J.

Mod. Entom.ol.

Vol. If,

DO.

10 February 1978

5: 545-552

THE FEEDING HABITS OF LABORATORY-BRED LUTZOMYIA LONGIPALPIS (DIPTERA: PSYCHODIDAE)1 By P. D. Ready2 Abstract: Female sand flies from a laboratory colony of Llltzoll!J'ill IOllgiPalpis Lutz & Neiva were offered various meals through membranes and from free solutions. Their feeding behavior was compared with that observed on living hosts. Flies fed sucessfully through membranes; over 50% ingested nearnormal amounts of blood fluids. Host breath was needed to stimulate host/membrane searching activity, but warmth alone then stimulated probing. Ingestion was initiated morc consistently through chick skin than Parafilm 'M.' The properties of the meal interacted with those of the membrane to maintain ingestion. Compared with the known membrane-feeding behavior of mosquitoes, that of L. IOllgiPalpis was unselective. Isotonic saline was ingested as avidly as whole blood through chick skin, and the addition of ATP to suspensions of serum proteins had no phagostimulatory effect. The method of feeding appears to be more important than the nature of the food in determining the destination of the meal in this sand fly. Food ingested through membranes was sent predominantly to the midgut, whereas that imbibed from free solutions was usually dispatcht.-d to the crop; a possible regulating mechanism is discussed. The effect of this control of food destination on the l'stablishment of Leishmallia in the fly is considered,

A colony of Lutzomyia longipalpis Lutz & Neiva (Psychodidae: Phlebotominae), a neotropical vector of dermal and visceral leishmaniasis (Lewis 1971), was established at Imperial College Field Station in 1972. Culture methods have been fully described (Killick-Kendrick et al. 1977). As part of a study of the fecundity of this species, a method was sought to feed test solutions to female flies artificially. At that time (spring 1975) Dr Boorman's team at the Animal Virus Research Institute, Pirbright, England, started experiments involving the infection of L. 107lgipalpis (from the above colony) with viruses. The membrane-feeding technique used routinely in their laboratory to offer blood meals to Culicoides (Mellor 1971) was adopted for both studies. Observations on the feeding habits of L. longipalpis made during the fecundity experiments and in the course of routine culture are reported in this paper. They form a basis for future investigations of sand fly ingestion, of which little is known (Friend & Smith 1977), although it may influence vectorial capacity. In view of the difficulty many investigators have experienced in feeding sand flies through membranes, 'This paper describes work carried out for a thesis approved for the Ph.D. degree of London University. 'Department of Zoology and Applied Entomology, Imperial College Field Station, Silwood Park, Ascot, Berks., England. Reprint requests to 6, Delme Drive, Fareham, Rants., England.

from Napier (1930) to Gemetchu the method are given in full. MATERIALS

(1976), details of

AND METHODS

Stock On the day of emergence adult flies were released into fine-gauze cages (18 cm3). Each cage was filled with 60-100 flies released over 1-3 days. The ratio of males to females was adjusted to 1.2: 1.0 per cage (the average ratio per generation). Each cage was sealed in a plastic bag containing damp cotton wool to maintain a high humidity. A 30% (wtjvol.) sucrose solution was provided on cotton wool in a dish placed on the floor of the cage. AIeasurement of feeding response This was measured in 4 ways: (a) proportion observed probing (a membrane); (b) proportion imbibing some of the test fluid, as measured by distension of the abdomen and increased postfeed weight; (c) quantity of test fluid ingested, where the fly was narcotized with CO2, then weighed on a Beckman microbalance shortly before and after a meal; and (d) location of ingested fluid in the fly, as observed after dissection under a binocular mIcroscope. Feeding methods Routine culture. Female flies were offered blood meals from a human arm or an anesthetized hamster (0.4 mg Nembutal per 10 g body weight, given intraperitoneally) placed in the rearing cage. Membrane feeds. Immediately after weighing, small groups of females of approximately equal weight (±0.005 mg) were introduced into a pill box (6-cm diam.) fitted with a nylon stocking top. Before feeding, these were left in an illuminated incubator at 25 100% RH for 15 min. to recover from the CO2 narcotization used to facilitate weighing. The recovery period at high humidity is essential if the majority are to feed. Preliminary observations showed that up to 7 females could engorge on human blood in 15 min., but groups of only 2-5 were used to ensure that there was ample time for all to feed. A IS-min. feeding period was chosen as the maximum possible without significant weight changes occurring as a result of

ac,

546

J. Moo.

diuresis. Little or no fluid was excreted by the females while feeding. The feeding unit was designed by Mellor (1971). It consisted of an outer glass cylinder (1.5-cm internal diam.) over the bottom of which a stretched membrane was attached. The test solution was introduced with a sterile pipette until it covered the membrane. A hollow glass "finger" rested inside the outer collar so that its tip (7-mm diam.) slightly stretched the membrane. An immersion thermostat with pump ("Thermomix II" by B. Braun, Melsungen, W. Germany) circulated hot water through the finger to warm the test solutions, whose temperature could be controlled to ± 1°C. Before use, the feeding apparatus was cleaned in a surfactant to remove proteins, washed several times in boiling water followed by methanol to remove fats and amino acids, and dried at 200°C. The feeding unit slightly dented the taut nylon gauze covering the top of the pill box. The human observer breathed into the box to activate the flies; without this few of them would move from the walls of the box where they rested. In an attempt to standardize this stimulus, each box was given only 3 gentle exhalations. Test suspensions were never left in the feeding unit for more than I hr (15 min. for warming and three I5-min. feeding periods), and, like all test solutions, were shaken vigorously between feeding periods to prevent settling. All membrane feeds were carried out in subdued and indirect light with an ambient temperature and humidity of 23°C and 60-80% RH, respectively. For all but I of the experiments the temperature of the test solutions was 37.5 °C.

Preparation of membranes. Two types of membrane were used. Ohick skin and Parafilm 'M' were chosen as the natural and artificial membranes that had given the best results with other hematophagous Nematocera (e.g., Rutledge et al. 1964 and Mellor 1971) . Freshly killed or deep-frozen day-old chicks were plucked and their skins peeled off. Subcutaneous fat was removed with forceps and the skin soaked for a total of I hr in 3 changes of distilled water and 1 of 0.85% saline. Further visible leachings from these leached chick skins (LCS) were slight. LCS were always attached to the feeding unit so that the epidermis was presented to the flies; if fitted the other way the skin soon dried. Parafilm 'M' was stretched to 2-1/2 times its normal area to form thin, uniformly translucent membranes. Care was taken not to touch the areas of the Parafilm used for feeding experiments, so

Vol. 14, no. 5

Entomol.

that no olfactory stimulants from the experimenter became associated with them. Parafilm membranes became dry and brittle if left on the feeding unit for more than 3 hr, whereas chick membranes retained their character even when left overnight.

Meals taken from cotton wool pads. Flies were deprived of sucrose and water for 2 days before an experiment. A petri dish (9-cm diam.) containing a cotton wool pad soaked in test solution was placed on the base of the gauze feeding cage (18 emS), which was left undisturbed for 2 hr in an illuminated incubator at 25 °0 and 70% RH. All flies were then dissected, and the proportion feeding and the location of their meals were noted. Test solutions

Isotonic saline (0.85%) was used as a diluent.

Mammalian

blood. Transfused,

whole human blood, with citrate phosphate dextrose (OPD) as an anticoagulant, was obtained from a local hospital on the day of its expiry (the first day when it is considered no longer fit for transfusion) and kept at 4 °0 for no more than a week before use. Hamster blood was obtained by cardiac puncture and heparin was used as an anticoagulant (300 USP units per 5 ml). The whole blood, and fractions from it, were used on the day drawn.

Red blood cell suspensions and plasma. Whole blood was centrifuged for 20 min. at 2500 rpm. The plasma was recovered and the cells washed in 3 changes of isotonic saline, in which they were finally resuspended to give a 45% (vol./vol.) suspension. Red blood cell extract. A freshly prepared 45% (vo1./vol.) suspension of red blood cells was twice frozen and thawed, and then centrifuged for 30 min. at 3500 rpm to remove the stroma of the ruptured cells. In addition, the supernatant was passed through a 1.2-(lm millipore filter to remove large cell fragments. Denatured human plasma proteins. Plasma from OPD human blood was heated at 60-90 °0 for 30 min. The coagulant was freeze-dried for 24 hr before being ground to a powder, which was suspended in 0.85% saline to give a final concentration 1/20 of that in plasma. Amino acid solution 1. This was the "Medium D" of Dimond et al. (1956), who found it to be the optimal adult diet for egg production in Aedes aegypti. Along with 12 "essential" amino acids, the solution contained (per 100 ml) 5 g of glucose, 5 g offructose, 0.15 g of salt mixture (in the propor-

1978

Ready:

Feeding habits of laboratory-bred

tions used for Mammalian Ringer's) and sufficient sodium hydroxide for acid neutralization. Amino acid solution 2. This differed from solution 1 only in the concentration of sugars, containing 0.09 g of glucose and 0.005 g of fructose per 100 ml, their concentration in human blood (Spector 1956). All solutions tested were prepared from Sigma grade chemicals. On occasion, cochineal red food coloring (Rayners Ltd, London, England) was added to test solutions (giving a concentration of 1% vo1./vol.) to facilitate identification of meals in dissected flies. The food coloring contained glycerin, isopropyl alcohol and potassium hydroxide, as well as El20 cochineal in aqueous solution. Standardizalion oj insect material Under normal rearing conditions, the proportion

TABLE

547

of newly emerged or old (> 16 days) female flies accepting a blood meal was always lower and more variable than that of other age groups. Four- to 6-day-old females were used for all experiments. As body size significantly affects the size of blood meal ingested by some mosquitoes (Clements 1963), exceptionally large or small flies were removed from experimental lots. The weights of the flies used ranged from 0.28 to 0.40 mg. Most of the experimental flies could not be sacrificed since they were needed to maintain a colony; therefore, sugar-feeding and mating were allowed to OCCur before the test feeds from membranes. However, females that had recently gorged on sucrose were identified (by their swollen abdomens) and removed from experimental lots in order to reduce variability in feeding response.

I. Feeding response of (N of L. longiPalPis to solutions offered in the membrane feeder.

TESTSOLUTION (PER VOL. OF ISOTONIC SALINE) Human origin Whole blood Whole blood 50% (vol./vol.) plasma 5% (vol./vol.) denatured plasma 5% (vol./vol.) denatured plasma I- 0.005 M ATP 45% (vol./vol.) red cell suspension 45t~~)(vol./vol.) red cell extract Hamster origin Whole blood 45"0 (vol./vol.) red cell suspension 45% (vol./vol.) red cell extract Other solutions 5% (wt/vol.) bovine serum albumen (BSA) 10% (wt/vol.) BSA 10% (wt/vol.) BSA 10% (wt/vol.) BSA 0.005M ATP 20% (wt/vol.) BSA Amino aeid solution 1

+

Amino acid solution 1 Amino acid solution 2 0.85% saline 0.85% saline 0.85% saline 10% (wt/vol.) sucrose cochineal red 10% (wt/vol.) sucrose 10% (wt/vol.) sucrose 30% (wt/vol.) sucrose

Lutzomyia longiPalpis

+

MEANWTOF STANDARD No. MEM- OFFERED % OFX % OFX No. SA- MEALTAKEN DEVIATION BRANE* FEED(x) PROBING FEEDINGTIATED** (MG)** FORMEAN**

RANGEOF MEALWT**

LeS P LCS

24 24 19

NR*" 54 NR

58 21 58

14 5 8

0.38 0.42 0.37

0.104 0.113 0.084

(0.18-0.55) (0.31-0.59) (0.26-0.46)

LCS

II

NR

45

5

0.28

0.064-

(0.21-0.36)

LOS

23

NR

56

13

0.23

0.099

(0.07- 0.38)

LCS

38

NR

58

20

0.40

0.342

(0.09-0.56)

LOS

34

NR

53

18

0.23

0.141

(0.09-0.36)

LOS

20

NR

65

10

0.36

0.070

(0.13-0.46)

LOS

35

NR

51

18

0.41

0.141

(0.16-0.62)

LOS

52

NR

40

21

0.27

0.044-

(0. I8-..Q.36)

LCS LOS P

37

40 35

70 33 63

70 33 57

24 13 19

0.30 0.38 0.20

0.118 0.132 0.145

(0.04-0.47) (0.12-0.56) (0.01-0.46)

P LOS P

37 42 IS

32 69 46

16 64 13

6 22 2

0.210 0.113

(0.05-0.45) (0.03-0.45)

LOS LOS OS LOS P

23 28 20 21 45

48 39 55 62 49

43 11 50 62 44-

10 3 8 10 18

0.24 0.25 0.15 and 0.16mg 0.07 0.05 0.42 0.36 0.21

0.028 0.012 0.072 0.056 0.121

(0.04-0.15) (0.04-0.06) (0.08-0.53) (0.18-0.53) (0.06-0.43)

LOS LOS P LOS

45 25 49 20

76 52 35 70

76 52 25 70

25 12 12 14

0.14 0.15 0.12 0.04

0.048 0.047 0.047 0.069

(0.03-0.23) (0.08-0.22) (0.03-0.19) (0.02-0.07)

OS=unleaehed

chick skin.

*LOS=leached chick skin, P=Parafilm, **Only uninterrupted meals. *"NR=not recorded.

J. Med.

548 RESULTS

Feeding behavior Normally, 40-60 females would take a blood meal from a human arm inserted into a cage for 20 min. Activated by the breath of the human host, both males and females congregated on the side of the cage nearest the man, where they hopped onto his arm. On alighting, a female usually made an exploratory walk, describing ever-decreasing circles about the point where she finally probed. Most found a "suitable" spot within 1 min. and engorged in 2-6 min. The softer areas of the hand and arm were preferred, but only regions with copious hair were avoided. There was no tendency to aggregate. When an anesthetized hamster was used, individual feeding times were the same, but females took longer to find the host. There was often an aggregation on the bare skin of the nose. Mating was observed during and after the feed. Males spent much of the feeding period dashing from one female to another, facing them and wing "fluttering" until copulation commenced. Over 23 generations, the mean proportion of 4to 6-day-old females feeding on either host was 80% (30-100%). Poor feeds were irregular and not the result of unfavorable physical conditions; 100% feeds occurred equally frequently over a range of temperatures (18-30°0) and humidities (60-100% RH) and at different times of the day (with or without

Vol. 14, no. 5

Entomol.

light). Unmated females fed as avidly as those kept with males from emergence. The average size of human and hamster blood meals was 0.41 mg (90 ,}bservationsj SD=0.05) and 0.46 mg (90 observations; SD=0.04), respectively. Ivlembrane feeds TABLE I gives details of the acceptability of various solutions offered across membranes. Preliminary experiments with human blood/chick skin showed that nearly all females probing fed. However, the percentage that probed varied from 30% to 82%. Therefore, when analyzing the results, "% feeding" was used only in comparison with "% probing"; mean weights of the freely ingested meals were more useful (FIG. I). Chick skins were leached to prevent possible phagostimulants from entering the test solution during an experiment. However, meals of isotonic saline from an unleached chick skin, tested on 1 occasion, were not significantly larger than those from leached skins. The destinations of some of the meals in the flies are recorded in TABLE 2. During the course of other experiments in this laboratory, many engorged L. longipalpis have been dissected, but only rarely has blood been found in the crop. The feeding behavior on membranes was similar to that of caged females offered living hosts. Females were satiated with most solutions well

0.4

0.3

~ 0.2 Cl

E

~ '00.1 or .c

Cl Q)

:. c

co

~ 0.0

Key:

~,

Mean meal weight

of A significantly

(]]=ill,

Mean meal weight

of A not significantly

BSA FIG. I.

=

bovine

serum albumen;

Relative acceptability

LeS

=

larger

than B

different

leached chick

('t'

test:

from C

('t'

p"0.05l test:

p">0.05l

skin

(schematic) of fluids ingested through membranes by ~~ of L. longiPalPis.

1978

Ready:

Feeding

habits

of laboratory-bred

TABLE 2.

Destination of meals ingested through membranes by ~~ of L. longiPalpis (solutions colored with I % (voL/voL) cochineal red].

red in 10% sucrose was neither an attractant nor a repellent when offered across chick skin (TABLE 1).

¥~ WITH MEALIN··

No. MEAl. (WT/VOI.. O.8S':;. SALINE)

BRANE·

20°" nSA··· 10'';. nSA 5"" nSA 0.85'':, saline 10'';, sucrose

LCS LCS LCS LCS LCS

1'\0. ~'i' TESTED

MEM-

10"" sucrose

P

30°:,

LCS

sucrose

15 15 12 10 21

R 14

DISCUSSION

------------

Crop

Midgut

(+)

++ ++ ++ ++ ++ ++ ++ ++ + + (+)t

15 15 12 10 18 2 I 5 3 9 I I 2 I

+ +

+ + +

Host-seeking behavior

+

(+)t

·LCS'-7Icached chick skin, P=Parafilm. ··Key for meal sizes: - empty, (+) trace, +