- Email: [email protected]
Aggregation pheromone released from the palps of feeding female Phlebotomus papatasi (Psychodidae)
J. /n.wr Ph.wio/. Vol. 30. No. 2. pp. 153-l 56. 1984 Printed in Great Bntain. All rights reserved.
Copyright
$’
0022.191Oi84 $3 00 + 0.00 1984 Pergamon Press Ltd
AGGREGATION PHEROMONE RELEASED FROM THE PALPS OF FEEDING FEMALE PHLEBOTOMUS PAPATASI (PSYCHODIDAE) YOSEF SCHLEIN, BOAZ YUVAL and ALLON WARBURC Department of Parasitology, The Kuvin Centre, Hebrew University-Hadassah Medical School, Jerusalem. Israel
(Receioed 31 May 1983; revised 29 July 1983) Abstract-Experiments with 2 feeding chambers: 1 new, the other having been used previously for feeding, showed that an aggregation and feeding pheromone had been secreted onto the chamber membranes by engorging females. Of the different extracts from parts of females or males tested, only the female palps and mouth parts exhibited pheromonal activity. The reaction to the pheromone was shown to be olfactory, and the pheromone itself is volatile. Reaction to the pheromone was dependent on chamber temperature. Pheromone-baited chambers at 28°C elicited a high response and the specific reaction to the pheromone was eliminated at 36iC. Large vacuolated cells in the 3rd and 4th segments and in Newstead’s organ in the palps of females are suggested as a source of the pheromone. K~JJ Word Index: Newstead’s organ
Female
Phlehotomus papatasi, olfactory
feeding
pheromone.
palp.
was 25 + 1°C and a high humidity was maintained. Series of 30 to 45 females and an uncounted number of males were introduced into each of the cages. The cages were flexible boxes, 10 x 10 x 7.5 cm, covered with fine plastic-mesh screening (3 holes/l mm), with a glass window on one side and a 12-mm diameter opening on another. The observation windows were made from microscope slides, attached with cellotape just below the plastic mesh. Sandflies were transferred by an aspirator through the openings. Feeding chambers were glass funnels enclosed in a glass casing. Extending from two sides of the casing were glass tubes that allowed temperature control by a water flow system. The opening of the funnel was 3.2cm. Shaved, chick-skin membranes were stretched on the wide side of the funnels 24 h before the experiments. The funnels were filled with saline (0.99/b NaCl) and two were placed, membrane side down, on the mesh cover of each experimental cage. The study consisted of groups of female sandflies that were given a choice between 2 different feeding chambers. Three variables were examined: (a) treatment of the feeding site, including chamber membrane and the mesh cover underneath; (b) the time interval between membrane treatment and the actual beginning of the experimental feeding; (c) temperature of the feeding chambers. Two alternative treatments were carried out before the tests. One was a preparatory feeding of a group (approximately 50) of sandflies on a chamber warmed to a temperature of 36°C. The chamber was removed and placed with a control chamber on another cage containing an experimental series. Treated chambers were tested 10 or 30 min after the preparatory feeding. The preparatory group was in contact with the mesh cover below the test chamber and its pheromonal activity was also tested. The location (site) of the fed chamber on the mesh was marked and the
INTRODUCTION role in Aggregation pheromones play an important . .. . the biology ot many coleopteran species. Their various functions have been discussed by Borden (1977). Among these are the observations that some species assemble at a suitable source of food (Anderson, 1948; Cross, 1973). Assembly and aggregation pheromones of haematophagous arthropods have been reported for the 2 families of hard and soft ticks (reviewed, Sonenshine et al., 1982). These pheromones are of 2 types. The first are assembly pheromones that attract ticks to the vicinity of other individuals. These occur in Argasid and Ixodid species, effect ticks at different life stages and, in some cases, have an interspecific effect (Leahy et al., 1975a,b; Graf, 1975; Treverrow et al., 1977). The second are aggregation and attachment pheromones that are species specific and are active only on the body of the host (Gladney. 1971). The presence of such pheromones has been demonstrated in hexane extracts of fed Amblyomma macdatum males and females (Gladney et al., 1974a,b). Observations on females of Phlebotomus papatasi indicated that they tended to aggregate when feeding. This study provides experimental evidence showing that aggregation is initiated by the emission of a pheromone. The source of the pheromone is located in the maxillary palps of females.
MATERIALS AND
aggregation,
METHODS
Sandflies from a colony of Phlebotomus papatasi originating from specimens collected in the Jordan Valley were used. Experimental insects were used before a blood meal and up to 7 days old. They were deprived of sugar solution and water 24 h prior to inclusion in experiments. The ambient temperature 153
YOSEF SCHLEIN et al.
154
was removed. The preparatory group was replaced by an experimental series of females and two unused chambers were placed on the cage, one on the treated site and one in another corner. In a variation of the first experiment. the two chambers were placed on plastic rings of 0.5 cm above the mesh to prevent direct contact with the sandflies. Chamber temperature was maintained at 28’C. The second treatment was the application of sandfly body parts extracts to the membrane of the chambers. Extract was prepared by placing the dissected palps, mouth parts, legless and headless bodies, or legs of 20 females on the chick-skin membrane of feeding chambers. The surface of each membrane was then flooded with 0.2 ml hexane, and dried in a cold air stream for 10 min. Five min after drying, the body parts were removed and two chambers, placed on each of the experimental cages, the temperature of the chambers being maintained at either 28 or 36°C. The observation period for the experiments was 10 min. Chi-square tests of the results comprising the number of feeding females in each experiment were carried out, excluding the ones that did not feed. The structure of the palps was observed by examining Bouin-fixed, 6 /1 thick, Mayer’s haematoxylin stained, serial sections of female heads, and stained or KOH-cleared whole preparations of heads.
earlier to feed a group of sandflies and a chamber with an unused membrane (Table ],a). Both chambers were at a temperature of 28°C. The number of females feeding on the used membrane (73) was more than three times greater than the control count [22]. The treated site on the cage cover (Table 1, b) had a similar effect under the same experimental conditions, with a count of 65 feedings compared to 22 on untreated sites. A significant effect on female sandflies was also observed (381 compared to 256, Table 1,d) when the feeding chambers were placed above the mesh cover on plastic rings and the test flies had no direct contact with the pheromone. The phenomenon described was short-lived, as there was no significant difference between test and control chambers when the tests were carried out 30 min after a preparatory feeding (Table 1,e. f, g).
chamber
RESULTS
Deposition and the duration qf activity omone on the feeding site
qf the pher-
The first experimental group was offered a choice between a feeding chamber that had been used 10 min
Table
The source of the pheromone perature
and the effect of tem-
The source of the pheromone in sandflies was located by comparing the effect of sandfly hexane extracts on feeding membranes, maintained at 28°C. Extracts from female palps and mouth parts were very effective, producing 95 feedings as compared to 33 feedings on a membrane treated with female leg extract (Table 2,a). All other extracts from females or males produced no effect (Table 2, b, c). Unwarmed feeding chambers did not elicit a reaction as only a random one or 2 females were observed to feed. Chambers at 36°C induced high activity but, extracts of female palps and mouth parts at this temperature exhibited no greater activity, producing 89 feedings as compared with 93 in the control group.
I. The aggregation of feeding Phkbotomus papatasi females on used (fed upon) chambers sites as compared to unused chambers at a chamber temperature of 28°C Tested after
Experiment* a.
Tested after 30min
10min
Test obJcCt
No females feeding
Used chamber Unused Used site Unused Total used Total unused Used, elevated chambert
b. c. d.
and
Test object
No females feeding
Used chamber Unused Used site Unused Total used Total unused
35 NS 35 18NS 15 53 NS 50
Experiment* e.
73f 22 651 22 1381 44
f. g.
3811
*The number of replicates for the experiments was a-3; b-3: c-6; d-5; e-2; f-2 and g-4. tChamber placed on a plastic ring 0.5 cm above the mesh cover of the cages and females were probing through the mesh. fChi-square test P < 0.001, NS-Not significant. (Experiments a, b. c. d started IOmin and e. f, g, 30min after the preparatory feeding). Table 2. A comparison of feeding females of Phleboromus paparasion chamber treated with different extracts of sandfly body parts Experiment* a.
Chambers temp.
Body parts Female palps and mouth
parts
legs
b. c. d.
Female headless bodies legs Male palps and mouth parts legs Female palps and mouth parts legs
*Each experiment was replicated three times. tChi-square tested P < 0.001. NS-not significant. Tested 15 min after extract application at a chamber
28°C 28°C 28’C 28°C 28°C 28°C 36°C 36 C
temperature
membranes
No feeding females 93
33 69 NS 66 58 NS 66 138 NS 132
of 28 or 36°C.
Aggregation pheromone of feeding female Phlehoromus papatasi
seg3
Fig. IA. Phlebotomus papatasi female, lateral view of head. Fig. IB. P. papatasi female, longitudinal section of 3rd and 4th right palp segments, dorsal view. Ic-large vacuolated cells; m-muscle; n-nerve; ns-Newstead’s sense organ; seg 3,4_palp segments 3,4. Sandfiy pulps (Fig. 1) The five-segmented palps of sandflies are held in an unusual position. In living sandflies, the first three segments are directed ventrally and are parallel to the mouth parts. The fourth segment is bent backwards and traverses the longitudinal axis of the head. The fifth segment is orientated backwards and upwards (Newstead, 1911, 1912). On the third segment, in addition to hairs and scales, there is a sense organ bearing the characteristic “Newstead spines”. This is present in males and females. In the Newstead organ on the anterior side of the third and fourth palpal segments of females there are groups of large vacuolated cells. These are not seen in the palps of males in which the organ is much smaller. Observations of feeding female sandflies show that the bent palps are in contact with the chick-skin membrane, when the mouth parts are embedded. In view of this distinct structural arrangement, it is possible that the large cells seen in histological sections are the source of the pheromone.
DISCUSSION
The initial observation that Phlebotomus papatasi are induced to feed in the vicinity of other engorging females was confirmed. The results showed that an aggregation pheromone is deposited on both the membrane of the feeding chamber and the mesh cover supporting it (Table l,a, b). The total number of females that engorged on the mesh-sites and membranes previously fed upon, I38 was significantly higher than the 44 that fed on the control membranes and sites (Fig. 1,~). The source of the secreted pheromone could only have been in the legs or palps and mouth parts, as only these were in contact with the substrate. A comparison of extracts from different body parts of the sandfly located the source of the pheromone in the female palps and mouth parts. Only an extract of these structures produced a significantly higher aggregation of sandflies (Table 2, b). The extract was made from the palps and mouth parts of unfed females, so that there would be no interference from
156
YOSEF SCHLEIN et al.
food or secretions from other organs. The pheromone function and secretion is limited to females. Males in the experimental cages were not attracted to feeding females, and females did not react to male palp extract. Female sandflies aggregated below a pheromone bearing funnel that was elevated above the mesh cover of the cage to prevent direct contact. These funnels had 381 female probings compared to 256 counted below a control funnel (Table I,d). Pheromone secreted on the membranes ceased to elicit excess probing within 30min after preparation (Table 1.e). These findings indicate that the pheromone is volatile and that the experimental series reacted to an olfactory cue. The response to the aggregation pheromone depended on the temperature of the feeding chambers. Thermal excitation was a necessary pre-condition for pheromone perception. Females were not stimulated by pheromone-baited funnels at room temperature. The pheromone was active at a chamber temperature of 28 ‘C. Sandflies were assembling to feed on both of the warmed funnels. but the numbers of feedings on the pheromone treated ones was three times higher (Table l,c; ?,a). The number of the gorging sandflies was very high at a chamber temperature of 36°C but the effect of the pheromone was eliminated. At this temperature, the count for pheromone-baited funnels was 138 compared to 132 for controls (Table 2,d). Warm-blooded host temperature is apparently a major attractant for P. papatusi. An increase in temperature of 0.2”C (i.e. 36.2’C compared with 36.0 ‘C) caused an increase of 40% in the number of sandflies feeding at the higher temperature compared to those on a funnel held at 36’C. (Schlein et al.. unpublished data). In the present study, 36C. the approximate external temperature of mammals. excited feeding in the sandflies but eliminated the expression of the pheromonal stimulus. P. puputusi in the Jordan Valley also feed on reptiles, as shown by blood meal identification (Schlein, unpublished data). Other species of Phlebotomus are also known to feed on cold-blooded animals (Thatcher and Hertig, 1966: Braack et al., 1981). Perhaps the role of the aggregation pheromone in nature is to facilitate the orientation of female sandflies to cold-blooded hosts over the short distance in which the pheromone operates. Extracts of female palps and mouth parts was the only extract exhibiting pheromonal activity. The mouth parts are cuticular structures which seem to contain no glandular cells. Histological examination of the palps showed that there are large vacuolated cells in the distal part of the third segment in Newstead’s sense organ and in the fourth segment (Fig. 1). These cells are not seen in the palps of males, where Newstead’s organ is much smaller and male palp extracts do not show pheromonal activity (Table 2,~). It is. therefore, suggested that the glandular cells found in female palps are the source of the aggre-
gation pheromone. The position of the palps is such that segments 4 and 3 would be pressed against the skin of the host, when the mouth parts are sunk into the tissue. This, apparently, is the way in which the pheromone is spread on the surface of the skin. It is also possible that Newstead’s sense organ plays a role in the release of the pheromone. .4cknowledgements-We
gratefully acknowledge grant support from the Leihsmaniasis Component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Project on Epidimiology and control REP-NIH-NIAID-AL-12669. for his help.
of vector borne diseases in Isreal We also thank Mr A. Cohen
REFERENCES Anderson R. F. (1948) Host selection by the pine engraver. J. econ. Ent. 41, 596602. Borden .I. H. (1977)Behavioral responses of Coleoptera to pheromones. In Chemical Control of Insect Behavior: *Theory and Application (Ed. by Shorey H. H. and McKelvev J. J. Jr). P. 169. John Wiley. New York. Braack H. k., Davidson 1. H.. Ledger J: A. and Lewis D. J. (1981) Records of sandflies (Diptera:Psychodidae: Phelbotominae) feeding on Amphibia. with a new record from the Kruger National Park. Koedoe 24, 187-188. Cross W. H. (1973) Biology control and eradication of boll weevil. A. Rea. Ent. 18;~17-46. Gladnev W. J. (1971) Mate seeking of female Amblvomma macu~atum (Acarina:Ixodidae) on a bovine. Nat&e 232, 401402. Gladney W. J., Ernst S. E. and Grabbe R. R. (1974a) The aggregation response of the Gulf Coast ticks on cattle. Ann. ent. Sot. Am. 67. 750-752. Gladney W. J., Ernst S. E. and Oehler D. D. (1947b) The Gulf Coast tick: Evidence of pheromone produced by males. J. med. Ent. 11, 303-306. Graf J. F. (I 975) Ecologic et ethologie d’lxodes ricinus L. en Suisse (1xodoidea:Ixodidae) Cinquieme note: Mise en evidence d’une pheromone sexuelle chez Ixodes ricinus. Acarologia 17, 43&441. Leahy M. G., Karuhize G., Mango C. and Galun R. (1975a) An assembly pheromone and its perception in the tick Ornithodoros moubata (Murray) (Acari:Argasidae). J. med. Ent. 12, 284287. Leahv M. G.. Sternbere S.. Mango S. and Galun R. (1975b) Lack of specificity in assembl; pheromones of soft ticks (Acari:Argasidae). J. med. Ent. 12, 413414. Newstead R. (I 9 I I ) The Papatasi flies (Phlebotomus) of the Maltese Islands. Bull. Enl. Res. 2, 4748. Newstead R. (1912) Notes on Phlebotomus with descriptions of new species-Part I. Bull. Enf. Res. 3, 361-367. Sonenshine D. E., Silverstein R. M. and Rechav Y. (1982) Pheromones of ticks. In The Ph.vsio1og.vof’ Ticks (Ed. by Obenchain F. D. and Galun R.). p. 439. Pergamon Press, Oxford. Thatcher V. E. and Hertig M. (1966) Field studies on the feeding habits and diurnal shelters of some Phlebotomus sandflies (Diptera:Psychodidae) of Panama. Ann. ent. Sot. Am. 59, 4652. Treverrow N. L., Stone B. F. and Cowie M. (1977) Aggregation pheromones in two Australian hard ticks I.xodes holocvclus and Aponoma concolor. E.xperientia 33, 68&683.
Copyright
$’
0022.191Oi84 $3 00 + 0.00 1984 Pergamon Press Ltd
AGGREGATION PHEROMONE RELEASED FROM THE PALPS OF FEEDING FEMALE PHLEBOTOMUS PAPATASI (PSYCHODIDAE) YOSEF SCHLEIN, BOAZ YUVAL and ALLON WARBURC Department of Parasitology, The Kuvin Centre, Hebrew University-Hadassah Medical School, Jerusalem. Israel
(Receioed 31 May 1983; revised 29 July 1983) Abstract-Experiments with 2 feeding chambers: 1 new, the other having been used previously for feeding, showed that an aggregation and feeding pheromone had been secreted onto the chamber membranes by engorging females. Of the different extracts from parts of females or males tested, only the female palps and mouth parts exhibited pheromonal activity. The reaction to the pheromone was shown to be olfactory, and the pheromone itself is volatile. Reaction to the pheromone was dependent on chamber temperature. Pheromone-baited chambers at 28°C elicited a high response and the specific reaction to the pheromone was eliminated at 36iC. Large vacuolated cells in the 3rd and 4th segments and in Newstead’s organ in the palps of females are suggested as a source of the pheromone. K~JJ Word Index: Newstead’s organ
Female
Phlehotomus papatasi, olfactory
feeding
pheromone.
palp.
was 25 + 1°C and a high humidity was maintained. Series of 30 to 45 females and an uncounted number of males were introduced into each of the cages. The cages were flexible boxes, 10 x 10 x 7.5 cm, covered with fine plastic-mesh screening (3 holes/l mm), with a glass window on one side and a 12-mm diameter opening on another. The observation windows were made from microscope slides, attached with cellotape just below the plastic mesh. Sandflies were transferred by an aspirator through the openings. Feeding chambers were glass funnels enclosed in a glass casing. Extending from two sides of the casing were glass tubes that allowed temperature control by a water flow system. The opening of the funnel was 3.2cm. Shaved, chick-skin membranes were stretched on the wide side of the funnels 24 h before the experiments. The funnels were filled with saline (0.99/b NaCl) and two were placed, membrane side down, on the mesh cover of each experimental cage. The study consisted of groups of female sandflies that were given a choice between 2 different feeding chambers. Three variables were examined: (a) treatment of the feeding site, including chamber membrane and the mesh cover underneath; (b) the time interval between membrane treatment and the actual beginning of the experimental feeding; (c) temperature of the feeding chambers. Two alternative treatments were carried out before the tests. One was a preparatory feeding of a group (approximately 50) of sandflies on a chamber warmed to a temperature of 36°C. The chamber was removed and placed with a control chamber on another cage containing an experimental series. Treated chambers were tested 10 or 30 min after the preparatory feeding. The preparatory group was in contact with the mesh cover below the test chamber and its pheromonal activity was also tested. The location (site) of the fed chamber on the mesh was marked and the
INTRODUCTION role in Aggregation pheromones play an important . .. . the biology ot many coleopteran species. Their various functions have been discussed by Borden (1977). Among these are the observations that some species assemble at a suitable source of food (Anderson, 1948; Cross, 1973). Assembly and aggregation pheromones of haematophagous arthropods have been reported for the 2 families of hard and soft ticks (reviewed, Sonenshine et al., 1982). These pheromones are of 2 types. The first are assembly pheromones that attract ticks to the vicinity of other individuals. These occur in Argasid and Ixodid species, effect ticks at different life stages and, in some cases, have an interspecific effect (Leahy et al., 1975a,b; Graf, 1975; Treverrow et al., 1977). The second are aggregation and attachment pheromones that are species specific and are active only on the body of the host (Gladney. 1971). The presence of such pheromones has been demonstrated in hexane extracts of fed Amblyomma macdatum males and females (Gladney et al., 1974a,b). Observations on females of Phlebotomus papatasi indicated that they tended to aggregate when feeding. This study provides experimental evidence showing that aggregation is initiated by the emission of a pheromone. The source of the pheromone is located in the maxillary palps of females.
MATERIALS AND
aggregation,
METHODS
Sandflies from a colony of Phlebotomus papatasi originating from specimens collected in the Jordan Valley were used. Experimental insects were used before a blood meal and up to 7 days old. They were deprived of sugar solution and water 24 h prior to inclusion in experiments. The ambient temperature 153
YOSEF SCHLEIN et al.
154
was removed. The preparatory group was replaced by an experimental series of females and two unused chambers were placed on the cage, one on the treated site and one in another corner. In a variation of the first experiment. the two chambers were placed on plastic rings of 0.5 cm above the mesh to prevent direct contact with the sandflies. Chamber temperature was maintained at 28’C. The second treatment was the application of sandfly body parts extracts to the membrane of the chambers. Extract was prepared by placing the dissected palps, mouth parts, legless and headless bodies, or legs of 20 females on the chick-skin membrane of feeding chambers. The surface of each membrane was then flooded with 0.2 ml hexane, and dried in a cold air stream for 10 min. Five min after drying, the body parts were removed and two chambers, placed on each of the experimental cages, the temperature of the chambers being maintained at either 28 or 36°C. The observation period for the experiments was 10 min. Chi-square tests of the results comprising the number of feeding females in each experiment were carried out, excluding the ones that did not feed. The structure of the palps was observed by examining Bouin-fixed, 6 /1 thick, Mayer’s haematoxylin stained, serial sections of female heads, and stained or KOH-cleared whole preparations of heads.
earlier to feed a group of sandflies and a chamber with an unused membrane (Table ],a). Both chambers were at a temperature of 28°C. The number of females feeding on the used membrane (73) was more than three times greater than the control count [22]. The treated site on the cage cover (Table 1, b) had a similar effect under the same experimental conditions, with a count of 65 feedings compared to 22 on untreated sites. A significant effect on female sandflies was also observed (381 compared to 256, Table 1,d) when the feeding chambers were placed above the mesh cover on plastic rings and the test flies had no direct contact with the pheromone. The phenomenon described was short-lived, as there was no significant difference between test and control chambers when the tests were carried out 30 min after a preparatory feeding (Table 1,e. f, g).
chamber
RESULTS
Deposition and the duration qf activity omone on the feeding site
qf the pher-
The first experimental group was offered a choice between a feeding chamber that had been used 10 min
Table
The source of the pheromone perature
and the effect of tem-
The source of the pheromone in sandflies was located by comparing the effect of sandfly hexane extracts on feeding membranes, maintained at 28°C. Extracts from female palps and mouth parts were very effective, producing 95 feedings as compared to 33 feedings on a membrane treated with female leg extract (Table 2,a). All other extracts from females or males produced no effect (Table 2, b, c). Unwarmed feeding chambers did not elicit a reaction as only a random one or 2 females were observed to feed. Chambers at 36°C induced high activity but, extracts of female palps and mouth parts at this temperature exhibited no greater activity, producing 89 feedings as compared with 93 in the control group.
I. The aggregation of feeding Phkbotomus papatasi females on used (fed upon) chambers sites as compared to unused chambers at a chamber temperature of 28°C Tested after
Experiment* a.
Tested after 30min
10min
Test obJcCt
No females feeding
Used chamber Unused Used site Unused Total used Total unused Used, elevated chambert
b. c. d.
and
Test object
No females feeding
Used chamber Unused Used site Unused Total used Total unused
35 NS 35 18NS 15 53 NS 50
Experiment* e.
73f 22 651 22 1381 44
f. g.
3811
*The number of replicates for the experiments was a-3; b-3: c-6; d-5; e-2; f-2 and g-4. tChamber placed on a plastic ring 0.5 cm above the mesh cover of the cages and females were probing through the mesh. fChi-square test P < 0.001, NS-Not significant. (Experiments a, b. c. d started IOmin and e. f, g, 30min after the preparatory feeding). Table 2. A comparison of feeding females of Phleboromus paparasion chamber treated with different extracts of sandfly body parts Experiment* a.
Chambers temp.
Body parts Female palps and mouth
parts
legs
b. c. d.
Female headless bodies legs Male palps and mouth parts legs Female palps and mouth parts legs
*Each experiment was replicated three times. tChi-square tested P < 0.001. NS-not significant. Tested 15 min after extract application at a chamber
28°C 28°C 28’C 28°C 28°C 28°C 36°C 36 C
temperature
membranes
No feeding females 93
33 69 NS 66 58 NS 66 138 NS 132
of 28 or 36°C.
Aggregation pheromone of feeding female Phlehoromus papatasi
seg3
Fig. IA. Phlebotomus papatasi female, lateral view of head. Fig. IB. P. papatasi female, longitudinal section of 3rd and 4th right palp segments, dorsal view. Ic-large vacuolated cells; m-muscle; n-nerve; ns-Newstead’s sense organ; seg 3,4_palp segments 3,4. Sandfiy pulps (Fig. 1) The five-segmented palps of sandflies are held in an unusual position. In living sandflies, the first three segments are directed ventrally and are parallel to the mouth parts. The fourth segment is bent backwards and traverses the longitudinal axis of the head. The fifth segment is orientated backwards and upwards (Newstead, 1911, 1912). On the third segment, in addition to hairs and scales, there is a sense organ bearing the characteristic “Newstead spines”. This is present in males and females. In the Newstead organ on the anterior side of the third and fourth palpal segments of females there are groups of large vacuolated cells. These are not seen in the palps of males in which the organ is much smaller. Observations of feeding female sandflies show that the bent palps are in contact with the chick-skin membrane, when the mouth parts are embedded. In view of this distinct structural arrangement, it is possible that the large cells seen in histological sections are the source of the pheromone.
DISCUSSION
The initial observation that Phlebotomus papatasi are induced to feed in the vicinity of other engorging females was confirmed. The results showed that an aggregation pheromone is deposited on both the membrane of the feeding chamber and the mesh cover supporting it (Table l,a, b). The total number of females that engorged on the mesh-sites and membranes previously fed upon, I38 was significantly higher than the 44 that fed on the control membranes and sites (Fig. 1,~). The source of the secreted pheromone could only have been in the legs or palps and mouth parts, as only these were in contact with the substrate. A comparison of extracts from different body parts of the sandfly located the source of the pheromone in the female palps and mouth parts. Only an extract of these structures produced a significantly higher aggregation of sandflies (Table 2, b). The extract was made from the palps and mouth parts of unfed females, so that there would be no interference from
156
YOSEF SCHLEIN et al.
food or secretions from other organs. The pheromone function and secretion is limited to females. Males in the experimental cages were not attracted to feeding females, and females did not react to male palp extract. Female sandflies aggregated below a pheromone bearing funnel that was elevated above the mesh cover of the cage to prevent direct contact. These funnels had 381 female probings compared to 256 counted below a control funnel (Table I,d). Pheromone secreted on the membranes ceased to elicit excess probing within 30min after preparation (Table 1.e). These findings indicate that the pheromone is volatile and that the experimental series reacted to an olfactory cue. The response to the aggregation pheromone depended on the temperature of the feeding chambers. Thermal excitation was a necessary pre-condition for pheromone perception. Females were not stimulated by pheromone-baited funnels at room temperature. The pheromone was active at a chamber temperature of 28 ‘C. Sandflies were assembling to feed on both of the warmed funnels. but the numbers of feedings on the pheromone treated ones was three times higher (Table l,c; ?,a). The number of the gorging sandflies was very high at a chamber temperature of 36°C but the effect of the pheromone was eliminated. At this temperature, the count for pheromone-baited funnels was 138 compared to 132 for controls (Table 2,d). Warm-blooded host temperature is apparently a major attractant for P. papatusi. An increase in temperature of 0.2”C (i.e. 36.2’C compared with 36.0 ‘C) caused an increase of 40% in the number of sandflies feeding at the higher temperature compared to those on a funnel held at 36’C. (Schlein et al.. unpublished data). In the present study, 36C. the approximate external temperature of mammals. excited feeding in the sandflies but eliminated the expression of the pheromonal stimulus. P. puputusi in the Jordan Valley also feed on reptiles, as shown by blood meal identification (Schlein, unpublished data). Other species of Phlebotomus are also known to feed on cold-blooded animals (Thatcher and Hertig, 1966: Braack et al., 1981). Perhaps the role of the aggregation pheromone in nature is to facilitate the orientation of female sandflies to cold-blooded hosts over the short distance in which the pheromone operates. Extracts of female palps and mouth parts was the only extract exhibiting pheromonal activity. The mouth parts are cuticular structures which seem to contain no glandular cells. Histological examination of the palps showed that there are large vacuolated cells in the distal part of the third segment in Newstead’s sense organ and in the fourth segment (Fig. 1). These cells are not seen in the palps of males, where Newstead’s organ is much smaller and male palp extracts do not show pheromonal activity (Table 2,~). It is. therefore, suggested that the glandular cells found in female palps are the source of the aggre-
gation pheromone. The position of the palps is such that segments 4 and 3 would be pressed against the skin of the host, when the mouth parts are sunk into the tissue. This, apparently, is the way in which the pheromone is spread on the surface of the skin. It is also possible that Newstead’s sense organ plays a role in the release of the pheromone. .4cknowledgements-We
gratefully acknowledge grant support from the Leihsmaniasis Component of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Project on Epidimiology and control REP-NIH-NIAID-AL-12669. for his help.
of vector borne diseases in Isreal We also thank Mr A. Cohen
REFERENCES Anderson R. F. (1948) Host selection by the pine engraver. J. econ. Ent. 41, 596602. Borden .I. H. (1977)Behavioral responses of Coleoptera to pheromones. In Chemical Control of Insect Behavior: *Theory and Application (Ed. by Shorey H. H. and McKelvev J. J. Jr). P. 169. John Wiley. New York. Braack H. k., Davidson 1. H.. Ledger J: A. and Lewis D. J. (1981) Records of sandflies (Diptera:Psychodidae: Phelbotominae) feeding on Amphibia. with a new record from the Kruger National Park. Koedoe 24, 187-188. Cross W. H. (1973) Biology control and eradication of boll weevil. A. Rea. Ent. 18;~17-46. Gladnev W. J. (1971) Mate seeking of female Amblvomma macu~atum (Acarina:Ixodidae) on a bovine. Nat&e 232, 401402. Gladney W. J., Ernst S. E. and Grabbe R. R. (1974a) The aggregation response of the Gulf Coast ticks on cattle. Ann. ent. Sot. Am. 67. 750-752. Gladney W. J., Ernst S. E. and Oehler D. D. (1947b) The Gulf Coast tick: Evidence of pheromone produced by males. J. med. Ent. 11, 303-306. Graf J. F. (I 975) Ecologic et ethologie d’lxodes ricinus L. en Suisse (1xodoidea:Ixodidae) Cinquieme note: Mise en evidence d’une pheromone sexuelle chez Ixodes ricinus. Acarologia 17, 43&441. Leahy M. G., Karuhize G., Mango C. and Galun R. (1975a) An assembly pheromone and its perception in the tick Ornithodoros moubata (Murray) (Acari:Argasidae). J. med. Ent. 12, 284287. Leahv M. G.. Sternbere S.. Mango S. and Galun R. (1975b) Lack of specificity in assembl; pheromones of soft ticks (Acari:Argasidae). J. med. Ent. 12, 413414. Newstead R. (I 9 I I ) The Papatasi flies (Phlebotomus) of the Maltese Islands. Bull. Enl. Res. 2, 4748. Newstead R. (1912) Notes on Phlebotomus with descriptions of new species-Part I. Bull. Enf. Res. 3, 361-367. Sonenshine D. E., Silverstein R. M. and Rechav Y. (1982) Pheromones of ticks. In The Ph.vsio1og.vof’ Ticks (Ed. by Obenchain F. D. and Galun R.). p. 439. Pergamon Press, Oxford. Thatcher V. E. and Hertig M. (1966) Field studies on the feeding habits and diurnal shelters of some Phlebotomus sandflies (Diptera:Psychodidae) of Panama. Ann. ent. Sot. Am. 59, 4652. Treverrow N. L., Stone B. F. and Cowie M. (1977) Aggregation pheromones in two Australian hard ticks I.xodes holocvclus and Aponoma concolor. E.xperientia 33, 68&683.