detrick MD 21702

4 Department of Life Sciences University of the West Indies StAugustine Trinidad and Tobago West Indies

0022-2585070111ETH0116$04000 2007 Entomological Society of America

THORNlariasis for mf enhancement to occur This was illus-trated in a study examining the ability of co-ingestedmf ofBrugiamalayi to affect the ability ofOchlerotatustaeniorhynchus (Wiedemann) (Diptera Culicidae)transmit Venezuelan equine encephalomyelitis virus(family Togaviridae genus Alphavirus VEEV)(Vaughan et al 1999) A 30-fold increase in VEEVtransmission rate was observed in the mosquitoes in-gesting both agents compared with mosquitoes ingest-ing VEEV alone yet ingested B malayi mf neverdeveloped to larval (L3) stage within Oc taeniorhyn-chus and were essentially all dead by 48 h after inges-tion (JAV unpublished data) Potentially therecould be innumerable combinations of parasiteETHvec-torETHvirus in nature where mf enhancement could ap-ply including combinations with mf not normally as-sociated with mosquitoes

Dengue is the most important mosquito-borne viraldisease of humans and involves cyclical transmissionof the virus between humans and mosquito vectors[primarily Aedes aegypti (L)] There are also severalhuman THORNlarial diseases that are sympatric with dengueincluding lymphatic THORNlariasis (Wuchereria bancroftiBrugia malayi and Brugia timori) loiasis (Loa loa)onchocerciasis (Onchocerca volvulus) and mansonel-liasis (Mansonella ozzardi Mansonella perstans andMansonella streptocerca) To understand the potentialthat these human THORNlarial infections may have on the mfenhancement of dengue transmission it is importantto understand the ability of these mf species to pen-etrate the midgut of Ae aegypti It is well establishedthatWuchereria and Brugia mf can readily penetratethe midgut of Ae aegyptimosquitoes (Ramachandranand Zaini 1967 McGreevy et al 1982 Lowichik andLowrie 1988 Zielke 1992 Calheiros et al 1998) In-deed laboratory studies have shown that co-ingestionof B malayimf and dengue 2 virus can enhance virustransmission by Ae aegypti (Turell et al 1987) Less isknown about the ability of the other nonmosquito-borne human THORNlarial species to penetrate the midgutof Ae aegypti mosquitoes Mansonella ozzardi is a be-nign THORNlarial infection of humans throughout tropicalLatin America that is transmitted by black szligies(Diptera Simuliidae Simulium spp) andor bitingmidges (Diptera Ceratopogonidae Culicoides spp)(Nelson and Davies 1976 Nathan 1978 Shelley et al1980 Shelley and Coscaron 2001) Although not gen-erally associated with mosquitoesMozzardioccurs inareas where several mosquito-borne arboviruses aretransmitted including dengue virus In this report wedescribe the ability of M ozzardi mf to penetrate themidgut of a co-indigenous strain of Ae aegypti mos-quitoes

Materials and Methods

Study Site The research was conducted on-sitewithin the village of Blanchisseuse (10 78 N 61 31W) a discrete rural community on the coastal foothillsof TrinidadOtildes forested Northern Range MountainsThe village is endemic for M ozzardi and has been astudy site for Mansonella research for many years

(Nathan et al 1982 Chadee et al 1995 Gonzalez et al1999)MosquitoesEngorgedAe aegypti females were col-

lected on-site in Blanchisseuse and returned to theInsect Control Division Ministry of Public Health inSaint Joseph Trinidad and Tobago where the femaleswere allowed to lay eggs and larvae were reared toadults To safeguard against the potential of usingmosquitoes that may have been vertically infectedwith dengue or other szligavivirus the parents of the F1mosquitoes (n 115) were frozen and shipped over-night to the University of North Dakota where theywere tested by polymerase chain reaction (PCR)against szligavivirus RNA by using the primer sets andmethodology described by Scaramozzino et al (2001)The RNA extracted from locally caught Culex tarsalis(Coquillett) (Diptera Culicidae) infected with WestNile virus (Bell et al 2005) served as positive controlsAll Ae aegypti parent mosquitoes tested negative en-suring that all F1 mosquitoes used in this trial werederived from szligavivirus-free stockMosquito Blood Feeding Before this research was

conducted the study protocol received approval fromthe Institutional Ethics Committee of the Ministry ofHealth Trinidad and Tobago and the Human SubjectsResearch Review Board of the University of NorthDakota Potential subjects (males older than 19-yrold) were recruited from villagers known from pre-vious surveys to harbor M ozzardi infections Twodays before mosquito blood feeding 200 l of bloodwas obtained from each of six subjects via THORNngerprickspread on a microscope slide air-dried overnight andstained with Giemsa stain and the total number of mfon each slide was counted to determine absolute den-sity of mf in the peripheral blood for each personMosquitoes were allowed 15ETH20 min to feed from ascreened cage based on procedures approved by thetwo Institutional Review Boards Most of the mosquitofeedings were conducted in the evening (2030ETH2230hours) Two of volunteers with the highest microTHORN-laremias allowed mosquitoes to feed on their arms thefollowing afternoon (1500ETH1700 hours) to test for pos-sible mf periodicity that might affect the level of mfuptake by a diurnal feeding mosquito such as AeaegyptiAssessment of Microfilarial Ingestion and Penetra-tionEngorged mosquitoes were held at ambient tem-perature Beginning 1 h after the feeding three people(two dissecting mosquitoes one counting mf) col-lected paired data for each mosquito (ie mf countsfor bloodmeal and carcass) Mosquitoes were aspi-rated into ethanol transferred to saline and individualmidguts were carefully excised with THORNne-tipped jew-elerOtildes forceps taking care not to rupture the midgutduring the process Only ldquocleanrdquo dissections wheremidguts were excised intact were included in the dataset Individual midguts were transferred to a glassslide compressed with a coverslip and examined formf at 100ETH400 magniTHORNcation The carcasses werethoroughly teased apart with needles and similarlyexamined for mf MicroTHORNlariae were relatively easy toTHORNnd when they were alive and wriggling but dead or

112 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

moribund mf often required careful inspection Foreach mosquito mf were counted as either being con-tained within the bloodmeal or in the hemocoel Thesum of the two counts represented the total numberof mf ingested by an individual mosquito To ensureaccuracy counts were rechecked by more than oneobserverStatistical Analyses MicroTHORNlarial counts for both

numbers ingested and numbers penetrating the mid-gut were not normally distributed (ShapiroETHWilk nor-mality tests W statistic 063 P 00001) nor didtransformation of data by logarithms or square rootsnormalize the data KruskalETHWallis tests were used tocompare medians Infection prevalences were com-pared using chi-square or Fisher exact tests Spearmanrank correlation (rs) was used to test for density re-lationships A 005 level of signiTHORNcance was used for allstatistical tests (Statistix version 8 Analytical Soft-ware Tallahassee FL)

Results

IngestionofMicrofilariaeBased on the results fromtwo volunteers that were fed upon twice there wereno differences among mosquitoes between nighttimeand daytime feedings with respect to either quantitiesof mf ingested (Wilcoxon rank sum tests P 027) orprevalences of mf ingested (Fisher exact tests P 063) (data not shown) Because there was no evi-dence to suggest that M ozzardi in Trinidad werenocturnally periodic data for the different times ofday were pooled for each of these volunteers in sub-sequent analyses MicroTHORNlaremias in the six volunteersranged from 3 to 394 mf per 200 l of blood (Table 1)Only three of a total of 87 mosquitoes dissected were

lost to ldquodirtyrdquo dissections Overall mf were detected in63 of the 84 engorged mosquitoes successfully dis-sected and examined The percentages of engorgedmosquitoes that ingested mf ranged from 0 (volunteer5) to 94 (volunteer 2) and were positively correlatedwith microTHORNlaremia of the volunteers (rs 083 n 6 P 003) The mean numbers ingested were mostlywithin the range expected based on host microTHORNla-remia and bloodmeal volumes and there was no in-dication that Ae aegypti concentrated M ozzardi mfduring feeding There was a signiTHORNcant differenceamong the six volunteers in the quantity of mf ingestedby the mosquitoes that fed on them (range 0ETH86 mfper mosquito KruskalETHWallis H 271 P 00001)and in general the quantity ingested increased withincreasing host microTHORNlaremia although the correla-tion was not statistically signiTHORNcant (rs 081 n 6P 006)Mf Penetration of the Mosquito MidgutOnly mos-

quitoes that had ingested mf were included in theanalyses of mf penetration of the mosquito midgutOverallM ozzardimf penetrated the midguts in 43of 63 mosquitoes that ingested mf (Table 2) Of the 539total mf ingested by the 63 mosquitoes 11 pene-trated the midgut and moved into the hemocoelThere was a signiTHORNcant positive correlation betweenthe numbers of mf ingested and the numbers of mf thatpenetrated the midgut (rs 045 n 63 P 0001)(Fig 1) Most mf found within mosquito hemocoelswere located in the vicinity of the thoracic szligight mus-cles Examination of mf penetration at selected inter-vals indicated that mf penetration of the mosquitomidgut increased over the THORNrst 2 h remained relativelyconstant up to 6 h and then decreased at 14ETH17 h(Table 3) Unlike mf observed wriggling in the hemo-

Table 1 Ingestion of M ozzardi mf by endemic F1 Ae aegypti mosquitoes fed on the forearms of six different microfilaremic adultmale human volunteers Blanchisseuse Trinidad 2004

Volunteer noand age (yr)

MicroTHORNlaremia(no mf200 l)

Expected no mfingestedmosquitoa

Actual no mfingestedmosquitob

ingesting mf(no examined)

2 (90) 394 59ETH98 86 (59 124) 94 (35)3 (62) 85 13ETH21 19 (11 29) 79 (24)4 (61) 73 11ETH18 16 (04 36) 75 (8)5 (58) 12 02ETH03 0 0 (4)6 (88) 10 01ETH02 03 (0 11) 40 (5)1 (62) 3 004ETH007 03 (0 07) 37 (8)

a Based on the mf density of volunteer blood per microliter times the average range of bloodmeal volumes measured for Ae aegypti (3ETH5l) as reported in Clements 1992bGeometric mean (upper lower 95 CL)

Table 2 Penetration of ingested M ozzardi mf through the midgut of resident Ae aegypti mosquitoes fed on microfilaremic humanvolunteers Blanchisseuse Trinidad 2004

Volunteer noNo mosquitoes

ingesting mfNo mosquitoes with mf

in the hemocoel ()Total no

mf ingestedTotal no

mf in hemocoelPenetrationefTHORNciency

2 33 16 (48) 453 48 113 19 10 (53) 64 13 204 6 0 (0) 17 0 06 2 0 (0) 2 0 01 3 1 (33) 3 1 33

Total 63 27 (43) 539 62 11

Only mosquitoes that ingested mf are included

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 113

coel during earlier time points most mf observed inthe hemocoel after 14 h were immobile and assumeda rigidly straight body position Melanized mf werenever observed in either the midguts or the hemocoel

Discussion

Mosquitoes are not considered to be involved in thetransmission of M ozzardi Nevertheless we demon-strated that ingestedM ozzardimf readily penetratedthe midguts of nativeAe aegyptimosquitoes Virtuallyall mf that had penetrated the midguts were eithermoribund or dead by 14 h after the bloodmeal con-THORNrming thatAe aegypti are not likely to be involved inthe natural transmission of this THORNlarial worm Thus theimportance of this study has nothing to do with trans-mission of M ozzardi but rather it addresses thebroader question of whether a nonmosquito borneTHORNlarial species like M ozzardi could enhance thetransmission of arboviruses from a vertebrate host(eg human) to mosquitoes (eg Ae aegypti)

It has been shown in laboratory studies that mos-quitoes that feed on vertebrates co-infected with mfand an arbovirus are signiTHORNcantly more efTHORNcient vec-torsof thevirus thanmosquitoes that ingest virus alone(Turell et al 1984 1987 Zytoon et al 1993 Vaughanand Turell 1996 Vaughan et al 1999) For this en-

hancing effect to happen the mf must be able topenetrate the mosquitoOtildes midgut To our knowledgethe only study examiningMozzardi in mosquitoes wasby Davis (1928) who dissected triatomine bugs andmosquitoes at various times after feeding them onmicroTHORNlaremic people and examined the insects fordeveloping THORNlariae in the hemocoel A small percent-age of some mosquito species (including Ae aegypti)contained stunted developmentally arrested or deadworms in the thorax Once it was discovered thatCulicoides were the vectors (Buckley 1934) no fur-ther studies were published on possible relationshipsbetween M ozzardi and mosquitoes Our study cor-roborates and expands that of Davis (1928) and showsthat M ozzardi mf are ingested during the course ofblood feeding by Ae aegypti mosquitoes and are ca-pable of penetrating the midgut and moving into thehemocoel Thus the potential exists forM ozzardi tobe involved in the mf enhancement of anthroponoticviruses such as the dengue viruses

There are other reasons to suspect that M ozzardiinfections could potentially interact with denguetransmission Throughout most of its distribution inthe Western Hemisphere dengue is sympatric withMozzardi Therefore it is not unreasonable to assumethat dually infected humans may exist wherever den-gue and mansonelliasis overlap particularly if theprevalence ofM ozzardi infection is high In Trinidadprevalence of mansonelliasis has been recorded ashigh as 11ETH31 (Nathan et al 1979 Gonzalez et al1999) Mansonelliasis is typically a rural disease due tothe distribution of its vector (ie biting midges)whereas dengue tends to be a more urban diseasebecause its principal vector Ae aegypti is synan-thropic However in Trinidad Ae aegypti abundance(as measured by Breteau index) and dengue inci-dence (ie per capita number of new cases) havebeen observed to be distributed equally between ruraland urban areas during recent dengue outbreaks(Brown et al 2004 Chadee et al 2005) In addition themf of M ozzardi are aperiodic (Nathan et al 1978)Thus M ozzardi mf are present in human peripheralcirculation during the time when Ae aegypti mosqui-toes are most likely to feed (ie during daytime)unlike the mf of most strains ofWuchereria bancroftiand Brugia spp that display nocturnal periodicity

It is still unknown whetherMozzardi infections canactually enhance dengue transmission Proof will re-quire vector competence studies by using M ozzardimf similar to studies described by Turell et al 1984who demonstrated enhancement of dengue virus in-fectivity to Ae aegypti by B malayi mf We hypoth-esize that the primary effect of mf enhancement ondengue transmission will be a reduction in the extrin-sic incubation period of dengue viruses within mos-quito species that are already vectors of dengue (egAe aegypti and Aedes albopictus Skuse) All evidenceto date indicates that intrathoracically inoculated den-gue viruses only replicate efTHORNciently in Aedes sppmosquitoes (Rosen et al 1985 Huang et al 1992)Therefore it seems unlikely that in the case of denguemf enhancement can alter the vector competence of

Fig 1 Density relationship between the number of Mozzardimf ingested by Ae aegyptimosquitoes and the num-ber of mf that penetrated the mosquito midgut and move intothe hemocoel (n 63) Some mosquitoes yielded the samevalues formf ingestedandmfescapedThus somedatapointshave associated superscripts that indicate the number ofmosquitoes having those values in common

Table 3 Penetration of ingested M ozzardi mf through themidgut of resident Ae aegypti mosquitoes at selected intervals afterfeeding on microfilaremic human volunteers Blanchisseuse Trin-idad 2004

Time afterfeeding (h)

of mosquitoeswhose midguts

were penetratedby ingested mf(no mosquitoes

ingesting mf)

of ingestedmf that penetrated

the midgut(no of mf ingested)

1 14 (7) 5 (20)2ETH3 54 (11) 14 (184)4ETH6 63 (19) 16 (122)

14ETH17 32 (25) 7 (213)

Only mosquitoes that ingested mf are included

114 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

otherwise refractory mosquito species ie mf en-hancement probably will not create new dengue vec-tors Even so shortening the extrinsic incubation pe-riod of dengue within the vector may still produce asigniTHORNcant impact on dengue epidemiology

Acknowledgments

We thank Robin Persad and Lester James Ministry ofHealth Saint Joseph Trinidad and Tobago for collecting andrearing of mosquitoes and invaluable help with the logisticsof this study Nathan Mickelson University of North Dakotaassayed mosquitoes for szligaviviruses This project was fundedin part by a University of North Dakota Faculty ResearchSeed Money Award (to JAV) and North Dakota EPSCoR(to JAB)

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Bell J A N J Mickelson and J A Vaughan 2005 WestNile virus in host-seeking mosquitoes from a residentialneighborhood in Grand Forks North Dakota VectorBorne Zoonotic Dis 5 373ETH382

Brown T U K Babb M Nimrod CVF Carrington R ASalas and M A Monteil 2004 A retrospective study ofthe 1996 DEN-1 epidemic in Trinidad demographic andclinical features Dengue Bull 28 7ETH19

Buckley JJC 1934 On the development in Culicoides fu-rens Poey of Filaria (Mansonella) ozzardi Manson1897 J Helminthol 12 99ETH118

Calheiros M L G Fontes P Williams and EMM Rocha1998 Experimental infection of Culex (Culex) quinque-fasciatus and Aedes (Stegomyia) aegypti withWuchereriabancrofti Mem Inst Oswaldo Cruz 93 855ETH860

Chadee D D C C Tilluckdharry S C Rawlins R Doonand M B Nathan 1995 Mass chemotherapy with di-ethylcarbamazine for the control of Bancroftian THORNlariasistwelve year follow-up in northern Trinidad includingobservations on Mansonella ozzardi Am J Trop MedHyg 52 174ETH176

Chadee D D FLR Williams and U D Kitron 2005Impact of vector control on a dengue fever outbreak inTrinidad West Indies in 1998 Trop Med Intl Health 10748ETH754

Chamberlain R W andW D Sudia 1961 Mechanism oftransmission of viruses by mosquitoes Annu Rev Ento-mol 6 371ETH390

Clements A N 1992 The biology of mosquitoes vol 1Development nutrition and reproduction Chapman ampHall New York

Davis N C 1928 A study on the transmission of THORNlaria innorthern Argentina Am J Hyg 8 457ETH466

Gonzalez A A D D Chadee and S C Rawlins 1999Ivermectin treatment of mansonellosis in Trinidad WestIndian Med J 48 231ETH234

Huang G E Vergne and D J Gubler 1992 Failure ofdengue viruses to replicate in Culex quinquefasciatus(Diptera Culicidae) J Med Entomol 29 911ETH914

KramerLD JLHardy SBPresser andE JHouk 1981Dissemination barriers for western equine encephalomy-elitis virus inCulex tarsalis infected after ingestion of lowviral doses Am J Trop Med Hyg 30 190ETH197

Lowichik A and R C Lowrie 1988 Uptake and develop-ment ofWuchereria bancrofti inAedes aegypti and HaitianCulex quinquefasciatus that were fed on a monkey with

low-density microTHORNlaremia Trop Med Parasit 39 227ETH229

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McGreevy P B N Kostrup J Tao M M McGreevy andT F de Marshall 1982 Ingestion and development ofWuchereria bancrofti inCulex quinquefasciatus Anophelesgambiae and Aedes aegypti after feeding on humans withvarying densities of microTHORNlariae in Tanzania Trans RSoc Trop Med Hyg 76 288ETH296

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Nathan M B 1978 Culicoides phlebotomus a vector ofMansonella ozzardi in coastal north Trinidad West IndiesTrans R Soc Trop Med Hyg 72 436ETH437

NathanM B C F Bartholomew and E S Tikasingh 1978The detection of Mansonella ozzardi microTHORNlariae in theskin and blood with a note on the absence of periodicityTrans R Soc Trop Med Hyg 72 420ETH422

Nathan M B E S Tikasingh G S Nelson A Santiago andJ B Davies 1979 The prevalence and distribution ofMansonella ozzardi in coastal north Trinidad WI TransR Soc Trop Med Hyg 73 299ETH302

NathanMBGBeckles E S Tikasingh P JHamilton andS Monteil 1982 Parasitological and clinical studies ofWuchereria bancrofti and Mansonella ozzardi in coastalnorth Trinidad West Indies West Indies Med J 31168ETH176

Nelson G S and J B Davies 1976 Observations on Man-sonella ozzardi in Trinidad Trans R Soc Trop Med Hyg70 16ETH17

Ramachandran C P and M A Zaini 1967 Studies on thetransmission of sub-periodicBrugiamalayibyAedes (Fin-laya) togoi in the laboratory I The intake and migrationof microTHORNlariae Med J Malaya 22 136ETH144

Rosen L L E Roseboom D J Gubler J C Lien and B NChaniotis 1985 Comparative susceptibility of mosquitospecies and strains to oral and parenteral infection withdengue and Japanese encephalitis viruses Am J TropMed Hyg 34 603ETH615

Scaramozzino N J Crance A Jouan D A DeBriel FStoll and D Garin 2001 Comparison of Flavivirusuniversal primer pairs and development of a rapidhighly sensitive heminested reverse transcriptase-PCRassay for detection of szligaviviruses targeted to a conser-vative region of the NS5 gene sequences J Clin Mi-crobiol 39 1922ETH1927

Shelley A J APA Luna Dias and MAP Moraes 1980Simulium species of the amazonicum group as vectors ofMansonella ozzardi in the Brazilian Amazon Trans RoyalSoc Trop Med Hyg 74 794ETH788

Shelley A J and S Coscaron 2001 Simuliid blackszligies(Diptera Simuliidae) and ceratopogonid midges(Diptera Ceratopogonidae) as vectors ofMansonella oz-zardi (Nematoda Onchocercidae) in northern Argen-tina Mem Inst Oswaldo Cruz 96 451ETH458

Turell M J P A Rossignol A Spielman C A Rossi andC L Bailey 1984 Enhanced arboviral transmission bymosquitoes that concurrently ingested microTHORNlariae Sci-ence (Wash DC) 225 1039ETH1041

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January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 115

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

THORNlariasis for mf enhancement to occur This was illus-trated in a study examining the ability of co-ingestedmf ofBrugiamalayi to affect the ability ofOchlerotatustaeniorhynchus (Wiedemann) (Diptera Culicidae)transmit Venezuelan equine encephalomyelitis virus(family Togaviridae genus Alphavirus VEEV)(Vaughan et al 1999) A 30-fold increase in VEEVtransmission rate was observed in the mosquitoes in-gesting both agents compared with mosquitoes ingest-ing VEEV alone yet ingested B malayi mf neverdeveloped to larval (L3) stage within Oc taeniorhyn-chus and were essentially all dead by 48 h after inges-tion (JAV unpublished data) Potentially therecould be innumerable combinations of parasiteETHvec-torETHvirus in nature where mf enhancement could ap-ply including combinations with mf not normally as-sociated with mosquitoes

Dengue is the most important mosquito-borne viraldisease of humans and involves cyclical transmissionof the virus between humans and mosquito vectors[primarily Aedes aegypti (L)] There are also severalhuman THORNlarial diseases that are sympatric with dengueincluding lymphatic THORNlariasis (Wuchereria bancroftiBrugia malayi and Brugia timori) loiasis (Loa loa)onchocerciasis (Onchocerca volvulus) and mansonel-liasis (Mansonella ozzardi Mansonella perstans andMansonella streptocerca) To understand the potentialthat these human THORNlarial infections may have on the mfenhancement of dengue transmission it is importantto understand the ability of these mf species to pen-etrate the midgut of Ae aegypti It is well establishedthatWuchereria and Brugia mf can readily penetratethe midgut of Ae aegyptimosquitoes (Ramachandranand Zaini 1967 McGreevy et al 1982 Lowichik andLowrie 1988 Zielke 1992 Calheiros et al 1998) In-deed laboratory studies have shown that co-ingestionof B malayimf and dengue 2 virus can enhance virustransmission by Ae aegypti (Turell et al 1987) Less isknown about the ability of the other nonmosquito-borne human THORNlarial species to penetrate the midgutof Ae aegypti mosquitoes Mansonella ozzardi is a be-nign THORNlarial infection of humans throughout tropicalLatin America that is transmitted by black szligies(Diptera Simuliidae Simulium spp) andor bitingmidges (Diptera Ceratopogonidae Culicoides spp)(Nelson and Davies 1976 Nathan 1978 Shelley et al1980 Shelley and Coscaron 2001) Although not gen-erally associated with mosquitoesMozzardioccurs inareas where several mosquito-borne arboviruses aretransmitted including dengue virus In this report wedescribe the ability of M ozzardi mf to penetrate themidgut of a co-indigenous strain of Ae aegypti mos-quitoes

Materials and Methods

Study Site The research was conducted on-sitewithin the village of Blanchisseuse (10 78 N 61 31W) a discrete rural community on the coastal foothillsof TrinidadOtildes forested Northern Range MountainsThe village is endemic for M ozzardi and has been astudy site for Mansonella research for many years

(Nathan et al 1982 Chadee et al 1995 Gonzalez et al1999)MosquitoesEngorgedAe aegypti females were col-

lected on-site in Blanchisseuse and returned to theInsect Control Division Ministry of Public Health inSaint Joseph Trinidad and Tobago where the femaleswere allowed to lay eggs and larvae were reared toadults To safeguard against the potential of usingmosquitoes that may have been vertically infectedwith dengue or other szligavivirus the parents of the F1mosquitoes (n 115) were frozen and shipped over-night to the University of North Dakota where theywere tested by polymerase chain reaction (PCR)against szligavivirus RNA by using the primer sets andmethodology described by Scaramozzino et al (2001)The RNA extracted from locally caught Culex tarsalis(Coquillett) (Diptera Culicidae) infected with WestNile virus (Bell et al 2005) served as positive controlsAll Ae aegypti parent mosquitoes tested negative en-suring that all F1 mosquitoes used in this trial werederived from szligavivirus-free stockMosquito Blood Feeding Before this research was

conducted the study protocol received approval fromthe Institutional Ethics Committee of the Ministry ofHealth Trinidad and Tobago and the Human SubjectsResearch Review Board of the University of NorthDakota Potential subjects (males older than 19-yrold) were recruited from villagers known from pre-vious surveys to harbor M ozzardi infections Twodays before mosquito blood feeding 200 l of bloodwas obtained from each of six subjects via THORNngerprickspread on a microscope slide air-dried overnight andstained with Giemsa stain and the total number of mfon each slide was counted to determine absolute den-sity of mf in the peripheral blood for each personMosquitoes were allowed 15ETH20 min to feed from ascreened cage based on procedures approved by thetwo Institutional Review Boards Most of the mosquitofeedings were conducted in the evening (2030ETH2230hours) Two of volunteers with the highest microTHORN-laremias allowed mosquitoes to feed on their arms thefollowing afternoon (1500ETH1700 hours) to test for pos-sible mf periodicity that might affect the level of mfuptake by a diurnal feeding mosquito such as AeaegyptiAssessment of Microfilarial Ingestion and Penetra-tionEngorged mosquitoes were held at ambient tem-perature Beginning 1 h after the feeding three people(two dissecting mosquitoes one counting mf) col-lected paired data for each mosquito (ie mf countsfor bloodmeal and carcass) Mosquitoes were aspi-rated into ethanol transferred to saline and individualmidguts were carefully excised with THORNne-tipped jew-elerOtildes forceps taking care not to rupture the midgutduring the process Only ldquocleanrdquo dissections wheremidguts were excised intact were included in the dataset Individual midguts were transferred to a glassslide compressed with a coverslip and examined formf at 100ETH400 magniTHORNcation The carcasses werethoroughly teased apart with needles and similarlyexamined for mf MicroTHORNlariae were relatively easy toTHORNnd when they were alive and wriggling but dead or

112 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

moribund mf often required careful inspection Foreach mosquito mf were counted as either being con-tained within the bloodmeal or in the hemocoel Thesum of the two counts represented the total numberof mf ingested by an individual mosquito To ensureaccuracy counts were rechecked by more than oneobserverStatistical Analyses MicroTHORNlarial counts for both

numbers ingested and numbers penetrating the mid-gut were not normally distributed (ShapiroETHWilk nor-mality tests W statistic 063 P 00001) nor didtransformation of data by logarithms or square rootsnormalize the data KruskalETHWallis tests were used tocompare medians Infection prevalences were com-pared using chi-square or Fisher exact tests Spearmanrank correlation (rs) was used to test for density re-lationships A 005 level of signiTHORNcance was used for allstatistical tests (Statistix version 8 Analytical Soft-ware Tallahassee FL)

Results

IngestionofMicrofilariaeBased on the results fromtwo volunteers that were fed upon twice there wereno differences among mosquitoes between nighttimeand daytime feedings with respect to either quantitiesof mf ingested (Wilcoxon rank sum tests P 027) orprevalences of mf ingested (Fisher exact tests P 063) (data not shown) Because there was no evi-dence to suggest that M ozzardi in Trinidad werenocturnally periodic data for the different times ofday were pooled for each of these volunteers in sub-sequent analyses MicroTHORNlaremias in the six volunteersranged from 3 to 394 mf per 200 l of blood (Table 1)Only three of a total of 87 mosquitoes dissected were

lost to ldquodirtyrdquo dissections Overall mf were detected in63 of the 84 engorged mosquitoes successfully dis-sected and examined The percentages of engorgedmosquitoes that ingested mf ranged from 0 (volunteer5) to 94 (volunteer 2) and were positively correlatedwith microTHORNlaremia of the volunteers (rs 083 n 6 P 003) The mean numbers ingested were mostlywithin the range expected based on host microTHORNla-remia and bloodmeal volumes and there was no in-dication that Ae aegypti concentrated M ozzardi mfduring feeding There was a signiTHORNcant differenceamong the six volunteers in the quantity of mf ingestedby the mosquitoes that fed on them (range 0ETH86 mfper mosquito KruskalETHWallis H 271 P 00001)and in general the quantity ingested increased withincreasing host microTHORNlaremia although the correla-tion was not statistically signiTHORNcant (rs 081 n 6P 006)Mf Penetration of the Mosquito MidgutOnly mos-

quitoes that had ingested mf were included in theanalyses of mf penetration of the mosquito midgutOverallM ozzardimf penetrated the midguts in 43of 63 mosquitoes that ingested mf (Table 2) Of the 539total mf ingested by the 63 mosquitoes 11 pene-trated the midgut and moved into the hemocoelThere was a signiTHORNcant positive correlation betweenthe numbers of mf ingested and the numbers of mf thatpenetrated the midgut (rs 045 n 63 P 0001)(Fig 1) Most mf found within mosquito hemocoelswere located in the vicinity of the thoracic szligight mus-cles Examination of mf penetration at selected inter-vals indicated that mf penetration of the mosquitomidgut increased over the THORNrst 2 h remained relativelyconstant up to 6 h and then decreased at 14ETH17 h(Table 3) Unlike mf observed wriggling in the hemo-

Table 1 Ingestion of M ozzardi mf by endemic F1 Ae aegypti mosquitoes fed on the forearms of six different microfilaremic adultmale human volunteers Blanchisseuse Trinidad 2004

Volunteer noand age (yr)

MicroTHORNlaremia(no mf200 l)

Expected no mfingestedmosquitoa

Actual no mfingestedmosquitob

ingesting mf(no examined)

2 (90) 394 59ETH98 86 (59 124) 94 (35)3 (62) 85 13ETH21 19 (11 29) 79 (24)4 (61) 73 11ETH18 16 (04 36) 75 (8)5 (58) 12 02ETH03 0 0 (4)6 (88) 10 01ETH02 03 (0 11) 40 (5)1 (62) 3 004ETH007 03 (0 07) 37 (8)

a Based on the mf density of volunteer blood per microliter times the average range of bloodmeal volumes measured for Ae aegypti (3ETH5l) as reported in Clements 1992bGeometric mean (upper lower 95 CL)

Table 2 Penetration of ingested M ozzardi mf through the midgut of resident Ae aegypti mosquitoes fed on microfilaremic humanvolunteers Blanchisseuse Trinidad 2004

Volunteer noNo mosquitoes

ingesting mfNo mosquitoes with mf

in the hemocoel ()Total no

mf ingestedTotal no

mf in hemocoelPenetrationefTHORNciency

2 33 16 (48) 453 48 113 19 10 (53) 64 13 204 6 0 (0) 17 0 06 2 0 (0) 2 0 01 3 1 (33) 3 1 33

Total 63 27 (43) 539 62 11

Only mosquitoes that ingested mf are included

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 113

coel during earlier time points most mf observed inthe hemocoel after 14 h were immobile and assumeda rigidly straight body position Melanized mf werenever observed in either the midguts or the hemocoel

Discussion

Mosquitoes are not considered to be involved in thetransmission of M ozzardi Nevertheless we demon-strated that ingestedM ozzardimf readily penetratedthe midguts of nativeAe aegyptimosquitoes Virtuallyall mf that had penetrated the midguts were eithermoribund or dead by 14 h after the bloodmeal con-THORNrming thatAe aegypti are not likely to be involved inthe natural transmission of this THORNlarial worm Thus theimportance of this study has nothing to do with trans-mission of M ozzardi but rather it addresses thebroader question of whether a nonmosquito borneTHORNlarial species like M ozzardi could enhance thetransmission of arboviruses from a vertebrate host(eg human) to mosquitoes (eg Ae aegypti)

It has been shown in laboratory studies that mos-quitoes that feed on vertebrates co-infected with mfand an arbovirus are signiTHORNcantly more efTHORNcient vec-torsof thevirus thanmosquitoes that ingest virus alone(Turell et al 1984 1987 Zytoon et al 1993 Vaughanand Turell 1996 Vaughan et al 1999) For this en-

hancing effect to happen the mf must be able topenetrate the mosquitoOtildes midgut To our knowledgethe only study examiningMozzardi in mosquitoes wasby Davis (1928) who dissected triatomine bugs andmosquitoes at various times after feeding them onmicroTHORNlaremic people and examined the insects fordeveloping THORNlariae in the hemocoel A small percent-age of some mosquito species (including Ae aegypti)contained stunted developmentally arrested or deadworms in the thorax Once it was discovered thatCulicoides were the vectors (Buckley 1934) no fur-ther studies were published on possible relationshipsbetween M ozzardi and mosquitoes Our study cor-roborates and expands that of Davis (1928) and showsthat M ozzardi mf are ingested during the course ofblood feeding by Ae aegypti mosquitoes and are ca-pable of penetrating the midgut and moving into thehemocoel Thus the potential exists forM ozzardi tobe involved in the mf enhancement of anthroponoticviruses such as the dengue viruses

There are other reasons to suspect that M ozzardiinfections could potentially interact with denguetransmission Throughout most of its distribution inthe Western Hemisphere dengue is sympatric withMozzardi Therefore it is not unreasonable to assumethat dually infected humans may exist wherever den-gue and mansonelliasis overlap particularly if theprevalence ofM ozzardi infection is high In Trinidadprevalence of mansonelliasis has been recorded ashigh as 11ETH31 (Nathan et al 1979 Gonzalez et al1999) Mansonelliasis is typically a rural disease due tothe distribution of its vector (ie biting midges)whereas dengue tends to be a more urban diseasebecause its principal vector Ae aegypti is synan-thropic However in Trinidad Ae aegypti abundance(as measured by Breteau index) and dengue inci-dence (ie per capita number of new cases) havebeen observed to be distributed equally between ruraland urban areas during recent dengue outbreaks(Brown et al 2004 Chadee et al 2005) In addition themf of M ozzardi are aperiodic (Nathan et al 1978)Thus M ozzardi mf are present in human peripheralcirculation during the time when Ae aegypti mosqui-toes are most likely to feed (ie during daytime)unlike the mf of most strains ofWuchereria bancroftiand Brugia spp that display nocturnal periodicity

It is still unknown whetherMozzardi infections canactually enhance dengue transmission Proof will re-quire vector competence studies by using M ozzardimf similar to studies described by Turell et al 1984who demonstrated enhancement of dengue virus in-fectivity to Ae aegypti by B malayi mf We hypoth-esize that the primary effect of mf enhancement ondengue transmission will be a reduction in the extrin-sic incubation period of dengue viruses within mos-quito species that are already vectors of dengue (egAe aegypti and Aedes albopictus Skuse) All evidenceto date indicates that intrathoracically inoculated den-gue viruses only replicate efTHORNciently in Aedes sppmosquitoes (Rosen et al 1985 Huang et al 1992)Therefore it seems unlikely that in the case of denguemf enhancement can alter the vector competence of

Fig 1 Density relationship between the number of Mozzardimf ingested by Ae aegyptimosquitoes and the num-ber of mf that penetrated the mosquito midgut and move intothe hemocoel (n 63) Some mosquitoes yielded the samevalues formf ingestedandmfescapedThus somedatapointshave associated superscripts that indicate the number ofmosquitoes having those values in common

Table 3 Penetration of ingested M ozzardi mf through themidgut of resident Ae aegypti mosquitoes at selected intervals afterfeeding on microfilaremic human volunteers Blanchisseuse Trin-idad 2004

Time afterfeeding (h)

of mosquitoeswhose midguts

were penetratedby ingested mf(no mosquitoes

ingesting mf)

of ingestedmf that penetrated

the midgut(no of mf ingested)

1 14 (7) 5 (20)2ETH3 54 (11) 14 (184)4ETH6 63 (19) 16 (122)

14ETH17 32 (25) 7 (213)

Only mosquitoes that ingested mf are included

114 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

otherwise refractory mosquito species ie mf en-hancement probably will not create new dengue vec-tors Even so shortening the extrinsic incubation pe-riod of dengue within the vector may still produce asigniTHORNcant impact on dengue epidemiology

Acknowledgments

We thank Robin Persad and Lester James Ministry ofHealth Saint Joseph Trinidad and Tobago for collecting andrearing of mosquitoes and invaluable help with the logisticsof this study Nathan Mickelson University of North Dakotaassayed mosquitoes for szligaviviruses This project was fundedin part by a University of North Dakota Faculty ResearchSeed Money Award (to JAV) and North Dakota EPSCoR(to JAB)

References Cited

Bell J A N J Mickelson and J A Vaughan 2005 WestNile virus in host-seeking mosquitoes from a residentialneighborhood in Grand Forks North Dakota VectorBorne Zoonotic Dis 5 373ETH382

Brown T U K Babb M Nimrod CVF Carrington R ASalas and M A Monteil 2004 A retrospective study ofthe 1996 DEN-1 epidemic in Trinidad demographic andclinical features Dengue Bull 28 7ETH19

Buckley JJC 1934 On the development in Culicoides fu-rens Poey of Filaria (Mansonella) ozzardi Manson1897 J Helminthol 12 99ETH118

Calheiros M L G Fontes P Williams and EMM Rocha1998 Experimental infection of Culex (Culex) quinque-fasciatus and Aedes (Stegomyia) aegypti withWuchereriabancrofti Mem Inst Oswaldo Cruz 93 855ETH860

Chadee D D C C Tilluckdharry S C Rawlins R Doonand M B Nathan 1995 Mass chemotherapy with di-ethylcarbamazine for the control of Bancroftian THORNlariasistwelve year follow-up in northern Trinidad includingobservations on Mansonella ozzardi Am J Trop MedHyg 52 174ETH176

Chadee D D FLR Williams and U D Kitron 2005Impact of vector control on a dengue fever outbreak inTrinidad West Indies in 1998 Trop Med Intl Health 10748ETH754

Chamberlain R W andW D Sudia 1961 Mechanism oftransmission of viruses by mosquitoes Annu Rev Ento-mol 6 371ETH390

Clements A N 1992 The biology of mosquitoes vol 1Development nutrition and reproduction Chapman ampHall New York

Davis N C 1928 A study on the transmission of THORNlaria innorthern Argentina Am J Hyg 8 457ETH466

Gonzalez A A D D Chadee and S C Rawlins 1999Ivermectin treatment of mansonellosis in Trinidad WestIndian Med J 48 231ETH234

Huang G E Vergne and D J Gubler 1992 Failure ofdengue viruses to replicate in Culex quinquefasciatus(Diptera Culicidae) J Med Entomol 29 911ETH914

KramerLD JLHardy SBPresser andE JHouk 1981Dissemination barriers for western equine encephalomy-elitis virus inCulex tarsalis infected after ingestion of lowviral doses Am J Trop Med Hyg 30 190ETH197

Lowichik A and R C Lowrie 1988 Uptake and develop-ment ofWuchereria bancrofti inAedes aegypti and HaitianCulex quinquefasciatus that were fed on a monkey with

low-density microTHORNlaremia Trop Med Parasit 39 227ETH229

MacDonald G 1952 The analysis of the sporozoite rateTrop Dis Bull 49 569ETH586

McGreevy P B N Kostrup J Tao M M McGreevy andT F de Marshall 1982 Ingestion and development ofWuchereria bancrofti inCulex quinquefasciatus Anophelesgambiae and Aedes aegypti after feeding on humans withvarying densities of microTHORNlariae in Tanzania Trans RSoc Trop Med Hyg 76 288ETH296

Mellor P S and J Boorman 1980 Multiplication of blue-tongue virus in Culicoides nubeculosus (Meigen) simul-taneously infected with the virus and the microTHORNlariae ofOnchocerca cervicalis (Railliet amp Henry) Ann TropMed Parasitol 74 463ETH469

Nathan M B 1978 Culicoides phlebotomus a vector ofMansonella ozzardi in coastal north Trinidad West IndiesTrans R Soc Trop Med Hyg 72 436ETH437

NathanM B C F Bartholomew and E S Tikasingh 1978The detection of Mansonella ozzardi microTHORNlariae in theskin and blood with a note on the absence of periodicityTrans R Soc Trop Med Hyg 72 420ETH422

Nathan M B E S Tikasingh G S Nelson A Santiago andJ B Davies 1979 The prevalence and distribution ofMansonella ozzardi in coastal north Trinidad WI TransR Soc Trop Med Hyg 73 299ETH302

NathanMBGBeckles E S Tikasingh P JHamilton andS Monteil 1982 Parasitological and clinical studies ofWuchereria bancrofti and Mansonella ozzardi in coastalnorth Trinidad West Indies West Indies Med J 31168ETH176

Nelson G S and J B Davies 1976 Observations on Man-sonella ozzardi in Trinidad Trans R Soc Trop Med Hyg70 16ETH17

Ramachandran C P and M A Zaini 1967 Studies on thetransmission of sub-periodicBrugiamalayibyAedes (Fin-laya) togoi in the laboratory I The intake and migrationof microTHORNlariae Med J Malaya 22 136ETH144

Rosen L L E Roseboom D J Gubler J C Lien and B NChaniotis 1985 Comparative susceptibility of mosquitospecies and strains to oral and parenteral infection withdengue and Japanese encephalitis viruses Am J TropMed Hyg 34 603ETH615

Scaramozzino N J Crance A Jouan D A DeBriel FStoll and D Garin 2001 Comparison of Flavivirusuniversal primer pairs and development of a rapidhighly sensitive heminested reverse transcriptase-PCRassay for detection of szligaviviruses targeted to a conser-vative region of the NS5 gene sequences J Clin Mi-crobiol 39 1922ETH1927

Shelley A J APA Luna Dias and MAP Moraes 1980Simulium species of the amazonicum group as vectors ofMansonella ozzardi in the Brazilian Amazon Trans RoyalSoc Trop Med Hyg 74 794ETH788

Shelley A J and S Coscaron 2001 Simuliid blackszligies(Diptera Simuliidae) and ceratopogonid midges(Diptera Ceratopogonidae) as vectors ofMansonella oz-zardi (Nematoda Onchocercidae) in northern Argen-tina Mem Inst Oswaldo Cruz 96 451ETH458

Turell M J P A Rossignol A Spielman C A Rossi andC L Bailey 1984 Enhanced arboviral transmission bymosquitoes that concurrently ingested microTHORNlariae Sci-ence (Wash DC) 225 1039ETH1041

Turell M J T N Mather A Spielman and C L Bailey1987 Increased dissemination of dengue 2 virus inAedesaegypti associated with concurrent ingestion of microTHORN-lariae of Brugia malayi Am J Trop Med Hyg 37 197ETH201

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 115

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

moribund mf often required careful inspection Foreach mosquito mf were counted as either being con-tained within the bloodmeal or in the hemocoel Thesum of the two counts represented the total numberof mf ingested by an individual mosquito To ensureaccuracy counts were rechecked by more than oneobserverStatistical Analyses MicroTHORNlarial counts for both

numbers ingested and numbers penetrating the mid-gut were not normally distributed (ShapiroETHWilk nor-mality tests W statistic 063 P 00001) nor didtransformation of data by logarithms or square rootsnormalize the data KruskalETHWallis tests were used tocompare medians Infection prevalences were com-pared using chi-square or Fisher exact tests Spearmanrank correlation (rs) was used to test for density re-lationships A 005 level of signiTHORNcance was used for allstatistical tests (Statistix version 8 Analytical Soft-ware Tallahassee FL)

Results

IngestionofMicrofilariaeBased on the results fromtwo volunteers that were fed upon twice there wereno differences among mosquitoes between nighttimeand daytime feedings with respect to either quantitiesof mf ingested (Wilcoxon rank sum tests P 027) orprevalences of mf ingested (Fisher exact tests P 063) (data not shown) Because there was no evi-dence to suggest that M ozzardi in Trinidad werenocturnally periodic data for the different times ofday were pooled for each of these volunteers in sub-sequent analyses MicroTHORNlaremias in the six volunteersranged from 3 to 394 mf per 200 l of blood (Table 1)Only three of a total of 87 mosquitoes dissected were

lost to ldquodirtyrdquo dissections Overall mf were detected in63 of the 84 engorged mosquitoes successfully dis-sected and examined The percentages of engorgedmosquitoes that ingested mf ranged from 0 (volunteer5) to 94 (volunteer 2) and were positively correlatedwith microTHORNlaremia of the volunteers (rs 083 n 6 P 003) The mean numbers ingested were mostlywithin the range expected based on host microTHORNla-remia and bloodmeal volumes and there was no in-dication that Ae aegypti concentrated M ozzardi mfduring feeding There was a signiTHORNcant differenceamong the six volunteers in the quantity of mf ingestedby the mosquitoes that fed on them (range 0ETH86 mfper mosquito KruskalETHWallis H 271 P 00001)and in general the quantity ingested increased withincreasing host microTHORNlaremia although the correla-tion was not statistically signiTHORNcant (rs 081 n 6P 006)Mf Penetration of the Mosquito MidgutOnly mos-

quitoes that had ingested mf were included in theanalyses of mf penetration of the mosquito midgutOverallM ozzardimf penetrated the midguts in 43of 63 mosquitoes that ingested mf (Table 2) Of the 539total mf ingested by the 63 mosquitoes 11 pene-trated the midgut and moved into the hemocoelThere was a signiTHORNcant positive correlation betweenthe numbers of mf ingested and the numbers of mf thatpenetrated the midgut (rs 045 n 63 P 0001)(Fig 1) Most mf found within mosquito hemocoelswere located in the vicinity of the thoracic szligight mus-cles Examination of mf penetration at selected inter-vals indicated that mf penetration of the mosquitomidgut increased over the THORNrst 2 h remained relativelyconstant up to 6 h and then decreased at 14ETH17 h(Table 3) Unlike mf observed wriggling in the hemo-

Table 1 Ingestion of M ozzardi mf by endemic F1 Ae aegypti mosquitoes fed on the forearms of six different microfilaremic adultmale human volunteers Blanchisseuse Trinidad 2004

Volunteer noand age (yr)

MicroTHORNlaremia(no mf200 l)

Expected no mfingestedmosquitoa

Actual no mfingestedmosquitob

ingesting mf(no examined)

2 (90) 394 59ETH98 86 (59 124) 94 (35)3 (62) 85 13ETH21 19 (11 29) 79 (24)4 (61) 73 11ETH18 16 (04 36) 75 (8)5 (58) 12 02ETH03 0 0 (4)6 (88) 10 01ETH02 03 (0 11) 40 (5)1 (62) 3 004ETH007 03 (0 07) 37 (8)

a Based on the mf density of volunteer blood per microliter times the average range of bloodmeal volumes measured for Ae aegypti (3ETH5l) as reported in Clements 1992bGeometric mean (upper lower 95 CL)

Table 2 Penetration of ingested M ozzardi mf through the midgut of resident Ae aegypti mosquitoes fed on microfilaremic humanvolunteers Blanchisseuse Trinidad 2004

Volunteer noNo mosquitoes

ingesting mfNo mosquitoes with mf

in the hemocoel ()Total no

mf ingestedTotal no

mf in hemocoelPenetrationefTHORNciency

2 33 16 (48) 453 48 113 19 10 (53) 64 13 204 6 0 (0) 17 0 06 2 0 (0) 2 0 01 3 1 (33) 3 1 33

Total 63 27 (43) 539 62 11

Only mosquitoes that ingested mf are included

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 113

coel during earlier time points most mf observed inthe hemocoel after 14 h were immobile and assumeda rigidly straight body position Melanized mf werenever observed in either the midguts or the hemocoel

Discussion

Mosquitoes are not considered to be involved in thetransmission of M ozzardi Nevertheless we demon-strated that ingestedM ozzardimf readily penetratedthe midguts of nativeAe aegyptimosquitoes Virtuallyall mf that had penetrated the midguts were eithermoribund or dead by 14 h after the bloodmeal con-THORNrming thatAe aegypti are not likely to be involved inthe natural transmission of this THORNlarial worm Thus theimportance of this study has nothing to do with trans-mission of M ozzardi but rather it addresses thebroader question of whether a nonmosquito borneTHORNlarial species like M ozzardi could enhance thetransmission of arboviruses from a vertebrate host(eg human) to mosquitoes (eg Ae aegypti)

It has been shown in laboratory studies that mos-quitoes that feed on vertebrates co-infected with mfand an arbovirus are signiTHORNcantly more efTHORNcient vec-torsof thevirus thanmosquitoes that ingest virus alone(Turell et al 1984 1987 Zytoon et al 1993 Vaughanand Turell 1996 Vaughan et al 1999) For this en-

hancing effect to happen the mf must be able topenetrate the mosquitoOtildes midgut To our knowledgethe only study examiningMozzardi in mosquitoes wasby Davis (1928) who dissected triatomine bugs andmosquitoes at various times after feeding them onmicroTHORNlaremic people and examined the insects fordeveloping THORNlariae in the hemocoel A small percent-age of some mosquito species (including Ae aegypti)contained stunted developmentally arrested or deadworms in the thorax Once it was discovered thatCulicoides were the vectors (Buckley 1934) no fur-ther studies were published on possible relationshipsbetween M ozzardi and mosquitoes Our study cor-roborates and expands that of Davis (1928) and showsthat M ozzardi mf are ingested during the course ofblood feeding by Ae aegypti mosquitoes and are ca-pable of penetrating the midgut and moving into thehemocoel Thus the potential exists forM ozzardi tobe involved in the mf enhancement of anthroponoticviruses such as the dengue viruses

There are other reasons to suspect that M ozzardiinfections could potentially interact with denguetransmission Throughout most of its distribution inthe Western Hemisphere dengue is sympatric withMozzardi Therefore it is not unreasonable to assumethat dually infected humans may exist wherever den-gue and mansonelliasis overlap particularly if theprevalence ofM ozzardi infection is high In Trinidadprevalence of mansonelliasis has been recorded ashigh as 11ETH31 (Nathan et al 1979 Gonzalez et al1999) Mansonelliasis is typically a rural disease due tothe distribution of its vector (ie biting midges)whereas dengue tends to be a more urban diseasebecause its principal vector Ae aegypti is synan-thropic However in Trinidad Ae aegypti abundance(as measured by Breteau index) and dengue inci-dence (ie per capita number of new cases) havebeen observed to be distributed equally between ruraland urban areas during recent dengue outbreaks(Brown et al 2004 Chadee et al 2005) In addition themf of M ozzardi are aperiodic (Nathan et al 1978)Thus M ozzardi mf are present in human peripheralcirculation during the time when Ae aegypti mosqui-toes are most likely to feed (ie during daytime)unlike the mf of most strains ofWuchereria bancroftiand Brugia spp that display nocturnal periodicity

It is still unknown whetherMozzardi infections canactually enhance dengue transmission Proof will re-quire vector competence studies by using M ozzardimf similar to studies described by Turell et al 1984who demonstrated enhancement of dengue virus in-fectivity to Ae aegypti by B malayi mf We hypoth-esize that the primary effect of mf enhancement ondengue transmission will be a reduction in the extrin-sic incubation period of dengue viruses within mos-quito species that are already vectors of dengue (egAe aegypti and Aedes albopictus Skuse) All evidenceto date indicates that intrathoracically inoculated den-gue viruses only replicate efTHORNciently in Aedes sppmosquitoes (Rosen et al 1985 Huang et al 1992)Therefore it seems unlikely that in the case of denguemf enhancement can alter the vector competence of

Fig 1 Density relationship between the number of Mozzardimf ingested by Ae aegyptimosquitoes and the num-ber of mf that penetrated the mosquito midgut and move intothe hemocoel (n 63) Some mosquitoes yielded the samevalues formf ingestedandmfescapedThus somedatapointshave associated superscripts that indicate the number ofmosquitoes having those values in common

Table 3 Penetration of ingested M ozzardi mf through themidgut of resident Ae aegypti mosquitoes at selected intervals afterfeeding on microfilaremic human volunteers Blanchisseuse Trin-idad 2004

Time afterfeeding (h)

of mosquitoeswhose midguts

were penetratedby ingested mf(no mosquitoes

ingesting mf)

of ingestedmf that penetrated

the midgut(no of mf ingested)

1 14 (7) 5 (20)2ETH3 54 (11) 14 (184)4ETH6 63 (19) 16 (122)

14ETH17 32 (25) 7 (213)

Only mosquitoes that ingested mf are included

114 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

otherwise refractory mosquito species ie mf en-hancement probably will not create new dengue vec-tors Even so shortening the extrinsic incubation pe-riod of dengue within the vector may still produce asigniTHORNcant impact on dengue epidemiology

Acknowledgments

We thank Robin Persad and Lester James Ministry ofHealth Saint Joseph Trinidad and Tobago for collecting andrearing of mosquitoes and invaluable help with the logisticsof this study Nathan Mickelson University of North Dakotaassayed mosquitoes for szligaviviruses This project was fundedin part by a University of North Dakota Faculty ResearchSeed Money Award (to JAV) and North Dakota EPSCoR(to JAB)

References Cited

Bell J A N J Mickelson and J A Vaughan 2005 WestNile virus in host-seeking mosquitoes from a residentialneighborhood in Grand Forks North Dakota VectorBorne Zoonotic Dis 5 373ETH382

Brown T U K Babb M Nimrod CVF Carrington R ASalas and M A Monteil 2004 A retrospective study ofthe 1996 DEN-1 epidemic in Trinidad demographic andclinical features Dengue Bull 28 7ETH19

Buckley JJC 1934 On the development in Culicoides fu-rens Poey of Filaria (Mansonella) ozzardi Manson1897 J Helminthol 12 99ETH118

Calheiros M L G Fontes P Williams and EMM Rocha1998 Experimental infection of Culex (Culex) quinque-fasciatus and Aedes (Stegomyia) aegypti withWuchereriabancrofti Mem Inst Oswaldo Cruz 93 855ETH860

Chadee D D C C Tilluckdharry S C Rawlins R Doonand M B Nathan 1995 Mass chemotherapy with di-ethylcarbamazine for the control of Bancroftian THORNlariasistwelve year follow-up in northern Trinidad includingobservations on Mansonella ozzardi Am J Trop MedHyg 52 174ETH176

Chadee D D FLR Williams and U D Kitron 2005Impact of vector control on a dengue fever outbreak inTrinidad West Indies in 1998 Trop Med Intl Health 10748ETH754

Chamberlain R W andW D Sudia 1961 Mechanism oftransmission of viruses by mosquitoes Annu Rev Ento-mol 6 371ETH390

Clements A N 1992 The biology of mosquitoes vol 1Development nutrition and reproduction Chapman ampHall New York

Davis N C 1928 A study on the transmission of THORNlaria innorthern Argentina Am J Hyg 8 457ETH466

Gonzalez A A D D Chadee and S C Rawlins 1999Ivermectin treatment of mansonellosis in Trinidad WestIndian Med J 48 231ETH234

Huang G E Vergne and D J Gubler 1992 Failure ofdengue viruses to replicate in Culex quinquefasciatus(Diptera Culicidae) J Med Entomol 29 911ETH914

KramerLD JLHardy SBPresser andE JHouk 1981Dissemination barriers for western equine encephalomy-elitis virus inCulex tarsalis infected after ingestion of lowviral doses Am J Trop Med Hyg 30 190ETH197

Lowichik A and R C Lowrie 1988 Uptake and develop-ment ofWuchereria bancrofti inAedes aegypti and HaitianCulex quinquefasciatus that were fed on a monkey with

low-density microTHORNlaremia Trop Med Parasit 39 227ETH229

MacDonald G 1952 The analysis of the sporozoite rateTrop Dis Bull 49 569ETH586

McGreevy P B N Kostrup J Tao M M McGreevy andT F de Marshall 1982 Ingestion and development ofWuchereria bancrofti inCulex quinquefasciatus Anophelesgambiae and Aedes aegypti after feeding on humans withvarying densities of microTHORNlariae in Tanzania Trans RSoc Trop Med Hyg 76 288ETH296

Mellor P S and J Boorman 1980 Multiplication of blue-tongue virus in Culicoides nubeculosus (Meigen) simul-taneously infected with the virus and the microTHORNlariae ofOnchocerca cervicalis (Railliet amp Henry) Ann TropMed Parasitol 74 463ETH469

Nathan M B 1978 Culicoides phlebotomus a vector ofMansonella ozzardi in coastal north Trinidad West IndiesTrans R Soc Trop Med Hyg 72 436ETH437

NathanM B C F Bartholomew and E S Tikasingh 1978The detection of Mansonella ozzardi microTHORNlariae in theskin and blood with a note on the absence of periodicityTrans R Soc Trop Med Hyg 72 420ETH422

Nathan M B E S Tikasingh G S Nelson A Santiago andJ B Davies 1979 The prevalence and distribution ofMansonella ozzardi in coastal north Trinidad WI TransR Soc Trop Med Hyg 73 299ETH302

NathanMBGBeckles E S Tikasingh P JHamilton andS Monteil 1982 Parasitological and clinical studies ofWuchereria bancrofti and Mansonella ozzardi in coastalnorth Trinidad West Indies West Indies Med J 31168ETH176

Nelson G S and J B Davies 1976 Observations on Man-sonella ozzardi in Trinidad Trans R Soc Trop Med Hyg70 16ETH17

Ramachandran C P and M A Zaini 1967 Studies on thetransmission of sub-periodicBrugiamalayibyAedes (Fin-laya) togoi in the laboratory I The intake and migrationof microTHORNlariae Med J Malaya 22 136ETH144

Rosen L L E Roseboom D J Gubler J C Lien and B NChaniotis 1985 Comparative susceptibility of mosquitospecies and strains to oral and parenteral infection withdengue and Japanese encephalitis viruses Am J TropMed Hyg 34 603ETH615

Scaramozzino N J Crance A Jouan D A DeBriel FStoll and D Garin 2001 Comparison of Flavivirusuniversal primer pairs and development of a rapidhighly sensitive heminested reverse transcriptase-PCRassay for detection of szligaviviruses targeted to a conser-vative region of the NS5 gene sequences J Clin Mi-crobiol 39 1922ETH1927

Shelley A J APA Luna Dias and MAP Moraes 1980Simulium species of the amazonicum group as vectors ofMansonella ozzardi in the Brazilian Amazon Trans RoyalSoc Trop Med Hyg 74 794ETH788

Shelley A J and S Coscaron 2001 Simuliid blackszligies(Diptera Simuliidae) and ceratopogonid midges(Diptera Ceratopogonidae) as vectors ofMansonella oz-zardi (Nematoda Onchocercidae) in northern Argen-tina Mem Inst Oswaldo Cruz 96 451ETH458

Turell M J P A Rossignol A Spielman C A Rossi andC L Bailey 1984 Enhanced arboviral transmission bymosquitoes that concurrently ingested microTHORNlariae Sci-ence (Wash DC) 225 1039ETH1041

Turell M J T N Mather A Spielman and C L Bailey1987 Increased dissemination of dengue 2 virus inAedesaegypti associated with concurrent ingestion of microTHORN-lariae of Brugia malayi Am J Trop Med Hyg 37 197ETH201

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 115

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

coel during earlier time points most mf observed inthe hemocoel after 14 h were immobile and assumeda rigidly straight body position Melanized mf werenever observed in either the midguts or the hemocoel

Discussion

Mosquitoes are not considered to be involved in thetransmission of M ozzardi Nevertheless we demon-strated that ingestedM ozzardimf readily penetratedthe midguts of nativeAe aegyptimosquitoes Virtuallyall mf that had penetrated the midguts were eithermoribund or dead by 14 h after the bloodmeal con-THORNrming thatAe aegypti are not likely to be involved inthe natural transmission of this THORNlarial worm Thus theimportance of this study has nothing to do with trans-mission of M ozzardi but rather it addresses thebroader question of whether a nonmosquito borneTHORNlarial species like M ozzardi could enhance thetransmission of arboviruses from a vertebrate host(eg human) to mosquitoes (eg Ae aegypti)

It has been shown in laboratory studies that mos-quitoes that feed on vertebrates co-infected with mfand an arbovirus are signiTHORNcantly more efTHORNcient vec-torsof thevirus thanmosquitoes that ingest virus alone(Turell et al 1984 1987 Zytoon et al 1993 Vaughanand Turell 1996 Vaughan et al 1999) For this en-

hancing effect to happen the mf must be able topenetrate the mosquitoOtildes midgut To our knowledgethe only study examiningMozzardi in mosquitoes wasby Davis (1928) who dissected triatomine bugs andmosquitoes at various times after feeding them onmicroTHORNlaremic people and examined the insects fordeveloping THORNlariae in the hemocoel A small percent-age of some mosquito species (including Ae aegypti)contained stunted developmentally arrested or deadworms in the thorax Once it was discovered thatCulicoides were the vectors (Buckley 1934) no fur-ther studies were published on possible relationshipsbetween M ozzardi and mosquitoes Our study cor-roborates and expands that of Davis (1928) and showsthat M ozzardi mf are ingested during the course ofblood feeding by Ae aegypti mosquitoes and are ca-pable of penetrating the midgut and moving into thehemocoel Thus the potential exists forM ozzardi tobe involved in the mf enhancement of anthroponoticviruses such as the dengue viruses

There are other reasons to suspect that M ozzardiinfections could potentially interact with denguetransmission Throughout most of its distribution inthe Western Hemisphere dengue is sympatric withMozzardi Therefore it is not unreasonable to assumethat dually infected humans may exist wherever den-gue and mansonelliasis overlap particularly if theprevalence ofM ozzardi infection is high In Trinidadprevalence of mansonelliasis has been recorded ashigh as 11ETH31 (Nathan et al 1979 Gonzalez et al1999) Mansonelliasis is typically a rural disease due tothe distribution of its vector (ie biting midges)whereas dengue tends to be a more urban diseasebecause its principal vector Ae aegypti is synan-thropic However in Trinidad Ae aegypti abundance(as measured by Breteau index) and dengue inci-dence (ie per capita number of new cases) havebeen observed to be distributed equally between ruraland urban areas during recent dengue outbreaks(Brown et al 2004 Chadee et al 2005) In addition themf of M ozzardi are aperiodic (Nathan et al 1978)Thus M ozzardi mf are present in human peripheralcirculation during the time when Ae aegypti mosqui-toes are most likely to feed (ie during daytime)unlike the mf of most strains ofWuchereria bancroftiand Brugia spp that display nocturnal periodicity

It is still unknown whetherMozzardi infections canactually enhance dengue transmission Proof will re-quire vector competence studies by using M ozzardimf similar to studies described by Turell et al 1984who demonstrated enhancement of dengue virus in-fectivity to Ae aegypti by B malayi mf We hypoth-esize that the primary effect of mf enhancement ondengue transmission will be a reduction in the extrin-sic incubation period of dengue viruses within mos-quito species that are already vectors of dengue (egAe aegypti and Aedes albopictus Skuse) All evidenceto date indicates that intrathoracically inoculated den-gue viruses only replicate efTHORNciently in Aedes sppmosquitoes (Rosen et al 1985 Huang et al 1992)Therefore it seems unlikely that in the case of denguemf enhancement can alter the vector competence of

Fig 1 Density relationship between the number of Mozzardimf ingested by Ae aegyptimosquitoes and the num-ber of mf that penetrated the mosquito midgut and move intothe hemocoel (n 63) Some mosquitoes yielded the samevalues formf ingestedandmfescapedThus somedatapointshave associated superscripts that indicate the number ofmosquitoes having those values in common

Table 3 Penetration of ingested M ozzardi mf through themidgut of resident Ae aegypti mosquitoes at selected intervals afterfeeding on microfilaremic human volunteers Blanchisseuse Trin-idad 2004

Time afterfeeding (h)

of mosquitoeswhose midguts

were penetratedby ingested mf(no mosquitoes

ingesting mf)

of ingestedmf that penetrated

the midgut(no of mf ingested)

1 14 (7) 5 (20)2ETH3 54 (11) 14 (184)4ETH6 63 (19) 16 (122)

14ETH17 32 (25) 7 (213)

Only mosquitoes that ingested mf are included

114 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

otherwise refractory mosquito species ie mf en-hancement probably will not create new dengue vec-tors Even so shortening the extrinsic incubation pe-riod of dengue within the vector may still produce asigniTHORNcant impact on dengue epidemiology

Acknowledgments

We thank Robin Persad and Lester James Ministry ofHealth Saint Joseph Trinidad and Tobago for collecting andrearing of mosquitoes and invaluable help with the logisticsof this study Nathan Mickelson University of North Dakotaassayed mosquitoes for szligaviviruses This project was fundedin part by a University of North Dakota Faculty ResearchSeed Money Award (to JAV) and North Dakota EPSCoR(to JAB)

References Cited

Bell J A N J Mickelson and J A Vaughan 2005 WestNile virus in host-seeking mosquitoes from a residentialneighborhood in Grand Forks North Dakota VectorBorne Zoonotic Dis 5 373ETH382

Brown T U K Babb M Nimrod CVF Carrington R ASalas and M A Monteil 2004 A retrospective study ofthe 1996 DEN-1 epidemic in Trinidad demographic andclinical features Dengue Bull 28 7ETH19

Buckley JJC 1934 On the development in Culicoides fu-rens Poey of Filaria (Mansonella) ozzardi Manson1897 J Helminthol 12 99ETH118

Calheiros M L G Fontes P Williams and EMM Rocha1998 Experimental infection of Culex (Culex) quinque-fasciatus and Aedes (Stegomyia) aegypti withWuchereriabancrofti Mem Inst Oswaldo Cruz 93 855ETH860

Chadee D D C C Tilluckdharry S C Rawlins R Doonand M B Nathan 1995 Mass chemotherapy with di-ethylcarbamazine for the control of Bancroftian THORNlariasistwelve year follow-up in northern Trinidad includingobservations on Mansonella ozzardi Am J Trop MedHyg 52 174ETH176

Chadee D D FLR Williams and U D Kitron 2005Impact of vector control on a dengue fever outbreak inTrinidad West Indies in 1998 Trop Med Intl Health 10748ETH754

Chamberlain R W andW D Sudia 1961 Mechanism oftransmission of viruses by mosquitoes Annu Rev Ento-mol 6 371ETH390

Clements A N 1992 The biology of mosquitoes vol 1Development nutrition and reproduction Chapman ampHall New York

Davis N C 1928 A study on the transmission of THORNlaria innorthern Argentina Am J Hyg 8 457ETH466

Gonzalez A A D D Chadee and S C Rawlins 1999Ivermectin treatment of mansonellosis in Trinidad WestIndian Med J 48 231ETH234

Huang G E Vergne and D J Gubler 1992 Failure ofdengue viruses to replicate in Culex quinquefasciatus(Diptera Culicidae) J Med Entomol 29 911ETH914

KramerLD JLHardy SBPresser andE JHouk 1981Dissemination barriers for western equine encephalomy-elitis virus inCulex tarsalis infected after ingestion of lowviral doses Am J Trop Med Hyg 30 190ETH197

Lowichik A and R C Lowrie 1988 Uptake and develop-ment ofWuchereria bancrofti inAedes aegypti and HaitianCulex quinquefasciatus that were fed on a monkey with

low-density microTHORNlaremia Trop Med Parasit 39 227ETH229

MacDonald G 1952 The analysis of the sporozoite rateTrop Dis Bull 49 569ETH586

McGreevy P B N Kostrup J Tao M M McGreevy andT F de Marshall 1982 Ingestion and development ofWuchereria bancrofti inCulex quinquefasciatus Anophelesgambiae and Aedes aegypti after feeding on humans withvarying densities of microTHORNlariae in Tanzania Trans RSoc Trop Med Hyg 76 288ETH296

Mellor P S and J Boorman 1980 Multiplication of blue-tongue virus in Culicoides nubeculosus (Meigen) simul-taneously infected with the virus and the microTHORNlariae ofOnchocerca cervicalis (Railliet amp Henry) Ann TropMed Parasitol 74 463ETH469

Nathan M B 1978 Culicoides phlebotomus a vector ofMansonella ozzardi in coastal north Trinidad West IndiesTrans R Soc Trop Med Hyg 72 436ETH437

NathanM B C F Bartholomew and E S Tikasingh 1978The detection of Mansonella ozzardi microTHORNlariae in theskin and blood with a note on the absence of periodicityTrans R Soc Trop Med Hyg 72 420ETH422

Nathan M B E S Tikasingh G S Nelson A Santiago andJ B Davies 1979 The prevalence and distribution ofMansonella ozzardi in coastal north Trinidad WI TransR Soc Trop Med Hyg 73 299ETH302

NathanMBGBeckles E S Tikasingh P JHamilton andS Monteil 1982 Parasitological and clinical studies ofWuchereria bancrofti and Mansonella ozzardi in coastalnorth Trinidad West Indies West Indies Med J 31168ETH176

Nelson G S and J B Davies 1976 Observations on Man-sonella ozzardi in Trinidad Trans R Soc Trop Med Hyg70 16ETH17

Ramachandran C P and M A Zaini 1967 Studies on thetransmission of sub-periodicBrugiamalayibyAedes (Fin-laya) togoi in the laboratory I The intake and migrationof microTHORNlariae Med J Malaya 22 136ETH144

Rosen L L E Roseboom D J Gubler J C Lien and B NChaniotis 1985 Comparative susceptibility of mosquitospecies and strains to oral and parenteral infection withdengue and Japanese encephalitis viruses Am J TropMed Hyg 34 603ETH615

Scaramozzino N J Crance A Jouan D A DeBriel FStoll and D Garin 2001 Comparison of Flavivirusuniversal primer pairs and development of a rapidhighly sensitive heminested reverse transcriptase-PCRassay for detection of szligaviviruses targeted to a conser-vative region of the NS5 gene sequences J Clin Mi-crobiol 39 1922ETH1927

Shelley A J APA Luna Dias and MAP Moraes 1980Simulium species of the amazonicum group as vectors ofMansonella ozzardi in the Brazilian Amazon Trans RoyalSoc Trop Med Hyg 74 794ETH788

Shelley A J and S Coscaron 2001 Simuliid blackszligies(Diptera Simuliidae) and ceratopogonid midges(Diptera Ceratopogonidae) as vectors ofMansonella oz-zardi (Nematoda Onchocercidae) in northern Argen-tina Mem Inst Oswaldo Cruz 96 451ETH458

Turell M J P A Rossignol A Spielman C A Rossi andC L Bailey 1984 Enhanced arboviral transmission bymosquitoes that concurrently ingested microTHORNlariae Sci-ence (Wash DC) 225 1039ETH1041

Turell M J T N Mather A Spielman and C L Bailey1987 Increased dissemination of dengue 2 virus inAedesaegypti associated with concurrent ingestion of microTHORN-lariae of Brugia malayi Am J Trop Med Hyg 37 197ETH201

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 115

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

otherwise refractory mosquito species ie mf en-hancement probably will not create new dengue vec-tors Even so shortening the extrinsic incubation pe-riod of dengue within the vector may still produce asigniTHORNcant impact on dengue epidemiology

Acknowledgments

We thank Robin Persad and Lester James Ministry ofHealth Saint Joseph Trinidad and Tobago for collecting andrearing of mosquitoes and invaluable help with the logisticsof this study Nathan Mickelson University of North Dakotaassayed mosquitoes for szligaviviruses This project was fundedin part by a University of North Dakota Faculty ResearchSeed Money Award (to JAV) and North Dakota EPSCoR(to JAB)

References Cited

Bell J A N J Mickelson and J A Vaughan 2005 WestNile virus in host-seeking mosquitoes from a residentialneighborhood in Grand Forks North Dakota VectorBorne Zoonotic Dis 5 373ETH382

Brown T U K Babb M Nimrod CVF Carrington R ASalas and M A Monteil 2004 A retrospective study ofthe 1996 DEN-1 epidemic in Trinidad demographic andclinical features Dengue Bull 28 7ETH19

Buckley JJC 1934 On the development in Culicoides fu-rens Poey of Filaria (Mansonella) ozzardi Manson1897 J Helminthol 12 99ETH118

Calheiros M L G Fontes P Williams and EMM Rocha1998 Experimental infection of Culex (Culex) quinque-fasciatus and Aedes (Stegomyia) aegypti withWuchereriabancrofti Mem Inst Oswaldo Cruz 93 855ETH860

Chadee D D C C Tilluckdharry S C Rawlins R Doonand M B Nathan 1995 Mass chemotherapy with di-ethylcarbamazine for the control of Bancroftian THORNlariasistwelve year follow-up in northern Trinidad includingobservations on Mansonella ozzardi Am J Trop MedHyg 52 174ETH176

Chadee D D FLR Williams and U D Kitron 2005Impact of vector control on a dengue fever outbreak inTrinidad West Indies in 1998 Trop Med Intl Health 10748ETH754

Chamberlain R W andW D Sudia 1961 Mechanism oftransmission of viruses by mosquitoes Annu Rev Ento-mol 6 371ETH390

Clements A N 1992 The biology of mosquitoes vol 1Development nutrition and reproduction Chapman ampHall New York

Davis N C 1928 A study on the transmission of THORNlaria innorthern Argentina Am J Hyg 8 457ETH466

Gonzalez A A D D Chadee and S C Rawlins 1999Ivermectin treatment of mansonellosis in Trinidad WestIndian Med J 48 231ETH234

Huang G E Vergne and D J Gubler 1992 Failure ofdengue viruses to replicate in Culex quinquefasciatus(Diptera Culicidae) J Med Entomol 29 911ETH914

KramerLD JLHardy SBPresser andE JHouk 1981Dissemination barriers for western equine encephalomy-elitis virus inCulex tarsalis infected after ingestion of lowviral doses Am J Trop Med Hyg 30 190ETH197

Lowichik A and R C Lowrie 1988 Uptake and develop-ment ofWuchereria bancrofti inAedes aegypti and HaitianCulex quinquefasciatus that were fed on a monkey with

low-density microTHORNlaremia Trop Med Parasit 39 227ETH229

MacDonald G 1952 The analysis of the sporozoite rateTrop Dis Bull 49 569ETH586

McGreevy P B N Kostrup J Tao M M McGreevy andT F de Marshall 1982 Ingestion and development ofWuchereria bancrofti inCulex quinquefasciatus Anophelesgambiae and Aedes aegypti after feeding on humans withvarying densities of microTHORNlariae in Tanzania Trans RSoc Trop Med Hyg 76 288ETH296

Mellor P S and J Boorman 1980 Multiplication of blue-tongue virus in Culicoides nubeculosus (Meigen) simul-taneously infected with the virus and the microTHORNlariae ofOnchocerca cervicalis (Railliet amp Henry) Ann TropMed Parasitol 74 463ETH469

Nathan M B 1978 Culicoides phlebotomus a vector ofMansonella ozzardi in coastal north Trinidad West IndiesTrans R Soc Trop Med Hyg 72 436ETH437

NathanM B C F Bartholomew and E S Tikasingh 1978The detection of Mansonella ozzardi microTHORNlariae in theskin and blood with a note on the absence of periodicityTrans R Soc Trop Med Hyg 72 420ETH422

Nathan M B E S Tikasingh G S Nelson A Santiago andJ B Davies 1979 The prevalence and distribution ofMansonella ozzardi in coastal north Trinidad WI TransR Soc Trop Med Hyg 73 299ETH302

NathanMBGBeckles E S Tikasingh P JHamilton andS Monteil 1982 Parasitological and clinical studies ofWuchereria bancrofti and Mansonella ozzardi in coastalnorth Trinidad West Indies West Indies Med J 31168ETH176

Nelson G S and J B Davies 1976 Observations on Man-sonella ozzardi in Trinidad Trans R Soc Trop Med Hyg70 16ETH17

Ramachandran C P and M A Zaini 1967 Studies on thetransmission of sub-periodicBrugiamalayibyAedes (Fin-laya) togoi in the laboratory I The intake and migrationof microTHORNlariae Med J Malaya 22 136ETH144

Rosen L L E Roseboom D J Gubler J C Lien and B NChaniotis 1985 Comparative susceptibility of mosquitospecies and strains to oral and parenteral infection withdengue and Japanese encephalitis viruses Am J TropMed Hyg 34 603ETH615

Scaramozzino N J Crance A Jouan D A DeBriel FStoll and D Garin 2001 Comparison of Flavivirusuniversal primer pairs and development of a rapidhighly sensitive heminested reverse transcriptase-PCRassay for detection of szligaviviruses targeted to a conser-vative region of the NS5 gene sequences J Clin Mi-crobiol 39 1922ETH1927

Shelley A J APA Luna Dias and MAP Moraes 1980Simulium species of the amazonicum group as vectors ofMansonella ozzardi in the Brazilian Amazon Trans RoyalSoc Trop Med Hyg 74 794ETH788

Shelley A J and S Coscaron 2001 Simuliid blackszligies(Diptera Simuliidae) and ceratopogonid midges(Diptera Ceratopogonidae) as vectors ofMansonella oz-zardi (Nematoda Onchocercidae) in northern Argen-tina Mem Inst Oswaldo Cruz 96 451ETH458

Turell M J P A Rossignol A Spielman C A Rossi andC L Bailey 1984 Enhanced arboviral transmission bymosquitoes that concurrently ingested microTHORNlariae Sci-ence (Wash DC) 225 1039ETH1041

Turell M J T N Mather A Spielman and C L Bailey1987 Increased dissemination of dengue 2 virus inAedesaegypti associated with concurrent ingestion of microTHORN-lariae of Brugia malayi Am J Trop Med Hyg 37 197ETH201

January 2007 VAUGHAN ET AL Mansonella MICROFILARIAE PENETRATE Ae aegypti MIDGUTS 115

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1

Vaughan J A and M J Turell 1996 Dual host infectionsenhanced infectivity of eastern equine encephalitis virusto Aedes mosquitoes mediated by Brugia microTHORNlariaeAm J Trop Med Hyg 54 105ETH109

Vaughan J A M Trpis and M J Turell 1999 Brugiamalayi microTHORNlariae enhance the infectivity of Venezu-elan equine encephalitis virus to Aedes mosquitoesJ Med Entomol 36 758ETH763

Zielke E 1992 Observations on the midgut penetration byWuchereria bancroftimicroTHORNlariae in mosquitoes of vary-

ing susceptibilities to infection Mosquito-borne Dis Bull9 60ETH63

Zytoon E M H I El-Belbasi and T Matsumura 1993Mechanism of increased dissemination of chikungunyavirus inAedes albopictusmosquitoes concurrently ingest-ing microTHORNlariae ofDirofilaria immitisAm J Trop MedHyg 49 201ETH207

Received 5 July 2006 accepted 12 October 2006

116 JOURNAL OF MEDICAL ENTOMOLOGY Vol 44 no 1