Factors influencing infection of the mosquito with Dirofilaria immitis (Leidy, 1856)

Factors Influencing with Dirofilaria

Infection immitis

of the Mosquito (Leidy, 1856)

Leo Kartmanl Department

of Parasitology,

The Johns Hopkins University, School of Hygiene Public Health, Baltimore,Md.

and

(Submittedfor publication, 7 November1951) A critical period of the filarial parasite’s life cycle undoubtedly occurs during its development in the arthropod host. Although microfilariae of the canine heartworm, Dirofilaria immitzs (Leidy, 1856), are known to be capable of metamorphosis in many speciesof mosquitoes, the history of experimental work with this parasite speaks eloquently of a contradiction between its wide experimental host range and the paucity of host species in which it may adequately reach the infective larval stage. Our knowledge of the mosquito’s role as host to filarial worms began with Manson’s (1878) observations on the infection of Culex fatigans by Wuchereria bancrofti. Calandruccio (1892) noted filarial larvae in the gut of a mosquito and presumed that these were D. immitis, but Grassi and NOB(1900) were the first to demonstrate experimentally the development of D. immitis in the mosquito. Since then, many workers have infected a number of species of mosquitoes with D. immitis and it has also been shown that some fleas may serve as hosts. The literature presents evidence that the same species of mosquito has shown varying susceptibility to D. immitis in different laboratories. Similarly in a survey of the world literature from 1901 through 1949 the experiments of over thirty workers show that Aedes aegypti varied in susceptibility to Wuchereria bancrofti from completely inhibiting the parasite’s development to allowing maturation of infective larvae (Kartman, 1950). Roubaud (1937) found that strains of aedes aegypti from Assam and Tanganyika allowed completely normal development of D. immitis. On the other hand, when a Cuban strain of aegypti was infected from the same host, completely arrested development occurred. Hybrids of Cuban and Assam aegypti showed some filariae with arrested development, 1 Now with the United States Public Health Service, Territorial of Health, Honokaa, Hawaii, T. H. 27

Department

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whereas most, of the microfilariae were able to complete t,heir metamorphosis. Roubaud concluded that, suscept,ibilit8y of t,his mosquito is an hereditary “racial” charact’eristic d&ermined by diverse biological races of Aedes aegypti. Newton, Wright, and Pratt (1945) tested various mosquitoes to determine potential vectors of Wuchereria bancrofti in the United States. It is of great interest to note that a Puerto Rican strain of Psorophora confinnis gave an infectibility of 12% as compared with an 80% rate for a U. S. strain. Other reports in the literature tend to support the hypothesis of a genetic basis for susceptibility of the invertebrate host to certain parasites. Huff (1941) found that susceptibility of Culex pipiens to Plasmodium cathemerium is a recessive character due to a single pair of genes and inheritable along lines of the classical Mendelian 3: 1 ratio. Trager (1942) developed by selective breeding a strain of A. aegypti more susceptible to infection with Plasmodium lophurae than the original colony strain. He concluded that the data did not support Huff’s views on the specific nature of this type of inheritance. A recent report by the Rockefeller Foundation (1948) indicates that it was possible to raise the susceptibility of Anopheles quadrimaculatus to Plasmodium gallinaceum from 20 to 100%. Similarly, Micks (1949) succeeded in elevating the susceptibility of Culex pipiens to P. elongatum from 13 to about 49yo within six generations of selective breeding. Contrariwise, Boyd and Russell (1943) could obtain no clearly defined results in attempting to select a strain of A. quadrimaculatus more susceptible to Plasmodium vivax. Jeffery (1944) failed to produce a strain of Aedes albopictus more susceptible to infection with P. lophurae and Hovanitz (1947) could obtain no genetic effects in six generations of A. aegypti selected for susceptibility to P. gallinaceum. Importance of the individual was elucidated by Huff (1930) when he studied the susceptibilit’y of Culex pipiens to three species of bird malaria parasites, Plasmodium cathemekm, P. elongatum, and P. relicturn. By means of double infectious feedings upon the same or different species of plasmodia he found that practically no correlation existed between susceptibility of individual females to one species of parasite and susceptibility to another species. Bahr (1912) infected Aedes pseudoscutellaris with two broods of Wuchereria bancrofti. Feng (1931) pointed out that different larval stages of W. bancrofti were found in specimens of Anopheles hyrcanus taken at the Woosung district of Shanghai and considered this evidence that the

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same mosquito may be repeatedly infected. A similar observation was made by Hu and Chang (1933) on nat’urally infected Culez pipiens pallens in the same district. Byrd, St. Amant, and Bromberg (1945) working with Aedes pseudoscutellaris and TV. bancrojti in the Samoan area state that many naturally infected individuals of t#his species carried double or triple infections. In experiments specifically designed to determine whether susceptibility to infection with filariae is an inherent characterisGc of the individual mosquito, Hu (1937) gate Culex pip&s pallens spaced double infectious feedings of W. bancrofti. He found that some of the females were susceptible to the first but not to the second infect’ive meal, and vice versa. Phillips (1939) found that an immunit’y to reinfection with Dirojilaria immitis would not develop in Anopheles punctipennis, Aedes cinereus, and A. triseriatus. The resistance of mosquitoes to parasites is apparently manifested in one manner by the formation of a dark-colored capsule around the invading organism. Well known instances of this phenomenon are the so-called “black spores” of Ross and the encapsulation of filariae. Bahr (1912) noted encapsulated larvae of Wuchereria bancrojti in Aedes (Stegomyia) variegatus and described them as enclosed in chitinous capsules. No6 (1901) had also noted encapsulated larvae of Dirofilaria immitis in Malpighian tubules. Fiilleborn (1929) similarly described the encapsulation of Dirofilaria repens larvae in Aedes aegypti. Brug (1932) suggested that encapsulat’ion of the filaria must be considered a means of defense since heavy infections injure or even kill the mosquito. He also indicated that filarial encapsulation occurs rarely and is therefore not a successful means of defense. It is not clear, however, whether encapsulation is of quantitative importance to the cyclical development of the parasite, and t’his remains to be determined as does the nature of the fact>ors inducing encapsulation. The experimental work reported here was designed to (1) obtain a quantitative estimat’e of infectibility of the mosquito host with DiroJiZaria immitis, and (2) to uncover factors affecting the developmental history of D. immitis wit,hin t,he mosquito host in order to establish a preliminary basis for underst’anding variation in susceptibility. MATERIALS

AND

METHODS

Microfilariae from the dog heartworm Dirofilariu immitis (Leidy, 1856) from a mongrel bitch obtained in St. Augustine, Florida in February, 1945 were used throughout the present studies. When these studies were initiated at the end of

30

LEO KARTMAN

December, 1948 the microfilaremia of this dog fluctuated from about 16,000 to 18,500embryos per cc of blood at between 2:30 to 5:00 P.M. Blood counts were made routinely at least once every two months and usually at the initiation of an experiment. No significant variation from the above microfilarial estimate was encountered. Microfilaremia was measured by the method of Franks et al. (1947). Although this dog, “S,” was used as the major source for microfilariae another dog, “D,” was also utilized in a few instances. This animal was obtained from a biological supply company in Sarasota, Florida and brought to the laboratory about November, 1949. Microfilaremia of this mongrel bitch indicated an infection of about 30,000 to 34,000 embryos per cc of blood throughout its experimental use. Some supplemental work was done with the frog filaria, Foleyella brachyoptera Wehr and Causey, 1939. The adults are found in the retroperitoneal spaces and on the intestinal mesenteries of frogs of the genus Rana and are thought to be restricted in their distribution to Florida (Kotcher, 1941). The mosquitoes used in these studies were Anopheles quadrimaculatus Say, 1824, A. jreeborni Aitken, 1939, Culex quinquefasciatus Say, 1823, C. pipiens Linn., 1758, Aedes aegypti (Linn., 1762) and A. albopictus (Skuse, 1895). Exotic strains of Aedes aegypti were obtained from Hawaii (Honolulu), South Africa (Durban), Anglo-Egyptian Sudan (Sennar), and Fiji (Suva). In addition, reciprocal hybrids of Culex pipiens and C. quinquejasciatus, and a hybrid of Aedes aegypti and A. albopictus were produced in the laboratory for experimental use. Anopheles quadrimaculatus was obtained from the Health and Safety Department, Tennessee Valley Authority, Wilson Dam, Alabama. A. jreeborni came from the colony maintained at the National Institutes of Health malaria laboratory in Columbia, S. Carolina. Culex quinquejasciatus was established from egg rafts sent from Galveston, Texas. C. pipiens had been originally collected in the vicinity of Baltimore. The insectary strain of Aedes aegypti is believed to have come originally from the Laboratory of Tropical Diseases, National Institutes of Health at Bethesda, Md., while A. albopictus was originally obtained from eggs laid in September, 1948 by females reared at the U. S. Public Health Service Laboratory in Manila, Philippine Islands and subsequently maintained in the insectary of the Army Medical Center at Washington, D. C. The exotic strains of Aedes aegypti were obtained as eggs which were sent by air from the regions indicated above. The rearing of larvae and maintenance of adults were accomplished in an airconditioned insectary with temperature held at approximately 27°C. The humidity affecting experimental lots of adults was maintained by the use of water-soaked cotton in a manner described below. Anopheles quadrimaculatus and A. freeborni were reared substantially by the method of Crowell (1940) with some modifications. The Culex quinquefasciatus and C. pipiens were fed on coarsely powdered dog biscuit, whereas the anophelines were fed finely powdered biscuit which was sprinkled upon the surface of the water as needed. The two species of Aedes were fed exclusively on rabbit chow (pellet type) and the amounts used varied with the number and instar of the larvae. The pupae of Aedes, and in some cases of Anopheles species, were placed in pint or half-pint ice cream cartons under glass chimneys, about 75 to 100 per carton. The pupae of Culex, and in some cases of the Anopheles species, were put into white bowls in a large cage used for overnight experimental feedings. In the glass chim-

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neys or in the cage the newly emerged females were fed slices of apple until needed for experiments. Colonies of certain experimental strains of C&x and Aedes mosquitoes were maintained in small cages approximately 12 X 12 X 12 inches whereas the stock colonies of all the species used were in cages approximately 18 X 20 X 24 inches. Adults of the culicines were routinely fed on chickens to secure eggs whereas the anophelines were fed on either human or rabbit blood. All experimental feedings of Aedes spp. on infected dogs were made with the females confined inside their emergence chimney and allowed to feed through bobbinet on the cleanly clipped skin of the vertebrate host. Some anopheline feedings were also done in this manner, but most of them, and all of the Culex feedings, were accomplished overnight) in a large cage, 3 X 3 X 236 feet, in which the clipped dog was confined in a smaller metal cage. After feeding, the fully engorged females were removed with an aspirator and confined to cardboard cages made from pint size ice cream cartons with netting at both ends and a tightly corked hole in the side. Up to 50 females were sometimes placed in t,hese cages but survival appeared best when the number was limited to 25. The experimental symbols were written on the outside of the cartons and these were placed in enamel pans lined with moist cotton. On the upper portion of each carton was placed a slice of apple covered by a large pad of watersoaked cotton. Thus the atmosphere within the cartons was kept in a saturated condition. A few experiments necessitated the use of in vitro feeding of mosquitoes on suspensions of microfilariae in dog or frog blood. The anticoagulant used was eit,her 2j$% sodium citrate in normal saline (1:lO with blood), or a “pinch” of heparin placed in the syringe. About 0.5 to 1 cc of infected blood was placed into glass tubes for feeding. These tubes were about 7.5 to 10 cm long and 1.5 cm in diameter. They were closed at one end by a membrane prepared from hog gut sausage casings which served admirably for feeding mosquitoes. Cleaned but not sterile glassware was used and procedures were so arranged that no more than 15 to 20 minutes ensued from the drawing of blood until feeding began. A detailed description of these feeding techniques is given by Eyles (1950). The prepared tubes were stoppered and placed in the artificial feeding unit devised by Greenberg (1949). Aedes aegypti was used almost. exclusivelp for in vitro feedings since it appeared .to respond best, up to 75% or more of the females usually taking blood meals. The mosquitoes were allowed to feed 30 to 50 minutes and, since they fed upon a membrane at the lower end of the tube, the tubes of blood were agitated every five to seven minutes to preclude possible uneven distribution of the microfilariae. After feeding, the fully engorged females were separated, placed in the ice cream carton cages, and maintained in the manner already described. In most experiments an attempt was made to feed as many mosquitoes as possible since some of these species showed considerable mortality after becoming infected. After the infective blood meal a certain number of females were dissected each day and their infections analyzed quantitatively over a period of twenty days. When sufficient numbers of mosquitoes were not available the females were given an infective blood meal and then dissected fifteen t.o sixteen days later for evaluation of their reaction to the par:tsit,es.

32

LEO KARTMAN

Dissections of infected females were done in a manner similar to that for malaria work. Needles were employed in the usual manner and the last two abdominal segments were cut and retracted so that the hind gut, Malpighian tubules, and the midgut were drawn into a drop of normal saline. The tubules were first examined intact and then by gentle pressure on the cover slip they were crushed to release the various stages of the filariae. This was found to have very little adverse effect on larval stages of D. immitis. Up to about eight days after infection only the midguts and Malpighian tubules were examined. At eight or nine days each mosquito was divided into head, thorax, abdomen, and tubules, each of these portions being examined separately in normal saline. During these dissections, when infective larvae were known or thought to be present, the saline containing tubules and gut was examined carefully for larvae which may have been in the abdomen. Ovaries were always taken out intact if possible since they obscured vision if broken up. The head was not macerated until the mouthparts had been separated from it. Usually, infective larvae in the labium could be induced to escape by subjecting them to the heat of the microscope lamp. If the labium and stylets were all in proper position and intact the larvae invariably escaped by penetration of the labella, but any twisting or break in the labium allowed escape of the larvae at some other point. Quite often the labium had to be broken gently with needles to obtain the larvae for examination. For measurements, a drop of 2% formalin was run under the cover slip especially to kill the vigorously moving third stage larvae or microfilariae; other stages were easy to measure while alive. Measurements were made with an ocular micrometer in a calibrated microscope. The following is the writer’s interpretation of some terms used in these studies: Microfilariae (Mf.)-precocious embryos found in the circulating blood of the vertebrate host and also in the arthropod after an infective blood meal. Larvae-developmental stages, beyond the typical microfilariae, found in the arthropod host; infective larvae are the 3rd stage larvae following the 2nd molt. Positive mosquito-a female containing filariae which are not necessarily developing. Infected mosquito-a positive female containing filariae undergoing development. Infective mosquito-a female with active 3rd stage larvae in the labium which are capable of rapid escape. Susceptible host-a mosquito in which the filariae are capable of developing beyond the microfilarial stage. Resistant host-a mosquito in which the filariae are inhibited from developing beyond the microfilarial state. Encapsulation-(“chitinieation” of authors)-formation of a brownish or blackish covering, either partial or complete, about any filarial stage in the mosquito. RESULTS Experiment

1. The Development of D. immitis Mosquitoes

in Thrre Species of

The prevalent method of determining whether a given species of mosquito may serve as a host for a filarial worm is to allow females of that

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species to engorge upon blood containing microfilariae after which the percentage of infected surviving females is recorded at a time deemed sufficient for the development of the larval stages. Although this is a type of estimate of host suitability, it is in no sense a quantitative evaluation of the host-parasite relations involved. Thus it was thought that a day to day analysis of the comparative development of D. immitis in three species of mosquitoes representing three different genera would form the basis for an evaluation of suscept#ibility both from the standpoint of the host and of the parasite. Females of Aedes aegypti, Culex quinquefusciatus, and AnopheZes quadrimaculatus were allowed to feed upon a dog infected with D. immitis and the fully engorged mosquitoes kept for a period of twenty days during which about ten females per species per day were dissected. The A. aeg?lpti females showed a predominance of unchanged microfilariae in their Malpighian tubules and this phenomenon was a daily finding throughout the experiment (Fig. I). On the other hand, in C. quinquejasciatus negative females were observed on the third and each succeeding day throughout, the period of study and practically none had unchanged microfilariae after the first two days (Fig. 2). With A. quadrimaculatus the majority of females dissected each day contained developing stages of the parasite (Fig. 3). The parasite also showed quantitative differences from day to day in the three mosquitoes under consideration (Table I). The majority of worms in A. aegypti failed to initiate growth and remained as undeveloped microfilariae in the Malpighian tubules, whereas most of the filariae that reached the tubules in t,he other two species developed to the larval stages. Some of the unchanged microfilariae in aegypti were still alive fifteen days after being ingested. The rate of growth of the filariae and the final size attained by them in the mosquito may be another index of reactions to different host environments. In Figure 4 the maximum, minimum, and mean lengths attained by the parasites are charted for each day of development over the twent,y day period and it is obvious that in all three species of mosquitoes there is a similar trend from the microfilaria to the short “sausage” form and then to t*he lengthening second and t,hird stage larvae. It is also seen that. various stages occur simultaneously, especially in A. aegypti and A. quadrimnculatus where great numbers of the parasites accumulat,e in the tubules. However, the picture present,ed by A. aegypti is one in which t.here is an erratic development and although C. quinquefawiakrs shows a more consist.ent growth the maximum lengths

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attained by the infective larvae in both these species is less than 950 P. In A. quadrimaculatus there is seen a steady and consistent growth in mean length of the worms and t,he infective larvae attain a maximum length of 1100 ~.r. The appearance of third stage larvae in the Malpighian tubules oc-

FIG. 1. The daily proportion of female Aedes aegypti (above) and A. albopictus exhibiting various degrees of development of D. i~~mitis during a twenty-day period. Each dot representas one mosquito and the same individual may be shown in more than one, or in all, of the squares for a particular day (unless in the “negative” square).

curred on the twelfth day aft,er the infective meal in A. quadrimaculatus and on the two succeeding days for C. quinquefasciatus and A. aegypti respectively. By the thirteenth and fourteenth days, C. quinquefasciatus and the anopheline showed infective larvae in the labium whereas these were not see11in A. aegypti until the sixteenth day. It should be noted that the rate of development of many individual worms in A. quadrinzaculatus was as rapid as that in C. quinquefasciatus despite the fact that over four times as many worms were recovered in 210 females of the former species as compared wit,h the same number of females in the

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latter. In the case of il. aegypti, which showed a similar intensity of invasion to A. quadrivnaculatu~s, t)he rate of filarial development was retarded. A distinctive feature of larval growth in A. quadrimaculatus was the finding of degenerate forms in the tubules beginning about the fifteenth

FIG. 2. The daily proportion of female Culcx quinquefasciatus (above) and C. pipiens exhibiting various degreesof development of D. immilis during a tmenty-

day period. Each dot represents one mosquito and the same individual may be shown in more than one, or in all, of the squaresfor a particular day (unless in the “negative” square). day after the infective meal and occurring each day thereafter until the experiment was terminated. These were mainly first or second stage larvae charact’erized by occasional vacuolation of the internal structure and more commonly with the cuticula covered by tubercle-like structures and pseudoannulations suggestive of arthropod larvae. These forms almost always showed activity and amounted to 0.477e of all filariae recovered in the 210 females dissected. No such larvae were found in A. aegypti or C. quinqu.ejusciatus. The invasion of A. aegypti by D. immitis was attended by a consistent

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LEO KARTMAN

encapsulation of the microfilariae, whereas C. quinquefasciatus showed no such phenomenon and A. quadrimaculatus presentNeda negligible encapsulation of the microfilariae and first, st)agelarvae (Tahle II). The percentage of encapsulation of all microfilariae found in A. aegypli was low yet it should he noted that females showing encapsulated em-

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FIG. 3. The daily proportion of female Anopheles quadrimaculatus (above) and A. jreeborni exhibiting various degrees of development of D. immitis during a twenty-day period. Each dot represents one mosquito and the same individual may be shown in more than one, or in all, of the squares for a particular day (unless in the “negative” square).

hryos were found on the third and each succeeding day following the infective meal. If only those females showing encapsulated microfilariae are taken into account, then 12% of t,he total worms found in these females were encapsulated. In addition to this, A. aegypti females showed 0.04 and 0.2’3, microfilariae encapsulated in the midgut and haemocoele respectively; those in the latter site being found exclusively among the tracheoles on the outer gut wall. The encapsulation of the microfilariae in A. aegypti appeared to follow a consistent chain of events. It began either at the anterior third or half,

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I,UJfITIS

t,he posterior t,hird or half, at both posterior and anterior tips, or only in t,he middle pm&on of the parasite. The microfilariae were invariably a,rtive at, this stage and the capsule was represent’edby a very thin coating TABLE I Location and Numbers of D. immitis in Aedes aegypti, Culex quinquefasciatus and Anopheles quadrimaculatus during a Twenty-Day Period -

No. 0 0 _ dissected

Mf in midgut

Day

UIldevejoyd hl. tubules

“,yy, M. tubules

AACQAQ

AACQAQ

Larvae 3 in M. tubules

:

--__ ACQAQ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 __Total

AACQAQ

9 8 10 147 334 11 46 7 192 0 0 10 10 10 92 342 6 186 0 98 7 5 7 11 10 51 116 0 176 0 35 3 1 0 0 461 1 10 6 8 13 10 10 62 14 10 10 30 0 0 289 0 9 3 7 0 0 175 0 12 14 5 8 11 10 0 0 0 256 0 14 28 8 13 10 10 0 11 10 10 0 0 0 293 0 4 18 3 0 0 356 0 1 11 5 14 11 10 0 0 0 253 0 4 5 3 7 14 10 0 13 10 10 0 0 0 347 0 3 14 7 11 10 10 0 0 0 171 0 0 9 8 0 0 154 0 2 6 5 11 10 10 0 0 0 143 0 1 3 2 11 15 10 0 0 0 127 0 0 4 0 14 10 10 0 15 10 10 0 0 0 153 0 0 4 1 0 0 184 0 0 5 3 16 10 10 0 0 0 134 0 0 4 1 15 10 10 0 0 0145 0 0 10 9 10 10 0 0 0 95 0 2, 1 0 8 10 10 0 --IOo-I--_________~ 129 210 210 382 792 17 4144 8 388,146 72 _____ __--8.183.10.488.10.89.2;3.27.658.3

I-

AACQAQ 0 174 248 209 138 188 181 209 217 208 148 107 220 132 19 13 14 9 4 10 3

0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 1 2 0 2 0 --

0 0 0 0 0 0 0 0 0 0 0 0 1 1 5 3 0 0 1 0

AACQAQ -.~-__ 0 0 0 0 0 0 0 0 0 0 0 34 47 72 1 15 23 12 3 10 3

AACQAQ -__

-----

---------

-

--

-I--

-

----

-----,--

-

---

-

-0 0 0

0 0 0

0 0 0 0 0 1 0 1

2 4 1 1 2 1 2 2

000 000 000

0 0 0

002 0 54 0 16 5 108 0 6 10 134 1 4 27 121 6 12 50 89 10 5 52 100 0 6 70 30 2 4 86 - 67 - 124 ____ -- --__ 2451 8 11 220 2 15 703 19 55 424 --____0.21.25.2 (b.04 1.6 16.7 0.4 5.8 10.2

* Haemocoele includes abdomen, thorax, and head.

with fine cross striations apparently corresponding to the striations on the cut&la. Occasionally, a worm was found to be half encapsulated and in this case the free portion, either anterior or posterior, was quite active. The majority of completely encapsulated forms evinced no movement whatsoever and were further characterized by a decided thickening and heavy encrustation of the capsule so that the cross striations were no longer visible. Although completely encapsulated microfilariae were found as early as the third day, these increased in number, beginning at

-

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.

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1100-1149 105olo99 Kso4J49

950~SBANOPHELES

QUADRIMACULATUS

t

llll

FIG. 4. Growth in length of D. immitis in Aedes aegypti, Cules quinquefasciatus, (See opposite page) and Anopheles quadrimaculatus. The central dot on each vertical line represents the approximate mean length of the filariae, with maximum above and minimum below. TABLE II of D. immitis in Three Species of Mosquitoes

Extent of Encapsulation

I Species

;

Encapsulation g&$(j

y=$&:y

_ / _.--__!----.

Aedes aegypti.. Culex quinquefasciatus Anopheles quadrimaculatus

I

229 210 210

-;$yzStage:jg __ .-. 2L

4701 953 4203

3rd stage -

-e

5.3 0 0.09

of:

I_ %

e

0.1 0 0.2

0 0 0

0 0 ~ O

about the seventh day. Most of the encapsulated forms were usually encountered lying together in an expanded sac-like structure at the distal end of an individual Malpighian tubule. At the end of the twenty day period, of 236 encapsulated microfilariae found in the tubules, 66 were partially and 170 were completely encapsulated.

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Thus far two main types of analyses of host differences have been present,ed,namely: (a) the infectibility of the female mosquito, and (b) the development of the parasite. It was thought that some simple numerical expression of the parasite’s developmental dynamics would serve as an index of host differences which might be utilized in place of the prevalent concept of per cent of females infected. This expression might be formulated in terms of a ratio between the number of developing filariae and the theoretical number of microfilariae originally ingested by a mosquito during its infective blood meal. The actual numbers of developing D. immitis in the three species of mosquitoes under consideration are the totals found over a period of twenty days when infected females are maintained under comparable conditions and similar numbers of females are dissected per day. The theoretical numbers of microfilariae ingested are based on the numbers of microfilariae ingested by thirty-five females per speciestaken from the lots of females used in the actual experiments. The product of the average number of microfilariae found in the midguts of these females and the number of females dissected gives the theoretical total number ingested. The theoretical number of microfilariae ingested by 229 A. aegypti females was 6183 while the total number of developing larvae found in these females over a twenty-day period was 175. If 175 is divided by 6183 the result is 0.03 which may be called the host ejkiency of these females. Using a similar relation involving only the 29 third stage larvae found, the figure derived is 0.004 or what might be termed the infective potential.

The theoretical number of microfilariae ingested by 210 C. quinquefusciatus was 7875 and the total number of larvae found in these females was 153 thus giving a host efficiency of 0.02. The 81 infective larvae found gave an infective potential of 0.01. In the case of A. quudrimacuZutus 210 females ingested a theoretical total of 6322 worms and 3798 larvae were actually found in these females, giving a host efficiency of 0.6. Since these females had 1347 infective larvae, their infective potential was 0.2. As the percentage of ingested filariae undergoing development increases, these ratios approach a value of 1. Experiment 2. The Development of D. immitis in Phylogenetically Host Mosquitoes

Related

Since certain species of mosquitoes represent morphologically related types, studies were undertaken to compare the development of the dog

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frlaria in forms taxonomically related to those used in Experiment 1. The species used were Aedes albopictus, Culex pipiens, and Anopheles freeborni.

In comparing the two Aedes speciesit is apparent from Fig. 1 that the TABLE III Location and Numbers of D. immitis in Aedes albopictus, Culex pipiens, Anopheles freeborni during a Twenty-Day Period

ond

Larvae 3 in Larvae 3 in haemocoele* labium

---

______~ !4LCPAF

--

-I-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 -Total __-

ALCPAF

ALCPAF

ALCPAF

______ 10 11 IO 10 10 10 10 10 10 10 10 10 10

10 171 1150 47 298 6 173 0 0 10 66 294 9 92 6 22 171 7 10 7 3 0 16 0 18 239 3 10 6 0 0 14 0 17 271 4 10 0 0 0 10 0 10 211 5 10 0 9 258 6 0 011 0 10 0 9 248 8 0 0 6 0 10 0 8 197 5 0 0 5 0 6 161 3 10 0 0 0 3 0 4 189 3 10 0 0 0 1 0 2 Ii2 4 10 0 0 0 6 0 10’ 0 3 160 0 0 0 0 0 3 151 0 10 0 0.0 1 0 0030 1 97 0 :: :i 3 i 40 0 0000 0 10 10 10 0 0010 1 13 1 10 10 10 0 6 0 0000 2 10 10 IO, 0 5 0 0000 0 10 10 10 0 0010 2 71 10 10 10 0 0000 0 50 ~. Ye101 201 2001240 1447 56 468 12 290 2601 50 ---I--___ -,5.1 93.1 2.119.7 0.8 10.71 55.5 3.2

--

%

--

10 10 10 10 10 10 10 10 9 10 10 11 10

ALCPAF I_-

AL CP AF

AL CP AF ___-

-0 () - ---l --0 I) -- --0 f) -- --0 [) -- --0 () -- --0 () 0 (1 --I ---- 0 I1 --~--O0 L) 0 LI 00 0 0 1 0 (1 02 0 0 2 1 !3 00 0 0 3 1 2:1 00 0 0 2 0 I3 81 1 47 3 5 1 2:1 132 0 7 40 3 0 !? 167 1 29 27 4 0 I5 123 0 45 37 5 1 I3 142 1 57 54 2 0 !5 133 0 60 65 3 I 93 2 42 74 4 _. ___1762 205 4 813 871 7 287 300 34 _. __--___ -65.01 4.3 0.3 3.12 19.8 0.5 10.5 5.6 2.1 0 0 217 0 125 0 123 0 118 0 128 0 138 0 129 0 119 0 128 0 122 0 128 10 105 64 62 34 64 26 19 18 21 22 10 13 9 10 7 80.

-

0 0 0 0 2 3 39 36 47 42 60 229 8.5

1

majority of female albopiclus exhibit a predominance of developmental forms of the parasite throughout a twenty day period whereas aeggpti females are refractory. The picture presented by albopictus females is much like that shown for females of Anopheles quadrimacukztus (Fig. 3). A comparison of C. pipiens with C. quinquejasciatus shows an essential similarity (Fig. Z), and Fig. 3 indicates a similar reaction of ,4. qua&inzaculatw and A. freeborni females to invasion by the parasit’e.

42

LEO EARTMAN

The data for development of the parasite in these related species are shown in Table III and should be compared with the corresponding information in Table I. The same general relations are borne out here as are evidenced by data on the host. The growth of the filariae in A. albopictus was almost identical to that shown by A. puadrimaculatus, the infective larvae in labium and head reaching a length of 1100 ~1.Also, TABLE IV Rating of Related Species of Mosquitoes I ‘2 Species

Theoretical No. of microfilariae ingested0

0-3 8s ski “02 .a z

Aedes aegypti. ............... Aedes albopictus.............. Culex quinquefasciatus ........ Culex pipiens ................

229 201 210 201

C&.x hybrid P. ............. Culexhybrid IIf. ............

210 244

Anopheles quadrimaculatus .... Anopheles freeborni. ..........

210 200

-

as Hostsfor D. immitis Total infective

II&C-

tive “$.’ 2% P;.$ciencyC in Xl-day period ___ __ .__

6183 4701 175 29 0.03 6800 4685 3977 1376 0.6 7875 953 153 81 0.02 12100 1554 95 45 0.008 5570 707 181 58 0.03 10980 749 262 110 0.02 6322 4203 3798 1347 0.6 4600 2710 2364 602 0.5

0.004 0.2

0.01 0.004

0.01 0.01 0.2 0.1

a Baried on the number of microfilariae ingested by 35 females taken from the lot of mosquitoes wed in the actual experiments; derived by: avg. no. mf. per 0 X no. ‘2 0 dissected. b Includes all larval stases; i.e.. 1st. 2nd. and 3rd. Total developing larvae . c Derived by: Theoretical total mf. ingested Total infective larvae . d Derived by: Theoretical total mf. innested e C. quinqueJasciatuscPd x C. pipiens 0 0 ; F2 generation. f C. quinqueJasciatvs 0 0 X C. pipiens cTd; Ft generation.

in contrast to aegypti, only one female in 201 of albopictus harbored encapsulated filariae. These were found on the eighth day after t,he infective meal and consisted of two encapsulated early first stage larvae, giving a total of 0.047o of all filariae seen in the females dissected. A comparison of parasite development in C. pipiens and C. quinguefasciatus shows an essential similarity, although there appears to be a substantial drop in numbers of developing D. immitis in pipiens as compared with quinquefasciatus (Tables I and III). The low intensity of infection in pipiens may account for the fact that the first infective larvae were noted in the labium on the tenth day in this species. In

INFWTION

OF

MOSQUITO

WITH

DZROFZLARZA

ZMMZTZS

43

pipiens the infective larvae attained a maximum length of 1100 P whereas those in quinquefasciatus were below 950 p. As a matter of fact, the maximum mean let&h attained by the parasite in pipiens was over 1000 p, whereas the corresponding figure for quinquejasciatus was about 900 p. Now of the filariae were found to undergo encapsulation in either of t,he C~tZrz species st,udied. Tables I and III show clearly that’ t’he development of the parasite proceeds almost identically in A. quadrimaculatw and A. jreeborni. The similarity is much more striking than in the Culex species just discussed. Besides comparable percent’ages of developmental stages, these species showed a similar rat,e of parasit#e growth in t,hat the infective larvae appeared in their t’ubules and reached their mout,hparts rapidly and attained similar maximum and mean lengths. Degenerate larval forms similar to those already described in quadrimaculatus were found in jreeborni females beginning with the fifteent,h and continuing until the t#wentieth day. These were mainly second stage larvae. Five encapsulated first st.age larvae mere noted between the fifth and sevent$h days in jreeborni, amounting to about 0.2% encapsulation in this species as compared wit,h an almost identical finding in quadrimaculatus (Table II), If the concept of host efficiency is applied the results obtained indicate t,hat A. aegypti and A. albopictus show profound differences in seaction to the parasite under the present circumstances. On the other hand, A. quadrimaculatus and il. freeborni are almost identical and C. quinquejasciatus and C. pipiens are within a similar range. Table IV summarizes these calculations for all the species already discussed and also for certain hybrids analyzed in Experiment 3. Experiment 3. The Development of D. immitis Aedes Mosquitoes

in Hybrids of Culex and

Since Culex pipiens and C. quinquejasciatus have been the subject,s of hybridization studies it was considered worthwhile t,o observe the reaction of hybrids of these species to invasion of D. immitis. The stock colonies of pipien.s and quinquefasciatus were given blood meals simultaneously on chickens and egg laying was synchronized by offering them water for this purpose eight days after the blood meal. About fifty to seventy pipiens and equal numbers of male quinquejasciatus were put into cages (12 X 12 X 12 inches) for mating. The reciprocal of this cross was produced in the same manner. All adults were obtained from previously sexed and isolated pupae. When adults of the

44

LEO KARTMAN

F1 generation had emerged they were kept in the cages, fed blood meals and the F2 generation thus obtained from brother X sister matings. The Fz females were allowed to feed overnight on the infected dog and all fully engorged individuals were collected the next morning and subsequently dissected daily over a twenty-day period as previously de-

FIQ. 5. The daily proportion of female Culez hybrid I (above) and hybrid II exhibiting various degrees of development of D. immitis during a twenty-day period. Each dot represents one mosquito and the same individual may be shown in more than one, or in all, of the squares for a particular day (unless in the “negative” square).Hybrid I-C. pipiens 0 0 X C. quinquefasciatus d 3 ; hybrid II-C. quinquefasciatus 0 9 X C. pipiens $3.

scribed. Females of both hybrid generations appeared to feed more avidly on the dog and in greater numbers than did either of the parent species. Hybrid I represented progeny of pipiens 9 9 X quinquefasciatus ~7$ and Hybrid II, of quinquefasciatus 9 9 X pipiens 8 d. The pertinent information for distribution of females in the various categories of parasite development during the twenty-day period is given in Fig. 5. The striking similarity of t,hesegraphic representations to t,he corresponding ones for the parent species (Fig. 2) is evident at a glance.

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

45

IMMlTlS

Development of the parasite in these Culex hybrids is shown in Table V. Infective larvae were noted in the Malpighian tubules on the tenth TABLE Location

V

of D. immitis in Reciprocal

and Xumbers

quint No. 9~ dissected

IKf in midgut

HI

HI -I_

I

Ulldev&qrd hf. tubules

D&Y

Hybrids

of Culex

(F2)

efasciatus and C. pipiens” -

Larvae 3 il M. tubules

b’

.arvae 3 in labium

--___

1

2 3 4 5 6 7 8 g 10 11

12 13 14 15 16 17 18 19 20

HI1

10 10 10 10 10

10 11 16 12 10

10

14

11 10 10 I1 10 10

13 I1

14 10 10 14

16 I1 16 11

10

20

10 11 I 10 1 10 10

10 12 10 10 10

I

Total i 210 244 -___--__ % i__

HII

HI

HI HI1 ~~ -.__

HII

5

5

0

10

0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

7 4 10 18 9

16 3 12

480

6

7

218 301

416 54

1 0 0 0 0 ‘0 0 0 0 0 0 0 0 0 0 0 0 0 --__ 520

73.5 64.1 -.__

0.8 0.9

_-

10 7

II 14

11 14

8 30 6 3 0 0

38 19 5 3 4 0

1 0

0 0

2 0 0 0 ___-

0 0 0 0

123

152

HI HI I ~-___ 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

1 2 12 0 0 0 0 0 0 1 0

1

0 0 0 0 0 0 0 0

5

5

2

-I_ 17.3 20.3

-

n Hybrid I = C. pipiens 0 0 X C. quinquefasciatus $8. pipiens $8. * H~omocoele includes abdomen, thorax, and head.

Hybrid

0.7 0.7

HI

HI. 1 HI

-------

---------

0 0

0 0

2 0

5 2

1

5

12 0 7 13 0 3 4 3

1 0

4 4

10 38 -- -__

HI1

0 0 1

0 0

1

8

9 3 7 6 5 4 4 3

7 7 10 4 5 6 6 5

43

67

9

_-

1.4 5.1

-- III = C. quinquefmxbtus

6.3 8.9 0 0 X C.

day and in the labium on the eleventh day for both hybrids and this rapid growth appears more like that found in pipiens t’han in quinquej’asciatus. Another similarity to pipiens shown by both hybrids is the fact t,hat infectfive larvae in them reached a maximum length of 1200 I.C.This

46

LEO

KARTMAN

is over 250 P longer than the maximum length of infective larvae in quinquefasciatus and 100 p longer than those in pipiens. As in both parent species, neither of the hybrids exhibited encapsulated filariae at any time. An analysis of host efficiency and infect,ive potential showed both hybrids to be almost identical with quinquefasciatus (Table TV). TABLE Susceptibility

VI

to D. immitis of a Hybrid from Aedes aegypti and A. albopictusa

Findings 16 days after infective blood meal

--I__. _______ % No. % No. % No. % No. % No. % -_.-_--. __

No. females fed. . No. females dissected. No. females positive. With unchanged Mf in tubules. With encapsulated Mf in tubules. . With larvae 1 and 2 in tubules. . . With larvae 3 in tubules and haemocoeleb. With larvae 3 in labium L Total females with developing larvaec. . Total females with infective larvae.. .

-

43 42 36 45 j6.2 30 i9.7 30 71.4 32 88.8 28 30.8 28 13.3 26 86.6 30 93.7 28

45 62.2 31 68.8 00.0 30 96.7

40.8 28 13.3 0

0

00.0

0

0

30.8

2

6.6

0

14.2

0

0

3.8

2

0 0

0

30 93.7 28 5 15.9

4

6.6 25 83.3

4 12.5

2

7.1 29 93.5

0 0

0 0

2 0

6.2 0

2 1

7.1 3.4

3.8’

2

6.6 26 86.6

4 12.5

2

7.1 30 96.7

0

0

0

2

7.1

3 10.0 3 10.0

3 10.0, 2

6.2

7 22.5 3 9.6

7 22.5

a Hybrid-A. aegypti 0 0 X A. albopietus$3. b Haemacoele includes abdomen, thorax, and head. E Developing larvae include I&, 2nd. and 3rd stage larvae.

The Aedes aegypti and A. albopictus strains available appeared to present another opportunity for this type of study since they were shown to constitute mutually exclusive host environments for D. immitis, and because the two species are capable of being crossed in the laboratory. The production of eggs by the aegypti and albopictus females was synchronized as already described in the preceding work with Culex hybrids. About 50 female aegypti and 50 male albopictus were put into a small cage (12 X 12 X 12 inches) for mating. Of four separate mating trials during the course of the work, sufficient numbers of hybrid progeny were

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

47

IMMITIS

obtained only twice since most of the eggs produced in the other trials failed to hatch. All hybrid adults seen (F1 and F2) appeared to resemble the aegypti female exclusively. It may also be noted here that an attempt to cross aegypli males with a2bopictzcsfemales failed entirely in six trials.

Susceptibility

TABLE VII to D. immitis of a Hybrid from Aedes aegypti and A. albopictusa; Parasite Data

-

! Hyttid Findings 16 days j after infective blood II---

1

Ada aegyfli

A. 1 albopictus I__

meal

No.

ii-j

I-

,-

Total filariae recovered. . . . . 104 -

Mean filariae per female. . 4.0 No. unchanged Mf in tubules. 83 No. encapsulated Mf in tubules.. . . . . . 20 No. larvae 1 and 2 in tubules.. 1 No. larvae 3 in tubules and haernocoeleb.. 0 No. larvae 3 in labium. . . . 0 Total develop1 ing larvaec.. Total infective, IMTW.. . . j 0

-

-

%

-

-I-

NO.

A aqjgti

A. albo~ictus

-__ % No. % No. % -__ --__ -

130 -

126

-

207 -

4.3

4.2

-

6.4

182 i7.9 161 8;6.5

79.8 125

-

-7

No. j % I No.

’

Hy::d I

6.1 IQ

0

0

0

0

19.3

3

2.3’

0.9

2

1.6 106 84.1i

0

0

0

11

8.; :

0

0

0

9

7.: 2

0.91 I 01

2

1.6’ 126 0o.c1’ 18

0

0 j 20

-I

-

-

7.3

-

6.6

0

0

0

0

12

4.8

3

1.6 194 85.1

8

4.0

9

4.8

26

11.3

0

0

1

0.7

8

3.6

8.8

8 4.0 $1 -

15.C

228

3.3

I’

- I

-

186 -

13

6.4

7.1 228 00.0

10 5.5

34

14.9

a Hybrid--A. aegypli 0 0 X A. albopictus dd. b Haemocoele includes abdomen, thorax. and head. c Developing larme include 1st. Znd, and 3rd stage ltlrvae.

Of the successful hybrids obtained, a batch of F1 females was fed on the infected dog and dissected sixteen days later. A batch of Fz females, produced by F1 brother X sister matings, was also dissected sixteen days after the infective meal. Control batches of aegypti and albopictus females were infected simultaneously with the hybrids and treated in an identical manner. Data for F1 and Fz Aedes hybrids are presented in Tables VI and VII.

48

LEO

KARTMAN

These data show that all of the F1 and F2 hybrid females reacted to D. immitis in a manner virtually identical with that of the refractory aegypti controls. The albopictus controls exhibited an infection rate the analysis of which has already been shown to constitute a high degree of host efficiency. Experiment 4. The Developmentof D. immitis in Geographically Separated Strains of Aedes aegypti As suggested earlier, the susceptibility of Aedes aegypti to D. immitis may be a physiological character tending to change from one region to another just as other adaptive characters have been shown to be modified by geographical and ecological influences (Huxley, 1943). In order to test this hypothesis experimentally an attempt was made to obtain eggs of A. aegypti from widely separated regions. Eggs were obtained and colonies successfully established of aegypti from Hawaii, South Africa, Anglo-Egyptian Sudan (AES), and Fiji. Rearing was synchronized so that females of all exotic strains and of the laboratory colony (U. S. strain) were available for simultaneous testing. A total of four trials were conducted with this material, the Fiji strain being tested only twice since it arrived late. Females of the various strains were fed on the same dog within a time range of about one and one half hours. The engorged individuals were separated and kept in the usual manner for fifteen days; they were then dissected and data recorded for both host and parasite. All of the strains, except the Hawaiian, showed similar numbers of females with maturing parasites in each of the separate trials. The total average percentage of females with developing larvae in the U. S. strain was 12.5; S. African, 7.9; AES, 7.1; Fiji, 7.7; and Hawaiian, 22.3. The total average percentage of females with infective larvae in the U. S. strain was 11.6; Hawaiian, 17.1; S. African, 6.7; AES, 7.1; and Fiji, 1.8. With regard to encapsulation of the microfilariae, the various strains showed the following total average percentages of females with these forms: U. S. 11.7; Hawaii, 14.9; S. Africa, 21.6; AES, 22.6; and Fiji, 52.0. Thus the Fiji females were shown to predominate in another refractory characteristic. The data for parasite development in females of these various strains indicate a similar situation with perhaps more clarity. The total average percentage of developing larvae for each strain in all the trials was found to be U. S. 7.0; Hawaii, 38.1; S. Africa, 6.4; AES, 11.4; and Fiji, 3.7. The

INFECTION

OF MOSQUITO

WITH

DIROFlLARl.4

IMMITIS

49

total average percentage of microfilarial encapsulation showed a distinct contrast between the Fiji strain on one side and all of the remainder on the other. The respective percentages for encapsulation were as follows: U. S., 3.1; Hawaii, 3.3; S. Africa, 10.1; AES, 7.2; and Fiji, 34.4. The total average percentage of infective larvae in the U. S. strain was 4.2; Hawaiian, 13.2; S. African, 3.7; AES, 9.4; and Fiji, 0.3. It should be noted that the high percentage of developing larvae in the Hawaiian strain is based mainly on the occurrence of several females whose reaction to the parasite resembled that of such susceptible mosquitoes as A. albopictus or A. quadrimaculatus. For example, one female in trial 2 had 55 pre-infective larvae in its Malpighian tubules and another female in trial 3 harbored 30 pre-infective and 6 infective larvae in its tubules and 5 infective larvae in its labium. Experiment 5. Double Infective Feedings of A. aegypti on Two Species of Filaria

The availability of the frog filaria, Foleyella brachyoptera, provided a means of t’esting the reaction of a mosquito species to infection with two species of filarial parasites. Experiments with a total of four lots of female aegypti lvere conducted. Lots 1 and 2 were given simultaneous feedings on suspensions of D. immitis and F. brachyoptera microfilariae, while lots 3 and 4 were first fed F. brwchyoptera in suspension and then fed D. immitis directly from the dog several days later. Lots 1 and 2 were dissected respectively at fifteen and twelve days after the infective feed, whereas lots 3 and 4 mere dissected respectively at fifteen and twelve days after exposure to F. brachyoptera (eight days after exposure to D. immitis). Two control lot’s were fed independently on D. immitis or F. brachyoptera and dissected t’hirteen days after the infective meal. For the in vitro feedings about 0.5 cc each of infected frog and dog blood were mixed and this appeared to have no adverse effect upon either species of filaria as far as could be determiued from microscopic observation. The heat of the artificial feeding apparatus (seeGreenberg, 1949) also appeared to have no ill effect upon the frog filaria since suspensions of these worms looked quite normal after having been subjected to the heightened temperature for at least fifty minutes. Heparin was used as anticoagulant throughout. No difficulty was encountered in distinguishing the two species of filaria in the mosquito since all stages of D. immitis occur in the Malpighian tubules whereas those of F. brachyoptera are found in the thorax

50

LEO KARTMAN

or other portions of the haemocoele. By the fifteenth day very few, if any, of the infective D. immitis larvae had migrated from the Malpighian tubules in aegypti and those that had were easily distinguished from the TABLE VIII Double Infective Feedings of Aedes aegypti with Two Species Simultaneously or at Intervalsa

I

Lot NO.

-

Days after

infective meal

-

T

-_15

12

15

25

12(FB) WI)

20

13

20

13

--

33

15(FB) 8(W

35

FB

--

-

1SO

%

15

25

1X0

-T

%

M. tubules

93.9

21

9

100.0

100.0

20 100.0

-

1r,, ItlO.

cOd -

63.6

60.0

14 56.0

7

20 100.0

-

-

-

--

-

31

Yo.filariae stage and locationfor eachsp.

0 0 positive for: DI

NO 99

of Filaria Fed

-

35.0

-

20 57.1

1c 58 mf. 11 encap 12b 5c

La. iur -

0

89 mf. 4 encap 0 5”

0

12 mf. 6 encap 15b

0

0

94 mf. 6 encap 9b

0

0

93 mf. 6 encap 7b

0

0

-

-

-

- a DI-D. immitis. FB-F. brachyoptera. C-controls. Haemoooele-includes head. Mf-miorofilsrise unchanged. Encap.-encapsulation of any stage. b 2nd stage larvae. e 3rd atrrge larvae.

FOl~y.dlO brachyoplcra

kin 98 mf. 0 encap 62b 82” 56 mf. 0 encap 61b 33”

73c

210

58 mf. 0 encap 330 78” 47” 31 mf. 0 encap 295 14c -

64 mf. 0 encap 84b 28=

at #doImen, thorax.

10”

-

32~

and

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

IMMITIS

51

F. brachyoptera infective larvae by their greater length and width and by the presence of terminal papillae not present in Foleyella. The pertinent data for this experiment are summarized in Table VIII. The most important observation derived here is that aegypti reacted independently to each species of filaria whether ingested simultaneously or at spaced intervals. The development of D. immifis appeared no different from the usual picture given in aegypti, i.e., a fairly refractory reaction by the mosquit’o resulting in a preponderance of unchanged microfilariae and in some microfilarial encapsulation, but with about 100% of the females positive. These same aegypti females showed a range of positives for F. brachyoptera of 35 to 637$ and the frog filariae exhibited a vigorous development since many infective larvae were found in the labium as early as the twelfth day. Many unchanged frog microfilariae were found in the abdominal and thoracic cavities, but of special interest is the absence of any encapsulation of t#he frog filariae in individuals with encapsulated D. immitis. Experiment 6a. Selection of the Mosquito Host (A. acgypti) to Establish Strain Differences in Susceptibility to D. immitis The nature of the reaction of A. aegypti to microfilariae of D. immitis indicated that in most females the embryos remained unchanged while in a few individuals larval development occurred. The presence of some females with developing larvae suggest’ed that a more susceptible strain might be developed through the medium of selective breeding. Male and female aegypti of the parent lot and succeeding selected lots were first allowed several days for copulation and t,hen the females were fed upon the infected dog. The engorged females were separated and placed singly in test tubes each of which was provided with a strip of filter paper kept moist by a few drops of water at the bottom of the tube. The eggs were usually deposited upon the moist filter paper. Each tube had a cotton plug holding a tooth pick inserted into t’he tube with a raisin impaled upon its lower tip to provide food for the mosquito. All tubes were numbered and held in a rack for fifteen or sixt*een days; the surviving females were then dissected. The extent of parasite development within these females determined whether previously laid eggs were retained or discarded. All experiments were checked with control lots of females taken from the laboratory colony. The major difficulty encountered was the high mortality rate of infected females in these test tubes. As an example, of 125 parent females

52

LEO

KARTMAN

isolated singly in test tubes only 20 or 16% survived to be dissected after oviposition. Attempts to maintain them in half-pint size milk bottles (Trager, 1942) indicated no essential difference in result. The high mortality dictated two essential methods of procedure: (1) the type of selection was primarily one of mass selection, and (2) certain filial generations were used solely to increase the available females in the selected strains by brother X sister matings. These experiments included eight generations of aegypti and infections with D. immitis were studied in the parent, Fz, Fd, Fe, and Fs generations. The initial selection of strains from the infected female parents resulted in the following: eggsfrom six females exhibiting unchanged microfilariae exclusively; eggs from six females showing unchanged and encapsulated microfilariae; and eggs from two females with normally developing larvae. Of these, five lots were finally retained as follows: strains # 30 and # 67 selected for susceptibility, and strains # 19, B 41, and # 87 selected for resistance. Strain # 19 was later lost since the Ft females refused to take blood meals, and strain B 30 was carried only through the Fa generation since eggs selected from it failed to hatch. Detailed analyses of both host reactions and parasite findings are presented in graphic form in Fig. 6. No consistent trend toward increased susceptibility of the females was noted in strain B30, whereas strain # 67 showed an increase in susceptibility from the Fz to the F8 without the fluctuations seen in strain # 30 and the controls. On the other hand, when the parasite data are analyzed there is seen a steady increase in percentage of developing larvae in strains Y% 30 and R 67 with the exception of a sharp drop in Fs for the latter strain. The controls exhibited no substantial trend of changes in parasite development. Females in strains # 4 1 and 187 showed a definite drop in numbers harboring developing larvae and both strains contained no females with larval stages in the Fe generation. This trend was not maintained since both of these strains exhibited larvae in the F8. Data on parasite development in these refractory strains show strain #41 absolutely negative for larvae in the Fq and Fe generations. A rise in larval development in the Fs, although equal to t$hat seen in susceptible strain B 67, is still far below all other percentages of development attained by the susceptible strains. If the total average percentage of females with filarial development and of developing parasites is calculated for each strain, the refractory strains are seen to fall below the susceptible ones with the pare& and

FIG. 6. Effect of selection on susceptibility of A. aegypti to D. immitis. AProportion of filariae in indicated stage of development. B-Proportion of females showing indicated development of filariae. A-Developing larvae include lst, 2nd, and 3rd stage larvae. Upper left-Strain So. 30, susceptible. Upper rightStrain No. 67, susceptible. Middle left-Strain No. 41, resistant. Middle rightStrain No. 87, resistsant,. Lower left---Controls for indicated generations of selected strains.

53

54

LEO

KARTMAN

controls occupying an intermediate position (Table IX). This table presents data for encapsulation of the microfilariae which suggest that the refractory strains led all others in this characteristic. It also should be noted that the refractory strains were generally devoid of infective larvae even during those generations in which pre-infective forms were noted in them. Thus although strain B 87 had developing larvae in the FP, Fq, and F8 generations, these included no third stage larvae whatsoever at the time of dissection. TABLE Summary

of Filarial

Development

IX

and Encapsulation

in Selected Strains

of

A. aegypti Total average % of:

30 67 19 41 87 Parents. . Controls, l

S-susceptibility.

S S R R R -

3 4 2 4 4 1 4

40.3 42.5 5.0 9.5 8.7 25.0 28.0

36.3 39.3 1.5 3.2 3.2

Total average % encapsulation:

9.6 19.2 51.5 40.0 44.2 65.0 25.2

1

3.3 7.2 23.5 28.5 35.0 10.8 7.0

R-resistance.

Experiment 6b. Selection of the Mosquito Host (Culex Hybrid) to Establish Strain Diflerences in Susceptibility to D. immitis Previous data have shown that the Culex species used in t,hese experiments gave a less complex reaction to invasion by D. immitis than did A. aegypti. Culex females were either completely negative or, if positive, they showed normal development of the parasite with no encapsulation or other unusual phenomena. Thus they segregated naturally into two groups which might lend themselves to more rapid selection. The Culex hybrids not only showed host efficiencies identical with that of C. quinquejasciatus but also exhibited more vigorous development of the parasite and a greater number of females willing to feed on the dog. For these reasons it was decided to attempt selection of a Culex hybrid, and the F, generation of hybrid II (C. quinquefasciatus 0 9 X C. pipiens 3 3) was used as the parent generation for subsequent selected strains.

INFECTION

OF MOSQUITO

WITH

DZROFZLARfA

IMMITIS

55

Procedures were much like those used in the A. aegypti selections except that the Culex females were fed on the dog overnight and were then held in carton-cages for fourteen days. Twenty-four hours before dissection, the females were confined to cotton-plugged test tubes, half full of water, for deposition of the egg rafts. Some females usually oviposited almost immediately, whereas most of the remainder had completed oviposition in about twelve to eighteen hours. All of the females were then dissected on the fifteenth day after their infective meal and their eggs retained, as desired, for selection. These experiments included seven generations, and infections with D. immitis were studied in the parent, Fz, Fa, F,, and Fr generations. The initial selection of strains from the parents resulted in two lots as follows: strain S (susceptible) composed of pooled eggs from nine females all of whom showed larval development; and strain R (resistant) composed of pooled eggs from nine females all of whom were completely negative for the parasite. During the course of the experiment strain S was divided into four substrains, S-12, S-28, S-12(19), and S-12(23). Four substrains were also selected from strain R and these were designated R-10, R-14, R-10(11), and R-10(12). Substrain R-14 was lost because its eggs failed to hatch. As in the case with A. aegypti, the type of procedure was primarily that of mass selection with certain generations used solely to increase the available females. All matings were brother X sister. Control lots from a stock colony of the same hybrid were used only during experiments with the F6 and F7 selections. Quantitative data for both host reaction and parasite findings show certain differences from the A. aegypti data which necessitatesa reorientation of approach. Dissection of the Culez hybrids presented the following general results: (1) the number of positive females were equal to the number of infected females (those exhibiting larval development); (2) practically no unchanged microfilariae were encount,eredwith the exception ;f 1.2% in strain Sl2; (3) no encapsulation was evident; and (4) the percentage of developing larvae was always 100% since no retarded forms were present. Thus selection of the Culex mosquito, in this instance, must be concerned primarily with t,he percentage of infected females and secondly, perhaps, with the number of females harboring infective larvae and the percentage of larvae which are infective at the time of dissection. The strains selected for susceptibility showed no trend toward increase of the number of infected females and the resistant strains also main-

56

LEO KARTMAN

tained a static position in this respect. The total average percentage of infected females for all susceptible lots was 36.8, for the resistant lots, 31.4 and for combined parent and control lots, 40.3. If infective larvae are used as an index, the total number of susceptible females with this stage was 27.6, of resistant females, 15.6, and of parents and controls, 40.3. Likewise, infective larvae in the susceptible females amounted to 66.6, in resistant females, 43.4, and in parents and controls 79.3. Experiment ?‘. The Fate of D. immitis in the Mosquito Midgut

In Experiments 1 and 2 a large number of negative females resulted after Culex quinquefasciatus and C. pipiens were fed on a dog infected with D. immitis. Furthermore, those Culex which becameinfected showed an extremely low intensity of infection when compared with the anopheline mosquitoes used. Aedes aegypti, although exhibiting poor development of the parasite, nevertheless had great numbers of microfilariae in the Malpighian tubules. These variations led to the hypothesis t,hat the passageof D. immitis microfilariae through the midgut of one mosquito may present a different problem for the pamsite than its contact with this environment in another mosquito species. In order to study the possible effects on microfilariae of passage through the midgut, observations were made on the parasite in the midgut of various mosquitoes at intervals after the infective blood meal. Dissections took place at the following hours after ingestion of the microfilariae: 1, 8, 12, 24, 48, 72, 96, and 120. Twenty females per speciesper period were dissected and the following mosquitoes were used: Aedes aegypti, A. albopictus, Culex quinquefasciatus, C. pipiens, reciprocal C&x hybrids, Anopheks quadrimaculatus, and A. freeborni. The observations were accomplished by dissection and separation of the female’s midgut into a drop of normal saline on a slide and examination of its contents. Every microfilaria found was placed into one ?f t,he following four categories: (1) normal; (2) alive but abnormal (i.e., the microfilaria moved very slowly by a sort of stiff bending rather than the normal serpentine undulations) ; (3) dead but intact> (internal structure apparently unchanged and cuticula intact) ; and (4) dead and disintegrating (body filled with many vacuoles, cuticula ragged and fragmenting, or parasite represented by a large vacuole surrounded by a f.ragmented cuticula). The results of this study are graphically represented by Fig. 7. Beginning with the dissections at the 48-hour period, not all the mosquitoes

FIG. 7. The fate of D. immilis microfilariae

after

various

periods

in the mitlguts

of several

species of mosquitoes.

58

LEO

KARTMAN

retained blood or parasites in their midguts, although a total of 20 midguts continued to be examined. In these cases,the figures for numbers of microfilariae found are based only on those midguts which contained parasites. The most striking fact brought forward by these data is the almost complete absence of normal microfilariae in the C&x mosquitoes at twenty-four hours after ingestion. By forty-eight hours many dead microfilariae could be seenin the hind guts of these mosquitoes apparently on the way out with their feces. With A. aegypti many microfilariae were still alive and normal at ninety-six hours after ingestion, and in A. aEbopictus many were still alive but abnormal at this time. The two anophelines retained negligible numbers of microfilariae in their midguts at forty-eight hours and the percentage of normal parasites in them at this time was between that shown by the Culex and Aedes spp. At the twenty-four hour period these Anopheles spp. showed a majority of normal microfilariae in their midguts. Experiment 8. The E$ect of Anticoagulants on the Migration Rate of D. immitis from the Mosquito Midgut to the Malpighian Tubules

Dissections of blooded midguts showed that a clot formed rapidly in the Aedes and Culex females whereas in t,he Anopheles spp. t,he erythro:ytes were agglutinated but no clot formed for at least several hours in most cases,Therefore, it was thought that the production of a blood clot in the midgut might act as a mechanical barrier to the migrat,ion of microfilariae into the Malpighian tubules. Observations were made on the rate of migration of microfilariae from the midgut to the Malpighian tubules in Aedes and Anopheles mosquitoes. Aedes aegypti, A. albopictus, Anopheles quadrimaculatus, and ,4. jreeborni were fed on the infected dog and twenty females of each species were dissected on each of the following periods after the infective meal: 1,8, 12, and 24 hours. The number of microfilariae present in the midgut and in the malpighian tubules was recorded for each mosquito at each time interval. These data are summarized in Fig. 8. It was found that an average of about 7.1 microfilariae per mosquito had already reached the Malpighian tubules in the Anopheles spp. at eight hours, whereas an average of 1.4 parasites had migrated to this location in the Aedes mosquitoes during the same interval. By twenty-four hours an average of 19.1 microfilariae per mosquito in the AnopheZes spp. were in the tubules

II’ZFECTIOIN

OF

MOSQUITO

WITH

DIROFILARIA

IMMITIS

59

whereas those in the Aedes tubules averaged 15.8 per female. Anopheles and A. freeborni showed similar percentages of microfilarial migration in each period of observation. On the other hand, Aedes aegypti and A. albopictus were quite similar except at twenty-four hours when the former species showed 42% less microfilarine in the tubules than did the latter. Further observations were then made on the speed of microtilarial migration when a mosquito was fed on an in vitro suspension of microquadrimaculatus

10080 -

A. ALEOPICTUS 20 3 A.QUADRIMACULATUS A. FREEBORN1 0

, 1

I 8

I I2

I 24

HOURS

8. The percentage of D. immitis microfilariae

in the midguts of several species of mosquitoes at various intervals after the infective meal. FIG.

filariae in blood containing an anticoagulant. In this case Aedes aegypti and A. albopictus females were fed on both in vitro suspensions of parasites and directly on the infected dog and the percentages of microfilariae in their midguts and Malpighian tubules estimated after twenty-four hours. Anopheles quadrimaculatus females were fed directly on the dog as further controls. Data resulting from these studies are presented in Table X. The major result of feeding A. aegypti on infected blood containing an anticoagulant was to increase the speed of migration of microfilariae from the midgut to the Malpighian tubules. At twenty-four hours after ingestion of the parasites over twice as many were in the tubules as were in the dog-fed aegypti controls. A. quadrimaculatus controls showed a majority of the

60

LEO

EARTMAN

microfilariae in the tubules during t.he same t,ime interval. The addition of an anticoagulant to the blood meal of A. nlbopictus substantially increased the number of mirrofilariae in the t~~bnks nfkr twent,y-four hours. TABLE X Effect of Anticoagulants in Blood Meal on Rapidity of Microfilarial Migration from Midgut to Malpighian Tubules of the Mosquito in Twenty-Four Hours 1

Species

%

Method of feeding

Anticoagulant

Total Mf in:” G

1 2 3 4 5

A. A. A. A. A.

quad. aegypti aegypti aegypti aegypti

20 Dog 20 Dog 12 Membrane 14 Membrane 10 Membrane

6 7 8 9 10 .-

A. A. A. A. A.

quad. aegypti albopictus aegyptib albopictus

10 20 10 20 12 -

-

a G-midgut. MT-Malpighiin b Dissected at 20 hours.

Experiment

Dog Dog Dog Membrane Membrane

M&l:

- ____ MT ___-

G

MT

G _-

MT

-

19 381 0.919.1 4.795.3 240 100 12.0 5.070.629.4 Heparin 22 88 1.8 7.320.080.0 S. citrate 15 82 1.1 5.815.484.6 Heparin am1 12 57 1.2 5.717.482.6 5. citrate 11 192 1.1’19.2 5.594.5 415 192 20.7 9.668.431.6 152 269 15.226.9 36.163.9 Heparin 144 346 7.217.329.370.7 S. citrate 1 44 315 3.7 26.2 12.387.7

tubules.

9. Variations in the Number of Microjilariae Ingested as Afleeted by Feeding Mechanisms in the Mosquito

There is evidence that the number of microfilariae ingested by a female mosquito is an index of the epidermal locus into which that female had inserted its fascicle. Observations on this problem consisted of a study of variations in numbers of ingested microfilariae when females fed directly on the dog and when they fed upon in vitro suspensions of parasites. Six batches of female A. aegypti were fed directly on the infected dog at different times of the day in order to obtain differences in mean numbers of microfilariae ingested (see periodicity study, Experiment 10). Five batches of aegypti were fed through membranes on in vitro suspensions of microfilariae in dog whole blood. The concentration of microfilariae was varied by dilution with sodium citrate solution, by use of heparin powder to prevent dilution, and by use of infect.ed blood from

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

IMMITIS

01

two dogs (S and D) whose microfilaremia \vas different. In t.his way average numbers of microfilariae ingested were obtained which were similar to averages in dog-fed females. C’entrifugat)ion of the blood for purposes of concentrating the microfilariae was not done since it was felt t’hat, any possible interference with normal activity of the parasite in the suspension should be avoided. When tlhe females had engorged either upon the dog or Z?Lvitro suspensions their midgut,s were dissected out and all microfilariae in t’hem reTABLE Variations

XI

in the 1Vurnber of D. immitis Microfilariae Ingested by Aedes aegypti When Fed on a Dog and on in vitro Suspensions of the Parasite*

-

Feeding method

No. 0 P

No. microfilariae

I Total

36 36 42 40 42 31 35 38 41 37 35 l

Dog Dog Dog Dog Dog Dog

in vitro in vitro

-

(citrate) (heparin) in vitro (heparin) in vitro (heparin) in vitro (heparin)

323 411 1000 1243 1431 1346 422 564 995 1220 1216

i-

MU.

ingested Mill.

Standard deviation

Meall -I-

27 28 75 77 104 105 18 29 46 51 50

1 3 2 2 4 7 3 9 12 21 20

8.9

11.4 23.8 31.2 34.1 43.4 12.1 14.8 24.1 32.9 34.7

-

6.5 7.1 16.9 16.2 21.9 23.6 3.6 4.2 7.6 9.4 9.2

All feedings, except the last two, on dog S or blood from S; the last two feedings on blood from dog D.

corded. These data are summarized in Table XI and have been presented as rising mean values of microfilariae ingested together with their standard deviations. The dog-fed females showed wide variations in numbers of parasites ingested as indicated by the extreme range presented by batches with over 20 microfilariae per individual. Even when the average number of parasites was as low as 8, the range was still over 25. On the other hand, the suspension-fed females showed a relatively contracted range since females with over 30 parasites per midgut varied only between 20 and 50 microfilariae. Thus a relation between the mean and the range is obvious. However, if the variance is calculated in terms of t,he standard deviation and then plotted against the mean values this contraction in variability of the suspension-fed mosquitoes is clearly

LEO KARTMAN

62

opposed to a reverse trend in the dog-fed individuals (Fig. 9). The application of a significance test indicat’es that the observed differences between two such degrees of variability are more than would be likely to have arisen by chance (P = <.OOl). O-DOG-FED A- MEMBRANE-FED

24 22 20

MOSQUITO

MIDGUTS

FIG. 9. Relation of the standard deviation to mean microfilariae ingested by Aedes aegypti when fed upon an infected dog and upon in vitro suspensions of D. immitis.

Experiment 10. Variations in the Number of MicroJilariae Ingested by a Mosquito as Afected by the Periodicity of the Parasite Although the daily periodicity of D. immitis is not as absolute as that shown by Wuchereria bancrofti, it is a commonly observed phenomenon and has been used for studies of filarial periodicity (Hinman, 1937). Since it has been shown that the number of microfilariae in the peripheral circulation of a dog may vary by as much as 30,000 per cc (Hinman, 1935) it was thought that this variation might be of some importance in determining the number of parasites available to the mosquito host. Six lots of A. aegvpti were fed on the infected dog, a lot being fed

INFECTION

OF MOSQUITO

WITH

DIROFILARIA IMMITIS

63

every four hours during a twenty-four hour period. The feedings were begun about ten minutes before the hour since it took fifteen to twentyfive minutes to obtain sufficient numbers of engorged females. As soon as about 80 to 100 females had completed feeding, the fully engorged ones were separated, their midguts dissected out and t’he ingested blood mixed with a drop of normal saline on a glass slide. Counts of all microfilariae in the midgut,s were recorded for each period as shown in Fig. 10. The variation in number of microfilaria,e ingested by individual fe-

2 EM.

6PM.

IO t?M.

2 A.M.

TIME FIG. 10. Availability of D. immitis microfilariae infected dog at 4-hour intervals.

6 A.M.

IO A.M.

4

to Aedes aegypti fed upon an

males during these periods shows significant differences when the frequency distributions are considered. It is clear that most females feeding at 6 and 10 P.M. obtained many more microfilariae than those feeding at 6 and 10 A.M. For example, at 10 P.M. only four females ingested less than 10 microfilariae each whereas at, 10 A.M. twenty-six females obtained less than 10 parasites each. Of those females which fed at 6 P.M. only one showed less than 10 parasites, while nineteen of the mosquitoes which fed at 6 A.M. had less than 10 microfilariae each. DISCUSSION

The principal objective of this invest,igation was to indicate the probable role of fact,ors affecting t,he infection of t,he mosquito with Diro-

64

LEO

KARTMAN

filaria immitis. However, before these factors could be measured it was necessary to determine, upon a quantitative basis, the details of the development of the parasite within the several species of mosquitoes available. Essentially, the latter objective constituted a reexamination of the index of experimental infection which, in the present study, is conceived as being based upon (1) the host reaction from day to day, (2) the parasite reaction from day to day, and (3) the host efficiency ratio. Thus the experimental evaluation of a mosquito species as host for D. immitis is here seen as a quantitative expression of the parasite’s cyclical development in mosquito hosts reacting in a particular manner under the conditions of the experiment. On this basis it was shown that the host efficiency of A. quadrimaculatus, as defined here, was approximately twenty times better than either A. aegypti or C. quinquefusciatus; and that the infective potential of quadrimaculatus was fifty times greater than that of aegypti and twenty times more than that of quinquefusciatus. The phenomenon of filarial encapsulation apparently was of little quantitative consequenceto the developmental ability of D. immitis in either of the three speciesunder consideration, although it appeared to be a consistent reaction to the parasite in the Malpighian tubules of A. aegypti. Likewise, the presence of degenerate larval forms of D. immitis in A. quadrimaculatus appeared to be of no quantitative significance. The efficiency of two or more mosquitoes as hosts for a particular parasite is not necessarily proportional to their phylogenetic relationships. The closely related Aedes albopictus and A. aegypti showed differences in their reactions to D. immitis infections which were as great as those occurring between the more distantly related A. aegypti and Anopheles quadrimaculatus. Recent studies by Farid (1949), Sundararaman (1949), and Barr and Kartman (1951) stress the close relationship between Culex pipiens and C. quinquefasciatus; in fact these authors contend that the two are nothing more than geographical subspecies.With these mosquitoes the host efficiency and infective potential were two and one half times greater for quinquefasciatus than for pipiens. This might be interpreted as indicating a specific status for the two insects, but on the other hand it may simply be another discontinuity exhibited by geographical subspeciesor strains. Anopheles freeborni and A. quadrimacblatus are so close to one another that they may be considered as sibling or cryptic species,and in this casethere was a correlation between taxonomic position and susceptibility to infection wit,h D. immitis since the responses of t,he two specieswere essentially identical.

INFECTI~S

OF MOSQUITO

IVITH

DIROFILARIA

IMMITIS

6.5

Because demonstrable differences existed between the two Aedes and the two Culex species, it was of interest to take advantage of the ability of t#hese mosquitoes to hybridize in order to determine whether the physiological characteristics responsible for t,he reactions to D. immitis infections would segregat.e especially in the F2 hybrid generations. Toumanoff (1939), Dowls a11(1Baker (1949), and Bonnet (1930) have shown that, hybrids of .-I. aegypti at1t1 *-1. nlhopictus are peculiar in that they always resemble the female parent.. The resuhs of the present, study are in agreement with the above iii that the resist,ance to t’he parasite shown by both F, and F, hybrids of aeyypfi 9 3 X nlbopictus 3 3 was almost identical to that exhibited by the czcgypti parent strain. C. pipiens and C. quinquejckatus hybrids behave in a more orthodox genetical manner and in Fz hybrids of these a physiological segregation is manifest,ed in the development of the parasite. The percentages of developing parasites were greater in the hybrids, t)hus resembling the condition in quinqwjasciatus, but the more vigorous grwth of the parasites in the hybrids was similar to that seen in pipienx. Bot,h hybrids showed host, efficiencies and infective potentials almost identical wit’h those of the quinqucfusciatus parent. It) is evident, therefore, that genetical factors do influence the susceptibility of a mosquito species to D. immitis infection, and that, although closely related species may respond in a similar manner, the genetical factors may opera,te independently of the phylogenetic position of the host. Furthermore, within a widespread species of mosquito, geographically or ecologically isolated strains or subspecies may arise which may manifest their distinctness through differences in suscept,ibility to parasitic infections as well as in other ways. For example, althoilgh the nZOopictlc.semployed in the present investigation was a most, suitable host, for I>. irnmitis, Galliard (1937) was unable to infect an Indochinese st>rain of nlbopicfus with D. imrnifis from the Eastern Pyrenees. Thus strains of the parasite as well as the host may exert an influence also. The experiments with the foul geographically isolated populations of A. aegypti were designed to t,est’ t,he possible influence of geographical strains. It is true t.hat as yet no one has proved the exist.ence of strains or subspecies of A. aegypli; nevertheless, marked differences have been reported from various parts of the world in the susceptibility of this species to filarial infections. The development of t,he parasite was quite similar in the four strains st,udied, except that in the Fiji strain t,here were fewer developing larvae and a far greater degree of microfilarial encapsulation, while t,he Hawaiian strain showed from 27 t,o X$?& more

66

LEO KARTMAN

developing larvae than any of the other strains, together with one of the lowest rates of microfilarial encapsulation. It should be noted that the high percentage of developing larvae in the Hawa.iian strain was due to the unusually high susceptibility of certain individuals, which may indicate the existence of susceptible and refractory strains among the Hawaiian population. The present observations do not substantiate the absolute nature of Roubaud’s (1937) results, which suggestedthe occurrence of a completely resistant strain of aegypti in Cuba and completely susceptible strains in Assam and Tanganyika. The present observations indicate that A. aegypti is a generally refractory host for D. immitis, but that in some regions there are especially susceptible individuals from which, through the process of selection, a population may arise that shows a superior host efficiency and a high incidence of infection. The phenomenon of individual response to infection thus becomes of particular significance, and in order to determine whether a mosquito is consistently refractory or susceptible to more t,han one species of filaria, A. aegypti females were allowed to ingest D. immitis and Foleyella brachyoptera either simultaneously or at intervals. Each species of filaria reached its normal location within the mosquito and developed there without interference from the other species. Almost all of the aegypti became positive for D. immitis, but only 35 to 63yo were positive for F. brachyoptera, so that the penetration of the gut wall by the latter seemsto be a more difficult operation than entrance into the Malpighian tubules by the former. Unchanged microfilariae of both species were found in the same mosquito, but this is a common finding in aegypti positive for D. immitis, and similar forms were reported by Kotcher (1941) for F. brachyoptera in Culex pipiens and C. quinquefasciatus, although these latter two speciesrarely exhibited unchanged D. immitis microfilariae. Encapsulation of the D. immitis microfilariae occurred, but not of the F. brachyoptera. It may be concluded that no correlation was evident between susceptibility of A. aegypti to D. immitis and to F. brachyoptera. This is in agreement with the observations of Huff (1930) on infection of C. pipiens with several speciesof avian plasmodia, and with those of Moorthy (1938) on the development of Camallanus in Cyclops previously infected with Dracunculus. Thus in line with the view of Huff (1941), susceptibility is bound up with the genetical constitOutionof t.he host and distinction must be drawn between the susceptibility of a speciesand that of its individual members.

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

IMMITIS

67

Attempts by various workers to modify artificially the susceptibility of mosquitoes to malarial parasites through the medium of selective breeding have been attended both by successesand failures. In the present study, artificial selection of A. aegypti resulted in the establishment of susceptible and refractory strains for D. immitis in which both the number of mosquitoes and the parasites showed either a quantitative increase or decrease in percentage of developing filariae. In general, this trend was not a steady one but was marked by occasional increase of resist’ance in the susceptible strains and decrease of resistance in the refractory strains. This may be att,ributed to the exigencies of procedure which was primarily one of mass selection; a similar situation was noted in selection of mosquitoes for susceptibility to avian plasmodia (Huff, 1929). The contrast between the susceptible strains and the refractory strains is striking in that the former showed a tendency toward reduction of unchanged microfilariae and encapsulated forms, whereas these forms increased in the latter. Thus the cumulative result appeared to be a decreaseof infective larvae in the refractory strains and an increase of these and all developmental stages in the susceptible strains. One interesting feature was an increase in intensity of infection in individual females of the susceptible strains. If the maximum number of developing larvae are counted for any one female in the selected generations of strains I30, B 67, and the controls, the figures are respectively: for Fz, 3, 4, and 1; for Fq, 18, 4, and 2; and for Fs, 14, 8, and 5. In contrast to t)he above, the number of developing larvae in the refractory strains was reduced more consistently than was the number of females harboring developing larvae. The maximum number of developing larvae in any one female for strains 141 and R 87 were respectively: Ff, 2 and 2; F4, 0 and 1; Fs, 0 and 0. In the FB generation these trends were not so marked; nevertheless, the percent,age of infect,ed females remained high in the susceptible st’rains, and low in the resistant ones. Another change was riotSedin the fact that whereas infective larvae in females of t,he controls and refractory strains never grew beyond a maximum of about 960 cc,this larval stage in the susceptible strains reached maximum lengths of 1000 to 1100 P wiDh an occasional larva of 1200 p. Similar effects on t,he growth of oijcysts of avian plasmodia in susceptible and refractory strains of mosquitoes have been noted (Huff, 1940; Trager, 1942; Rockefeller Foundation, 1948; and Micks, 1949). The increase of bot,h the number of females showing microfilarial encapsulat,ion and the number of encapsulated microfilariae in t,he re-

68

LEO

KARTMAN

fractory strains indicates t’hat encapsulation may have some genetic basis in the mosquito. If a comparison is made between the selected and exotic strains of aegypli we find that susceptible strains #30 and $67 had averages of 40.3 and 42.5% respectively, and the Hawaiian strain 22.3% of females with developing larvae. Furthermore, 36.3 and 39.3’$$ respectively of the larvae in the susceptible strains, and 38.1% in the Hawaiian &rain, were undergoing development. On t’he ot#her hand, refractory strains #41 and ~$87 had averages of 9.5 and 8.7°jo respeet’ively of females with developing larvae, and an average of 7.7yo of the Fiji strain females showed comparable stages of the parasite. Finally, the two refractory selected strains showed averages of 3.2 and 3.2yo respectively of developing larvae, whereas the Fiji strain exhibited an average of 3.77, of these forms. These data give the impression that selection of aegypti for differential susceptibility to D. immitis accomplished arGficially what had already been established t’hrough rlatural selection. It seems possible that a higher susceptibilit,y rat’e to D. immitis might be attained in selective breeding of aegypti if the work were conducted with a xt,raill, such as the Hawaiian, which seems to contain individuals of high susceptibilit,y. The present selection experiments substantiate the suggestion that hereditary fact’ors undoubtedly play a role in the susceptibility of A. aegypti to D. immitis. The data, however, do not allow an analysis commensurate with that of Huff’s (1934) whose conclusion was that susceptibility of Culex pipiens to Plasmodium cathemerium behaved as a simple recessive. The Mendelian nature of susceptibi1it.y has been shown more conclusively wit’h arthropods in which both sexes are disease vectors (Storey, 1932; and Black, 1913). However, in the present case, more work needs to be done before such an analysis may be possible. Trager (1942) and Royd and Russell (1943) were forced to a similar conclusion in t,heir work with selection of mosquit,oes for susceptihilit,y to avian plasmodia. Selective breeding of a Culex hybrid for susceptibility to D. immitis gave an entirely different picture than that obtained in the Aedes selection. The results showed that total average percentages of infection of females in both susceptible and refractory strains were below t#hose of t,he parent and cont,rol lot,s. The percentage of females harboring infective larvae exhibited a drop of about’ 13% in t’he susceptible strain and about 25% in the resistant st’rain as compared wit,h parents and controls. h somewhat similar result was obtained by Jeffery (1944) with selection of

INFECTION

OF MOSQUITO

WITH

DIROFILARIA

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69

Aedes albopictus for susceptibility to Plasmodium lophurae, although t,he decrease in percentage of infection in the susceptible strain was not as great as in the present instance. It should be noted that susceptible strain # 12 showed an increase in Dhe F5 of about 19% of infect,ed females over the control lot, and an increase of about 48yo over the number of infected females in refractory strain #R-10. Although this may have been due to genetic factors rather than to mere chance variation, it does not alter the impression that select,ion of a Culex hybrid for susceptibility to D. immitis failed to increase significantly the percentage of either susceptible or refractory females of which the parental stock appeared to be constituted. It has been suggested above that mosquito species, strains, and individuals respond to infection by D. immitis in various ways, and that these responses are probably determined in part by the genetical constitution of the host’. Several mechanisms may be involved in such responses to invasion by the parasites. The first of these which would be encountered by the microfilariae is the digestive process in the midgut of the mosquito. There is evidence which indicates that “feeble” microfilariae of W. bancrofti in the midgut of C. quinquefasciatus are most’ probably unable to ext’ricate themselves from t,he blood clot (O’Connor and Beatty, 1938). Wuchereria bancrofti has been noted to be unable to pass through the gut of Tripteroides bambusa (Yamada, 19273, and high densities of this species in the stomach of C&x quinquejascialus were thought to lead to a high mortality of the parasit’es in that organ (Basu and Sundar Rao, 1939). O’Connor and Beatty (1938) showed that of 2880 W. bancrofti found in 100 wild C. quinquefasciatus, 880 or 30~57~ died in the stomach blood. The present work has indicated that the fate of the microfilariae of D. immitis in the mosquito midgut is undoubtedly a crit’ical one for the completion of the parasite’s life cycle. The preponderance of negative females in the Culex species is explained by the fact that the majorit’y of microfilariae are killed in the midguts during the first twenty-four hours after the infective meal. On the other hand, the majority of microfilariae ingested by the Aedes and Anopheles species successfully migrat’e through the midgut and enter the Malpighian tubules. In the Anopheles midgut, which contains uncoagulated blood, the migration of microfilariae to the tubules is so rapid that adverse factors in the gut may not have sufficient1 time to operate. Those which remain behind are dead or moribund after forty-eight hours. The present work demonstrates that some microfilariae of D. immitis

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remain alive in the midgut of Aedes aegypti and A. albopictus for at least seventy-two hours after the infective meal in contrast to their rapid destruction in Culex species. Travis (1947) similarly noted that D. immitis microfilariae remaining in Culex stomachs on the second day were dead, whereas a high percentage were still alive in Aedes species. Other workers have also made passing mention of the destruction of D. immitis in the midgut of Culex mosquitoes (O’Connor and Beatty, 1938) ; however, the exclusiveness of Culex as a destroyer of microfilariae in the midgut is not always borne out since Aedes trivittatu.s, il. taeniorhynchus, and A. vexans have been mentioned in this connection (Yen, 1938; and Newton, Wright, and Pratt, 1945). With frog filariae, Foleyella spp., Causey (1939) noted active microfilariae in the stomach of A. aegypti twenty-four hours after the infect’ive feeding, whereas Kotcher (1941) indicated that’ the majority of microfilariae are killed in the ventriculus of C. pipiens and C. quinquefasciatus. Although the mechanism of destruction of the microfilariae in the mosquito midgut would appear to be a process of digestion, it should be noted t,hat certain parasitic nematodes have been shown to succumb to digestive enzymes only after death. This subject has been reviewed by Bueding (1949), but it may not apply especially to the present problem as it concerns the reactions to digestion primarily of Ascaris, which is a normal inhabitant of t,he intest#ine while the sojourn of the microfilaria in the mosquito’s midgut is brief. However, if microfilariae, like Ascaris, cannot be digested by enzymes unless they are first killed, we may adduce the hypothesis that some factor in the midgut or salivary glands of C. pipiens and C. quinquefasciatus, not present in A. aegypti and A. albopictus, is capable of rapidly killing the microfilnriae prior to t,heir digestion. Whether the microfilariae are destroyed by digestive enzymes or another substance, their speed of passage through the midgut is of extreme importance to their survivztl. Rapid blood clot format,ion in the midgut may impede the migration of the microfilariae in Aedes and Culex, while in Anopheles the slow clot formation is accompa,nied by a rapid disappearance of the microfilariae from t,he midgut. The simplest and most obvious explanation is that, rapid migration of the microfilariae occurs most efficiently in mosquitoes possessing salivary anticoagulants. Critical studies with A. quadrimaculatus have shown that the salivary glands contain an anticoagulating agent for mammalian bloods which is active at dilutions of more than 1: 10,000. Similar agents were found in the glands of A. punctipennis and A. crucians, whereas these factors

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were completely absent in Aedes aegypti, A. vexans, Culex salinarius, C. quinquefasciatus, C. restuans, and Psorophora discolor (Metcalf, 1945). Thus the above hypothesis is supported by the present study in that the addition of an anticoagulant to the infective meal produces a rate of microfilarial migration in Aedes aegypti quite comparable t.o that in Anopheles quadrimaculatus. Another limiting factor may be the variation in number of microfilariae ingested by individual mosquitoes. In work on mosquito host’s for D. immitis, Hu (1931) found variable intensit,ies of infection in individual mosquitoes. Extreme variations in the number of microfilariae ingested by individual mosquitoes fed simultaneously on the same host were observed by Hinman (1935), Galliard (1936), and O’Connor and Beatty (1937). The possible reason for this variation in microfilarial uptake by individual mosquitoes was not clearly defined until the work of Gordon and Lumsden (1939) showed that when Aedes aegypti fed on the web of a frog’s foot blood was taken either directly from a capillary or from an extravasation of blood derived from a previously lacerated capillary. These two methods of feeding were then correlated with the number of microfilariae taken up from a frog infected with Foleyella dolichoptera and it was found that feeding from ext’ravasated blood resulted in a lower concentration of microfilariae than when blood was taken directly from a capillary. It was also pointed out that the microfilarial concentration in different capillaries varies considerably. The present experiments with A. aegypti and D. immitis appear to substantiate the findings of Gordon and Lumsden, though in an indirect manner. The data suggest that individual variations in microfilarisl intake by mosquitoes can be reduced considerably when t’he females are fed upon in vitro suspensions of the parasites as compared with dog-fed mosquitoes. Presumably, the suspensions presented a more uniform distribution of microfilariae and resulted in a more uniform ingestion of the parasites by individual females. The extreme degree of variation in the dog-fed females may be account’ed for by the exigencies of feeding either directly from capillaries or upon intercapillary hemorrhages. Since individual variation appears to be somewhat lessened when mosquitoes are fed either on dogs with lower numbers of circulating microfilariae or upon in vitro suspensions of the parasites, this may be of some import>ance in transmission or other experiments where uniformly infected mosquitoes are needed. The number of microfilariae ingested by a mosquito is also affected by

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the periodicity of the parasite. The periodicity of D. immitis microfilariae during a twenty-four hour period shows a minimum of circulating embryos between 8 A.M. and 12 noon, and a maximum between 10 P.M. and 2 A.M. (Underwood and Wright, 1933; Hinman, 1935; Inoue, 1935; and Kubo, 1938). The form of the periodicity curve, however, may show considerable fluctuations in the same dog from time to time (Faust, 1937; and Kubo, 1938) and a definite seasonal periodicity apparently has been demonstrated for D. immitis (Kubo, 1938). When mosquitoes are fed on an infected dog at intervals during a twenty-four hour period the resulting curve of mean number of ingested microfilariae is similar to the periodicity curve based on embryos present in measured volumes of peripheral blood. Thus the present work has indicated that A. uegypti ingest a maximum mean number of microfilariae between 6 P.M. and 10 P.M. and a minimum between 6 A.M. and 10 A.M. from the particular dog used. Microfilarial periodicity may thus affect the quantitative character of the parasite’s life cycle. For instance, a day-biting mosquito with physiological propensities similar to those of C. quinquefasciatus would ingest very few D. immitis microfilariae most, or all, of which might be killed in the midgut. On the other hand, a day-biting mosquito with a host efficiency similar to that of A. quadrimaculatus would ingest few embryos all of which might complete their development, whereas if this species had fed at night the great number of invading parasites might eventually kill a significant percentage of the females. Thus the successfulmaturation of sufficient numbers of infective filarial larvae and the survival of sufficient numbers of infective mosquitoes may depend, to some extent, upon when the mosquito takes its blood meal. &JMMARY

1. A quantitative evaluation of the development of Diro$laria immitis in Aedes aegypti, Cukx quinquefasciatus, and Anopheles quadrimaculatus was established. The types of evidence used in t’his work consisted of (1) the host reaction from day to day, and (2) the parasite reaction from day to day. A third category of analysis was the concept of host e$Tciency defined as a ratio between the theoretical number of microfilariae ingested by a group of mosquitoes and the total number of developing larvae found in them during a specified period. A similar ratio involving only infective larvae was suggested as basis for the supplementary concept of infective potential.

2. As one of the measures of a genetic basis underlying susceptibility of the mosquito to a filarial parasite, taxonomically related mosquito

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species were compared as host,s for D. immitis. Infection of the strains of Aedes aegypti and rl. albopictus used here showed that the host efficiency of albopictus was twenty times better, and the infective potential was fifty times greater t’han the corresponding ratios for aegypti. Infection of Fr and Fz hybrid females from a cross bekeen aegypti females and albopictus males showed no segregation of susceptibility in either generation and resulted in complete dominance of the aegypfi parent female characteristics. Infection of Culex pipiens and C. qdnquefasciatus showed that both the host efficiency and infective potential of ql~inquefasciatzls were two and one half times greater than those for pipiens. Infect.ion of FS reciprocal hybrids of quinquefasciatus and pipiens showed some physiological segregation in that host efficiencies and infective pot.emials were close to those of the quinquefasciatus parent, whereas t,he gron-th of the larvae most resembled that seen in pipiens. Infection of Anopheles quadrimaculatus and A. freeborni indicated almost iderrtiwl physiological reactions to D. immitis ant1 comparable development of the parasite. 3. As another measure of t,he genetics of susceptibility, several geographical strains of Ades aegypti were infected with D. immitir and the resulting infections quantit~atively evaluated. A. aegypti from the T’nited States, 8. Africa, and hnglo-Egyptian Sudan were shown t,o give similar host, and parasit,e reactions, whereas a Hawaiian strain n-as shown to be more susceptible and a Fiji st.rain to be more refractory to the dog filaria. 4. Individual susceptibilit,y of Aedes aegypti to infection with filarial parasites was tested by feeding females on D. immitis and Foleyella brachyoptura either simultaneously or at intervals. Each species of tilaria reached its particular site of development in the mosquito with 110 apparent, mmual antagonism and their subsequent development was tylk~l for the species. 5. Selective breeding of mosquit’oes for differential susceptibility to D. immitis was conducted to evaluate further genet#k and helminthic interactions in Dhe intermediate host. With Aedes aegypti, art,ificial selection resulted in the establishment of susceptible and refractory st’rains for D. immitis. Selective breeding of a Cu2e.r hybrid for differential susceptibility to D. immitis failed to establish eit,her suscept,ible or reftwtory strains. The pewetrtage infection of the selected strains was below that of both the parems and controls. A. The ability of D. immitis microtilariae to survive passage t~hrough

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the midgut of several species of culicine and anopheline mosquitoes was studied. It was found that all 6he microfilariae in midguts between 24 and 48 hours are killed and digested in Culex quinquefasciatus, C. pipiens, and their reciprocal hybrids, whereas some microfilariae may remain alive in the midguts of Aedes aegypti and A. albopictus for over 72 hours and in Anopheles quadrimaculatus and A. freeborni for at’ least 48 hours. 7. The active migration of D. immitis microfilariae from the mosquito’s midgut to its Malpighian tubules was found to be mechanically inhibited by the rapid formation of blood clots in the midgut of the Aedes species used in these experiments, whereas no such barrier to migration was noted in the Anopheles spp. By twenty-four hours an average of about 91% of the parasites in A. quadrimaculatus and A. freeborni were in the tubules whereas t’hose in A. aegypti and A. albopictus tubules averaged 45 per cent. The addit’ion of an anticoagulant to in vitro suspensions of microfilariae fed t,o the Acdes spp. caused an increase in t’he rat,e of parasite migration to the t’ubules comparable to that’ seer1in the Anopheles spp.

8. Variations in number of microfilariae ingested by individual mosquitoes were studied by allowing Aedes aegypti t)o feed on iin infectfed dog and upon in vitro suspensions of D. immitis microfilariae. Dog-fed females showed wide individual variations in numbers of ingested microfilariae, whereas suspension-fed females showed significantly less individual variation. 9. Variations in the number of ingested microfilariae were shown to be affected by periodicity of the microfilariae in t,he dog. The present work indicated t’hat A. aegypti ingested a maximum mean number of D. immitis microfilariae between 6 P.M. ad 10 P.M. and a minimum between 6 A.M. and 10 A.M. &KNOWLEDGMENT

It is a pleasure to thank Dr. Lloyd E. Rozeboom for his critical judgments on these studies and Dr. Gilbert F. Otto for his enthusiastic interest and penetrating criticisms. The writer also wishes t,o thank t,he following for their generous aid: Dr. Eli Chernin for technical assistance in Experiment 10; Mr. E. R. Sasscer, Bureau of Entomology and Plant Quarantine, for permits to obtain exotic strains of Cedes aegypti; Dr. D. J. Lewis, Anglo-Egyptian Sudan, Dr. S. M. K. Hu, Hawaii, Dr. Botha De Meillon, S. Africa, and Mr. S. Nelson, Fiji, for eggs of A. aegypti; Robert Traub, Lt. Col. MSC, AUS and Dr. C. G. Huff for eggs of A. albopictus; and Professor W. G. Cochran for applicat,ion of statistical tests to the data in Experiment 9.

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