TRYPANOSOMES
IN CALIFORNIA
SANDFLIES
Chao (personal communication) found many trypomasti-
gotes in his cultures of T . scelopori. Four of the 6 infections
showed local concentrations of flagellates: 2 with concen-
trations in the anterior stomach as with trypanosomes from
alligator lizards, and 2 with concentrations in the posterior
stomach.
Fourteen sandflies that had fed upon an infected fence
lizard were examined after 6-10 days. Eleven of the flies
were infected, and 7 had infections limited to the cardia
alone, with long, thin epimastigotes structurally indis-
tinguishable from those of T . gsrrhonoti. In 4 of the in-
fected flies flagellates occupied the entire midgut; both
stomach and cardia were filled with thousands of epimasti-
gotes in different stages of development.
Experimental Transfer
Limited attempts to transfer both T . gerrhonoti and T .
scelopori were mostly unsuccessful. A few trypanosomes
were found in wet and dry smears of one of 3 fence lizards
inoculated 4 weeks earlier with blood from an infected
fence lizard. No trypanosomes were found in 3 hatchling
alligator lizards inoculated with blood from infected alli-
gator lizards, nor in 3 other hatchling alligator lizards
inoculated with blood from an infected fence lizard.
Five hatchling alligator lizards were each inoculated
with flagellates from the intestines of 5-10 sandflies fed on
an infected alligator lizard 11-12 days previously. The
hatchlings had been in the laboratory for 4 months without
showing natural infections. Two of the 5 lizards had de-
J. PROI-OZOOI..
18(3), 4 3 3 4 3 6 (1971).
Trypanosomes in Wild California Sandflies, and
Extrinsic Stages of Trypanosoma bufophlebotomi
STEPHEN C . AYALA*
Div. of Parasitology, Univ. of California, Berkeley
SYNOPSIS. Trypanosomes were found in 94 of over 1,600 wild
Lutzomyia vexatrix occidentis, a common phlebotomine sandfly
in central California; 25% of the infected sandflies harbored T .
bufophlebotomi, identifiable by its peculiar kinetoplast and body
structure. The toad trypanosome was cultured from insect isolates
ILD phlebotomine sandflies are often found naturally
W infected with trypanosomes from unknown verte-
brate hosts 10,
(e.g., 6, 11,12, 13). Sandflies transmit
described trypanosomes of toads (2, 7 ) and lizards ( 1, 14),
and are implicated in the life cycles of other trypanosomes
from lizards and mammals (e.g., 8, 13).
Several trypanosomes of poikilotherm vertebrates from
California undergo extrinsic development in sandflies
(Fig. 1 ) . Trypanosoma bufophlebotomi (Fig. 2) was de-
scribed from the California toad Bufo boreas ( 3 ) . It was
* Present address: Seccibn de Parasitologia, Facultad de Medi-
cina, Universidad del Valle, Cali, Colombia.
433
veloped trypanosome infections when they were examined
3 weeks later. Attempts to transfer T . scelopori to 3 year-
ling fence lizards by the same route and using similar num-
bers of sandflies were unsuccessful, Infections via an oral
route were not attempted. A more extensive study of this
type will be required in order to decide whether one or 2
trypanosome populations are involved.
This study was supported in part by U.S. Public Health Service
fellowship No. 5-FOl-GM-43,295-02. We are grateful for the
help of Dr. John R. Anderson, who initiated the study, Drs.
Clarence J. Weinmann, Dean P. Furman and William Balamuth
for the use of their facilities, and to Dwayne Lee for his help
in the field.
REFERENCES
1. Adler, S. & Theodor, 0. 1935. Investigations on Mediter-
ranean kala-azar. X. A note on Trypanosoma platydactyli and
Leishmania tarentolae. Proc. R o y . SOC.Lond. 116, 543-4.
2. Ayala, S. C. 1970a. Two new trypanosomes from California
toads and lizards. J . Protozool. 17, 370-3.
3. ___ 197013. Protozoan parasites associated with California
sandflies. Ph.D. dissertation. Univ. Calif. (Berkeley). 99 pp.
4. -- 1971. Trypanosomes in wild California sandflies,
and extrinsic stages of Trypanosoma bufophlebotomi. J. Proto-
zool. 18, (this issue).
5. Chaniotis, B. & Anderson, J. R. 1968. Age structure, popula-
tion dynamics and vector potential of Phlebotomus in northern
California. Part 11. Field population dynamics and natural
flagellate infection in parous females. J. M e d . Ent. 6, 273-92.
6. McKay, J. G. 1936. Trypanosoma gerrhonoti sp. nov. from
the lizard Gerrhonotus scincicauda. (Unpublished M. S . thesis).
Univ. Calif. (Berkeley). 19 pp.
7. Shortt. H. E. & Swaminath. C. S. 1931. Life historv and
morphology of Trypanosoma phlebotomi (Mackie 1914).' Ind.
J. M e d . Res. 19, 541-63.
and freeze-preserved. Growth in culture occurred at 23 C, but
not at 15 C or 30 C. The other 75% of trypanosomes from
wild sandflies remain unidentified, altho some were probably T .
scelopori or T . gerrhonoti of lizards.
transmitted in the laboratory by feeding young toads
sandflies that had fed 10 days earlier upon an infected
toad ( 2 ) . I hoped to conduct a longer study, but I was
unable to locate additional infected toads (altho infected
sandflies were occasionally collected). I n 1970, the area
around the type location was being developed for resi-
dential housing.
This article provides a supplementary description of the
peculiar insect stages and in vitro development of T .
bufophlebotomi. I t also describes the flagellate forms
found in wild Lutzomyia vexatrix occidentis captured in
the central California coastal mountain range. Extrinsic
434 TRYPANOSOWES
IN CALIFORNIA
SANDFLIES

Fig. 1. The sandfly Lutzomyia vexatrix occidentis supports ex- California toad. Fig. 3. Extrinsic stages of T . bufophlebotomi from
trinsic development of alligator lizard, fence lizard and toad the hindgut of an infected sandfly. Figs. 2, 3, x 1200.
trypanosomes. x 13. Fig. 2. Trypanosoma bufophlebotomi of the

development of 2 lizard trypanosomes in California sand- lates lining the intestinal wall. Final digestion of the
flies is described in a companion article ( 5 ) . blood meal was marked by the excretion of a brown residue.
This material was examined twice; in both instances
MATERIALS AND METHODS flagellates were seen, altho the insect remained infected.
Wild sandflies were caught in light traps and funnel traps Motion of the flagellates in the hindgut was restricted until
upon emerging in the evening from ground squirrel burrows where it was dissected open, whereupon several thousands of
they breed ( 6 ) . They were maintained in the laboratory in active flagellates spilled out into the surrounding solution
wooden boxes at room temperature (24-26 C ) , with a relative
(Figs. 3, 4 ) . The trypanosomes final development in the
humidity above 75% until examined. Most observations were
made on L. v. occidentis since it was the most abundant species hindgut was described previously ( 2 ) .
in my Solano County collection areas. L. stewnrti and L. cali- T . bufophlebotornis structure in the hindgut (Fig. 5)
fornicus were also examined when captured. Wild sandflies with does not correspond to any of the currently accepted cate-
pre-existing infections have usually completed one ovarian cycle gories of flagellate structure. They are not promastigotes
and seldom survive more than 3-4 days in the laboratory, even
if they do take a 2nd blood meal. All flies that had not fed in
because the kinetoplast occurs adjacent to the nucleus in
the laboratory within 2 days after capture were then dissected the middle third of the 5-12 p body (it may be anterior,
in sterile Ringers solution and examined for physiologic state and lateral or posterior to the nucleus). Epimastigotes are
parasites. defined (9) as flagellates in which the axoneme emerges
from the side of the body and lies along an undulating
RESULTS AND DISCUSSION membrane. Forms with undulating membranes were not
Trypanosoma bufophlebotomi seen at any stage of the extrinsic cycle, and at the light
microscope level it appeared that the axoneme emerged
1. Development and structure in sandflies: Sandflies were from the anterior end rather than from the side. Neither
dissected at various intervals after feeding on a large adult were epimastigotes found among the culture forms (de-
toad with a peripheral parasitemia of approximately 4 scribed below). This serves to emphasize that the new
trypanosomes per cu mm ( 2 ) . I found multiplying amas- terms (9) serve as useful categories, but should not be
tigote stages (5-7 p ) in smears made from the stomach of too rigidly interpreted.
21 sandflies examined after 1-2 days. Nests of 50-100
multiplying parasites were seen in serial sections of infected 2. Laboratory observations : T . bufophlebotomi was iso-
flies, especially around the periphery of the semi-digested lated from a sandfly hindgut and cultured on Senekjies
blood meal. Finally, multiplication was arrested in 18 re- Leishmania agar ( 2 ) for over 3 years. I was unable to
cently-fed flies by holding them for 3 days at 7 C. Within get the bloodstream form directly into culture and after
a few hr after the flies were returned to room temperature, the first few months of isolation culture forms failed to
hundreds of small, pear-shaped (8 by 2 p ) flagellates were infect young toads. Transfers were made every 2-3 weeks,
found undergoing binary fission in the stomach contents. altho motile forms still occurred in some cultures from
After 2 days, I found long forms (5-12 by 1.5 p ) with which transfers were made after 26 months. Typically,
15-20 p flagella in the stomach of all of 12 flies examined, there was abundant growth of amastigote, aphaeromas-
in addition to the rounded amastigote forms. The rec- tigote and promastigote forms during the first weeks follow-
tangular kinetoplast and the nucleus overlapped in the ing transfer (Fig. 7 ) .
center of the body. Within a month after transfer, the entire fluid surface
Four days after engorgement, the blood meal was well of the culture tubes was covered with rosettes, each com-
digested. As residues passed back thru the hindgut, the posed of several hundred flagellates. As the cultures aged,
infections became concentrated in that area, with flagel- forms with long cytoplasmic tails appeared and became
 TRYPANOSOMES
IN CALIFORNIA
SANDFLIES
Figs. 4-7, Extrinsic stages of Trypanosoma bufophlebotomi: 4.
colonization of the posterior hindgut of L. u. occidentis; 5.
flagellates from posterior hindgut as in Fig. 3 ; 6. culture forms
abundant. Flagellates grown in NNN medium, fixed in
osmic acid fumes and stained with Giemsa's stain were
often distended with granules (Fig. 6 ) . Distended forms
were seldom found among flagellates grown on Senekjie's
Leishmania agar (Fig. 7 ) .
Several culture tubes of T . bufophlebotomi were success-
fully freeze-preserved for 4 weeks at -70 C after the ad-
dition of 7.5% glycerol. Other cultures were maintained
at various temperatures for several weeks, simultaneously
with cultures of Blastocrithidia culicis, Leishmania taren-
tolae and Herpetomonas muscarum, organisms with wide
temperature tolerances. Comparable growth occurred at
435
-- ..*. '
..:.':'.::. .
grown in NNN medium; 7. culture forms grown in Senekjie's
Leishmania agar medium.
23 C. However, the narrow temperature tolerance of T .
bufophlebotomi became evident when no growth occurred
at 15 C and cultures were killed at 30 C. The other
flagellates continued to grow at these temperatures.
Trypanosomes in W i l d Sandflies
Chaniotis and Anderson (6) found a 4.6% natural
flagellate infection rate in 1,016 sandflies from Capay,
Yo10 County. When they considered only flies that had
taken a blood meal, they found a natural infection rate
of 20%. I found flagellates in 94 of over 1,600 (6%) wild
female sandflies examined from Solano County between
436 TRYPANOSOMES
IN CALIFORNIA
SANDFLIES

1967 and 1970. The bipolar-staining kinetoplast and dis-
tinctive structure made it possible to identify T . bufo-
phlebotomi from the stomach and hindgut of 24 of the
infected sandflies.
There was a different type of flagellate in the hindgut
of 2 parous sandflies. A few small rosettes of 20 individuals
or less occupied the space around the rectal ampullae. In
both instances, the flagellates died almost immediately once
the intestine was opened. They were small and short,
about 15 p long including flagella, and were clearly epi-
mastigotes. I found monoxenous trypanosomatids in the
hindguts of several other insects caught in the burrow
traps, but with the unlikely possible exception of the above
2 cases, there were no such trypanosomatids in the fe-
male sandflies or in over 200 males examined for grega-
rines (4).
I was unable to find any characteristic features to dis-
tinguish extrinsic stages of T . scelopori or T . gerrhonoti in
sandflies ( 5 ) . Four wild parous sandflies had heavy cardia
infections of thin epimastigotes like those of the 2 lizard
trypanosomes.
Heavy flagellate infections of unidentified epimastigote,
sphaeromastigote and amastigote forms occurred in the
stomach of 36 parous or gravid sandflies that died en route
to the laboratory after capture. In 12 cases the infection
extended forward into the cardia and twice it included
the stomach and the entire hindgut. Sometimes a dark
residue of partially digested blood remained in the stomach
of such infected flies long after it should normally have
been excreted.
The remaining 28 naturally infected sandflies had lighter
infections, usually limited to the stomach. It seems certain
that other trypanosomes were involved, in addition to T .
bufophlebotomi, T . scelopori and T . gerrhonoti.
This study was supported in part by a US. Public Health
Service fellowship, No. 5-FO l-GM-43,295-02. Grateful acknowl-
edgment is made to Dr. John R. Anderson, who initiated the
study and provided the sandfly traps, Dr. Rosalie Watkins, who
provided the culture medium, and Drs. Clarence J. Weinmann,
Rex Dadd, Dean P. Furman and William Balamuth for their
help, companionship and the use of their facilities.
REFERENCES
1. Adler, S. & Theodor, 0. 1935. Investigations on Mediter-
ranean Kala-azar. X. A note on Trypanosoma platydactyli and
Leishmania tarentolae. Proc. Roy. SOG.Lond. 116, 543-44.
2. Anderson, J. R. & Ayala, S. C. 1968. Trypanosome trans-
mitted by Phlebotomus: first report from the Americas. Science
161, 1023-5.
3. Ayala, S. C. 1970. T w o new trypanosomes from California
toads and lizards. J . Protorool. 17, 370-3.
4. --. 197 1. Gregarine infections in California sandflies.
J . Invert. Path. (in press).
5.
~ . 1971. Trypanosoma gerrhonoti n. sp., and extrinsic
development of lizard trypanosomes in California sandflies. J .
Protozool. 18, (this issue.)
6. Chaniotis, B. & Anderson, J. R. 1968. Age structure, popula-
tion dynamics and vector potential of Phlebotomus in northern
California. Part IT. Field population dynamics and natural
flagellate infection in parous females. J . M e d . Ent. 5, 273-92.
7. Feng, L. C. & Chung, H. L. 194.1. Phlebotomus squamirostris
(Newstead), transmitter of Trypanosoma bocagei (FranGa) in the
toad Bufo bufo gargarizans. Chin. M e d . J . Suppl. 3, 198-210.
8. Herrer, A. 1942. Trypanosoma phyllotis n. sp. e infecciones
asociadas en una titira, el Phlehotomus noguchii. Rev. M e d . ESP.
(Peru) 1, 40-55.
9. Hoare, C. A. & Wallace, F. G. 1966. Developmental sta es
of trypanosomatid flagellates, a new terminology. Nature 2 h
1358.
10. Johnson, P. & Hertig, hl. 1970. Behavior of Leishmania in
Panamanian Phlebotomine sandflies fed on infected animals. ESP.
Parasit. 27, 281-300.
11. McConnell, E. & Correa,. M. 1964. Trypanosomes and other
microorganisms from PanamanIan Phlebotomus sandflies. J . Para-
sit. 50, 523-8.
12. Sherlock, I. & Pessoa, S. B. 1966. Leptomonas infectando
naturalmente Phlrbotomus em Salvador (Bahia, Brasil) . Rev.
Lat.-Amer. Microbiol. Parasit. 8, 47-50.
13. Shaw, J. J. 1969. The hernoflagellates of sloths. Lond. Sch.
Hyg. T r o p . Med., Memoir 13. 132 pp.
14. Shortt, H. E. & Swaminath, C. S. 1931. Life history and
morphology of Trypanosoma phlebotomi (Mackie 19 14). Znd. J .
M e d . Res. 19, 511-63.
15. Williams, P. 1970. Phlebotomine sandflies and leishmaniasis
in British Honduras. Trans. Roy. Soc. Trop. M e d . Hyg. 64,
317-64.

BOOK REVIEW.. .
Alexander, M. 1971. Microbial ecology. John Wiley & the microbial community, dispersal, colonization, succes-
Sons, New York, N. Y . x +
511 pp. Price $12.95. sion and climax, nutrition, tolerance range, geography and
microenvironment of microorganisms, and natural selec-
This well-written book, with its ecologic orientation, is
tion. Part 2 (Interspecific relationships) has chapters on
representative of the new generation of texts. The author homeostasis, commensalism and protocooperation, symbi-
is a specialist in soil microbiology, and his book is more osis, competition, amensalism, parasitism and predation.
authoritative in the area of bacteriology than in that of
Part 3 (Effect of microorganisms on their surroundings)
protozoology. For instance, he says (p. 331) that Ent-
has chapters on microorganisms and biogeochemistry, and
amoeba histolytica occurs in man, dog and rabbit; this is
on the effect of microorganisms in animals and plants.
true in a way, but the dog is an extremely rare natural
host and, while the rabbit can be infected experimentally, There is a taxonomic list of microorganisms referred to in
I know of no natural cases. Further, Kudos classification the text, and a 19-page index.
of the Protozoa is used rather than the Society of Proto- This book applies the general principles of ecology to
zoologists modern one. Nevertheless, protozoologists should microbial interactions and relationships. Protozoologists
be grateful that the objects of their affection are included could learn a great deal from it about other microorga-
at all. nisms, and I recommend it to them for this purpose.-
The book is organized into 3 parts and 18 chapters. Part Norman D. Levine, College of Veterinary Medicine, Univ.
1 (The community and its development) has chapters on of Illinois, Urbana.