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Pathogenicity of Trypanosoma rangeli Tejera, 1920 in the invertebrate host
Volume
6, No. 2, March
1957
Prankain [I. s. /i. EXPERIMENTAL
PARASITOLOGY
6, 12%130
(1957)
Pathogenicity
of Trypanosoma rangeli Tejera, 1920 in the Invertebrate Host Manohar
Depurtment
Singh
Grewal
of Parasitology, London School of Hygiene University of London, England
and Tropical
Medicine,
(Submitted for publicatiorl, 16 July 1956) The pathogenic effects of protozoan parasites have heen studied extensively in vertebrate hosts, hut, there is little information on the pathogencitiy of such organisms in invertehrates. The aim of this paper is to discuss and put forward results on the pathogenicity of T. ranyeli for its invertebrate host. Insects are commonly infected with flagellates in nature. The parasites may be present only in the gut and in such instances may be in enormous numbers. They may, however, occupy the gut, body cavity and salivary glands simultaneously; Leptomonas pyrrhocoris, for example, occurs in these sites within the plant bug, Pyrrhocoris apterus, while Leptomonas pyruustae (Paillot, 1928), a flagellate parasite of the corn-borer, Pyrausta nubilalis, is found in t,he gut and t,he Malpighian t,ubulcs. Similar sites of infection are occupied by Leptomonas ctenocephali whkh infects the dog flea, Ctenocephalides can,is. It is diffk& to decide whcthcr these three parasites, with single hosts, have any pathogenic effect on the insects. When considering the parasitic flagellates of vertebrates, whirl1 undergo part of their developmental cycle in an invertebrate, there is a little more evidence to suggest variable degrees of pathogenicity while in the latter host. Smith, Halder and Ahmed (1940) found that in sandflies infected wit,h Leishmaniu donovani a huge aggregation of the parasites was found in the mid gut, while the proventriculus was lined with sessile leptomonad forms many layers deep. Later in the infection, a forward migration of the parasites caused dilation of the esophagus and massive parasitic growth in the pharynx and buccal cavity. With the alimentary canal 123
124
MANOHAR
SINGH
GREWAL
thus blocked, attempts on the part of the insects to feed resulted in the expulsion of a plug of parasites. Sandflies in this condition remained alive and no particular mortality was noted. Some species of trypanosomes invade the cells of their insect vectors during their development in these invertebrates. According to Minchin and Thomson (1915) T. Zewisi actually invades the cells of the stomach wall of the flea, Ceratophyllus fasciatus (= Nosopsyllus fasciatus) which transmits the infection. As the flagellate penetrates the cell, a vacuole is formed about it. This trypanosome then undergoes further development and multiplication. Wenyon (1926) noted that the invaded epithelial cell of the host’s stomach is reduced to a mere membrane enclosing the actively moving developing organisms (T. Zewisi). Eventually, the cell ruptures; the trypanosomes escape into the stomach of the flea and from there they may invade other epithelial cells. Rodhain (1942, a and b) described the developmental intracellular stages of T. lewisi and T. pipistrelli in the gut of the tick, Ornithodorus moubata, but no mention was made about the pathogenic effect of these flagellates on the tick. Garnham (1955) carried out experiments using Xenopsylla cheopis infected with T. lewisi and found that there was a higher mortality in the infected fleas during the earlier part of the infection, when the trypanosomes were penetrating the epithelial cells of the stomach. Macfie and Thomson (1929) showed that in mites fed on birds infected with trypanosomes the infection became so intense that the parasites became injurious and even killed the mites. On the other hand, as pointed out by Garnham (1955), T. cruzi, developing in the ‘posterior station’ of the reduviid bugs appears to be harmless, although the organisms may be very numerous. Duke (1928) believed that T. brucei, T. gambiense and T. rhodesiense in Glossina were actually beneficial to the fly. Although he could not confirm his view by experiment, he found that the presence in the intestine of developing forms of the polymorphic trypanosomes is not to any noticeable extent injurious to the fly. Burtt (1946) found only two instances when a fly infected with T. rhodesiense failed to salivate on three consecutive probes, and in both cases the fly died by the following day. He also found a significantly higher proportion of bacteria in flies infected with trypanosomes. Bacteria persisted throughout the fly’s life and their presence seemed to exert no harmful effect on the insect.
PATHOGENICITY
OF TRYPANOSOMA
IN
INVERTEBRATES
125
Finally, Tejera (1920) studied the reduviid bug, Rhodnius prolixus, infected with Trypanosoma rangeli (= T. ariarii) and found that they appeared undamaged. Various other authors (Pifano, 1949 and 1954; Groot, 1952 and 1954; and Zeleddn, 1954) who worked on T. rangeli did not discuss the pathogenicity of this human trypanosome in R. prolixus. MATERIALS
AND
METHODS
The strain of Trypanosoma rangeli was brought back from Dr. H. Groot by the late Professor P. A. Buxton and was isolated by Dr. F. Pifano in Venezuela. It was kept in N.N.N. and W.N. media. Rhodnius prolixus bugs were fed on the abdomen of animals 3-24 hours after inoculation with culture.
During the course of the work on T. rangeli, it was found that some infected bugs were unable to molt, while others died. On examination, all these bugs were found to be heavily infected. The infection was either in the gut alone or both in the gut and hemolymph; but the majority of the bugs so affected had infection in their hemolymph. If the infection were heavy the nymphs died .before molting. If the bugs were able to survive until the last molt, this period was completed with great difficulty, if at all, and often the insect came through the molting process in a crippled condition. Heavily infected bugs were sluggish and slow in movement; the color of the bodies of the infected bugs was translucent and paler than ‘controls.’ When the bugs observed in front of the light were turned from side to side, the alimentary canal of the infected bugs could be seen rolling in all directions in the infected hemolymph. The volume of hemolymph was greater in t,he infected bugs than in the ‘clean’ bugs of the same larval instars. The folowing experiments were performed with R. prolixus: One hundred and twenty first nymphal instar bugs were fed on a rat with a heavy infection (2-3 trypanosomes per field in wet preparation); the majority of the bugs died as soon as the hemolymph infection was established, there being more deaths in the first nymphal instar bugs than in the following instars. Only the bugs which could molt were able to feed, the rest either died before molting or during the act of molting. The majority of them were given many chances to feed; some of them did feed, partially, but ultimately died (Table I). Out of 120 first nymphal instar bugs, 35 died after their first feed, 8 died after the second, and 19, 7, and 10 after the third, fourth, and fifth feeds, re-
126
MANOHAR
SINGH
GREWAL
TABLE
I
Pathogenicity of T. rangeli in R. prolixus
Number of bugs
120 85 77 58 51 41
Date of feed (dw/mdyr)
5/4/55 3014155 25/5/55 23/6/55 20/7/55 15/g/55
Deaths
Number of bugs with Fat infection
Number of bugs with hemolymph infection
Number of bugs with infected salivary glands
22 6 12 5 7 -
13 2 7 2 3 -
35 8 19 7 41
25 6 19 4 10 1
4 1 11 3 8 1
TABLE
II
Number of bugs molted
Number of bugs unmelted
85 77 58 51 41 Adult
Pathogenicity of T. rangeli in R. prolixus Number of bugs 49
34
74
Parasitemia in the vertebrates
1-2 parasites per field in wet preparation l-2 parasites per 5 or 6 fields in wet preparation 2-3 parasites per field in wet preparation
Deaths after each feed First
Second
Third
Fourth
Fifth
11
3
I
I
4
4
1
-
1
2
21
4
3
discontinued
spectively. Only 41 bugs were able to molt up to the adult stage, and of these only one had infection in the hemolymph. In three more experiments 49, 34, and 74 first nymphal instar bugs were fed on animals with a variable intensity of infection (Table II). Results obtained were similar to those of the first experiment and the death rate in the first nymphal instar bugs was higher when they were fed on animals with heavy infections. In order to determine the pathogenicity of T. rangeli in the bed bug, Cimex Zectuhius, 8 experiments were performed (Table III). The bed bugs have a semi-transparent body so that much of their internal anatomy can be seen whilst they are alive and uninjured; it is therefore possible to examine the bugs without dissecting them. Even when engorged with blood, the head, a portion of the thorax, the legs and the periphery of the body remain clear and can be searched for parasites. It was thus
PATHOGENICITY
OF TRYPANOSOMA
TABLE Pathogenicity Parasitemia in animals on which bugs had their first infective feed
High Low
Number of bugs
178 138
127
INVERTEBRATES
III
of T. rangeli Days after which hemolymph infection
4-6 7-11
IN
in Cimex
lectularius
Days after which the bugs started dying
8-10 10-12
Days for which the infected bugs remained alive
17-40 61-128
observed that the invasion of the hemolymph started very early in the bed bugs, and immediately after this the bugs began to die. It was observed in the case of R. prolixus that the infected bugs which survived and molted as far as the adult stage, continued to survive for a long time in spite of the high mortality rate during molting. This parasite was found to be more harmful to bed bugs; more than 80% of the bugs died before reaching the adult stage and the rest died after feeding three times at the most. It appears that the insects die when the infection is heavy, the entire system being blocked by flagellates. In the earlier infection of the hemo-
FIG. 1. Hemolymph cell of Rhodnius prolixus infected with T. rangeli. The nucleus of the cell is pushed to one side. The cell phagocytoses many flagellates from the infected hemolymph, until it becomes very swollen.
128
MANOHAR SINGH GREWAL
coele it is seen that some parasites are phagocytosed by the hemolymph cells, but when the parasites increase in number they form rosettes around the hemolymph cells and some of the cells are found to contain about 8 to 10 parasites in leishmania form (Fig. 1). In the still older and heavy infections, when the bugs succumb, the number of hemolymph cells is considerably reduced. When ‘clean’ bugs, R. prolixus, were inoculated with culture or infected hemolymph the mortality rate due to the heavy infection was, once again, higher. This gives a reason to believe that even when flagellates do not penetrate through the intestinal canal they are equally pathogenic. DISCUSSION
Steinhaus (1949) stated that the penetration of a parasite through the intestinal wall might harm the insect. In the case of the bugs infected with T. rangeli it has been found that the invasion of the infected bug’s hemolymph by flagellates depended upon the intensity of infection, and the heavily infected bugs succumbed to infection much earlier. ReyMatiz (1941) described some intestinal forms in the bug, R. prolixus, infected with T. rangeli which he considered to be intracellular. Although I could not demonstrate intracellular forms, they probably exist and may constitute the point of entry of flagellates into the hemolymph, bursting out of the infected intestinal cells. With a heavy infection in the hemolymph, flagellates invade the salivary glands, which lose their pink color and become whitish (Grewal, 1956). The hemolymph at this stage is also white and flagellates are present in every portion of the body of the infected insect. A heavily infected salivary gland was found to contain approximately 10,000,000 metacyclic trypanosomes. A transverse section of the infected salivary gland has shown that flagellates form intracellular stages before entering the lumen (Grewal, 1957, in press). It is probably the heavy infection in the gut and hemolymph, invasion of the hemolymph cells and the entry of the parasites into the salivary glands which prevents the bugs from molting and finally kills them. Harington (1955) found in R. prolixus that there is a drop in concentration of amino acids during the third week after feeding and that this may be due to their utilization in preparation for the molt. Preliminary results show that the concentration of certain amino acids in the hemolymph may be lowered by the presence of flagellates and that this deficiency interferes with the molting process (Grewal and Ormerod, 1957, in press).
PATHOGENICITY
OF TRYPANOSOMA
IN
INVERTEBRATES
129
It is interesting to note that of the two human trypanosomes of the New World, T. cruzi Chagas, 1909 is pathogenic to its vertebrate but non-pathogenic to its invertebrate host; while 7’. rangeli Tejera, 1920 is non-pathogenic to its vert’ebrate but pathogenic t,o its invertebrate host. SUMMARY
1. The pathogenicity of flagellat,es to their invertebrate hosts is discussed. 2. The pathogenicity of’ T. rangeli to K. prolixw is found to be heavy in the first nymphal in&am. Rugs mit,h infect,ion in t,he hemolymph were unable to molt. 3. The pathogenicity of T. rangeli in the bed bug, C. bectubarius, was found to be greater than in R. proliaus. Invasion of the hemolymph was fairly rapid. 4. Bugs with a heavy infection in the hemolymph carried a higher mortality than those with lighter infection. 5. Mortality ia probably not connected with the penetration of the gut wall, since it occurs in bugs infected directly into the hemolymph. Preliminary studies indicate that the deficiency in the concentration of’ certain amino acids interferes with the making procaessin the infected bugs. I tender my thanks to Professor P. C. C. Garnham, Director of the Department of Parasitology, London School of Hygiene and Tropical Medicine, for placing at my disposal the facilities for doing this work in the laboratory. I also wish to thank Dr. W. I<. Ormerod for his helpful advice and criticism, and Mr. C. R. Hill for helping me in taking the photomicrograph.
BIJRTT, E. 1946. Salivation by Glossina morsitans on to glass slides: A technique for isolating infected flies. Ann. Trop. Med. Parasitol. 40, 141-144. DUKE, H. L. 1928. On the effect on the longevity of G. pulpalis of trypanosome infections. Ann. Trop. Med. Parasitol. 22, 25-32. GARNHAM, P. C. C. 1955. The comparative pathogenicity of protozoa in their vertebrate and invertebrate hosts. No. 5, Mechanisms of microbial pathogenicity. Symposium Sot. Gen. Microbial. 191-206. GREWAL, M. S. 1956. Trypanosoma rangeli Tejera, 1920, in its vertebrate and invertebrate hosts. Trans. Roy. Sot. Trop. Med. Hyg. 60, 301-302. GREWAL, M. S. 1957. Development of Trypanosoma rangeli in the salivary glands of its invertebrate host, Rhodnius prolixus. In press GREWAL, M. S., AND ORMEROD, W. E. 1957. In press
130
MANOHAR
SINGH
GREWAL
GROOT, H. 1952. Further observations on Trypanosoma ariarii of Colombia, South America. Am. J. Trap. Med. Hyg. 1, 585-691. GROOT, H. 1954. Estudios sobre 10s trypanosomas humanos. (T. rangeli y T. ariarii). Ann. sot. biol. BogoM, 6, 109-126. HARINGTON, J. S. 1955. Certain aspects of the comparison of the haemolymph of Rhodnius prolixus St&l. Thesis for the Ph. D. Degree, University of London. MACFIE, J. W. S., AND THOMSON, J. G. 1929. A trypanosome of the canary (Serinus canarius Kock). Trans. Roy. Sot. Trap. Med. Hyg. 23, 185-191. MINCHIN, E. A., AND THOMSON, J. Il. 1915. The rat-trypanosome, Trypanosoma lewisi, in its relation t,o the rat flea, Ceratophyllus fasciatus. Quart. J. Micro-
stop. Sri. 60, 463492. PAILLOT, A. 1928. On the natural equilibrium of Pyrausta nubilalis Hb. Intern. Corn Borer Invest., Sci. Repts. 1, 77-106. PIFANO, F. 1949. Estado actual de las investigaciones en Venezuela sobre una
nueva trypanosomiasis humana de la region neotropica producida por el Trypanosoma rangeli. Arch. venez. pat. trop. y parasit. med. 1, 135-152. PIFANO, F. 1954. Nueva trypanosomiasis humana de la region neotropica producida por el Trypanosoma rangeli, con especial referencia a Venezuela. Arch. venez. pal. trap. y parasit. med. 2, 89-120. REY-MATIZ, H. 1941. Observaciones sobre trypanosomas en Colombia. Rev. jar. med., Bogot4 10, l-25. RODHAIN, J. 1942a. Au sujet du d&eloppment intracellulaire de Trypanosoma lewisi chez Ornithodorus moubata. Acta Biol. Belg. 2, 413-415. R~DHAIN, J. 194213.Au sujet du developpment intracellulaire de Trypanosoma pipistrelli (Chatton et, Courrier) chez Omithodorus mouhata. ncta Biol. Be/g. 2, 416-420. SMITH, R. 0. A., HALDER, K. C. ANI) AHMED, I. 1940. Further investigations on the transmission of Kala-azar. Part II. The phenomenon of the ‘blocked’ sandfly. Indian J. Med. Res. 28, 581-584. STEINHAUS, E. A. 1949. Principles of Insect Pathology. McGraw-Hill Book Company, Inc. New York, Toronto, London. 1st Ed. TEJERA, E. 1920. Un nouveau flagellb de Rhodnius prolixus, Trypanosoma (OU Crithidia) rangeli, n.sp. Bull. sot. pathol. Exotique 13, 527430. WENYON, C. M. 1926. Protozoology. BailliBre, Tindall and Cox, London, p. 471. ZELED~N, R. 1954. Trypanosomiasia rangeli. Rev. Biol. Trap. 2, 231-268.
6, No. 2, March
1957
Prankain [I. s. /i. EXPERIMENTAL
PARASITOLOGY
6, 12%130
(1957)
Pathogenicity
of Trypanosoma rangeli Tejera, 1920 in the Invertebrate Host Manohar
Depurtment
Singh
Grewal
of Parasitology, London School of Hygiene University of London, England
and Tropical
Medicine,
(Submitted for publicatiorl, 16 July 1956) The pathogenic effects of protozoan parasites have heen studied extensively in vertebrate hosts, hut, there is little information on the pathogencitiy of such organisms in invertehrates. The aim of this paper is to discuss and put forward results on the pathogenicity of T. ranyeli for its invertebrate host. Insects are commonly infected with flagellates in nature. The parasites may be present only in the gut and in such instances may be in enormous numbers. They may, however, occupy the gut, body cavity and salivary glands simultaneously; Leptomonas pyrrhocoris, for example, occurs in these sites within the plant bug, Pyrrhocoris apterus, while Leptomonas pyruustae (Paillot, 1928), a flagellate parasite of the corn-borer, Pyrausta nubilalis, is found in t,he gut and t,he Malpighian t,ubulcs. Similar sites of infection are occupied by Leptomonas ctenocephali whkh infects the dog flea, Ctenocephalides can,is. It is diffk& to decide whcthcr these three parasites, with single hosts, have any pathogenic effect on the insects. When considering the parasitic flagellates of vertebrates, whirl1 undergo part of their developmental cycle in an invertebrate, there is a little more evidence to suggest variable degrees of pathogenicity while in the latter host. Smith, Halder and Ahmed (1940) found that in sandflies infected wit,h Leishmaniu donovani a huge aggregation of the parasites was found in the mid gut, while the proventriculus was lined with sessile leptomonad forms many layers deep. Later in the infection, a forward migration of the parasites caused dilation of the esophagus and massive parasitic growth in the pharynx and buccal cavity. With the alimentary canal 123
124
MANOHAR
SINGH
GREWAL
thus blocked, attempts on the part of the insects to feed resulted in the expulsion of a plug of parasites. Sandflies in this condition remained alive and no particular mortality was noted. Some species of trypanosomes invade the cells of their insect vectors during their development in these invertebrates. According to Minchin and Thomson (1915) T. Zewisi actually invades the cells of the stomach wall of the flea, Ceratophyllus fasciatus (= Nosopsyllus fasciatus) which transmits the infection. As the flagellate penetrates the cell, a vacuole is formed about it. This trypanosome then undergoes further development and multiplication. Wenyon (1926) noted that the invaded epithelial cell of the host’s stomach is reduced to a mere membrane enclosing the actively moving developing organisms (T. Zewisi). Eventually, the cell ruptures; the trypanosomes escape into the stomach of the flea and from there they may invade other epithelial cells. Rodhain (1942, a and b) described the developmental intracellular stages of T. lewisi and T. pipistrelli in the gut of the tick, Ornithodorus moubata, but no mention was made about the pathogenic effect of these flagellates on the tick. Garnham (1955) carried out experiments using Xenopsylla cheopis infected with T. lewisi and found that there was a higher mortality in the infected fleas during the earlier part of the infection, when the trypanosomes were penetrating the epithelial cells of the stomach. Macfie and Thomson (1929) showed that in mites fed on birds infected with trypanosomes the infection became so intense that the parasites became injurious and even killed the mites. On the other hand, as pointed out by Garnham (1955), T. cruzi, developing in the ‘posterior station’ of the reduviid bugs appears to be harmless, although the organisms may be very numerous. Duke (1928) believed that T. brucei, T. gambiense and T. rhodesiense in Glossina were actually beneficial to the fly. Although he could not confirm his view by experiment, he found that the presence in the intestine of developing forms of the polymorphic trypanosomes is not to any noticeable extent injurious to the fly. Burtt (1946) found only two instances when a fly infected with T. rhodesiense failed to salivate on three consecutive probes, and in both cases the fly died by the following day. He also found a significantly higher proportion of bacteria in flies infected with trypanosomes. Bacteria persisted throughout the fly’s life and their presence seemed to exert no harmful effect on the insect.
PATHOGENICITY
OF TRYPANOSOMA
IN
INVERTEBRATES
125
Finally, Tejera (1920) studied the reduviid bug, Rhodnius prolixus, infected with Trypanosoma rangeli (= T. ariarii) and found that they appeared undamaged. Various other authors (Pifano, 1949 and 1954; Groot, 1952 and 1954; and Zeleddn, 1954) who worked on T. rangeli did not discuss the pathogenicity of this human trypanosome in R. prolixus. MATERIALS
AND
METHODS
The strain of Trypanosoma rangeli was brought back from Dr. H. Groot by the late Professor P. A. Buxton and was isolated by Dr. F. Pifano in Venezuela. It was kept in N.N.N. and W.N. media. Rhodnius prolixus bugs were fed on the abdomen of animals 3-24 hours after inoculation with culture.
During the course of the work on T. rangeli, it was found that some infected bugs were unable to molt, while others died. On examination, all these bugs were found to be heavily infected. The infection was either in the gut alone or both in the gut and hemolymph; but the majority of the bugs so affected had infection in their hemolymph. If the infection were heavy the nymphs died .before molting. If the bugs were able to survive until the last molt, this period was completed with great difficulty, if at all, and often the insect came through the molting process in a crippled condition. Heavily infected bugs were sluggish and slow in movement; the color of the bodies of the infected bugs was translucent and paler than ‘controls.’ When the bugs observed in front of the light were turned from side to side, the alimentary canal of the infected bugs could be seen rolling in all directions in the infected hemolymph. The volume of hemolymph was greater in t,he infected bugs than in the ‘clean’ bugs of the same larval instars. The folowing experiments were performed with R. prolixus: One hundred and twenty first nymphal instar bugs were fed on a rat with a heavy infection (2-3 trypanosomes per field in wet preparation); the majority of the bugs died as soon as the hemolymph infection was established, there being more deaths in the first nymphal instar bugs than in the following instars. Only the bugs which could molt were able to feed, the rest either died before molting or during the act of molting. The majority of them were given many chances to feed; some of them did feed, partially, but ultimately died (Table I). Out of 120 first nymphal instar bugs, 35 died after their first feed, 8 died after the second, and 19, 7, and 10 after the third, fourth, and fifth feeds, re-
126
MANOHAR
SINGH
GREWAL
TABLE
I
Pathogenicity of T. rangeli in R. prolixus
Number of bugs
120 85 77 58 51 41
Date of feed (dw/mdyr)
5/4/55 3014155 25/5/55 23/6/55 20/7/55 15/g/55
Deaths
Number of bugs with Fat infection
Number of bugs with hemolymph infection
Number of bugs with infected salivary glands
22 6 12 5 7 -
13 2 7 2 3 -
35 8 19 7 41
25 6 19 4 10 1
4 1 11 3 8 1
TABLE
II
Number of bugs molted
Number of bugs unmelted
85 77 58 51 41 Adult
Pathogenicity of T. rangeli in R. prolixus Number of bugs 49
34
74
Parasitemia in the vertebrates
1-2 parasites per field in wet preparation l-2 parasites per 5 or 6 fields in wet preparation 2-3 parasites per field in wet preparation
Deaths after each feed First
Second
Third
Fourth
Fifth
11
3
I
I
4
4
1
-
1
2
21
4
3
discontinued
spectively. Only 41 bugs were able to molt up to the adult stage, and of these only one had infection in the hemolymph. In three more experiments 49, 34, and 74 first nymphal instar bugs were fed on animals with a variable intensity of infection (Table II). Results obtained were similar to those of the first experiment and the death rate in the first nymphal instar bugs was higher when they were fed on animals with heavy infections. In order to determine the pathogenicity of T. rangeli in the bed bug, Cimex Zectuhius, 8 experiments were performed (Table III). The bed bugs have a semi-transparent body so that much of their internal anatomy can be seen whilst they are alive and uninjured; it is therefore possible to examine the bugs without dissecting them. Even when engorged with blood, the head, a portion of the thorax, the legs and the periphery of the body remain clear and can be searched for parasites. It was thus
PATHOGENICITY
OF TRYPANOSOMA
TABLE Pathogenicity Parasitemia in animals on which bugs had their first infective feed
High Low
Number of bugs
178 138
127
INVERTEBRATES
III
of T. rangeli Days after which hemolymph infection
4-6 7-11
IN
in Cimex
lectularius
Days after which the bugs started dying
8-10 10-12
Days for which the infected bugs remained alive
17-40 61-128
observed that the invasion of the hemolymph started very early in the bed bugs, and immediately after this the bugs began to die. It was observed in the case of R. prolixus that the infected bugs which survived and molted as far as the adult stage, continued to survive for a long time in spite of the high mortality rate during molting. This parasite was found to be more harmful to bed bugs; more than 80% of the bugs died before reaching the adult stage and the rest died after feeding three times at the most. It appears that the insects die when the infection is heavy, the entire system being blocked by flagellates. In the earlier infection of the hemo-
FIG. 1. Hemolymph cell of Rhodnius prolixus infected with T. rangeli. The nucleus of the cell is pushed to one side. The cell phagocytoses many flagellates from the infected hemolymph, until it becomes very swollen.
128
MANOHAR SINGH GREWAL
coele it is seen that some parasites are phagocytosed by the hemolymph cells, but when the parasites increase in number they form rosettes around the hemolymph cells and some of the cells are found to contain about 8 to 10 parasites in leishmania form (Fig. 1). In the still older and heavy infections, when the bugs succumb, the number of hemolymph cells is considerably reduced. When ‘clean’ bugs, R. prolixus, were inoculated with culture or infected hemolymph the mortality rate due to the heavy infection was, once again, higher. This gives a reason to believe that even when flagellates do not penetrate through the intestinal canal they are equally pathogenic. DISCUSSION
Steinhaus (1949) stated that the penetration of a parasite through the intestinal wall might harm the insect. In the case of the bugs infected with T. rangeli it has been found that the invasion of the infected bug’s hemolymph by flagellates depended upon the intensity of infection, and the heavily infected bugs succumbed to infection much earlier. ReyMatiz (1941) described some intestinal forms in the bug, R. prolixus, infected with T. rangeli which he considered to be intracellular. Although I could not demonstrate intracellular forms, they probably exist and may constitute the point of entry of flagellates into the hemolymph, bursting out of the infected intestinal cells. With a heavy infection in the hemolymph, flagellates invade the salivary glands, which lose their pink color and become whitish (Grewal, 1956). The hemolymph at this stage is also white and flagellates are present in every portion of the body of the infected insect. A heavily infected salivary gland was found to contain approximately 10,000,000 metacyclic trypanosomes. A transverse section of the infected salivary gland has shown that flagellates form intracellular stages before entering the lumen (Grewal, 1957, in press). It is probably the heavy infection in the gut and hemolymph, invasion of the hemolymph cells and the entry of the parasites into the salivary glands which prevents the bugs from molting and finally kills them. Harington (1955) found in R. prolixus that there is a drop in concentration of amino acids during the third week after feeding and that this may be due to their utilization in preparation for the molt. Preliminary results show that the concentration of certain amino acids in the hemolymph may be lowered by the presence of flagellates and that this deficiency interferes with the molting process (Grewal and Ormerod, 1957, in press).
PATHOGENICITY
OF TRYPANOSOMA
IN
INVERTEBRATES
129
It is interesting to note that of the two human trypanosomes of the New World, T. cruzi Chagas, 1909 is pathogenic to its vertebrate but non-pathogenic to its invertebrate host; while 7’. rangeli Tejera, 1920 is non-pathogenic to its vert’ebrate but pathogenic t,o its invertebrate host. SUMMARY
1. The pathogenicity of flagellat,es to their invertebrate hosts is discussed. 2. The pathogenicity of’ T. rangeli to K. prolixw is found to be heavy in the first nymphal in&am. Rugs mit,h infect,ion in t,he hemolymph were unable to molt. 3. The pathogenicity of T. rangeli in the bed bug, C. bectubarius, was found to be greater than in R. proliaus. Invasion of the hemolymph was fairly rapid. 4. Bugs with a heavy infection in the hemolymph carried a higher mortality than those with lighter infection. 5. Mortality ia probably not connected with the penetration of the gut wall, since it occurs in bugs infected directly into the hemolymph. Preliminary studies indicate that the deficiency in the concentration of’ certain amino acids interferes with the making procaessin the infected bugs. I tender my thanks to Professor P. C. C. Garnham, Director of the Department of Parasitology, London School of Hygiene and Tropical Medicine, for placing at my disposal the facilities for doing this work in the laboratory. I also wish to thank Dr. W. I<. Ormerod for his helpful advice and criticism, and Mr. C. R. Hill for helping me in taking the photomicrograph.
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