LABORATORY B!0NOMICS, COLONIZATION. 7
AND MORPHOLOGY OF THE
IMMATURE STAGES OF
THE SANDFLY LUTZOMYIA PANAMENSIS
(SHANNON) (DIPTERAnPSYCHOOIDAE)

Dissertation for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
JOHN HARVEY ZIMMERMAN
1975

It";

Echigan 3?;th
. l "remit
.,, Um. l y c?“

This is to certify that the
thesis entitled
Laboratory Bionomics, Colonization, and
Morphology of the Immature Stages of the Sandfly
Lutzomyiagpanamensis (Shannon) (Diptera:

Psychodidae)
presented by

John H. Zimmerman

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Entomology

X/ ¢ 7WA,‘

H. D. Newson

 

Major professor

J 16, 1975
Date une

 

0-7 639

    

HOAB & SUIS'
; 800K BINDERY INC.

RY BINDERS

m: BINDING ay ‘7'

ABSTRACT

LABORATORY BIONOMICS, COLONIZATION, AND
MORPHOLOGY OF THE IMMATURE STAGES OF
THE SANDFLY LUTZOMYIA PANAMENSIS
(SHANNON)(DIPTERA:PSYCHODIDAE)

 

BY

John Harvey Zimmerman

The laboratory bionomics, colonization, and
morphology of the immature stages of the anthropophilic

phlebotomine sandfly Lutzomyia panamensis (Shannon) were

 

studied. Wild—caught blood-engorged females were captured
from a horse near Achiote, Canal Zone, Panama, from March
1973 to January 1974.

In an experiment to evaluate the feeding preference

of E. panamensis larvae, four experimental food sources

 

(yeast, liver powder, hemoglobin and beef blood serum) were
placed on the same plaster lined petri dish. The first
three instars preferred yeast to the other food sources.
The fourth instar preference for yeast declined with an
increased preference for the other food sources, especially
hemoglobin. The four food sources were also compared indi—
vidually on petri dishes to determine the effect of these
single diets on the duration of the life cycle, pupation,

and adult emergence. The yeast and standard larval food

John Harvey Zimmerman

combination dishes produced the shortest developmental time
from the first instar to pupa, with yeast, liver powder,
beef blood serum, and hemoglobin, respectively, increasing
deve10pmental time. When the deve10pmental times of the
larvae reared on single food sources were compared with
those of larvae reared on the combination dishes, the
combination produced faster and more desirable development.
Larvae reared on the combination dishes survived to produce
more pupae and adults than those reared on individual diets.
The plaster lined petri dish was very satisfactory as a
rearing chamber and general observations of larval behavior
could be made with ease.

The effects of various saturated sugar solutions on
the longevity of laboratory reared females and males of E.

panamesis were examined. Fructose and sucrose were the most

 

efficient in prolonging the survival of the females. Fruc-
tose, sucrose, honey, Karo syrup and dextrose appeared to
provide the same male life expectancy. The styrofoam test
vessel was adequate in studying the longevity of these
males and females.

Longevity studies also conducted on the wild-caught
blood-engorged females indicated that the saturated solu-
tions of fructose, sucrose, and dextrose were the most
efficient in promoting longevity. In an analysis of the

effects of these saturated sugar solutions on oviposition,

John Harvey Zimmerman

no difference could be detected between the soaked raisin,
fructose, sucrose, and dextrose groups.

Four laboratory generations of L. panamensis were

 

reared with 37.7 days being the average length of the life
cycle from oviposition to adult. Larval and adult behavior
were described including pupation, emergence and mating.
The unglazed Boston bean pot was the most satisfactory for

rearing L, panamensis in large numbers. The mixture of

 

liver powder, hemoglobin, and beef blood serum added during
the third instar to the already present yeast in the pots
was shown to be the best larval medium tested. The spiny
rat was used successfully as the laboratory host. The
double feeding method of using the cloth releasing cage
and the plastic feeding cage was the most successful in
feeding the largest number of laboratory reared adults with
little injury to the females.

The morphology of the immature stages of L.

panamensis was redescribed and clarified. The dorsal and

 

ventral aspects of the first and fourth instars and the
lateral aspect of the pupa were illustrated. Measurements
of the prominent setae were very useful in differentiating
changes between instars and other closely related species.
Scanning electron photographs of the fourth instar served
as an aid in describing the nature of the cuticular surface.

The scanning electron microscope was used to describe the

John Harvey Zimmerman

egg surface ultrastructure of L. panamensis and other

 

anthropophilic species L. pessoana, L. gomezi, L.

sanguinaria, L. trapidoi, and L. ylephiletor.

 

 

LABORATORY BIONOMICS, COLONIZATION, AND
MORPHOLOGY OF THE IMMATURE STAGES OF

THE SANDFLY LUTZOMYIA PANAMENSIS

 

(SHANNON)(DIPTERA:PSYCHODIDAE)

BY

John Harvey Zimmerman

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Entomology

1975

To Many

ii

ACKNOWLEDGMENTS

I would like to extend my sincere appreciation to
my advisor Dr. Harold D. Newson for his guidance and moral
support throughout my Ph.D. program.

I am indebted to Drs. James E. Bath, Roland L.
Fischer, and Herbert W. Cox for serving on my committee
and for continued assistance during this project.

Special gratitude is extended to Dr. John M. Hunter
of the Latin American Studies Center for providing the
financial support during my stay in Panama. Also, a special
thanks goes to Drs. Gordon E. Guyer and James E. Bath for
their continued financial assistance during the remainder
of my program.

A special thanks goes to Drs. Howard A. Christensen
and Aristedes Herrer of the Leishmaniasis Department,
Gorgas Memorial Laboratory, Republic of Panama for their
guidance and technical assistance, use of laboratory facil-
ities and equipment, and logistical support during my stay
in Panama. I am especially grateful to Dr. Martin D. Young,
the director of the Laboratory, for his continued support
and encouragement. Special recognition goes to the follow-
ing technicians of the Leishmaniasis Department: Angélica

Ceballos, Anita Velasquez, Millie Vieto, Belinda de Merel

iii

Choy, Marta Chacén, Aurelio Powers, Roberto Rojas, Samuel
Scott, and Enrique London for their friendship and
assistance while in Panama.

A very special thank you is extended to Enrique Van
Horn who accompanied me on my jungle sandfly collecting
expeditions. His enthusiastic willingness to work with me
and to share his knowledge of sandflies and the jungle, was
greatly appreciated. My stay in Panama was enriched for
having known him.

Thanks goes to Dr. Byron Chaniotis and Major Louis
Rutledge (U.S. Army, Ret.) for their support and assistance
early in my sandfly research efforts.

Two other individuals deserve recognition for their
assistance during this project: Dr. Gary Hooper for his
guidance in scanning electron microscopic techniques and
Mrs. Natalie Knobloch for her instruction in drawing

techniques for my illustrations.

iv

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . .
INTRODUCTION . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . .

Taxonomic Status . . . . . . .

Morphology of the Immature Stages . . . .

Geographical Distribution . . .
Disease Relationships . . . . .
Bionomics--Adult . . . . . . .
Vertical Distribution . . .
Seasonal Distribution . . .
Diel Periodicity and Biting
Diurnal Resting Habitats .
Host Preference . . . . . .
Sugar-Feeding Behavior . .
Bionomics--Immature Stages . .
Rearing and Colonization . . .
Ultrastructure of Egg Stage . .

MATERIALS AND METHODS . . . . . . .

Collection Site . . . . . . . .
Collection Procedure . . . . .
Laboratory Handling . . . . . .
Rearing Procedure . . . . . . .
Adult Feeding . . . . . . . . .
Larval Experimental Techniques
Adult Experimental Techniques .
Ultrastructure of the Egg Stage

Behavior

Morphology of the Larvae and Pupa . . . .

RESULTS AND DISCUSSION . . . . . .

Species Composition . . . . . .
Feeding Preferences--Larvae . .

Page

vii

Page

Sugar Feeding Studies--Laboratory Reared

Females and Males . . . . . . . . . . . . . . . 54
Sugar Feeding Studies--Wild-Caugh Female . . . 61
Colonization . . . . . . . . . . . . . . . . . . 68
Egg Morphology . . . . . . . . . . . . . . . . . 78
Morphology of the Larvae and Pupa . . . . . . . . 87

Fourth Instar . . . . . . . . . . . . . . . . 87
Third Instar . . . . . . . . . . . . . . . . 97
Second Instar . . . . . . . . . . . . . . . . 97
First Instar . . . . . . . . . . . . . . . . 98
Pupa . . . . . . . . . . . . . . . . . . . . 102
Discussion . . . . . . . . . . . . . . . . . 105
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 108

APPENDIX

A. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON FRUCTOSE . . . . 112

 

B. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON SUCROSE . . . . 113

 

C. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON HONEY . . . . . 114

 

D. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON KARO SYRUP . . . 115

 

E. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON DEXTROSE . . . . 116

 

F. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES FEEDING ON MALTOSE . . . . 117

 

G. LONGEVITY OF LABORATORY REARED L. PANAMENSIS
FEMALES AND MALES WITHOUT SUGAR SOURCE . . . 118

 

H. BODY AND CHAETOTAXAL MEASUREMENTS OF THE
IMMATURE STAGES OF L. PANAMENSIS . . . . . . 119

 

LITERATURE CITED . . . . . . . . . . . . . . . . . . 122

vi

LIST OF TABLES

Page

Seasonal occurrence of phlebotomines--
Achiote, Canal Zone, 1973-74 . . . . . . . . . 44

Comparison of larval diets and their
effects on pupation and adult emergence
of L. panamensis . . . . . . . . . . . . . . . 50

 

The effects of experimental diets on
the development of L. panamensis . . . . . . . 52

 

Longevity of laboratory reared L.
panamensis females and males feeding
on various saturated sugar diets . . . . . . . 56

 

A summary of the effects of various

saturated sugar solutions and soaked

raisins on oviposition of wild-caught

blood-engorged L. panamensis females . . . . . 65

 

Comparison of various sugar diets on
the mean percentage of eggs oviposited
by L. panamensis . . . . . . . . . . . . . . . 67

 

Developmental times of L. panamensis in
the laboratory . . . . . . . . . . . . . . . . 70

 

vii

Figure
l.

2.

4,5.

12.

13.

LIST OF FIGURES

g. panamensis, pump and genital filaments .

 

Same, male genitalia, inner aspect; the two
long hairs on the coxite are apparently the
same as the deciduous hairs on the outer
surface, but one or two commonly persist

in this position in our series of

specimens . . . . . . . . . . . . . . . . .

Same, spermathecae, ducts and genital fork,
dorsal aspect; note asymmetrical terminal
knob bent to one side . . . . . . . . . . .

Same, female cibarium entire, showing
armature, chitinous arch, pigment patch,
salivary pump; detail of armature at higher
magnification. The central rows or patch
of very erect teeth are characteristic of
most of the group; two different females .

Same, head, female . . . . . . . . . . . .

Same, antennal segments II-IV and palps,
male and female; antennal segment IV with

ascoids enlarged . . . . . . . . . . . . .
Same, wing, male, costal portion . . . . .
Same, sternites I and II, female . . . . .
Sandfly collection from a horse . . . . . .

Collection vials; also used for longevity
studies with wild-caught blood-engorged
L. panamensis females . . . . . . . . . . .

 

Plastic feeding cage and laboratory host
spiny rat . . . . . . . . . . . . . . . . .

Holding and test vessel for laboratory
reared adults . . . . . . . . . . . . . . .

viii

Page

32

32

36

36

Figure

14.

15.

16.

17.

18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.

33.

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
The percentage of larvae on each food source
during the days when the proportion feeding
was approximately constant . . . . . . . . . 47
Longevity of L. panamensis females feeding
on various saturated sugar diets . . . . . . 58
Longevity of L. panamensis males feeding
on various saturated sugar diets . . . . . . 60
Longevity of wild-caught blood-engorged
females of L. panamensis feeding on
various saturated sugar diets . . . . . . . . 73
Egg L. panamensis 200 X . . . . . . . . . . . 80
Egqu. panamensis 1000 X . . . . . . . . . . 80
Egg L. panamensis 2000 X . . . . . . . . . . 80
Egg L. panamensis 200 X . . . . . . . . . . . 80
Egg L. pessoana 1000 X . . . . . . . . . . . 80
Egg L. pessoana 5000 X . . . . . . . . . . . 80
Egg L. sanguinaria 200 X . . . . . . . . . . 83
Egg L. sanguinaria 1000 X . . . . . . . . . . 83
Egg L. sanguinaria 5000 X . . . . . . . . . . 83
Egg L. trapidoi 200 X . . . . . . . . . . . . 83
Egg L. trapidoi 1000 X . . . . . . . . . . . 83
Egg L. ylephiletor 200 X . . . . . . . . . . 83
Egg L. ylephiletor 1000 X . . . . . . . . . . 86
Egg L. ylephiletor 5000 X . . . . . . . . . . 86
Egg L. gomezi 1000 X . . . . . . . . . . . . 86
Egg L. gomezi 1000 X . . . . . . . . . . . . 86

ix

Figure

34.

35.

36.

37.

38.

39.

40.

Head, thorax, and abdominal segments 1, 8,

and 9 of the fourth larval instar; dorsal
aspect . . . . . . . . . . . . . . . . .

Head, thorax, and abdominal segments 1, 8,
and 9 of the fourth larval instar; ventral

aspect O O I O O O O O O O O O O O O O O

Caudal setae, fourth instar. L. panamensis

 

1000 x O O I C O O 0 O O O O I O O O O O

Dorsal accessory seta, fourth instar, L.
panamensis and cuticular surface, 1000 X

 

Head, thorax and abdominal segments 1, 8,
and 9 of the first larval instar; dorsal
aspect . . . . . . . . . . . . . . . . .

Head, thorax and abdominal segments 1, 8,
and 9 of the first larval instar; ventral
aspect O O O O O O 0 O O O I O C O O O 0

Head, thorax, and abdominal segments 1-6
of the pupa; lateral aspect . . . . . . .

Page

89

91

96

96

100

100

103

INTRODUCTION

At present 68 phlebotomine sandfly species are known

to occur in Panama, of which Lutzomyia panamensis (Shannon),

 

L. gomezi Nitzulescu, L. ylephiletor Fairchild and Hertig,

 

L. sanguinaria Fairchild and Hertig, L. trapidoi Fairchild

and Hertig, L. pessoana Barretto and L. olmeca-bicolor

 

Fairchild and Hertig are the most abundant anthropophilic
species. Experimental studies on these species have been
limited primarily to attempts at laboratory rearing. Of

these species only L. gomezi and L. sanguinaria were main-

 

tained in colonies at the Gorgas Memorial Laboratory (GML),
Republic of Panama, when the present study was initiated
in March 1973. Studies by Chaniotis (1974) on L. trapidoi
dealt primarily with the sugar feeding behavior of this
species under experimental conditions and were a significant
contribution to the very limited knowledge of the behavior
and bionomics of sandflies.

The purpose of this study was to contribute to the

existing information of the man-biting species Lutzomyia

 

panamensis by laboratory investigations of its biology and

 

external morphology of its immature stages. This sandfly

is an important species in Panama and other areas of Central

and South America. Leishmania braziliensis g. lat., the

 

causative agent of cutaneous leishmaniasis, has been

isolated from naturally infected L. panamensis in Panama

 

(Christensen et al., 1969).

L. panamensis has been reared in the laboratory by

 

Mirsa (1952) and Pifano et a1. (1960) through one generation.
Efforts to colonize this species by Hertig and Johnson (1961)
were hampered by the sandflies' reluctance to feed on a
laboratory host (man and spiny rat), thus preventing
colonization beyond the second generation. The description

of the external morphology of L. panamensis immature stages

 

has been limited to the fourth instar and brief statements
about the first instar and pupa (Hanson, 1968). The egg

stage of L. panamensis and other man-biting species has not

 

been adequately described. The scanning electron microscope
(SEM) has been used successfully to differentiate the egg
surface ultrastructure of mosquitoes but has not previously
been used to compare the surface structure of sandfly eggs.
The specific objectives of this study were to
determine: (1) the biological parameters needed for

successfully rearing and colonizing L. panamensis; (2) the

 

effects of various diets on the longevity and egg production
of wild-caught blood-engorged females and laboratory reared
males and females of this species; and (3) the times during

the female life cycle when various sugar diets were imbibed;

and (4) the effects of different feeding regimens on larval
growth. In addition, the external morphology of the larval
and pupal stages were redescribed and clarified and the egg

surface ultrastructure of L. panamensis and other anthro-

 

pophilic sandflies including L. pessoana, L. trapidoi,

L. ylephiletor, L. sanguinaria, and L. gomezi were studied

 

by means of the SEM.

LI TERATURE REVIEW

Taxonomic Status

 

Phlebotomus panamensis was first described in 1926

 

by Shannon from specimens he collected in Cano Saddle, Canal
Zone, Panama. At that time there were no other published

records of Phlebotomus from Panama. He described both the

 

male and female terminalia in detail. Dyar (1929) could not
find the subdivision in the middle (upper) claspers of the
male terminalia as pictured in Shannon's figures and created

a new subgenus, Shannonomyia, thinking it was a peculiar

 

species that required a separate subgenus. This was later

changed to Shannonomyina by Pratt (1947) because the sub-

 

genus Shannonomyia had already been prOposed for a genus of

 

Tipulidae. Root (1934), after examining both Dyar's and
Shannon's specimens, disagreed with Dyar and had no doubt
that Shannon's figure and description of the middle (upper)
clasper were correct. Barretto (1946) examined the type

specimens of L. panamensis and described additional details

 

of the median (upper) claspers. Ortiz (1950) described the
setae on the parameres from specimens collected in
Venezuela and he compared his drawings with those drawn

by Stone in Barretto's paper.

The most complete and accurate set of figures and

description of L. panamensis were made by Fairchild and

 

Hertig (1951) and are as follows (Figures 1-9):

A medium sized sandfly with markedly infuscated
mesonotum and the abdomen clothed with flat white
recumbent scales. The male genitalia have not
previously been adequately figured, the original
sketch by Shannon being quite misleading. Stone's
sketch in Barretto (1946) shows the proportions,
but is admittedly diagrammatic and does not indicate
the broad blade-like character of the setae on the
parameres, ascoids simple, not quite reaching the
ends of their respective segments, paired on all
segments, but absent from the terminal three seg-
ments in the female, the terminal six in the male.
Terminal three segments in both sexes noticeably
shortened, pearshaped. Newstad's scales scattered
sparsely over the distal two-thirds of palpal segment
III. Eyes rather large in both sexes. Palpi as
figured, those of the male about one-third shorter
than the female. Stem of genital fork rather slender
and pointed in dorsal View. GonapOphyses of the
eighth sternite short and slender. Spermathecae as
figured, the common duct mostly very thin-walled and
difficult to see. Annulations of spermathecae vari-
able in number, 10 to 11 in our material, though
others show up to 13, the terminal annulation mark-
edly asymmetrical. Cerci rather slender. Cibarium
as figured. Pharynx fairly broad and well sclerotized,
its posterior end provided with numerous fine trans-
verse wrinkles, apparently beset with minute spinules.
Venation as figured, the veins and margin bearing
numerous fine long hairs but no scales, though the
hairs at the extreme base of the costa are somewhat
lorate.

While describing the Panamanian sandflies, Hertig and
Fairchild (1950) found a new character (the second sternite)

that was useful in the taxonomy of Phlebotomus (Figure 9).

 

Its size and proportions were very similar in both sexes.

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

l.

2.

4.

P. panamensis, pump and genital filaments.

 

Same, male genitalia, inner aspect; the two long
hairs on the coxite are apparently the same as
the deciduous hairs on the outer surface, but
one or two commonly persist in this position

in our series of specimens.

Same, spermathecae, ducts and genital fork,
dorsal aspect; note asymmetrical terminal
annulation, terminal knob bent to one side.

5. Same, female cibarium entire, showing armature,

chitinous arch, pigment patch, salivary pump;
detail of armature at higher magnification.
The central rows or patch of very heavy erect
teeth are characteristic of most of the group;
two different females.

Same, head, female.

Same, antennal segments II-IV and palps, male
and female; antennal segment IV with ascoids
enlarged.

Same, wing, male, costal portion.

Same, sternites I and II, female.

Fairchild (1955) attempted to subdivide the New

World Phlebotomus into subgenera as Theodor (1948) had

 

proposed for the Old World species but was hindered in
his classification because less than half of the described
species were known in both sexes. He placed L. panamensis

in the subgenus Psychodopygus Mangabeira (=Shannonomyia Dyar,

 

Shannonomyina Pratt), group Panamensis (=Shannbnomyina

 

 

Fairchild and Hertig, 1951). Barretto (1955) revised the
systematics of the subfamily Phlebotominae (Rondani) and

placed 2. panamensis in the genus Sergentomyia, an Old

 

 

World genus. His paper had many errors and contained no new
characters. Theodor (1965) proposed a new classification

of American Phlebotominae because the evolution of these
phlebotomines has apparently followed different lines from
those in the Old WOrld. Barretto (1962) elevated the sub-

genus Lutzomyia Franca to generic status which included

 

most of the New World sandflies. In this classification

Lutzomyia panamensis was retained in the subgenus Psycho-

 

dogygus where it remains today. Forattini (1971) elevated
PsychodOpygus to generic status, but as of this writing his

classification has not been generally accepted.
Morphology of the Immature Stages

Morphological studies of the immature stages of New

‘WOrld phlebotomine sandflies have been undertaken by only

a few investigators, largely because they are difficult to
find in nature and to rear in the laboratory. Barretto
(1940, 1941) described the immature stages of ten neo-
tropical species and established the taxonomic nomenclature
of the larvae and pupae. Other scientists have since
described the immature stages: Mangabeira (1942a-e),

Addis (1945), Sherlock (l957a,b), Mangabeira and Sherlock
(1962), Sherlock and Carneiro (1963), Carneiro and Sherlock
(1964), and Guitton and Sherlock (1969). Hanson's (1968)
dissertation described the immature stages of the subfamily
Phlebotominae of Panama, primarily the fourth instars and
pupae of 32 species including L. panamensis. Ward (1972)
drew and described the four larval and pupal stages of
Psychodopygus wellcomei Fraiha, Shaw, and Lainson and was
the first to include comparative measurements of the setae.
He did not mention Hanson's work and apparently was not

aware of its existence.

Geographical Distribution

Lutzomyia panamensis has a wide geographical
distribution: Mexico through Central America and South
America (Venezuela, Colombia, Peru, and Brazil). Barretto
(1947, 1950) in his critical analysis of the known sandfly
literature up to 1950 reported L. panamensis from Colombia,

Panama, Peru, Venezuela, and the Canal Zone. Pifano and

10

Ortiz (1952) and Iriarte (1952) listed the Venezuelan

states in which L. panamensis had been collected.

 

Vargas and Néjera (1953) reported L. panamensis
from three localities in Mexico. Fairchild and Hertig
(1959, 1960) stated that this sandfly occurred in Mexico,
Costa Rica, Nicaragua, British Honduras (Belize), and
Panama. Lewis and Garnham (1959) collected L. panamensis
in several localities in British Honduras. Najera's (1963)
studies extended the range of this sandfly in Mexico to
Veracruz. De Biagi (1966) and de Biagi et a1. (1966) found
this species in Quintana Roo, Mexico. Martins et a1. (1963)

collected L. panamensis for the first time in Brazil in an

 

area close to Venezuela. The geographic distribution of
this phlebotomine in Colombia was discussed in articles by

Osorno-Mesa et a1. (1967), Barretto (1969), and Morales et

a1. (1969).
Disease Relationships

Phlebotomine sandflies comprise an important group
of hematophagus Diptera because they can transmit certain
bacterial, viral, and protozoal disease agents. In the
INew‘World, sandflies have been incriminated as vectors
of bartonellosis, various viral diseases, and forms of
leishmaniasis. Pifano and Ortiz (1952), in their epide-

iniological investigations of tegumentary leishmaniasis in

.hs‘

11

Venezuela, suspected that L. panamensis was a vector of
Leishmania braziliensis Vianna. Five of the 72 specimens
they captured while feeding on the border of leishmania
lesions of man revealed leptomonad forms which were very

similar to cultured L. braziliensis. In 1959 Pifano et a1.

 

continued their investigations in northern Venezuela and
demonstrated the transmission of tegumentary leishmaniasis
to a human by the bite of L. panamensis.

Using sandflies found in nature and infected
experimentally in the laboratory, several groups have
attempted to demonstrate flagellate forms of L. braziliensis
in the digestive tract of L. panamensis. The time from
feeding to the detection of flagellate forms was 8-20 days.
Pifano et a1. (1960) dissected 75 specimens of L. panamensis
collected in the Yaracuy Valley, Venezuela, during an out-
break of cutaneous leishmaniasis and found three sandflies
infected with leishmanial and leptomonad forms morphologi-
cally indistinguishable from the cultured L. braziliensis.
Johnson et a1. (1962, 1963) in studies of natural infections
in Panama found leptomonad infections in 11 out of 579

specimens of L. panamensis dissected. These were confined

 

tetthe hind-gut, with the hind triangle usually the only
area infected. Biagi et a1. (1965) reported Lutzomyia

:flayiscutellata (Mangabeira) naturally infected with

 

leptomonad flagellates but found no infections in

(tissected specimens of L. panamensis.

12

Christensen et a1. (1969) found that four out of

306 dissected L. panamensis from several localities in

 

Panama had flagellate infections. One out of four infec-
tions was, by its morphology and behavior Lg YEEEQI
indistinguishable from those parasites responsible for
human cutaneous leishmaniasis in Panama. This strain was
considered to be Leishmania braziliensis s. Lag. and was
maintained in laboratory culture and in the golden hamster.
As mentioned previously, phlebotomines are of
increasing importance in the transmission of various viral
agents. Chaniotis et a1. (1974) reported an isolation of
virus of the Phlebotomus Fever group from L. panamensis.
Sabin et a1. (1944) and Velasco (1973) have reviewed the
existing literature on sandfly transmitted viruses with
special attention given to sandfly fever virus, which does

not occur in Panama.

Bionomics--Adult

Vertical Distribution
In British Honduras, Williams (1970a) studied the
vertical distribution of phlebotomine sandflies and found

that L. panamensis was the only common man-biting species

 

haying greatest biting activity at ground level (0-3 m).
Only a few specimens of this species were caught at 25 feet

and even fewer at 40 feet. The specimens at these levels

13

were collected only when feeding also occurred at ground
level. These findings confirmed those of Disney's (1968)

in which L. panamensis appeared to search for blood meals

 

near the ground, though an occasional specimen took its
blood meal above the ground. Resting flies were taken by
Disney during the day in greatest numbers near the ground.

In light trap collections, L. panamensis was the most

 

numerous species at 25 and 40 feet with very few flies
captured at ground level. Williams (1970b) postulated
that between blood meals females remained close to their
preferred resting places high above ground in tree foliage
and the physiologically hungry females descended to the
forest floor for a blood meal and then remained in close
contact with the breeding site for oviposition.

Studies were also conducted in Panama by Chaniotis,
Neely et a1. (1971) and Chaniotis, Correa et a1. (1971).
Their light trap collections showed that L. panamensis
was the second most numerous sandfly captured at the ground
level and in the canopy (28 meters). Daytime collections
from the tree trunks rarely yielded L. panamensis. A total
of 420 specimens were captured biting man of which 90.7%
‘were taken at ground level. Christensen et a1. (1972)

reported that 18 females of L. panamensis were collected

 

by'a light trap at ground level and 125 females at 35 feet.

14

Seasonal Distribution

 

The seasonal distribution of sandfly populations
was studied by Chaniotis, Neely et a1. (1971) in Panama
to discern some of the factors important in the complex
interplay between the biotic potential of various sandfly
species and the "environmental resistance," consisting of
a number of physical and biotic variables. Their data
suggested that the amount and distribution of rainfall
might affect the sandfly density by transforming the breed-
ing conditions on the ground. For their study they divided
the year into three climatological periods. L. panamensis
showed sharp density peaks during the early wet season (May
to August) with decreasing abundance during the late wet
season (September to December). Christensen et al. (1972)

indicated that L. panamensis was a wet season species having

 

the highest densities in June and September. Fairchild and
Hertig (1951), Hertig et a1. (1968), Biagi and de Biagi

(1953), and Disney (1968) stated that L. panamensis was

 

a rainy season species.

Diel Periodicity_and Biting Behavior
Chaniotis, Correa et a1. (1971) determined the
daily man-biting activity of anthropophilic sandflies.

L. panamensis was found to feed on humans throughout the

 

24 hour period with primary activity peaks occurring from

dusk to 2200 hours and at dawn; thus, its primary biting

15

activity was crepuscular. In Panama they also found L.

panamensis feeding on man during the daylight hours.

 

Williams (1966a) studied the biting rhythms of some
anthropophilic sandflies in British Honduras and found L.

panamensis to be the most numerous fly biting man. It was

 

collected during all hours of the day and night, with peak
biting activity at dusk from 1800 to 1859 and from 2100 to
2159. Day biting activity was greatest at sunrise and least
in the early afternoon when the temperatures were the high-
est and the relative humidity the lowest. To discern what
part of the human body was preferred by sandflies, Williams

(1966b) collected L. panamensis feeding on the ear, but

 

subsequent observations by Williams (1970b) revealed that
when collectors worked without shirts most L. panamensis
landed on the arms and torso. The comparative flying and
biting activity of Panamanian sandflies in a mature forest
and adjacent Open space was studied by Chaniotis and Correa

(1974). Fewer L. panamensis and other anthropophilic

 

species were taken in the open space than in the forest

by both light trap and human bait collections.

Diurnal Resting Habitats
In Panamanian forests the diurnal resting sites of
;ph1ebotomine sandflies were studied (Chaniotis et al.,

1972). The habitats studied included animal burrows,

16

tree hollows, leaf litter, green plants, tree trunks and
buttresses (ground level to 0.6 meters and 0.6 to 2.0 m),
and tree trunks (5.0 m, 9.0 m, and 15.0 m). Specimens of

L. panamensis were found on tree trunks (0.0 to 0.6 m) and

 

green plants and in leaf litter and tree hollows. Females
and males were most frequently collected in leaf litter.

Hertig et a1. (1960) flushed L. panamensis from green leaves

 

within a few feet from the ground. Hanson (1961) and John-
son and Hertig (1961) collected this phlebotomine from the
dead leaf microhabitat on the forest floor. Disney (1968)

collected L. panamensis from under leaves in British

 

Honduras. Williams (1965, 1970b) did not often find this
sandfly on tree buttresses but frequently collected it

beneath leaves on the forest floor.

Host Preference

 

A number of studies have been conducted to determine
the natural host of phlebotomine sandflies. Several traps
have been used utilizing various animals as bait. Disney
(1966) developed an animal-baited cage trap situated over
a pan of castor oil so that after a fly had fed, it usually
dropped off into the oil. He noted that only a few speci-

mens of L. panamensis were collected in the traps baited

 

with the rat Ototylomys phyllotis Merriam but that he and

 

his assistant were attacked by many more L. panamensis than

 

‘were caught in the rat-baited traps during the collection

17

period. Williams (1965) also used the Disney fly traps

with different bait animals. L. panamensis was rarely

 

 

taken and then only in the traps baited with Ototylomys.

Disney (1968) collected L. panamensis in traps

 

baited with the opossums Didelphis marsupialis L. (common

 

opossum) and Philander opossum L. (four-eyed opossum) and

 

the rats Heteromys desmarestianus Gray, Oryzomys cousei

 

 

 

(Alston), Tylomys nudicaudus (Peters), 9. phyllotis, and

 

 

Sigmodon hispidus Say and 0rd. He questioned whether or not

 

sandflies fed on all animals to which they were attracted.

He found only one freshly engorged L. panamensis in a trap

 

baited with Didelphus and none in rat baited traps. To

 

determine if sandflies showed any preference for a partic-
ular bait he erected traps in pairs, baiting one trap with

Ototylomys and the other trap with the alternative bait.

 

The numbers of L. panamensis trapped were too low to have

 

any significance.
Fairchild and Hertig (1951) and Hertig et al. (1959)

stated that L. panamensis fed on horses and pigs. Thatcher

 

and Hertig (1966) compared the baited trap technique with
the direct collection of sandflies from animals and pre-
ferred the latter method in their host distribution studies.

They collected L. panamensis from Philander and Potos flavus

 

 

 

(Schreber) (kinkajou) with the kinkajou the most attractive

for all sandfly species collected. They found that maximum

18

feeding took place on warm nights with no wind and that an
8-10 mph wind inhibited sandfly activity. Thatcher (1968)

studied the sandfly host preference of L. panamensis in the

 

same general area as my project. He found that L. panamen—
§L§_was the second most abundant species captured, with 95%
being taken in traps at ground level and only a few trapped
at the 10 to 13 meter level. This phlebotomine was more

attracted to Didelphis than to Potos and was the only sand-

 

fly attracted to all the bait animals used, including the
chicken. Christensen et al. (1972) indicated that L;
panamensis was the second most numerous sandfly trapped
by the animal-baited (spiny rat or rice rat) castor oil
trap method; however, it represented less than 1% of the
total catch.

Tesh et a1. (1971) tried to determine the natural
hosts of Panamanian sandflies by the precipitin test.

Seven freshly blood-engorged specimens of L. panamensis

 

collected from the field were tested with three class-
specific antisera. They all reacted with mammal antiserum.
Tested with six order-specific mammalian antisera these same
flies reacted positively to one rodent and one marsupial
antisera. In studies continued and expanded by Tesh et al.

(1972) only six L. panamensis were tested against nine

 

order-specific mammalian antisera. One reacted with rodent,
two with edentate, two with carnivore, and one with peri-

sodactyl antisera. Most flies used in the above two studies

19

were collected from tree buttresses early in the morning,

a resting place usually not sought by L. panamensis.

 

Sugar-FeedingyBehavior

 

The feeding habits, including the sugar—feeding
habits of biting flies belonging to Nematocera, were well
documented by Downes (1958). Both sexes feed on sugars
from various sources in nature such as plant juices, nectar,
and ripe fruits. In the laboratory they feed on raisins,
fresh fruit, honey, solutions of assorted commercial syrups,
and solutions of pure sugars, as well as the pure sugars
themselves. The literature contains very little information
concerning the sugar-feeding behavior of tropical phleboto-
mine sandflies. Lewis and Domoney (1966), Chaniotis (1974),
Barretto (1942), and Downes (1958) have reviewed the exist-
ing literature on this subject.

Lewis and Domoney (1966) examined the sugars in the
dissected crOps (esophageal diverticulum) of sandflies from
British Honduras by thin-layer chromatography and found
sucrose, fructose, and glucose. If there were other sugars
present they were indistinguishable with this method because
of their small quantities. They concluded that the liquid
normally present in the crop always contains some type of
sugar.

Chaniotis (1974) used the sandfly Lutzomyia trapidoi

to determine some of the Optimal laboratory sugar-feeding

20

conditions necessary to maintain this sandfly. Out of the
11 sugars screened sucrose, fructose, maltose, raffinose,
and glucose were preferred by the wild-caught sandfly
females, Chaniotis concluded that the rate of sugar
acceptance was unaffected by concentration, pH, NaCl
content, color of the solution, or temperature. In an
experiment using laboratory reared L. trapidoi, the sucrose
solution concentration had no appreciable effect on sandfly
longevity. The highest feeding rates were obtained using
the styrofoam cup holding vessel developed by Chaniotis.

He described the optimal sugar meal as a highly concentrated
solution of sucrose or fructose in distilled water, with or

without added coloring.
Bionomics--Immature Stages

Very little information is known about the natural
breeding habitat of immature phlebotomine sandflies. This
is largely due to the difficulty in finding and isolating
the immature stages from the soil. Very few randomly taken
soil samples contain the immature stages because of their
discontinuous breeding habitat. Early investigations of
the breeding sites of New World phlebotomines by Ferreira
et al. (1938), Coutinho and Barretto (1941), Pifano (1941),
Hertig (1942), Forattini (1954), and Deane and Deane (1957)

uncovered only about 60 specimens.

21

In an effort to further the knowledge of the
immature stages of New World sandflies, Hanson (1971)
conducted an investigation in Panama to discern the breeding
habitats of these flies, especially the anthropophilic
species. A total of 370 soil samples were processed by
the screening flotation method. This method, in combination
with direct examination of the soil and debris in the field,
yielded 2,258 larvae and pupae, of which 600 were reared to
the adult stage and identified. The breeding places inves-
tigated were soil between tree buttresses, from burrows,
under roots, at base of trees, and dead leaves in forest

litter. The immature stages of L. panamensis were found

 

only among the dead leaves of the forest floor habitat.

The larvae were on moist areas of either the upper or the
lower surfaces of decaying leaves when the leaves were
lying loosely. Hanson speculated that the feces of various
animals along with fragments of insects and other dead
arthropods formed part of the natural food supply. He
mentioned that various bacteria, fungi, and algae might
also contribute to the diet. Two other studies were
conducted in Panama to learn more about the immature
habitats, but they did not yield any more information

about L. panamensis (Thatcher, 1968b; and Rutledge and

 

Mosser, 1972).

22

Rutledge and Ellenwood (1975c) used emergence traps
to determine the species composition of sandflies on the
open forest floor in Panama. They reported that populations

of L. panamensis reached their maxima during the early part

 

of the wet season (May to August), when the soil moisture
conditions were relatively moderate, and reached their
minima during the late wet and dry season (September to
April) when the moisture conditions were at the extremes

in wetness and dryness. They found that L. panamensis bred
regularly in the soil or litter of the open forest floor.
Rutledge and Ellenwood (1975a) also determined the hydro-
logic and physiographic features of this species’ breeding
habitat. They indicated that the forest litter tended to

be removed from steep slopes and valleys and accumulated on
gentle sepes and prominences. The forest litter occurred in
stable, residual deposits on hilltops, while relatively
unstable, alluvial deposits were more common on stream banks

and hillsides. L. panamensis tended to be more abundant in

 

the hilltop regions. No effects of non-destructive
inundation or of physiographic aspects could be detected.
The forest vegetation and its local effects on the
sandfly breeding habitat on the open forest floor was
studied by Rutledge and Ellenwood (1975b). L. panamensis
was most abundant in association with large trees of the

<genus Ancardium (Sapindales, Ancardiaceae). They were also

 

23

:Eound in association with litter of Oenocarpus (Principes,

 

iPalmae) a tall columnar tree; Scheelia (Principes, Palmae)
tall columnar tree; L333 (Rosales, Leguminosae) medium-sized
branching tree; Croton (Geraniales, Euphorbiaceae) medium-
sized branching tree; Ourouparia (Rubiales, Rubiaceae) large
liana; and Sabicea (Rubiales, Rubiaceae) small liana. L.
panamensis was especially abundant in palm forest of
Oenocarpus and Scheelia. They concluded that sandfly-plant
interactions largely determine the sandfly composition in

the forest floor breeding habitat.
Rearing and Colonization

Phlebotomine sandflies have been reared and
colonized by several individuals, with varying degrees
of success, in connection with various investigations
concerning sandfly-borne diseases. Most of the early
rearing techniques were developed by individuals working
with Old WOrld sandflies: Waterston, 1922; Whitingham and
Rock, 1922; Smith, 1925; Shortt et al., 1926; McCombie Young
et al., 1926; Christophers et al., 1926; Ashner, 1927;
Roubaud and Colas Belcour, 1927; Adler et al., 1938; Young
and Hertig, 1941; Unsworth and Gordon, 1946; Eldridge et al.,
1963; Schmidt, 1964; Hafez and Zein e1 Dine, 1964; Safianova,
1963; and Mesghali and Lofti, 1968. Some of the more

important studies which dealt with the laboratory

24

(development of New World sandflies were done by Hertig,
1942; Addis, 1945; Sherlock and Sherlock, 1959; Chaniotis
and Anderson, 1964; Chaniotis, 1967; Christensen, 1972;
Sherlock and Sherlock, 1972; and Ward, 1972. The essential
basic requirements for sandfly rearing determined in their
studies were: (1) a moist surface for oviposition for the
gravid females, (2) a moist environment for development of
eggs and immature stages, (3) suitable food for the larvae,
and (4) containers or cages for the emerging adults and
their subsequent confinement, feeding, and experimental
manipulation (Hertig and Johnson, 1961).

The first attempt at rearing L. panamensis was made

 

by Mirsa (1952) using flat-bottomed test tubes (9 cm by 2.5
cm) with paper wadding in the bottom two centimeters. Two
circles of filter paper placed over the wadding provided
the rearing substrate. A mixture of dry pulverized rabbit
and sheep excrement was placed on the filter paper after
the appearance of the first instar. The food source was
sterilized to prevent the establishment of the fungus

Aspergillus niger Tieg. and mites. Water was added with

 

an eyedropper to maintain the humid conditions of the

rearing container. Development of L. panamensis from

 

egg to adult took 29 days at 26-28° C. No attempt was

made to colonize this sandfly.

25

Pifano et a1. (1960) utilized the technique
developed by Young and Hertig (1926) of earthen pots lined

with plaster of Paris for rearing L. panamensis. Dried

 

pulverized sterile rabbit feces mixed with dry pulverized
soil from the sandfly habitat was used as the food source.
Laboratory developmental temperature was 26-28° C with the
relative humidity above 65%. The period of development
from egg to adult was 42 days. After emergence, adults
were maintained at a temperature of 21-23° C at a very high
humidity and lived for two weeks on a diet of the juice in
boiled raisins.

The observations made by Johnson and Hertig (1961)
on the develOpment and behavior of Panamanian sandflies in
laboratory culture provided the background information for
the present study. Their rearing techniques were generally
the same as those used in this study although certain modi-

fications were necessary to successfully rear L. panamensis.

 

Collected gravid females were kept at 25.5° C in an air
conditioned room until oviposition. Larval development was
successful at 26.5° C but both hatching and development were
more satisfactory at higher temperatures. Daily tempera-
tures ranged from 26-29° C and occasionally rose to 30-31° C
in the rearing room. During the dry season (January-April)
the relative humidity ranged from 54-92% and in the rainy

season (May-December) it was usually above 90%.

26

Johnson and Hertig (1961) observed that larvae of

L. panamensis were attracted to the soft, pigmented material

 

of the inner surface of dipteran eye capsules, which were
part of the larval food mixture. The larvae were always
found on the surface of the food material perhaps because
they had long caudal bristles which caused difficulty in
movement if they crawled under and into the substrate
material. The eggs were dark, thick-shelled, and had a
sticky substance which purportedly cemented them to the
substrate. Certain larval reactions to some external
stimuli were noted. The sandfly larvae did not react to
changes in light intensities. When the caudal bristles
were touched, the larvae quickly flattened them down to
the substrate on the same level as the body and would then
remain motionless for several seconds. They were observed
to clean their caudal bristles of debris by bending backward
dorsally, grasping the base of the bristle between their
mandibles and pulling the bristle through. This process

was repeated with each bristle. L. panamensis larvae were

 

regarded as slow-moving and would remain in one place for
prolonged periods. They were observed to crawl as far up
the pot walls as possible before pupating, but not to the
point of leaving the moistened substrate.

For sandflies in general, it is known that a higher

proportion of females feed on a laboratory host six to seven

27

days after the first adults emerged in a given culture.
Johnson and Hertig (1961) speculated that a slight change
in the environment such as light intensity, temperature, or
humidity, might lead to a feeding response in physiologi-

cally hungry females. Although the spiny rat (Proechimyes

 

semispinosus (Tomes)), gUinea pig (Cavia porcellus (L.)),

 

and man were used as laboratory hosts, L. panamensis females

 

did not readily feed on any of these animals under any con-

ditions tried. The reluctance of L. panamensis to feed in

 

these studies prevented the species from being carried
beyond the second generation.

They designed an experiment to show the length of
time from engorgement to oviposition, and found that ten

laboratory fed L. panamensis females laid eggs four days

 

after feeding. In three of 127 pools of wild-caught blood-
engorged females, one or more of the females laid eggs one
and two days after feeding, while in the other 124 pools,
eggs were laid after three to five days. This indicated
that the females which laid eggs after one or two days had
had a previous blood meal because normal developmental time
was four days.

Johnson and Hertig (1961) observed quiesence in one

culture of L. panamensis that had produced adults and then

 

was overlooked and allowed to become very dry. Viable

fourth instars and pupae were still present two weeks after

28

adults first appeared and after the pot was moistened they
continued deve10pment and normal adults were produced.
Uneven rates of hatching were also noted in some cultures

of L. panamensis started in the dry season. The first

 

hatching took place within nine to ten days, but three

to four weeks later, with most of the larvae in the fourth
instar, newly hatched larvae were discovered. Batches of
eggs that did not hatch within 21 days were routinely
discarded.

They reported that the life cycle of L. panamensis

 

averaged 38 days from oviposition to adult emergence. Wild-
caught females laid an average of 28 eggs. The larvae of
all stages had very long caudal bristles. The first instars
were a dark grey in color and flattened dorsoventrally. The
first and second instars had dark brown heads, but the heads
of the third and fourth instars became lighter than the body.
The heads of the last two instars bore greatly enlarged
conical antennal bases which, they suggested, resembled

a profile of a cat's head. They noted almost all larvae,
while on leaves in the culture, constantly manipulated their
mandibles, presumably scraping the leaf surface. The pupae
were very slender and dark brown in color. Larval mortality
in the cultures was high and pots started with 200-300 eggs

might only yield 30-50 adults.

29

Ultrastructure of Egg Stage

 

Until now no attempt has been made to use the SEM
to demonstrate the fine ultrastructure of anthropophilic
phlebotomine sandfly eggs. Barretto (1941) used the light
microscope to obtain photographs of the eggs of 12 sandfly
species but they were difficult to interpret because of the
low magnification and light glare.

The SEM has been used by many authors to study the
fine surface structure of mosquito eggs. Hinton (1968a,b)

and Hinton and Service (1969) used the SEM to characterize

 

the function and structure of Anopheles, Culex, and Aedes
egg surfaces. Matsuo et a1. (1972) described the egg sur-
face structure of five species of Aedes and one species of

Armigeres from Japan and Malaysia. Matsuo et a1. (1974)

 

continued their study of the egg surface structure with

13 Aedes species from Taiwan.

MATERIALS AND METHODS

Collection Site

 

Field collections of L. panamensis were initiated

 

March 26, 1973, in an area near Achiote, Colon Province,
Panama, inside the Canal Zone boundary. This site was
selected for two reasons: (1) past collections conducted
by Gorgas Memorial Laboratory (GML) personnel showed that

L. panamensis was the most abundant species in man-biting

 

counts; and (2) the Atlantic drainage of the Canal Zone was
considerably wetter than the Pacific, resulting in minimal
fluctuations of the sandfly populations. The collection
site was adjacent to the large forested area which forms
part of the Fort Sherman jungle training preserve. The
town of Achiote and the Fort Sherman training area in the
Canal Zone were known to be endemic for human cutaneous

leishmaniasis (Thatcher, 1968).

Collection Procedure

 

Plaster of Paris lined 5-dram glass vials (55 x 27
1m“) . used to collect sandflies, were modified in this study
frtnn the original design of Hertig and Johnson (1961). The

inSide walls were coated with plaster to a distance of

30

31

approximately 10 mm from the shoulder, leaving an Opening
for observing the sandfly without removing the cap. Two
small notches were cut through the glass opposite each other
on the bottom edge of the vial to allow moisture from the
substrate to penetrate the plaster in the vial.

The method employed to collect wild-caught blood-

engorged females of L. panamensis was the standard procedure

 

adopted by the Leishmaniasis Department Of GML. On collec-
tion mornings, approximately 150 vials were moistened and
placed in lunch boxes described by Hertig and Johnson (1961).
Late in the afternoon shortly before dusk, a horse was
tethered at the collection site. Sandflies soon began

to feed on the horse and shortly before a fly had fed to
repletion, a moistened plaster lined vial was placed over
the fly (Figure 10). The sandfly would then fly up into

the vial permitting the rapid placement of the cap. The

vials were placed in the kits for return to the laboratory.

Laboratory Handling

 

The next morning each vial was examined. Dead
specimens were discarded and live specimens were placed in
individual vials. The vials were then placed in clay dishes,
approximately 36 per dish (Figure 11). Moisture was main-
tained in the vials by placing about one-half inch of water

in the dish every other day for three minutes or until the

Figure

Figure 11.

 

10.

32

3.4'

e.
“(at .
1%

Sandfly collection from a horse.

 

Collection vials; also used for longevity
studies with wild-caught blood-engorged

L. panamensis females.

33

plaster was visibly moist. The excess water was then
dumped out. The vials were examined every day at the

same time and the following information recorded: (1)
whether or not oviposition had occurred, and (2) the number
of females surviving.

Early in the study, when a female died, she was
removed from the vial and placed on a well-slide containing
92% phenol for clearing, identified to species, and the
number of eggs retained in the abdomen recorded. The
number of eggs deposited in the vial was also recorded
to give the total number of eggs developed by each female.
The vials were then numbered and placed according to the

day oviposition occurred.

Rearing Procedure

 

The standard breeding vessel used at GML, the
unglazed clay pot (Boston bean pot, outside dimensions
80 mm high by 85 mm in diameter at widest part, 70 mm at
lip, 64 mm at constriction below lip, flat bottom 65 mm
in diameter), described by Hertig and Johnson (1961), was
employed in this study as the principle rearing chamber.
Three other rearing chambers were tested for colonization
attempts but only one (plaster of Paris lined petri dish)
proved to be satisfactory and is discussed in the larval

experimental techniques section. The other two chambers

34

are described as follows. Chaniotis (1974) used an 8 ounce
styrofoam cup as a sugar feeding vessel for L. trapidoi and
also as a rearing chamber (Chaniotis, personal communica-
tion). Both techniques were adopted in the colonization

procedures of L. panamensis in this study. Approximately

 

80% of the inside cup surface was lined with plaster and
a small hole placed in the bottom of the cup permitting the
plaster to contact the moist sponge on which the cup was
placed. A plastic petri dish half served as a cover for
the rearing chamber. Pressed peat moss disposable potting
cups with the same dimensions as the styrofoam cups (8 cm
high and 7.5 cm in diameter at the top) were also used.
Plastic petri dish halves were used as covers. These cups
were placed on sponges to maintain the moisture content.
Eggs from the same oviposition day were transferred
to the rearing chambers in the following manner. They were
carefully brushed from the sides and bottom of the plaster
in each vial and washed into a beaker. Care was taken not
to injure the eggs during this process. The eggs were
washed three times with sterile water and transferred to
the rearing chamber with a pipet. Approximately 500 eggs
were placed in the center of the bean pot and 200 into the
styrofoam or peat moss cups. In initial rearing attempts,
a water-yeast solution was given to the first instars and

increasing amounts of a mixture of autoclaved ground dried

35

leaves and animal feces (standard larval food) were

provided beginning with the third instar.

Adult Feeding

 

The cloth releasing cage described by Hertig and
Johnson (1961) was utilized in this study to transfer
adults from one holding vessel to another and for some
feeding trials. A new feeding cage was developed along
the same design of the WOhlbach louse feeding cage (WOhlbach

et al., 1922) to feed up to 50 L. panamensis females in

 

close proximity to the host. The round plastic feeding
cage was 7 cm in diameter by 3 cm in depth with openings

of 4.5 cm in diameter in the cover and the bottom. The
opening in the bottom was covered by a piece of nylon cloth
(9 meshes per linear centimeter) and fastened tightly into
place by acetone placed on the plastic. Another piece of
nylon cloth not fastened to any plastic surface (22 meshes
per linear centimeter) having an outside dimension 4 cm
larger than the cover, was used as a covering under the
plastic top (Figure 12). To facilitate the easy entry of
adults into the feeding cage, a small plastic entry tube,
approximately 25 mm in length was placed half way through
the center of the cloth and glued into position. The inner
plastic surface of the bottom portion of the cage was coated

with plaster, which was moistened before use to provide the

36

 

Figure 12. Plastic feeding cage and laboratory
host spiny rat.

 

Lgfir ._7 *V _

 

Figure 13. Holding and test vessel for laboratory
reared adults.

37

humid conditions necessary during feeding. The cage was
assembled by placing the topcloth over the plastic bottom
and placing the cover over the cloth, securing the cloth
tightly in place. After feeding was completed, the feeding
chamber was placed in the releasing cage and the top removed
allowing the fed adults to be transferred to the holding
chamber.

The aspirator used to transfer the adult flies from
one container to another was made from a disposable pipet
with the small end filed off, leaving a hole just large
enough for the entry of the adults (Figure 13). A 20 inch
piece of rubber tubing (6 mm in diameter) was attached to
the pipet, with a piece of cloth placed between the glass

and tubing.

Larval Experimental Techniques

 

The food sources used were Bacto-Beef Blood Serum,
Bacto-Liver Powder, Bacto-Hemoglobin (Difco Laboratories,
Detroit, Michigan), Fleishmann's package yeast (Standard
Brands Inc., N.Y., N.Y.) and the standard larval food
(ground leaves and rabbit feces). These substances were
autoclaved prior to use.

A rearing chamber, similar to the one used by
Gemetchu (1971), was utilized to study the feeding

preferences of L. panamensis larvae. A small hole

 

38

(6 mm in diameter) was made in the bottom half of a 9 cm
plastic petri dish. Plaster of Paris was poured into the
bottom half to a depth of about 7 mm three hours prior to
the placement of the eggs. The plaster seeped through the
hole and became even with the underside of the plastic
bottom. Test dishes were made in the following manner.
Eggs selected were from wild-caught females who had ovi-
posited approximately 25 or 50 eggs on either the fifth or
sixth day after blood engorgement. The amount of food
initially placed on the dish accommodated about 25 larvae
so those females who deposited 50 eggs had their complement
divided in half. Eggs were placed in the center of the
plaster 24 hours before the anticipated emergence of the
first instars. The foods, mixed with sterile water, were
placed in the dishes with a microcapillary tube about six
hours before the anticipated first instar emergence. Small
mounds of food (24), between 4-5 mm in diameter, were placed
in rows (6), alternating each food source, equidistant from
each other. When only one food source was used per dish,
care was taken to place the same number of rows and mounds
on each dish. The dish cover was then positioned with a
rubber band fastened around it and the dish was placed on

a watch glass in a pan of water to protect it from ants

and mites. Daily, after recording the pertinent data, the

dishes were placed on a moist sponge for about three minutes

39

to maintain the proper humidity. The Chi-square test was

used to evaluate the feeding preferences of the larvae.

Adult Experimental Techniques

 

The carbohydrate sources used in these experiments
were: fructose and sucrose (General Biochemicals, Chagrin
Falls, Ohio); dextrose (Allied Chemicals, Morristown, N.J.);
maltose (Fisher Sci. Co., Fair Lawn, N.J.); honey, Karo
syrup and soaked raisins. Saturated sugar solutions were
made fresh each week from reagent grade sugars mixed with
distilled water, placed into screw-cap vials and refriger-
ated. All sugar sources were colored with green food
coloring to facilitate easy detection in the ventral
diverticulum of the sandfly.

A holding and test chamber was used to determine
the effects of various carbohydrate sources on the longevity

of laboratory reared adult L. panamensis (Figure 13). The

 

styrofoam plaster lined bottom used was described previously.
The tOp for the holding chamber was constructed from a nine
ounce plastic cup (9 cm in diameter at the top, 5 cm in
diameter at the bottom and 7.5 cm in depth). A hole 3.5 cm
was made in the bottom of the cup and a small hole (9 mm)

was placed about midway between the top and bottom. The
bottom hole was covered with a nylon cloth (22 meshes per

linear centimeter) and glued to the plastic by acetone.

40

A No. 0 cork was used to plug the hole in the side. The
plastic cup was inverted and placed on top of the styrofoam
cup and held together by two opposing strips of masking tape
to form the holding or test chamber.

Five drops of each sugar were spotted on the nylon
mesh of each test chamber. Five females and five males,
less than 24 hours old, were aspirated into each container
via the side hole. The chamber was then situated on a moist
sponge to maintain the humidity in the vessel. Standard
procedure was to keep up to 50 or 60 adults in these holding
containers when they were not being used for test purposes.
Life tables were used to determine observed life expect-
ancies of the males and females feeding on the various
sugar sources (Barretto, 1942).

The plaster lined vials were used as test chambers
for the wild-caught blood-engorged females to determine
longevity and egg production. Specimens from each field
collection were divided into groups and placed in the clay
dishes (Figure 11) with one drop of a sugar or a soaked
raisin spotted on the nylon mesh. The maintenance proce-
dures and data collected are outlined in the Laboratory
Handling section. The comparison of various sugar diets

on mean percentage of eggs oviposited by L. panamensis was

 

determined by an analysis of variance with replications of
unequal size and an arcsin transformation (Snedecor and

Cochran, 1973).

41

The ambient rearing temperature throughout the
colonization and experimental studies varied from 22° to

28° C with the relative humidity averaging 75%.

Ultrastructure of the Egg Stage

 

Eggs were obtained from wild-caught blood-engorged
females captured from a horse near Achiote, Canal Zone,
Panama. The collected females were placed in moistened
plaster of Paris lined glass vials, described previously,
and allowed to oviposit. Two days after oviposition the
eggs were fixed in 5% glutraldehyde and then passed through
an alcohol dehydration series and allowed to dry. The
specimens were then sent to the Center for Electron Optics,
Michigan State University, East Lansing, Michigan, for
further processing. The eggs were mounted on specimen
stubs, sputter coated with approximately 300 A of gold,
and examined in an AMR-900 SEM at varying beam currents,
voltages, and tilt angles.

The eggs are very fragile and if allowed to dry
before eclosion, will collapse. Although it was difficult
to keep the eggs from collapsing with the techniques
employed in this study, the surface structure remained

unchanged.

42

Morphology of the Larvae and Pupa

 

The immature stages described in this study were
obtained from the laboratory colony, mounted in Hoyer's
medium and ringed with nail polish. Measurements of the
body structures and setae of ten specimens of each instar
and pupa were made with the Wild ocular micrometer and
camera lucida tracings. Drawings of the dorsal and ventral
aspects of the first and fourth instars and the lateral
aspect of the pupa were made with the aid of the Wild

drawing tube.

RESULTS AND DISCUSSION

Species Composition

 

Twenty-seven collections were made beginning
March 26, 1973 and ending January 15, 1974. The species
distribution of anthropophilic phlebotomine sandflies on
a horse in the primary forest near Achiote, Canal Zone,

is shown in Table l. Lutzomyia panamensis was the most

 

numerous species with an average of 135.6 per collection,

followed by L. trapidoi, L. pessoana, L. gomezi, L.

ylephiletor, and L. sanguinaria in decreasing order

 

 

of abundance.

The numbers of sandflies in the collections
increased with the onset of the rainy season (early May)
and decreased late in the wet season (late September).
This decrease in the late wet season has been reported

by others (Chaniotis and Neely et al., 1971).

Feeding Preferences--Larvae

 

Four experimental foods (yeast, liver powder, beef
flood serum, and hemoglobin) were placed together, but
separately, on plaster of Paris lined petri dish bottoms

to determine which were preferred by L. panamensis larvae.

43

44

 

 

 

 

 

 

 

 

m0m.m m.0 HN 0.m 5HH 0.0 NN 0.H mm 0.0 mm 0.00 000.m HOUOB
13 m; «I 93 N: «A M. o W. 0 .ol 46s «.2 EITmH
50 0 0 m.vN vN H.v v 0 0 0 0 H.H5 00 v5IHIm
NmH 0 0 0.0 0H 0.N v m.m m 0 0 m.5m MMH m5IHHXImH
0m 0 0 0 0 0 0 0 0 0 0 0.00H 0m m5IHHxIHH
00H 0 0 0.0 H 0.0 H 0.0 H 0 0 H.m0 mmH m5IHHxIv
50H 0 0 0 0 0.0 H 0 0 0 0 v.00 00H m5IHxI5N
00 0 0 0.N H 0.v N 0 0 0 0 0.v0 5v m5IHxI0N
5MH 0 0 0 0 0 0 N.N m 5.0 H H.50 mmH m5IHxIm
00 v.v v 0 0 H.H H m.m m 0 0 H.H0 N0 M5IxINN
00H m.v m 0.H H 0 0 0.H H 0 0 m.m0 m0 m5IxHIVN
m0H 0 0 H.N w 0 0 0.0 H 0.0 H 0.00 00H m5lel0
OHN 0 0 H.5 mH 0 0 v.N m 0 0 0.00 00H m5IHHH>IMN
00H 0 0 0.H N 0 0 0.N v 0 0 0.50 NOH m5IHHH>I0
mmH 0 0 0 0 0 0 0 0 0 0 0.00H mmH m5IHH>I0N
HmH 0 0 0 0 0 0 0 0 0 0 0.00H HmH m5IHH>INH
mNN 0 0 0 0 0 0 0 0 0 0 0.00H mNN m5IHH>Im
m5H 0 0 0 0 0 0 0 0 0 0 0.00H M5H m5IH>IvH
50N 0 0 0 0 0 0 0.H N 0 0 0.00 00N m5IH>I5
00N 0 0 0.0 N 0 0 0.N 5 0 0 0.00 5mN M5I>I0N
mMN 0 0 «.0 H 0 0 5.H v 0 0 0.50 OMN M5I>ImH
00H 0 0 0 0 0 0 0 0 0.0 H 0.00 50H M5I>Im
v0 0 0 0 0 0 0 0 0 0.5 m N.N0 0m m5I>HImH
Hv 0.v N 0 0 0 0 0 0 0.v N N.00 5m m5I>HI0H
HMH H.m v N.NH 0H 0.H N 0.0 H 0.0 0 0.05 00 m5I>Htv
m0H 0.H N 0.H H 0.H H 0 0 0.H N N.¢0 50 m5I>HIN
5HH 0 0 0.0 5 v.m v 0.0 H H.HH MH 0.05 N0 m5IHHHI0N
0m 0 0 0 0 0 0 0 0 5.H H m.m0 mm M5IHHHI0N
HmUOB w .02 w .02 w .02 w .02 w .02 w .02 OHMQ
MODOHH£QOHN HopHmeuu mHHmcHsmcmm accommom .mmmmmm mHchachm
g
vSIMhmH .ocou ngmo .OOOHnomuimmsHEOOOQOHsm mo monounsooo Hmcommom .H manna

45

A total of ten plastes and 236 eggs were used in the
experiment.

The proportion of first, second, third, and fourth
instar larvae feeding on each food was observed on each day
following eclosion. The assumption was made that the number
of larvae feeding on each of the four food sources was equal
and that they had no preference for a particular food source
during the feeding period of each instar. This hypothesis
was tested by the following method. Since the feeding of a
larva on one day may not be independent of its feeding on
other days, the mean number (N) of larvae and the mean
proportion (P1, P2, P3, P4) of larvae feeding on each food
was computed for the days that each proportion was approx-
imately constant. The number of larvae feeding on each food
per day was then computed by multiplying N'by each Pi
(i = 1,2,3,4). Using the values of Pi specified by the
null hypothesis (i.e., P1=P2=P =1’3

3 4
number of larvae feeding on each food was computed. The

==0.25), the expected

Chi-square test was used to evaluate the hypothesis for

the first, second, and third instars. The tests showed
that yeast was significant at the P<<.01 level for the
first, second, and third instars. This clear preference
for yeast is demonstrated further in Figure 14 which shows
the percentage of larvae feeding on each food source during
the days when the proportion feeding was approximately

constant.

46

Figure 14. The percentage of larvae on each food source
during the days when the proportion feeding
was approximately constant.

47

«H musmHm

m><0

0NVNMNNN_NONQQ_5_0.Bin—N. : 0.00 5 0

 

d

d) d d d d it

11 1

 

Eaton oooB Loom Ii.
590.095: ......
B033 35 '1
$2.» I

TIL—SSE alliance. 51.963501 1.528;...

 

0.

0.

ON
mm
00
0m
0?
0?
00
00
00
00
05
05
00
00
00
00

bugpaag ammo-1 moored

48

There was no period when the proportion of fourth
instar larvae feeding on each food source was nearly
constant. Those feeding from day 15 through 18 on yeast
decreased linearly at a rate of about 9.2 larvae per day;
the number feeding on liver powder and beef blood serum
generally increased in a non-linear trend. The number
feeding on hemoglobin decreased from day 13 through 16
but then steadily increased through day 20. These results
differed from those of a similar study conducted by Gemetchu

(1971) in which the Phlebotomus longipes first through third

 

stage larvae preferred yeast and liver powder equally and
were less attracted to beef blood and rabbit feces. However,
no clear preference could be detected for a particular food
source by the fourth instar in his study which substantiates
the results obtained for the fourth instar in this study.
Based on the findings of these two studies, the larvae of
sandfly species appear to have different food preferences
early in their development but have no clear preference
during the fourth instar. Therefore, before colonization

is attempted, studies should be conducted to find the best
possible larval medium for each developmental stage.

Rearings of L. panamensis using only one food source

 

were conducted to determine the effects of specific foods on
the duration of the life cycle, the pupae and adults result-

ing from first instar larvae, and the percentage of adults

49

emerging from pupae. In addition to these individual
comparisons, studies were made to determine at what stage
in their life cycle larvae actually started to feed on the
standard larval food rather than on yeast. Approximately
25 eggs were used in each of eight petri dishes for each
individual food source experiment except for beef blood
serum in which only six dishes were used.

The development from the first instar L. panamensis

 

to pupa varied with the diets provided: 19.6 days (yeast
and standard larval food); 21.6 days (yeast); 22.4 days
(liver powder); 40.7 days (beef blood serum); and 50.5 days
(hemoglobin), compared with 18.7 days for the combination of
the four foods.

For rearing sandflies in the laboratory, it is
important that a larval diet be developed that will assure
maximum pupation and adult emergence. Table 2 summarizes
the larval diets tested in this study and their effects on

pupation and adult emergence of L. panamensis. The combi-

 

nation of the four foods was most efficient in producing the
maximum number of pupae (91.5%) and adults from pupae
(94.6%), whereas liver powder, yeast, hemoglobin, and beef
blood serum showed decreasing efficiency, reSpectively.

The larvae reared on the yeast and standard larval food plus
yeast alone seemed to develop normally through the first

three instars, but during the late developmental period of

50

.Oe>umH HmumcH umuHm 00 “mass: Hmuoeo

.muHspm mo Hogan: Hmuoa

.mmmsm mo Hogan: Hmuoam

 

 

 

 

Q
AOmHIOV
AmHHV o.mn A00 m.5 ANHV v.0H Eamon OOOHQ mmmm
AemHnmv
loose m.we real 0.0H Ammo 6.0m canoamosmm
AmHmimv
Ammav 0.vm Aomv m.5m A500 0.0m ammo»
Amomimv
A5wHV m.mm AHHHV 0.0m AONHV N.¢0 poow Hm>HmH
pumocmum one ummmw
Ammaumv
ANOHV v.0m AOHHV 0.50 AmNHV 0.m5 Hopzom uo>Hq
AOMNIOHV
cHnonoson
.Esuom OOOHQ moon
.uop3om HO>HH .ummom
oAvmmv 0.vm nAvav 0.0m mAmomv m.Hm "OOHumcHnEoo
ommsm Eonw OO>HOH umpmcH OO>HOH HoumcH Ammmo .OCtmoumHm .ocv
mchanO muHDOm umHHm 02H>H>Hsm Eoum umuHm msH>H>H5m Eouw umHo
mo ommucoouom mUHspm mo wmmucoouom mmmsm mo ommusoouom
mHmcoEHcmm

am no oocomuoao

uHspm can coHummsm so muowmwm HHonu can ODOHO HO>HMH mo sowHHmmEou .N OHQMB

51

the fourth instar some developed a darkening of the eighth
and ninth abdominal segments and died. One can only spec-
ulate at this time as to the cause. It may have been due
to the lack of some nutrient in the yeast or standard larval
food or a bacterial or viral infection. This phenomenon was
also noticed when these same two foods were used in my early
colonization attempts but was not seen in the larvae feeding
on the other test food sources.

The larvae reared on the yeast and standard larval
food plates showed a clear preference for yeast through
the third instar. No larvae began feeding on the standard
larval food until the second or third day of the fourth
instar, and then only to a limited extent. Although it
seemed that the larvae derived some nutritive value from
the standard larval food, they did not do so until the
fourth instar when the mouth parts might have been better
developed to handle the larger pieces of leaf and feces.

The effectiveness of the combination food source
as a larval medium is demonstrated further in Table 3,
which shows the developmental time range of each stage of
L. panamensis utilizing the different experimental diets.
This table shows that the combination and yeast and standard
larval food have approximately the same developmental ranges,
with yeast, liver powder, beef blood serum, and hemoglobin

increasing in developmental time, respectively.

52

.muHspm HHO mo monomumem OUOHQEOO ou pmustou oEHBo

.OOHMOQ HousmSQOHo>op some sH mwmp mo noses: Hmuoa

n

.mmmum 30mm How mwmp OH OOGOH 08H» HmusmEQOHO>Oom

 

 

 

Isms asuma lame «summ Ammo mmumm Aves Hausa Isms mmuaa Ammo smug anneamosmm
AMNV oo-sm Ammo mm-om lave NG-HN Ammo ev-ma lags mm-m lame HN-H wooflmswwwm
Ammo omukm lone mmuom Ammo ov-qa Ammo «mum lmac mauo less «H-H season Hm>fla
ANAL mmuom lags omtma Ammc emuma real «mum lmav saum Amav NHIH ummms
doom HO>HOH
Asst omumm ANAL om-mH Iago Hm-mH lose maum has «film lass Hana cumecmum
one ammo»
okay emumm loge mmnma less mmuaa AHHS maum Ame maum gloacmoaufl conumcflnsoo
032 mesa >H HHH HH H .63
HmumcH

 

mHmcoamcmm am mo ucosmoHo>op can so ODOHO HmucoeHuomxm mo muoowmo one

.m OHQOB

53

Some general observations were made on the behavior
of these larvae during their development. The first instars,
upon eclosion, seemed to move randomly over the surface of
the plaster for about six hours before they began to feed.
Usually when the first instar has located a preferred food
source it either climbed on the food source or fed around
the edges, frequently remaining there during its entire
first instar feeding period. Shortly before ecdysis, it
stopped feeding and usually moved away from the food and
did not start to feed again until the moulting process
had been completed. The second, third, and fourth instars
tended to wander from food mound to food mound during their
developmental periods. The only time that the larvae left
the plaster substrate and crawled on the plastic was shortly
before pupation. This occurred either on the plaster, on
the plastic side of the dish bottom, or on the plastic
surface of the top. The pupae apparently did not require
a semi—moist substrate since the moist environment of the
petri dish appeared to be adequate.

Covered petri dishes lined with plaster of Paris
were very satisfactory as rearing vessels for Observing the
feeding behavior and feeding preference of L. panamensis.
The advantages of using this technique for rearing and
colonization are: (1) the larvae can be observed at any

time without removing the cover; (2) the desired amount of

54

food can be placed on the plates prior to eclosion; and
(3) the plates are free of mites and fungi that would
compete for food and ensnare the larvae, as long as the
dishes remain on glass plates in a pan of water. This
technique would be ideal for rearing larvae hatched from
eggs produced from blood-engorged wild-caught females,
where only the female was known and the larvae and the
male needed to be reared and described.

Sugar Feeding Studies—-Laboratory
Reared Females and Males

 

 

The styrofoam holding and testing vessel developed
by Chaniotis (1974) proved to be very satisfactory in
studying the effects of various saturated sugar solutions
(fructose, sucrose, dextrose, maltose) and commercial honey
and Karo syrup on the longevity of laboratory reared L.

panamensis. The complete life expectancies of the females

 

and males for each sugar source are shown in the life tables
in Appendices A-G. The average life expectancies for adults
feeding on each sugar are summarized in Table 4. Of the
sugars tested, females feeding on fructose and sucrose
resulted in the longest average female life expectancies
(13.9 and 13.5 days, respectively). There were no apparent
differences between females which fed on honey (10.6 days),
Karo syrup (10.7 days), and dextrose (9.8 days). Maltose

(4.6 days) was the least effective diet for the females and

55

also the males (3.0 days). No difference was observed
between fructose (10.0 days), sucrose (9.1 days), honey
(8.8 days), Karo syrup (8.4 days), and dextrose (7.9 days)
in the average observed life expectancies of the males.
The females and males that received no sugar had average
life expectancies of 1.8 and 1.3 days, respectively.

The percentage of females surviving on each day
is shown in Figure 15, with the number of days to 100%
mortality listed in Table 4. The survival time ranged
from 22 days (maltose) to 29 days (dextrose). The per-
centage of males surviving on each day is shown in Figure 16,
with the maximum number of days to 100% mortality shown in
Table 4. Survival time ranged from 19 days (maltose and
Karo syrup) to 23 days (dextrose). Males and females with-
out sugar lived a maximum of three days. Barretto (1942)
stated that females reared in his laboratory lived up to
seven days without a sugar source.

Female L. panamensis lived longer on each sugar

 

diet than did the males. The females imbibed more of the
sugar solutions than the males. Both females and males
began to feed on the sugar solutions immediately after the
drOps were placed on the nylon mesh, averaging two minutes
per feeding. The green color of the sugar solutions was
observed through the body wall of the sandflies and it

persisted until shortly before death. The flies extracted

56

Table 4. Longevity of laboratory reared L. panamensis females and
males feeding on various saturated sugar diets

 

 

 

 

 

Maximum no. of days Average length of life
to 100% mortality at emergence (days)

Sugar diets Fa M F M

Fructose 26 (70)b 22 (70) 13.9 10.0
Sucrose 28 (70) 21 (69) 13.5 9.1
Honey 24 (61) 21 (72) 10.6 8.8
Karo syrup 27 (67) 19 (70) 10.7 8.4
Dextrose 29 (70) 23 (70) 9.8 7.9
Maltose 22 (64) 19 (69) 4.6 3.0
No. sugar 3 (65) 3 (66) 1.8 1.3

 

aF = female; M = male.

bTotal number of sandflies.

additional moisture from the plaster of Paris in the test
vessels. The reagent grade saturated sugar solutions did
not visibly change in consistency during the experiment.
However, the honey and the Karo syrup did appear to become
less viscous as the experiments progressed and became par-
tially fouled with mold after the eighth day. This did not
seem to interfere with feeding unless the feeding surface

was completely covered with the mold.

57

Figure 15. Longevity of L. panamensis females feeding on
various saturated sugar diets.

 

58

mH musmHm
mm mm H mm mm 4 m><o
.4241... u eiwmm mm a ow m._ m. m. w. m. s m. N_ __ o. m m e o m
I. .0.0. IOIIonI000000000000. a a q a 1 a . . q . W M
fl .lol.l.’.0 K x I K I 00000
”cl. 0” x 00.00000
// ’ol

 

1 [1—
I 0 0,0,0, / 0 O

 

1 0_
. m_
00 ‘
.0. x co 1 ON
/ / I.’ / o... ‘
/ // 0.0 X 00.. 1 mm
/. / .... . om
/ / I! oo
a. a; .. mm
/ / . .. .. i
// // 0.0. X at A a 0? w
i. o. J
/ / .... g .. . 9. m
. I .
/./ / .I// o. 1 on I?
. .0 x . S
/ z . . n
. 0.0 .— 1 B J
/. / .I .. M.
. o A
.Irfll. / w .oomw
//./. . .... .mo
0’ .
603m 0: / /. it . i
/ / . . O5
800 6:80 if. / . / I. ..
A500 0.3. .20.: //. 0.; .. .0 5
3400 08:02 .3... J 1.1 .. .Om
MO5V 000.580 le .I/ .ll.’ ..
050 39.030 II / . .. .
“050 000.03“. I3! ./'.I/.// /, . 0.; .. 00
.MHUI/hV m .00
,. .0, .

 

00

 

_ 00.

59

Figure 16. Longevity of L. panamensis males feeding on
various saturated suagr diets.

 

60

mm ON

0H wusmHm

w><o
m. t m. m. S m. N. __

H

mm mm _N ON 0.
if: I“

' o'c’o..-o"o-a.o.o.o -o'o.o-o.o-o-l'

. 00 I
/ '0' o
o
a

503m 0:
.8200
9.3.
at :5:
800 01.02.2).
at 39:25
300 08.85
at 0383...... II.|

A000
.050

 

 

 

 

L111413161r16L$6656
888839680848m8~N—— O
bugnwns tuaorad

 

. 00.

61

Sugar-Feeding Studies--Wi1d-Caught
Females

 

The effects of various saturated sugar solutions
and the water-soaked raisin fluid on longevity and egg
production of wild-caught blood—engorged females of L.

panamensis are presented in this section. The females were

 

maintained in five dram plaster of Paris lined glass vials
for the entire test period except when they were transferred
to another vial to permit the eggs to be counted and trans-
ferred to rearing chambers. Two main sugar—feeding periods
were observed with the aid of green food coloring in the
sugar diets. The first was the day after the sugar was
added to the nylon mesh of the vial cap and the other was
the day after oviposition. Many flies started to feed
immediately after the sugars were made available, even
though they had fed to repletion on blood less than 24
hours previously. Once a female had fed on a sugar the
green coloring could be Observed in the abdomen usually
until she died.

This method of maintaining wild-caught blood-
engorged females for experimental purposes proved to be
very satisfactory. The adults could be observed in the
vials at all times and the number of eggs deposited were
counted without difficulty. The sugar sources, except for

the raisin, could be placed on the nylon mesh of the cap

62

and did not have to be replaced for the duration of the
test. As mentioned previously, the reagent grade sugar
sources fructose, sucrose, dextrose, and maltose did not
lose their consistency or develop any visible mold or
fungal deposits on the drop of sugar during the test.

Oviposition began on the third day after blood-
engorgement, but the majority of females oviposited on
days four, five, and six and an occasional female did not
oviposit until day ten. Some split their complement of
eggs and oviposited on two different days, waiting as long
as nine days after the first oviposition period of 18 days
from blood—engorgement.

Figure 17 shows the longevity of wild-caught

blood-engorgement females of L. panamensis feeding in the

 

laboratory on various saturated sugar sources and soaked
raisins. The females survived for 39 days (dextrose), 37
days (fructose), 29 days (sucrose), 26 days (maltose), 12
days (raisin), and 6 days (control). No sugar source was
given to the control group. Female mortality increased
rapidly with the onset of oviposition, especially in the
control group and those feeding on raisins and maltose.
Table 5 summarizes the effects of the sugar solu-
tions on the oviposition of the wild-caught females. The
percentages of females surviving long enough to oviposit

were 92.1% (fructose), 92.9% (sucrose), 88.5% (dextrose),

63

Figure 17. Longevity of wild-caught blood-engorged
females of L. panamensis feeding on
various saturated sugar diets.

 

64

5H onumHm

  

 

 

 

m><o
comm mm em on on em mm N». .n Onaw om hm 8mm 3 mm «N _N ON 0. o. t 0. n. v. m_ N. or
!'.1.I.I I II II III 4 . a 4 . a . C
.l. .l" 100:!
a.o.o.n.a.:.oul/..U, Inc: A
3!. 1...! m
a; I. /
Iii I.//. so.
i; .m.
. Om
.mm
.On
.nm
. 0v
. no.
.on
_ Ann
85395 _

_ .00

3.200 I .I.
:56: N no

32...: a}...
39:60 nu .- ox.

8903 III
322:... 1.1 mi.
00
mm
om
mm

 

 

bugAuuns moored

65

.mouMOHHmwu uano muOHO ummsm umnuo

.mOHSOm nmmsm on OO>HOOOH msoum Houucou

Q

“mmUMOHHmou Hsom mo Ommuo>m on» so OOmOmm

 

 

m.~v m.m H.m~ H.NH H.ma 0.ma mamsmm\emcflmumu momm w
v.sm R.Hm m.os m.sm m.0m «.0m mamsmm\emuflmomfl>o mama H
«.mm m.ov «.mm m.av «.ma m.sm mamsmm\mmsm “when: Hmuoe
o.mH e.m m.w o.m m.m m.v mamsmm\amcflmumu momm .oz
~.om m.om m.mm m.mm m.om «.mm mamsmm\emuflmodh>o mama .oz
N.HmoH H.Hmm m.ooma m.smma «.msma m.vv~H meow manage Hobos
H.mmw m.ms m.msm m.mva o.msa o.mOH emcwmumu meow umnssz
H.mmm m.mom o.smm m.omoa m.moma o.msoa emuflmomfl>o mama umnsaz
o.vs 6.4» H.mm m.mm m.~m H.mm mcauflmomfl>o mmamsmm mo 0
H.0N 0.mN 0.Hm 0.0N 0.Nm v.mm 0CHuHmOmH>O mmHmEOm .Oc .O><
m.mm m.mm m.mm m.mm H.mm «.0m mmflmsmm .oc mmmum><
«Hm «ma smm can Hmm omm mmamsmm mo .0: Hmuoe
QHOHHGOU MGHWHMM meUHMZ meHuXOQ wwOHODm OmOUODHm

 

muOHO Hmmdm

 

 

mOHmswm mHmsOEmcmm um OmmuochtOooHn yamsMOIOHH3 an soHuHmomH>o
so mchHmu OOxOOm paw mGOHusHom woman Omumnsumm mSOHum> mo muommwo on» mo mumsasm a .m OHQMB

66

88.1% (maltose), 74.6% (raisin), and 74.0% (control). More
than 25% of the females in the control and raising groups
died either shortly before oviposition or at the beginning
of the oviposition period. None of the females in the
control group lived beyond the sixth day while some females
receiving sugar lived beyond their oviposition period.

An analysis of variance with replications of unequal
size and an arcsin transformation of the mean percentages of
eggs oviposited per female was conducted testing results
obtained with the various sugar sources. The mean per-
centages of eggs oviposited per female presented in Table 6
were separated with the LSD test at the 5% level of error.
The analysis of variance test revealed a significant dif-
ference in the effects of the various sugar solutions on
the percentage of eggs deposited by each female. The
soaked raisin, fructose, sucrose, and dextrose solutions
were superior to maltose, which in turn outperformed the
control group in promoting a high percentage of oviposition.
Although the soaked raisin appeared to promote the highest
percentage of eggs to be deposited, it did not increase
female longevity past the oviposition period to any
appreciable degree. It is speculated that the females
imbibed only small quantities of the sugar from the raisin
prior to oviposition and did not have the energy upon com—

pletion of egg deposition to refeed. The raisin, unless

67

Table 6. Comparison of various sugar diets on the mean percentage of
eggs oviposited by L. panamensis

 

 

 

Variants: Raisin Fructose Sucrose Dextrose Maltose Controla
Mean:b 91.7 86.4 86.9 87.9 76.9 57.4
Relationship: a a a a

b

 

a . .
Control group received no sugar diet.

bMeans with same letter are not significantly different at P = 0.05.

changed daily, promoted mold growth on the surface of the
nylon mesh which might have inhibited the feeding of the
adults and decreased longevity.

The average number of eggs oviposited was 32.4 for
females fed on fructose, 36.9 on sucrose, 36.3 on dextrose,
29.3 on maltose, and 20.2 control. The range for all
groups was 1 to 110. Johnson and Hertig (1961) stated

that L. panamensis females laid an average of 28 eggs

 

with a range of l to 90. Each group of females that was
provided a sugar diet in this study produced, on the aver-
age, a higher number of eggs than that observed by Johnson

and Hertig.

68

Colonization

 

The colonization of L. panamensis was an extremely

 

difficult project. The unglazed Boston bean pot proved to

be the only vessel in which L. panamensis could be reared

 

in large numbers. The plaster of Paris lined styrofoam
cup and the compressed peat moss cup were unsatisfactory.
Moisture could not be regulated in the styrofoam cups so
the plaster substrate became too dry or too moist. Also,
these cups could not be autoclaved prior to use, conse-
quently fungus enveloped the first instars so they became
entangled and died. The peat moss cups also became over-
grown with fungus and bacteria as soon as they were
moistened and did not provide a smooth substrate for
the first instars.

In this study early attempts at rearing L.

panamensis in the bean pots met with little success. Though

 

the bean pots were standard in size and shape, each one
apparently had certain unknown features that affected the
rearing of a particular culture. As was noted earlier in
the Larval Experimental section, the first instars would
wander randomly for about six hours before they began to
feed. Thus, if the yeast, which was usually added the day
before the eggs hatched, was not placed at convenient loca-
tions on the pot bottom and sides the larvae wandered away

from the food source and died. Nevertheless, some larvae

69

died because of this wandering habit no matter how much
care was taken in the placement of the yeast.

Moisture and a smooth substrate were also problems.
The first pots used were too moist and the first instars
drowned or became stuck in the very moist yeast. To cor-
rect this, the pots were placed on moistened cotton in glass
petri dish bottoms. Certain modifications had to be made
for each pot because the porosity of the clay varied from
pot to pot. The first instars required a relatively smooth
plaster substrate. After several cultures had been reared
in one pot, the substrate became rough and pitted, which
proved to be detrimental to the first instars, so it had
to be removed and replaced.

The life history of laboratory reared generations

of L. panamensis are presented in Table 7. The four lab—

 

oratory generations were completed during this study with
very little variation in the total number of days for each
generation (blood meal to adult). The average length of
time for the four generations to develop from egg deposition
to adult (37.7 days) is almost identical to the results
obtained by Johnson and Hertig (1961) of 38 days (average)
for two generations. However, the average developmental
time, oviposition to adult, differed from the 29 days
reported by Mirsa (1952) and the 42 days of Pifano et al.
(1960). They reared only single generations of this spe-

cies, using different laboratory conditions and techniques.

70

.mmmum 3000 How mxmv 00 000mm

0

.mmmum 30mm How mxnw mo HOQEOG mmmum>4o

.mmHSUHso mo umnadz

n

.GOHumuOcmm some OH mmmw mo amass: Hmuoem

 

 

Ase Anuov Avv Amumv Amuov AOHumV Avnmv AN.ov0
o.n N m.0 N o.v m m.N 0.0 m m.m 0 m.m 0 Que
Amn50 A5nm0 Honmv Avumv Hmumv HOHImV Amuvv H0.Hv0
0.5 m 0.0 0H m.v NH H.m NH v.0 NH N.m mH H.v mH Ohm

AHHumv ANHan Amumv ARINV Amumv AOHnmv Amnmv Am.Nv0
0.0 Hv m.5 mv v.v 5m v.m v0 0.0 50 N.m v5 0.v vs ch
ANHIMV AnHuvv AOHINV H5IH0 H5|v0 AMHimV OHOHImV mam.Nv0
m.5 va N.n va m.m mmH m.m 0mH m.m 0mH m.m mmH ov.v nmmH umH
mmsm >H HHH HH H 00m 000 on mcoHumumcom
, Hews OOOHm Nuoumuonmq

 

mum.“ mGfl HMEMH

 

muoumuoan man

 

CH mHchEncwm am no 05H» HmucosQOHo>oo .5 OHOOB

71

The developmental times of the egg and the fourth
instar were the most variable in the life cycle; the
developmental times of the egg stage have already been

discussed. Quiescence in the fourth stage of L. panamensis,

 

observed by Johnson and Hertig (1961), was not seen in this
study. However, the extreme variability, especially in the
developmental time of the first generation fourth instar
reared in the laboratory, may have been due to adverse
conditions in some pots. This variability was demonstrated
very clearly in the larval feeding experiments in which
different food sources were used. They showed extremes

in deve10pmental times in all immature stages.

The behavior of the larvae in the pots was
essentially the same as that observed in the petri dishes.
All stages were very sensitive to the touch of other larvae.
If contact was made between two larvae, very violent flexing
actions of the body occurred in an effort to escape. How-
ever, larvae that fed in very close proximity to each other
without touching did not show any type of avoidance reaction.
Second, third, and fourth instars were observed to clean
their caudal setae from time to time with their mandibles.
The larva reared backwards, dorsally grasped one of the
caudal setae with its mandibles and gradually drew the
entire seta through the mandibles. This process was
sometimes repeated with the remaining setae but more often

only the one seta irritating the larva was cleaned.

72

Pupation took place anywhere on the inner surface
of the pot, usually in the drier regions along the sides
around the rim and was occasionally observed on the dry
clay surface. These pupae remained viable because of the
humid environment of the pot. When the fourth instar was
ready to pupate a substance was apparently secreted from
the anal region of the larva anchoring it to the substrate.
A split in the dorsum of the thoracic segments allowed the
pupa to break through the larval integument. The pupa,
which was white in color during this process, forced the
cast integument of the fourth instar to the base of the
pupa by peristaltic movements. Two large prominences on
the mesonotum (mesonotal tubercles) were also used to push
the cast integument downward by rearing backwards and rub-
bing the tubercles against the receding integument. The
pupal position after this process was perpendicular to the
substrate. The pupal stage reacted violently to the touch
of a foreign object by snapping its body forward to the
substrate, then returning to its original position.

The adult emergence from the pupal skin began by
the protrusion of the mesothorax through the dorsal split
in the thoracic region of the pupa. The thoracic region
of the adult was gradually freed from the pupal skin by a
peristaltic—like action. The head was the next body part

freed, along with the mouth parts and the antennae and

73

followed by the wings and legs. The body of the adult
extended backwards pulling the long femur and tibia out

of the exuvia. The adult remained in this position for
about two minutes. Then the tarsal segments were pulled
out freeing the legs completely. A sudden jerk of the body
freed the remaining abdominal segments and the adult landed
on the substrate next to the pupal exuvia. The adult was
able to crawl immediately but could not fly for a few
minutes. The whole process from the break in the pupal
skin to the complete freedom of the adult took about 15
minutes.

The adults reared from the cultures were placed
in the styrofoam holding vessels and drops of the saturated
sugar solutions were placed on the nylon mesh to provide a
carbohydrate source for the newly emerged adults. Before
the experimental sugar feeding studies were completed,
fructose and sucrose were used for this purpose because
of the success Chaniotis (1974) had with them. After the
completion of the experimental tests, fructose and sucrose
were selected as the primary carbohydrate sources.

Early attempts at feeding the adults on a laboratory
host (suckling mice and hamsters), suspended in the holding
vessels were unsuccessful. The few adults that were
obtained from early larval rearing attempts severely
limited initial efforts to find a successful host,

feeding technique, and feeding chamber.

74

In the initial first generation laboratory cultures,
adults were obtained when yeast and the standard larval food
were used as the larval medium. The average number of eggs
per pot in all cultures was 550. In 41 cultures using this
food combination adult emergence averaged 10.3% (>.l%-34.1%).
Because of the low production of adults, the larval feeding
experiments, cited previously, were set up to develop a

better larval medium for L. panamensis. In the meantime,

 

liver powder added to the yeast and standard larval food
in the first generation cultures during the third instar
stage increased the average adult yield in 53 cultures to
15.4% (.1%-36.4%). Upon completion of the larval medium
tests, a mixture of liver powder, hemoglobin, and beef
blood serum was added to the yeast and standard larval
food already present during the third instar stage. This
enriched larval mixture increased the average adult yield
in 49 first generation cultures to 23.2% (l.4%-55.3%).
Not only did the mixture increase the number of adults
produced in each culture but the time period of maximum
adult emergence was shortened to about six days. Some
cultures produced as many as 30 females and-30 males a
day which was more than adequate to maintain the colony
and also provide sufficient adults for experimental

purposes.

75

At this time adult feeding attempts again were
initiated. Adults approximately four days old were
released into a damp, cloth-covered releasing cage.
Mating was observed almost immediately upon the release
of adults into the cage. The adults were left in the cage
for about one-half hour until mating was completed. No
mating was ever observed in the holding vessels even if
more than 100 males and females were placed in the same
vessel. The transfer from a small container to a larger
cage might have been the stimulus needed for mating.

The mating process was similar to that described

by Mukerji (1931) for Phlebotomus argentipes Ann. and Brun.

 

The male usually approached the female from the front,
moving its abdomen from side to side flexing its claspers.
Once the female was reached he swung his abdomen toward her
abdomen, clasping it firmly, remaining in a horizontal
position facing each other. The female was now in charge
and any movement occurring during the mating process was
done by the female. COpulation usually lasted two minutes
or longer. The same male often copulated with one or more
females.

The first successful feeding attempts occurred in
the small metal framed feeding cage of the WOhlbach design
used by Hertig and Johnson (1961). The females were

aspirated from the releasing cage and placed into the

76

feeding chamber. A moist cloth then was placed over the
feeding cage and the cage placed on the laboratory host
which was either man or a spiny rat. If the spiny rat was
used, the belly was shaved and the animal was restrained,
belly side up (Figure 12). After one-half hour the cage

was taken off the host and the flies were freed in the
releasing cage. Only a small number of flies could be

fed at one time with this method. It was determined that
one-half hour was sufficient time for the flies to feed
since most of the females fed within the first ten minutes.
The females were then aspirated back into another holding
vessel and allowed to oviposit in that vessel. No males
were placed with the females after blood engorgement because
preliminary tests showed that no mating occurred after blood
engorgement.

Placing the adults in close proximity to the host
seemed to be the key in successfully feeding the females.
However, the metal cage was too small to allow large numbers
(30-40) of females to be fed at the same time. The plastic
cage (Figure 12) was very successful in feeding large
numbers of females at one time, thus decreasing the labor
involved in manipulating these flies. Using the spiny rat
as the host an average of 59% (0%-93%) of the females fed
in 66 feeding trials which included first, second, and
third generation adults. The lower percentages always

occurred when ten or less flies per attempt were used.

77

To minimize the handling of the flies by aspiration,
another method of feeding the females was initiated. After
the flies had mated, a restrained belly shaven spiny rat
was inserted into the releasing cage. The flies were
allowed one-half hour to feed on the rat, after which
it was removed from the cage. The engorged females were
then transferred into the new holding vessel while those
that had not fed were aspirated into the plastic cage and
placed directly on the rat's belly. The females in the
cloth releasing cage were handled once, while the females
aspirated into the plastic cage were handled twice. This
method reduced injury and trauma to the fragile blood-
engorged females. This double feeding attempt method was
successful and an average of 62% (l7%-100%) of the females
fed in 28 feeding trials. These feeding trials also in-
cluded batches of females from the first, second, and third
laboratory generations. Very slight differences occurred in
the percentage of feeding successes between the generations.
In only two out of the 28 trials did all the females in the
plastic cage fail to feed on the host.

The human host, my arm, was used only when a spiny
rat was not available for feeding. The results were as
successful as with the spiny rat, and, I must add, very

painful.

78

Egg Morphology,

Sandfly eggs are generally elongate and ellipsoidal
in shape. The sculptured outer layer (exochorion) is sticky

which allows a species like L. panamensis to lay its eggs

 

singly on the oviposition substrate. The eggs remain in
place and are difficult to dislodge even with considerable
jarring. The term chorion used in the following descrip-
tions refers to the outer layer (exochorion). The described
results are presented according to the format of Matsuo et
a1. (1974). Egg measurements are in microns. The number
(N) measured is followed by the range of variation and

the mean in parenthesis. L indicates the length and W

the width at the widest point. The descriptions follow

the nomenclature set forth by Barretto (1941).

Lutzomyia panamensis<Shannon), 1926.

 

Figures 18-20.

Size: N = 43; L 364-429 (398); W 102-136 (122).

Chorion: Reticulation composed of flattened irregular raised areas
shaped like mesas, approximately equidistant from each other;
surface of mesas uneven and pitted; the area between mesas
very rugous at 2000 X magnification.

Remarks: The chorion is unlike any of the descriptions by Barretto
(1941). The egg is similar to but longer and wider than
L. pessoana or L. wellcomei. The adults and larvae of

these three species are also very similar morphologically.

Figure
Figure
Figure
Figure
Figure

Figure

18.
19.
20.
21.
22.

23.

E99
Egg
Egg
Egg
Egg

Egg

79

IL"

It"

It‘

II."

II."

II."

panamensis 200 X.

 

panamensis 1000 X.

 

panamensis 2000 X.

 

pessoana 200 X.
pessoana 1000 X.
pessoana 5000 X.

80

 

Figures 18-23

Lutzomyia

81

pessoana Barretto, 1955.

 

Figures:

Size:

Chorion:

Remarks:

Lutzomyia

21-23.
N = 9; L 357-391 (377); W 95-129 (103).

Similar to L. panamensis except mesa-like prominences

 

elongated; not as rugous as L. panamensis with irregular

 

shaped cells formed between lower irregular ridges at
5000 X magnification.

The egg is similar but usually shorter than L. panamensis

 

or E. wellcomei.

sanguinaria Fairchild and Hertig, 1957.

 

Figures:
Size:

Chorion:

Remarks:

Lutzomyia

24-26.

N = 8; L 344-388 (372); W 95—122 (108).

Conspicuous narrow longitudinal ridges more or less parallel;
connecting crossridges form pentagonal, hexagonal or polygonal
cells; ridges generally continuous except where damaged.

The eggs are similar to L. trapidoi. The surface structure

is closely related also those eggs described by Barretto

(1941).

trapidoi Fairchild and Hertig, 1952.

 

Figures:
Size:

Chorion:

27-28

N = 14; L 327—378 (350); W 92-122 (104).

Conspicuous narrow longitudinal ridges more or less parallel;
crossridges of the same consistency forming generally elon—

gated rectangular cells; ridges seem more discontinuous than

L. sanguinaria.

 

Figure
Figure
Figure
Figure
Figure

Figure

24.
25.
26.
27.
28.

29.

E99
Egg
Egg
Egg
Egg

E99

It' It" up It‘ up

It."

82

sanguinaria 200 X.

 

sanguinaria 1000 X.

 

sanguinaria 5000 X.

 

trapidoi 200 X.

trapidoi 1000 X.
ylephiletor 200 X.

 

9
2
.
4
2
S
e
r
u
g
.1
F

 

Remarks:

Lutzomyia

84

The egg is similar to L. sanguinania but generally smaller

 

in size.

ylephiletor Fairchild and Hertig, 1952.

 

Figures:
Size:

Chorion:

Lutzomyia

29-31.

N = 6; L 333-374 (250); W 92-106 (101).

Conspicuous flattened longitudinal ridges more or less
parallel formed from flattened globular mounds discontinuous
in pattern at 2000 X; crossridges formed the same way; ridges
may be discontinuous, cells somewhat elongate rectangular to

polygonal, no distinct pattern to cells.

gomezi Nitzulescu, 1931.

 

Figures:
Size:

Chorion:

Remarks:

32-33.

N = 18; L 300-347 (332); W 95-122 (104).

Very conspicuous wide flattened longitudinally directed
ridges; crossridges narrow and oblique to longitudinal
ridges; cells are elongate polygons at 1000 X magnification.

The egg is similar to Lutzomyia monticola as described by

 

Barretto (1941).

Figure
Figure
Figure

Figure

30.
31.
32.

33.

Egg
Egg
Egg

E99

IL"

IL"

It‘

It"

85

ylephiletor 1000 X.

 

ylephiletor 5000 X.

 

gomezi 1000 X.
gomezi 1000 X.

86

 

Figures 30-33

87

Morphology of the Larvae and Pupa

 

The egg stage of L. panamensis has been described

 

previously. The setal nomenclature used in this study
follows that of Barretto (1941). Hanson's (1968) descrip-

tion of the fourth instar of L. panamensis supplements the

 

data presented in this work. The average measurements and
ranges of the body structures and setae of ten specimens

of each instar are presented in Appendix H, with the aver-
ages included in the text. The morphology of the fourth
instar is described in full and the first, second, and

third instars are described where they differ from each
other. Only the dorsal and ventral aspects of the first

and fourth instars and the lateral aspect of the pupa are
figured because the second and third instars are essentially

the same as the fourth, except for size.

Fourth Instar

 

Body length from front of head to base of caudal
setae 2.975 mm; flattened dorsoventrally, grey in color,
head darker grey; body covered with barnacle-like projec-
tions (Figure 37); prominent sclerotized protuberances bear
conspicuous setae (Figures 34 and 35).

Hegd.--Head capsule longer (262p) than wide (246p),
frontal area prominent, frontoverticular angle approaching

90°. Gular region strongly inflected. Antennal tubercles

88

Figure 34. Head, thorax, and abdominal segments 1, 8, and
9 of the fourth larval instar; dorsal aspect.

Setal Nomenclature-~Lutzomyia panamensis—-Larvae

 

Head

ant t antennal tubercle

ant 1-2 antennal segments
ap antennal apical process
af anterior frontals
pf posterior frontals
1v lateral vertical
dv dorsal vertical
c clypeal
dg dorsal genal

Prothorax

dsa dorsal shoulder accessory
aid anterior internal dorsal
aed anterior external dorsal
pid posterior internal dorsal
ped posterior external dorsal
avl anterior ventrolateral
pvl posterior ventrolateral
d1 dorsolateral

as anterior spiracle

Meso and Metathorax and Abdominal Segments 1-8

id internal dorsal

ed external dorsal

da dorsal accessory
Remainder of nomenclature as above

Abdominal Segment 9

 

ps posterior spiracle
ec external caudal
c caudal
Remainder of nomenclature as above

Pro.

mes.

met.

0b.!

89

I ‘ '~ d
"'—\\ g I! an: I

\V —ant t

”we I i _ dv
aid . ,3. aed

\ a . dsa
()9 :1 \

dl \ —— E .. p"

as l'. 6%,, (gig g ”(.3 ‘32? .u't‘lii

ovl \,2¢_ ____,9‘

. do

4

”Ii—rt. ..

>12XJL .

 

avl Q~___:
—\ V dc!"

H1K%L

pvl

dl

 

 

dors.
(WI
T
E‘
s
'0.
-L

90

Figure 35. Head, thorax, and abdominal segments 1, 8, and
9 of the fourth larval instar; ventral aspect.

Setal Nomenclature--Lutzomyia panamensis--Larvae

 

Head

lg lateral genal
vg ventral genal

Prothorax

aiv anterior internal ventral

aev anterior external ventral

piv posterior internal ventral

pev posterior external ventral

pvi posterior ventral intermediary
vma ventral median accessory

Meso and Metathorax and Abdominal Segments 1-9

iv internal ventral

ev external ventral

vi ventral intermediary
Remainder of nomenclature as above

Abdominal Segment 9

 

i pre a internal pre anal
e pre a external pre anal
i post a internal posterior anal
e post a external posterior anal
ea external anal
vc ventral caudal
Remainder of nomenclature as above

91

vent

pni

mes.

rnet

0“

pH

abJ

 

ab13

obf)

Figure 35

 

92

as long as antennae (7lu), situated posterior to anterior
frontals. First and second antennal segments nearly

equal in length, second segment directed dorsad at a

slight angle from first segment; second segment slightly
wider. Mandibles dark brown; upper margin straight curving
at apex; lower margin possesses four rounded teeth. Mentum
dark brown, toothed. Clypeus rounded in profile protruding
over labrum-epipharynx. All setae simple, slightly curved
anterad; anterior (af) (172u) and posterior (pf) (121u)
frontals and lateral (1v) (164u) and dorsal (dv) (90p)
verticals possess minute branches along apical 3/4, darker
than head integument; clypeals (c) (97p) long and slender;
dorsal (dg) (80p) and lateral (1g) (88p) and ventral (vg)
(38p) genals long and slender, no branches present.

Prothorax.--Symmetrical sclerotized patches present

 

dorsally and ventrally. Lighter in color than following
body segments, fewer cuticular prominences. Anterior
internal (aid) (54u) and external (aed) (65p) dorsals
proclinate, on prominent tubercles, sparsely branched,
pigmented; dorsal shoulder accessories (dsa) (18u) laterad
to anterior dorsals, short spine-like; posterior external
(ped) (32p) and internal (pid) (29p) dorsals almost equal
in length, brush-tipped, pigmented, directed caudad;
dorsolaterals (d1) (65p) laterad to posterior dorsals

and anterad to conspicuous anterior spiracles, curved,

93

brush-tipped, situated on prominent tubercles; anterior
ventrolaterals (avl) (ll7u) large, stout, directed slightly
anterad and ventrad, anterad to spiracle, pigmented, brush-
tipped; posterior ventrolaterals (pvl) (lSu) small, spine-
like, directly below dorsolaterals, lightly pigmented.
Ventral setae lightly pigmented; anterior internal (aiv)
(56p) and external (aev) (65u) ventrals simple, projecting
anterad; posterior external ventrals (pev) (68u) arise from
large tubercles, stout, brush-tipped; posterior internal
ventrals (piv) (43p) shorter than posterior external ven-
trals, comb-tipped; posterior ventral intermediaries (pvi)
(22p) short, spine-like, lie anterad between posterior
internal and external ventrals; ventral median accessories
(vma) (20p) small, comb-tipped.

Meso and Metathorax.--Dorsal and lateral setae

 

pigmented and brush-tipped except for dorsal accessories.
Internal (id) (34h) and external (ed) (37p) dorsals larger
than those of prothorax, directed caudad and mesad; dor-
solaterals (720) slightly larger than those of prothorax,
arise from prominent tubercles, projected caudad; anterior
ventrolaterals (125u) longer than those of prothorax,

curved ventrad at apex and directed slightly cephalad;
posterior ventrolaterals (114p) slightly smaller than
anterior ventrolaterals, curved ventrad and directed caudad;

dorsal accessories (da) (19u) slightly dorsad and mesad of

94

ventrolaterals, spine-like, inconspicuous. Ventral

setae lightly pigmented; internal ventrals (iv) (41p)
almost equal to those of prothorax, comb-tipped, directed
caudad and ventrad; external ventrals (ev) (72u) almost
equal to those of prothorax, brush-tipped, arise from
prominent tubercles, directed caudad and ventrad; ventral
intermediaries (vi) (24p) same as prothorax; ventral median
accessories (12u) smaller than those of prothorax otherwise
similar.

Abdominal segments l-7.--Dorsal and lateral setae

 

pigmented, brush-tipped; internal (28H) and external (29p)
dorsals equal in length, directed caudad, become smaller
progressing from first to seventh segment, shorter than
those of thorax; dorsoventrals (109u) long, stout, arise
from large sclerotized tubercles, directed caudad, curved
apically, longer than those of thorax; dorsal accessories
(16u) (Figure 37) small, directed caudad, different shaped
than those of meso and metathorax; anterior ventrolaterals
(llBu) slightly smaller than posterior ventrolaterals (124u),
both setae long, stout, anteriors directed laterad curved
ventrad, posteriors directed caudad curved ventrad. Ven-
trals long and slender, lightly pigmented; internal ventrals
(SZu) on edges of large abdominal prolegs, directed caudad,
curved apically, longer than those of thorax; external

ventrals (42p) much shorter than those of thorax, 1% times

95

longer than those of third instar, curved apically, directed
caudad; ventral intermediaries absent.

Abdominal segment 8.--Heavily sclerotized dorsally

 

and laterally, bearing numerous small spines directed caudad.
Dorsal and lateral setae pigmented, brush-tipped; internal
dorsals (l6u) small directed caudad; external dorsals (lZlu)
long, stout, directed caudad, curved apically ventrad, much
longer than those of other abdominal and thoracic segments;
dorsolaterals (94p) ventrad to large posterior spiracles,
shorter than those of other abdominal segments, directed
caudad, curved apically ventrad; dorsal accessories and
posterior ventrolaterals absent; anterior ventrolaterals
(36p) medium in length, stout, directed caudad, shorter than
those of other abdominal and thoracic segments. Ventral
setae short, lightly pigmented, directed caudad, curved
apically; ventral intermediaries (Sp) present.

Abdominal segment 9.--External caudals (ec) (63u)

 

long, stout, brush-tipped, directed caudad, curved. Caudal
segment bears two bifurcated caudal setae (c) (3.509 mm),
each setae bears a point of separation (node) at the base
(Figure 36). Ventral setae simple, unbranched, varying in
length and pigmented; ventral caudals (vc) (62p) same length
as external caudals, situated ventral to caudal setal fork,
directed dorsad; external anals (ea) (72p) long and slender,

directed ventrad, curved apically; internal (i pre a) (35p)

96

 

Figure 36. Caudal setae, fourth instar, E. panamensis,
1000 X.

 

!

Figure 37. Dorsal accessory seta, fourth instar, E.
panamensis and cuticular surface, lOOOX.

97

and external (e pre a) (50p) pre-anals fine and
inconspicuous, directed ventrad, curved apically; external
posterior anals (e post a) (lSlu) very long and slender,
directed ventrad, curved apically; internal posterior anals

(i post a) (42u) slender and inconspicuous, directed ventrad.

Third Instar

 

Body length 2.118 mm, body grey, head slightly
darker; body flattened somewhat dorsoventrally.

gggg,--Somewhat longer (180u) than wide (163u);
first antennal segment darkly pigmented, antennal length
53u.

Prothorax.-—Symmetrical sclerotized patches on

 

cuticle of dorsum.

Abdominal segments l—7.--Posterior ventrolaterals

 

(68p) equal to anterior ventrolaterals (70u) in length.

Abdominal segment 8.--External dorsals (76p) longer

 

than dorsolaterals (67p).

Abdominal segment 9.--Four caudal setae (2.48 mm).

 

Second Instar

 

Body length 1.423 mm, round in shape, light grey.

figgd.--Somewhat square in shape, length l40u and
width 134u, brown in color, sclerotized. Antennae (40u)
brownish, arising from prominent sparsely spined tubercles.

Setal structure same as first instar.

98

Prothorax.--Anterior internal dorsals (24u) present,

 

slightly shorter than anterior external dorsals (32p), both
sets proclinate; posterior external (9p) and internal (lou)
dorsals small, brush-tipped, directed caudad; posterior
ventrolaterals (Su) small. Ventral setae same as other
instars.

Meso and Metathorax.--Anterior ventrolaterals (41p)

 

large, stout with small bristles on apical 3/4, directed
anterad; posterior ventrolaterals (21p) present, large,
same as anterior ventrolaterals, directed laterad.

Abdominal segments l-7.--Posterior ventrolaterals

 

(38p) present, larger than same meso and metathoracic setae,
slightly smaller than anterior ventrolaterals (46p); exter-
nal ventrals (lOu) smaller than same in first instar,
directed ventrad.

Abdominal segment 8.--External dorsals (49p),

 

longest setae, much larger than same in first instar,
directed caudad.

Abdominal setment 9.--External caudals (30p)

 

shorter than those of first instar; pair of caudal setae

(1.764 mm) bifurcated making four.

First Instar

 

Body lengths 0.927 mm, round in shape (Figures 38

and 39).

99

Figure 38. Head, thorax and abdominal segments 1, 8, and
9 of the first larval instar; dorsal aSpect.

Figure 39. Head, thorax and abdominal segments 1, 8, and
9 of the first larval instar; ventral aspect.

Setal nomenclature same as fourth larval
instar Lutzomyia panamensis.

 

100

 

  

     
     
   
     

     

r0. “
P ‘C/ \. 33’
‘\\‘ ‘
rrMaS. ;u€§;2§159
'
met

ab.|

 

ab.8

0 b9

 

 

200,.

Figure 38 Figure 39

101

gggd.--Head capsule lightly sclerotized, almost
square in shape, length 94H and width 87p. Antennae (33p)
erect and prominent on erect tubercles. Dorsal setae simple
but anterior (64p) and posterior (29p) frontals and lateral
(45p) and dorsal (22p) verticals possess minute spare
branches (brush-tipped) almost to base of setae. Ventral
setae simple and delicate. Prominent, heavily sclerotized
egg burster.

Prothorax.--Anterior spiracles small, indistinct.

 

Anterior interior dorsals absent along with dorsal acces-
sories mentioned by Ward (1972); anterior external dorsals
(29p) directed cephalad to head capsule; dorsal setae
minutely branched apically; anterior ventrolaterals (29p)

as long as anterior external dorsals. Ventral setae simple,
no branches; posterior external ventrals (l6u) longest.

Meso and Metathorax. Dorsolaterals (29p) stout,

 

minute branches on apical 3/4; posterior ventrolaterals
absent along with anterior dorsals; external (7p) and
internal (6p) dorsals larger than prothoracic; anterior
ventrolaterals (22p) smaller than prothoracic, directed
anterad. Ventral setae similar to those of prothorax.

Abdominal segments l-7.-—Dorsa1 accessories (4p)

 

small, directed caudad; external (7p) and internal (6p)
dorsals directed caudad, progressively smaller from first

to seventh segment; ventral intermediaries and ventral

102

median accessories absent; external ventral (l9u) larger
than those of thorax, curved; directed laterad; posterior
ventrolaterals absent.

Abdominal segment 8.--Dorsal accessories absent;

 

anterior ventrolaterals (16u) smaller than those of other
abdominal segments; dorsolaterals (31p) directed caudad;
internal dorsals (8p) larger than external dorsals (5p),
situated on small chitinized area; posterior spiracles
larger than anterior spiracles. Ventral setae small,
inconspicuous, directed caudad; ventral intermediaries
present.

Abdominal segment 9.--External anals (26p) simple,

 

curved, directed caudad; external (llu) and internal (8n)
pre-anals simple small, curved ventrad; external (64p) and
internal (150) post-anals directed caudad; external caudals
(33p) larger than same in second instar extending past anal
protuberance. Caudal setae (two) (1.288 mm) longer than

body, conspicuous.

Pupa
Body length of pupa from pronotum to base of

abdominal segment nine 2.810 mm (2.609-3.259 mm); color
light to dark brown; lateral aspect Figure 40. Respiratory
horn and prealar lobe prominent; pre—alar setae 90p
(82-1090), darkly pigmented, tip blade—like, divided

(trifid). Mesonotal tubercles long. Wing sheath slender.

103

Figure 40. Head, thorax, and abdominal segments 1-6
of the pupa; lateral aspect.

Setal Nomenclature--Lutzomyia panamensis--Pupa

 

h head
mp mouthparts
ant antennae
pr prothoracic setae
pro prothorax
pa pre alar setae
mt mesontal tubercle
mes mesothorax
ms mesothoracic setae
met metathorax
ab 1-6 abdomen
w wing
1 leg
abs dorsal abdominal setae

 

105

Abdomen without tubercles. Dorsal setae similar in
length but different in shape; pro and mesothoracic setae

blade-like; dorsal abdominal setae spine-like.

Discussion

 

Contrary to Hanson's (1968) short description

of the first instar of E. panamensis, based on a single

 

specimen, the anterior frontals appeared to be the longest
setae on the head instead of the posterior frontals. In

his description of the fourth instar, based on two specimens,
the prominent tubercles, on which the anterior prothoracic
dorsals were situated, were stated to be one-half the length
of the setae; they were only one-sixth as long in the spec-
imens examined in this study. The prothoracic anterior
ventrolaterals were almost twice as long as the dorso-
laterals instead of equal in length. Hanson mentioned

that the meso and metathoracic dorsals were equal in length
to those of the prothorax but they were longer in the spec-
imens measured in this study. He also stated that a single
pair of anterior prothoracic dorsals were present but two
pair were found. Hanson used this character to differen-

tiate E. panamensis from Lutzomyia apicalis Floch and

 

 

Abonnenc. He stated that E. panamensis fourth instar

 

can hardly be distinguished from £- pessoana. Specimens
of E. pessoana were not available for comparison in this

study, but setal measuremens of g. pessoana might prove

106

to be the distinguishing characteristics needed to
differentiate these two closely related species. It
appears that Hanson was in error in some of his setal
comparisons; however, if more than two specimens had been
available to him these oversights might not have been made.
Ward (1972), who was not aware of Hanson's dis-

sertation, compared his six specimens of E. wellcomei to

 

Lutzomyia arthuri Fonseca as described by Barretto (1941).

 

If Hanson's work had been available, Ward undoubtedly would
have compared his fourth instar larval descriptions with

those of E. panamensis and E. pessoana since the four

 

species belong to the same subgenus Psychodopygus and

 

are very similar in many respects.
Ward also used measurements of setae in his

descriptions of B. wellcomei which were compared with

 

those of E. panamensis. The average body length of E.

 

panamensis fourth instar was smaller than 3. wellcomei

 

 

but the range was similar. The antennae of E. wellcomei

 

 

were larger (97p) than those of E. panamensis (71p) but
the shape was the same. Head setae were similar in size

and shape. Prothoracic dorsal accessories of P. wellcomei

 

were absent in E. panamensis. The prothoracic dorsolaterals

 

of E. panamensis were more than twice as long as those of

 

E. wellcomei. The dorsal setae of the meso and metathorax

 

of E. panamensis were more than twice as long as those of

 

107

g. wellcomei. The dorsolaterals were also longer in

 

L. panamensis. The ventral setae of E. panamensis were

 

 

in general longer than those in E, wellcomei. The caudal

 

 

setae of E. panamensis were longer than those of 2. well-

 

comei. The point of separation (node) at the base of E.

panamensis has not been mentioned by other sandfly inves-

 

tigators but is probably present in all larvae and in all
instars that possess these long caudal setae. Often larvae
were observed with one or two setae missing. The easy
removal of these setae might serve as an escape mechanism
when the caudal setae become caught or to escape from
predators.

The pupal stage of E. panamensis was very similar

 

to g. wellcomei. The pre—alar setae were similar in size

 

and were both trifid. However, the thoracic setae of E.

panamensis were blade-like which differ from the spine-like

 

setae of E. wellcomei. The dorsal abdominal setae of both

 

species were spine-like. Hanson indicated that the color

 

of E. panamensis pupae was grey but those in this study

were brown to dark brown.

SUMMARY AND CONC LUS IONS

The laboratory bionomics, colonization, and
morphology of the immature stages of the anthropophilic

phlebotomine sandfly Lutzomyia panamensis (Shannon) were

 

studied. In addition, the SEM was used to examine the egg

surface ultrastructure of E. panamensis as well as those

 

of the other common Panamanian anthropophilic sandflies

E. pessoana, E, trapidoi, E. gomezi, E. ylephiletor, and

E. sanguinaria.

 

In an experiment to evaluate the feeding preference

of E. panamensis larvae, four experimental food sources

 

yeast, liver powder, beef blood serum and hemoglobin were
placed on the same plaster lined petri dish. The first
three instars preferred yeast to the other food sources.
The fourth instar preference for yeast declined with an
increased preference for the other food sources, especially
hemoglobin. The four food sources were also compared
individually on petri dishes in order to determine the
effect of these single diets on the duration of the life
cycle, pupation, and adult emergence. The yeast and
standard larval food combination dishes produced the

shortest developmental time from the first instar to pupa,

108

109

with yeast, liver powder, beef blood serum and hemoglobin,
respectively, increasing developmental time. When the
developmental times of the larvae reared on single food
sources were compared with those of larvae reared on the
combination dishes, the combination produced faster and
more desirable development. Larvae reared on the combi-
nation dishes survived to produce more pupae and adults
than those reared on individual diets. The plaster lined
petri dish was very satisfactory as a rearing chamber and
general observations of larval behavior could be made with
ease.

The effects of various saturated sugar solutions
on the longevity of laboratory reared females and males of

E. panamensis were examined. Life tables were constructed

 

for each of the six sugars tested and the observed life
expectancies at emergence were computed. Fructose and
sucrose were the most efficient in prolonging the survival
of the females. Fructose, sucrose, honey, Karo syrup and
dextrose appeared to provide the same male life expectancy.
The styrofoam test vessel was very satisfactory in studying
the longevity of these females and males.

Longevity studies also conducted on the wild-
caught blood-engorged females indicated that the saturated
solutions of fructose, sucrose and dextrose were the most

efficient in promoting longevity. In an analysis of the

110

effects of these saturated sugar solutions on oviposition,
no difference could be detected between groups feeding on
the soaked raisin, fructose, sucrose and dextrose groups.
Both longevity and maximum oviposition are important
factors in rearing and colonizing sandflies. With extended
longevity it is possible to refeed the wild-caught females
on a laboratory host following the first oviposition period
and have them produce a second batch of eggs. This would
make maximum use out of the sometimes hard to collect
wild-caught blood-engorged females. If no sugar source

is provided the females do not live to feed a second time
and lay only about one-half of their complement of eggs.

In all the experimental tests conducted with both labora—
tory reared adults and wild-caught females, fructose and
sucrose were consistently preferred by the adults.

Four laboratory generations of E. panamensis were

 

reared. The average length of the life cycle from ovi-
position to adult was 37.7 days. The unglazed Boston bean

pot was the most satisfactory for rearing E. panamensis in

 

large numbers. The Optimum condition of the plaster sub-
strate was damp and smooth in texture. The mixture of
liver powder, hemoglobin and beef blood serum added during
the third instar to the yeast already present in the pots
was shown to be the best larval medium tested. The spiny

rat served very well as the laboratory host. The double

111

feeding method of using the cloth releasing cage and the
plastic feeding cage was the most successful in feeding
the largest number of laboratory reared adults with little
injury to the females.

The morphology of the immature stages of E.

panamensis was redescribed and clarified. Hoyer's medium

 

proved to be satisfactory as a mounting and clearing medium.
Ink drawings of the dorsal and ventral aspects of the first
and fourth instars and the lateral aspect of the pupa were
illustrated. Measurements of the prominent setae were very
useful in differentiating changes between instars and other
closely related species. Scanning electron photographs of
the fourth instar served as an aid in describing the nature
of the cuticular surface. The use of the scanning electron
microscope in describing the egg surface structure of the
anthropophilic sandfly species proved to be very useful.

Two distinct patterns were shown. E. panamensis and E.

 

pessoana eggs had a surface structure composed of raised
mesa-like prominences with the remainder of the surface
very rugose in appearance. The egg surface structures of

E. trapidoi, E. ylephiletor, E. gomezi and E. sanguinaria

 

 

were made up of cells of varying shapes bordered by ridges

of varying thicknesses.

APPENDICES

112

 

 

 

 

 

 

 

 

 

m.o 0.H H m m mm
m.H o.m m o N mm
m.H 0.0 m N v em
H.N m.OH m.v H m mm
m.o ¢.N m m.mH m o v N v 5 mm
m.H m.m m.o o.mm m.v m.m H m m 0H Hm
v.H o.m o.vH 0.0m m.5 HH m m 0H NH om
o.m v.m m.mm o.mm m.HH 5H m 0H MH mm mH
m.m m.m o.ov o.m5 m.vH mm m 9 pH mm mH
o.m m.m m.5m 0.00H m.5H Hm m o mH vm 5H
m.m o.v o.m5 o.va m.om mm m m mm @m 0H
m.v 5.v o.OOH m.HmH mm m.5m o m mm mm mH
5.v m.m o.mmH m.m- mm Hv o v mm mv VH
o.m 0.0 m.me o.mwm m.mm m.mv m H Hm vv MH
0.0 5.0 m.mmH 0.0Hm Hm mv o m Hm we NH
m.o v.5 m.5Hm 0.5mm mm 5v m m mm mv HH
0.5 o.m o.Nmm m.©ov m.vm m.mv m m mm Hm 0H
0.0 H.m 0.Hmm m.oov mm gm 0 o «v 5m m
0.5 v.w m.mmm 0.0mm m.mv m.mm H m mv mm m
m.m ¢.m m.o5m o.mmm me mm o 0 mg mo 5
m.@ N.OH m.omv m.wvm vv m.mo m H mv mm o
5.0 H.HH 0.5wv o.m05 m.mv m.mm m H mv we m
H.0H m.HH m.on m.m55 m.mv m.vo m H Hm mm v
m.OH m.mH 0.05m o.mmm m.mm m.mm m H mm mm m
w.m m.NH 0.0mm 0.00m 00 mm w v we 05 m
o.oH m.MH 0.5mm 0.05m 50 05 o o 05 05 H
memz monEmm memz mmHmem mewz meMEmm mmHmz mmHMEmm mmHmz mmHmEmm Axe
m>MU
.“NHme mocmuommxw x9 pm>HH mama xnx..xvnuxq pm>HH xv "x no xv "x um Hm>umucH
BmMHH om>ummno mo Hogans Hmuoe mwmp mo Hmnfisz mcHMG umnEdz m>HHm quEsz 0mm
mmOBUDmm ZO UZHQmmm mmH§ O72 magma mHnggm .uH. 03mm wmagog mo >6H>mwzoq Jm xHszmmd

113

 

 

 

 

 

 

 

 

 

m.o m.o m.o H H mN
m.H m.H H o H 5N
m.H o.m m.H H N oN
m.N o.m N o N mN
m.N m.5 m.N H m vN
o.N o.NH m.w m 0 MN
v.N o.mH 5 N m NN
m.o 5.N m.o mN m.o m H N H OH HN
0.H o.m o.N m.mm m.H m.HH m m N mH 0N
m.H v.m m.v o.mm m.N m.mH H m m 0H mH
5.H m.m m.m m.N5 v m.5H N m m mH mH
m.N o.v o.vH m.mm m.m HN H v 0 MN 5H
5.H N.v o.vN o.mHH 0H m.mN m m VH mN 0H
v.N o.v o.mm o.mVH mH om N v 0H Nm mH
5.N o.v m.5m o.mmH m.mH mm m m HN ow vH
o.m m.v m.m5 o.mNN NN mv N 0 MN ow mH
m.m ®.m m.vOH m.m5N mN m.5w v m 5N av NH
N.v v.0 o.VMH m.mNm m.mN om m N Nm Hm HH
m.w m.m 0.5oH m.m5m mm mm N v «m mm 0H
v.m m.5 o.mON o.mmv mm m.om v m mm mm m
N.m m.m m.mVN m.va m.Nv m.5m m H 5v mm m
m.m N.m o.va m.Hmm m.mv mm m N om oo 5
m.m 0.0 o.vvm m.NHm om Ho 0 N om No 0
m.5 5.0H o.vmm o.m5o om m.No o H om mm m
o.m m.HH o.mvv m.mm5 Hm m.mo N H Nm we v
m.m m.HH m.mmq m.vom m.vm co m v 5m mm m
m.m m.NH o.Nwm m.m5m m.N® mm H N mo 05 N
H.m m.MH m.omo m.mvm m.mo 05 H 0 mo 05 H
monz memswm mmHmz moHMEmm mmHmz monEmm monz mmHmemm mmHmz mmHmEmm Axe
x m>MU
xNuxo xosmuommxm x9 ow>HH mummy xpwl anxa Uw>HH xv "x no xw "x um Hm>uoucH
BmMHH Um>ummno mo Honfidc Hmuoe m>mn mo Hmnfisz mcva Honesz m>HHm quEsz mod
mmomUDm ZO OZHDmmnH mqufi 02¢ mmqgmm mHnggm .M Ogdmm Nmoemmog mo NBH>MOZOH .m xHozmmm¢

114

 

 

 

 

 

 

 

 

 

0.0 0.0 0.0 H H 0N
0.H 0.H H o H MN
0.H o.M 0.H H N NN
0.0 0.N 0.0 0.0 0.0 N H o H N HN
0.H 0.M 0.H 0.5 H N o o H N 0N
0.N 0.N 0.N 0.0H H M o N H 0 0H
0.H N.N 0.0 0.0H 0.M 0.0 0 M 0 5 0H
0.H N.N o.MH 0.vN 5 0 N v 0 HH 5H
v.N 0.N 0.HN 0.0M 0.0 NH H N 0 MH 0H
5.N o.M o.NM 0.H0 0.0H 0H M 0 NH 5H 0H
0.N N.M 0.0V o.H5 vH 0.0H v 0 0H NN vH
0.N v.M 0.00 0.00 0N 0N 0 0 «N 0N MH
0.M 0.M 0.00 o.0NH VN Om v v 0N NM NH
0.M v.v 0.0HH o.o0H 0.0N «M 0 0 HM 0M HH
v.0 N.0 0.HOH 0.50H 0.NM 5M M N VM 0M 0H
v.v 0.0 0.00H o.0MN 0.0M 0M 0 N Mv ov 0
H.0 0.0 o.VMN o.55N 0.vv Hv M N 00 N0 0
0.0 M.5 o.HON o.ONM 5v Mv N N 0v we 5
5.0 0.5 0.0NM 0.00M 0.0V 0.0V H 0 00 00 0
H.5 0.5 o.NOM 0.5Hv 0.N0 H0 0 v 00 M0 0
0.5 5.0 0.5Mv 0.H5v 0.00 0.M0 M H 50 V0 v
0.5 0.0 0.50v 0.5N0 00 0.00 0 0 M0 00 M
0.5 0.0 0.000 0.000 0.50 00 0 0 N5 00 N
0.0 0.0H 0.5M0 0.000 N5 00 o N N5 H0 H
mmHmz mmHmEmm mmHmz mmHmemm mmHmz mmHmEmm mmHmz mmHmEmm mmHmz mmHmEmm Axe
aw x 0000
.mmnu m xocmuommxm x9 00>HH mama x0x..anuxH Um>HH x0 "x um xv "x um Hm>uwucH
mMHH cm>ummno mo umnfidc Hmuoa m>00 mo umnfisz 0GH>0 umnadz w>HHm uwnasz 00¢
xmzom zo UZHDmmnH 0502 072 mMHgmm mHngzdm .m. Qmmdmd wmoeéomfi mo wBH>mOZOH .0 xHDzmmmd

115

 

 

 

 

 

 

 

m.O m.O m.O H H 0N
m.Hlm.m m.Hlm.m H O H QNIVN
m.H mom N N m MN
m.N 0.0 m.m H V NN
m.m o.MH V O V HN
o.M O.mH m N 0 ON
m.O m.N 0.H m.mN H m.h N m N m 0H
N.H o.M m.m 0.0m m.N m.OH H m m NH mH
m.H m.m O.m 0.0V m.V MH m N o VH 0H
0.H V.m 0.0H m.m® m m.®H V m OH mH 0H
O.N m.m O.mN 0.0m NH m.ON V m VH NN mH
N.N H.V m.mV m.OHH m.hH m.VN h m HN 0N VH
O.N H.m m.m® m.hMH VN EN 0 O 0N 0N MH
m.m V.m m.hm m.®©H m.hN mN H V mN Hm NH
O.V h.m m.bNH m.mmH Om mm V V NM mm HH
h.V N.® m.O®H O.QMN mm m.®m N m Vm mm OH
©.m N.® O.mmH m.th m.Vm m.HV H 0 mm mV m
N.@ N.N 0.HMN m.NNm om mV N 0 0m mV m
0.0 m.h 0.0hN m.m®m mm 0V V N HV NV 0
M.5 N.m O.NHm m.hHV NV mV N V mV Hm m
w.h h.m m.©mm 0.0BV m.VV m.Nm m m 0V Vm m
N.@ V.@ O.VOV O.mNm m.hV mm m N mV mm V
V.m 0.0 m.mmV m.mmm m.Hm m.mm m m Vm Ho m
m.h 0.0 m.hHm m.hV® No V0 m o 00 no N
V.m b.0H m.mmm m.VHh mm 50 V 0 On 00 H
x m0Hmz 00120.0 m0H02 03080.0 m0Hmz 00Hmé0m m0Hmz m0Hme0rm 00H02 03050.0 CC
INux0 mocmuo0mx0 x x x 00.00
x8 .H. 00>HH 0000 0» wan .H 00>HH x0 "x 00 xv ”x 00 H0>H0ucH
00HH 00.30000 00 H0355 H0009 0000 no .0852 09.26 .0852 0>HH0 H0852 00¢

 

 

momwm omdx ZO UZHQmmm mmddz 02¢ mmqflzmm mHmzmS¢z¢m um ammdmm MMOBdmomdq ho WBH>MUZOH .Q xHQmemfl

116

 

 

 

 

 

 

0.0 0.0 0.0 H H 0N
0.HI0.0 0.Hn0.0 H o H 0N|0N
0.0 0.0 0.0 0.0 0.0 H H o H H MN
0.H 0.5 0.H 0.5 H H o o H H NN
0.H 0.0 o.M 0.0 0.H H H o N H HN
0.N 0.N 0.0 0.HH N 0.N o M N 0 ON
0.N 0.M 0.5 0.0H 0.N 0 H o M 0 0H
M.N 0.0 0.HH 0.0H 0 0 N o 0 0 0H
H.N 5.0 0.0H 0-MN 5 0.0 0 H 0 0 5H
0.N 0.N 0.0N 0.HM 0.0H 0 M 0 NH HH 0H
0.0 0.N 0.H0 0.00 NH 0.MH o 0 NH 0H 0H
M.M 0.M 0.00 o.N0 0.0H 5H 0 N 5H 0H 0H
0.M H.0 0.M5 o.H0 0H 0H N N 0H oN MH
H.0 0.M 0.00 0.00H HN 0.0N 0 HH MN HM NH
5.0 N.0 0.0HH 0.0MH 0N NM N N 0N MM HH
0.0 5.0 0.00H 0.M5H 5N 0M 0 0 0N 5M 0H
0.0 0.0 0.05H 0.0HN 0.0N H0 H 0 0M 00 0
0.0 M.0 0.HHN 0.HON 0M 50 NH 0 N0 00 0
0.0 0.0 0.MON 0.NHM 0.N0 H0 H 0 M0 M0 5
0.5 0.0 0.00N 0.50M M0 00 o 0 M0 50 0
5.5 H.5 0.00M 0.0N0 0.M0 0.00 H M 00 00 0
0.0 0.5 0.00M 0.500 00 H0 N N 00 N0 0
5.0 M.0 o.MMO 0.H00 00 00 0 0 o0 00 M
0.5 0.0 0.000 0.0H0 0.00 0.50 MH M M0 00 N
0.5 0.0 0.000 0.000 0.00 0.00 5 H 05 05 H
m0H02 m0H0E0m m0H0z m0H050m m0H0z m0H0E0m m0H0z m0H0E0m m0H0z 00H0E0m Axv
.MN.ux0 0oc0uo0mww 0000
x9.. x9 00>HH 0000 x0w..xv;uxq 00>HH x0 "x 00 xv "x 00 H0>H0ucH
00HH 00>u0mno 00 M0QESG H0009 0000 00 u0nfisz 0cH00 “00502 0>HH0 u0nEdz 00¢

 

 

mmomemQ ZO UZHDmmh mums/NS QZd MMHgmm mHmZMZ/wzdmm hm 090de 0mOB§0mS ."HO 09H>WOZOH .m XHQmeg

117

 

 

 

 

 

 

 

 

 

0.0 0.0 0.0 H H NN
0.H|0.N 0.Hu0.N H O H HNuON

0.0 0.M 0.0 0.M 0.0 H H O H H 0H
O.H 0.N O.N 0.0 0.H 0.H H H N N 0H
O.N 0.M 0.0 O.5 N N O O N N 5H
o.M 0.0 0.0 0.0 N N O O N N 0H
0.0 o.M 0.0 O.NH N M O N N 0 0H
0.0 0.0 0.0H 0.0H N 0 O O N 0 0H
0.0 H.0 O.NH 0.0N N 0.0 O H N 0 MH
O.5 0.M 0.0H 0.0N N 0 O N N 5 NH
M.0 M.0 O.5H 0.0M M 0.5 N H 0 0 HH
M.0 M.0 o.HN O.N0 0 0 O O 0 0 OH
M.0 M.0 0.0N 0.00 0 0 N O 0 0 0
0.0 0.0 0.NM 0.00 0.0 0 H N 5 OH 0
0.0 H.0 0.0M O.H5 5 NH O 0 5 0H 5
0.0 0.0 O.50 O.50 0.5 0H H 0 0 0H 0
0.0 0.0 0.00 0.00H 0 HN N 0 OH 0N 0
5.0 M.0 0.00 0.0MH 0.NH 0N 0 0 0H NM 0
0.N 5.M 0.N0 0.05H 0N 0.0M 0H 0H MM 50 M
N.N 0.M 0.00H O.HMN 00 0.00 OM 5H M0 00 N
O.M 0.0 0.00N 0.00N 00 00 0 O 00 00 H
00H0z 00H0E0m 00H0z 00H0E0m 00H0z 00H0E0m 00H0z 00H0E0m 00H0z 00H0E0m Axv

xv x 0000

.Inn" 0 0oc0uo0mx0 x x x x x x

x9 9 00>HH 0000 00.. NJ" A 00>HH 0 "x 00 v "x 00 H0>u0ucH
00HH 00>u0mno 00 00985: H0009 0000 00 H0nfisz 0GH00 “00652 0>HH0 H0nfisz 000

“000.0442 20 UZHDmmm 0mg 02.4 mmgmm mHmszdz<m hm 05.0mm 0m08<mom§ mo 0BH>muzOH .m xHszmm<

118

 

 

 

 

 

 

 

 

 

0.0 0.0 0.0 0 0.0 0 H NH H NH M
0.0 0.0 0.0N 00 0N 00 00 NM 00 00 N
M.H 0.H 0.00 0.0HH 0.00 0.00 5H H 00 00 H
00H02 00H0E0m 00H0z 00H0E0m 00H02 00H0E0m 00H0z 00H0E0m 00H0z 00H0E0m CC
x
.lvux0 000000090 x x x 0000
x9 x9 00>HH 0000 000 I vu .H 00>HH x0 "x H0 xv "x H0 H0>H0HGH
00HH 00.50000 00 H0850 H0009 0000 00 H052 0GH00 H052 0>HHO H052 004
000000 00000 90009:» 00052 02.4 00199200 302020240 .m 0.00de 000940005 .00 09H>m02QH .0 03020ng

"L
r. 0 5.4 ...IF...n. h I..$w.u III

 

.0H5000E 00 HH080 000 050 0c000Hm 000000 .000H0HE CH 00H5000E 00000 0000 H0000 HH<0

.0H000EHHHHE 0H 00H5000E 00000 H00s00 000 £0000H 00000

 

119

 

 

0-- Anumv 0 “00-00 m AmH-HHV mH 00000 H00000Houuc0> .0000
Ammuomv mm 200-000 00 Amnumov Hm Aanummv FHH 00000 H00000Houuc0> .000
Amumv m AHaumv m AmH-VHv 0H onuomv mm 00000 H00000 .0x0 0000
“o-Mv m AmHumv OH AmHumHv 0H Aomnwmv mm 00000 000000 .000 .00om
“HH-0V 0 AMHumv OH 00H-OHV MH A0NIOHV 0H 00000 .00000 000Hsonm .0000
Ammuvmv mm Anm-0mv mm Avvummv 0m Amnunmv m0 00000 Hmmuo0 .0x0 .000
.. AnmumHv 0N onummv mm Amoumvv 00 00000 H00000 .0cH .004
"x0Hon00Hm
AMHuoHV HH Amm-mHV 0H Amm-Hmv mm Anv-mmv 0m 00000 H0200 .0c0>
2mm-nmv 0N Amvummv m0 200-000 00 AMHHumnV 00 00000 H0000 .000
Amvummv ow Aomumvv 00 Amoummv mm 200-000 om 00000 00000 .0000
Amm-m~v mm Aomuovv 00 AHmummv 00 AMHHumnv 00 00000 H0000H0
Ammumav mm 20m-mmv mm Ammumqv om AmOHquV om 00000 H000000> .moo
Avmuomv mm Anvummv 00 “00-000 H5 AvaumOHv HNH 00000 H0uaoum .0000
200.000 m0 AHnumov 00 AHHHnmmv 00H AmanHmHv 00H 00000 H000000> .000
“00-000 00 200-000 00 AvaumHHv 0H0 AmmHlooHv NFH 00000 Hmucoum .000
Amm-0mv mm Amq-0mv ow Anmummv mm 000-000 H» 000:0H Hmac00c0
“00-000 n0 AmmHumHHv 0mH AmmH-HmHv moH AmomumHmv 00m 00003 0000
AHOH-000 00 Aan-0HHV 00H AHHN-MOHV 00H Ammmumvmv mom QAJV 000000 000m
"000m
“000.0-nmm.ov 20mm.H-mmm.Hv AmHv.~-om0.Hv Amm0.mummm.~v
“mm.o m~0.H mHH.m mnm.m 0Aesv 000:0H 0000
00HHm 000000 0HH£B £0Hnom

 

AH0000H £000 H00 00H0000E 000EH0000 OHV H0000H
A0000HV £0mC0H 0m0H0>4

 

mHmzm2¢Z¢m um m0 mmwflfim mmDB€SZH mmB m0 mezmzmmbmdmz Adxmeoemdmo Q24 000m .3 anzmmmd

.mudmmme on HHmEm oou won ucmmmum mmummm

 

120

 

 

 

 

Hmumv v Anumv o AHHan 0H AmHumHv mH mmumm .mmoom .muoo
AmmuHmv «m ANqumv mm AHNummv om ANNHummV moH mmumm HmumumHOmuoa
Amnov N AVHumV HH AanvHv mH AvquHNv mN mmumm Hmmuov .uxm
ANIVV o AmHumv HH AmHuHHV mH AnmumHv mN mmumm Hamuoo .ucH
"NIH mucmemmm .cHEO©Q¢
mu- Anumv m AqHqu m HmHumv NH mmumm .mmoom cmvas .ucm>
Annmv m AMHumv 0H ANHuHHV «H AHmumHv «N mmumm .wmaumucH .ucm>
AmHanv oH AmmnoNv mN Avvnva mm HHmnmov NN mmuwm .ucm> .uxm
AVHuov HH AoNumHv NN HomumHv mN Homuva Hv mmumm .ucm> .ucH
u- AmNuNHV HN Amonva mm ANNHuHmV vHH mmumm HmumumHoupcm> .umom
AoN-NHv NN Amvummv Hv Amnummv mo ANvHuVOHV mNH mmumm HmumumHouucm> .u:<
Avmuva mN Aomuva mN Avv-mmv mm Anmnmmv NN mmuwm HmumumHomyoo
Amumv N AmH10HV NH AmNuoHv mH ANVIHNV hm mmumm Hmmuoc .uxm
Hulmv o AmH-0HV NH AmNIoHV mH Ammuva vm wmumm Hmmuou .ucH
Hmumv m AOHuNV m ANHIOHV NH AwNumHv NH mmumm .mmoum .muoo
"XMHOSumuwS Ucm Omwz
mu- Amuvv o AHNumv NH ANNanv 0N wmumm .mmoom :chme .ucw>
Ahuvv m HNHumv m AmHIOHV NH ANNumHv NN mmumm .wmeumuaH .ucm> .umom
HmHumHv oH ANmumNV NN Hov-NNv mm Homnmqv mo mmumm .ucw> .uxm .umom
HmHan NH AmNumHV NN ANNuHNV mN “HouHmv Nv mmpmm .ucm> .ucH .pmom
HoHumv NH AVN-HNV 0N ANmuvmv ow Annummv mo wmumm .u:m> .uxm .uca
AHHnov m ANNumHV HN onnva Nm Ammuovv om mmumm .ucm> .ucH .ucm
HmHunv o AHN-oHv mH Ammuomv mm HNmnmmv mo mmumm HmumumHomuoo
.uCOO meOSUOHm
umufim ocoomm GHHQB Suusom

 

umumcH
Hmmcmuv mnumcmH mmmum>d

 

wmscHHGOUIIm xHozmmmd

.mndmmme ou HHmEm oou pan ucmmmnm mmummm

 

Ahmm.HlonH.Hv Ammm.alhmm.av Ammb.mlmmm.mv AvhH.vlmom.mv

121

 

 

 

OON.H vom.H vmv.N mom.m mmumm Hawsmu
AOmIHmO mm HNmumNO Om Hmvummv mv Amoummv mo mmumm Hmcsmu .uxm
AmNuOHV NN AOmumNO mm HmqumO mm Ammumvv NO mmumm Hmwsmu .ucm>
AmH-OO HH AvNumHv ON HovumNO vm AHmuOmO Om mmumm Hmcm mum .uxm
AHHumO O AmHsNHO OH HHmuOHO vN Hmvuva mm mmumm Hmcm mum .ucH
Ammummv «O ANOuOmO mm HOHHuNOHO mHH AmmHumNHO HmH mmumm Hmcm umom .uxm
AOHuHHO mH AONIOHO HN AmmuHNO ON AmmuHmv Nv mmumm Hmcm umom .ucH
HomuHNv ON Amm-ONV Hm AOmummO mq AmmuNmO Nm mmumm Hmcm .uxm

mun AmaNO N AmnNO w Amumv m mmumm .OmeumucH .ucm>

Honmv v AHHuOO O HOHuOO NH AmNuHHO ON mmumm .ucm> .uxm
Aquv m Amuvv O HmHumO m AOHuNHO mH mmuwm .ucm> .ucH
AOHuOHO mH AmNumHO OH AHmumHv mN AvvuONO Om mmumm HmumumHouucm> .uc4
HOmumNO Hm vaummv NO Ammuvmv NO AmOHquO vm mmumm HmumumHomuoO
Amnmv m Avmumvv mv AHOnHmv Om AOVHINOHV HNH mmumm Hmmuoc .uxm
AHHIOV O AmHuOO OH AOHuHHO OH HOHumHV OH mmumm Hmmuoo .ucH
“mam mucwemwm .cHEownm

AmN-OHO OH AmHan OH HmNuNHO OH HmqumO Nv mmumm .ucm> .uxm
AmHumv HH AvmuNNO ON AmmnHNO Nov AmmuOmO Nm mmumm .u:m> .ucH
u: ANqumO mm HHmummv mo AOVHnOOHO «NH mmumm HmumuMHouucm> .umom
HON-OHO NN Homivvv ow AHmummv Om ANvHuvmv OHH mmumm HmumumHouucm> .uc4
.ucoouIOIH mucmEbmm .Eovnd
umuHm vsoomm UHHse suusom
HmumcH

Ammcmuv nuocmH mmmum>¢

 

mmscflucoollm xHozmmm<

LITERATURE CITED

LITERATURE CITED

Addis, C. J. 1945. Laboratory rearing and life cycle
of Phlebotomus (Dampfomyia) anthophorus Addis.

J. Parasit. 31: 319-322.

 

 

Adler, S., O. Theodor, and G. Witenberg. 1938.
Investigations on Mediterranian Kala-azar.
XI. A study of leishmaniasis in Canea (Crete).
Proc. Roy. Soc. B. 125: 491-516.

Ashner, M. 1927. Observations on the breeding of
Phlebotomus papatasii. Trans. Roy. Soc. Trop.

Barretto, M. P. 1940. Morphologia dos ovos das larvas e
das pupas de Phlebotomus intermedius Lutz e Neiva,
1912 (Diptera, Psychodidae). An. Fac. Med. Univ.
Sao Paulo 16: 91-105.

1941. Morphologia dos ovos e das pupas de alguns
flebétomos de Sao Paulo. An. Fac. Med. Univ.
Sao Paulo 17: 357-427. '

1942. Contribuicao para o estudo da biologia dos
flebotomos em condicoes experimentais. Tese de
Don toramento, Facultade de Medicina da Universidade
de Sao Paulo, Cadeira de Parasitologia 162 pp.

1946. Uma nova especie de flebétomos da Colunbia, e
chave para a determinacao das especies afins
(Diptera, Psychodidae). An. Fac. Med. Univ.

Sao Paulo 22: 279-293.

1947. Catalogo dos flebotomos americanos. Arq. Zool.
Est. Sao Paulo 5: 177-242.

1950. Nova contribuicao para o estudo da distribuicao
geographica dos flebotomos americanos (Diptera,
Psychodidae). Arq. Hyg. Safide. Pub1., Sao Paulo
15: 211-226.

122

123

1955. Sabre a sistematica da subfamilia Phlebotominae
Rondani. Rev. Brazil Ent. 3: 173-190.

1962. Novos subgéneros de Lutzomyia Franca, 1924
(Psychodidae, subfamilia Phlebotominae). Rev.
Inst. Med. Trop. Sao Paulo 4: 91-100.

 

1966. Estudos sobre flebotomineos americanos.
I. Notas sdbre espécies de Lutzomyia Franca,
subgénero Psychodopygus Mangabeira 1941, com
a descricao de uma nova especie (Diptera,
Psychodidae). Papéis Avulsos 18: 133-146.

 

 

Barretto. P. 1969. Artropodos hematéfogos del Rio Raposo,
Valle, Columbia. IV. Psychodidae. Caldasia
10:459-472.

Biagi-F., F., and A. Ma de B. de Biagi. 1953. Algunos
flebétomos del area endemica de leishmaniasis
tegumentaria americana del estado de Campeche
(México). Medicina (México) 23: 315-319.

Biagi-F., F., A. Ma de B. de Biagi, y H. F. Beltran. 1965.
Phlebotomus flaviscutellatus, transmisor natural de
Leishmania mexicana. Medicina (México) 30: 267-272.

 

 

Carneiro, M., and I. A. Sherlock. 1964. Estudo
morfologico sébre as pupas de Phlebotominae (Diptera:
Psychodidae). Rev. Bras. Malariol. e Doenc. Trop.
16: 311-317.

Chaniotis, B. N. 1967. The biology of California
Phlebotomus under laboratory conditions.
J. Med. Ent. 4: 221-233.

 

1974. Sugar-feeding behavior of Lutzomyia trapidoi
(Diptera:Psychodidae) under experimental condi-
tions. J. Med. Ent. 11: 73-79.

1974. Personal communication.

Chaniotis, B. N., and J. R. Anderson. 1964. Notes on
the morphology and laboratory rearing of Phlebotomus
vexator occidentis (Diptera:Psychodidae). Pan-Pac.
Entomol. 40: 27-32.

 

Chaniotis, B. N., and M. A. Correa. 1974. Comparative
flying and biting activity of Panamanian Phleboto-
mine sandflies in a mature forest and adjacent open
space. J. Med. Ent. 11: 115-116.

124

Chaniotis, B. N., M. A. Correa, R. B. Tesh and K. M.
Johnson. 1971. Daily and seasonal man-biting
activity of phlebotomine sandflies in Panama.
J. Med. Ent. 8:415-420.

Chaniotis, B. N., R. B. Tesh, M. A. Correa and K. M.
Johnson. 1972. Diurnal resting sites of
phlebotomine sandflies in a Panamanian tropical
forest. J. Med. Ent. 9: 91-98.

Chaniotis, B. N., J. M. Neely, M. A. Correa, R. B. Tesh
and K. M. Johnson. 1971. Natural population
dynamics of Phlebotomine sandflies in Panama.
J. Med. Ent. 8: 339-352.

Christensen, H. A. 1972. Colonization of Lutzomyia
trinidadensis and Lutzomyia vespertilionis
(Diptera:Psychodidae). Ann. Ent. Soc. Amer.
65: 683-686.

 

Christensen, H. A., A. Herrer and S. R. Telford, Jr. 1969.
Leishmania Braziliensis 5. lat., isolated from
Lutzomyia panamensis in Panama. J. Parasit.

55: 1690-1091.

 

 

1972. Enzootic cutaneous leishmaniasis in eastern
Panama. II. Entomological investigations. Ann.
Trop. Med. Parasit. 66: 55-66.

ChristOphers, S. R., H. E. Shortt, and P. J. Barraud. 1926.
Technique employed in breeding Phlebotomus argentipes
in Assam. Ind. Med. Res. 4: 173-175.

Coutinho, J. 0., and M. P. Barretto. 1941. Dados
bionomicos sdbre o Phlebotomus fisheri Pinto 1926.
Rev. Brasil. Biol. 1: 423-429.

Deane, L. M., and M. P. Deane. 1957. Observacoes sobre
abrigos e criadouras de flebotomos no noreste do
Estado do Ceara. Rev. Brasil. Malariol. Doencas
TrOp. 9: 225-246.

De Biagi, A. Ma de B. 1966. Clave para identificacion
rapida de 1as hembras de Phlebotomus anthropofilos
del area endémica de leishmaniasis cutanea en
México. Rev. Invest, Salud. Pfibl. (México)

26: 367-373.

 

125

De Biagi, A. Ma de B., H. F. Beltran y F. Biagi-F. 1966.
Nuevos conocimientos sobre los flebotomus del area
endémica de leishmaniasis cuténea en Yucatan. Rev.
Invest. Salud. Publ. (México) 26: 139-153.

Disney, R. H. L. 1966. A trap for phlebotomine sandflies
attracted to rats. Bull. Ent. Res. 56: 445-451.

1968. Observations on a zoonosis:Leishmaniasis in
British Honduras. J. Appl. Ecol. 5: 1-59.

Downes, J. A. 1958. The feeding habits of biting flies
and their significance in classification. Ann.
Rev. Ent. 3: 249-266.

Dyar, H. G. 1929. The present knowledge of the American
species of Phlebotomus Rondani. Amer. J. Hyg.
10: 112-124.

 

Eldridge, B. F., J. E. Scanlon and I. M. Orenstein. 1963.
Notes on the laboratory rearing of sandflies
(Diptera:Psychodidae). Mosq. News 23: 215-217.

Fairchild, G. B. 1955. The relationships and classifi-
cation of the Phlebotominae (Diptera:Psychodidae).
Ann. Ent. Soc. Amer. 48: 182-196.

1951. Notes on the Phlebotomus of Panama (Diptera:
Psychodidae). VII. The subgenus Shannonomyina
Pratt. Ann. Ent. Soc. Amer. 44: 399-421.

 

1959. Geographic distribution of the Phlebotomus
sandflies of Central America (Diptera:Psychodidae).
Ann. Ent. Soc. Amer. 52:121-124.

 

1961. Three new species of Phlebotomus from Mexico and
Nicaragua (Diptera:Psychodidae). Proc. Ent. Soc.
Wash. 63: 22-28.

 

Ferreira, L. C., L. Deane and F. O. Mangabeira. 1938.
Sabre a biologia dos flebotomos no noreste do
Estado do Ceara. 0 Hospital (Rio de Janeiro)
14: 3-6.

Forattini, O. P. 1954. Algumas observacoes sébre a
biologia de flebotomos (Diptera:Psychodidae)
em regiao da bacia do rio Parana (Brasil).
Arq. Fac. Hig. Safide Publ. Univ. Sao Paulo
8: 15-136.

126

1971. Sabre a classificacao da subfamilia Phlebotominae
nas Américas (Diptera:Psychodidae). Pap. Avulsos
2001., Sao Paulo 24: 93-111.

Gemetchu, T. 1971. Liver and yeast as larval diets in
colonization of a sandfly (Phlebotomus longipes).
Trans. Roy. Soc. Trop. Med. Hyg. 65: 682-683.

 

Guitton, N., and I. A. Sherlock. Descricao das fasas
imaturas do Phlebotomus longipalpis Lutz e Neiva,
1912 (Diptera:Psychodidae). Rev. Bras. Biol.
29: 383-389.

 

Hanson, W. J. 1961. The breeding places of Phlebotomus
in Panama (Diptera:Psychodidae). Ann. Ent. Soc.
Amer. 54: 317-322.

 

1968. The immature stages of the subfamily
Phlebotominae in Panama (Diptera:Psychodidae).
Ph.D. Dissertation, Univ. of Kansas. 104 pp.

Hafez, M., and K. Zein-el-dine. 1964. Culturing
Phlebotomus papatacci (Scopoli) in the laboratory.
Bull. Ent. Res. 54: 657-659.

 

Hertig, M. 1942. Phlebotomus and Carrion's disease.
Suppl. Amer. J. Trop. Med. 22: 1-81.

 

Hertig, M., and G. B. Fairchild. 1950. Notes on the
Phlebotomus of Panama. V. The second sternite
as a taxonomic character. Proc. Ent. Soc. Wash.
52: 91-95.

 

Hertig, M., G. B. Fairchild and C. M. Johnson. 1959.
Leishmaniasis transmission-reservoir project.
Rep. Gorgas Mem. Lab. 31: 11-15.

Hertig, M., G. B. Fairchild, C. M. Johnson, P. T. Johnson,
E. McConnell and W. J. Hanson. 1960. Leishmaniasis
transmission--reservoir studies. Rep. Gorgas Mem.
Lab. 32: 5-11.

Hertig, M., and P. T. Johnson. 1961. The rearing of
Phlebotomus sandflies (Diptera:Psychodidae).
I. Technique. Ann. Ent. Soc. Amer. 54: 753-764.

 

Hinton, H. E. 1968a. Observations on the biology and
taxonomy of the eggs of Anopheles mosquitoes.
Bull. Ent. Res. 57: 495-508.

 

127

1968b. Structure and protective devices of eggs of
the mosquito Culex pipiens. J. Insect Physiol.
14: 145-161.

 

Hinton, H. E., and M. W. Service. 1969. The surface
structure of aedine eggs as seen with the scanning
electron microsc0pe. Ann. TrOp. Med. Parasit.

63: 409-411.

Iriarte, D. R. 1952. Lista de los flebotomos sefialados
en Venezuela, hasta ahora. Bol. Lab. Clin. Luis
Razetti 12: 487-490.

Johnson, P. T., and M. Hertig. 1961. The rearing of
Phlebotomus sandflies. II. Development and
behavior of Panamanian sandflies in laboratory
culture. Ann. Ent. Soc. Amer. 54: 764-776.

 

Johnson, P. T., E. McConnell and M. Hertig. 1962. Natural
and experimental infections of leptomonad flagel-
lates in Panamanian Phlebotomus sandflies. J.
Parasit. 48: 158.

 

1963. Natural infections of leptomonad flagellates
in Panamanian Phlebotomus sandflies. Exp. Parasit.
14: 107-122.

 

Lewis, D. J., and P. C. C. Garnham. 1959. The species of
Phlebotomus (Diptera:Psychodidae) in British
Honduras. Proc. Roy. Ent. Soc. Lond. (B) 28: 79-89.

 

Lewis, D. J., and C. R. Domoney. 1966. Sugar meals in
Phlebotominae and Simulidae. Proc. Roy. Ent. Soc.
Lond. (A) 41: 175-179.

McCombie Young, T. C., A. E. Richmond and G. R. Brendish.
1926. Sandflies and sandfly fever in the Peshawar
District. Ind. J. Med. Res. 13: 961-1021.

Mangabeira, F. O. 1942a. 8a. Contribuicao ao estudo
dos Flebotomus (Diptera:Psychodidae). Flebotomus
(Brumptomyia) avellari Costa Lima, 1932. Mem. Inst.
Oswaldo Cruz 37: 225-240.

 

 

 

1942b. 9a. Contribuicao ao estudo dos Flebotomus

(Diptera:Psychodidae). Flebotomus (Pressatia)
tricanthus Mangabeira, 1942. Mem. Inst. Oswaldo
Cruz 37: 241-250.

 

 

 

 

128

1942c. 10a. Contribuicao ao estudo dos Flebotomus
(Diptera:Psychodidae). Flebotomus longispinus
Mangabeira, 1942. Mem. Inst. Oswaldo Cruz 37:
251-257.

 

 

1942d. 11a. Contribuicao ao estudo dos Flebotomus
(Diptera:Psychodidae). Flebotomus oswaldoii
Mangabeira, 1942. Mem. Inst. Oswaldo Cruz 37:
287-295.

 

 

l942e. 13a. Contribuicao ao estudo dos Flebotomus
(Diptera:Psychodidae). Flebotomus (Brumptomyia)
travassosi Mangabeira, 1942. Mem. Inst. Oswaldo
Cruz 37: 375-381.

 

 

Mangabeira, F. 0., and I. A. Sherlock. 1962. SObre o
Phlebotomus brasiliensis Costa Lima, 1932
(Diptera:Psychodidae). Mem. Inst. Oswaldo Cruz
60: 311-319.

 

Martins, A. V., A. L. Falcao y J. E. da Silva. 1963.
Notas sabre os flebotomos do territorio de Roraima,
com a descricao de trés novas espécies (Diptera:
Psychodidae). Rev. Brasil. Biol. 23: 333-348.

Matsuo, K., Y. Yoshida, and I. Kunou. 1972. The scanning
electron microscopy of mosquitoes. I. The egg

surfaces of five species of Aedes and Armi eres
subalbatus. J. Kyoto Pref. Univ. Med. 81: 358-363.

 

Matsuo, K., Y. Yoshida and J. C. Lien. 1974. Scanning
electron microscopy of mosquitoes. II. The egg
surface structure of 13 species of Aedes from
Taiwan. J. Med. Ent. 11: 179-184.

Mesghali, A., and M. Lofti. 1968. The rearing of sandflies
in the laboratory. Bull. Soc. Path. Exot. 61:
797-800.

Mirsa, A. 1952. El desarrolo de Phlebotomus panamensis
Shannon, 1926, y Phlebotomus gomezi Nitzulescu,
1931, (Diptera:Psychodidae) en condiciones de
laboratorio. Rev. Sanidad Assist. Soc. Caracas
16: 561-572.

 

Morales-Alarcén, A., E. Osorno-Mesa y F. de Osorno. 1969.
Phlebotominae de Columbia (Diptera:Psychodidae).
II. Sobre algunos Phlebotomus de los llanos
orientales. Caldesia 10: 377-382.

 

129

Mukerji, S. 1931. The process of copulation in
Phlebotomus argentipes Ann. and Brun. Parasit.
23: 443-444.

 

Néjera, A Diaz. 1963. Phlebotomus de México descripcion
de la hembra de Phlebotomus (Brumptomyia) alindoi
Fairchild and Hertig, 1947; Datos de distr1buc1 n
geogréfica (Diptera:Psychodidae). Rev. Inst.
Salubr. Enferm. Trop. (México) 23: 193-199.

 

 

 

Ortiz, I. 1950. Notas en Flebotomus. I. Sobre 1a armadur
a genital del macho de Flebotomus panamensis
Shannon. Arch. Venez. Patol. Trop. Parasit. Med.
2: 83-88.

 

 

Osorno-Mesa, B., A. Morales-Alarcon y F. de Osorno. 1967.
Phlebotominae de Columbia (Diptera:Psychodidae).
I. Distribucién geogréfica da especies de
Phlebotomus registradas con algunas anotaciones
biologicas y descripcion de una nueva. Caldasia
10: 27-38.

 

Pifano, F. C. 1941. La leishmaniasis tegumentaria en el
Estado Yaracuy, Venezuela. Gaceta Med. Caracas
48: 292-299.

Pifano, F. C. y I. Ortiz. 1952. Representes venezolanos
del género Phlebotomus Rondani, 1840. Rev. San.
Asis. Soc. 7: 135-151T

 

Pifano, F. C., I. Ortiz y A. Alvarez. 1960. La ecologia,
en condiciones naturales y de laboratorio, de
algunas especies de Phlebotomus de la region de
GuatOpe, Estado Miranda, Venezuela. Arch. Venez.
Med. Trop. Parasit. Med. 3: 63-71.

 

Pifano, F. C., A. Alvarez, I. Ortiz, C. Dagert y J. V.
Scorza. 1959. Phlebotomus panamensis Shannon,
1926: transmisor de la Leishmaniasis tegumentaria
en Venezuela. Gaceta Medica 67: 229-235.

 

Pratt. H. D. 1947. Shannonomyina new name for Shannonomyia
Dyar (not Alexander) (Diptera:Psychodidae). Proc.
Ent. Soc. Wash. 49: 86.

 

 

Root, F. M. 1934. Some American species of Phlebotomus
with short terminal palpal segments. Amer. J. Hyg.
20: 233-246.

 

130

Roubaud, E., and J. Colas-Belcour. 1927. Recherches
biologiques sur les phlebotomes de la Tunisie
de Nord. Arch. Inst. Pasteur Tunis 16: 59-80.

Rutledge, L. C., and H. L. Mosser. 1972. Biology of
immature sandflies (Diptera:Psychodidae) at the
bases of trees in Panama. Environ. Ent. 1:300-309.

Rutledge, L. C., and D. A. Ellenwood. 1975a. Production
of phlebotomine sandflies on the open forest floor
in Panama: Phytologic and edaphic relations.
Environ. Ent. (In press.)

1975b. Production of phlebotomine sandflies on the
open forest floor in Panama: Hydrologic and
physiographic relations. Environ. Ent. (In
press.)

1975c. Production of phlebotomine sandflies on
the open forest floor in Panama: The species
complement. Environ. Ent. (In press.)

Sabin, A. B., C. B. Philip, and J. R. Paul. 1944.
Phlebotomus (pappataci or sandfly) fever.
J. Amer. Med. Assoc. 125: 603-606.

Safianova, V. M. 1963. Laboratory cultivation of
sandflies. Bull. Wld. Hlth. Org. 31: 573-576.

Schmidt, M. L. 1964. A laboratory culture of two
Phlebotomus species, P. papatasii and P. orientalis.
Bull. Wld. Hlth. Org. 31:577-578.

 

 

 

Shannon, R. C. 1926. The occurrence of Phlebotomus in
Panama. J. Wash. Acad. Sci. 16: 190-193.

 

Sherlock, I. A. 1957a. Sdbre o Phlebotomus lenti
Mangabeira 1938 (Diptera:Psychodidae). Rev.
Brasil. Biol. 17: 77-88.

 

1957b. Sobre o Phlebotomus renei Martins, Falcao and
da Silva 1956 (Diptera:Psychodidae). Rev. Brasil
Biol. 17: 547-556.

Sherlock, I. A., and V. A. Sherlock. 1959. Criacao e
biologia, em laboratorio do Phlebotomus longipalpis
Lutz and Neiva, 1912 (Diptera:Psychodidae). Rev.
Brasil. Biol. 19: 229-250.

 

 

131

1972. Métodos précticos para criacao de flebotomineos
em laboratorio. Rev. Brasil. Biol. 32: 209-217.

Sherlock, I. A., and M. Carneiro. 1963. Descricao das
fases imaturas do Phlebotomus bahiensis Mangabeira
and Sherlock, 1961 (Diptera:Psychodidae). Mem.
Inst. Oswaldo Cruz 61: 491-494.

 

Shortt, H. E., P. J. Barraud and C. S. Swaiminath. 1926.
Further observations on the breeding behavior of
Phlebotomus argentipes in Assam. Ind. J. Med. Res.
13: 943-946.

Smith, R. O. A. 1925. Note on a simple method of breeding
sandflies. Ind. J. Med. Res. 12: 741-742.

Smith, R. O. A., K. C. Halder and I. Ahmed. 1940. Further
investigations on the transmission of Kala-azar.
Part I. The maintenance of sandflies Phlebotomus
argentipes on nutriment other than blood. Ind. J.
Med. Res. 28: 575-579.

 

Snedecor, G. W., and W. G. Cochran. 1973. Statistical
Methods. The Iowa State University Press, Ames,
Iowa. 593 pp.

Tesh, R. B., B. N. Chaniotis, M. D. Aronson and K. M.
Johnson. 1971. Natural host preferences of
Panamanian phlebotomine sandflies as determined
by precipitin test. Amer. J. Trop. Med. Hyg.
20: 150-156.

Tesh, R. B., B. N. Chaniotis, B. R. Carrera and K. M.
Johnson. 1972. Further studies on the natural
host preferences of Panamanian phlebotomine
sandflies. Amer. J. Epidemiol. 95: 88-93.

Thatcher, V. E. 1968a. Studies of phlebotomine sandflies
using castor oil traps baited with Panamanian
animals. J. Med. Ent. 5: 293-297.

1968b. Arboreal breeding sites of phlebotomine
sandflies in Panama. Ann. Entomol. Soc. Amer.
61: 1141-1143.

Thatcher, V. E., and M. Hertig. 1966. Field studies on
the feeding habits and diurnal shelters of some
Phlebotomus sandflies (Diptera:Psychodidae) in
Panama. Ann. Ent. Soc. Amer. 59: 46-52.

 

132

Theodor, O. 1948. Classification of the Old World species
of the subfamily Phlebotominae (Diptera:Psychodidae).
Bull. Ent. Res. 39: 85-115.

1965. On the classification of American Phlebotominae.
J. Med. Ent. 2: 171-197.

Unsworth, K., and R. M. Gordon. 1946. The maintenance of
a colony of Phlebotomus papatasii in Great Britain.
Ann. Trop. Med. Parasit. 40: 219-227.

 

Vargas, L., and A. Diaz Najera. 1953. Lista de flebotomos
mexicanos y su distribudion geografica (Diptera:
Psychodidae). Rev. Inst. Salubr. Enf. Trop. Mexico
13: 309-314.

Velasco, J. E. 1973. The phlebotomine sandflies of the
Los Yungas Region of Bolivia. Master's Thesis.

Louisiana State University. 204 pp.

Ward, R. D. 1972. Some observations on the biology and
morphology of the immature stages of PsychodOpygus
wellcomei Fraiha, Shaw and Lainsom, 1971 (Diptera:
Psychodidae). Mem. Inst. Oswaldo Cruz 70: 15-28.

 

Waterston, J. 1922. A contribution to the knowledge
of the bionomics of sandflies. Ann. Trop. Med.
Parasitol. 16: 69-92.

Whittingham, H. E., and A. F. Rook. 1922. Demonstration
of the life history of Phlebotomus papatasii and
its maintenance in captivity. Trans. Roy. Soc.
Trop. Med. Hyg. 16: 262-266.

Williams, P. 1965. Observations on the phlebotomine
sandflies of British Honduras. Ann. Trop. Med.
Parasit. 59: 393-404.

1966a. The distribution on the human body of bites
inflicted by phlebotomine sandflies in British
Honduras. Ann. Trop. Med. Parsit. 60: 219-222.

1966b. The biting rhythms of some anthropophilic
phlebotomine sandflies in British Honduras.
Ann. Trop. Med. Parasit. 60: 357-364.

1970a. On the vertical distribution of phlebotomine
sandflies in British Honduras. Bull. Ent. Res.
59: 637-646.

133

1970b. Phlebotomine sandflies and leishmaniasis in
British Honduras (Belize). Trans. Roy. Soc. TrOp.
Med. Hyg. 64: 317-368.

Wolbach, S. B., J. L. Todd and F. W. Palfrey. 1922.
Etiology and Pathology gf_Typhus. Cambridge,
Mass.

 

 

Young, C. W., and M. Hertig. 1926. The development of
flagellates in Chinese sandflies (Phlebotomus)
fed on hamsters infected with Leishmania donovoni.
Proc. Soc. Exptl. Biol. Med. 23: 611-615.

 

 

    

T

”'Ttililfit7lmflfl(TuLi):i@fy)))fl@fia)l“

L