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1954 Relationships Between the Stomach Wall of Mosquitoes and the Exogenous Development of Malaria. Augustus Burns Weathersby Louisiana State University and Agricultural & Mechanical College
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Recommended Citation Weathersby, Augustus Burns, "Relationships Between the Stomach Wall of Mosquitoes and the Exogenous Development of Malaria." (1954). LSU Historical Dissertations and Theses. 8092. https://digitalcommons.lsu.edu/gradschool_disstheses/8092
This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. RELATIONSHIPS BETWEEN THE STOMACH WALL OF MOSQUITOES AND THE EXOGENOUS DEVELOPMENT OF MALARIA
A dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Keehanioal College In partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Zoology, Physiology, and Entomology
by Augustus Burns Weathersby B* A., Louisiana S tate U niversity, 1958 M. S., Louisiana State University, 1940 May, 1964 UMI Number: DP69470
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LOUISIANA STATE UNIVERSITY LIBRARY
1 1 9 -a aokbqwlbdgment
the Author wlahaa to expreaa hit aiaoere appreoiation to Dr*
Clay a* Huff, guest committee member and advisor* for hi a corneal and guidance inthii study. Ha la Indebted to Dr* J, A* Roberta*
Dr. H. J. Bennett* and Dr* 0* V* Boaeeall for helpful oritloiama and encouragements to the Sami ttedloal Research Institute Photo* graphic Laboratory for aid in preparing the illuatrationai and to
Hlaa Taugl Shiroishi for aid in maintenance of the paraaite atralna* TABLE* OF CONTENTS
PAGE
I . INTRODUCTION ...... X
I I . MATERIALS AND METHODS...... 8
III. RESULTS...... 1?
(A) The Plasmodium gal linaoeum * Aedes aegypti *• Cuiex 'piplens combinations ...... 17
1. The susceptible host, Aedea aegypti . . . 17
2. Demonstration of the eotopic development of oocysts ...... ID
5. The refraotory host, Culex pipiena . . . 21 (B) The Plasmodium fallax - Aedes alboplctus - Culex pipienscombinations ••••... 24
1. The susceptible host, Aedes albopictus • 26
2. The refractory host, Culex pipiens . . . 27
XV. DISCUSSIGS...... 28
V. SUMMARY AND CONCLUSIONS...... 38
BIBLIOGRAPHY...... 40
i l l LIST OF TABLES
TABLE PAGE
I. Comparison of the sporosoite development in salivary glands of susceptible Aedes aegypti end refraotory Culex pipiens, injected with various stages of the sexual cyole of Plasmodium gallinaoeum **•*«•* 45
II* Tabulation of infection in serial sections of sus ceptible Aedes aegypti which had been injected with g&metooytes from a bird infected with Plasmodium gallinaoeum ...... *••*•*•*» 44
III* Comparison of the sporosoite development in salivary glands of susceptible Aedes alboplctus and refractory Culex pipiens, injected Wii'h various stages of the sexual cycle of Plasmodium fallax 46
i v LIST OF ILLUSTRATIONS
FIGURE PAGE
1. Vacuum flask for harvesting pupae . • . 46
2. ICioropipette needle for mosquito injections ...... 46
3. Apparatus for injecting mosquitoes . * ...... * 4?
4. Injection of Aedes aegypti ...... 46
6. Comparison of oocyst development in normally infected and injected mosquitoes ...... 49
6* Oooyst (8-day) near striated muscle ...... 50
7. Oooyst (Q-day) in striated muscle ...... 50
8. Two oocysts (7-day) in thorax of Aedes aegypti . . . . 51
9. Oocyst (7-day) in fat body in abdomen ...... 51
10. Oooyst (8-day) on ventral nerve ganglion ...... 52
11. Two oocysts (8-day) on salivary gland ...... 62
12. Oocyst (8-day) on traoheole and fat body ...... 65
15. Oocyst (8-day) on integument ...... 65
14* Oooyst (8-day) on ventral diverticulum ...... 54
15. Oocyst (8-day) on malpighian tubule ...... 54
16. Oocyst (8-day) in head, near osDoatidia ...... 66
17. Oocyst (8-day) in third segment of maxillary palp • . . 56
18. Oocyst (8-day) in haemocoel of abdomen...... 66
19. Oocyst (8-day) in fat body near traoheole . 56 v ABSTRACT
RELATIONSHIPS BETWEEN THE STOMACH WALL OF MOSQUITOES AND THE EXOGENOUS DEVELOPMENT OF MALARIA
Numerous species of mosquitoes are innately immune to certain malarial parasites, although they appear to be morphologically and biologically capable of serving as vectors* Earlier investigators traced the development of the parasite to its death in the stomach nail of refractory mosquitoes* In susceptible species, oocysts de- veloped only on the stomach! thus the stomach nail eras considered
the critical tissue in determining the susceptibility of a species.
This investigation has endeavored to determine whether or not the
forces responsible for the development of the parasites in suscep*
tible mosquitoes or for their death in refractory species were con*
fined to the stomach wall*
The critical stomach wall phase was bypassed by injecting the
various exogenous stages (gametooytes, oocysts of various ages, and
sporosoites) directly into the haemoooels of both susceptible and
refractory mosquitoes* The criterion of development was the demon*
stration of viable sporosoites in the salivary glands of the expert*
mental mosquitoes* The infeotivity of the sporosoites was proved in
susceptible vertebrate hosts* Serial sections were prepared from
injected mosquitoes for demonstrating the sites of oooyst development,
vi Two host-parasite combinations ware en^loyed so as to preelude results that applied to unique associations only.
The exogenous stages of Plasmodium gallinaoeum, injected into the haemoooela of susceptible Aedes aegypti, developed and produced
infective sporosoites in the salivary glands* Of the 2704 injected mosquitoes, 22*7 per oent of the 348 individuals that survived were
infected* Mo infection resulted in 476 refractory Culex pipiens which
survived from 6438 individuals injected with the same parasites*
Yiable sporosoites were produced in 13*1 per oent of the salivary
glands of 466 susceptible Aedes albopiotus which survived from 2404
individuals whioh were injected with the various exogenous stages of
Plasmodium fallax* None of the 489 refractory Culex pipiens surviv
ing from 2458 individuals injected with the same parasites were
infected*
Oocysts were demonstrated developing in all body regions and
In most tissues of A* aegypti whioh were serially sectioned 7 and 8
days following haemoooel injections of P« gallinaoeum* This develop*
ment was independent of the stomach wall*
In view of these results it was concluded that the tissues of
the stomach wall were not essential to the development of the para
sites in the susceptible mosquitoesf neither were the forces that
caused the death of the parasites in refractory species confined to
the stomach wall* The tissues of the stomach wall were merely the
first intimate relationship between the parasites and the mosquito*
v i i INTRODUCTION
The relationship* existing between a parasite and its host are as conplex as life itself* Any investigation direoted toward expos ing the factors involved In such an association is doubly complicated in that the fundamental biology of not one but two organisms* with all their complexities* must be analysed* The problem is further involved in that most combinations which are of concern to man are
genealogically widely separated (Christophers* 1954)* It is diffi
cult enough to study the biology of the host* but even the phases of life cycles are obsoure* and some are unknown* in many of our common and important endoparasitie organisms * Once a parasite has entered
the host* its development* activities* physiological and chemical
responses* and life cycle are extremely difficult to determine or measure. Much of our knowledge regarding parasites has been deter mined from clinical ohanges exhibited by the host and not from direct
observations of the living parasite. The evolutionary history of
parasites could be of value to investigators* but even this story is
based primarily on hypotheses* since parasites have left little or no
fossil record* as was true of higher animals (Kellogg* 1913j Metcalf*
1923* Huff* 1938 and 1943).
Very little is known regarding the specificity displayed by
most parasites in the selection of their host} also little is known about the susceptibility of the host to the parasite. The problem of
innate immunity and susceptibility of animals to disease has intrigued and perplexed students of biology since the time of Pasteur and Koch, yet no adequate explanation has been made fo r th is phenomenon except
in single and uncomplicated associations. Kuhn (1939) explained the
immunity of rabbits to Oiyptooocous ho minis on the basis of the ele~
rated normal and fever temperature of the rabbits. Six strains of
C. hominis did not survive in broth at rabbit body and fever tempera
tures (102° - 107° F), whereas viable cells increased readily at mouse body temperatures (95° 00 101° F). Mice produced no fever from the
experimental infections. Angell et al (1990) found that the presence
of protooateohuic acid in certain varieties of onions was responsible
for their resistance to smudge, caused by Colletotriohum cireinaus.
This occurrence of acidity was found to follow the Ifendelian pattern
of inheritance. An exposition of the forces or mechanisms involved
in natural immunity or susceptibility would be of great importance in
devising methods of control directed against parasitic diseases.
Although this constitutes one of the greatest unsolved problems in
parasitology, few investigators have been concerned with such investi
gations, especially in animal parasites.
Organisms of the genus Plasmodium are unsurpassed as subjects
for studies on host-parasite relationships and have been popular
experimental subjects because of their economic importance. They
attack a great variety of vertebrates, many of whioh are well adapted
to the laboratory! they attaok many species and varieties of arthropods} 3 they have no free-living cycle to complicate the maintenance of the strains; they invade the tissues of both types of hosts; and yet they display a specificity of host selection equaled by few organisms (Huff,
1929).
Parasite specificity is adequately demonstrated by the mosquito- malaria relationship. It has long been known that mosquitoes of the genus Anopheles are the only vectors of human malaria, and for that same period of time the question has been asked, "Why are other mos quitoes immune to the human malarias?". Most avian malarias are trans mitted by mosquitoes of the tribe Culicini (Hewitt, 1940). Within these groups there are many species which exhibit extremely complicated susceptibility patterns. Some are highly susceptible to one or more speoies of malaria yet may be naturally immune to closely related strains (Huff, 1927, 1930 and 1931). Others may be refractory to all the species of malaria although they appear to be well adapted, morpho logically and biologically, to serve as a host. What then are the reasons for these varying susceptibility adaptations? What are the factors that control susceptibility and immunity? It is possible that any information on the forces controlling infactivity of the defini tive host would be of considerable help in understanding the poten tialities of the parasite in the intermediate host. Some authorities express the view th a t the two cycles may be more a lik e than has been considered. Tersian et al (1949) showed that prophylactio drugs had similar actions against the parasites in both types of host. Huff
(1954) summarized the observations on the cytology of exoerythrocytic 4 schisonts in aviea Plasmodium together with an evaluation of recent morphological and chemotherapeutic findings whioh pointed to sim ilari ties between schizogony and sporogony*
Soon a f te r the discoveries o f Roes (1398) and Grass! (1801) th a t malaria was transmitted by mosquitoes, Stephens and Christophers (1902) noted that not all mosquitoes were equally susceptible to the disease*
Darling (1910) noted that a few individuals of a susceptible species failed to become infected even under the most favorable conditions*
Be believed they possessed an active immunity toward the parasite*
Many hypotheses were given as to the cause for certain mosquitoes escaping infection but only a few investigators have attempted to explore the causes* Huff has made extensive studies of susceptibili ties and immunities of the mosquitoes* In 19M he traced the develop ment of the parasite to its death in the tissues of the stomach wall of refractory mosquitoes* The report of lilliamson and Zane (1937)
Cu*ex bltaeniorhynohus serving as a host for Plasmodium vivax and
Plasmodium falciparlum is the only record of a suspected refractory
species permitting the parasite to develop beyond these tissues* This
report has not been oonfimod by other workers and no vertebrate host
challenge was made of the infection* There seems to be no record o f
oocysts developing in tissues other than those of the stomach wall*
Ball (1948) reports having kept oocysts alive for 20 days on dissected
stomachs in vitro* but little if any development was noted although
they remained attached to the stomach*
After the discovery by Boss (1393) of the role played by mosquitoes in the transmission of Plasmodium, the details of the life oyoles of the parasite in both the vertebrate and invertebrate host became so well known and widely accepted that the literature now taoltly gives the impression that the stomach wall is essential to the develop** meat of the ooeysts and that the parasite can not deviate from the routine pattern* The stomach wall has been given a role of prime impor tance in the development or the inhibition of the parasite in its exogenous cycle* In suoh a role it is the most important determining factor in the susceptibility of mosquitoes* Under natural conditions this is essentially truei the stomach wall seems to determine whether the mosquito becomes infected or not. Since this critioal tissue plays such an Important part in the susceptibility of mosquitoes, an evalua tion of its role was indicated as the first step in studying natural immunity to malaria* Several possible explanations regarding the funotion of the stomach wall were considered* In refractory mosquitoes, the tissues might have formed a simple mechanical barrier, or they might have possessed local or tissue immunity factors, confined to the stomach wall* Death of the parasite might have been caused by the lack of essen tial metabolites, action of antagonistic forces, or combinations of these forces* In susceptible nosquitoes, the forces responsible for the devel opment of the parasite might have been confined to the stomaoh wall, making this tissue essential to the parasite*
The present investigation has been concerned with determining the role of the stomach wall in the development of the exogenous cycle of malaria* In seeking an approach to the problem, several questions arose 6 which determined the experimental procedures. Could the parasite con* tinue its development if the oritioal tissues of the stomach wall were by-passed? Could oocysts of varying ages survive in the haemocoel of refractory mosquitoes? Could sporosoites, with no further development to undergo, survive in such an environment? Could any stage, from gametocyte to mature sporosoite, survive in the haemoooels of either susceptible or refractory species?
The forces responsible for susceptibility or non-susceptibility have not been exposed in experiments reported here. This is but one step in a series of experiments designed to investigate these forces.
The experiments of Huff, which were prerequisite to this investigation, traced the development of the parasite in refractory mosquitoes as far as was possible under natural conditions of infection. This work attempts to extend that development, experimentally, beyond the criti
cal point of the stomach wall and give the parasite a chance to develop without passing through this tissue.
Briefly, the re stilts of these experiments show that, although the forces required for parasite development in susceptible mosquitoes and those responsible for death in refractory species are present in the tissues of the stomach wall, they are not confined to this organ but are present in other body tissues. The stomach wall is merely the first I intimate association between the parasite and the new host. For the
^Preliminary results of the first host-parasite combinations have been published. Weathereby (1952). 7 first time, oocysts were demonstrated developing In tissues other than those of the stomach m il of susceptible mosquitoes. Oocysts were found in all body regions and in most tissues of the experimental mosquitoes. MATERIALS AMD METHODS
Th© mosquitoes used in these experiments were not merely tools for the propagation and transfer of parasites but were truly l&bora- tory animals. They were the subject of the research problem and as such were required to be normal, vigorous, and healthy individuals.
Every effort was made to produce adult mosquitoes whioh were maximum in sise and capable of surviving the rigorous treatment required for the experimental animals. Since the experimental animal was so impor tant in this investigation, the methods used in the in sectary are described in more than the usual d e ta il.
Laboratory strains of Aedes aegypti (Linn.), Aedes albopiotue
(Skuse), and Culex pipiens Linn, were used throughout these experi ments. They were maintained at a constant temperature of 80° F. and a relative humidity of 70 per cent.
Aedes aegypti have been maintained in this laboratory continu ously for the past seven years. The stock colonies were kept in screen wire cages 12 x 14 x 14 inches and were populated w ith about 1000 individual mosquitoes, approximately equally divided as to sex. Oranges, one quarter per cage, were supplied to the mosquitoes on alternate days and a constant supply of water was provided in a crystallising dish 9 cm. in diameter and 6 cm. deep. The stock mosquitoes were allowed
8 9
to food on a normal chick twioe per week* Oviposition began the third
day following the blood meal and the eggs were oviposited on two-inoh
strips of paper toweling whioh lined crystallizing dishes, one-half
full of water. The egg dishes were removed on the sixth day following
the blood meal. The w ater was removed and the dish was covered w ith a petri dish top. The eggs were allowed to dry gradually in the dish, after which the egg strips were stored. The dry eggs remained viable
for long periods of time, up to 4 months or longer. The eggs hatched
in a matter of minutes after being placed in water. The addition of a
few oubio centimeters of old culture water aooelerated the hatching.
The f i r s t in s ta r larvae were se t up in one-gallon mouse Jars to which two quarts of tap water, one guinea pig food pellet, and 10 oc.
of old culture water were added. Approximately 300 larvae were added
to each Jar. Care was taken not to overpopulate the cultures. An addi
tional food pellet was added to each Jar on alternate days until the
larvae attained the 4th Instar stage, at which time the food pellets
were increased to two. Pupation began the 7th day following hatching.
The pupae were harvested on alternate days until pupation was completed.
The cultures were poured into white enamel pans for easier harvesting.
The pupae were picked with a special flask (fig. 1) connected to a
vacuum line or aspirated by mouth. The pupae were set up in crystal
lising dishes and the imagos allowed to emerge into lucite cylinders,
4 inches in diameter and 6 inches high. These were covered at one end
with bobbinet and secured to metal stands by a strong rubber band.
Usually 100 females and an equal number of males were kept in a xo cylinder* These adult mosquitoes were maintained on a 4 per oent sugar solution diet, supplied in a wide-mouth medicine dropper, Inserted through a hole in the bobbinst. The solutions were changed daily.
The excess pupae and adults, not used for experiments, were added to stook oages.
Oulex pipiens has been maintained here for the same length of time as A. aegypti. The stock mosquitoes were maintained as described
^or £.* *«gyP*i except for the blood meals. These mosquitoes were more difficult to induce to feed. The techniques of Tate and Vincent (1932!) were used to induce gorging. The cages of mosquitoes were illuminated during the night preceding the day they were to be fed. The influence of the light inactivated the mosquitoes and they would gorge readily when placed in the dark. Oviposition occurred 3 days following the blood meal and the egg rafts were oviposited on the surface of the water rather than on paper towel strips. Since the eggs were not adapted to drying and storing, they were used immediately. The eggs were hatched in the crystallising dishes and the larvae were set up in white enamel pans 10 x 16 x ^ inches. The culture water and feeding schedule were the same as for A. aegypti. The harvesting of pupae was ouch faster and easier with this species. The larvae and pupae were strained from the culture water and placed in water at 4° C. The larvae sank immediately, while the pupae floated and were drawn off with the pupae flask. The larvae were returned to the culture pans. There was no apparent damage to the mosquitoes subjected to the cold water,
Aedes albopictus was obtained from the Army Medical Center, 11
Washington, D*C*, and haa been colonised in this laboratory for the past three years* This is believed to be the only colony in this country at the present time* The culture and handling was identical with the techniques used for A* aegypti.
The 8A s tra in o f Plasmodium gallinaoeum Brumpt was used in the first bost-parasite combination experiments* It was maintained in
Rhode Island Red chickens and propagated either by blood passage from infected bird to normal bird or by sporosoites from infected mos quitoes* Infected blood used for mosquito injections was obtained from birds showing a parasitemia of at least 20 per oent and a gams* toeyte count of from 6 to 20 gametocytes per 10 oil immersion fields*
In general, birds showing an ascending or precrisis parasitemia were used as gametocyte sources*
The ISA strain of Plasmodium fallax Schwets was used in experi ments with A, albopiotus and £• pipiens * It was isolated from the
Uganda tufted guinea fowl, Rural da meleagris major, from the Anglo-
Rgyptian Sudan, in 1948 by Huff* It has been maintained in Beltsville white turkeys and was propagated and used for mosquito injections in the same manner as described for P* gallinaoeum* The infected blood to be used for injections was heparinlzed, mixed with 0*88 per oent saline and injected into the mosquitoes immediately*
The exogenous stages of the parasite of both P* gallinaoeum and JP* fallax were obtained and prepared in the same manner* The donor mosquito hosts were allowed to feed on an infected bird showing a high gametocyte count* At appropriate intervals following the blood 12 meal, oocysts of varying ages were obtained for injections by dissect** ing the infeoted stomach in 0.35 per oent saline* Those stomachs showing a heavy oooyst infection were transferred to fresh solutions*
The stomachs were cleared of all debris by aspirating them in and out of a aioropipette, leaving only the thin outer envelope (the tunica elastioo-nueoularis, Grassi, 1901), and attaohed oocysts* The oooysts were not easily ruptured by such manipulation, but a few were found free in the fluid* The cleared tissues with oocysts were transferred to fresh saline in a hanging drop slide and cut into strips or sections containing 15 to 20 oocysts each* Oocysts of all ages* from 3 days old to mature ones, were used for injections*
The sporosoites used for injections into the mosquitoes were derived from salivary glands of infeoted mosquitoes* These mosquitoes were dissected in 0*85 per oent saline and the salivary glands removed
Immediately to fresh saline in a hanging drop slide. From 2 to 4 pairs of infected glands were dissected for each mosquito to be injected*
The sporosoites were liberated from the glands by forcing them in and out of a micropipette.
The injection needles (fig* 2) were made by heating a small area of 6^ mm* pyrex glass tubing and drawing it out to the sise of melting** point capillaries (0*08 mm,). The tubing was then out approximately
1^ inches from the point of the draw* The cap ilia ly section was then redrawn to approximately 50 ^ in diameter by passing a small section across a minute flame while exerting tension in opposite directions on the capillary* The resulting long glass "fiber" was left attached 13 until the needle mas to bo used. The point was then broken at the desired diameter.
The m icroneedles were attached to a rubber tube vacuum lin e in which two glass "Ts* had been inserted and mounted on the left base of a stereoscopic dissecting microscope (fig. 3). A rubber bulb (fig. 3B) was placed on the open end of the ”TW nearest the injection needle.
The other ”T* (fig. 3A) was plaoed ahead of the bulb, and the open end
remained exposed. The vacuum was adjusted to a very slight negative
pressure. The materials to be injected were draim into the needle by
closing the vacuum line with a series of light taps with the finger on
the open "T* (fig . SA), being oareful not to remove the needle from
the so lu tio n s while the vacuum was closed. The flu id was expelled by
pinching the vacuum line ahead of the rubber bulb (fig. 3B) with the
left forefinger and exerting pressure on the bulb. Several different
types of injection apparatus were tried before a satisfactory one was
devised. In the first experiments, the needle was activated by mouth
aspiration.
The experimental mosquitoes were deprived of food and water 12
to 24 hours prior to injecting so that they would be able to receive
the maxiimim amount of injeotion solutions. In most of the experiments
the mosquitoes were immobilised in a refrigerator at 4° C, for 5 to 10
minutes. Ether and carbon dioxide gas were used as anesthetics in a
few preliminary experiments but required a little t o o re care in handling.
Approximately 20 mosquitoes, usually equal numbers from each species,
were plaoed on a dear lucite stage (fig. 3G), 1 Inch thick and 3 14 inches square* that had been kept in the freesing compartment. Several stages were kept cold so that a cold stage was ready for each batch of mosquitoes to be injected. The cold stage kept the mosquitoes Immobi lised for sufficient time to permit injections.
The injections were effected manually* since micro manipulators were time-consuming and the procedure was not so delicate as to demand such exacting instruments. This was performed under the dissecting microscope in bright illumination* After determining the sise of the needle and the most suitable site of insertion* it was found that few mosquitoes died because of injury when needles under 80 /x in diameter were used. Although several anatomical areas were found suitable for injection* less damage* less chance of puncturing diverticula and foregut* and easier manipulation was noted when the needle was inserted in the membranous p o stsp ira cu lar area* p o ste rio r to the mesothoraoio spiracle (fig. 4). The direction of the insertion was dorsal and posterior. Many of the later experiments using blood stages were not
Injected* a f te r i t was noted th a t a drop of the solu tio n was drawn in to the mosquito as i f by vacuum when the membrane was broken or pierced with the needle. This technique insured no mechanical injury to the internal organs.
The mosquito to be injected was impaled on the needle (fig. 2 and 4) and the fluid was forced into the haemoooel until the abdomen was fully distended. The injected mosquito was then dropped into a
cylinder cage* each species to its respective cage. Approximately
100 mosquitoes could be injected with blood within 50 minutes* Oooyst 15
Injection required a longer period. When sporosoites were being in
jected, a small drop of the suspension was examined under the micro
scope. This was then allowed to dry and was stain ed w ith Giemsa fo r a permanent record of the inooulum. At the end of the injections a normal chick, 70-100 grams, was inoculated with the suspension as a
test for infectivity.
The hanging drop slide containing the cleared stomach sections and oocysts was plaoed on the outer area of the cold stage so as to accelerate the injections, Only the sections intended for injection
into a single mosquito were drawn into the needle at one time, since
the sections tended to cling together, bqual inoculation of the mos quitoes was not possible if all sections were picked up together,
ftithin 20 to 30 minutes after injections, 95 per cent of the mosquitoes recovered and were able to fly. The mortality was low for
the first day but was very high 24 to 36 hours after injection. Those
individuals surviving 36 to 40 hours usually recovered completely and
their longevity compared favorably with that of normal mosquitoes.
In many of the early experiiaanta the mortality was 100 per cent. It was difficult to account for the high mortality in some of the experi ments.
Serial sections were made from mosquitoes 7 and 3 days following
injections of chick blood containing approximately 20 gametocytes per
10 oil immersion fields. The legs, wings, heads, and terminal segments were removed from most mosquitoes to permit better fixation. They were
injected with Bouin's solution, plaoed momentarily in 8b per cent 16 alcohol as a wetting agent, and then dropped into the Boilin's. The mosquitoes were fixed according to the following schedule:
Boilin' s overnight 85$ alcohol 24 hours 50$ w 24 hours 65$ * 24 hours 80$ * 24 hours 96$ ** 24 hours Absolute alcohol 24 hours Xylol (2 changes) 1 to 2 hours Xylol and 62° C. melting-point Paraffin (equal parts) overnight Xylol (25$). Paraffin (76$) overnight Paraffin (2 changes) 24 hours
The mosquitoes were imbedded in a high melting-point paraffin
(62° C.) in paper boats in the usual manner. They were sectioned
longitudinally with a microtome at 6 m thickness. Some few sections
were out as thin ae 5/x with very little distortion or overlapping
of tissues. The ribbons were fixed to the microscope elides with
egg albumen, stained with Delafield's hematoxylin and eosin stain
and technique, and mounted with balsam. RESULTS
(A)* The Plasmodium gallinaoeum - Aedes aegypti - Culex plpiens combination a
These host-parasite combinations were chosen as subjects for the first step in studies on innate immunity of mosquitoes because they represented ideal susceptibility associations* Under favorable conditions, A. aegypti approached an infeotivity rate of 100 per oent with P* gallinaoeum, while £. pip lens had never been shown to become infected with this parasite and, therefore, its known infactivity rate was sero* Both mosquitoes were well adapted to laboratory cultures and P* gallinaoeum was easily propagated and maintained in the domestic chioken*
The criterion of development of the parasite in the haemocoela of the injected mosquitoes was the microscopic demonstration of motile sporosoites in the salivary glands and the challenge of their viability by inoculations into susceptible chicks*
1* The susceptible host, Aedes aegypti.- It was necessary to demonstrate the ability of the exogenous stages of the parasite to develop, after transplantation, in the haemocoals of suoeptible mos quitoes* This species served both as controls and as experimentals*
If the stomach wall were essential to the development of the parasites, bypassing this organ in either the susceptible or refractory host
17 la would preolude further development.
The results of this phase of the Investigation are olear-out and way he stated briefly. Viable sporosoites were recovered from the salivary glands of unfed A. aegypti that had received haemoooel inoeola tions of gametooytes, oocysts of various ages, and mature sporosoites,
A total of 467 A, aegypti In 16 lots was injected with spore* soites obtained from salivary glands of mosquitoes of the same species.
After a period of 3 or more days, the glands of the surviving mosquitoes were removed and examined for sporosoites. The survival rate for these mosquitoes was 30,4 per cent, and of the 142 individuals that survived,
29 (20.4/4) possessed sporosoites (Table X), Sporosoites were not pres
ent in these glands in large numbers, probably because of difficulties
in concentrating them in numbers equal to those found in natural in fections. Although these sporosoites appeared nomal and active, there were insufficient numbers for chick inoculations, as was performed with the other stages.
There were 31 lots (separate batches) of A. aegypti, totaling
973 mosquitoes, which were injected with oocysts, ranging in age from
3 days to mature oooysts. Of these, 166 (16$) mosquitoes survived until
dissection, and 37 (23,7$ of the survivors) showed sporosoites in their
salivary glands. The sporosoites were more numerous in these glands than those in the sporosoite*injeoted mosquitoes. Two lots, not dls*
sected, but fed on and inoculated into susceptible chicks, produced
typical infections*
Infected chicken blood having a high percentage of gametocytes 19
(5 to 20 p er 10 fie ld * ) was in je c te d in to 1264 mosquitoes in 20 separate lots* The infected ohioken blood was the most toxic of the materials injected. Only 50 (5*9^) blood-injected mosquitoes survived for the time required for the development of sporosoites (12 to 15 days). Some increased mortality was expected, since the time required for develop ment from gametecyto to sporosoite was much longer than that with the other stages. The slow rate of breakdown of the blood and elimination of the waste may be responsible, in part, for this toxicity. The over loading of the circulatory system with so many cells capable of absorb ing oxygen may have had a role in the mortality. The survival rate increased as the experiments progressed, probably because of improved techniques. There were 12 (24/&) mosquitoes positive for sporosoites.
In each mosquito the glands were heavily infected and the sporosoites were proved viable by inoculations into chickens. All chicks developed typical infections.
2. Demonstration of the ectopic development of oocysts.- The criterion of development by the microscopic demonstration of sporo soites in the salivary glands and proof of their viability in sus ceptible chicks were not sufficient evidence that the stomach wall tissues were not essential to the developing parasite. It was neces sary to date mine the sites of the developing oocysts. Were they attaching to any particular organ or tissue or were they floating free in the haemolymph? I t was possible th a t the ookinetes were attaching to the stomach and that only these parasites were developing and producing the viable sporosoites. If this were true it would 20
indicate the essential nature of the stomach wall* Examinations of
stomachs of mosquitoes being tested for sporosoites failed to reveal evidence that oocysts developed on this organ* There were no immature parasites or old oooyst "shells*” Developing oocysts, even mature ones, would have been difficult to find in gross dissections of fresh mos
quitoes* Serial sections seemed to be the procedure indicated*
Infected chicken blood with high percentages of gametooytes was
injected into 182 mosquitoes in 5 separate lots* From each lot* IS to
55 mosquitoes were fixed and prepared for sectioning on the seventh and
eighth days following injection* At this age the oocysts would be about
maximum size and be easier to find* The remaining mosquitoes in the
cage were dissected on the 12th day and the salivary glands examined
for sporosoites* The first series presented such a light sporosoite
Infection that the fixed specimens were not sectioned. The dissected
mosquitoes from the second series presented 7 positive glands out of
17 survivors* There had been 33 mosquitoes from this series fixed and
imbedded 7 and 8 days following injection* These mosquitoes were then
s e ria lly sectioned* stained* and mounted* Of these* 17 (51*6$) con*
tained oocysts (Table II)* Oocysts were found in 6 out of 19 mosquitoes
sectioned from the third series*
Oocysts were found developing in most tissues and in all three
regions of the mosquito (fig. 6). The oocysts appeared normal. They
were compared with 7- and 8-day old oocysts in mosquitoes which were
naturally infected (fig* 6)* There seemed to be no histological evi
dence that the mosquito developed any kind of cellular response to the 21 parasite* All oocysts were clear and there ms no indication of a
"walling off** response*
Oooysts were seen in and on bundles of striated muscles in the thorax (fig. 6 and 7), in fat bodies (fig. 8 and 9), on the ventral nerve ganglion (fig. 10), on salivary glands (fig. 11), on trachea
(fig. 12), next to the integument (fig* 15), on the ventral divertiou- l \ m (fig. 14), and on malpighian tubules (fig. 15). A few oooysts were apparently not attached to any tissue but were free in the haemo- ooel. Ho oooysts were found attaohed to the stomach, although one was developing on the foregut and two on the ventral diverticulum (fig.
14). These were the only ones found on any p a rt of the digestive system* The majority were found in or attaohed to fat bodies, especially near tracheoles* More than half were developing in the thorax* The head was included in the sections in only two mosquitoes, and oooysts were seen in this region in both individuals. One was de veloping in the area between the brain and the ommatidla (fig. 18).
Some were attached to the brain, and one in the third segment of the maxillary palp (fig. 17)* Sporosoites were demonstrated in several oooysts (fig . 18 and 19)*
5* The refractory tost, Culex piplens.- It is difficult to determine the value of an investigation presenting negative results*
One individual mosquito showing an infection is sufficient evidence to indicate susoeptibility, but in dealing with refractory individ uals, it is impossible to observe a sufficiently large number to arrive at the conclusion that under the same conditions they will 22 always present negative susceptibilities. It night be possible with inproved techniques, a better understanding of the parasite, and much higher concentrations of parasites to have one or a few individuals present a mild infection. The report of Williamson and Zone (1950), which indicated that a Oulex was susceptible to human malarias, is an indication of this possibility. Negative results in many experiments are often as significant as are results that are positive. Valuable information has been obtained from experiments presenting negative results, especially in experiments with adequate controls.
As was the case with susceptible mosquitoes, the survival rate for the injected refractory mosquitoes was very low, but sufficient nus&ers survived to be disseoted and inoculated into susceptible chicks to indicate certain conclusions.
Concentrated sporosoites, obtained from salivary glands of A* aegypti, were injected into the haemocoels of 851 £. pipiens in 26 separate lotsi of these, 176 (21.1$) survived to b® dissected. All
surviving mosquitoes in 5 of the experiments were ground up in 0.86 per cent saline and inoculated into susceptible chicks. The injected mosquitoes were allowed to feed on normal susceptible chicks before they were dissected or ground up for inoculations. The disseoted
glands were injected into another susceptible chick after the micro
scopic examination. No sporosoites were found in the salivary glands of the experimental mosquitoes and none of the chicks, either inocu
lated with or bitten by the mosquitoes, became infected. Typical
infections were produced in these chicks when they were exposed to 25 normal sporosoites from A. aegypti. Since birds acquire immunity during
primary attacks, this challenge would determine whether or not the ex
posure to the experimental mosquitoes had resulted in infection and would also establish the faot of susceptibility of the bird* Most of
these mosquitoes were injected with sporosoites from the same suspension
as was injected into the susceptible mosquitoes* In the few lots in which only refractory mosquitoes were injected, a sample of the sus pension was challenged as to viability by inoculations into susceptible
chicks which produced typical infections*
There were 963 £• plpiens in 34 lots that received kaemocoel in
jections of oocysts in the same manner as did the A. aegypti * These
mosquitoes seemed to tolerate the oooysts better than the A. aegypti,
and the survival rate was higher* In all, 221 individuals (ZZ%) sur
vived to be dissected or inoculated into susceptible chicks* Sporo
soites could not be found in the glands of those dissected and no
infeotion was produced in the inoculated chicks* The chicks were
shown to be susceptible by subsequent inoculations with nonoal sporo
soites. Whether or not the injected oocysts were able to develop beyond
the stage at which they were injected was not determined. At any rate
they were unable to develop sufficiently to produoe sporosoites* Some
of the oocysts were fully mature and ready to rupture when they were
injected*
A total of 1644 £. piplens in 32 lots was injected with infected
chicken blood with a high percentage of gamstooytes* These mosquitoes
were lighter colored than A. aegypti and the blood could be seen in all 24 parts of the body as the fluid was injected. Only 78 (6.0$) mosquitoes survived the time required for the completion of the asexual cycle.
Sporosoites could not be demonstrated in the dissected salivary glands and no infections were produoed in chicks injected with these glands or exposed to the bite of these mosquitoes before dissection. Sub-
inooulations were mad© from these chicks but with negative results.
They were also proved to be susoeptibl© by subsequent inoculations with normal sporosoites.
(B). The Plasmodium fall ax - Aedes al bop ictus - Culex pip lens
combinations
It was possible that the host-parasite combinations of P.
gallinaoeum with A* aegypti and with C. pipions were unique and that
the results pertained only to these combinations. Several other com
binations were considered. Experiment with a human strain was pre
ferred but the only strain available was the St. Elizabeth strain of
Plasmodium malariae* and this had been blood-passaged for so many
years that gametooytes were no longer present in sufficient numbers
to produce reliable infeotion in the mosquito* even from normal feed
ings.
Of the host-parasite combinations maintained in this laboratory*
those of Plasmodium fallax with Aedes albopiotus and P. fallax with
Culex pipiena seemed to fu lfill the experimental requirements best.
From experience in routine infections here* only about 61 per oent
of A. albopiotus exposed to P. fallax became infected. This* of 26
course, does not approach the susceptibility of A. aegypti to P.
gallinaoeum* As far as is known, £. pipiens has never been Infected with P. fallax, and, In this respect, is as well suited to this series
as it was with P. gallinaoeum. m m OVMNIMMIIMWWMHM* The ISA strain of F. fallax is well adapted to the Beltsville
white turkey. The common chicken is susceptible but requires about
twice the inoculum of parasites (blood) to produce an infection equal
to that produced in turkeys*
The experimental procedures for these experiments were the same
as were used in the P* gallinaoeum series* There was a gradual improve*
ment in the injection apparatus and the handling of the experimental
animals which was evidenced in a gradual increase in rate of survival*
The survival rate in this series was 21,3 per cent, conpared to 16*5
per cent in the first experiments, but the survival rate was approach
ing the former figure at the end of the first series* The percentage of
infection for all surviving mosquitoes in this experiment was 13*1,
compared to 22*7 for the P* gallinaceum experiment* Since A. albopiotus
was little more than one-half as susceptible to P* fallax as A* aegypti
was to P. gallinaceum, the infeotivity rate in the experimentals was in
accord* The individual infeotivity was much milder, as was evidenced
in the microscopioal examinations and in the smaller number of verte
brate hosts which became in fected from challenging bites*
The susceptible host, Aedes albopiotus*- There were 446 A*
albopiotus in 16 lots which were injeoted with sporosoites from salivary
glands of normally infected mosquitoes of the same species* Of these, 26
142 (31.8$) survived to be disseoted* Eighteen (12*6$) were positive for sporosoites in the salivary glands* Sporosoites were not numerous in the glands* and in some oases could be detected only in the stained preparations which were made after the examination of the fresh materi als* Bo challenge was made of the viability of the fresh glands. The birds that were exposed to these mosquitoes before dissection failed to become infected*
Oocysts* varying in age from 4 to 6 days following blood meal* were in je c te d in to 666 (25 lo ts ) A. albopiotus. Of these* 119 (11.8$)
survived the time required for sporosoites to be present in the salivary
glands* Sporosoites were demonstrated in 19 (15*9$) of these mosquitoes.
The infection was heavier in these glands than in those of the sporo-
soite-injested mosquitoes* but was light compared to normally infected mosquitoes. Two birds exposed to these insects before dissection pre
sented a very mild parasitemia 18 and 22 days following exposure.
Infected blood having a high peroentage of gametooytes was
injeeted into 1292 A. albopiotus in 27 lots* The survival rate for
these mosquitoes was 16*0 per cent (194 individuals)! of these* 21
(10*8$) were positive for sporosoites. Although the sporosoites were
more numerous than in the above injected mosquitoes, the infection was
lighter than was noted in mosquitoes infected normally* Three birds
exposed to the bites of the experimental mosquitoes before they were
disseoted developed mild parasitemias. Several of the birds used to
challenge the first experiments were chickens• Since turkeys were
more susceptible to P* fallax than were chickens* the number of
infections resulting from exposure to experimental mosquitoes might 27 have been higher if turkeys had been used exclusively as test animals,
2. The refractory host, Culex pipiens,- The refractory host ms injected with sporosoites from the same suspension as ms used in the susceptible mosquitoes, A total of 526 C. pip lens in 16 lots ms in* jected and 126 (23,9$) survived to be dissected, Susceptible birds were bitten by these mosquitoes before dissection, No sporosoites could be found in the salivary glands of the experimental mosquitoes and no parasitemia resulted in the exposed birds,
Oooysts, disseoted from A, albopictus, were injected into both the susceptible and refractory mosquitoes, usually in paired lots.
There were 604 (24 lots) £. plpiens that were injected with oocysts, varying in age from 4 to 8 days following blood meal. Of these, 158
(25,9$) survived the time required for sporosoites to be present in the salivary glands. The surviving mosquitoes were allowed to bite sus* eeptible birds before the mosquitoes were dissected. No sporosoites were found in the dissected glands and none of the birds became infected,
A total of 1328 £, pipiens in 25 separate lots was injected with infeeted blood from the same suspensions as were used in the susceptible mosquitoes. There were 225 (16,9$) individuals which survived the period of time required for sporosoites to be present in the salivary glands.
As was the case with other experimentals, these were allowed to feed on susceptible birds. Other birds were inoculated with the salivary glands after they had been examined microscopically, Sporozoitee could not be found in the salivary glands, and none of the birds either exposed to the bites of the experimental mosquitoes or inoculated with their sa liv a ry glands became in fected . DISGUSSXOH
At the present time, most of the details of the exogenous life cycles of most Plasmodium are known. The cycles for the various species are so nearly alike that it is most difficult, if not impossible, to differentiate between the species on the basis of morphology or inter vals in life cycles# Most species undergo changes in form at approxi mately the same interval of time, in the same tissues, and follow identical courses in the mosquitoes. These facts are so well known and taken for granted that many workers have assumed that little more information could be gained in studies of the sexual cycles. A great deal of work has been devoted to determining the relative abilities of various srcsquitoes (especially Anopheles) for serving as vectors of malaria. Much has been learned of their biting habits, flight range, and host preferences# Although many of the factors that are necessary for a given species of mosquito to become an efficient or important vector of malaria are known (Gill, 1921* King, 1911; Mayne,
1917* Siddons, 1944), one important phase remains unsolved, i,#e#, the
factors controlling the susceptibility or natural immunity of certain
species of laasquitoes to malaria. In view of the importance of this problem, it was surprising to find that only a few investigators have been concerned with trying to date mine these factors.
23 Since the early studies on the susceptibility of mosquitoes to
Plasmodium have a direct bearing on this investigation, and since the findings were prerequisite to the experimental approach used in this problem, & brief review of the most important results is to b© desired*
In his first investigations on the infeotivity of avian malarias to mosquitoes, Euff (1927) disclosed that Plasmodium oathemerium could develop to the ookinete stage in refractory A. aegypti as well as in susceptible p ip ie n s* This work also showed no apparent difference in powers or rates of digestion of blood in the stomachs of the two species of mosquitoes* The failure of certain individuals of the susceptible species to become infected was not due to in su ffic ie n t or unequal distribution of the gametocytea, since they received from
21 to 449 gametocytes per 10,000 red blood cells, but rather to an individual difference in mosquitoes themselves. The viability of the asexual forms, taken from the stomachs of the two species at varying intervals of time, were the same* Kicolaew and Yakowlewa (1929) found the rate of digestion of asexual forms of Plasmodium vivax in the stomachs of C* pipiens, Culex alaakaensia, Aedes salinellus, and
Anopheles aaculipennls to be the same, and the ookinete formation agreed with the experiments of Huff*
Micks et al (1948) found no correlation between exflagellation and jgH in the stomachs of several apeoi©3 of mosquitoes, but referred to a "particular chemical factor" present in tho stomachs of C, pipiens which greatly activated the gametooytes of Plasmodium reliotum to exflagellate and complete fertilization, and a similar factor in A. 30
aegypti and Anopheles quadrimaoulatus which Inhibited this process.
Huff (1929 and 1931) showed that inherent hereditary character istics were important factors in the susceptibility of an individual mosquito to a given species of malaria. By selection, he was able to influence the susceptibility of £. pipiens to P. oa theme rium. An in crease in the percentage of susceptible individuals followed selection from infected females and a decrease followed selections from uninfected ones. Had bisexual selections been possible, he believed that entirely refractory strains could have been derived from the original stock showing 40 per cent infeotivity. The susceptibility behaved as a simple recessive Hendelian character. Storey (1932) demonstrated that the ability of an insect to transmit a plant disease was inherited.
In 1927 Huff noted that some individuals of susceptible species of mosquitoes failed to become infected with P. oathemerium even when they received thousands of gametocytes. Any chance combination of environmental conditions responsible for this escape from infection was ruled out, since all were maintained under identical conditions.
He studied th is individual immunity in 1930 by means of consecutive infective feedings on the same species of malaria, with the results that each individual either became infected from each feeding or failed entirely to beoome infected (2 exceptions out of 60). When several species of parasites were used, little or no correlation existed between the susceptibility of individuals to one species and their susceptibility to others. The Individual susoeptlbility to a given parasite was im mutable but was subject to change, through selection, in the case of 31 the speciea as & whole.
The work reported here has neither been concerned with indi vidual immunity nor with acquired immunity, if there be such in mos quitoes. Roubftud and Meager (1934) reported that rural strains of
C. pipiens, constantly exposed to P. relictum, acquired an immunity not apparent in urban strains which rarely, if ever, were exposed.
This might well have been attributed to the natural selection of
Huff (1929 and 1931). nevertheless, investigations on natural im munity or susceptibility of species as a whole are concerned with factors responsible for individual susceptibility, since these same factors may be involved in species infeotivity. The factors that are inherited which cause a susceptible species of mosquito to in crease or doorcase in susceptibility through selection also are of great importance to anyone seeking the oause of innate immunity of species. The factors that are inherited could be the answer.
Huff (1934) substantiated the findings of some of his earlier works by more elaborate and detailed studies using histological pro cedures. In this study he found that the zygote degenerated and died in the stomach wall of refractory mosquitoes and no development past this stage was ever noted. It was this experiment that was prerequi site to the experimental approach of the investigation reported here.
Since he found degenerating zygotes in susceptible mosquitoes also, he concluded that the same mechanism might be concerned in killing the zygote in each species of mosquito, the difference in action being one of degree. He believed that the death of the zygote in the stomach 32 v ail was n ot duo to looal immunity, Th® result® reported here preyed that point.
It was evident that the studies above have revealed several factors that contribute to susceptibility, they have ruled out other agents, and they have demonstrated the end point in the normal develop ment of the parasite in refractory mosquitoes. Since the ultimate point of development had been ascertained in the refractory host, it was evident that the problem should be attaohed by other methods. Huff
(1934) indicated serologioal studies, in vitro studies of culicid blood, and bleeding and injections of mosquitoes as possible avenues of approach, but concluded that suoh procedures awaited development of new techniques.
Since the sygote had been shown to degenerate and die in the stomach wall of refractory mosquitoes, it seemed logical to approach the problem of innate immunity by determining the role of the stomach wall. If this tissue were by-passed or eliminated from the cycle of the parasite by injecting the parasite direotly into the haeraocoel of the mosquito, an estimation of its value in either the development or the inhibition of the parasite could be determined. The tissues of the stomach of refractory mosquitoes could effect the death of the parasite but, were the causes localized in this tissue? It was also evident that the stomach tissues of susceptible mosquitoes nourished the para site but, were they essential to itB development? Were the nutritive elements confined to these cells? If the causes of death were in effect only in this tissue, parasites introduced beyond this organ might be able to develop in refractory mosquitoes. If the stomach nail of susceptible mosquitoes wore essential to the parasite, para sites introduced directly into the h&emocoel would either attach to this tissue or die*
It seemed reasonable to assume that sporosoites from salivary glands of normally infected mosquitoes could live and remain viable when transplanted into the h&emocoel of a mosquito of the same species* the sporosoites undergo no further division or development, they are the mature stage or end product of the sexual cycle, and, to a degree, may be comparable to the resting or infeotious forms of other parasites*
The other stages, from gametocytas to mature oooysts, undergo suoh vast changes that their requirements and also tolerances are probably much more exacting* The sporozoite was past the stages that were supported by the stomach wall. In the h&emocoel of the new host it had only to retra ce i t s m igration to the sa liv a ry glands* There seemed to be only one thing that might prevent an infection in th© new host* Had the salivary glands of the original host altered the sporozoite in any way?
The results show that th© sporosoites were able to survive and remain viable in the new host, and evidently no change had been effected by the salivary glands of the donor that would prevent their ability to reinfect the glands*
If any one stage of the exogenous cycle could have tolerated the environment in the h&emocoel of refractory mosquitoes, the sporo- soite seemed best qualified* The results indicated that they were unable to survive. Apparently the forces that adversely affeoted the parasite in the stomach wall were active in other parts of the 34
body. This failure to survive was a slight indication of the type of immunity that might be present. If the immunity had been caused by the laoic of required nutritive elements, the sporozoite -would have had a better chance of survival than if antagonistic forces were responsi b le .
Since it was shown that the parasites were capable of develop ing from gametooytes to infectious sporosoites in both species of susceptible mosquitoes, it would appear that all of these stages found approximately the same conditions in the injected susceptible hosts as were present in the donor or normally infected mosquitoes. Therefore, they were able to complete their oyoles and produce infectious sporo soites in the salivary glands. The experiments indicated very little, if any, retardation in parasite development in the injected mosquitoes.
Transplanted oooysts produced sporosoites in the salivary glands at the same time they were first evident in the glands of the donor's cage mates. The time required to produce sporosoites from infected blood which had been injected into haemocoels of susceptible hosts was the same as required for natural infections. This demonstrated the ability of a parasite to adapt to unusual or abnormal conditions. Because an organism responds in a constant manner or follows a definite course under normal circumstances, its behavior is frequently taken for granted. After the normal activities are known, additional information is frequently exposed by observations of abnormal and of normal activ ities in different environments. In these experiments the mosquitoes were the "test tubes" and the parasites were subjeoted to abnormal (renditions. The results have given a better understanding of both the parasite and the host, w hich may be of value in attacking the major problem of natural immunity.
The demonstrations of the oocysts in the various body tissues of the mosquito in serial sections eliminated a source for criticism of the experiments employing the production of sporosoites in the salivary glands as evidence of development, jUe** that some part of the digestive system, either fore gut or diverticula, might have been accidentally damaged during in je c tio n s in the few mosquitoes presenting p o sitiv e results* Had this been true, oooysts would have been found only on the stomach wall as in natural infections* This evidence also ruled out the possibility that ookinetes in the haemocoel sought out and developed only on the stomach wall, thereby indicating its significant nature* In many instances it becomes necessary for an investigator to perform difficult or elaborate experiments to demonstrate a fact of which he has been convinced* It was known that the injection needles were not damaging the digestive system* Experimental mosquitoes, dissected immediately follow ing in je o tio n s, showed no damage to th is system and no red blood cells could be found in thia system* Mos quitoes injected with blood have not been able to produce eggs, indicating that blood did not enter the stomach* This, together with careful observations while the mosquitoes were being injeoted, was assurance that the digestive system was by-passed.
The results of the injections of gametocytes and oooysts in refractory hosts were more difficult to interpret than were the results 36
in susceptible mosquitoes* It is exceedingly difficult to evaluate negative results* Since there are reasons for such results just as there are reasons for those that are positive, they should be analysed*
All of the stages in the exogenous oyole of the parasites were given the opportunity to live and develop beyond a point whioh prevented such development under natural conditions, but none was able to develop.
The only positive deductions that oould be made were that no stage developed sufficiently far to produce viable sporosoites in the sali vary glands and that the mechanisms denying this development were not localised in the stomach wall of the insects* No attempt was made
during this investigation to determine whether the mechanisms responsi ble for the failure of the parasites to develop were atreptic or antiblastic types of immunity, or combinations of both. It was not
shown whether the stages that were injected were able to extend their
development to any degree or whether development was arrested immedi
ately, The survival rate of the experimental mosquitoes was so low
that it was considered unwise to sacrifice the few that lived to
determine such development, but to await the production of sporosoites
as evidence of complete development.
In view of the development of all exogenous stages of the para
sites without stomach wall associations in the haemoooels of suscepti
ble mosquitoes and the visual proof of oocysts developing in various
body tissues, it was evident that the stomach wall was not essential
to the parasite. In natural infections the stomach wall was eimply
the first important association between the parasite and the host. 37
To the same extent, since no parasites were able to survive in the
environment of the haemoooel of the refractory mosquitoes even
though there was no direot association with the stomach wall, it was olear that the forces responsible for the death of the parasites
were not confined to the stomach wall. In natural infections, the
stomach wall was merely the first contact between the parasite and
the inhibitory forces. SUMMARY AND CONCLUSION
The relationships between the stomach well of mosquitoes and the exogenous stages of m alarial parasites were evaluated by eliminating the possibility that the stomach wall could participate in the develop mental eyele of the parasite* the various stages of Plasmodium gallinaceum from gametooyte to sporosoite were injected d irectly into the haemocoels of both susceptible Aedes aegypti and refractory Pules pipiens* All of these stages were able to develop and produce in fective sporosoites in the susceptible host* Of the 2704 susceptible mosquitoes that were injected* 22*7 per cent of the 248 individuals that survived were infected* Ho infection resulted in 478 individuals surviving from 9488 refractory mosquitoes which were injected. Since the results obtained in the host-parasite combinations of P* gallinaceum with A. aegypti and with jC* pipiens could have been due to unique relationships* Plasmodium fallax with Aedes albopiotus and with £* pipiens was tested by the same methods* A total of 2404 sus ceptible A* albopiotus was injected with the various exogenous stages of P. fallaxj of these* 18*1 per cent of the 455 individuals that survived were infected* None of the 489 refractory £* pipiens surviving from 2458 injected individuals became infected* Serial sections were made from susceptible A* aegypti which had
38 39
been injected with gametooytes of £• gallinaceum in order to demon strate the site of the developing oooysts In the haemocoel. Oooysts were found in all body regions and in most tissues of the mosquitoes, independently of the stoma.oh wall*
In view of the complete development of the exogenous stages in the haemocoels of susoeptible mosquitoes without direct association with the stomach wall, and the demonstration of the oocyst in other body tissues, it is evident that the relationship between the parasite and the stomach wall is not essential to the development of the para site, In refractory mosquitoes the factors that oause the death of the parasites are not confined to the stomach wall, since the exogenous stages failed to develop even though the natural critical tissues were by-passed. The cells of the stomach wall were merely the first inti mate association between the parasite and the new host. BIBLIOGRAPHY
Amgell, H. R., Walker, J* C*, and Link, K* P. 1930, The relation of protoeatechulc acid to disease resistance in the onion* Phytopathology 80 1 431-438 . Ball, G* H* 1948, Extended persistence of Plasmodium rellctum la cultures* Am* Trop* Med* 28< 533-53§* r Cantaeusene, J. 1925, Le problems do l ’inmunite ohes les invertebres. Celeb* 75* Anniv* Soc* Biol*, P aris, 48*119* Christophers, R. 1954, Malaria from a soological point of view, Proc* Roy* See* Med* 27* 991*1000* Darling, S* T* 1910a, Studies in relation to malaria* Isthmian Canal Commission* Laboratory of the Board of Health, Depart* nent of Sanitation* Washington, U# S* Government Printing Office* 58pp* » 1910b, Factors in the transmission and prevention of malaria in the Canal Zone* Ann* Trop* Med* 4i 179*225* Gill, C* A* 1921, The influence of humidity on the life history of mosquitoes and on their power to transmit infection* Tr* Roy* Soc* Trop* Med* and Hyg* 14* 77*82* Grass!, G* B* 1901, Die Malaria* Studien eines Zoologen* Jena, G* Fischer* 250pp* Hewitt, R* 1940, Bird malaria* (Am* J* Hyg* Monog, Ser* Ho* 15), Baltimore, The Johns Hopkins Press* 228pp*
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TABUS I. Comparison of the sporozoite development in salivary glands of susceptible Aedes aegypti and refractory Culex piplens, injeoted with various stages of the sexual cycle of Plasmodium gallinaoeum.
Aedes1 aegypti Culex piplens
Stage of Sporo- p a ra site Gama- Sporo Gaiue- so ite Oooyst zo ite Oooyst in jected tooyte tooyte
Sumber of 16 31 20 26 34 32 lo ts
Number in jected 467 973 1264 831 963 1644
Humber surviv 142 166 60 176 221 78 ing * (30.4/i) ( 1 6 % ) (3.9^) (22.9?S) ( 3%) Number p o sitiv e 29 87 12 0 0 0 fo r spo (20.4^) ( 2 3 , 7 % ) (24?b) re so i tee
* All surviving mosquitoes were dissected. TABli. II* Tabulation of infection In serial sections of susceptible Aedes aegyptl which had been in looted wit'h gamotooytes from a bird infected with Plasmodium galltnaoeuou
Number Number Case Number of of Number Age Per mates p o sitiv e mosquitoes mosquitoes w ith of cent surviv for spo ro in jeo ted sectioned oocysts oocysts p o sitiv e ing s e tte s
56 18 1 (6.6£ )
19 9 7 47*5 64 17 7 (41.154) 14 8 8 57,1
62 19 6 8 51,5 19 6 (31.650 IABIE I I I . Comparison of the sporoioite development in salivary glands of susceptible Aedes albbplotus and refractory Culex piplens. Injected with various stages of the sexual oyoXe of Pluniodj/upi
Aedes albopictus Culex piplens
Stage of II Sporo- Sporo- Game- parasite soite Oooyst © Oocyst injeoted zoite tooyte Number of 15 25 27 16 24 25 lo ts Humber Injected 446 666 1292 526 604 1326 Number surviv 142 119 194 126 138 225 ing • (SI. 855) (1T.8J5) (16#) (23, 9%) (22*8#) (16*9#) Humber positive 18 19 21 0 0 0 for sporo- (12.8J&) (16.9J4) (10*8#) soites
* Surviving mosquitoes were dissected* 46
Fif. 1. Vacuum flask for harresting pupae*
*
Fig. 2. ilicropipette needle for moeqiuito injections.
48 Inaction of Aedes aegyptl CONTROL EXPERIMENTAL Injection of Oocyst on Infected Blood Striated Muscle O ocyst in Head
Natural Blood Meal O ocyst on Ventral Ganglion
Oocyst on Stomach Wall Oocyst on Malpighian Tubule
Ovanes -Oocyst on Integument
Fig* 5* Comparison of oocyst development in normally infected and injected mosquitoes (diagrammatic)*
Fig 6. Qooyst (8-day) near striated muscle. X 520. / V ' * < t Fig. 7. Oocyst (8-day) in striated muscle. X 312. SI Pig* S. Two oocysts (7-day) in thorax of Aedes aegyptl X 160* —— ■' “ Fig* 9* Oocyst (7-day) in fat body In abdomen* X 312* 62 I A*M Fig. 10. Oocyst (8-day) on ventral nerve ganglion. X 912 Fig. 11. Oocyat (8-day) on salivary gland. X 312. 68 n * . 1 2 , O ooyst ( 8-day) on traoheol® and fat body. X 512. Fig. 13. Oooyst (8-day) on intogumont. X 620* 64 Fig* 14. Oooyst (8-day) on ventral diverticulum. X 620. Fig. 16. Oooyst (8-day) on malpighian tubule# X 620. W*. 16* Oooyst (8-day) In head, near ommatidia. X 812, Pig# 17, Oocyst (8-day) in third segment of maxillary palp. X 812. 66 m m ® , g t # * • I 1 Fig* 18* Oooyst (8-day) in haemocoel of abdomen* X 520* Fig. 19* Oooyst (8-day) in fat body near traoheole; note sporosoites* X 520* BIOGRAPHY Augustus Burns Weathersby was bora May 19, 1913, in Simpson County, Mississippi. Most of his boyhood was spent in Texas, where he attended elementary and junior high schools. He graduated from the fylertown, Mississippi High School in 1931. His first two years of college work were taken at Copiah-LincoIn Junior College, Wesson, Mississippi. In 1938 he received his Bachelor of Arts degree from Louisiana State University, Baton Route, Louisiana, and in 1940 a Master of Science in Entomology from the same University, he was employed by the Louisiana Department of Agriculture as an Entomologist from 1940 to 1942. He e n liste d in the Ikiited S tates Navy in 1942 and was commissioned Ensign in 1943. He served as Entomologist in Epide miology Unit ho. 17, attached to the Third Marine Division in the Pacific Theater from 1943 to 1945. Upon returning to the United States in 1945, he married Miss Olive Hammons of Ruston, Louisiana. He was Entomologist for Naval Medical Research Unit No, 3 in Cairo, Egypt and Tehran, Iran during 1946 and 1947, From 1947 to 1949 he was engaged in malaria research at the Naval Medical Research Institute, Bethesda, Maryland. For three semesters in 1949-1950 he was under instruction at Louisiana State University, He returned to the Naval Medical Research Institute in 1950, continuing work in malaria and studying at The George Washington University, Washington, D*C, He is a member of the American Society of Tropical Medicine and Hygiene* The An to mo logical Society of Washington* and The American Mosquito Control Association* He was elected in 19b0 to associate membership in Sigma Ai. EXAMINATION AND THESIS REPORT Candidate: A* B* We&theraby Major Field: Snto&ology Title of Thesis: “Relationships B©tween the Stomaeh Wall of Mosquitoes and tha Exogenous Development of Malaria* 11 Approved: Major Professor and Chairman Graduate School EXAMINING COMMITTEE: Date of Examination: Monday, May 10, 1954