biological factors influencing transmission of trypanosoma rangeli by rhodnius prolixus

Biological Factors Influencing Transmission of Trypanosoma rangeli by Rhodnius prolixusAuthor(s): Eleanor Johnson TobieSource: The Journal of Parasitology, Vol. 51, No. 5 (Oct., 1965), pp. 837-841Published by: The American Society of ParasitologistsStable URL: http://www.jstor.org/stable/3276172 .

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THE JOURNAL OF PARASITOLOGY Vol. 51, No. 5, October 1965, p. 837-841

BIOLOGICAL FACTORS INFLUENCING TRANSMISSION OF TRYPANOSOMA RANGELI BY RHODNIUS PROLIXUS

Eleanor Johnson Tobie U. S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014

ABSTRACT: Certain biological factors which might influence transmission of a Venezuelan strain of Trypanosoma rangeli by Rhodnius prolixus are presented with statistical evaluation of their significance. T. rangeli must invade the salivary glands of R. prolixus before transmission is possible. When 1st instar nymphs were exposed to an infected meal, survival during the nymphal stages decreased. This decrease was statistically significant and indicates that T. rangeli is pathogenic for R. prolixus nymphs. There was no indication that blood from different vertebrate hosts influenced survival. In adult survivors exposed as nymphs as well as in those exposed as adults the cycle progressed to the salivary glands in a significantly greater percentage of males than females. There was no significant difference in the percentage of mature infections between those exposed as 1st instar nymphs and those exposed as adults. There appeared to be no advantage in multiple infective feedings.

Following the ingestion of Trypanosoma rangeli by Rhodnius prolixus, the cyclical process begins with the trypanosomes chang- ing form and multiplying in the alimentary canal. This first stage of development is a constant feature; the second, passage into the hemolymph, is not. However, once the hemolymph is invaded, development progresses to the salivary glands. Transmission of Trypano- soma rangeli by the invertebrate host is not possible until salivary gland invasion has occurred. Groot (1954) in a comparative study of the biological behavior of four strains of T. rangeli in Rhodnius prolixus reported that in one strain (ariarii) invasion of the salivary glands always occurred when flagellates were found in the hemolymph and his figures indicated at least 33% with salivary gland involvement. This high percentage of invasion was not observed in all the strains. Tobie (1961), working with a cyclically maintained strain, obtained only a 12.5% invasion of the salivary glands. The percentage, however, increased after a number of cyclical passages (Tobie, 1964). Grewal (1957) studying pathogenicity of T. rangeli to R. prolixus reported that most of the bugs which died or could not molt had

hemolymph infections. Only 41 out of 120 survived to become adults and only one of these had a salivary gland infection. This great loss he attributes to heavy parasitosis of the insect, "the entire system being blocked by flagellates."

Received for publication 26 February 1965.

837

Intestinal infections without subsequent hemolymph-salivary gland invasion are com- mon in R. prolixus exposed by bite, but once the hemolymph is involved, the infection in- vades the glands. It seemed, therefore, of interest to consider factors which might have some influence on the completion of this cycle in the insect host.

MATERIALS AND METHODS

The strain of Trypanosoma rangeli used in this study was derived from the Venezuelan El Tocuyo strain received from Dr. Felix Pifano in 1958 (Tobie, 1961). Five- to 6-day-old Sprague-Daw- ley rats served as the vertebrate host and the reduviid, Rhodnius prolixus as the invertebrate host. The rats were infected by the bite of R. prolixus and reduviids were exposed individually by feeding on the rats (Tobie, 1964). Infection in the rats was verified by microscopic examina- tion of fresh drops of blood or by blood culture, and in the reduviid by transmission of the infection or by dissection (Tobie, 1961).

When 1st instar nymphs were used they were observed for salivary gland infection when they fed, which as a rule was once between each molt and for at least two feedings as adults. Whenever possible, exposed nymphs, which could not complete a molt or were unable to feed, were dissected and carefully examined to determine the stage of the infection. When infective feedings were begun after the last molt, each of the adults was observed for approximately 3 months, through six or seven feedings. This approximated the length of time those exposed as nymphs were observed as well as the number of feedings they received. Since the sex of R. prolixus cannot be determined before the 3rd instar (Gillett, 1935) it was not possible to start infective feedings of 1st instar nymphs with equal numbers of males and females. This, however, was done when adults were used.



838 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 5, OCTOBER 1965

TABLE I. Adult survivors among Rhodnius prolixus, unexposed and exposed to Trypanosoma rangeli.

No. fed No. of survivors R. prolixus1 st mstnr R. p sst instar Adult Males Females

Unexposed Fed on adult rabbits 100 84 (84.0)2 31 (36.9)3 53 (63.1)3 Fed on baby rats 54 45 (83.3) 17 (37.8) 28 (62.2) Total 154 129 (83.8) 48 (37.2) 81 (62.8)

Exposed Fed on baby rats 237 147 (62.0) 74 (50.3) 73 (49.7)

1 All fed on uninfected animals except for the one infective meal of the exposed group. 2 Percentage of number fed. 3 Percentage of adult survivors.

Unexposed triatomids were fed on two different hosts. The first lot was fed in groups, following each molt, on the shaved back of a rabbit, the same manner in which our colony of R. prolixus is fed. Because the question arose as to the possible influence of blood from two different hosts each triatomid in a second lot was fed individually on young rats just as the exposed ones were. All the reduviids were maintained in an incubator at 27 C with approximately 67% relative humidity.

RESULTS

As shown in Table I, 84% of the unexposed reduviids, observed from the 1st instar stage until they molted the fifth time, survived to become adults. During the nymphal period inability to molt or feed, which resulted in mortality, was due to unknown causes since environmental conditions were controlled. The vertebrate host, rat or rabbit, had no apparent influence on mortality.

When an infected meal was supplied to 1st instar nymphs survival decreased to 62%.

Thirty-eight per cent of the 237 fed were lost

during the nymphal period regardless of the

stage to which the infection had progressed. Comparing this with the total unexposed group (Table I), we have a statistically significant decrease in survival due to infective feeding (x2 = 20.2 P < 0.001).

TABLE II. Mortality distribution during nymphal to Trypanosoma rangeli.

The infection interfered with the molting process itself in that many bugs could not

complete a molt. This interference was particularly noticeable during the first two and the last two molts. Table II shows how the losses were distributed during the five instars and molts in 100 bugs from the last ten groups exposed, as compared to the losses in the two

unexposed groups. There were longer inter- vals between feeding and molting in exposed nymphs than in unexposed nymphs, particularly in the latter stages. In the unexposed groups the first nymph molted at the end of each succeeding instar on days 7, 7, 9, 10, 16, while in two groups totaling 22, exposed in the 19th cyclical passage, the first nymph molted on days 8, 9, 10, 12, 21.

There were indications that T. rangeli is more pathogenic for nymphs destined to become females rather than males. Table I shows the difference in the percentage of male and female survivors in unexposed and exposed groups. When these percentages are compared statistically the difference is significant (x2 = 4.29 P = 0.04). In evaluating these figures it was assumed that initially both groups had the same ratio of males and females, since, as

previously mentioned, sex cannot be determined in the early nymphal stages.

stages in Rhodnius prolixus, unexposed and exposed

No. fed No. of deaths during indicated instar and molt (Percentage) R. prolixus 1st instar 1st 2nd 3rd 4th 5th Total

Unexposed Fed on adult rabbits 100 3 (3.0) 5 (5.0) 4 (4.0) 0 4 (4.0) 16 (16.0) Fed on baby rats 54 2 (3.7) 2(3.7) 1 (1.8) 0 4 (7.4) 9 (16.7) Total 154 5(3.2) 7(4.5) 5(3.2) 0 8(5.2) 25 (16.2)

Exposed Fed on baby rats 100 13 (13.0) 11 (11.0) 1 (1.0) 2 (2.0) 7 (7.0) 34 (34.0)

1 All fed on uninfected animals except for the one infective meal of the exposed group.



TOBIE-FACTORS INFLUENCING TRANSMISSION OF TRYPANOSOMA RANGELI 839

Of the 237 1st instar nymphs given infective feedings, 147 survived to become adults. In 28 of these survivors, or 19%, the infection progressed to the salivary glands. When the infected meal was provided by the first feeding after the final molt, 4 out of 44 or 9.1% developed salivary gland infections. Although there was a higher percentage with salivary gland involvement in survivors when the infection was acquired in the 1st nymphal stage the difference is not of statistical significance (x2 = 1.75 P = 0.19).

Among the 90 lost during the nymphal period in the group given infective feedings as 1st instar nymphs, 11 were known to have had salivary gland infections. These plus the 28 survivors with salivary gland involvement bring to 39 or 16.5% the number in which T. rangeli completed its developmental cycle. As compared to the total number with mature infections in those exposed as adults the difference again is not significant (x2 = 1.5 P > 0.20).

Of the 147 adult survivors exposed as nymphs, 74 were males and 73 females. In 21 of the male survivors, or 28%, T. rangeli completed its development. In only seven, or 10%, of the female survivors did the infection progress to completion. Male and female survivors were evenly divided but the chance of male survivors being able to transmit the infection because the cycle of development was completed was significantly greater (x2 =

7.24 P < 0.01). Of the total number of adults (survivors exposed as nymphs and those exposed as adults) 24 of 96 or 25% of the males and only 8 of 95 or 8.4% of the females developed salivary gland infections. When salivary gland involvement among all males and females is compared statistically greater involvement in the males is again highly significant (x2 = 8.26 P < 0.01). Sex of the triatomid is apparently a significant factor influencing development of T. rangeli in R. prolixus.

Since mature infections developed in a rela- tively low percentage of insects, multiple infective feedings were considered as a factor which might influence completion of the developmental cycle of T. rangeli in R. prolixus. Twenty-two adults, males and females equally divided, were given two to five infected meals

and compared with the same number given one infected meal. Salivary gland infections developed in two of each group. It is possible that the first feeding would have been suffi- cient for the two which received two and three infective meals respectively. These results confirmed incidental observations made on nymphs during the course of these studies. It appears that there was no particular advantage to multiple infective feedings.

When reduviids were exposed as adults there was no mortality due to the infection during the 3 months of observation. Intestinal infections were poor in all those exposed as adults compared to those exposed as nymphs. Of the four exposed as adults in which there was salivary gland involvement two were able to feed and transmit the infection for 6 months, one for 7 months, and one for 8 months. Pre- viously, one adult, 75 F, had been used 9 months after exposure for cyclical passage of the strain (Tobie, 1964). During this 9-month period it fed and infected a rat every 2 weeks. When nymphs with salivary gland infections became adults and could feed they survived with the infection for at least 4 and even up to 9 months. Their exposure occurred in the 1st nymphal stage approximately 3 months before they became adults.

DISCUSSION

Criteria for determining pathogenicity in arthropods are limited. It is usually possible to estimate pathogenic effects only by noting an increase in mortality. Gamham (1955) has discussed pathogenicity of protozoa to invertebrate hosts. It varies considerably depending on whether the arthropod is the sole host or whether two hosts are involved. Protozoa can be very virulent if an insect is the only host. Where two hosts are involved and the protozoan is pathogenic to the vertebrate, it seems to have little or no effect on the invertebrate. Garnham attributes the mechanism causing pathogenicity in insects to some gross change leading to compression, blockage, or total re- placement of tissue. Rhodnius prolixus infected with Trypanosoma rangeli, as Grewal (1957) also observed, can show a heavy infection in the gut, increased hemolymph which has become cloudy, and salivary glands which have lost their red color and are alive with



840 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 5, OCTOBER 1965

moving flagellates. Fisher and Sanbor (1962) reported experiments which provided evidence that the microsporidian, Nosema, exhibited an- other mode of pathogenesis in several insect hosts. Excessive amounts of the juvenile hormone were apparently produced which in- duced more rapid growth and inhibited meta-

morphosis. T. rangeli inhibited growth of R.

prolixus, rather than accelerated it, as evi- denced by the longer periods between each ecdysis. The molting process, however, was interfered with in that many could not com-

plete a molt. In either case growth and meta-

morphosis were abnormal. Environmental factors affecting the develop-

ment of parasites in invertebrates can easily be controlled in the laboratory, and temperature, particularly, has been found to be an

important factor. Biological factors are less

readily controlled. They can, however, be of

great importance in the development of a parasite. Wijers (1958) found that the age of the tsetse fly, Glossina palpalis, at the time of the infected meal was an important factor affecting the number of flies with mature infections, i.e., showing metacyclic trypanosomes in the salivary glands. Flies fed within 24 hr of emerging were more readily infected than older ones. R. prolixus will not feed for at least 3 days after hatching or molting, and

exposing them in the 1st nymphal stage did not produce a significantly greater number in which mature infections developed. It is possible that young adults which we used were more readily infected than older ones would have been. This we did not investigate.

Wood (1954), during a study on environmental temperature as a factor in the development of T. cruzi in a reduviid, noted that certain bugs which received fewer parasites in their infected meal showed as many parasites in a fecal droplet as others which received

greater numbers. T. rangeli parasitemia in the vertebrate is always low. Many times trypanosomes could not be found by microscopic ex- amination. Regardless of the low level of parasitemia in the vertebrate the 1st stage of the infection in the insect was a constant feature and heavy intestinal infections developed particularly in nymphs.

The number of R. prolixus males with mature infections of T. rangeli was greater than

the number of females. It is of interest that the male G. morsitans is also more readily in-

fected, as Fairbaim and Culwick (1950) and Ashcroft (1959) reported for T. brucei and T. rhodesiense.

Several species of vertebrates have been found infected with T. rangeli in nature and various laboratory rodents and chickens have been used as a source of blood for feeding reduviids. In our experience R. prolixus fed

willingly on either the rat or the rabbit. The vertebrate host apparently has no influence on

development or survival of these insects. It would be interesting to know whether the

observations on the various factors presented in this study would be similar if the insect were observed in nature.

ACKNOWLEDGMENTS

The author is indebted to Dr. David W.

Alling for the statistical analysis of the data. She also wishes to acknowledge the valuable technical assistance of Mrs. Flora C. Gilliam.

LITERATURE CITED

ASHCROFT, M. T. 1959. The sex ratio of infected flies found in transmission experiments with Glossina morsitans and Trypanosoma rhodesiense and T. brucei. Tr. Roy. Soc. Trop. Med. Hyg. 53: 394-399.

FAIRBAIRN, H., AND A. T. CULWICK. 1950. The transmission of the polymorphic trypanosomes. Acta Trop. Basel 7: 19-47.

FISHER, F. M., AND R. C. SANBORN. 1962. Pro- duction of insect juvenile hormone by the microsporidian parasite Nosema. Nature 194: 1193.

GARNHAM, P. C. C. 1955. The comparative pathogenicity of protozoa in their vertebrate and invertebrate hosts. No. 5, Mechanisms of microbial pathogenicity. Symposium Soc. Gen. Microbiol., p. 191-206.

GILLETT, J. D. 1935. The genital sterna of the immature stages of Rhodnius prolixus (Hemip- tera). Tr. Roy. Ent. Soc. London 83: 1-5.

GREWAL, M. S. 1957. Pathogenicity of Trypano- soma rangeli Tejera, 1920 in the invertebrate host. Exp. Parasit. 6: 123-130.

GROOT, H. 1954. Estudios sobre los trypano- somas humanos clasificados como T. rangeli con especial referencia a su evoluci6n en Rhodnius prolixus y a su comparacion con T. ariarii. An. Soc. Biol. Bogota 6: 109-126.

TOBIE, E. J. 1961. Experimental transmission and biological comparison of strains of Trypano- soma rangeli. Exp. Parasit. 11: 1-9.

1964. Increased infectivity of a cyclically maintained strain of Trypanosoma



TOBIE-FACTORS INFLUENCING TRANSMISSION OF TRYPANOSOMA RANGELI 841

rangeli to Rhodnius prolixus and mode of transmission by invertebrate host. J. Parasit. 50: 593-598.

WIJERs, D. J. B. 1958. Factors that may influence the infection rate of Glossina palpalis with Trypanosoma gambiense. I. The age of

the fly at the time of the infected feed. Ann. Trop. Med. Parasit. 52: 385-390.

WOOD, S. F. 1954. Environmental temperature as a factor in development of Trypanosoma cruzi in Triatoma protracta. Exp. Parasit. 3: 227-233.

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