Title: Host microflora relationship of vectors of canine heartworm disease
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00098672/00001
 Material Information
Title: Host microflora relationship of vectors of canine heartworm disease
Physical Description: x, 138 leaves : ill. (some col.) ; 28 cm.
Language: English
Creator: Hamilton, Dale Rey, 1942-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Insects as carriers of disease   ( lcsh )
Mosquitoes   ( lcsh )
Canine heartworm disease   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 113-137.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Dale R. Hamilton.
 Record Information
Bibliographic ID: UF00098672
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000355488
oclc - 02162604
notis - ABZ3723

Downloads

This item has the following downloads:

PDF ( 7 MBs ) ( PDF )


Full Text



















HOST MI~CROFLORA RELATIONSHIP~ OF
VECTORS OF CANINE HEARTWIORM DISEASE












By

Dale R. Hamnilton








A Dissertation Presented to the G-lradute Councl o
the University of Florida in Partial: Ful-fillmicgn of
the Requirem~ents -for the Degree of DC~~tor of P~i losophiy


Univer-si iy of Florida
1973



































Copyright
1975






Dale R. Hlamilton































dedicatedl ci my mrke:-ir and dasd,
whose patien~ce hiad nzo i~mitn,

furo their' son iJho -Is le-ss for~tunLte.










ACKNOWLEDGEMEN~TS


I am proud to embark upon my scientific career, distinguished by

the influence of Dr. R.E. Bradley. Dr. Bradley has my gratitude and

my admiration for having brought me to this moment in my professional

life.

I am grateful to Dr. H.L. Cromroy, Chairman of by Supervisory

Committee, who has exemplified what to me are the best qualities of

scholarship and teaching. Dr. J.F. Butler deserves my lasting thanks.

His door and his mind were always open.

Dr. Donald Weidhaas generously opened the door to the USDA Agri-

cultural Research Service, Insects Affecting M4an Laboratorty, of which

he is its director. I appreciate the laboratory facilities, office

space, and most of all, the vast resources of experience made available

to me.

Dr. Jai K;. Nayar of the Florida Medical Ent;om;ological Laboratory

took time from his own research to recount his invaluable experience

with gnotobiotic insect culture.

I am~ obliged to my friend and lab technician, Louis Ergle, who

tried in vain to impart the virtues of laboratory cleanliness. Barbara

Hudgens engaged in a heroic one-wdoman struggle to interpret the tangled

sk~ein kn-own as "a first draft".

I ami also grateful for my place at the Red Lion bar where I re2-

ceived much valuable counsel throughout myv tenure of study.

Finally, a special thanks to Ginny. To her, I dedicate all mly efforts

This investigation was supported by NIH training grant number 5 TO1

AIOO 383-04Sa from the Institute of Allergy and Infectious Diseases.


















TABLE OF CONTENTIS

Page

A.CKNOWLEDGEM4ENTS .................. ... .. ............ iv

LIST OF TABLES .............................. ........... Vi

LIST OF FIGURES ....................... ... ........ .......... vii

ABSTRACT ................ .................. .................. viii

INTRODUCTION .. .............. ......... ............ ..... 1

LITERATURE REVIEW ................ .. . ..... ..... ......... 10

Dual etiology of parasitic disease ................... ...... 24

Microflora associated writh mosquitoes ................... ....26

Statement of the problems ............ .... ............... 37

RESUILTS ..................... 39

I. Bioassy of Early Vector Miortali-ty Following
Dirofita~r~ia immzitici Leidy Infection ..................** 39

II. An Integrated System for the Production of
Gnotabiotic Anopheles qua~drifmacu~latus Say ............ 47

III. The N~ative and Midgut Bacterial Flora of Several
Wild and Colonized Mosquito Spcies ....... ............ 60

IV. Effects of Bacteriological Flora on the Ear~ly
Development of Divofilaria immi~tic Leidy in
Alnopheles qua~drirm,cula,~tus Say .......... ... ......... 89

DISCUISSION AND CONCLUSIONdS ........... .... ... ......__. '105

BIBLIOGRAPHY ...., ............. . .. ._ ............,113

BIOGRAPHICAL SK:ETCH .............. ..........................13














LIST OF TABLES


Table Page

1-1. Effects of Diff:erential Filaremia on
Mortality of An~opheles qjuadrim~acouZatus3 Say .............. 42

I-2. Effects of Feed-ing Dead Intact, and Homogenized
Microfilaria to Femnale Anropheles3 quadrimaculatus Say .... 43

I-3. Effects of Feeding Homogenized Body Parts from
Females Fed Infective Meals 24 Hours Previously ......... 44

II-1. Composition of Diet for Rearing An~opheless
quadr~imaLcuilaus Say ................... ...............~... 55

III-1. Species Com~position of Midgut Bacterial Flora
in 5 Groups of Mosquitoes ............................... 66

III-2. Correlation Coefficients of the Frequency of
Occurrence of M~idgut Bacterial Species .............----- 82













LIST OF FIGURES


Figure Page


II-1. Gnotobiotic Arthropod M~odule (GAM) Used for the
Maintenance of Blood-Feeding Aniophele c quad?rfrimaculatu/s
Say .................................... ........ 52

II-2. A Battery of 4 GA~s in Position for Blood-Feeding
in Heating M4anifold ....................................... 54

III-1. Procedures in the Presumptive Determination of
Bacterial Genera ......................................... 65

IV-1. Type I-Ib Larva in Malpighian Tubule "Squash" from a
Conventional Nlosquito 48 Hours After Taking an
Infective Blood Meal ................... .................. 95

IV-2. Typically Unchanged Type I Larva Recovered After 48
Hours in a Mosquito Mionocontaminated with Salm~onella sp. ... 96

IV-3. Early Type II Larva Recovered After 48 Hours in a
Mosquito Monocontaminated with Acinetobacter calcoacetious.. 97

IV-4. Type II Larvae Recovered After 48 Hours in a Mosquito
Mlonocontarninated with BacilZurs cereus ................... ... 98

IV-5. Early Type III Larvae Recovered After 48 Hours in a
Mosquito Mionocontaminated with LactobaciZllue sp. .......... 99

IV-6. An Encapsulated Type I Larva Recovered After 48 Hours
in a Mosquito Monocontaminated with Non-pathogenic
coryneb5acterl:um sp. .......................................10

IV-7. Typical "Sausage" Stage Type IV Larva Recovered After
48 Hours in a Gnotobiotic Mosquito ........................101

IV-8. Tw~o "Sausage" Stage Type IV Larvae Hlithin a Tubule of a
Gnotobiotic Mosquito .......... ......... ..................102













Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requiremnents
for the Degree of Doctor of Philosophy





HOST MICROFL.ORA RELATIONSHIP OF
VECTORS OF CAN~IIE HEARTWORMV DISEASE


By

Dale R. Hamliltton
August, 1975



Chairman: H.L. Cromroy
Co- cha irma n: R.E. Bradl ey
Major department: Entomology and Nematology


The phlenomnenon of early vector mortality of Dir~oji~iLaric. imitici

L~eidy infected mosquitoes wJas investigated by means of a bioassay -tech-

nique. Anoplheles quadrima,~3cuZabuss Say w~ere fed differentially filaremic

blood meals, living and dead microfilariae, and special adjuncts such

as homogeniz~ed mosquito body fractions. At least 25"! mortality wajs

produced r inmosquitoes ini the first 3 days following ingestion of living

miicrofila riae. Nlei their d-ad, intact, nor- homogenized larvae, whole

mosqui to bodies, nor body fractions refproduced this miortal ity, suggesting

that ear-ly vector death is a function of the kinetics of living micro-

filariae.

An integrated systemn of diet, technique, and hardwairee was developed

for the productionl of gno0tabiotic blond--feeding arthropods. This system









produced the fir-st successful rearing of An,. qua"dr~iiimr;L7atulaba under

bacteria-free conditions.

A quantitative and qualitative bacteriologic evaluation was

accomplished on the midguts of several hundred adult female specimens

of colonized in. qu~adrjimacuiZatu-s, wild An. quladri-macuilatus, A-nopheles

crurcian-s Weidemanrl Anophel~ec cruciansr complex, and Aedea ii~jrnfcirmt

Dyar and K~nab.

The total midgut bacterial count, and the species composition of

midgyut flora was distinctive for each group of mosquitoes. The most

frequently isolated bacterial species for each group of mosquitoes were:

A. inir.-zmatis, non-pathogenic Coirynebacteri.:m sp., wild An. qu~adrimaculAiatus3,

Bnterobacte~r cloacae! (Jordan), and Ac~inetobater calcoace~t~ions (Beijerinck),

colonized Ani. quaodrimaev7Yat lue Bacill:L20 ctereuo Franklanld and Frankland,

An. cruciiians, Allealigenzes ~faeccali~s Castellani and Chalmers, and An.

cr~-ucian complex, Sa~n0trone!Za sp. Data analysis revealed several signi-

ficant (P .001) positive and negative correlations among the various

bacterial species comnpris-ing thle normal flora of these 5 mosquito groups.

A proportion of~ bacteriologically sterile specimens were encountered

in 4 of the 5 mosquito groups. Sixty-one per-cent of w;ild An. quaclh~rimcun-

latvos were sterile while 410% of the colonized An. qua~d2ima7culati.u s wer'e

sterile. An. crutciann and An. crucians complex were 36% and 43;: sterile.

All specilenss of Ai. infiine alz~ examined w~ere bacteriologically sterile.

Pure cultures of bacteria isolated froml these mnosquito-s wJere used

a s mnon ocontami;i na7nts of gjno tobi oti c ;ln. quadi~macula;7lln tus. Thiese mosqui toes

as well as conventional and gnotobiotic mosquitoes were subsequently: in-









fected with D?. immithii infective larvae. Forty-eight hours post-

i nfecti on all mosqui toes w~ere ki lled. The degree of filarial develop-

mnent w~as recorded by light and phase photomicroscopy, and typed ac-

cording to their gross dimensions.

The early development of D. irmmi~tis larvae proceed independently

of bacteria or bacterial mediation in the midgut. Sausage stage larvae

appeared within the first 48 hours in gnotobiotic mosquitoes, but re-

quired at least 72 hours in conventional mosquitoes. Nematode de-

velopment appeared to be marginally improved in the complete absence

of bacteria and differentially retarded in the presence of pure cultures

of certain bacterial species. Pure cultures of LactoaSiZelvjs sp. and

B. cerceui, were found associated with more advanced larvae, while SaZ-
monella sp., Al. calcacetiouls and Corynebacterium~hn sp. monocontarminarnts

were found in the presence of less developed larva. If the presence of

midgut bacterial flora is inversely related to the development of D.

immritic in the mosquito, it is suggested that the "naturally" sterile

specimens in the field are the most effective vector of D. immritis.













INTRODUCTION


Caninle heartwor-m disease research in the last decade has evolved

into tw~o distinct efforts. The first approach has been towlards develop-

ment of adequate chemotherapy to replace the potentially hazardous

arsenicals presently in use. Heartw~orm prophylaxis, although effective,

involves considerable risk as well as bothersome clinical management.

Development of a vaccine or imrmunizing agent against canine heartw~orm

disease, although promising (Otto, 1970), is not imminent and miust aw~ait

fuller understanding of the mechan-ism of imm;unity to filarial infection.

The second approach, and historically the miost suiccessful, is

direccted against the arthropod vector. Di?ofitar~ia fronit Le'idy, the

etiological agent of canine heartworm disease, depends upon passage

through a mosquito to th-e infective stage. The traditional methods of

controlling such arthropod borne diseases such as filar-iasis, yellow fever,

malaria, and trypanosomiasis have been to release sufficient pesticide

into the endanger-ed area. This has been justifiable, because it is most

certainly DDT and its descendants that hvav done the most to reduce, and

in places eradicate, malaria, typhus, filariasis, trypanosomiasis, and

many other of the world'ss worst killer diseases (Provest, 1972).

Inl recent years, it has become dramatically apparent that the

traditional netihod of vector control using insecticides is no longer

effective. The Universitiy of Califor-nia's Mosquito Control laboratory

recently reported that the common pasture mosquito Asda!! nig:i.-naculia;

(Ludlow) is now~ resistant to every available insecticide (Kendrick, 1970).









Boardman (1973) quoted a report of the Agricultural Experiment Station

at the University of California which reported the fact that the two miost

important species of mosquitoes in California had developed near total in-

secticide resistance. Elsewhere, the use of organic insecticides against

mosquito larvae has led to so mluch resistance, in Cuter f~atigans Wi edemann

especially (Brown, 1967), that control of filariasis and several arbo-

viruses is now one of the greatest problems of vector control. According

to a recent review (Busvine and Pal, 1969), resistance to DDT, BH!C, and

dieldrin is severely affecting control, in one place or another, of malaria,

yellow fever, filariasis, typhus, and plague, while certain other vector-

borne diseases have so far not been affected by pesticide resistance, e.g.,

onchoceriasis, Chagas disease, typanosmiasis, and le-ishmnaniasis.

To compound the problem, killing mosquitoes with insecticides is

becoming prohibitively expensive. The logic of pest control demands more

specific chemicals. However, the more specific a compound is the more

limited its use, and the more costly it is to produce. A decade ago, it

cost less than half a million dollars to market a new~ insecticide. The

same development job in 1970 cost from 3 to 5 million dollars (Kendrick,

1970), and today even more. At a time~ when mosquito abatement districts

are demand-ing new insecticides to replace those of declining value,

manufacturers have less incentive to produce then and particularly the

narrow~ spectriml m;aterialls designed for limited uses.

The persistence of pesticides in thle environment, now thoroughly

documented (Woodwell, 1970; Mriller and Berg, 1969), has led to increasingly

miore stringent regulations to govern their uses aInd appl-ication. The

Federal Environmental Pesticide Control Act of 1972 for example, established

the Environmental Protection Agency which promptly banned the use of DDT.

The net result of increasing insecticidal resistance, obsolete.









pesticides, and legislative restraints is that mcosquitoes are increasing

in number, and w~ith themi, canine hefar-tworm disease. Results of a nation-

wide geographical survey of canine filariasis taken in 1955, have made it

abundantly clear that canine heartworm disease is expanding its range

(Young, 1955). The response of 2,337 veterinary practitioners throughout

the United States indicated that the distribution of heartworm was primarily

in the states bordering the Gulf of Me~xico and the Atlantic Ocean, but ex-

tending northward to illinois and central lowa (Young, 1955). Marquardt and

fabian (1966) collected reports of D. iirnwilino in Florida, Georgia, Alabama,

Louisiana, Mississippi, Ne~w Jersey, Tennessee, Maryland, Arkansas, Pennsyl-

vania, Rhode Island, and Hawaii. Subsequently, enzootic dirofilariasis has

been reported in Indiana, Michigan, Ohio, Canada (Otto and Buaman, 1959),

Mininesota (Schlotthoauer and Griffiths, 19)64) and Connecticut (Hirth et al.,

1966). In a 1973 statewide survey of Florida, Bradley* reported that 27

north Flor-ida veterinarians diagnosed 4,648 cases of caninie heartworm in

1973, an increase of 22% over the previous year. Seventy-seven central

Flor-ida veterinlarians and 84 south Florida practitioners reported 23,212

and 8,658 cases, respectively, 1973, an increase of more than 50% over

the 1972 case level. Eighteen west Florida veterinarians diagnosed 3,927

cases in 1973 which consti-tuted a 33% increase for the year.

Nlot all mosquito species, however, are capable o-f transmitting

;. ?IIII.cibS. Demirick and Sandholn (1966) list 50 mosquito species known to

be successful vectors in the United Statefs. Ma~cnonald (1971) has compiled

a list of 107 species in 6 genera of Culicidae which support the development

of filarial larvae. Ludlam and Jachlowsk~i (1970) report 64 species of

mo~squitoes in 5 genera in wihichh comcplete larval development of s. immi~si


* R.E. Bradley, Personal Com~munication, 1973









can take place. Yen (1938) described differences in relative susceptibility

to infection in 12 mosquito species. Travis (1947) demnons-trated the relative

efficiency of 6 species of mosquitoes from Guam, and Intermill and Frederick

(1970) reported a similar comparison of Ryruku Island species. Kartman (1952)

also reported a quantitative evaluation of the developmentt of D. immiiis in

7 common mosquito species. Furthermore, evidence has accumulated that indi-

cates that the same species of mosquito may, exhibit varying susceptibility

to D. imnit~ic in different laboratories. K~artmran's (1950) monumental survey

of the world literature from 1901 to 1949 indicated that the experiments of

over 30 workers show that Ac~des aegypti (Linnaeus) varied in susceptibility

to Wutchrereia bannerofti (Cobbold) from completely inhibiting the! parasites'

development of allowing maturation of infective larvae.

Eveni within successful species and refractory species, there are

individuals who show varying susceptibility to infection with D. inuritis.

A recent epizootiologic study carried ouit by Bradley (1971), surveyed Hills-

borough County, Florida, an area heavily populated by known vectors of

D. iinvitis. Only small numbers of parasites were found in resident dogs,

which Bradley (1971), concluded may indicate some factor being exerted on

the vector population to limit the exposure of dogs to infective larvae.

Miccreevy et al. (1970) in a survey of 5 counties in northern California

failed to find a single case of D. im~mitis inl 515 pound dogs. The same

authors surveyed the clinical records of dogs examined for heartworm at the

University of California's School of Veterinary Medicine from 1957-1968.

Only 13 cases of D. irnvitifi infection could be con-firmied. Of the known

species of mosquito vectors, 9 species werne reported to occur in Calif~ornia,

and 6 were reported in counties where D. i!mmais was known to occur

(M-c~reevy et al., 1970). A survey of 50 wild foxes trapped within the








geographical limits of a hyperenzootic area in Georgia proved negat-ive for

D. 7jwnitis microfilariae (Walton et al., 1963).

Regional differences in relative susceptibility to infection had

also been noted. R~oubaud (1937) reported that Assam and Tanganyika strains

of Ai. aegypti permitted normal development of D,. wnimmics buit Cuban strains

infected from the same host were entirely refractory. Newton, W~right and

Pratt (1945) reported that Puerto Rican strains of Peorophioia con~finiz had

a 12% infectivity potential for W. bancroffi, whereas United States strains

had an 80% rate.

The basis of vector susceptibility fo filarial larva has been hypothe-

sized since Noe (1908) first reported the destruction of microfilariae of

Dipebtnonzema grransi N~o~e, in thie gut of its tick host during the latter

stages of feeding. Rozeboom (1973) had observed that filariae exhibit a

strict vertebrate host specificity, but the insect vectors for a given para-

site may include many species among two or more genera. These parasite-

vector associations, he noted, have arisen as the parasites evolved and ex-

panded their geographic distribution and have depended less upon the taxo-

nomic position of the insect species than upon the fortuitous existence of

genetically controlled factors for susceptibility. WJeinstein (1973) has

made the following generalizations regarding the specificity of development

of filariae in arthropod hosts.

1. There is generally a higher degree of specificity in the
vertebrate phase of the cycle than in the invertebrate.

2. Most recognized vectors are mosquitoes.

3. Filarial development in the arthropod takes place in only
one of 3 types of cells (Mialpighian tubule, thoracic flight
muscle, and fat body), and the specificity of cell type is
virtually abDsoute fur a particular species of filariid.

4. Development is intracrellular.








A genetic explanation for susceptibility of invertebrate hosts to

certain parasites has received substantial support. Huff (1941) determ-ined

that the susceptibili'ty of cular piipices LinlnaEu( s to P~oameld iumn cazthemerrium

Hartman is a recessive character due to a single pair of genes and in-

heritable along the lines of thle classical M~endelian 3:1 ratio. Trager

(1942) produced a strain of A. aegypti mrore susceptible to infection wi th

PZasmodiium Zophurae Coggeshall than the original colony strain. A report by

the R~ockefeller Foundation (1948) indicated that it w~as possible to raise

the susceptibility of Anopheles quLad~r-imacu7Y atus Say to Pla::mod-iu galiamesw n

Brumpt from 20% to 100%. By selective breeding Nicks (1949) elevated the

susceptibility of c. pipien~s to PZa;emodCium elongatium Huff and Bloomr from 13%/

to 49% within 6 generations.

Evidence to the contrary ha:: been advanced by Boyd and Russell (1943)

who could obtain no clearly defined results in attempting to select a strain

of Anz. quadrimnaculatus more susceptible to Plasmodiurm vivrw: (G:-assi and

Feletti). Jeffrey (1949) similarly failed to produce a strain of Anophelea

albopictus (Skuse) more susceptible to infection- withC P. Zophuroae and
Horvanitz (1947) could obtain no genetic effects in 6 generations of A.

aegypti selected for susceptibility to P. ga2%iincea~.

A genetic basis of mosquito susceptibili ty to filarial nematodes was

first suggested by Roubaud it al. (19353) and Roubaud (1937) w!ho noted

differences in the susceptibility rate between colonies of AI. aegy~ptC

originating from different geographical areas and exposed to the same strain
of D. imanitis. Ramachandran et al. (1960) reported similar variations in

susceptibility of Al. aegyp~ti for^ BrG1h- m77,cai (Brug). The response of a

colony of mosquitoes to artificial selection pressure for changed suscepti-

bilit~y was noted by Kartman (19533i)wi-th D. imnitics and by Thomias and









Ramiachandran (1970) for Wuzche~rcria~ sp. in: DAlrx pipit'" ns frabigno Wiedman.

N~acDonald (1962a, 1962b, 1963a, 1963b) determined that susceptibility

to infection of A. aegyipti w~ith sub-periodic B. malayi was controlled by

a sex-linked, recessive gene called FM which exhibited incomplete dominance.

Subsequently, MacDonald and Ramachandran (1955) reported that this gene

controlled development of 5 str~ainls of Wu~chereviai sp. and DvGi~:a sp. in

the thoracic muscles of Al. aegypbi, but apparently had no effect on the

development of D. inmitic or Divoufitarila repensc Rail let and Henry in~ the

Malpighian tubules. McGreevy (1972) finally demonstrated that susceptibility

of A. aegypti to D. jmmitis was also controlled by a sex-linked, recessive

gene and that penetrance of this genie was incomplete. Moreover, the gene

that controls the development of Dn. inside~i in the M~alpighian tu!bules is

not identical to the FM gene which controls the development of Wuichercria sp.

and Brugia sp. in the thoracic muscles. M~acDonald (1973) cited unpublished

woirk by Oblamine and MacDonald that indicates the susceptibility of c. p.

fatigans to D. pahzangii was also controlled by a sex-linked, recessive gene.

Kartman (1952) considers that genetic factors may operate independently

of the phylogenetic position of the host. The efficiency of 2 closely re-

lated mosquitoes as vectors for D. imm-itis is not necessarily comparable.

Instead, susceptibility seems to be an inherent characteristic of the in-

dividual mosquito. Hu (1937) gave C. p. pallena double spaced infectious

feedings of W!. !:aw;rofi' in an effort to determine whe!ther susceptiblity

to infection wJith filaria in an inherent characteris-tic of the individual.

Hie reported that some2 of the fem-ales were susceptible to the first but not

to the second infective meal, anid vice versa. Similarly, imm~unity to re-

infection w~ith D. i:mitis would not develop ill Ausph :-,Zes I;uncT;ti enni: (Say),

Aedes cineru~s Mleigen, and ZedeZn: eviiseratus Say (Phlillips, 1935). Ka~tma-n









(1953) noted an unusually high susceptibility for D,. i;-r.itic drmong certain

individuals of Haw~aiian str-ain of A. cogyp'ti, a species generally refractory
to ths filriae.Furthermore, individual respione of a mosquiohoti

distinctly specific to the species of parasite. Kartma7n (1952) could

demonstrate no correlation between sulsceptibility of A. aegypti to mixed

infections of D. i:~nvitic and FoZeye~ll sp. Each species of filaria reached

its normal location within the mosquito without interference from the other

species. This is consistent with the observations of Huff (1930) on in-

fection of C. pipienc with several species of avian plasmodia, and with

those of Moorthy (1938) on the development of Canattanlusu sp. in cyclops

previously infected with D~-racen-Lutus sp.

A more critical examina-tion of the individual mosquito, rather than

a species as a unit, seems to be warranted. The factors and circumstances

which serve to distinguish the -individual mosquito are manifold, and may

have an important effect on the individual's susceptibility to ma~ny parasitic

agents. One of these distinguishing factors may be quantitative and quali-

tative differences in the composition of intestinal microflora.

Mosquito species tend to select very specific nesting sites and

subsequent rearing sites for the larvae. Considerable diversity exists

between species in their choice of sites which range from vernal ponds

(Wills and Fish, 1973), pools and flood w~at:ers (Jamezs and Harwood, 1969)

to tree holes and automobile tires (W~omack, 1971), and inl certain da-ter

holding plants suich as bromeliads, heloconids and pitcher plants (Gillette,

1972). The bacterial and fungal populations of these selected microhabitats

reflect the diversity of their environment.

Newly hatched mosquito larvae are indiscriminant filter-feeders which

voraciously invest anything of a specific particle size. Since this








particle size is usually less th~an 100 microns, bacteria make up a sub-

stantial proportion of thle larval diet. This fortuitous acquisition of

maicroflora apparently established the individual's lifetime comnplemlent

of internal bacteria, which is both pspcies specific and individually

distinct. Quali tative and quantitative differences in the composition

of this microflora may represent one of the factors mediating vector

susceptibility to filarial infection. The passage of filarial larvae

into and through the gut of suiscep~tible mosquitoes evidently r-equires a

critical set of conditions since attempts to culture infective filaria

larvae have me-t withh little success (Weinstein, 1970, 1972; Rothstein and

Brown, 1960; Sawyer and W~einstein, 1933.4, 1963b; Taylor, 1960b;Wood and

Suitor, 1966; Cupp, 1972). Residen-t microflora may indirectly interact

with the infective larvae, or merely mediate an intestinal climate

favorable to survival of the parasite.

The demonstration of an interaction between larvae of D,. i~mmtis

and resident bacteria, may provide a future means of biocontrol for both

anthropod and niematode.














LITERATURE REVIEW


Subsequent to feeding on a filaramic dog, 3 stages in the development

of D. inmniti8 occur within the mosquito (Taylor, 19360). A moul-t marks the

onset of each stage terminating in the third stage infective larva. M~icro-

filariae taken in with the blood remain in the stomach of the mosquito for

the first 24 hours. During the next 24 hours they migrate to the Malpighian

tubules. Further development occurs at thie distal end of the tubules until

the fifteenth or sixteenth day. The larvae spent the first 6 or 7 days in-

side the cells of the M~alpighian -tubules. Suguri et al. (1969) have de-

scribed this zone on the Malpighian tubules as rich in mitochondria and

course endoplasmic reticulum. The brush border has a multitude of vertically

arranged protoplasmic processes each containing a mitochondria whose cristae

run parallel to the process. Fromn thle ninth to the fifteenth day, larvae
are found in the lumen of the tubules. By the fifteenth day the larvae are

ready to break out of the tubules and migrate through the mosquito, to occupy

the cephalic spaces of the head and proboscis (Taylor, 1960). Transmissionn

of infective larvae occuISrs with the next blood meal. Al-though many arthro-

polds have been evaluated as vectors of D. ZinwZith,, only mnosquitoes appear to

be entirely competent (Ludham and! Jachowski, 1970).

Kartman (1952) observed that althoulgh microfilariae of D. C-invitic are

knownl to be capable of metamonrphosis in many spcies of m~osquito-s, there is

a dist-inct pauicity of host species in whic-ih it maiy adequately reach the in-

fective larval stage. The same writer introduced thec concept of "host

efficiency," a ratio between theoretical numbers of microfilariae ingested









by a group of mosquitoes and the total number of developing larvae found in

them during a specified period, and "infective potential," a similar ratio

involving only infective larvae. Kar~tmian (1952) reported that the host

efficiency of Anl. qua~dritaimculatu:; was approximately 20 times better than

either Al. aegypti or Culex qutnquefus-rc auSnllr Say; and that the infective

po-tential of An. quzadr:imacuZabus was 50 times greater than A. ae~gypti and

20 times more than C:. quinquefansciatus. Infection of C:. pipiene and C.

quinquefas~!ciatus showed that both the hiost efficiency and the infective po-

tential of C. quvinquef,~aceiatus~. were 2-1/2 times greater than those for C.

pipiens. Ewart (1965) correlated variations in vector potential of CuZe-,

Aln3,he~ine, and lede-s mosquitoes to failure of Brugia. pahangi Buckley micro-

filaria to exsheath. Weatherby et al. (1971) reported a similar case of host

efficiency in susceptibility to P. galinazcewnm by feeding mosquitoes whole

body extracts of both susceptible and refractory mosquitoes. CuiZez pipiens

extract (refractory) fed to susceptible A. aegypti reduced the number of
oocysts developing by 50% while concentrations of 75%etatreue h

numbers to only 34% of the number in the controls. Clearly, some factor

present within refractory mosquitoes, and transferable,w~as capable of reducing

the host efficiency of susceptible mosquitoes.

A problem fundamental to the production of experimental Di. im~ibia in-

fections has beeni survival of the mosquito -following uptake of m~icrofilariae

fromn infected caninle blood. Various authors including Yen (1938), Kershaw

ot al. (1953), Bradley (1953a, b), Hiusain and Kershaw (1956), Wefbber and

HawJking (1953), Pistey (1959), Symes (1960) and Weiner and Dr-adley (1970)

have reported vctor- mortality approaching7 1003 in the first fewi days follow-

ing the blood m~eal. A common antecedenti to death appears to be distenision of

the abdomen persistinig 2 to 3 days, whiich Weiner and Bradley (1970) interpret









as a result of the improper digestion of blood. Additionally, it appears

that vector mortality increases proportionately with the number of micro-

filariae ingested (Rosen, 1954; Kershaw et al., 1955; Wlharton, 1957; Kartman,

1952). Kartman (1952) suggested that variations in vector survival w~ere

sometimes dependent upon genetic strains of the same species, and later

(19534)observed a direct relationship between numbers of microfilariae fed

to mosquitoes and vector survival. Diet (LeCorroler, 19J57), environmental

temperature and humlidity~J (Galliard, 1957), but not atmnospher-ic pressure

(Williams, 1959) have been shown to affect survival of filarial-infected

m~osquitoes. Laurence (1963) had estimated the natural mortality of 2 infected

mosquito populations in the field. c. fa;tigans and An~opihelrs pediitae~nia~tus

(Leichenster) in south Inidia had a daily miortality of from 14% to 24% during

a season favorable for survival.

Kershaw et al. (1953) investigated the effect of microfilaria of D.

immjitia~ on the longevity of A. aegypti under controlled laboratory conditions.

Infected mosquitoes had a constant but very low survival rate during the

first 5 days corresponding to the migration of microfilaria from the midgut

to the Malpighian tubules of the mosquito: a constant and high survival

rate from the fifth to the twentieth day, corresponding to the development

in the tubules; anid a very low but constant survival ra7ie flrom the 21st to

the 25th day, corresponding to the presence of infective larvae in the hemro-

coel and in the head and! mouthpartss of the mosquito.

Septicemia induced by emigrating microfilarid may be another contribution

to vector mortality in the first few dayvs -following infection. Intestinal

miicroflora, fortu-itously acquired in the larval stage, ar~e unable to multiply

in the conditions prevailing in the gut of healthy insects (Bucher, 1960).

Substantial mortality from bacterial infections in laboratory colonies had









been reported w~hen environmental conditions favor thel multiplication of one

or more of the bacterial species already present inl the guit (Prinsloo, 1967).

Chu and Toumanoff (1965),orepote that most of the bacteria isolated from

laboratory colonies are Gram-negative which K~alucy and Daniel (1972) concluded

are "potential pathogens" which require predisposing factors such as damage

to the cuticle or gut wall in order to penetrate the hemocoel. The bacteremia

resulting from multiplication in the hemocoel caused death of the host.

Nematode-intro3duced septic conditions are not unknown in the literature.

Most of the infective juveniles of the DD-136 strain of Necapteebana car~poca-

sae W~eiser contain cells of a specific bacterium, identified and described as

Alchromobacter -nemato;/h~ilis in the ventricular portion of their intestinal

lumen (Poinar, 1972). Soon after the nem~atode reaches the hemocoel of a para-

sitized insect, such as G02Zleria larvae, the bacterial cells are passed fromn

the intestine out through the anuis into the hemolymph. The bacteria then

rapidly multiply in the hosts' body causing a fatal septicemia. In mos t in-

stances it is the bac-teria that actually kills the host-usually wiithin 48

hours; however, most nematodes without their associated bacteria are also

able to kill the host within a short period. Without the Achro-?oba~ter

bacteria Poinar (1972) reports, N;. carp~oozpnea is unable to gain access into

the insect hemriocoel, thus the relationship between A. LneatoyhZi lic and

N,. carpocapa~ce m~ust be considered mu~talistic (Poinar and Thiomas, 1967). Such

bacteria ar-e undoubtsd ly as:sociated w~it-h all of the 11 knlownr species of

WNyop~chanazi (Wdelch:, 1965).

Large numbers of m~icrofi'lariae ingested wi th blood iieal have been

shown to interfere wi, th the formation of thle p-tritrophic mem;brane with

fatal r-esuilts (Lew~is, 1953). Wadnson (1950) noted that the blood contained

in thie stomach of Sier2Zin ;um downocu Th-obald killed by heavy m~icrofilariae









infection, appeared to be undigested. Similar observations w~ere recorded

by Roubaud et al. (1938) and Mlackecrrar. (1953) on other filar-iae-host com-
binations. Lavo-ipierre (1958) regarded the mos-t likle~ly cause of death in

heavily infected arthropods as "indigestion" due to extensive damage to the

peritrophic membrane rendering thle digestion of blood almost impossible.
The same author found it impossible to demonstrate the peritrophic membrane

in dying heavily filaremic A. aeSYpti with und-igested blood meals, whereas
thle membrane wdas present in the controls in which the blood meal was in an

advanced state of digestion. Bain and Philippon (1970) found the peritrophic

membrane intact in AniOpheles3 stdphnC~1i Liston and S. dan':osum; given a blood

meal containing reptile microfilariae. Furthermore, they concluded that the

peri-trophic membrane did not prevent the crossing microfilariae from the
blood meal to the midgut. Lewis (1953) reported that peritrophic membrane

formation in S. dawnmocumn following the blood meal was critically important

to thle survival of onzchocerieavolvuilus Leuchart. Only the filariae which re-

main in the anterior portion of the midgut are able to pass backward (after

formation of the membrane) into the space between the membrane and the gut

wall in order to pierce the stretched waills of the distended stomach.

Wigglesworth (1965) described two different methods of peritrophic
membhrane formation around the food bolus. The membrane may be delaminated

from most or all of the midgut epithelial cells, or it may be secreted from

specialized cells in the anterior mnidgut. The mridgut opithelium of mouse-
fed A. asegypti produces a granular secretion for up to 15 hours after feed-

ing and the peritrophic mlembranee is apparently formed from this (Bertr~am
anrd B-ird, 1961). The membrane formed by the first method generally acquires

the shape of the food, bo'lus, while that formed by the second method is

usually cylindrical. In larval mosquitoes, the membrane is formed by the









second method (Wigglesworth, 1930; Richards and Richards, 1971). The mem-

brane in adult mosquitoes is formed by the fiirst method (Freyvogel and Staubli,

1965; Freyvogel and Jacquet, 1965; Stcher, 1957). Romoser andr Rothman (1973)

published the first report of the presence of peritrophic mem~branee around

the meconium of a pupal mosquito. Romoser (1974) subsequently demonstrated

a second membrane, closely applied to the first which is formed somewhat

later in the pupal period. Howard (1962) reported that the peritrophic mem-

brane appears about 12 hours after At. aegypt-i feeds, and increases in size

up until 36 hours, and becomes brit-tle by 48 hours. As the amount of blood

in the midgut decreases, the peritrophic membrane is fragmented by the con-

traction of the midgut muscles.

Zhuzhilov (1962) reported that the peritrophic membrane was apparent

in A. aegypti within 20 minutes of taking a blood meal. In contrast, Frey-

vogel and Staubli (1965) could not discern the membrane in this species

before 8 hours after -feeding. Richardson and Ram~oser (1972) reported that

the membhrane was visible in Ai. twiseriauer 20 minutes after taking the

blood meal.

Stohler (1957) noted that the memabrane adhieres to the blood parcel,

not to the midgut cells. Although then membrane in Aeder; sp. passes through

stages described by Freyvogel and Staubli (1965) as viscous, elastic, solid,

and finally fragile, the membrane in Anroph-?en sp. never develops beyond a

fragile membranes. The same authors also report::d that 3 species (A. cofuPi;~

rinopho~leS sawh71i*., Gilals, Ani ScOIphem:1 i) whilch no-nmally formn a pe~ritrophic

nemb~-rane do nlot do so comnpletely if the2y ingesjt onlly a small quan:t~ity of

blood. Richairds and Richards (1971) found n~o tradce of a m~embranez in adult

male or unfed adult -female A;. aaegyphi 1 to 3 days post emergence. Older

adult fem~ales given a blood meal produce a peritrophic mem~brzne from material









secreted by a ring of midgut cells in the anterior half of the proventricular

pouich.

The rate of destruction miicrofilar-iae during the first 48 hours after

ingestion determines vector mortality to a considerable extent. Hughes (1950)

observed that miicrofilaria of rLitomociozdes; ingested by or~nithon;iuccuIs ba~coti

(Hirst) mites w~ere phagocytosized by certain cells in the midgut. Kartman

(1953) regarded the initial destruction of microfilariae in the midgut as a

process of digestion, although, as he p~ointed out, death may not be caused

by digestive enzymes but rather by other factors ini the midgut or salivar~y

secretions. Digestive destruction of inappropriate parasites is an early

hypothesis to explain vector-parasite specificity (N~uttall. 1908) which re-

mains an attractive explanation. Digestion of the blood meal by mosquitoes

begins at the outer edge of the meal and proceeds inward (Davis and Phillip,

1931). This has been observed in C. pipiens (Hfuff, 1934), A. aegypti (Stohler,

1957), Ain. st-ephkensi, An~. goinbiac and Ainophales macutlpipenns M4eigen (Frey-

vogel and Staubli, 1965). Before engorgement they become squamnous w~ith con-

vex internal borders (How~ard, 1962). Bertram and B-ird (1961) observed in

the midgut cells of Aedes after a blood meal, that w~horles of granular endo-

plasmic reticulum condensed around the nucleus unfold to form a complex

system "ramifying throughout the cytoplasm" which revert to whorles when

blood digestion is complete. These changes, confirmed by Staubli et al.

(1966), apparently facilitate the liberation of proteolytic enzymles for

digestion of the blood meal, and Hector and Freyvogel (1971) noted that whorl

formation wdas found only in female mosquitoes.

The anterior midgut cells of Ain. garmbian secrete fair-ly large amounts

of mucous-like material within 7 minutes of a blood meal. Al-though this

mucous forms a plug at both ends of the stomach and of ten completely surrounds









the meal, Freyvogal and Staubli (19635) have concluded that it exerts no

important effects onl d-igestion.

The presence of a! microfilaricide in the dlige-stive secretion of the

mosquito host has yet to be verified. Duncan (1926) de-termined thle bac-

teericidal activity of the gut contents andl feces of several bloodsucking

ar~thropods includilnD d~l-,haeled bi/'ureatusi (L~innaleus), A. cineran,~i~ StOmoge~~i

caleitr~ans (Linnaeus) and blood fed Muse2~r d~omesaican (Linneaus). Activity

was found against 8 of 18 species o-f bacteria used. The bac trial material

fromi S. calcit~ranti was heat stable (1000C for 30 minutes) and wias not

destroyed by trypFsin. St. John et al., (1930) found no evidence of bac-

tericidal material in the digestive tract.

Kartman (1952) also considered that th~e sped of microfilarial passage

thr-ough the midgut to be of critical importance to lar\'al survival, and was

probably related to the pr~esence or absence of salivary anticoagulants.

Salivary/ anticoagulants have beenl d~emonstrated in Ani. m:iaultipei:s (Yorke

and I-acfie, 1924i), An2. qluadrimaCulatusln and Aln. pun~chipennis (Mletcalf, 1945),

AnzophcZes subpicius Grassi (Cornwell and Patton, 1945). They are absent in

Ai. aegypriti (Mletcalf, 1945), Aiedec den!tL'itls Haliday (Shute, 1935), An,.

steph~ensi (Shute, 1948), C. quin~quefa~isciatus!: and C'. pip,~ien (Shu~te, 1936).

Salivary anticoagulins appear to prevent the premature clotting of the

blood me~al (Lloyd, 1928).

Salivary aggiutinins are absent in A. ccepy bi, A?. dense ~'- jo: (S~hute,

1935), As stephens,-I: (Shutle, 1935), and C. p:,ipiere (Shute, 1936). Sal-ivary

aggjlutinins have been demronlstrated in~ ::. r::zu iZir,:niic (Shlute, 1948), Ajn.

quadvimou7 abnL 1s (Metcalf, 194;5), and Anii. subpi:-1- as (Cornw~ell an i Pa tton,

1914). It should be noted that salivaryj aggllutininr -fromr An. quadrimacu-

Latu:: are effective against red cells o amlcw idg abt








and mouse, but not chicken or turtle (Metcalf, 1045).

The addition of an anticongulanlt to the infective blood mieal produces

a rate of microfilarial migration in I. na:7Y~ti, a poor vector, which is

quite comparable to that in An,. quadrZjima~nu7latus, an efficient hos~t (Kartman,

1953). Zaini et al. (1961) reported 911 of B. molayi microfilariae ingested

by Aedes F'inl~a::a) t~oloi (Theobald), which lacks an antiicoagulin, were trapped
in the stomach blood clot after 24 hour~s.

The speed of blood digestion by the miosquito may affect mnicrofilaria

survival and is often quite variable between species. Variations in digestive

rates are related to such factors as photoperiod (0'Gower, 1956), temperature

(Sella, 1920), season (Guelmino, 1951), and the amount of blood ingested

(Deeoissezon, 1930). De Buck et al. (1933) suggested that refractoriness of

Anr. macu-Zipennis to Plasmodiumi was correlated w~ith slow digestion of the

blood meal in certain varieties during overwintering.

Thayer et al. (1971) have advanced the interesting hypothesis that host

speci ficity of the insect and its abi lity to transmi t certain parasites may
be related more to components of the blood meal itself rather than differ-

ences in digestive power. In a comparison of the free amino acid levels

following a human blood meal to those after an avian blood meal revealed

significant differences in the concentrations of aspartic acid, isoleucine,
and carinosine.

One of the most consistently demonstrated phenomena regarding thre

resistance of mosquitoes to invadiny microfilaria is that of encapsula-tion,

a process by which the parasite becomes imnpregnated w~ith a brown deposit
which eventually forms a capsule. Encapsurlation or chitinization, is

currently viewed as a defense mechanism (Salt, 1957), which may occur

around living or dead microfilaria (Kartman, 1950). Yen (1938) reported









encapsulation of all larval stages of D. immitle in the stomach, Malpighiani

tubules, and the body cavity of mosquitoes. Schacher (1962) reported the

recovery of encapsulated larval i. pahan-.iS from Anr. qarjn!imaonuZabtus and

P. confi~nis.

Salt (1955) reported two defense reactions of M!icrolepidopteraer against

experimentally injected parasitaid eggs, which consist of encapsulation and

the deposition of melanin. The deposition of me~lanin, a well-known reac-tion

of the tyrosinase complex of enzymes on tyrosine, or an intermediate chromo-

gen in the presence of oxygenr, acts as defense reaction only fortuitously,

when the deposit is so cited as to prevent a vital activity, such as the

hatching or the feeding of the parasite (Salt, 1957). A similar reaction

was noted in parasitoid-infected Carausinc in which Salt (1956) described

the melanin reaction to be as strong in confined spaces as in the main blood

stream and progressing to encase the parasite in a brittle black sheath.

Salt (1960) described a rapid reaction of the haemocytes of the tomato moth

caterpillar against infected parasitoid eggs or larvae, which led to encap-

sulation within 4 hours and the subsequent death by asphyxiation of encapsu-

lated parasites. The haemnocyte reaction was similarly invoked by other

species of' parasites, by dead parasites, by glass rods, pieces of nyloni

thread, by injured pieces of the caterpillar itself, and by organs trans-

planted from other species of caterpillar. Salt (1960) concluded that sur-

face properties of the parasite or implant determine whether or not it will

be encapsulated. Goodwin (1958) previously noted that the action of diethyl-

carbamazine on microfilariae and diaminodiphenoalkane derivatives on schisto-

somies was "to change the surface of the parasite in w:uch a way that it becomes

recognizable to the host as an intruder."








Bronskill (1962) noted an iden-tical reaction in the larvae of A1.

an:!Urpti, ieAes schautb ans (Walke-r), and AndecZ twic~hu!ri. (Dyar), wh!ien chab-

dita-id DD136 juveniles (an undescribed species of the Nieoaplactanida? with

its associated bacterium) penetrated the blood sinus and entered thle body

cavity. By 5 hours, a thick capsule developed about many of the ensheathed

imm~ature forms as a result o-f a rapid defense reaction of the mosquito which

had both a melanin and a cellular manifestation. He further noted that this

type of host resistance resembles that of adult mosquitoes to filarial nema-

todes. Poinar and Leutenegger (1971) have recently shown that this reaction

in mosquitoes is a humoral type of melanization and unlike typical melani-

zation reactions in insects, does not depend on the direct presence of

hemocytes as a precursor stage of melanization. They describe a deposit

which slowly became pigmented, originated from components in the noncellular

portion of the hemolymph that coagulated out on the surface of 4 neoplectanid

nematodes in C. pipiens.

Whether the "gut barrier to infection" is due to the enzymes of the

digestive processes or to mechanical factors of movement through the ali-

mentary tract remains unknown. Chamberlain and Sudie (1961) have advanced

several hypotheses including virus inactivation by digestive Fluiids and the

impermeabili ty of the peritrophic membrane, but emphasize that no mechanism

has been completely proven. Lavoipierre (1958) reviewerd the relationships

between filarial nematodes and their arthropod vectors including a brief

discussion of the possible role of digestive physiology of the vector in

limiting the intensity o-f infection.

The endocrine physiology of thle arthropod vector has also been impli-

cated in the mediation of filarial competence. The insect endocrine system

is remarkable in its complexity and its ability to integrate development and









physiological events wi~th the environment. The pdfaSitiC nematOde mUSt re-

spond appropriately to this changing mnilieu of hormones because they reflect

the external environment in which the host finds itself. Davey and Hominick

(1973) observed that where the nematode-arthropod association is obligatory,

thle long evolutionary history of host and parasite mnay have resulted in a

dependence of the nematodes on the? endocrines of the host. They suggest

that such a dependence might take the form of host endocrines providing cues

to the nematode to sense the hiormone molecule itself; an indirect cue would

have the nematode respond to a hormone induced change in the physiology of

the host.

Numerous reviewsof insect endo~crinology have appeared recently in the

literature (Highnam and Hill, 1969; W~iggleswvorth, 1964, 1970). At least

6 enldocrine tissues have been described in insects. Neurosecretory cells

of the pars inte~rcerebralis of the brain have axons which terminate in the

corpus cardiacuim, a storage and release organ closelyv associated with the

heart. These cells secrete "activation hormone" which activate the thoracic

(prothoracic) gland (Wigglesworth, 1970), and also a factor that is involved

in the control of egg formation (Davey, 1965), protein synthesis, and the

mocbilization of lipids (Wigglesworth, 1970). The hormonal cycle in blood-

suckers is initia-ted by distention of the m~idgut wiall following engorgement

(Certram and Bird, 1961), wJhich stimuilatess the neurosecretory cells in the

braini to release a hormone into the~ hem;ioccel (Lea, 1967). Thiis in tulrn

stimulates the corpuis cardiacum to secrete the gonadotropic hormone; oocysts

begin micropinocytossi (Roth and3 Porter, 1964; Anderson andl Spielman, 1971),

vitellogenin is synthes-ized (Hage;lorn and Judson, 1972), and eggs subsequently

mature. The corpus cardiacum also contains its own intrins-ic cells which

produce and release a variety of other hormonal factors chief among them a








peptide active inr the con-trol of he!art andl other muscles (Davey, 1964).

The paired corpora allata produce juvenile hormone (Wigglesworth,

1954, 1964, 1970) which is active in development, and is essential to the

full expressioni of egg produc-tion. Juivenile hormone dietermines the dir-ection

of the development initiated by ocdysone; whether toward another larval form,

a pupal form, or the adult form (Wigglesworth, 1954, 1964, 1970). It also

governs the titre in the hemnolymph of the yolk protein by regulating its

synthesis in the fat body (Engelmnann, 1969). Juvenile hormone has been iso-

lated from a wide variety of plant and animal sources (Schneiderman and Gil-

bert, 1958, Wlilliams, M~oorhead and Pulis, 1959). Schneiderman, Gilbert and

Weins-tein (1960) reported that cell-free broth of Eacherichia~ coZi and whiole

broth from Proteus species had a very high juvenile hormone activity in the

polyphymus test.

The principle remaining endocrine organ, is that thoracic gland which

through the production of ecdysone or moulting hormone, initiates those

developmental events culminating in the production of ecdysone or moulting

hormone. It also initiates those events culminating in the production of a

new cuticle and the shedding of the old. Although insect gonadal tissue

has been considered as nonendocrine, in somne insects at least, the testis

(Naisse, 1966), and the ovary (Pratt and Davey, 1972) have been shown to

produce hormones. Even the hinrdgut has been implicated as a source of hor-

mones in at least 1 species (Beck and Alexander, 1964).

The role of hormo~nes in m~osquitoes is of special interest since

Bhattacharya and Chowduiry (1964) reported that certaini vertebrate steroids

appeared to increase the susceptibility of A. aegypti7: to infection with

w. banicrofti. Spielman and others (1971) reported that 1 steroid hormnone

ecdysteronie, stimulates ovarian development in adult mosquitoes, an effect









not observed after the administration of various vertebrate steroids. Howi-

ever, administration of massive quantities of ecdysterone did not influence

the development of B. Pahan)1i. Gwai~rdz and Spielmnan (1974) reported that

application of synthetic ecdysonea~nd juvenile hormone to AI. aegyabti did not

affect the development of Bi. pahiang-i.

Certain oxyurid parasites of roaches appear to require hormones from

the hosts' corpora allata (Nadakal and Nayer, 1968), and median neurosecre-

tory cells (Gordon, 1968, 1970) for developmennt. Even nonentomophilic nemia-

todes may be influenced by the presence of such exogenous insect hormones

as juvenile hormone and ecdysterone (Hitcho and Thorson, 1971; Johnson and

Viglierch~io, 1970; M~eerovitch, 1965; Shanta and Me~erovitch, 1970; Wiebster

and Craig, 1969; Davey, 1971).

Davey and Hominick (1973) have concluded that however attractive the

notion that development of a parasitic nematode is under the control of the

host endocrine system may be for many authors, the cases in which this has

been demonstrated are very rare. Yoeli and others (1962) found that micro-

filariae of D. ilnv~itiS developed to the sausage stage in Al. qualtimac=ulatus

decapitated immediately after the infective blood meal. Since the vitello-

gen-ic hormone is secreted and stored in the head of the mosquiito, this find-

ing established that the inritial stages of larval differentiation are not

con-trolled by the same hormone systemn that regulates host oogenesis. Gw~ardz

and Sp~ielman (1974) showedc thati the allatu:n secretion does; not influence

the development of larval B. pshangi. Such larvae3 nature in allactomized

juvenile-hormone "free" females of both suisceptible and refractory mosquito

species. These authors also concluded that larval development of B. paFhangir

wa~s independent of the insect hosts' enldocrine milieu.








Dual Etiology of Parasitic Diseases


The relationship of normal intestinal microbiota to the vectoring

capacity of insects is an idea that has not yet been adequately investigated.

Insects are not particularly unique among animals in their relationships in-

volving bacteria and fungi. Parasites passing through the host's alimentary

tract are exposed to both the microorganisms that reside there and to the

intestinal climate which may, in part, be influenced by them. Indigenous

microorganisms present at the host-parasite interface must be regarded with

as much consequence as the host itself.

Gnotobiotic studies have shown that the pathology of many parasitic

diseases depend not only upon the etiological agent, but also upon the host's

intestinal microflora. Phillips and Gorstein (1966) have repor-ted that the

association of ameba Fntamoeba histotytica Schaudinn with living bacteria

such as Escherichia coZi (Migula), Aerobacter aerogenes (Kruse), and

Clostridiumn perfringens (Vellon and Zuber) was necessary to produce typical

hepatic abscesses in hamsters. Wittner and Rosenbaum (1970) confirmed this

study and observed that the participation of bacteria with ameba must be

more than just maintenance of a suitable intestinal environment, but might

involve a resistance transferring factor described by Anderson (1965) passed

from bacteria to ameba. Phillips (1964) considered that the bacterial flora

acted by "providing a suitable environment, physical and chemical, for excys-

tation and establishment of lumen infection until such time as the ameba can

enter the tissue."

Infectious enterophepatitis in turkeys has been shown to reflect a dual

etiology requiring Histonomas moeoegridlis (Smith) and a single species of

bacteria (E. co~i, C. per~fr~ingeno, OF Bacilluts subtilis Cohn) (Bradley et al.,

1964, Bradley and Reid, 1966). Franker (1965) produced a moderate rate of








infection with this protozoan and a species of infection w~ith this protozoani

and a species of Eimar~in in comb~inationn with 3,~ZwIn C~u a ciru Frankland and

FrankZland or I~actoba~cithe fe medir;~zi. Deijerinick. Springer et al. (1970)

noted that the bacterial requirements for producing this disease in bacteria-

free chickens were different from those for the disease in bacteria-free

turkeys. The essential contributing factor of bacteria in the pathogenesis

of infectious enterohepatitis was determined to be neither a favorable pH,

nor an oxidation-reduction potential value within the intestine of the host,

but rather th make the cecal environment more suitable for survival of the

transport host, liateratkis gaZZinavuzm (Schrank).

Wlescott (1971) noted in gnotobiotic meice infected with H~ematospir2oide~s

c-1ubiuso Baylis and Nirppostrongyi~ue brasilctie is ravassos that mo~re parasite

developed, infections were of greater duration, and of a higher reproductive

potential in the conventional rather than in germ-free hosts.

Feline infectious enteritis was shown by Johnson and others (1967) and

Rohovsky and Griesemer (1967) to be almost asymptomatic in germ-free cats,

but severely symptomatic in speci~fic-pathogen-free (SPF) cats. Visco and

Burns (1972) reported a parallel situation in gnotobiotic chickens infected

writh Eimenia tenclza (Railliet and Lucet). It w~as previously demonstrated

by Radhakrishnan (1971) that E. tenetta could produce typical cecal pathology

in qnotobiotic chicks only wihen associated with certain bacteria or combi-

nations of bacteria. Johnson (1971) noted severely depressed growth and

development of Alccurid2ia gazZEi (Schrank) in germ-free chicks and Bosmak

(197'1) reported that Hy't"na~no!;lpic: nanae (von Sifbold) in mice appears depen-

dent upon host bac~teria, and that the variety of bacteria present changes

withl increasing intensities of tapeworm burden. Conversely, the absence

of bacterial associations may be a factor in disease transmission. For









successful transmission of leishmania, the PhZeboornu.; fly must have a

sterile alimentar-y tract because human leishmanias do not tolerate bac--

terial contamination. Infection of A. stophensi mosquitoes with a micro-

sporidian parasite Nosemae al~gorae Vavra and Undeen, 1970, reduces their

susceptibility to infection with the simian malarial parasite, Plasmodiumn

cynomolgi Mayer as measured by mialarial oocyst counts 6 days after the

infective meal (Ward and Savage, 1972). Mortality from nosematosis was so

great that 90-95% of the exposed mosquitoes died by sporozoite levels

could be assessed. Fox and Weiser (1959) pointed out that there is

apparently no evidence that Njosema is physiologically antagonisitc to plas-

modium, or that the former attacks the latter. These investigations con-

cluded that the miidqut wall of heavily infected mosquitoes was so dis-

integrated that suitable sites were not available for oocyst development.

From these and similar studies, it is apparent that the intimacy of host

bacterial flora with parasites appears to be more significant than cir-

cumstantial.


Microflora Associated with Mosquitoes


Mitchell's (1907) report that larval A. aegypti were predominantly

bacteria feeders was based on the observation that larvae develop more

rapidly in water contaminated by sewage. The same year Putter (1907) re-

ported that organic colloids and solutes serve as the primary food source for

aquatic invertebrates. Hinmlan (1930, 1932) andi Trager (1936) considered

that var-ious substances in solution ser-ved as the sole food sour-ce, buit

Shipitzinia (1930), and Rozebloom (1935) and Buddington (1941) showed

that pond water passed through a bacterial filter- could not suppor-t growth

beyond the further instar. Trager (1935), however, reported that larva could








utilize liver extract and whole killed yeast in solution.

Lamborn (1921) claimed that some mosquito larvae feed on specific

living organisms suchl as Si~irro:7r~a sp., Eugnl~an sp. and JVolvox~ sp., but

M~etz (1919) showed that it made little difference whether a variety of animal

or vegetable matter fed to anopheline larvae was living or dead. Hi nman

(1930) reported that bacteria, algae, and protozoa although ingested by

mosquito larvae, were not digested and hence, contributed little to the

nutrition of larvae.

Laird (1956) concluded that algae and protozoa are the primary sources

of food for mosquitoes breeding in permanent ponds and lakes in mosquito

breeding areas of the South Pacific. Coggeshall (1926), Hamlyn-Harris (1928),

Senior-White (1926), and Howland (1930) have all reported that algae is the

primary food source of at least some mosquito species.

Atkin and Bacot (1917) raised larvae of Al. aegypti in pure cultures of-

Sacchar~omyces cerviiciae? Meyen and Ba-iZllus coZi courauniior (Dur-hamn) and Bacot

(1917) noted that mosquito larvae kept in water originally turbid with bac-

teria, was rapidly cleared. The intestinal contents of such larvae were

relatively free of bacteria, which Bacot (1917) considered a result of com-

plete digestion. GldSser (1924) considered that liver and yeast supplied

certain accessory growth factors to the larvae of the several species of

flies he maintained in culture. Bragina (1926) nourished newly hatched A.

menuipenison bacteria (unidentifieId) and Barber (1927) found that algae

alone, bacteria alone, or infusoria alone consti tutes a suffricient food

source for Ainopheles? cru ;ianci andi A. criuadei:;:culatus.n Thlese infusorinsi~

wer-e chosen as r-epresent;ative of the or~ganisms commonly found in the plankton

of Anloph~els-producing water and included: Cotpidiumn sp. isolated from

cultures of rotting algze, a motile, unlicellular, grass-green algae, possibly









ChiZ~mndomnonacs sp. or a related form:, anld a lar-ge and a small variety of~

SpirlZElum sp. Barber (1928) reported similar results using these organisms

as food for culicine larvae.

Rozeboomn (1935) reported that bacteria, to a certain extent, can be

utilized as food by A. aegypti, C7. pipien~s, Ciculex te2riritnr s Walker and

Cu~e- salinaruss Coquillet larvae. Environmental bacteria, associated writh

the different natural breeding places of the mosquitoes, promoted optimum

development when bacteria were the only source of food. Rozeboom (1935)

found that E. coli, Bacilluis subtilis Cohn, BaciZllus myrcoides Fluigge, A.

aetrog~enes, and Pseud~omonays aeiruginzosa Migula were of equal value, but that

Sarciina Zubea Schroeter was inferior. Pseudomnonas fluoremen3 s M~igula had

a toxic effect on young larvae. Bridges (1965) reported that B. subtiis,

Bi. mega~therium, and Staphylococcus aureuls Rosenbach were unsatisfactory as

a sole source of food for C'. qu~77inquef~j~atlscau larvae. The yeast S. ce?e-

visiae provided the best food source. E. colL and A. aerogenes w~ere about

equally indifferent in food value. P. aeruginosa and S. Lusta were utilized

as food only to a limited extent. Buddington (1941) reported that E. co~i,

B. su~btilis, and BaCiZtuS meantheriumr DeBary in B~erk~efeld-W filtered pond

water supplied sufficient nutrients for the complete development of Ai. ac,~-

Beattie and Howland (1929) considered that bacteria play some part in the

nutrition of orthopodomyia sp. and Rudolfs and Lackey (1920) reported that

microorganisms fr-om decomposed vegetable matter w~ere responsible for miosqua;

breeding. Laird (1956) concluded that bacteria were the primary food of

rain pool mosquitoes because bacteria were thle most abundant organisms !1row1

ing in these transient ponds.

Yeasts have been shown to contain most of the nutrients needed by

mosquitoes for complete growth (Atkin and Bacot, 1918; Trager, 1935; Fred









et al., 1936; Subbarow and Trager, 1940; Buddington, 1941, Goldberg and

de Meillon, 1948; Akov, 1962). Alcohol sterilized, but not heat sterilized

yeast will support complete development of mosquito larvae (Buddington, 1941).

An artificial media supplemented with autoclaved yeast has been used to rear

normal adult mosquitoes (Lea et al., 1956; Akov, 1962).

Infusions of organic material have been used to successfully produce

adult mosquitoes of many species (Atkin and Bacot, 1917; Barber, 1927;

Hertig, 1936) in the ovaries and testis of C. pipiens. Brumpt (1938) described

Ricketecia curlicis Brumpt which had been fed 12 days previously on a subject

with filariasis. Sellards and Sites (1928) frequently noted masses of ricket-

tsiae in the lumen and epithelial calls of the hind gut of Ai. aegypti.

These rickettsiae were present in specimens known to be infected with dengue

fever virus, whbereas they wvere not observed in control mosquitoes. More

recently, Venters et al., (1971) reported the presence of rickettsia-like

organisms in the midgut cells of 25% of A~n. stephe~s~i examined. Since they

observed such organisms only in stomachs free of malarial cocysts, they

considered mosquito susceptibility to malarial infection can be influenced

by the presence or absence of this organism. Yen and Barr (1971a) considered

that the cause of cytoplasmic incompatibility in C. pipiens was due to

rioZbachia-like rickettsia which are strain specific and localized in the

follicles of the eggs. These microorganisms, especially abundant near the

mnicropyle of recently laid eggs, lie in close proximity to sperm cells entering

the micropyle in their passage to the femlalee nucleus (Yen an~d Barr, 1971b).

These workers suggest a deleterious effect by these microoi-ganisms on pre-

sumably incompatiblel" sperms so that they are unable to participate with

the oocyte nucleus in -the -formation of a zygote. Rickettsia-l-ike organisms

are not novel to blood-sucking arthropods, however, and new x~amlpless continue









to be reported in such insects as Rhipicephp~alus bursa Canestrini and Fan-

Zdngo (Friedhoff, 1970).

The bacteria associated writh mosquitoes have only begun to be docu-

mented. Star and Micks (1957) (quoted by Ferguson and M~icks, 1961) isolated

22 different bacteria cultures from 3 to 5 intact adult females of c. fa~tigans~

and 35 different strains from a homogenate of 49 specimens. Eighty percent

of these were Gram negative rods.

Hinman (1932a)found bacteria as w~ell as yeast within the eggs of

A. aegypti and Kellen and W~ills (1962) reported evidence of transovarial

transmission of Thetlobanis sp. in various species of California mosquitoes.

Chao et al. (1963), however, reported their failure to isolate microorganisms

from within the eggs of 2 species of Cu!Ze, 2 of Anopheles and 2 of Aedes.

Mlany workers have since reported the successful axenic rearing of adult

mosquitoes from surface-sterilized eggs (Dougherty, 1959; Akov, 1964; Nlayer,

1966; W~allis and Lite, 1970, Rosales-Ronquillo et al., 1972, 1973).

Chao and Wistreich (1959) isolated bacteria from the midguts of 46

adult CuZex barsatis Coquillett selected from a colony fed on apple slices

for at least 2 days after emergence. Results of these isolations showed the

presence of Allcatigen~es gutactus (Zimmerman). Achromobarcter ap., and Aero~-

bacter clocae Jordon, Eschzerichia Intermedia W'orkman and Gillen, F'lavn-

bacteriumn sp., M~icrococcus sp., MsocrococcuN vivi~ano Nligula, Pr~oteus sp.,

and Pseud~omonaa sp. Cultures of unidentified Gram-negative rods as well as

Sacchnromlices sp. and Geotirichum sp. were also reported. Chao and Wistreich

(1959) also noted a higher ratio of midlut sterility in males than in females,

and reported that males, after a forced blood meal could not digest the blood

and died shortly after feeding. Wistreich and Chao (1960) isolated bacteria

from midgut sections of 14 fourth-instar larvae of c. tar~salis that had been








raised in fresh tapwater and fed brewers yeast. Results of these isolations

showed the presence of Ach,?;~roobact:er sp., L~a-tobaci~Z~ue sp., Mi:cro-coccu

candidus Cohtn, Micrci'7OeCuS sp., Pr~oteus: rattge~i Hadley et l., and

P7seudomonas sp. Again, cultures of unidentified Gram negative rods and

Sacchar~omyces sp. and Geotrichycrm sp. were observed. Isolations from the

larval rearing water showed the presence of A. eloacae, Klebcietta sp.,

Sacchacromy:~cetee sp. and Geotrichim sp. Fourth instar larvae were found

to contain examples of all the genera of bacteria isolated from adult C.

tarsatiis w~ith the exception of Aerobraectr sp., Escherichia sp., and Flanvo-

bacte~riu~m sp., whereas isolations from adults did not show the presence of

Lactobaciltlus sp. or the coral pigmoented form of Micr~ococcus sp.

Bacterial isolates from the midguts of adult C. quinquefasciatus fed

apple slices for 2 days after emergence showed the presence of lch~romobacter

sp., Flavobacteriium sp., KlebuieZla sp., Mi~croocus ocaseoi~ytious Evans, and

Pseudomonas sp. (Chao and Wistreich, 1960). Midgut sections from 11

fourth instar larvae of the same species produced only BacitZus sp. and

the yeast Saccharonyces sp. No apparent difference in the microbial flora

from male and female adults were noted, with the exception of Miicrococcus

sp. and Saccharomyices sp. which were isolated from female mosquitoes only.

Wistreich and Chao (1961) extended their research to microbial iso-

lation from larvae and adult Aetdeos sierrensis Ludlow and Anzopheles albimialus

W~eidemramfed on a larval diet of yeast and adult diet of apple slices. Lar-

val isolation fr-om A. a~hbimanue Weidemarnfed on a larval diet of yeast and

adult diet of apple slices. Larval isolation from A. athtman~us showed the

presence of Alcaligecnas sp., Racsittue and unidentified Gram-negative rods.

Adult A. aZbim~anusi showed Ach:~i~r,~romebactrsq>., Ahealigena s sp., and corynle-

bacterium sp. Saccaharamyces sp. w~as isolated from both larvae and adults.









A. siervenclsis larvae contained Bu~illus circuzlanoi: Jordan, Paracolobact!rum?

-in-tennedi~um Barman and Prettger. Alchelomobac~ter sp. and E'. inte~~irmdia were

found in adults. Sach:aromyjces sp. were isolated From the larvae. W~istreich

and Chao (1961) also reported that microorganisms isolated from the adult

and larval environment were distinctly different from isolates of the mid-

guts.

Wistreich and Chao (1963) reported microbial isolations from larvae

showed the presence ofl the following bacteria: AZealigenes sp., Coryne-

barcterium sp., Escherichia fraunditii (Braak<), Ku~irthia beasoni Hauduroy et al.,

Sarcin~a flava de Bary, as well as fungi belonging to the general Geotrichzum,

Penzic~iumi, anld Saiccharomylces. Isolations fromn adult mosqjuitoes showed

the presence of A~chromobacter sp., Alcaligenes sp., and Saicca~romyces sp.

Furthermore, they reported an especially high rate of midgut sterility in

the adults of this species. Microorganisms were isolated from 5 to 21 fe-

males and 4 of 9 males. Alealigenes sp. wiere found only in adult females.

Ferguson and Micks (1961a) instituted a similar series of experiments

using adult female C. fatigans from a colony that had been denied access to

food. By means of a sterile riicro-dissection technique, they demonstrated

the presence of LactobacEiZtus sp., AlZealigneszc sp., PoeudZ~omonas sp., and 2

unidentified cultures of Gram-negative rods. Bacterial isolates from thle

midgut o-f similarly tr-eated adult female Ani. quandr;;maculatuszi proved to be

2 strains of Strenoto-ooccls sp. and a species of Ac~~orobcter (M'icks and Fergu-

son, 1963). Ai. accypt~i': produced only a species of CorynobaSrcctr~iwnj and no

microorganisms could be cultured from the mnidguts of adult female CutZe:c

m:olestus~ F-orskal.

The midguts of newly emnergjed adults of i'. frtlatigan, C. m~les~tus, An?.

quadrj,,iimacultus. and A. aegypliti which w~ere not allowed access to either








blood or sugar were examined by electron microscopy (Micks et al., 1961a).

Ultrathin sections of C. fa~tigans midgut revealed the presence of intra-

cellular microorganisms which M~icks et al. (1961a) described as Gramn-

negative rickettsiae. Since these organisms did not appear to produce

pathologic changes within the cells and were occasionally found in mycetome-

like bodies. Micks et al. (1961a) suggested they might be symbiotes.

The question of a symbiotic relationship between mosquitoes and

microorganisms has yet to be satisfactorily answered. Brooks (1965)

generalized that arthropods possess symbiotic microorganisms only if they

feed on nutritionally inadequate (i.e., incomplete) diets during their en-

tire life cycle. Blood or serum is considered inadequate since it is

deficient in B vitamins. Furthermore, since mosquitoes undergo complete

metamorphosis from a larval stage spent as an omnivorous or scavenger-

feeder, they are considered to pick up an adequate amount o-f vitamins and

intestinal microflora (Brooks, 1964) in that stage to suffice for their

entire life. Blood-feeders with incomplete metamorphosis which feed on

blood during all immature stages, do have symbiotic microorganisms present,

and as a rule, their alimentary tracts are otherwise sterile. Such arthro-

pods are represented by bed bugs, sucking and chewing lice, and ticks and

mites. Removal of symbiotes by axenic rearing procedures causes severe

growth impairment, body malformations, lack of reproduc-tion and death

(Baines, 1956). Wdigglesworth (1929) considered that symbionts provide some

accessory food sutstances to tsetse flies that are similar to the vitamins

necessary for mammalian growth. B vitamin supplementation of the blood

med1 fed to axenic blood-feeders does, in fact, alleviate the growth factor

deficiency but fails to restore normal reproduction (Pucha, 1955). Aschner

(1934) found that sumbionts are a source of food for the body louse Pediculus









hom~inus hominu:- Linnlaeus. Young lice died within 5 or 6 days when deprived

of the mycetomes that contain symbionts. Actinemy~cr e I'hodnri~ Erickson in

the gut of Rhodninea sp. are essential for the maturation of the insect

(Brechner and Wigglesworth, 1944).

In order to obtain further evidence of a possible symbiotic relation-

ship between C. fabtigans and its associated microorganisms, M~icks and Fer-

guson (1961b) placed a number of females on a chloroamphen-ical -dihydrostrep-

tomy~cin-sugar solution diet -for periods of 5 to 12 days. Mosquitoes relieved

of their microorganisms by such means were subsequently allowed to take a

blood meal from canaries infected writh Plasmodium~ relictum Grassi and

Feletti to ascertain possible effects on host immunity. Antibiotic treated

females became inac-tive, lacked coordination, were unable to fly, and when

dissected, their midguts were found to contain large masses of undigested

blood as long as 10 days after the blood mieal. The normal rate of digestion

of human blood, although variable with -the species and day--length, is re-

ported to be 31 to 48 hours (0'Gower, 1956). Eighty-five percent of the

treated mosquities and 74% of the untreated controls were infected with P.

2elictuw. In general, antibiotic treated individuals contained about twice

the number of oocysts per midgut as did controls.

M~icks and Ferguson (1961b) concluded that microorganisms play a

significant role in the susceptibility or resistance of the mcosquito to the

mnalarial parasite. They mention the possibility of competition between the

miicroorganisms anid the malarial parasites for essential nutrients, in w~hichi

case, the antibiotic would work in favor of thle P-lasmod~iumi. Alternatively,

they suggested that certain strains of these m~icroorganisms could supply

factors needed by the host for metabolic processes and defense mechanisms,

and that killing thle organisms increases host susceptibility to malaria.









Unfortunately, their work appears to have been discontinued at this stage.

Furthermore, the feeding of antibiotic diets, besides killing mrost, but not

all, internal microorganisms, may have produced additional e-ffects on host

physiology not considered by these workers.

Shymala et al. (1960) reported that chloromycetin in the diet of the

silkworm influenced the digestion and utilization of protein, fat, and

minerals. Tetracycline, oxytetracycline, and chlorotetracycline have been

shown to combine specifically within the mitochondria of living monkey kidney

cells and with the cells of salmon?1c:~ byphoc-a Wlhite (Dubuy and Showracre,

1961). Although the mammalian cells remained alive for days, there is much

that remains unexplained about the effects on metabolism and nutritional

requirements of antibiotic-treated cells.

The roles of microbiotic symnbiotes within mosquito midguts are manifold.

Hinson (1933) found within the bacterial cell, a factor which stimulated

growth of mosquito larvae. Rozebloom (1935) considered that different

species of bacteria possessed varying nutritional properties for mosquitoes.

F. coli, B. subtii's, B. mi~eoides and P. flo2scens were equally satis-factory

as a sole source, while S. Zutea and Pseudomonas pyeaneta Migula (P. aerug~inosa

Schroeter) were detrimental. Arnal (1950) considered that specific symbiotes

were essential for the digestion of erythrocytes in mosquitoes and described

intracellular organisms in midguts of C. p~ipien which secreted enzymes

capable of hemolyzing erythrocytes. Micks and Ferguson (1961b) found un-

digested blood only in those mosquitoes in which the microbial flora had been

reduced or eliminated by the antibiotic diet. Ferguson and M~icks (1961b)

also noted that one of the bacterial str-ains isolated from the midgut of

C. fatigans produced hemolysis of human blood. The work of Chao and W~istreich

(1959) support this relationship between midgut microorganisms and the proper









digestion of blood. They found a higher ratio of midgut sterility in males

of C. tarcatio and also noted that males could not digest blood with which

they had been fed. Terzian et al. (1952) reproduced the "indigestion"

syndrome in A2. aegypti: by feeding calcium and calcium-antibiotic combinations

before and after engorging. Only diges-tion of the hemoglobin fraction of the

blood was inhibited. The effect of the antibiotic (they reported) was to

potentiate the inhibi-tory effect of the cations upon the particular enzyme

system involved. Terzian (1953) also reported that streptomycin and chloro-

mycetin increased susceptibility of Al. aeigypti to Plasmodium gallinacewr!

(Brumpt). The author apparently did not consider that this increase in sus-

captibility might be due to antibiotic mediated alteration in host microbial

flora.














Statement of the Problem


This dissertation is a series of 4 investigations on the relationship

between filarial vector and its resident microflora in the midgut. The

participation of these microflora in the transmission of parasitic disease

is of special interest. The a priori premise is that mosquitoes acquire

their life-long complement of midgut bacteria as indiscriminate filter-

feeding larvae. During growth, competition among microorganisms establishes

a microecosystem of bacterial types which are retained throughout life.

The arthropod midgut is thle only part of the alimentary tract which is not

shed during molting. This bacterial complement is individually distinct be-

cause of its fortuitious acquisition, yet species specific because of the

consistencies of egg-laying site selection among mosquito species.

The influence of this internal microhabitat upon potential parasites

passing through the alimeintary tract may be manifold. It may be of

sufficient importance ot determine the fate of the pathogen, and thus, the

efficiency of the vector. The recently developed techniques in gnotobiology

presented the opportunity of selectively separating the arthropod vector

from its indigenious microflora.

The first problem was to titrate levels of filaria infection in the

mosquito, determine their effect on the mosquito, and the ultimate fate of

the larvae. Of parallel interest wJas the phenomenon of early death in in-

fected mosquitoes. This investigation is reported in Results, I.

Secondly, a system had to be devised whereby a very efficient filaria









vector such as An?. quadirim~aculatusl could be raised, maintained, and infected

under gnatobiotic conditions. Previously reported techniques were not

appropriate to this species. Results, Section II describes this effort.

The problems of defining the "normal" bacteriologic flora of mosquitoes

was also unique. Nlo reports of bacteriological evaluations of wild-caught

mosquitoes have appeared in the literature. Furthermore, such reports as

have appeared, were not quantitative. Results III reports a quantitative

and qualitative evaluation of 5 groups of wild and colonial mosquitoes.

Finally, the effect of the normal flora on the establishment of

filarial larvae in a susceptible mosquito is investigated. Pure cultures

of bacteria isolated from wild mosquitoes are "added back" into gnatobiotic

mosquitoes as monocontaminants. These mosquitoes, as well as conventional

and bacte ia-free specimens were infected w~ith D. immitis larvae.

For-ty-eight hours later, all mosquitoes were terminated and the degree of

filarial development assessed. This is reported in Results,Section IV.














RESULTS

I. BIOASSAY OF EARLY VECTOR MORTALITY
FOLLOWIING DoIROFILARIA ~IMIIs LEIDY INFECTION


Introduction


The m~ost sal-ient problems attending experimental attempts to transmit

dirofilariasis through laboratory mosquitoes is the initial high vector

mortality. Various authors including Bradley (1953a), Kershaw, Lavoipierre

and Chalmers (1953), Webber and Hawkins (1953), and Weiner and Bradley (1970),

have reported mosquito mortality appr-oaching 100%: in the first days follow-

ing the blood meal. A common antecedent to death appears to be distension

of the abdomen persisting 2 to 3 days, which W~einer and Bradley (1970)

interpret as a result of improper digestion of blood.

Mortality subsequent to the ingestion of large numbers of infective

larvae in the blood meal has been shown to be a result of mechanical de-

struction in the insect tissue (Pistey, 1959; Villavaso and Steelmian, 1970).

Larval destruction of the Malpighiann tubules as they leave these organs

on the fifteenth day is an obvious cause of mortality. Esslinger (1962)

documented three pathological stages consequent to infection of Anop7heles

quadvrimaouiatus Say with Brug-ia pah~angi Buckley. These include perforation

of the peritrophic membrane, destruction of the gut epithelial cells and

puncture of the basement membrane, and finally, destruction of the fat

body. However, death due to mechanical destruction after infection of low

or moderate numbers of microfloriae seems inappropriate in such successful

filarial vectors as mosquitoes are known to be.








The incidence of mosquito mortality during the first 3 days after

infection with D. inimitic may be the result of (A) mechanical action

of the larvae, (b) a toxic factor elaborated by the larvae, or in response

to the larvae, or (c) disturbances in endocrine function occasioned by

the larvae. Even subtle physiologic insults produced by filaria larvae

miay be greatly magnified in the female mosquito as all the endocrine

machinery of egg-laying begins to function.

The following bioassay is an effort to determine which of these 3

possibilities, if any, is the cause of vector mortality in the first 3

days following infection with D. immoitis larvae.


Materials and Methods


Anl. quradrimaculatus (Gainesville strain) were reared and maintained

in an insectary at the Insects Affecting Mlan Research Laboratory, Agri-

cultural Research Service, U. S. Department of Agriculture, Gainesville.

Ambient conditions were maintained at 26oC, 80% RH and 12 he~ LD. Adult

females were fed 5 days on a 2.5% sugar solution after emerging; starved

for 2 days (Terzian et al.,1957) and fed on various preparations through

either a capillary tube or a heated gutta percha membrane. Such preparations

included live microfilariae, dead heat-killed microfilariae, dead homoge-

nized microfilariae, and either the homogenized midguts, heads, or re-

mainders of the bodies of previously infected female mosquitoes.

As R.oskpi LoEs completed -feeding, they w~ere transferred to holding

cages in groups of 20 where m~ortality observations were made twice daily.

Nioribund mosquitoes were dissected and examined for the presence and con-

dition of D. immith larvae. ilicrofilariae weire collected from filuremic

dogs using the K'lein and Bradley (1973) modification of the K~nott technique.









The mnicrofilariae w~ere separated from -the cellular components concentrated

by centri-f-ugation, adjusted to a 2,000 microfilariae per 0.5 ml concentration

with normal saline (Wong, 1964). The cellular components were then reintro-

duced to bring the volume up to 1 ml. Hiomogenized material wlas sonicated

in cold saline, brought up to 0.5 ml volume with saline, and up to 1 ml

volume with canine red cells obtained from an uninfected donor dog.


Results


The mortality of mosquitoes taking blood meals with differential

filaremias is shown in Table I-1. A concentration of 1,000 microfilariae

per ml of the blood meal permits optimum mosquito survival and results in

an average of 12 microfilariae per mosquito. Gross dissection of mosquitoes

succumbing from this level of infection revealed no notable tissue destruc-

tion in the 3 day critical period. The Malpighian tubules contained active

microfilariae in the cells at the distal end. The tubules were intact, and

entirely normal in appearance. Digestion of infective blood meals wras con-

sistently retarded in comparison to digestion in control mosquitoes.

Table I-2 shows that no significant mortality was produced b~y feed-

ing either dead intact microfilariae or sonicated microfilariae bodies to

newly emerged female mosquitoes. This finding eliminates consideration of

a toxic factor produced by, or within the bodies of microfilariae.

Table I-3 shows the effect of feeding the homogenized body parts

from mosquitoes fed 24 hours previously on infective blood meals. Neither

midgut preparations, head preparations, nor remaining body parts contain

factors which duplicate the high vector mortality experienced w~hen viable

microfilariae are fed.


















Table I-1 Effects of Differential Filaremia on
Mortal ity of AnopheZen quacdiimaculatus SAY


Concentration
La~rvadelm1




1,000

5,200

9,600

10,000

20,000

29,000

30,000

66,000


Number
Females Feeding


Cumul ati ve Mlortal ity
in 3 days


Percent
Mortal1i ty























Dead Intact Larva Number Females Cumulative Mortality in
(1,000 Larva~e:al) Feeding 3 Days (%)


Trial 1 24 0

2 23 2

3 20 0

4 20 0


Sonica~ted Larvae
(1,300 Larvay/mir)


Trial 1 30 1

2 30 2


Table I-2 Effects of Feeding Dead Intact, and
Homogenized Microfilaria to Female
Alnopheales qu~adrimaculatu~s SAY

















Table I-3 Effects. of Feeding Homogenized Body Parts
From Females Fed Infective Meals 24 Hours
Previously






Adjunct to Number Femal es Cumulative Nlortal ity in
Blood Meal Feeding 3 Days (%)



Homogenized Midgut 58 5.1


Homogenized Remainder
of Body 73 4.1


Homnogenized Heads 20 0









Discussion


Results of this study show that living microfilariae produce at

least 25% mortality in infected AIn. quadErimnr iaiulatu during the critical

first 3 days post infection. This mortality could be the result of either

occult destruction of mosquito tissue, or some factor produced by, or in re-

sponse to the microfilariae. Dead, intact larvae do not stimulate a comparable

mortality reinforcing the explanation of mechanical destruction. When sus-

pensions of dead homogenized microfilariae were fed, a comparable level of

mortality was not achieved, indicating that a lethal factor could still be

intrinsic t the mosquito. Feeding homogenized midguts of previously in-

fected mosquitoes failed to reproduce mortality suggesting the female endo-

crine system as a possible source of the factor.

Blood feeding in non-autogenous female mosqui toes initiates a neuro-

endocrine arc involving endocrines from the brain which stimulate the

ovaries to proceed with egg formation (Larsen, 1958). Neither head prepa-

rations, nor whole-body homogenates from previously fed mosquitoes contained

factors which reproduced the high mortality in newly emerged mosquitoes. An

endocrine related factor is not supported b~y these findings.

Developmental studies by Kartman (19535)and Pistey (1957) have shown

that prelarvae migrate to the Malpighian tubules very soon after ingestion,

and Pistey (1959) infers that the sudden invation of the tubules is selec-

tivity fatal. Pistey (1959) reports that the most critical period in

Div~ofitaria tenuis Chandler infected mosquitoes was the initial 3 days when

microfilariae enter the M4alpighian tubules. Intermill1 (1973), however,

describes the migration of D. irmnitis larvae to the M~alpighian tubules of

Aedec-s triseriatus Say and notes that it proceeds without any apparent








histological damage to the gut or tubules.

Inasmuch as no obvious destruction of the M~alpighian tubules wras

apparent in the present study by gross dissection, anid no toxic factors

could be demonstrated, the cause of vector mortality remlainss unexplained.

Viable, pre-larvae of D. iimmitis, however, are extremely active and subtle

alterations in Malpighian tubule function could be produced by their pres-

ence with little physical evidence. The destruction of tubule cell cyto-

plasm and cellular membranes by invading larvae has been known since the

earliest authors (Noe, 1901). Sub-tle destruction of the cellular components

of the tubules may impair nitrogenous excretion, or the retention or re-

absorption of dietary components necessary for life. Even a minute perfo-

ration of the tubule integrity could allow leakage of uric acid and other

nitrogenous constituents into the haemocoel with terminal results for the

mosquito.

It would seem appropriate to implicate "indigestion" as a factor in

the early death of filarial vectors. Lewis (1952) noted that the presence

of microfilaria of anchooera volou~lus Leuchlart interfered w~ith the formation

of the peritrophic membrane of Simuliumn damnosumn Theobald and Wianson (1950)

reported that blood contained in the stomach heavily infected Similiumn

which died a few days after an infective meal appeared to be undigested.

Similar observations on other filaria host combinations were reported by

Roubaud et al. (1936) and M~acKerras (1953). Lavoipierre (1958) concluded

that the most likely cause of vector death was extensive damage to the peri-

trophic membrane rendering digestion almost impossible. The peritrophic mem-

brane was impossible to demonstrate in dying, heavily infected flies with un-

digested blood meals, whereas thle membrane w~as present in controls in which

the blood meal was in an advanced state of digestion.












RESULTS

II. AN INTEGRATED SYSTEM FOR THE PRODUCTION
OF GNOTOBIO0TIC Azoph2eles quadriii~macuulatus SAY


Introduction


A host of investigators beginning with Schottelius in 1899 have

demonstrated the fallacy of Pasteur's assumption that the host-microflora

relationship is obligate. Since then, the germ-free animal has given the

biologist one of his most valuable models to study the interactions of host

and microorganisms.

The development ofgniotabioticar~thropods was initia-ted by Trager

(1935a, b, 1936, 1937) who reported the growth of larval Aedes aegypti (L)

in sterile media containing essential nutritional compounds. In the 4

decades subsequent to that, the dietary requirements of A. aegy/pti in

axenic culture have been thoroughly studied by a number of workers (Goldberg

and DeMeillon, 1948a, b; Lea et al., 1956; Lea and D~eLong, 1958; Singh and

Brown, 1957; Akov, 1962; Lang et al., 1972). However, only a few species

other than A. aegyPti have been successfully cultured in sterile media.

They include: Cu.Eiseta -Incidens (Thom) (Frost et al., 1936), cutben pipienzs

pipicen (L) (Buddington, 1941; Dadd et al., 1973), Cutlex molectus Forskal

(Leichenstein, 1948), Aedeaz taeniorhky~chucs (W~iedeman) (Niayar, 1966), Cu1Zexn

saZin~arius Coquillett (Wallis and Lite, 1970), and Anioph~lses ctephzsna

Liston (Rosales-Ronquillo et al., 1973).

The objective of most of these studies has been to define the nutri-

tional requirements of mosquitoes, rather than to investigate the microflora-

host relationship. Of equal interest is the role of bacteria in the biology








of the host and in the pathogenesis of certain disease mechanisms.

The public health significance of the mosquito makes it one of the

most appropriate arthropods to receive such attention. An~ophecles quad2i-

malcuclatusi Say is a mlajor vector of canine heartw~orm disease in thle eastern

United States. Thils paper reports the first successful rearing of An.

quadr~imacutlatus under bacteria-free conditions and the successful use of

a lucite capsule specifically designed for gnotobiotic studies w~ith blood-

feeding arthropods.


Materials and Methods




Ain. qu~adrimalcu!?atu~s eggs were collected from colonies maintained b~y

standard procedures (Anon., 1973) at the USDA Agricultural Research Service,

Insects Affecting M~an Laboratory in Gainesville, Florida. Egg sterilization

wras accomplished in 3 cm long cylinders made from the tr~uncated upper half

of a disposable plastic syringe barrel. Organdy netting was cemented over

the lower end to form a deep non-w~ettable dipping cylinder for egg handling.

These cylinders fit neatly through the neck of 25 mm x 10 mm screw cap tubes

and w~ere autoclaved as a unit before use. With several hundred eggs in the

cylinder, the cylinder was dipped in the first tube containing 70% ethyl

alcohol for 15 seconds to facilitate wetting the egg surface (Hinton, 1968).

Thle cylinder was then transferred to a tube containing 105 Zephiran chloride

for 15 minutes w~ith occasional agitation. [Wdhite's solution (Whnite, 1931) for

15 minutes is also an effective germicide.] Eggs usually sank to the bottom

of the cylinder during immersion in thle germicide. Thle cylinder was next

transferred through 2 tubes of sterile wiater for 5 minutes each. An aliquot

Wluinthrop Laboratories, Nlew York, N.~Y.








broth. The culture flasks were incubated with a 12-hour light cycle in

an incubator. Pupation begin at the 8th day, and female pupa, recognized

by their larger head, were w~ithdrawin with a syringe fitted to a long 14 g

hypodermic needle.


The gnotobiotic arthropod module

The gnotobiotic arthropod module or GAM (Figure II-1) is a sealed

lucite capsule allowing sugar feeding at one end, and blood feeding through

a heated artificial membrane at the other. Mosquito pupae were injected

through the rubber stopper (#3) with a small amount of water, which allowed

adult emergence and humidified the capsule when held at 27.C.

The GAM was constructed from .3 cm lucite cylinders measuring 7.5 cm
i.d. x 10 cm and cemented with PVC industrial solvent. About 2/3 of the

inner surface of the cylinder was covered with plastic screen to provide

resting sites. A Gelman~filter support (#5,9) was bonded to the GAM floor

(#8) and interposed a stretched gutta percha membrane (#r6). The outer

collar (#9) of this filter support was threaded so it can be inverted and

screwed tightly into the floor (#10) of the heating manifold. A tefloR

seal (#7) insured watertight integrity. The GAM wras sterilized writh the

membrane in place in an ethylene oxtide chamber, or autoclaved if the membrane

was to be inserted later (30 minutes at 15 1bs and 120"C).

Figure II-2 shows a battery of 4 GAMs in the blood feeding mode in

place in the heating manifold. The blood meal was injected through the
rubber stopper of the blood tube (#11) and was kept at a constant temperature

of 32oC. Heated w;ater was pumped from a laboratory water bath into the

manifold inlet (#3) causing an overflowJ (#2) return to the water bath.

Nutritional Biochemicals, Cleveland, Ohio
3Gelman Instrument Co., Ann Arbor, M~ich.








of 25-50 eggs was taken up in a small amount of water in a sterile pipette

and transferred into a sterile dry tube containing 1.5 cm x 10 cm strip of

filter paper. The eggs were dispersed over the str-ip in an amount of water

just sufficient to thoroughly wet the paper strip and -the tubes wJere incu-

bated on their sides at 27oC. Eggs hatched in 24 hours and the larvae were

seen crawling on the surface of the paper. Within the next 3 hours, the

tubes were filled witih sterile water, causing the empty egg shells to sink,

and the sterile larvae to float to the surface for recovery.


The larval diet

Formulation of the larval diet met the following requirements: good

growth and adult emergence after autoclave sterilization, standardization,

and ease of preparation. The composition of this diet is illustrated in

Table II-1. The RNA component and the casein wrere dissolved in 250 mil of

water over heat. Dilute KOH was added to maintain pH at about 7.0 as solu-

tion proceeds (Dadd et al., 1973). The hog supplements wias previously

pr-epared by drying at 140OF for 1 hour, ground in a mill, and sieved twice

through a 50 mesh sieve. The remainiing ingredients were added and final

volume brought up to 1 liter. The pH was adjusted to 7.0 with KOH and dis-

pensed to flat-sided 16 ounce screw~ cap prescription battles in volumes

sufficient to produce a depth of .5 cm: (about 50 m~is). Each culture bottle

contained a 5 cm x 15 cm strip of plastic screen miaterial for emerging adults

to rest. on when cultures w~ere continued to that stage. Finally, then culture

bottles were auto0claved for 15 minutes.

Newly-hatched larvae were inoculated 1 larva per 2 ml of mediaa using

a sterile Pasteur pipette. Sterili ty was moni tored by i nocul ati ng an

appropriate larval sample into tubes of thioglycollate2 broth and nutrient2

:Ralston Purina, St. Louis, Mlissouri
Difco Laboratories, Detroit, ilichigan




































Gnotobiotic Arthropod Module (GAM) used for the Maintenance
andi Blood Feeding of Anopheles quadr~imaeulabuis: 1. Sugar
delivery tube and wick, 2. Cylinder top, 3. Rubber stoppers,
4. Lucite cylinder, 5. Outer collar of Gelman filter
support, 6. Gutta percha membrane, 7. Teflon seal,
8. Cylinder floor, 9. Inner Gelman filter support,
10. Floor of heating manifold, 11. Blood delivery tube.


Figure II-1.















6

































Figure II-2. Battery of 4 GAbls in Position for Blood Feeding in Heating
Manifold: 1. Top of manifold, 2. Overflow into waterbath,
3. Inlet from waterbath, 4. Manifold tank.








54














Table II-1 Composition of Diet for Rearing
Alnophzelec quadr~-iimaea~atuIs Say




SIngredient mg/1,000 ml

Purina S.E. Hog Supplementl 40% 1,000

NBC Liver Powder2 500

Casein2 50

Brewer's Yeast2 500

Ribonucleic Acid2 1,000

Vitamin Diet Fortification Mlix2 1,000

Salt Mixture W (Wesson)2 250



Ralston Purina Co., St. Louis, Missouri


2
Nutritional Biochemicals Corp., Cleveland, Ohio









Survival of mosquitoes in the GAMl was quite good provided leakage of blood

and sugar solution was prevented. Normally, 12-15 adult mosquitoes were

maintained in each GAM.


Results


Egg~51-striiato method
Several mlethodss for the surface sterilization of mosquito eggs wecre

tried including peracetic acid (Doll ~et al., 1963), antibiotics (Epps

et al., 1950), LysolR (Adkin and Bacot, 1917), C10roxR (Lea, 1957), iodine,

alcohol, ZephiranR (Dadd et al.,1973) and White's solution (W~hite, 1931).

Only the latter 2 germicides consistently produced viable sterile An4.

quadrlimaculabus lar-vae, and then, only when the eggs w~ere held on moist

filter paper. Effective egg sterilization deprived the egg of its ability

to float, and eggs which sink are non-viable. An extended embryonationn

period is another result of several of the germicides tested. Bacteria-free,

viable larvae could be consistently produced in 24 hours, using the sterili-

zation procedure described.


The larval diet

The larval diet was a composite of the diets suggested by several

workers. Liver powder and brewer's yeast1 are traditional protein sources

(Trager, 1935) which, in the present study, were included as first food for

first instar larvae. Purina S.E. hog supplement 40%2 is an excellent protein

source for older larvae and was used as the standard diet for conventional

Anopheles sp. Dadd et al. (1973) reported that ribonucleic acid, and

soluble casein, because it contained an essential steral as a contaminant,

1Difco Laboratories, Detroit, Michigan
Ralston-Purina, St. Louis, Missouri









were both critical components of their miosquito diet. For that reason,

both were included! in the present diet.

The 8 vitamnins essential to growth and development of mosquitoes

(Akov, 1962) were more than supplied by the addition of 1 gm/11ter NBC1

Vitamin Fortification Mixture. Although probably not utilized (Clements,

1963), it also contains vitamins A, 812, C. D, and E. Furthermore, it

is compounded in dextrose which, as Nayar (1966) and Johnson (1969) found,

contributed to maximum adult survival when included in Acdes sp. diets.

Originally, this vitamin component was sterilized by Millipore filtration,

but like Dadd et al. (1973), no adverse effect could be detected when the

vitamin component was autoclaved along wlith the rest of the media. Use of

the Saltlnixture-W2 very conveniently approximated Trager's (1935b) salt

mixture and evidently provides excellent buffering capacity in this appli-

cation.

Immoderately high osmotic pressure of the culture media can be re-

sponsible for poor growth and development on otherwise adequate chemically

defined media (Nayar, 1966). An. qu/adim-i~~aclluatus larvae are highly sen-

sitive to high osmotic pressure which can cause larval death in the first

25 hours of culture. For that reason, the diet described here was very

dilute in comparison to other diets (Rosales-Ronquillo et al., 1973).

Developmental time of germ-free mosquitoes was approximately the

same as for conventionally-reared mosquitoess. Approximately 75%/ of the

first stage larvae survived to theo pupal stage on the 8th day. N~o reduction

of vigor in pupae or adults was noted.


Nutritional Biochemicals, Cleveland, Ohio
MFillipore Corporation, Bedford, Mlass.








Use of the G~notobiotic Arthropod Miodule

The GAN provided an air-tight sterilizable capsule to maintain 12-15

gnotobiotic arthropods. Some practice was required in determining how much

water is injected with the pupae. In general, as little as possible will

suffice to humidify the capsule but will not entrap adults. Leakage of

sugar solution from the wick and blood seepage around the membrane are other

problems which may be encountered. Both can entrap mosquitoes if allowed

to develop.


Discussion


Considerable flexibility is possible with the GAM and it should pro-

vide appropriate experimental units for a variety of blood feeding arthro-

pods. The GAM may be successfully employed in comparative nutritional

studies of related arthropods, the identification of growth factors, trace

elements, and the effects of environmental variations. The influence of

contaminating microorganisms may be selectively eliminated for the study

of host metabolism, growth and aging under a variety of conditions.

Insect pathogen studies would be an appropriate application of the

GAM. Pathological material may be safely isolated, and its effects evaluated

without the influence of bacterial contamination. Pathological effects of

insect microsporidian parasites may be evaluated in the sterile intestine

and in conjunction with various combinations of indigenous microflora.

The GAM may have wide application in basic research on the relation-

ship between an arthropod host and the parasites it verters. Previous

gnotobiotic studies have shown that the success of many parasitic diseases

depends not only on the etiological agent, but also on the host's intestinal

microflora (Phillips and Gorstein, 1966; Mlicks and Ferguson, 1961;









Bradley et al., 1964). Parasites and potential parasites passing through

the arthropod host's alimentary tract are exposed to both the microorganisms

that reside there, and to the intestinal climate which mnay, in part, be

influenced by them. Bacteria present at the host-parasite interface mray

influence the ability or disability of the arthropod to vector parasitic

disease.











RESULTS


III. THE NATIVE MIDGUT BACTERIAL FLORA OF
SEVERAL WILD AND COLONIZED MOSQUITO SPECIES


Introduction


The ability of mnosquitoes to vector disease is of such significance

to world-wide public health, that a better understanding of their bio-

logical relationships with microorganisms in general is critical. The

intimacy existing between the host and i ts mnidgut population of mnicro-

organisms suggests more than chance environmental contamination. In-

stead, years of conjunct evolution mnay have produced a highly integrated

gastrointestinal ecosystem which may ultimately participate in the ca-

pacity of an insect to transmit disease.

On the basis of a long series of compar-ative studies of albino mice

from different colonies, Dubos et al. (1965) have concluded that the

indigenous (or normal) microbiota of these animals exerts a profound

influence on their rate of growth, their efficiency in the utilization of

food, and their resistance to infection, toxic substances, and other stress-

ful agencies. Many attributes of miicE which are characteristic of the

colonies from which the animals were derived are in reality determined

not by genetic endowment, but by the microbiota prevailing in the colony.

There is no reason to suppose that insects should be unique among animals

in their association with microorganisms.

In the course of another study, it became of interest to define the

midgut bacterial population of wild filaria vectors. Two pairs of inde-









pendent wJorkers (Chao and W~istreich, 1959, 1960; W~istreich and Chao,

1960, 1961; Ferguson and Mick~s, 1961; Miicks et al., 1961; Miicks an~d

Ferguson, 1961, 1963), have produced a series of reports on colonized

mosquitoes which indicate that the flora of adults is different from

that of larvae; it varies among the different species, and it is

distinct from bacteria in the specimen's environment. Although there

w~as always a high percentage of sterile individuals, the microorganisms

recovered from contaminated specimens are generally non-pathogens widely

distributed in food, water, soil, and intestines of animals.

These reports, however, w~ere qualitative evaluations of laboratory

insects maintained under standard conditions in established colonies.

No reports have appeared which define the microbial composition of wild

naturally-feeding mosquitoess. The following report is a qualitative

and quantitative evaluation of several hundred adult specimens of

colon-ized Anophelec quladi-~nrcimatcult Say, wild An. quadimaiculabust~s wild

Anophl~c crucciians Wiedemrann,wild Aedes inf~irmaictu Dyar and Knrab, and

Al. erucians complex, trapped at 6 stations in Gainesville, Florida.


Materials and Methods


One-hundred adult femnale specimecns of An2. quadtEl~inc~ulatus (Gaines-

villo str-ain) were selected over a period of time froml the colonies at

the USDA's Agricultural Research Service, Insects Affectin~g Man Laboratory

in Gainesville, Florida. They had been fed a liverl:yeastl miixture


1
Nutritional Biochemicals, Cloveland, Ohio









for- the first 3 larval days and ground S.E. Hogl Supplementl for the

balance of the larval period. Adults were held 5-7 days after emer-

gence, fed a 2.5'; sugar solution, and starved 24 hours prior to dis-
section.

Several hundred wiild mosquitoes w~ere trapped from 6 field stations

around Gainesville, Florida, with a CDC2 type light trap using dry ice

as a carbon dioxide source. The species composition of the wild females

selected for study w~as: An. qjuad~rimaculatus, 25 specimens; An?. orulcian,lc

80 specimens; Al. infi2rmatus, 12 specimens; An. orucian~s complex, 20

specimens. These mosquitoes were also held 24 hours without food before

dissection.

Both oral and anal openings of each insect were sealed wyith aucoR

cement by bonding the insect at these points to a plastic cover slip.

Seepage of the germicide into the digestive tract was prevented, and

the coverslip effectively anchored the insect for dissection beneath the

surface of sterile water. The mounted insect was immersed in 70% ethanol

for 30-60 seconds, rinsed and dissected under sterile water. A section

of abdominal integument was removed and placed in a tube of thioglycollate

broth3 as a sterility check. Gentle distention of the lower abdomen ex-

posed the digestive tract, and the midgut wads retracted and removed w~ith-

out puncture. The midgut was then homogenized in .4 ml sterile water in

a glass tissue homogenizer. One-tenth ml aliquots of the homogenate

were then streaked for isolation onto duplicated! Eugon3 agar plates, 1 Eugon



Ralston Purina Co., St. Louis, Mo.
2 iiational Communicable Disease Center, Atlanta, Ga.
Difco Laboratories, Detroit, Mich.









agar plate fortified with 5% defibrina-ted sheep blood, and I tube of

fluid thioglycollatel. Incubation took place at 370C, 250C, 370C, and

370C, respectively. A total count of organisms present in the midgut

w~as made by counting all of the colony-forming units, appearing in

Eugon agar after 24 hours, and multiplying by a factor of 4. Each

different type of colony appearing on the 3 agar plates was counted,

sampled, assigned an isolate number and streaked for pure culture on

Eugon 1 agar. When growth appeared inl the thioglycollatel tube, ino-

culations were made into veal infusion agar tubes' to differentiate

obligate anaerobes. Yeasts and fungi were inoculated into Sabouraud
maltose agarl

When pure cultures were apparent, inoculations w~ere made into triple

sugar iron agarl (TSI) and brain-heart infusion agarl (BHI) slants which

were maintained as stock cultures at 90C. Colony morphology, chromo-

genesis, and Gramn-reaction were determined at this time. Table 1

lists the procedure in the presumiptive determination of bacterial genera.

Hydrolytic abilities were determined in agar supplemented w~ith 5%:

soluble starch, 5% gelatin, 5% Wesson oil, or jjZ alphacell. Oxygen

relations were demonstrated in duplicate tubes (one overlaid with sterile

mineral oil) of OFI media and incubated f-or 5 days. The presumptive

identification of Gr'am-negative enteric bacteria as w;ell as the catalase

test were performed on TSI. Decarboxylase production w~as tested in

lysine, arginine, ornithine, and control tubes. An I.N~.V.i.C. series

(indol production, methyl red pH, Voges-Proskauer, and citrate utiliza-

tion) was performed in appropriate media. Both the nitrate reduction


Difco Laboratories Detroit, Mich.










test and motility were determined in nitrate agar1 slants. The type

of milk reaction w~as demonstrated in litmnus milk1 media anid the hydroly-

sis of urea was determined on urea agarl. Carbohydrate formentations

were demonstrated on cystine trypticase agar1 plates overlaid with

any 10 carbohydrate differentiation discs listed in Table 1.

Data analysis wras by means of a Statistical Analysis System (SAS)

program written by Barr and Goodnight (1972). A frequency distribution

of the species composition of midgut bacterial flora was computed.

Correlation coefficients and probability statements were calculated on

the frequency of occurrences of all pair combinations of bacterial isolates.




Results


~Ades infirmatzue Dyar & Knab

The average midgut bacterial count of 8 specimens of A. infir-matis

was 263. No bacteriologically sterile specimens were encountered in

the sample. Table III-1 illustrates the species composition of midgut

bacterial flora isolated from all 5 groups of mosquitoes. The most

frequently isolated bacterial species in this mosquito group was Bacill~as

cereus Frankland and Frankland, non-pathogenic Coryniieba~ctrii- sp., anld

Micrococcu~s iuteus (Schroeter).


Ano~pheles quadr-ncimazculatus Say, (ild)

Sixty-one percent of the w~ild An,. quad~~rimculatuss examined w~ere

found to be bacteriologically sterile, which was the highest rate of


1
Difco Laboratories, Detroit, Mich.












FIGURE III-1



Procedures in the Presumptive Determination of Bacterial Genera


1. Hydrolysis Reactions:


Starch Hydrolysis
Gelatin Hiydrolysis
Lipid Hydrolysis
Cellulose Hydrolysis


2. Oxidative Fermentation Reaction

3. Catalase Trest

4. Triple Sugar Iron Slant


5. Growth Study:


Blood Agar
Eugon Agar @ 250C
Eugon~ Agar @ 37oC


6. Decarboxylase: Lysine
Arginine
Ornithine
Control


I.M.V.i.C. Series

Motility

Nitrate Reduction Test

Litmus Milk Reaction

Urease


12. Fermentation Reactions (any 10):


Arabinose
Cellobiose
D~ulci-tol
Fr-uctose
Galatose


Glucose
Glycerol
Inositol
Inulin
Lactose


Mal tose
Mlannose
~a n n itol
Raffinose
Salicin


Sorbitol
Sucrose
Trohalose
Xylose
Control





TADLE III-1

Spe~cis Co-iposition of Mijdgut Bacterlial Florii in 5 Groups of Mosquitoes


SPECIES CO;PO;ITIONI PERCENT OF TOTAL?)


.i;
'
~
c o. r
a
3


~ ~
ub21~~i:
'-1 C
' ~ -; L~ ; L'-~:L


(i rl
~ F 9
~iaS?~ n ~ r,
i-" ~ "" ~'~
I'Ef
Yi r;
t a ~~ N ~~ N
U _a ;? ~ ri
~ '" 6
` 4 .& 5
... m
: ;i S .Z
SP;C;S C:L:.T h ~ u u o
"' ~ 3 B
~ v. .. i.


i;Z 2
i.'.7 1 C3
ii, :5 3 ~2
;ia 1C:


3 37 16 4

40


47 23

32


89
33: 23
7?


4
5
Ii ''
r.
0 i,.
" ~ B B ,,
i: i i. ~ r-j vl
09 a BS
r. ,, P, i
~~a~~GsoaC
~~"4~8~~~8
X S '~ ~ P .~
~ ~ $ ~ iS U
r 1: ~J it ?j C1
~Q ij o iI n t~ ~
'' ;; r;~ ~: X
i; u u r:








TABLE III-1 (continued)

SPECIES COMPOSITIONS PERCENTT OF TOTAL)











318 94 6
20 100 :


19 CO i "a 9~ Bf

210 1001
340 :00

































__ ~I __


TArBLE III-1 (continued)


SPECIES CO;MFOSITION (PERCENT OF TOTAL.)


j If
g ''
'"
a ~ '''
i-.
3. i ~ a n .-!
q ~ ` ' ~? ~ ~
- jqi. -r :1
ii O C .I~ ;' ~ ~
i u
ri F
u ~ t-
J
rl ~ U U C? U U u '-r w ii ~


i,

1
a
vl



-:


C :I
,
,i
, ~ ., N ii
"
r
"
?
u rj j ,., ~,
: c
.. . h r. r:
i i ~ ii c : u 8
-.1 ., II i. ~~ -, 3
; .o
I -. -~ a g
c, ir x ,,


ED 16
3333


17 8
70 23
'L0
53


;346


423


5:"0



133


6
7
51
6j


50
65
93
55 10





TABLE :iI-1 (continued)



SPECIES COMPOSITION PERCENT OF TOTAL)


~ o
~
-
i:

"


vinu
e, J
o ;1 o

2 '" 'L
ii ~ d ~
c


1 Z
c. .~
8 "
&P~"I i

ood~ a a

gma3
&
is ~ ~~ r~ ~ '' B
lr ') W
~4 i .i
lu f rl R


~ "
c~ 9
p

"J "
~ o

n ~,
N (i e
-a-
~I ~1 F:
i u -~
''
n d


TPs c:aL


ii
Y~ C
Y1
Ii
I ;
vi o
; -
~1 u :;
i

i.



$
o,
N s



c.
i
o u
31


Y1


'"

~g
o ~


Aophelf;s 382



200S


(continued) 2
C"C

300



;50


78 22
3


44 53


50
87 10

41 5?
50 50
100
100
82 15











;~e~E III-I (continued)


SPECIES CGI1POS:TION I~ERCE~T OF TOTAL)


9
- -,
I: ? ei ~ a ~
e U '-' O '"
i) E3 .~
:i
3 ii' c, ~ -I: c ~
h U ~ ~ .I ~ L1 ii 3 ii c
:: ,,,
'li o ~ o r. g g n
, "
Y~ 6~ ii
_:~ ~ 5 ~ U -: i Li V i.
c PJ ~ L3
3
'. ii ~ ~~ t : ~
;. u ~ L" C i LI
:1 X :
r- ~ r, ! ' -i i
. ~ tIO Y U ii :. U . r)
Tj-? i ii i, `1
~ i: ii '' ;. ~
' L ;; 2 ''' :: ''
I~ i ~; .
4 ~ Y

;':71 ;:.: Z-:
G;j i3 ? i9
(~~ntis,~cJ jC0


I O",





TA8LE II-I- (continued)



SPECIES COMPOSITIONI PERCENT OF TOTAL)


I


C)

~
u

~

" ~

I;
c7

M e
Z


00







o~ u


:
rJ

r
vl c
U
ii C
'1
ii :, ~-
'1
I:j
i. :I
r. U


(1
B
~ 8
:1 .,
o ~
a c, o
~ w L
ij , C L
o -,
1. ~I u
N j h II
\ e~
"' c
C E:
'i P,

C n og g ~YILI


~,.~ m ~
yiN jl
"g~
N C 3
b
o ~ c -I e


3
ruvzip
8"`
uuoo


::s ullo
SI'Ec:Es ConT ;


309)


2C'"


(Continuecd) 0
142





203
j30



































calatu 13 100

2 100
continued) 100 5
CC
-60 --5 5
103


15? 20i go
:00 :Cn
iC: 100
25'J 100
312i 96 $


TABLE III-1 (continued)


SPECIES COMPOSITIONI (PERCENT OF: TOTAL)


y
r
:: ,I
R
? '~J V)
,,
C I c
Y i) ~ ?. I ^
~ ~ "
'' '* r
~
II ~. '' ~' '' ~ ~ L:
~ u
; or u i
-~ u u -i~ ~ -
L) I. L1 i.


'' 3
,
F n ~-, ;,
rr r vi N ~

" d 1~
P3 :jl ~ ii rj i.

"
1 ~~` H E: E


:'



:'~5'J:T~







































*:::Ca ?;3 5 IC')
(colony) ;00 10
130 100
120 tCC
(con:tJnue) 90 100
173 80;


TABLE iII-1 (continued)



SPECIES CODPOSITION (PERCENT OF TOTAL)


:i
X "
P, UU C
u .~as
B 9
a S ;'"
~ "' D a 4 ~,.,
g ~ g ;j
i- L; o ~
o""Y":~"#~
,~ ~, o*IN ~g ,, ~,!
h ii 1 N
~ ~ B f~ u a
$on i a
( ~ % t-~ ir Lj C) i:


rlcs:u!rc! ~ ii
ri;Ei!ES


U r,
:
pi YI a
;C"
~ ;
~1
i'
" i:
o r: y ~ e
1~ ~;
u . i
C: s -
re .r ~ ; c


; a
~ S g
o
G '"
d :: o
v i, u.a
O O
- ~Y N O i~
C' O
B U '*
a U, .,
,,
c. c~ :~ B ~,


o r, o
':"8~'
" g - 0

~-~ FI Y)
osg

Sar:j~





TkALE III-1 (continued)


SPECIES COMIPOSITION [PERCENT OF TCTAL)


r: "]
~ ;

I
a u N
P '''
F: B ,; ;~
'% :
ild "" I; -e
a:: n~
r.) m 1 n r
L~ aU r; , c_
YI ; L) U i, g ., Y1 YI ~ .I:
~ a u i~ rl n I .I u~
~,~~g i.
r~ u~g"~"S~' 3
B :i
n $ ~~ )J .I.i P C
C 1? i;
;
r, @ I
--
R " a : ` 6 ~
i i


FZ o
'"
'" '5 F c
ii "
~
~ i: - i; s "
i;
t
i~ X ~"
c u
~1 T y 3 C:
TATI 3 ... i B
u r;; - 3
:ICSLI!TD eiL I~?:~L ,, ~ L r~
S'EZIES C~i"lT ii r; i~ P; U
rj ,: o X i,


C3
r: ,~ s o

~ ?,~
SN r~ N ~
~ ., .e ~ g
.9 ,
Q 3 :: ~'
"~"';6
"


^124


2320





23;
312
411
22?


20
10
29J

35 C5

11


C0 3





































<:4 c:ca 10 99
:aic: 2537 82 58
00O 1 9
2000 70 30

1100
:7C 100



-)CC100)


TABLE III-1 (continued)


SPECIES COMPiOSITION PERCENTT OF TOTAL)


"
ri

i..

B

"
i! d.




L:


rj C
6 ~-
7 'j
Li .. L 7: 1--.
g il~ 'P L~ i.
'i C ri ii vi ri v r-
n
r? o V " 1'~ 'O Y~
* i. C
~rryJ~~e;m; ~? "
gs c` "3
4 a c! ~, P r:
o~ sS~~i
-r -I
P ~ '' 3 :'
ii i '~ '~ ':
C "


ii F
X 9 ,
~~ c? u
VIN i)
P
no io O.;q. 'c,
't-, c, 'i

_i i 9 - ? r
ZYc~ ~1
"' ~ ~~ G ~ '" '"
e~ i: g X
N - E L1 '" ~ ~~ ~i ~ B
i-
,, .:~"38"? E:
':""' LL ;-









midgut sterility encountered. Twenty-five specimens however, had an

average midgut bacterial counit of 666. The most frequently isolated

bacterial species were Enterobactel cloacaet (Jordan) followed by

B. cereus.


^nopheles cr~c-ia a compl ex

Forty-three percent of this mosquito group were bacteriologically

sterile. The average midgut bacterial count of 25 specimens was 272

which was usually composed of Sa~lmone.Lla sp., Alcatigenes fa~ecaic

Castellani and Chalmers, and E. cloncae.


AF.nopheles crucian~a Wiiedemann

The midgut sterility rate of An. crucians wdas 36%. Eighty specimens

however, had an average midgut bacterial count of 377. The most fre-

quently isolated bacterial types were B. cereus, Salmonella sp., A.

faecal-ie, and Klebsietta. pnzemioniae (Schroeter).


Anophzieles quadrimacul~abus Say (colony)

Colonized An. quadrimaculatus had a midgut sterility rate of 40%.

Sixty individuals had an average midgut bacterial count of 214, which

w~as composed of B. cereus and the yeast, SaEccharmyices sp.



The Normal Flora

Sa7,mone.lla sp. appeared most frequently in An~. c~ruciana7 and Anr.

arucians complex. They produced very small (1-2 mm) mnucoid, discrete

colonies on Eugon agar and blood. A negative Gram stain demonstrated

m~otile, non-encapsulated rods. Miost isolates were catalase positive









and all were able to utilize citrate. Decarboxylase reactions were

variable, but most isolates carboxylated lysine and ornithine, and

produced H2S on TSI. Carbohydrate fermentations w~ere quite variable.

Antigenic typing of Sal~monella sp. was not attempted.

The Actinomycetales wdere represented by a group of non-pathogenic

Corynebacteriu~m and the genus Arthroba-"ter. The former were frequently

isolated from A. infirmatus~, while the latter appeared only in a

single isolate from a wild Ani. qu~1ad~~rim~aculatus The Coryn~jebacterfuml~

sp. produced grey to white star-shaped colonies on Eugon agar and

frequently produced luxuriant growth on blood agar without hemolyzing

it. These isolates were characterized by their high degree of pleo-

morphism which usually took the form of a non-spore forming Gram-

positive rod of irregular outline. Most isolates were catalase positive

and produced strong oxidative reactions on OF media. These isolates

were presumed to be non-pathogenic coryneforms because of their ap-

parently non-pathogenic nature.

A. faecalis w~as isolated most frequently fr~om Aln. oruccians. They

produced pale yellow, wrinkled colonies on Eugon's agar. They usually

occurred as motile coccoid Gram-negative rods. Mlost isolated produced

no change on TSI slants and negative reactions on indole, H2S and urea

sl ants. Carbohydrate fermentations were also negative. Simmons-citrate

reactions were variable, but all isolates produced oxidative type OF

reactions.

E. e loacae wa s i sol ated from nea rly 50% of wilid An.? quad-rimacul~t atu~s

examined. These Gramn-negative motile rods usually produced smooth,

round, rose-pigmented colonies on Eugon agar. They wiere distinctive









in producing an acid TSI, positive citrate and a positive VP test.

Most isolates produced moderate to strong hydrolytic reactions on

starch agar, lipid agar, and gelatin agar. An occasional strain hydro-

lyzed cellulose (5% alphace0)~. Catalase reactions were positive and

most carbohydrates were actively fermented. nitrates were reduced by

most isolates.

B. cercus, was the domiinant midgut bacterial species of 95% of the

colonial Ain. qualdrirnaculaitus. that we!re contaminated. It appeared oc-

casionally in all other mosquito species. B. cereusE produced rough,

white lobate or round colonies on Eugon' agar. Their long, thick

rods usually occurred in long chains and produced cylindrical spores.

All isolates were catalase positive and produced strong hydrolytic reac-

tions on starch agar and gelatin agar. Decarboxylase activity was

usually not present. Carbohydrate fermentations were variable, but

arabinose, xylose, and mannitol typically w~ere not fermented. B. cereus

was differentiated from Bacilluts suibtilisi (Ehrenberg) which was occa-

sionally isolated from colonized An. q7uadr'imaclatu!Z 2s by their inability

to form chains and their characteristic litmus milk reaction.

KC. pn~eumonriseo was isolated most frequently fr~om Anz. crucians. They

produced 1-2 mm brown colonies on Eugon agar and appeared as non-

mnotile capsulated rods which occur singly or in short chains. Occasionally,

the fimbriae wJere recognized. This group typically produced negative
reactions in ornithine, and positive reactions in gelatin.Teypoud

acid and gas from glucose, utilized citrate, and produced a positive VP'

reaction. These isolate; often produced luxuriant growth with a green

non-hemolytic sheen on blood agar.

1-
Nutritional BiochemicalsCo., Cleveland, Ohio









Mlicrocovenel! ro0selS Flugge and M, Zubea~si were isolated most fre-

qluently from A. iLafirma7tcs and An?. criiiucias, respectively. They are

Gram-positive cocci that produced large, opaque growth on Eugon's

agar under aerobic conditions. Cell division occurred in 2 or more

planes forming irregular clusters. Both groups were strongly catalase

positive and produced acid without gas from glucose. M~. roaeus. hydro)-

lyzed starch and gelatin, and produced bright red colonies on lipid

agar. Smooth, convex rose-colored colonies were produced on Eugon'

agar. Metabolism was of the respiratory type and decarboxylose activity

was negative. M~. luteus produced yellow, glossy colonies on Eugon

agar and was distinguished by its ability to hydrolize Fats. An un-

identified non-pigmented Miciococcus sp. was occasionally isolated from

wiild An. quladrima~culatuls which failed to hydrolize starch and gelatin,

and produced variably positive decarboxylase reactions.

PSEUdZomIonaS aerugin2osa (Schroeter) and Pseudao~n-nas au~rofaciene

Kluyver were isolated in low frequency from A. infirmnal lue and An. crucianis,

respectively. An unidentified Psuedoonanss sp. was isolated 3 times from

A. cru~cians. This family of bacteria are short, motile, Gram-negative rods

that are highly pigmented. P. aureofacienss produced bright orange dis-

crete colonies while P". a.-ruginosa developed red pigment. Neither species

gave a positive MR, VP, or H2S reaction. Negative decarboxylase reac-

tions occurred in lysine, ornithine, and arginine medias. Both species

gave alkaline milk; reactions and produced strong catalase refactions.

Nitrate reduction was variable.

Se'rat-ia mancsconzs Bizio w~as isolated from wild An. c,ucJians, complex.

These bacteria were motile, Gram-negative rods that produced smooth 2-3









mm colonies on Eugon agar. They gave strong catalase reactions and

reflected a greenish-i ridescenrce in oblique light. Most isolated pro-

duced brigh red colonies on lipid agar- and hydrolized starch agar.

They produced positive VP reactions and wer-e able to utilize citrate.

Metabolism was of thle fermentative type.

Es-cher-ich~ia co~i- (Migula) was isolated frequently from Anl. orucians

and An. oru~cians complex. These Gram-negative rods produced acid and

gas but no H2S on TSI. Nlo capsules or fimlbriae were noted, but isolates
were strongly catalase positive. Ornithiine, lysine, and arginine are

decarboxylated. Citrate was not utilized but acid and gas w~ere produced

from xylose, dextrose, maltose, lactose, and mannitol. Antigenic typing

was not performed.

Frequent isolates of Staphylococcus caprlophytiou~s (Fairbrother) were

made from An. crucia~s.. These Gram-positive cocci occurred in clusters

and produced smoothh, white convex colonies on Eugon agar. They were

facultatively anaerobic w~ith strong catalase reactions and variable de-

carboxylase activity. Acid was produced from glucose, lactose, and

sucrose. Nitrates wdere reduced and gelatin, lipids, and urea wdere

hydrolyzed. Citrate could be utilized as a sole source of carbon.

Streptcc~culs fan:calis Andrew~s and Harper and an unidentified Strepto-
coc.cu~s sp. wlere isolated fromn An. ecuicianz. These Gram-positive cocci

typically occurred in pairs or in short chains. Metabolism was of the

fermentative type alnd catalase tests w~ere negative. Colonies appeared

as discrete, white and muccid on Eugon agar and luxuriant on blood agar.

S'. faecalis did not hemnolyze blood, but the unidentified Streptococaves

produced strong beta he-molysis. An acid curd w~ith reduction appeared in










litmus milk and nitrates were not reduced. Both citrate and glucose

were actively fermented. Both cultures grew in the presence of .04%

tellurite. Antigen typing was not accomplished.

An unidentified isolate showing the characteristics of L~ctoacrcilZ1o

sp. 'was consistently isolated from An. cru~cians complex. These Gram-

positive, non spore-forming rods or coccobacilli form minute colorless

colonies on Eugon agar. They were consistently catalase negative

and failed to reduce nitrates. Indole, H2S, acetyl methylcarbinal, and

urea were not produced. They produced arginine dehydrolase but did not

split lysine or ornithine. Proteolysis was often produced in litmus

milk. Carbohydrate fermentation was variable.

Acinetobacter catcoaceticuis (Beijerinck) was isolated consistently,

but in low numbers from all 5 mosquito groups. These short, thiick Gram-

negative rods produced discrete smooth mucoid colonies on Eugon agar

and brown hemolytic colonies on blood agar. These isolates produced

a pr~oteolytic reaction in litmus milk, were catalase positive, and failed

to produce indole, H2S, reduce nitrate or liquify gelatin. All isolates

were oxidase negative and demonstrated weak OF reactions.

Three isolates from Ain. c2rucinans were tentatively identified as a

species of soil Niaisseria. These Gram-negative cocci w~ere aerobic anrd

formed large, yellow~ colonies on Eugon agar and were hemolytic on blood.

Thely wEre catalase and oxidase positive, and they produced indole, arrdacety

mefthylcarbinal. They u-tilized citrate as a sole carbon source but failed

to decarboxylate lysine, ornithine or arginine. Glucose and maltose

werle weakly attacked.

























Positive Correlation

Species R P2


Neisseria sp.; SalmnellcZa sp. 0.334 .0002

Azomonas sp. ; S. facealis 0.272 .001

P. aureofaciens; A. faecalis 0.468 .0001

S. faecalis; A. faecalis 0.215 .001

Al. faecealis; M. iZutea 0.214 .012

S. Zu~tca; Salmonella sp. 0.379 .0001

A. faecalis; P. aeru2ginosa 0.177 .037



Negative Correlation


Ei. cereus; Salmonlella sp. -0.199 .001

B. eereus; A. faecalic -0.264 .002

Br. cereus; E'. clZoaeae -0.173 .004

B. cereus; K. pnewoioniae -0.180 .003


TABLE III-2




Correlation Coefficients of the Frequency
of Occurrence of Midgut Bacterial Species


1
Correlation coefficient
2
Probabil ity








The remain-ing bacterial species consisted of lowJ frequency isolations

or solitary isolations. These bacteria were Protceus vu~lgari~s Hauser,

Flavobacter~~-oiw ilutc~enis (Migula), Cibtrobacter frau;ndlii (Braak,), and un-

identified species of Planococcus, Alzobachce7ium, anld Amomr~ona. The yeast

Saccheromyces sp. was identified by morphological characteristics and its

ability to grow on Sabouraud maltose agar.


Correlation in Bacterial Occurrence

Table III-2 represents correlation coefficients and their probability

statements calculated for each combination of bacterial species in the

midgut flora. Salmnonella sp. is positively correlated (P>.001) with both

Micrococuis Zu.teuts and the unidentified Neisser-ia sp. Stre~ptoocous

fe7ecaZis is positively correlated (P>.001) with Azohaobersp. and A.

faecais although these combinations occurred in only 3 insects. A.

faea~is is positively correlated with M~. Zuteus- (P>.001) and P. aerugin~osa

(P>.005) .

The negatively correlated bacterial pairs all involved B. cereus.

b'. cercuo is negatively correlated with Sa~lmoneZZa sp. (P>.001), Al.

f'aecais (P>.001), E. cloacae (P>.005) anld P. pneumon~iae (P>.005).


Discussion


The large proportion of bacteriologically sterile individuals is

not surprising. Chac and Wdistreich (1959) reported a sterility rate of

8% and 23%, respectively, for adult female and male Cu:~ex tar;aia

Coquillette. Wiistrich and Chao (1963) reported a midgut ster-ility rate

of 24% and 40Z, respectively, for female and male adult naeds iaogypti L.

Mlicks et al. (1961) reported mnidgut sterility rates of 50:5 for An!. quadri-

macuZatus females, 91%: for Al. ategy!pti, and 78% and 89X, respectively,









for Culex rat-igansz W~iedemamand cuLZey~ motestus2! Forskal. M~icks and

Ferguson (1963) in a later investigation, reported the midguts of all

C. molestus females examined wer-e uniformly sterile.

The presence of a bactericidal and/or bacteriostatic substance

in the midgut of certain mosquito species has some support. Duncan

(1926) demonstrated the presence of a bactericidal substance present in

the gut contents and feces of Aledes cincreu~s M~eigen and Anlopheles bi-

furcatuis (L) which was effective against 3 species of Ba~cillu*s. More

recently, the activity of antibacterial factors present in other blood-

sucking arthropods has been largely associated with and limited to Gram-

positive bacteria (Duncan, 1926, Anigstein et al., 1950).

The fact that microorganisms could not be isolated from a number of

the specimens in this and other studies should not be taken to mean that

microorganisms were not there. The standard cultural procedures em-

ployed in these studies are not adequate to demonstrate intracellular or

exquisitely fastidious organisms.

The species composition of the microflora isolated from the S groups

of mosquitoes in this study compares favorably w~ith other work~ers. The

isolates from adult C. tarsatis (Chao and Wistreich, 1959) showed the

presence of Achromobacter gutatus (Zimlmerman). Achromoajcter sp., E.

cloacae, c~itrobseber. intrmiedia (Braak), Fl~avobatterriumn sp., M~icroolccus

sp., M~icrco ccusf vivir-anzs M~igula, ProbetaLs sp., and Pseudomona~s sp. Cul-

tures of unidentified Gr~am-negative rods as well as SaccharomycesR sp. and

Geotirichu:n sp. wer~e also reported. The same investigators, Wistreichh and

Chao (1960), isolated bacteria from midgu-t sections of 14 fourth-instar

larvae of 0. tarsais that had been raised in fresh tap waiter and fed









brewer's yeast. Results of these isolations showed the presence of

Achrom~obancter sp., Tsactobacittlus sp., M.IC.'OsocL'uS CanizZd'I Cohn, M~icro-

coccuis sp., pr'oteus resttgel't Hadley et a., and P~seudomoarcr, sp. Again,

cultures of unidentified Gram-nega~tive rods and Saxchchromyces sp. and

Geotrichum; sp. were observed. Isolations from the larval rearing water

showed the presence of E. cloaczae, Kr~ebsiella sp., Sa~cchaxramuce sp. and

Geotr-ichum7 sp. Fourth instar larvae were found to contain examples of

all the genera of bacteria isolated from adult C. tarsai3 w~ith the

exception of Fnierobacter, Citrobatcter, and Flavobacterium,, whereas iso-

lations from adults did not show~ the presence of Lactobacillus or the

coral pigmented form of Micrococa.!

Bacteria isolated from the midguts of adult CuZez quinqjuefasciatus

Say fed apple slices for 2 days after emergence showed the presence of

Achromobac-ter sp., Flavobac~teriumi sp., KlecBielZra sp., Microcccurs cazseo-

lyticus Evans, and Poeudomnras sp. (Chao and W~istreich, 1960). M~idgut

sections from 11 fourth instar larvae of the same species produced only

Baociltis sp. and the yeast Sacchiammryces sp.

Bacteriologic evaluation of the moidguts of larval anid adult Aedes

sicervensic Ludlowi and Anzophece a'bimanus Wiedemannfed on a larval diet

of yeast and adult diet of apple slices was reported by Wlistreich and

Chao (1961). Larval isolation f~rom Al. albimanus showed the presence of

Alcaligenea sp., Bac~%i~ll sp. anid unidentified Gram-negative rodls. Adult

Al. alb~imian~u: showed Ah ~'iomoacter's~p., Alcal~igrene sp., and Corynebabcte,1:iu

sp. Sacchurequesli~r was isolated from both larvae and adults. A. sierrancsis

larvae contained RalciZue~ cimulous:: Jordan, and P~arac-colobactkum, intermedi

Bor-man. Alc~iacroba:cter sp. and c. itr~er~media were found in adults. Sac-










charomyeces sp. were isolated only from the larvae. Wlistreich and Chao

(1961) also repor-ted that mlicroorganisms isolated from the adult and

larval environments were distinctly different from isolates of the

midguts.

Wlistreich and Chao (1963) reported bacteriologic evaluations on the

midguts of larval and adult A. aegypti which had been fed yeast and

apple slices, respectively. Isolations from larvae showed the presence

of the following bacteria: Alcaiigeneis sp., Cory~nsbceba-Cteiu sp., c.

freundii, urbhialz beesonii Hauduroy et al., M. luteus, as well as fungi

belonging to thle general Geotlichumv, Peniiciiuim, and Saccharomyces.

Isolations from adult mosquitoes showed the presence of Achromobacter

sp., AZealigenzes sp., and Saccharomyce~s sp.

Ferguson and Micks (1961) reported additional experiments using

adult female C. fatigans from a colony that had been denied access to

food. By means of a sterile micro-dissection technique, they demon-

strated the presence of 7,actobacitus sp., Aiealigeness sp., Pseuidomoza~s

sp., and 2 unidentified cultures of Gram-negative rods. Bacterial iso-

lates from the midgut of similarly tr-eated adult female An1. quaSdlvima:cuSa~tus

proved to be 2 strains of Screpiococzus and a species of Aerobaeber (Micks

and Ferguson, 1963). Al. aegypti produced only a species of Colyneb:3cacterium

and no microorganisms could be cultured -from the m~idguts of adult female

C. moiestus.

Generally, these bacteria represent common soil, water, or enteric

or-ganisms widely distributed in nature. Four of the 6 collection sites

from which Anz. crucians~, Ain. cr~caians complex and w~ild An. quladrimacul~atus










were collected w~ere locations around a large lake on the University of

Florida campus. This lake receives a number of sewe~rage effluent. The

presence of such enteric organisms as P. vi'lga1'ri, C.
fas~cais in the mnidguts of detritis feeders is not surprising.

Colonial mosquitoes tend to exhibit a microFlor-a made up of 1 or

2 bacterial types which are consistent for the colony. WJild mosque toes

usually have a variety of microorganisms which are generally comparable

within the species.

A notable exception proved to be the isolation of Net~s~cori sp.

from 3 specimens of An. crudenc~. They r~esemnbled thle N~icrococcaceae.

somewhat, but bore an even closer resemblance to 2 types of Ieisneria

described in grasshoppers by Blucher andi Stephens (1959). One isolate

was tentatively identified as N. catalrshalic (Frosch and Kolle). The

taxonomic position of these 2 types is uncertain and it is questionable

if they belong to this family or if they should be considered a member

of thle M:icrococcaceae that had lost their ability to retain the Gram

stain or to ferment carbohydrates.

The significance of the positively correlated bacterial combinations

is difficult to interpret. S. ~faecais and Ai. farccaicci might be ex-

pected to occur together because? of their enteric origin. The other

combinations mlay be the result of mutually satisfying metabolic relation-

ships.

Significance of the negatively correlated comibinat-ions favoring

thle presence of P. comm~u is moore obvious. The family Cacillaceae produce

a variety of extracellular products. Those products secreted by B. cerease







88


include hemnolysin, soluble toxin lethal for mice, enzymles lytic for

bacterial cells, proteolytic enzymies and phospholipase C. (Buchanan

and Gibbons, 1974). R. cerlius has been incrimninated in food poisoning

where extensive multiplication has occurred in foods. By wiay of its

secreted products, this bacterial species apparently competitively

inhibits most other intestinal microorganrisms, w~ith th~e yeast Sacchzaro-

myces being a notable exception.














RESULTS
IV. EFFECTS OF BACTERIOLOGICAL
FLORA ON THE EARLY DEVELOPMENT OF
DIROFILARIA IMITIlrS LEIDY IN
ANOPELE QUA\DRIMACULATUS SAY


Introduction


The relationship of the normal intestinal microflora to the vectoring

capacity of mosquitoes has not been adequately investigated. Insects are

not unique among animals in their relationships with microor-ganisms.

Parasites and potential parasites, passing through the arthropod hosts

alimentary tract are exposed to both the microorgansism that reside there,

and to the intestinal climate which may, in part, be mediated by them.

Indigenous bacterial flora present at the host-parasite interface must be

regarded w~ith as much consequence as the host itself.

Dirofitarias inn7i;tic Leidy, the etiological agent of canine heartworm

disease, requires passage through thle mosquito to the L3 or infective

stage. Although susceptible or resistant mosquitoes species are genetically

determined reviewedd by MacDonald, 1973), the physiologic mechanism

whereby the insect accepts or rejects the infection has not been elucidated.

The presence or absence of host micr-oflora, or the species composition of

micr~oflora, mnay be one of the factors mefdiating the establishment of D.

ilmmithi in susceptible mnosqu itoes.

Gnotobiotic studies have shown that the success of many parasitic

diseases depends not only on the etialogic agent, but also upon the host's









intestinal mnicroflora. Phillips and Gorstein (1966) reported that the

association of the amioeba Entamoeba ~h~i a-totytica Kruse) with one of 3

species of bacteria was necessary to produce typical hepatic abcesses

in hamsters. Phillips (1964) considered the bacteria acted by pro-

viding a physically and chemically suitable environment favorable to

survival of the amoeba, but W~it-tner and Rosenbaum (1970) proposed the

existence of some critical factor passing from bacteria to pathogen.

Mlicks and Ferguson (1961) concluded that microorganisms play a signifi-

cant role in the susceptibility of mosquitoes to malarial parasites.

They suggest either a competition between the microorganism and ma-

larial parasite for essential nutrients, or that certain strains of

microorganisms supply factors needed by the host for metabolic processes

and defense mechanism~s. Elimination o-f the microorganisms in the latter

case would presumable increase host susceptibility to malaria.

This paper reports the possible effects of midgut bacterial flora

isolated from 5 groups of wild and colony mosquitoes on the ability of

gnotabiotic mosquitoes to support development of first stage D. iinitis

larvae.



Materials and Methods


Eggs of Alnophelces quair~imnaculatis~ Say were obtained from the colonies

of the USDA Agricultural Research Service, Insects Affecting Man Labora-

tories in Gainesville, Florida. Egg sterilization procedures, larval diet,

and germ-free rearing techniques have been previously described (Results

II). Eggs w~ere surface sterilized in 10%J Zephiran; allowed to hatch,

and the sterile larvae injected into 16 ounce prescription bottle rearing




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs