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Observations on the development of Trypanosoma rangeli in the hemocoel of Rhodnius prolixus
JOURNAL
OF
IXVERTEBRATE
Observations
15, 118-125
PATHOLOGY
(1970)
on the Development of Trz~~an~s~ma in the HemocoeI of Rhodnius prolixus
rungeli
ELEANOR JOHNSON TOBIE Department Institute
of Health, of Allergy
Education and
and Welfare, National Institutes of Health, National Diseases, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014 Infectious
Receirjed
July
8, 1969
The development of Trypanosoma rangeli in the hemocoel of Rho&us prolixus was studied with the idea of elucidating the function of the forms found there. When flagellates from cultures of T. rangeli that have decreased in virulence are introduced into the hemocoel of R. prolixus the salivary glands are not invaded regularly or, if invaded, the infection does not persist. However, the parasite lives and multiplies in the hemocoel. Crithidial forms enter the plasmotocytes where they roll up and divide. This hemocyte becomes greatly enlarged due to the dividing leishmania forms within its cytoplasm. Eventually the cell ruptures releasing numerous crithidias, some of which transform into trypanosomes as evidenced by the presence of metacyclic as well as long forms. These observations also help explain the pathogenic action of T. rangeli to its invertebrate host.
observed that no trypanosomes were found in the salivary glands unless they were found in the hemolymph. For some time after this discovery transmission was thought possible with forms from both the rectal ampule and salivary glands. However, more recent experimental transmission attempts with the forms found in the feces were not successful (Tobie, 1964). Multiplication occurs in the hemolymph and intracellular forms have been observed in the hemocytes (Coutinho and Nussenzweig, 1952; Grewal, 1957; Groot, 1952; Tobie, 1968), but the development of these forms and their function has not been fully elucidated. The percentage of naturally infected R. pruliws in which T. rangeli passes through the intestinal wall, permitting development in the hemocoel, is low (D’Alessandro, 1963; Tobie, 1965). In order to have sufficient material to review, it was necessary to initiate the infection in the hemocoel. This also provided additional observations on
The natural development of Tr~~panosom~ rangeli in its invertebrate host begins with the ingestion of blood containing mature trypanosomes, and the change of host provokes morphological variations in these trypanosomes. Aflagellated and flagellated spherical and pyriform stages in the stomach, short and long crithidial forms in the intestine, and trypanosomes mixed with crithidias in the rectal ampule were described by Tejera (1920) and Rey-Matiz ( 1941). For many years the cycle was thought to be confined to the digestive tube, as in the case of Trypanosoma cruzi. Transmission through the bite of the insect was suggested by Pifano and Mayer (1949) when they investigated a report of trypanosomes in the proboscis of a triatomid, Rhodnius prolixus. The course of the cycle in the insect became apparent when Groot ( 1952) reported flagellates in the hemolymph of R. prolixus infected with T. rangeli, and 118
Trypanosoma
DEVELOPMENT IN Rhodnius
the pathogenicity of T. rang& to R. pro&us, a phenomenon previously studied by Grewal ( 1957), Tobie ( I965), and Gomez (1967).
119
diluted with a drop of 0.85 NaCl, pH 7.0, and injected into the hemocoel of the fresh triatomids. RESULTS
MATERIALS AND METHODS The strains of Trypanosoma rangeli were substrains of the Venezuelan El Tocuyo strain isolated from rats infected by the bite of triatomes approximately 12 years after the original isolation of the strain from a human by Prof. Felix Pifano C. When used the substrains had been maintained in vitro on a diphasic blood agar medium for not more than 10 months. The colony of Rhodnius prolixus was maintained in the laboratory and adults were selected for inoculation as previously described (Tobie, 1968). Each triatomid was infected by injecting a drop of rich culture material into the coelomic cavity through the connexivum by a syringe fitted with a short 27-gauge needle as described by Tobie ( 1968). At designated intervals a leg was severed from an infected specimen and the drop of hemolymph which oozed was collected on a slide. After quickly examining the drop microscopically the slide was tilted to allow the drop to spread before drying. After drying the smear was fixed in methyl alcohol, and stained 15-20 min with Giemsa’s stain prepared with distilled water buffered to pH 6.5. The same bug could not be used day after day since the infection was reduced when a drop of hemolymph was lost, The volume of hemolymph also needed to be replenished. The insects were saved, however, and from a few hemolymph was taken a second time weeks later, near the end of the experiment. A substrain of T. rangeli was also maintained in R. prolixus by the transfer of infected hemolymph from one bug to another. The drop of hemolymph for transfer was
When culture forms of Trypanosoma rangeli are introduced into the hemocoel of Rhodnius prolixus the salivary glands are not invaded regularly or, if invaded, the infection does not persist. This is caused by some change occurring in vitro affecting the infectivity of the strain. However, multiplication and interesting variations of the flagellate can be observed in the hemocoel. Short, stiff crithidial forms (Fig. 1) are present but not in as abounding numbers as the long crithidial forms (Fig. 2). Fewer trypanosomes, including both long (Fig. 3) and metacyclic forms (Fig. 4), are found. Leishmanialike forms occur free in the hemolymph (Fig. 5) as well as within the plasmatocytes (Figs. 7-13). In the substrain used only a rare flagellate was seen in drops of hemolymph on the first 2 days, after which many short, stumpy crithidias (Fig. 1) appeared. By day 5 numerous long, slender crithidias forming huge masses were present (Fig. 6). Some of the short crithidias show signs of division but this cannot account for the rapid increase in free flagelIates. During the early days the intracellular stages usually are rather indistinguishable (Fig. 10) as compared to those in later infections (Fig. 12) which might indicate some intracellular destruction of the parasite, but as can be seen by the enormous number within the hemocytes in advanced infections, multiplication is occurring within the cell. On the other hand, it may be merely a staining problem that makes the rare inclusion difficult to recognize since normal hemocytes are more difficult to stain than those filled with parasites. One can only speculate on the exact sequence in the development of these forms.
120
TOBIE
FIGS. l-5. Extracellular forms of Trypanosoma range& in hemolymph of Rhodnius prolixus. 1000 X . 1. Short, stumpy crithidias with very short free flagellum. 2. Long slender crithidias with long free flagellum. Kinetoplast anterior to nucleus. 3. Large trypanosome form. Kinetoplast posterior to nucleus. 4. Metacyclic trypanosome. Double kinetoplast indicates that this form may be capable of division. 5. Leishmania forms, probably released by hemocyte rupturing prematurely. Arrow indicates leishmania form in division. 5-month infection.
All can be found on the same slide as the infection progresses but, of course, their proportion varies. A scheme is presented in Fig. 14. The particular crithidial form which penetrates the digestive tube (B), and the area in which penetration takes place, are not known1 However, some changes must occur in the parasite when it begins life in 1 Recently R. dup. Watkins, (PhD Thesis, University of California, Berkeley, 1969) visualized the intracellular development of T. rungeli in the cells of the gut wall as well as in the wall of the salivary glands of R. pro2iru.s. Development in the wall of the salivary glands has also been observed by 0. E. Sousa, (Libro de Resumens, 2nd Cong. Centroamer. y 1st Nat. de Microbial., P. 55 Panama, 1968) in R. p&exerts.
the hemocoel. Certain crithidias invade the plasmatocytes ( C ), round up, and multiply (D). The plasmatocytes become enlarged and full of dividing parasites. Eventually they rupture releasing into the hemolymph numerous small crithidis (Fig. 13) some of which develop into metacyclic and long trypanosomes ( G) . Crithidias released by the plasmatocytes may invade the salivary glands (E ) where transformation to metacyclic trypanosomes takes place. It is also possible that the same crithidias that enter the plasmatocytes may invade the salivary glands (F) without going through a hemocyte phase. The process of the hemocytes engulfing
128
SCHWARTZ,
JR.,
TABLE 1 OF JAPANESE-BEETLE LARVAE INJECTED WITH SPORES OF Bacillus popilliae PRODUCED IN vrr~o (STRAIN NRRL B-2309M) OR IN vrvo (DRIED BLOOD FILM FROM INFECTED LARVAE )
INFECTION
Concentration spores/larvaa 100 1,000 10,000 0 Twenty-five tration for each
of
% Milky diseased larvae produced by spores in vivo
in vitro
0 24 40
0 0 24 larvae were used type of spore.
at each
concen-
than strain NRRL B-2309, but only 60% as viable as the spores from the dried blood film. By Dutky’s (1963) Equation 4, (% infection) l/2 = 3.051ogroN-5.2 the percentage of larvae infected by the dried blood film should have been 49% for 10,000 spores and 16% for 1,000 spores. Thus, these spores had not lost their infectivity (Table 1). Also, the percentage of larvae infected by injection of strain NRRL B-2309M spores proved that the strain had not lost infectivity by aging. However, we could not be sure that the failure of these spores to infect larvae in the feeding dust tests was not caused by such factors as the weak solution of formaldehyde or the soil pH. To eliminate these factors as a cause of loss of viability and to assure ourselves that each larva was getting a dose of spores that should initiate infection, we administered droplets of spore suspension containing 209,000 spores of strain NRRL B-2309M to each of 50 larvae, droplets containing 280, 000 spores from a dried blood film in the same way to each of 50 larvae and droplets of water to each of 25 larvae. The B-2309M spores did not infect any larvae, but spores from the blood film infected 60% of the larvae, and one larva fed a water droplet became infected (as a result of a natural infection present in the larvae). During the 28day test, 52% of the
AND
SHARPE
larvae were fed water only, 58% the droplets containing spores of B-2309M, and 20% the droplet containing spores of a blood film survived to the prepupal stage. It appeared that the larvae fed 260,000 spores of B-2309M were not adversely affected. Since the dose of B-2309M spores administered to the larvae in this test was 20 times greater than the dose that caused 24% infection when it was injected, we felt that it had been sufficient to induce an infection. B. popilliae strain NRRL B-2309M was therefore not infectious to Japanese beetle when it was administered to larvae via the alimentary canal at the doses tested. The reasons for the lack of infectivity were not apparent. REFERENCES DUTKY, S. R. 1940. Two new spore-forming bacteria causing milky diseases of Japanese beetle larvae. J. Agr. Res., 61, 57-68. DUTKY, S. R. 1942. Method for the preparation of spore-dust mixtures of Type A milky disease of Japanese beetle larvae for field inoculation. U.S. Dept. Agr., BUT. Entomol. Plant Quarantine, ET-192, 10 pp. (Processed ). DUTKY, S. R. 1963. The milky diseases. In “Insect Pathology, An Advanced Treatise,” (E. A. Steinhaus, ed.), Vol. 2, pp. 75-115, Academic Press, New York. DUTKY, S. R., AND FEST, W. C. 1942. Microinjector. U.S. Patent No. 2,270,804. PRIDHAM, T. G., ST. JULIAN, G., JR., ADAMS, G. L., HALL, H. H., AND JACKSON, R. W. 1964, Infection of Popillia japonica Newman larvae with vegetative cells of Bacillus popilliae Dutky and Bacillus lentimorbus Dutky. I. Insect Pathol., 6, 204-213. RHODES, R. A. 1965. Symposium on microbial insectides. II. Milky disease of the Japanese beetle. Baoteriol Reo., 29, 373-381. ST. JULIAN, G., AND HALL, H. H. 1968. Infection of Popillia japonica larvae with heatactivated spores of Bacillus popilliue. J. Invertebrate Pathol., 10, 48-53. SKARPE, E. S. 1966. Propagation of BaciUus popilliae in laboratory fermentors. Biotech. Bioeng., 8, 247-258.
I22
TOBIE
Intracellular development of Trypanosom rangeli in plasmatocytes of Rhodnius proFIGS. ‘7-12. zixus. 1000 x . 7. Long crithidia entering plasmatocyte. Several flagellates are in the process of rolling up within the cell. 8. Single crithidia rolling up inside plasmatocyte. 9. Invaded hemocyte with flagella of crithidias still evident outside cell membrane. 10. Hemocyte with intracellular forms from a 4day infection. 11. Crithidias apparently ready to enter cell. 12. Ruptured plasmatocyte with numerous dividing leishmania. 5-month infection.
duviids but most often this has been interpreted as phagocytosis (Groot, 1952; Zeledon, 1954; Grewal, 1957). Multiplication in the hemocoel was thus thought to take place outside the cell. Zeledon (1954) reported thick forms in division during the first 48 hr which is more or less in agree-
ment with the present observation of the presence of division in the short, stiff crithidias which appeared early in the infection. However, Coutinho and Nussenzweig ( 1952) interpreted the intracellular forms they saw as rolled-up crithidias in the process of evolution. Intracellular multiplica-
Trypanosoma
FIG. S-month
13. a. Intact plasmatocvte infection. 1OOo’X. ’
full
DEVELOPMENT
of
parasites. I
tion of T. rung& in plasmatocytes of R. prokrus was noted by Tobie (1968) while phagocytosis with destruction of the parasite occurred when several other hemoflagellates were introduced into the hemocoel. It may be that the hemolymph of R. proZixus is an excellent “tissue culture” for strains of T. rangeli that no longer have the ability to invade the salivary glands. It is of interest that development can progress to the final metacyclic trypanosome without involving the glands which are normally the site of the final stage in the cycle. The fact that metacyclic trypanosomes are found free in the hemolymph indicates that the bug can be infective to animals through contamination by the hemolymph if the insect is eaten or crushed. T. rangeli is a unique protozoan in that it is pathogenic to its invertebrate host and, so far as is known, has no adverse effect on its vertebrate host. Excessive multiplication in the hemocoel of R. prolixus increased
IN
b.
Rho&Gus
Ruptured
plasmatocyte
123
releasing
crithidias.
the quantity of hemolymph and inhibited molting with resulting deformities or death of a large percentage of bugs (Grewal, 1957; Tobie, 1965). Similar observations were made by Harington (1955) in uninfected nymphs artificially fed mixtures deficient in essential amino acids. Ormerod (1967) reported an increase in certain amino acids but a fall in the overall concentration of amino acids in the hemolymph of R. prolixus infected with a pathogenic Venezuelan strain of T. rangeli. This he interpreted as indicating that the large numbers in the hemolymph, as in a culture, used up nutrients which could not be replaced. This may help to explain the pathogenic action of T. rangeli to R. pro&us. However, in spite of this, the overwhelming parasitemia which can develop destroys the phagocytic plasmatocytes, and obstructs the circulation of the hemolymph, causing the death of the infected host.
124
TOBIE
FIG. 14. Development of Typanosoma rangeli in the hemocoel of Rho&us prolixus: Schematic. A. Reduviid sucks blood from vertebrate host and acquires trypanosome infection. B. Flagellates penetrate wall of digestive tube to enter hemocoel. C. Crithidias enter plasmatocytes (posterior end first) divide, change to crithidias, and are reand round up, D. Leishmania forms within plasmatocytes, leased into hemolymph. E. Crithidias penetrate wall of salivary gland and transform into metacyclic trypanosomes. This infective form is injected into a new host when feeding. F. In a natural infection it is possible that certain crithidias may enter the salivary glands directly. G. Infective metacyclic and noninfective large trypanosome forms in the hemolymph. 1. Stumpy crithidia-short free flagellum. 2. Short dividing crithidial form. 3. Long crithidial form. 4. Rounding-up crithidial form. 6. Dividing leishmania form. 7. Emerging crithidial forms. 8. Metacyclic 5. Leishmania form. trypanosomes. 9. Large trypanosomes.
ACKNOWLEDGMENTS The author is grateful to Mrs. Flora for her technical assistance, and to Gertrude H. Nicholson for the drawing
C. Gilliam artist Mrs. of Fig. 14.
REFERENCES D’ALESSANDRO Trypanosoma
B.,
A. 1983. The life cycle of rungeli in triatomid bugs as it
occurs in nature. Fat., 23, 2130.
Bull.
TuZune
Univ.
COUTINHO, J. O., AND NUSSENZWEIG, V. InfecTa experimental de triatomineos Trypanosomu rungeli Tejera, 1920. Fob Biol. Scio Z’auZo, 18, 181-188. G6MU,
I. 1967. Neuvas de la aecion patogena geli Tejera, 1920 sobre
Med. 1952. pelo Clin.
observaciones acerca de1 Trypanosoma ranRhodnius prolixus Stal,
Trypanosoma
DEVELOPMENT
1859. Heu. Inst. Med. Trap. S&o Paulo, 9, 510. Pathogenicity of TrypanGREWAL, M. S. 1957. osoma Tangeli Tejera, 1920 in the invertebrate host. Exptl. Parasitol., 6, 123-130. GIIOOT, H. 1952. Further observations on Trypanosoma a7iarii of Columbia, South America. Am. J. TI+o~. Med. Hgy., 1, 585-592. HARINGTON, J. S. 1955. Certain aspects of the haemolymph of Rho&us prolixus StX Ph.D. Thesis, University of London, England (cited by Ormerod, W. E., 1967). 1967. The effect of TrypanoOWEROD, W. E. soma Tangeli on the concentration of amino acids in the hemolymph of Rho&us prolixus. J. Invertebrate Pathol., 9, 247-255. PIFANO, F., AND MAYER, M. 1949. Hallazgo de formas evolutivas de1 Trypanosoma rangeli en el jugo de la trompa de Rho&us proZixw de Venezuela. Arch. Venezolanos. Patol. Trap. Pam&tot. M&d., 1, 153-158.
IN Rhodnius
125
REY-MATIZ, H. 1941. Obsewaciones panosomas en Colombia. Reu. Bogotd, 10, 25-19.
sobre tryFat. Med.
TEJERA, E. 1920. Un noveau flagellb de Rhodnius prolixus, Trypanosoma (ou Cri~hidia) rangeli n. sp. Bull. Sot. Pathol. Exotique, 13, 527530. TOBIE, E. J. 1964. Increased infectivity of a cyclically maintained strain of Typanosoma rangeli to Rho&us prolixus and mode of transmission by invertebrate host. J. Parasitol., 50, 593-598. TOBIE, E. J. 1965. ing transmission Rho&us prolixus.
Biological factors influencof Trypanosoma rangeli by J. Parasitol., 51, 837-841.
TOBIE, E. J. 1968. flagellates in the lixus. J. Parasitol.,
The fate of some culture hemocoel of Rhodnius pro54, 1040-1046.
ZELED~N, Biol.
1954. Tripanosomiasis R. Trap., 2, 231-268.
rangeli.
Reu.
OF
IXVERTEBRATE
Observations
15, 118-125
PATHOLOGY
(1970)
on the Development of Trz~~an~s~ma in the HemocoeI of Rhodnius prolixus
rungeli
ELEANOR JOHNSON TOBIE Department Institute
of Health, of Allergy
Education and
and Welfare, National Institutes of Health, National Diseases, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014 Infectious
Receirjed
July
8, 1969
The development of Trypanosoma rangeli in the hemocoel of Rho&us prolixus was studied with the idea of elucidating the function of the forms found there. When flagellates from cultures of T. rangeli that have decreased in virulence are introduced into the hemocoel of R. prolixus the salivary glands are not invaded regularly or, if invaded, the infection does not persist. However, the parasite lives and multiplies in the hemocoel. Crithidial forms enter the plasmotocytes where they roll up and divide. This hemocyte becomes greatly enlarged due to the dividing leishmania forms within its cytoplasm. Eventually the cell ruptures releasing numerous crithidias, some of which transform into trypanosomes as evidenced by the presence of metacyclic as well as long forms. These observations also help explain the pathogenic action of T. rangeli to its invertebrate host.
observed that no trypanosomes were found in the salivary glands unless they were found in the hemolymph. For some time after this discovery transmission was thought possible with forms from both the rectal ampule and salivary glands. However, more recent experimental transmission attempts with the forms found in the feces were not successful (Tobie, 1964). Multiplication occurs in the hemolymph and intracellular forms have been observed in the hemocytes (Coutinho and Nussenzweig, 1952; Grewal, 1957; Groot, 1952; Tobie, 1968), but the development of these forms and their function has not been fully elucidated. The percentage of naturally infected R. pruliws in which T. rangeli passes through the intestinal wall, permitting development in the hemocoel, is low (D’Alessandro, 1963; Tobie, 1965). In order to have sufficient material to review, it was necessary to initiate the infection in the hemocoel. This also provided additional observations on
The natural development of Tr~~panosom~ rangeli in its invertebrate host begins with the ingestion of blood containing mature trypanosomes, and the change of host provokes morphological variations in these trypanosomes. Aflagellated and flagellated spherical and pyriform stages in the stomach, short and long crithidial forms in the intestine, and trypanosomes mixed with crithidias in the rectal ampule were described by Tejera (1920) and Rey-Matiz ( 1941). For many years the cycle was thought to be confined to the digestive tube, as in the case of Trypanosoma cruzi. Transmission through the bite of the insect was suggested by Pifano and Mayer (1949) when they investigated a report of trypanosomes in the proboscis of a triatomid, Rhodnius prolixus. The course of the cycle in the insect became apparent when Groot ( 1952) reported flagellates in the hemolymph of R. prolixus infected with T. rangeli, and 118
Trypanosoma
DEVELOPMENT IN Rhodnius
the pathogenicity of T. rang& to R. pro&us, a phenomenon previously studied by Grewal ( 1957), Tobie ( I965), and Gomez (1967).
119
diluted with a drop of 0.85 NaCl, pH 7.0, and injected into the hemocoel of the fresh triatomids. RESULTS
MATERIALS AND METHODS The strains of Trypanosoma rangeli were substrains of the Venezuelan El Tocuyo strain isolated from rats infected by the bite of triatomes approximately 12 years after the original isolation of the strain from a human by Prof. Felix Pifano C. When used the substrains had been maintained in vitro on a diphasic blood agar medium for not more than 10 months. The colony of Rhodnius prolixus was maintained in the laboratory and adults were selected for inoculation as previously described (Tobie, 1968). Each triatomid was infected by injecting a drop of rich culture material into the coelomic cavity through the connexivum by a syringe fitted with a short 27-gauge needle as described by Tobie ( 1968). At designated intervals a leg was severed from an infected specimen and the drop of hemolymph which oozed was collected on a slide. After quickly examining the drop microscopically the slide was tilted to allow the drop to spread before drying. After drying the smear was fixed in methyl alcohol, and stained 15-20 min with Giemsa’s stain prepared with distilled water buffered to pH 6.5. The same bug could not be used day after day since the infection was reduced when a drop of hemolymph was lost, The volume of hemolymph also needed to be replenished. The insects were saved, however, and from a few hemolymph was taken a second time weeks later, near the end of the experiment. A substrain of T. rangeli was also maintained in R. prolixus by the transfer of infected hemolymph from one bug to another. The drop of hemolymph for transfer was
When culture forms of Trypanosoma rangeli are introduced into the hemocoel of Rhodnius prolixus the salivary glands are not invaded regularly or, if invaded, the infection does not persist. This is caused by some change occurring in vitro affecting the infectivity of the strain. However, multiplication and interesting variations of the flagellate can be observed in the hemocoel. Short, stiff crithidial forms (Fig. 1) are present but not in as abounding numbers as the long crithidial forms (Fig. 2). Fewer trypanosomes, including both long (Fig. 3) and metacyclic forms (Fig. 4), are found. Leishmanialike forms occur free in the hemolymph (Fig. 5) as well as within the plasmatocytes (Figs. 7-13). In the substrain used only a rare flagellate was seen in drops of hemolymph on the first 2 days, after which many short, stumpy crithidias (Fig. 1) appeared. By day 5 numerous long, slender crithidias forming huge masses were present (Fig. 6). Some of the short crithidias show signs of division but this cannot account for the rapid increase in free flagelIates. During the early days the intracellular stages usually are rather indistinguishable (Fig. 10) as compared to those in later infections (Fig. 12) which might indicate some intracellular destruction of the parasite, but as can be seen by the enormous number within the hemocytes in advanced infections, multiplication is occurring within the cell. On the other hand, it may be merely a staining problem that makes the rare inclusion difficult to recognize since normal hemocytes are more difficult to stain than those filled with parasites. One can only speculate on the exact sequence in the development of these forms.
120
TOBIE
FIGS. l-5. Extracellular forms of Trypanosoma range& in hemolymph of Rhodnius prolixus. 1000 X . 1. Short, stumpy crithidias with very short free flagellum. 2. Long slender crithidias with long free flagellum. Kinetoplast anterior to nucleus. 3. Large trypanosome form. Kinetoplast posterior to nucleus. 4. Metacyclic trypanosome. Double kinetoplast indicates that this form may be capable of division. 5. Leishmania forms, probably released by hemocyte rupturing prematurely. Arrow indicates leishmania form in division. 5-month infection.
All can be found on the same slide as the infection progresses but, of course, their proportion varies. A scheme is presented in Fig. 14. The particular crithidial form which penetrates the digestive tube (B), and the area in which penetration takes place, are not known1 However, some changes must occur in the parasite when it begins life in 1 Recently R. dup. Watkins, (PhD Thesis, University of California, Berkeley, 1969) visualized the intracellular development of T. rungeli in the cells of the gut wall as well as in the wall of the salivary glands of R. pro2iru.s. Development in the wall of the salivary glands has also been observed by 0. E. Sousa, (Libro de Resumens, 2nd Cong. Centroamer. y 1st Nat. de Microbial., P. 55 Panama, 1968) in R. p&exerts.
the hemocoel. Certain crithidias invade the plasmatocytes ( C ), round up, and multiply (D). The plasmatocytes become enlarged and full of dividing parasites. Eventually they rupture releasing into the hemolymph numerous small crithidis (Fig. 13) some of which develop into metacyclic and long trypanosomes ( G) . Crithidias released by the plasmatocytes may invade the salivary glands (E ) where transformation to metacyclic trypanosomes takes place. It is also possible that the same crithidias that enter the plasmatocytes may invade the salivary glands (F) without going through a hemocyte phase. The process of the hemocytes engulfing
128
SCHWARTZ,
JR.,
TABLE 1 OF JAPANESE-BEETLE LARVAE INJECTED WITH SPORES OF Bacillus popilliae PRODUCED IN vrr~o (STRAIN NRRL B-2309M) OR IN vrvo (DRIED BLOOD FILM FROM INFECTED LARVAE )
INFECTION
Concentration spores/larvaa 100 1,000 10,000 0 Twenty-five tration for each
of
% Milky diseased larvae produced by spores in vivo
in vitro
0 24 40
0 0 24 larvae were used type of spore.
at each
concen-
than strain NRRL B-2309, but only 60% as viable as the spores from the dried blood film. By Dutky’s (1963) Equation 4, (% infection) l/2 = 3.051ogroN-5.2 the percentage of larvae infected by the dried blood film should have been 49% for 10,000 spores and 16% for 1,000 spores. Thus, these spores had not lost their infectivity (Table 1). Also, the percentage of larvae infected by injection of strain NRRL B-2309M spores proved that the strain had not lost infectivity by aging. However, we could not be sure that the failure of these spores to infect larvae in the feeding dust tests was not caused by such factors as the weak solution of formaldehyde or the soil pH. To eliminate these factors as a cause of loss of viability and to assure ourselves that each larva was getting a dose of spores that should initiate infection, we administered droplets of spore suspension containing 209,000 spores of strain NRRL B-2309M to each of 50 larvae, droplets containing 280, 000 spores from a dried blood film in the same way to each of 50 larvae and droplets of water to each of 25 larvae. The B-2309M spores did not infect any larvae, but spores from the blood film infected 60% of the larvae, and one larva fed a water droplet became infected (as a result of a natural infection present in the larvae). During the 28day test, 52% of the
AND
SHARPE
larvae were fed water only, 58% the droplets containing spores of B-2309M, and 20% the droplet containing spores of a blood film survived to the prepupal stage. It appeared that the larvae fed 260,000 spores of B-2309M were not adversely affected. Since the dose of B-2309M spores administered to the larvae in this test was 20 times greater than the dose that caused 24% infection when it was injected, we felt that it had been sufficient to induce an infection. B. popilliae strain NRRL B-2309M was therefore not infectious to Japanese beetle when it was administered to larvae via the alimentary canal at the doses tested. The reasons for the lack of infectivity were not apparent. REFERENCES DUTKY, S. R. 1940. Two new spore-forming bacteria causing milky diseases of Japanese beetle larvae. J. Agr. Res., 61, 57-68. DUTKY, S. R. 1942. Method for the preparation of spore-dust mixtures of Type A milky disease of Japanese beetle larvae for field inoculation. U.S. Dept. Agr., BUT. Entomol. Plant Quarantine, ET-192, 10 pp. (Processed ). DUTKY, S. R. 1963. The milky diseases. In “Insect Pathology, An Advanced Treatise,” (E. A. Steinhaus, ed.), Vol. 2, pp. 75-115, Academic Press, New York. DUTKY, S. R., AND FEST, W. C. 1942. Microinjector. U.S. Patent No. 2,270,804. PRIDHAM, T. G., ST. JULIAN, G., JR., ADAMS, G. L., HALL, H. H., AND JACKSON, R. W. 1964, Infection of Popillia japonica Newman larvae with vegetative cells of Bacillus popilliae Dutky and Bacillus lentimorbus Dutky. I. Insect Pathol., 6, 204-213. RHODES, R. A. 1965. Symposium on microbial insectides. II. Milky disease of the Japanese beetle. Baoteriol Reo., 29, 373-381. ST. JULIAN, G., AND HALL, H. H. 1968. Infection of Popillia japonica larvae with heatactivated spores of Bacillus popilliue. J. Invertebrate Pathol., 10, 48-53. SKARPE, E. S. 1966. Propagation of BaciUus popilliae in laboratory fermentors. Biotech. Bioeng., 8, 247-258.
I22
TOBIE
Intracellular development of Trypanosom rangeli in plasmatocytes of Rhodnius proFIGS. ‘7-12. zixus. 1000 x . 7. Long crithidia entering plasmatocyte. Several flagellates are in the process of rolling up within the cell. 8. Single crithidia rolling up inside plasmatocyte. 9. Invaded hemocyte with flagella of crithidias still evident outside cell membrane. 10. Hemocyte with intracellular forms from a 4day infection. 11. Crithidias apparently ready to enter cell. 12. Ruptured plasmatocyte with numerous dividing leishmania. 5-month infection.
duviids but most often this has been interpreted as phagocytosis (Groot, 1952; Zeledon, 1954; Grewal, 1957). Multiplication in the hemocoel was thus thought to take place outside the cell. Zeledon (1954) reported thick forms in division during the first 48 hr which is more or less in agree-
ment with the present observation of the presence of division in the short, stiff crithidias which appeared early in the infection. However, Coutinho and Nussenzweig ( 1952) interpreted the intracellular forms they saw as rolled-up crithidias in the process of evolution. Intracellular multiplica-
Trypanosoma
FIG. S-month
13. a. Intact plasmatocvte infection. 1OOo’X. ’
full
DEVELOPMENT
of
parasites. I
tion of T. rung& in plasmatocytes of R. prokrus was noted by Tobie (1968) while phagocytosis with destruction of the parasite occurred when several other hemoflagellates were introduced into the hemocoel. It may be that the hemolymph of R. proZixus is an excellent “tissue culture” for strains of T. rangeli that no longer have the ability to invade the salivary glands. It is of interest that development can progress to the final metacyclic trypanosome without involving the glands which are normally the site of the final stage in the cycle. The fact that metacyclic trypanosomes are found free in the hemolymph indicates that the bug can be infective to animals through contamination by the hemolymph if the insect is eaten or crushed. T. rangeli is a unique protozoan in that it is pathogenic to its invertebrate host and, so far as is known, has no adverse effect on its vertebrate host. Excessive multiplication in the hemocoel of R. prolixus increased
IN
b.
Rho&Gus
Ruptured
plasmatocyte
123
releasing
crithidias.
the quantity of hemolymph and inhibited molting with resulting deformities or death of a large percentage of bugs (Grewal, 1957; Tobie, 1965). Similar observations were made by Harington (1955) in uninfected nymphs artificially fed mixtures deficient in essential amino acids. Ormerod (1967) reported an increase in certain amino acids but a fall in the overall concentration of amino acids in the hemolymph of R. prolixus infected with a pathogenic Venezuelan strain of T. rangeli. This he interpreted as indicating that the large numbers in the hemolymph, as in a culture, used up nutrients which could not be replaced. This may help to explain the pathogenic action of T. rangeli to R. pro&us. However, in spite of this, the overwhelming parasitemia which can develop destroys the phagocytic plasmatocytes, and obstructs the circulation of the hemolymph, causing the death of the infected host.
124
TOBIE
FIG. 14. Development of Typanosoma rangeli in the hemocoel of Rho&us prolixus: Schematic. A. Reduviid sucks blood from vertebrate host and acquires trypanosome infection. B. Flagellates penetrate wall of digestive tube to enter hemocoel. C. Crithidias enter plasmatocytes (posterior end first) divide, change to crithidias, and are reand round up, D. Leishmania forms within plasmatocytes, leased into hemolymph. E. Crithidias penetrate wall of salivary gland and transform into metacyclic trypanosomes. This infective form is injected into a new host when feeding. F. In a natural infection it is possible that certain crithidias may enter the salivary glands directly. G. Infective metacyclic and noninfective large trypanosome forms in the hemolymph. 1. Stumpy crithidia-short free flagellum. 2. Short dividing crithidial form. 3. Long crithidial form. 4. Rounding-up crithidial form. 6. Dividing leishmania form. 7. Emerging crithidial forms. 8. Metacyclic 5. Leishmania form. trypanosomes. 9. Large trypanosomes.
ACKNOWLEDGMENTS The author is grateful to Mrs. Flora for her technical assistance, and to Gertrude H. Nicholson for the drawing
C. Gilliam artist Mrs. of Fig. 14.
REFERENCES D’ALESSANDRO Trypanosoma
B.,
A. 1983. The life cycle of rungeli in triatomid bugs as it
occurs in nature. Fat., 23, 2130.
Bull.
TuZune
Univ.
COUTINHO, J. O., AND NUSSENZWEIG, V. InfecTa experimental de triatomineos Trypanosomu rungeli Tejera, 1920. Fob Biol. Scio Z’auZo, 18, 181-188. G6MU,
I. 1967. Neuvas de la aecion patogena geli Tejera, 1920 sobre
Med. 1952. pelo Clin.
observaciones acerca de1 Trypanosoma ranRhodnius prolixus Stal,
Trypanosoma
DEVELOPMENT
1859. Heu. Inst. Med. Trap. S&o Paulo, 9, 510. Pathogenicity of TrypanGREWAL, M. S. 1957. osoma Tangeli Tejera, 1920 in the invertebrate host. Exptl. Parasitol., 6, 123-130. GIIOOT, H. 1952. Further observations on Trypanosoma a7iarii of Columbia, South America. Am. J. TI+o~. Med. Hgy., 1, 585-592. HARINGTON, J. S. 1955. Certain aspects of the haemolymph of Rho&us prolixus StX Ph.D. Thesis, University of London, England (cited by Ormerod, W. E., 1967). 1967. The effect of TrypanoOWEROD, W. E. soma Tangeli on the concentration of amino acids in the hemolymph of Rho&us prolixus. J. Invertebrate Pathol., 9, 247-255. PIFANO, F., AND MAYER, M. 1949. Hallazgo de formas evolutivas de1 Trypanosoma rangeli en el jugo de la trompa de Rho&us proZixw de Venezuela. Arch. Venezolanos. Patol. Trap. Pam&tot. M&d., 1, 153-158.
IN Rhodnius
125
REY-MATIZ, H. 1941. Obsewaciones panosomas en Colombia. Reu. Bogotd, 10, 25-19.
sobre tryFat. Med.
TEJERA, E. 1920. Un noveau flagellb de Rhodnius prolixus, Trypanosoma (ou Cri~hidia) rangeli n. sp. Bull. Sot. Pathol. Exotique, 13, 527530. TOBIE, E. J. 1964. Increased infectivity of a cyclically maintained strain of Typanosoma rangeli to Rho&us prolixus and mode of transmission by invertebrate host. J. Parasitol., 50, 593-598. TOBIE, E. J. 1965. ing transmission Rho&us prolixus.
Biological factors influencof Trypanosoma rangeli by J. Parasitol., 51, 837-841.
TOBIE, E. J. 1968. flagellates in the lixus. J. Parasitol.,
The fate of some culture hemocoel of Rhodnius pro54, 1040-1046.
ZELED~N, Biol.
1954. Tripanosomiasis R. Trap., 2, 231-268.
rangeli.
Reu.