- Email: [email protected]
Development of Blastocrithidia triatomae (Trypanosomatidae) in Triatoma infestans after vitamin B-supplementation of the blood-diet of the bug
Europea n Journa l of
Europ.J.Protistol. 27, 17- 20 (1991) March 28, 1991
PROTISTOLOGY
Development of Blastocrithidia triatomae (Trypanosomatidae) in Triatoma infestans after Vitamin B-Supplementation of the Blood-Diet of the Bug Cord Jensen and Gunter A. Schaub Institut tur Biologie I (Zoologie), Albert-Ludwigs-Universitat, Freiburg, FRG
SUMMARY Investigati ons of the inter relatio nships o f the trypan osom at id Blastocrithidia triatomae and the redu viid bug Triatoma infestans ind icat ed an int erferen ce o f B. triatomae with bug symbionts or with their vitamin supply. For an initial study of this possibility, B. triatomae-infected bugs were fed two or three times at monthly intervals on defibrin at ed sheep blood with or without B-group vitamin supplementa tion (folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, and thiamin ). The tot al number of flagellates in the sma ll intestine and the rectum of the bug were then determ ined with a hemo cytom eter. Vitami n B-suppleme ntati on supported the initial development of B. triatomae in the small intestine of most th ird and four th instar larvae which had been infected in the first instar, In the small intestines of fifth instars and adults, bot h which had been infected as th ird instar lar vae, most populat ions of B. triatomae were not affected by vitami n B-suppl ement at ion. Similarly, recta l populations in all bugs were never increased by supplementa tion.
Introduction The homoxenous trypanosomatid Blastocrithidia triatom ae develops in the intestine and occasionally in the excretory system of hematophagous reduviid bugs, the vecto rs of Chagas' disease [11, 23]. B. triatomae causes a numb er of pathological effects on the reduviid Triatoma infestans [summarized in 17, 18,21], some of which were also observed in Rh odniu s prolixus infected with Trypanosoma rangeli [4] and in aposymbiotic bugs fed on hum ans, guinea pigs or rabbits [2,3,6, 14]. Watkins [26] suggested that T. rangeli might induc e such path ological effects by reducing the number of symbiotic bacteria in the insect midgut. Symbiotic bacteria pre sumably supply the bugs with B-group vitamins [7, 8, 9] and when rabbit blood was supplemented with these vitamin s, the development of apo symbiot ic larvae, which are particularl y affected in the late instars, was significantly improved [e.g., 12]. The pathologic similarities between apo symbiotic and B. triatomae-infected bugs can be explained either by a © 199 1 by Gustav Fischer Verlag, Stuttgart
redu ction in the number of symbionts caused by the flagellates or by competition betw een bug and flagellates for the B-vitamins. Preliminary studies showed great variation in the numb er of bacteria in uninfect ed and B. triatom ae-infected T. infestans, [Schaub & Fischer unpub\. data]. We ther efore decided to investigate the path ological effects of B. triatom ae in bugs fed on norm al blood supplemented with B-group vitamins. Since this supplement might cau se increased trypanosom atid popul ations which could compete with the insect for essential nutrients, it was necessary to study the effects of B-group vita mins on popul ations of B. triatomae. So far, the effects of vita mins on development of insect trypanosomatids have only been investigated with in vitro cultures [24], not with parasites in the insect host.
Material and Methods Th e original strain of Blastocrithidia triatomae Cerisola et aI., 1971 was maint ained in our laboratory in Triatoma infestans 0932 -4739/9 1/002 7-001 7$3 .50/0
18 . C. Jensen and G. A. Scha ub [20], which were used to initia te pr imar y in vitro cultures [15]. Cysts from the second passage were used after cryopreser vat ion a t -80 °C for six months [11]. Th e cyst suspension wa s diluted with defibr inat ed sheep blood to a final concent ration of 10 6 cysts/ern! blood. Th e Triatom a infestans (Klug, 1834 ) strain orig inates from Cac hiyuyu, Chile [20]. Bugs we re fed ro utinely on hen s and reared at ca. 26 °C and ca. 60 % relat ive humidity on a 16h/8h light/dark rhythm. T wo batches of first (Ll) and two of third insrar (L3) larvae were infected with the cyst suspension [19]. Th ose fed to cap acity were divided into 8 gro ups, maintained in 1 I beakers [17], and received un infected L5 to allow an upt ake of bacterial syrnbion ts. At mo nthly intervals four gro ups of bugs were fed on defibr inated sheep blood (Gesellschaft fiir Lab ordiagnostika, Mulheim-Ruhr, F.R.G. ) and four other gro ups of bugs with sheep blood , supplemented with B-group vita mins in th e same concentration s as in the FNPPRT diet of Lak e and Friend [12]: 3 mg folic acid , 10 mg nicotinic acid, 10 mg pantothenic acid-Ca salt, 10 mg pyrid oxine-HCI, 2 mg riboflavin-5-phosphate and 10 mg thi amin e-H CI (all from Sigma, St. Lo uis) was added to 100 em! defibrin ated sheep blood. All diets contai ned 1.1 mM ATP as a ph agostimulant. O ne or 2 weeks after feed ing, S-15 bugs we re dissected: L3 a nd L4 originating from th e two ba tches which had been infected as L1; L5 a nd adults from those infected as L3. Th e first L3 a nd L5 were dissected 9 weeks post infectionem (p.i.). Some of the bugs did not moult after on e feeding and needed a second bloodm eal; they were thu s infected for four addit iona l weeks when dissected as L3 or L4. Dissection and macerat ion of the small intestin e (fo ur bat ches) and the rectu m (two batches) were as pr eviou sly described [16]. N umber of B. triatom ae in the se tissues was det ermin ed in N eubauer hemocytom eters. Stu dent's t-rest, using th e softwa re of SAS Institute Inc. (Ca ry, USA), was used for sta tistica l compa riso ns of dat a.
insta r and at the same time after feeding with the same diet (Ta ble 1). When data from these two groups were combined, we found a conspicuo us increase in the number of flagellate s in succeeding instar s (Ta ble 2). The initial developm ent of flagellates was significantly bett er in both batch es of L3 fed upon the diet supplement ed with B-group vitamins (Ta ble 2). This improvement was also found in the L4 of the first batch. Differences also occurred betw een the two batches which had been infected as L3. In the first batch , in which none of the vitamin-deficient L5 moulted to the adult stage, a significant increase in the numb er of flagellates was observed in the first dissections, made 1 week after feeding L5 on supplemented blood ; in the second batch of L5 and adults, population densities did not differ significantly.
Rectum At each dissection time, about 10 % of the tot al popul ation was found in the rectum (Table 3). In individual bugs the difference between tr ypano somatid populations in both regions varied between 1 % and 25 % of the tot al population; the tot al number of flagellate s in the small intestine did not show any correlation with that in the rectum. The number of flagellates in the rectum also increased in the succeeding instar s, but no growth support by vitamin s was observed. A sta tistically significant differen ce was only obtained for the adults, two weeks after feeding (t-rest; p ~ 0.01 ).
Discussion Results Great variation was found in the number of flagellates per intestine or per rectum ; minim al and maximal values could differ by a factor of ten. Intestine
The number of flagellates in the small intestine did not differ statistically significantly between long-term and sho rt-term infected bugs which were dissected in the same
Since endoparasites ob tain their food from the host, they can be influenced by the vitamin content of its diet. So far, such studies with tryp anosom atids have only consid ered mammalian parasites and their hosts. Expecting a po sitive effect of vitamins on developm ent of parasites, we at first found the reduced growth of trypanosomes in mice which fed on cod-liver oil and the increased growth in hosts which fed on a vitamin -deficient diet [5, 27] surprising. However, flagellates were not affected directl y, but rather by increased or decreas ed immun e reactions .
Tabl e 1. Number o f Blastocrith idia triato maes (x l 0 S ) in the sma ll int estin e of sho rt- and lon g-term infected th ird and fourth insta rs of Triatom a infestans after feeding on pu re or vitamin B-supplemented sheep blood Batch No.
2
Stage
W.aJ.
Pur e sheep blood . Infection peri od : sho rt lon g
Vita min B blood . Infection per iod : sho rt lon g
L3
1 2
6.2 ± 4.3 12.1 ± 4,4
8.8 ± 2.3 11. 0 ± 2.8
11.6 ± 3 .6 22 .7 ± 7,4
10. 2 ± 2.2 23 .1 ± 9.0
L4
1 2
16.5 ± 4.0 21. 0 ± 11.1
23.5
16.6 ± 3.7 ± 4.0
27.5 ± 10.6 41.4 ± 6.7
45.7
1 2
15.3 ± 4.6 41. 2 ± 15. 7
27.1 ± 20,4 b 48.3 ± 24 .8 b
15.3 ± 3.9 46.3 ± 10.6
18.8 ± 9.9 b 53.2 ± 12,4b
L4
mean and SD of five bugs b mean and SD of ten bugs
a
27.7 ± 6.6 ± 8.0
W.a.f. weeks after first feedin g in th e respective stage
Vitamin B and Development of Blastocrithidia in Bugs' 19 Table 2. Number of Blastocrithidia triatomae» (x10 5 ) in the small intestine of Triatoma infestans after feeding on pure or vitamin B-supplemented sheep blood Batch No.
Stage
W.a.f.
n
1
L3
1 2
10 10
7.5 ± 3.5 11.5 ± 3.5
10.9 ± 2.9 22.9 ± 7.8
0.05 0.001
L4
1 2
10 10
16.6 ± 3.6 22.2 ± 8.0
27.6 ± 8.3 43.5 ± 7.3
0.01 0.001
L3
1 2
10 10
5.6 ± 1.8 8.2 ± 2.8
9.9 ± 2.7 22.9 ± 7.9
0.001 0.001
L4
1 2
15 15
23.2 ± 17.5 45.9 ± 21.8
17.6 ± 8.4 50.9 ± 11.9
n.s. n.s.
3
L5
1 2
10 10
68.2 ± 27.4 142.0 ± 43.8
116.8 ± 31.8 128.5 ± 32.2
0.01 n.s.
4
L5
1 2
6 6
44.5 ± 9.0 86.5 ± 14.5
36.4 ± 18.0 92.1 ± 17.5
n.s. n.s.
Adults
1 2
6 6
216.0 ± 53.2 301.1 ± 116.2
240.5 ± 171.9 243.9 ± 72.1
n.s, n.s.
2
Pure sheep blood
Vitamin B blood
p:S (t-test)
mean and SO W.aJ. weeks after first feeding in the respective stage
n number of bugs n. s. not significant
A direct influence of B-group vitamins on mammalian trypanosomes can be shown by in vitro cultivation. Media often contain vitamins or supplements such as liver extract or serum which supply vitamins [e.g., 24]. Several B-group vitamins were needed for cultivating both the insect trypanosomatid Crithidia [asciculata and the mammalian parasite T. cruzi [1, 10]. The Drosophila medium used for B. triatomae requires supplementation with fetal calf serum and a reduviid cell line [15]. The vitamin supply in the host has not been determined for mammalian trypanosomes or for trypanosomatids of insects. One week post infectionem (p.i.), Miihlpfordt [13] found a significantly better development of T. cruzi in R.prolixus which contained symbionts than in those developing in aposymbiotic bugs. This difference could be caused by B-group vitamins supplied by the symbionts. Surprisingly, two and three weeks p.i., significantly more T. cruzi were found in aposymbiotic bugs. This can only be
explained by alterations of a factor limiting the population development, e.g. attachment sites. In B. triatomaeinfected bugs, disturbances of digestion occur, as well as reduction of extracellular membrane layers [11]; these layers, typical for Hemiptera, are similar to the peritrophic membranes of other insects [25]. If disturbed digestion in aposymbiotic bugs also resulted in similar ultrastructural alterations, better attachment by the parasite might account for greater development of T. cruzi in the aposymbiotic R. prolixus. However, other factors such as disturbed digestion or absorption have to be considered. Unfortunately, no detailed studies are available comparing the digestion of different instars of triatomines which might offer an explanation of the differences between batches in the present study. The initial development of B. triatomae was supported by the addition of B-group vitamins in bugs infected in the first instar. Since B. triatomae attaches to the microvillar border of the midgut epithelium even during initial development [Schaub & Neukirchen, unpubl. data], the extracellular membrane layers should not restrict the attachment in bugs fed sheep blood or in those fed vitamin B-supplemented blood. However, long- and short-term infected L3 and L4 showed a stage-specific level of population densities which were also observed in T. cruzi-infected T. infestans [16]. The similar population densities in the second batch of L4 indicate that established populations were limited by a factor which was not influenced by vitamin supplementation. This could have been the availability of attachment sites. In addition, effects in aposymbiotic bugs are evident in late instars. Therefore, a higher demand for vitamins and a better absorption is possible by the bug at the stomach wall - upstream of the major B. triatomaepopulation in the small intestine. After infection of third instars, the initial development was significantly increased by the vitamin supply in only
a
Table 3. Number of Blastocrithidia triatomae- (xl 05) in the rectum of Triatoma infestans after feeding on pure or vitamin B-supplemented sheep blood Batch No.
Stage
W.aJ.
n
Pure sheep blood
Vitamin B blood
1
L3
1 2
10 10
0.8 ± 0.9 1.3 ± 0.9
0.7 ± 0.5 0.7 ± 0.5
L4
1 2
10 10
2.6 ± 1.6 3.0 ± 1.1
2.3 ± 1.2 3.8 ± 1.9
L5
1 2
6 6
6.3 ± 2.2 13.4 ± 7.2
4.3 ± 2.7 9.6 ± 4.8
Adults
1 2
6
6
19.7 ± 8.6 46.0 ± 17.1
23.9 ± 16.4 19.4 ± 2.5
4
abbreviations see Table 2.
20 . C. Jensen and G. A. Schaub
one batch. In addition to the possibility of differential vitamin absorption, as mentioned above, individual infection dose was different. Older larvae ingested more blood and thereby more cysts of B. triatomae. Thus more cysts passed from the storage region of the midgut, i.e., the stomach, where B. triatomae remains encysted, into the digestive intestine, where excystment occurs [22]. Further studies are necessary to determine whether the initial phase of rapid multiplication is shorter after infection of older larvae and whether it could be stimulated by B-group vitamin supplementation including biotin. Investigations comparing the digestion and vitamin metabolism of different instars of Triatominae might explain the interesting result in the present study that vitarnin-B supplementation of the insect diet supports at least the initial development of B. triatomae in the young instars. Acknowledgements We wish to thank Mrs. M. Walter for typing the manuscript and we are most grateful to Dr. P. Billingsley, London, for correcting the English version of the manuscript. The support of the Deutsche Forschungsgemeinschaft (project no. Scha 339/1-5) is gratefully acknowledged.
References 1 Adroher F. J., Jimenez-Ortiz A. y Osuna A. (1987): Influencia de algunas vitaminas en eI crecimiento y diferenciacion de Trypanosoma cruzi. Rev. Iber. Parasitol., 47, 223-227. 2 Bewig F. und Schwartz W. (1956): Untersuchungen ~ber die Symbiose von Tieren mit Pilzen und Bakterien. VII. Uber die Physiologie der Symbiose bei einigen blutsaugenden Insekten. Arch. Mikrobiol., 24, 174-208. 3 Brecher G. and Wigglesworth V. B. (1944): The transmission of Actinomyces rhodnii Erikson in Rhodnius prolixus Stal (Hemiptera) and its influence on the growth of the host. Parasitology, 35, 220-224. 4 D'Alessandro A. (1976): Biology of Trypanosoma (Herpetosoma) rangeli Tejera, 1920. In: Lumsden W. H. R. and Evans D. A (eds.): Biology of the Kinetoplastida, vol. 1, pp. 327-403. Academic Press, London. 5 Godfrey D. G. (1958): Influence of dietary cod liver oil upon Trypanosoma congolense, T. cruzi, T. vivax and T. brucei. Exp. Parasitol., 7, 255-268. 6 Gumpert J. und Schwartz W. (1962): Untersuchungen iiber die Symbiose von Tieren mit Pilzen und Bakterien. X. Die Symbiose der Triatominen. 1. Aufzucht symbiontenhaltiger und symbiontenfreier Triatominen und Eigenschaften der bei Triatominen vorkommenden Mikroorganismen. Z. Allgem. Mikrobiol., 2, 209-225. 7 Gumpert J. und Schwartz W. (1963): Untersuchungen iiber die Symbiose von Tieren mit Pilzen und Bakterien. X. Die Symbiose der Triatominen. 3. Panrothensaurelieferung als Funktion der Symbionten. Z. Allgem. Mikrobiol., 3, 1-14. 8 Halff L. A. (1956): Untersuchungen iiber die Abhangigkeit der Entwicklung der Reduviide Triatoma infestans Klug von ihrem Darmsymbionten. Acta Trop., 13, 225-253. 9 HaringtonJ. S. (1960): Synthesis ofthiamine and folic acid by Nocardia rhodnii, the micro-symbiont of Rhodnius prolixus. Nature, 188, 1027-1028.
10 Hutner S. H., Bacchi C. J. and Baker H. (1976): Nutrition of the Kinetoplastida. In: Lumsden W. H. R. and Evans D. A. (eds.): Biology of the Kinetoplastida, vol. 2, pp. 653-691. Academic Press, London. 11 Jensen c., Schaub G. A. and Molyneux D. H. (1990): The effect of Blastocrithidia triatomae (Trypanosomatidae) on the midgut of the reduviid bug Triatoma infestans. Parasitology,
100,1-9. 12 Lake P. and Friend W. G. (1968): The use of artificial diets to determine some of the effects of Nocardia rhodnii on the development of Rhodnius prolixus. J. Insect Physiol., 14, 543-562. 13 Miihlpfordt H. (1959): Der Einfluf der Darmsymbionten von Rhodnius prolixus auf Trypanosoma cruzi. Z. Tropenmed. Parasitol., 10,314-327. 14 Nyiradi S. A. (1973): The germfree culture of three species of Triatominae: Triatoma protracta (Uhler), Triatoma rubida (Uhler) and Rhodnius prolixus Stal. J. Med. Entomol., 10, 417-448. 15 Reduth D., Schaub G. A. and Pudney M. (1989): Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae). Parasitology, 98, 387-393. 16 Schaub G. A. (1989): Trypanosoma cruzi: quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans. Exp. Parasitol., 68, 260-273. 17 Schaub G. A. (1990a): The effect of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: influence of group size. J. Invertebr. Pathol., 56, 249-257. 18 Schaub G. A. (1990b): Influence of starvation of the reduviid bug Triatomae infestans on the pathological effects of Blastocrithidia triatomae (Trypanosomatidae) and the coprophagic infection rate of the bug. Z. Angew. Zool. (in press). 19 Schaub G. A. (1990c): Membrane feeding for infection of the reduviid bug Triatoma infestans with Blastocrithidia triatomae (Trypanosomatidae) and pathogenic effects of the flagellate. Parasitol. Res., 76, 306-310. 20 Schaub G. A. and Boker C. A. (1986): Scanning electron microscopic studies of Blastocrithidia triatomae (Trypanosomatidae) in the rectum of Triatoma infestans (Reduviidae). J. Protozool., 33, 266-270. 21 Schaub G. A. and Jensen C. (1990): Developmental time and mortality of the reduviid bug Triatoma infestans with Blastocrithidia triatomae (Trypanosomatidae). J. Invertebr. Pathol., 55,17-27. 22 Schaub G. A. and Pretsch M. (1981): Ultrastructural studies on the excystment of Blastocrithidia triatomae (Trypanosomatidae). Trans. R. Soc. Trop. Med. Hyg., 75, 168-171. 23 Schaub G. A. and Schnitker A. (1988): Influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: alterations in the Malpighian tubules. Parasitol. Res., 75, 88-97. 24 Taylor A. E. R. and Baker J. F. (1968): The cultivation of parasites in vitro. Blackwell Scientific Publications, Oxford. 25 Terra W. R. (1990): Evolution of digestive systems of insects. Annu. Rev. Entomol., 35, 181-200. 26 Watkins R. (1971): Histology of Rhodnius prolixus infected with Trypanosoma rangeli. J. Invertebr. Pathol., 17, 59-66. 27 Yaeger R. C. and Miller O. N. (1960): Effect of malnutrition on susceptibility of rats to Trypanosoma cruzi. IV. Pyridoxine deficiency. Exp. Parasitol., 10,238-244.
Key words: Vitamin B - Blastocrithidia triatomae - Triatoma infestans - Development - Insect host Giinter A. Schaub, Institut fur Biologie I (Zoologie), Albert-Ludwigs-Universitat, Albertstrafe 21 a, D-7800 Freiburg, FRG
Europ.J.Protistol. 27, 17- 20 (1991) March 28, 1991
PROTISTOLOGY
Development of Blastocrithidia triatomae (Trypanosomatidae) in Triatoma infestans after Vitamin B-Supplementation of the Blood-Diet of the Bug Cord Jensen and Gunter A. Schaub Institut tur Biologie I (Zoologie), Albert-Ludwigs-Universitat, Freiburg, FRG
SUMMARY Investigati ons of the inter relatio nships o f the trypan osom at id Blastocrithidia triatomae and the redu viid bug Triatoma infestans ind icat ed an int erferen ce o f B. triatomae with bug symbionts or with their vitamin supply. For an initial study of this possibility, B. triatomae-infected bugs were fed two or three times at monthly intervals on defibrin at ed sheep blood with or without B-group vitamin supplementa tion (folic acid, nicotinic acid, pantothenic acid, pyridoxine, riboflavin, and thiamin ). The tot al number of flagellates in the sma ll intestine and the rectum of the bug were then determ ined with a hemo cytom eter. Vitami n B-suppleme ntati on supported the initial development of B. triatomae in the small intestine of most th ird and four th instar larvae which had been infected in the first instar, In the small intestines of fifth instars and adults, bot h which had been infected as th ird instar lar vae, most populat ions of B. triatomae were not affected by vitami n B-suppl ement at ion. Similarly, recta l populations in all bugs were never increased by supplementa tion.
Introduction The homoxenous trypanosomatid Blastocrithidia triatom ae develops in the intestine and occasionally in the excretory system of hematophagous reduviid bugs, the vecto rs of Chagas' disease [11, 23]. B. triatomae causes a numb er of pathological effects on the reduviid Triatoma infestans [summarized in 17, 18,21], some of which were also observed in Rh odniu s prolixus infected with Trypanosoma rangeli [4] and in aposymbiotic bugs fed on hum ans, guinea pigs or rabbits [2,3,6, 14]. Watkins [26] suggested that T. rangeli might induc e such path ological effects by reducing the number of symbiotic bacteria in the insect midgut. Symbiotic bacteria pre sumably supply the bugs with B-group vitamins [7, 8, 9] and when rabbit blood was supplemented with these vitamin s, the development of apo symbiot ic larvae, which are particularl y affected in the late instars, was significantly improved [e.g., 12]. The pathologic similarities between apo symbiotic and B. triatomae-infected bugs can be explained either by a © 199 1 by Gustav Fischer Verlag, Stuttgart
redu ction in the number of symbionts caused by the flagellates or by competition betw een bug and flagellates for the B-vitamins. Preliminary studies showed great variation in the numb er of bacteria in uninfect ed and B. triatom ae-infected T. infestans, [Schaub & Fischer unpub\. data]. We ther efore decided to investigate the path ological effects of B. triatom ae in bugs fed on norm al blood supplemented with B-group vitamins. Since this supplement might cau se increased trypanosom atid popul ations which could compete with the insect for essential nutrients, it was necessary to study the effects of B-group vita mins on popul ations of B. triatomae. So far, the effects of vita mins on development of insect trypanosomatids have only been investigated with in vitro cultures [24], not with parasites in the insect host.
Material and Methods Th e original strain of Blastocrithidia triatomae Cerisola et aI., 1971 was maint ained in our laboratory in Triatoma infestans 0932 -4739/9 1/002 7-001 7$3 .50/0
18 . C. Jensen and G. A. Scha ub [20], which were used to initia te pr imar y in vitro cultures [15]. Cysts from the second passage were used after cryopreser vat ion a t -80 °C for six months [11]. Th e cyst suspension wa s diluted with defibr inat ed sheep blood to a final concent ration of 10 6 cysts/ern! blood. Th e Triatom a infestans (Klug, 1834 ) strain orig inates from Cac hiyuyu, Chile [20]. Bugs we re fed ro utinely on hen s and reared at ca. 26 °C and ca. 60 % relat ive humidity on a 16h/8h light/dark rhythm. T wo batches of first (Ll) and two of third insrar (L3) larvae were infected with the cyst suspension [19]. Th ose fed to cap acity were divided into 8 gro ups, maintained in 1 I beakers [17], and received un infected L5 to allow an upt ake of bacterial syrnbion ts. At mo nthly intervals four gro ups of bugs were fed on defibr inated sheep blood (Gesellschaft fiir Lab ordiagnostika, Mulheim-Ruhr, F.R.G. ) and four other gro ups of bugs with sheep blood , supplemented with B-group vita mins in th e same concentration s as in the FNPPRT diet of Lak e and Friend [12]: 3 mg folic acid , 10 mg nicotinic acid, 10 mg pantothenic acid-Ca salt, 10 mg pyrid oxine-HCI, 2 mg riboflavin-5-phosphate and 10 mg thi amin e-H CI (all from Sigma, St. Lo uis) was added to 100 em! defibrin ated sheep blood. All diets contai ned 1.1 mM ATP as a ph agostimulant. O ne or 2 weeks after feed ing, S-15 bugs we re dissected: L3 a nd L4 originating from th e two ba tches which had been infected as L1; L5 a nd adults from those infected as L3. Th e first L3 a nd L5 were dissected 9 weeks post infectionem (p.i.). Some of the bugs did not moult after on e feeding and needed a second bloodm eal; they were thu s infected for four addit iona l weeks when dissected as L3 or L4. Dissection and macerat ion of the small intestin e (fo ur bat ches) and the rectu m (two batches) were as pr eviou sly described [16]. N umber of B. triatom ae in the se tissues was det ermin ed in N eubauer hemocytom eters. Stu dent's t-rest, using th e softwa re of SAS Institute Inc. (Ca ry, USA), was used for sta tistica l compa riso ns of dat a.
insta r and at the same time after feeding with the same diet (Ta ble 1). When data from these two groups were combined, we found a conspicuo us increase in the number of flagellate s in succeeding instar s (Ta ble 2). The initial developm ent of flagellates was significantly bett er in both batch es of L3 fed upon the diet supplement ed with B-group vitamins (Ta ble 2). This improvement was also found in the L4 of the first batch. Differences also occurred betw een the two batches which had been infected as L3. In the first batch , in which none of the vitamin-deficient L5 moulted to the adult stage, a significant increase in the numb er of flagellates was observed in the first dissections, made 1 week after feeding L5 on supplemented blood ; in the second batch of L5 and adults, population densities did not differ significantly.
Rectum At each dissection time, about 10 % of the tot al popul ation was found in the rectum (Table 3). In individual bugs the difference between tr ypano somatid populations in both regions varied between 1 % and 25 % of the tot al population; the tot al number of flagellate s in the small intestine did not show any correlation with that in the rectum. The number of flagellates in the rectum also increased in the succeeding instar s, but no growth support by vitamin s was observed. A sta tistically significant differen ce was only obtained for the adults, two weeks after feeding (t-rest; p ~ 0.01 ).
Discussion Results Great variation was found in the number of flagellates per intestine or per rectum ; minim al and maximal values could differ by a factor of ten. Intestine
The number of flagellates in the small intestine did not differ statistically significantly between long-term and sho rt-term infected bugs which were dissected in the same
Since endoparasites ob tain their food from the host, they can be influenced by the vitamin content of its diet. So far, such studies with tryp anosom atids have only consid ered mammalian parasites and their hosts. Expecting a po sitive effect of vitamins on developm ent of parasites, we at first found the reduced growth of trypanosomes in mice which fed on cod-liver oil and the increased growth in hosts which fed on a vitamin -deficient diet [5, 27] surprising. However, flagellates were not affected directl y, but rather by increased or decreas ed immun e reactions .
Tabl e 1. Number o f Blastocrith idia triato maes (x l 0 S ) in the sma ll int estin e of sho rt- and lon g-term infected th ird and fourth insta rs of Triatom a infestans after feeding on pu re or vitamin B-supplemented sheep blood Batch No.
2
Stage
W.aJ.
Pur e sheep blood . Infection peri od : sho rt lon g
Vita min B blood . Infection per iod : sho rt lon g
L3
1 2
6.2 ± 4.3 12.1 ± 4,4
8.8 ± 2.3 11. 0 ± 2.8
11.6 ± 3 .6 22 .7 ± 7,4
10. 2 ± 2.2 23 .1 ± 9.0
L4
1 2
16.5 ± 4.0 21. 0 ± 11.1
23.5
16.6 ± 3.7 ± 4.0
27.5 ± 10.6 41.4 ± 6.7
45.7
1 2
15.3 ± 4.6 41. 2 ± 15. 7
27.1 ± 20,4 b 48.3 ± 24 .8 b
15.3 ± 3.9 46.3 ± 10.6
18.8 ± 9.9 b 53.2 ± 12,4b
L4
mean and SD of five bugs b mean and SD of ten bugs
a
27.7 ± 6.6 ± 8.0
W.a.f. weeks after first feedin g in th e respective stage
Vitamin B and Development of Blastocrithidia in Bugs' 19 Table 2. Number of Blastocrithidia triatomae» (x10 5 ) in the small intestine of Triatoma infestans after feeding on pure or vitamin B-supplemented sheep blood Batch No.
Stage
W.a.f.
n
1
L3
1 2
10 10
7.5 ± 3.5 11.5 ± 3.5
10.9 ± 2.9 22.9 ± 7.8
0.05 0.001
L4
1 2
10 10
16.6 ± 3.6 22.2 ± 8.0
27.6 ± 8.3 43.5 ± 7.3
0.01 0.001
L3
1 2
10 10
5.6 ± 1.8 8.2 ± 2.8
9.9 ± 2.7 22.9 ± 7.9
0.001 0.001
L4
1 2
15 15
23.2 ± 17.5 45.9 ± 21.8
17.6 ± 8.4 50.9 ± 11.9
n.s. n.s.
3
L5
1 2
10 10
68.2 ± 27.4 142.0 ± 43.8
116.8 ± 31.8 128.5 ± 32.2
0.01 n.s.
4
L5
1 2
6 6
44.5 ± 9.0 86.5 ± 14.5
36.4 ± 18.0 92.1 ± 17.5
n.s. n.s.
Adults
1 2
6 6
216.0 ± 53.2 301.1 ± 116.2
240.5 ± 171.9 243.9 ± 72.1
n.s, n.s.
2
Pure sheep blood
Vitamin B blood
p:S (t-test)
mean and SO W.aJ. weeks after first feeding in the respective stage
n number of bugs n. s. not significant
A direct influence of B-group vitamins on mammalian trypanosomes can be shown by in vitro cultivation. Media often contain vitamins or supplements such as liver extract or serum which supply vitamins [e.g., 24]. Several B-group vitamins were needed for cultivating both the insect trypanosomatid Crithidia [asciculata and the mammalian parasite T. cruzi [1, 10]. The Drosophila medium used for B. triatomae requires supplementation with fetal calf serum and a reduviid cell line [15]. The vitamin supply in the host has not been determined for mammalian trypanosomes or for trypanosomatids of insects. One week post infectionem (p.i.), Miihlpfordt [13] found a significantly better development of T. cruzi in R.prolixus which contained symbionts than in those developing in aposymbiotic bugs. This difference could be caused by B-group vitamins supplied by the symbionts. Surprisingly, two and three weeks p.i., significantly more T. cruzi were found in aposymbiotic bugs. This can only be
explained by alterations of a factor limiting the population development, e.g. attachment sites. In B. triatomaeinfected bugs, disturbances of digestion occur, as well as reduction of extracellular membrane layers [11]; these layers, typical for Hemiptera, are similar to the peritrophic membranes of other insects [25]. If disturbed digestion in aposymbiotic bugs also resulted in similar ultrastructural alterations, better attachment by the parasite might account for greater development of T. cruzi in the aposymbiotic R. prolixus. However, other factors such as disturbed digestion or absorption have to be considered. Unfortunately, no detailed studies are available comparing the digestion of different instars of triatomines which might offer an explanation of the differences between batches in the present study. The initial development of B. triatomae was supported by the addition of B-group vitamins in bugs infected in the first instar. Since B. triatomae attaches to the microvillar border of the midgut epithelium even during initial development [Schaub & Neukirchen, unpubl. data], the extracellular membrane layers should not restrict the attachment in bugs fed sheep blood or in those fed vitamin B-supplemented blood. However, long- and short-term infected L3 and L4 showed a stage-specific level of population densities which were also observed in T. cruzi-infected T. infestans [16]. The similar population densities in the second batch of L4 indicate that established populations were limited by a factor which was not influenced by vitamin supplementation. This could have been the availability of attachment sites. In addition, effects in aposymbiotic bugs are evident in late instars. Therefore, a higher demand for vitamins and a better absorption is possible by the bug at the stomach wall - upstream of the major B. triatomaepopulation in the small intestine. After infection of third instars, the initial development was significantly increased by the vitamin supply in only
a
Table 3. Number of Blastocrithidia triatomae- (xl 05) in the rectum of Triatoma infestans after feeding on pure or vitamin B-supplemented sheep blood Batch No.
Stage
W.aJ.
n
Pure sheep blood
Vitamin B blood
1
L3
1 2
10 10
0.8 ± 0.9 1.3 ± 0.9
0.7 ± 0.5 0.7 ± 0.5
L4
1 2
10 10
2.6 ± 1.6 3.0 ± 1.1
2.3 ± 1.2 3.8 ± 1.9
L5
1 2
6 6
6.3 ± 2.2 13.4 ± 7.2
4.3 ± 2.7 9.6 ± 4.8
Adults
1 2
6
6
19.7 ± 8.6 46.0 ± 17.1
23.9 ± 16.4 19.4 ± 2.5
4
abbreviations see Table 2.
20 . C. Jensen and G. A. Schaub
one batch. In addition to the possibility of differential vitamin absorption, as mentioned above, individual infection dose was different. Older larvae ingested more blood and thereby more cysts of B. triatomae. Thus more cysts passed from the storage region of the midgut, i.e., the stomach, where B. triatomae remains encysted, into the digestive intestine, where excystment occurs [22]. Further studies are necessary to determine whether the initial phase of rapid multiplication is shorter after infection of older larvae and whether it could be stimulated by B-group vitamin supplementation including biotin. Investigations comparing the digestion and vitamin metabolism of different instars of Triatominae might explain the interesting result in the present study that vitarnin-B supplementation of the insect diet supports at least the initial development of B. triatomae in the young instars. Acknowledgements We wish to thank Mrs. M. Walter for typing the manuscript and we are most grateful to Dr. P. Billingsley, London, for correcting the English version of the manuscript. The support of the Deutsche Forschungsgemeinschaft (project no. Scha 339/1-5) is gratefully acknowledged.
References 1 Adroher F. J., Jimenez-Ortiz A. y Osuna A. (1987): Influencia de algunas vitaminas en eI crecimiento y diferenciacion de Trypanosoma cruzi. Rev. Iber. Parasitol., 47, 223-227. 2 Bewig F. und Schwartz W. (1956): Untersuchungen ~ber die Symbiose von Tieren mit Pilzen und Bakterien. VII. Uber die Physiologie der Symbiose bei einigen blutsaugenden Insekten. Arch. Mikrobiol., 24, 174-208. 3 Brecher G. and Wigglesworth V. B. (1944): The transmission of Actinomyces rhodnii Erikson in Rhodnius prolixus Stal (Hemiptera) and its influence on the growth of the host. Parasitology, 35, 220-224. 4 D'Alessandro A. (1976): Biology of Trypanosoma (Herpetosoma) rangeli Tejera, 1920. In: Lumsden W. H. R. and Evans D. A (eds.): Biology of the Kinetoplastida, vol. 1, pp. 327-403. Academic Press, London. 5 Godfrey D. G. (1958): Influence of dietary cod liver oil upon Trypanosoma congolense, T. cruzi, T. vivax and T. brucei. Exp. Parasitol., 7, 255-268. 6 Gumpert J. und Schwartz W. (1962): Untersuchungen iiber die Symbiose von Tieren mit Pilzen und Bakterien. X. Die Symbiose der Triatominen. 1. Aufzucht symbiontenhaltiger und symbiontenfreier Triatominen und Eigenschaften der bei Triatominen vorkommenden Mikroorganismen. Z. Allgem. Mikrobiol., 2, 209-225. 7 Gumpert J. und Schwartz W. (1963): Untersuchungen iiber die Symbiose von Tieren mit Pilzen und Bakterien. X. Die Symbiose der Triatominen. 3. Panrothensaurelieferung als Funktion der Symbionten. Z. Allgem. Mikrobiol., 3, 1-14. 8 Halff L. A. (1956): Untersuchungen iiber die Abhangigkeit der Entwicklung der Reduviide Triatoma infestans Klug von ihrem Darmsymbionten. Acta Trop., 13, 225-253. 9 HaringtonJ. S. (1960): Synthesis ofthiamine and folic acid by Nocardia rhodnii, the micro-symbiont of Rhodnius prolixus. Nature, 188, 1027-1028.
10 Hutner S. H., Bacchi C. J. and Baker H. (1976): Nutrition of the Kinetoplastida. In: Lumsden W. H. R. and Evans D. A. (eds.): Biology of the Kinetoplastida, vol. 2, pp. 653-691. Academic Press, London. 11 Jensen c., Schaub G. A. and Molyneux D. H. (1990): The effect of Blastocrithidia triatomae (Trypanosomatidae) on the midgut of the reduviid bug Triatoma infestans. Parasitology,
100,1-9. 12 Lake P. and Friend W. G. (1968): The use of artificial diets to determine some of the effects of Nocardia rhodnii on the development of Rhodnius prolixus. J. Insect Physiol., 14, 543-562. 13 Miihlpfordt H. (1959): Der Einfluf der Darmsymbionten von Rhodnius prolixus auf Trypanosoma cruzi. Z. Tropenmed. Parasitol., 10,314-327. 14 Nyiradi S. A. (1973): The germfree culture of three species of Triatominae: Triatoma protracta (Uhler), Triatoma rubida (Uhler) and Rhodnius prolixus Stal. J. Med. Entomol., 10, 417-448. 15 Reduth D., Schaub G. A. and Pudney M. (1989): Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae). Parasitology, 98, 387-393. 16 Schaub G. A. (1989): Trypanosoma cruzi: quantitative studies of development of two strains in small intestine and rectum of the vector Triatoma infestans. Exp. Parasitol., 68, 260-273. 17 Schaub G. A. (1990a): The effect of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: influence of group size. J. Invertebr. Pathol., 56, 249-257. 18 Schaub G. A. (1990b): Influence of starvation of the reduviid bug Triatomae infestans on the pathological effects of Blastocrithidia triatomae (Trypanosomatidae) and the coprophagic infection rate of the bug. Z. Angew. Zool. (in press). 19 Schaub G. A. (1990c): Membrane feeding for infection of the reduviid bug Triatoma infestans with Blastocrithidia triatomae (Trypanosomatidae) and pathogenic effects of the flagellate. Parasitol. Res., 76, 306-310. 20 Schaub G. A. and Boker C. A. (1986): Scanning electron microscopic studies of Blastocrithidia triatomae (Trypanosomatidae) in the rectum of Triatoma infestans (Reduviidae). J. Protozool., 33, 266-270. 21 Schaub G. A. and Jensen C. (1990): Developmental time and mortality of the reduviid bug Triatoma infestans with Blastocrithidia triatomae (Trypanosomatidae). J. Invertebr. Pathol., 55,17-27. 22 Schaub G. A. and Pretsch M. (1981): Ultrastructural studies on the excystment of Blastocrithidia triatomae (Trypanosomatidae). Trans. R. Soc. Trop. Med. Hyg., 75, 168-171. 23 Schaub G. A. and Schnitker A. (1988): Influence of Blastocrithidia triatomae (Trypanosomatidae) on the reduviid bug Triatoma infestans: alterations in the Malpighian tubules. Parasitol. Res., 75, 88-97. 24 Taylor A. E. R. and Baker J. F. (1968): The cultivation of parasites in vitro. Blackwell Scientific Publications, Oxford. 25 Terra W. R. (1990): Evolution of digestive systems of insects. Annu. Rev. Entomol., 35, 181-200. 26 Watkins R. (1971): Histology of Rhodnius prolixus infected with Trypanosoma rangeli. J. Invertebr. Pathol., 17, 59-66. 27 Yaeger R. C. and Miller O. N. (1960): Effect of malnutrition on susceptibility of rats to Trypanosoma cruzi. IV. Pyridoxine deficiency. Exp. Parasitol., 10,238-244.
Key words: Vitamin B - Blastocrithidia triatomae - Triatoma infestans - Development - Insect host Giinter A. Schaub, Institut fur Biologie I (Zoologie), Albert-Ludwigs-Universitat, Albertstrafe 21 a, D-7800 Freiburg, FRG