Oviposition Attractants and Stimulants for the Sandfly Lutzomyia longipalpis (Diptera: Psychodidae) DIA-ELDIN A. ELNAIEM AND RICHARD D. WARD Department of Medical Entomology, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA, Liverpool, U.K.
KEY WORDS Insecta, Lutzomyia longipalpis, oviposition attractants, oviposition preferences
DESPITE RECENT PROGRESS in the colonization of phlebotomine sandflies, the methods remain laborious and time-consuming. One problem is the high mortality of females before or during oviposition (Killick-Kendrick et al. 1977, Chaniotis 1986) which restricts many basic studies on sandfly biology and their vectorial competence for Leishmania and arboviruses. Like many other insects, oviposition by sandflies probably is controlled through a combination of environmental and physiological factors, of which very little is known. Of the few oviposition studies, most have dealt with the regulation of physical factors such as temperature and relative humidity (Foster et al. 1970, Chaniotis 1986). Recently, Schlein et al. (1990) found that egg laying by Phlebotomies papatasi Scopoli is promoted by many factors including the presence of cow manure. In addition, we have demonstrated evidence for the presence of an oviposition attractant-stimulant pheromone associated with the eggs of Lutzomyia longipalpis Lutz & Neiva (Elnaiem & Ward 1990). In our study, the effect on oviposition by L. longipalpis of old colony remains (frass), fresh larval rearing medium, and rabbit feces is described. The study was designed to determine if the sandfly female lays more eggs on surfaces containing frass or laboratory larval food than on controls. If so, it was considered important to establish which portion of the larval food was responsible for the increased egg laying and
whether the response was mediated or induced by chemical or physical factors or both. Furthermore, we attempted to determine if rabbit feces acted as an oviposition attractant or stimulant and if it affected postoviposition survival. Materials and Methods Sandflies. The two laboratory colonies of L. longipalpis used in this study, "J a c °bina" and "L'Aguila," originated from females collected in Salvador (Brazil) and Tolima (Colombia), respectively, and were maintained using the methods described by Modi & Tesh (1983). Before use in experiments, females that had fed on a hamster anesthetized with a 12 mg/ml solution of sodium pentabarbitone were isolated with approximately equal numbers of males at 95% RH, 27°C, and 12:12 (L:D) photoperiod for 3 d to allow complete defecation and egg maturation. Influence of Frass, Larval Food, and Rabbit Feces on Oviposition by L. longipalpis. The effects of frass, larval food, and rabbit feces on oviposition by L. longipalpis were investigated using oviposition choice chambers. These chambers were constructed by drilling 30 equally spaced 0.5-cm-diameter holes in the base of polymethyl pentane pots (11.0 cm diameter, 7.0 cm high) (Nalgene, BDH Apparatus, BDH Ltd., Speke, Liverpool, England) which then were lined with a 1-cm-thick layer of plaster of paris on the bottom of each pot. Four oviposition sites
0022-2585/92/0005-0012$02.00/0 © 1992 Entomological Society of America
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
J. Med. Entomol. 29(1): 5-12 (1992) ABSTRACT Oviposition preferences of Lutzomyia longipalpis Lutz & Neiva for surfaces containing frass (colony remains), larval rearing medium, and rabbit feces were investigated in the laboratory. In oviposition choice chambers, significantly more eggs were laid on sites containing frass, larval rearing medium, or rabbit feces than on untreated control sites. Experiments using unwashed and washed materials indicated, for the first time, the presence of chemical oviposition attractants or stimulants or both in larval medium (consisting of equal weight proportions of rabbit feces, potting compost, sand, and Daphnia). In an olfactometer, aroma from rabbit feces strongly attracted ovipositing females, and significantly higher numbers of eggs were laid in the olfactometer test chamber containing the rabbit feces than in the untreated control chamber. In comparisons of oviposition responses to hexane and distilled water extracts of rabbit feces, only the water extract was attractive. Similarly, the distilled water extract of rabbit feces proved to be an oviposition stimulant. Females kept individually with filter papers impregnated with the extract of feces oviposited earlier and survived longer than females kept with untreated papers.
JOURNAL OF MEDICAL ENTOMOLOGY
(4 cm 2 area) then were delineated at the sides of the plaster surface of each pot by a 3-mm-deep groove and quartered by a shallow groove. Two of the oviposition sites adjoined each other on one side of the chamber; the two others were situated on the other side. The former were the "T" or test sites, the latter were the "C" or the control sites (Fig. 1). Immediately before use in experiments, the plaster bottom of the chamber was wetted with distilled water, the excess moisture was removed with laboratory paper toweling, and test and control surfaces were treated as described below. A group of 20 gravid females then were introduced into the chamber, and the chamber was transferred to a sealed plastic box lined with damp laboratory paper toweling. The chamber was kept in complete darkness for 3 d under the insectary conditions described above. At the end of this period, numbers of eggs laid on test and control oviposition sites were counted. Eggs laid outside the test and control sites were ignored. The results were analyzed statistically using Wilcoxon's matched-pairs signed-rank test. The treatments of test and control sites were as follows: Frass. Frass was collected from larval rearing pots from which adults had emerged. A paste of 0.2 g frass/0.3 ml distilled water was applied only in the grooves of the two test oviposition sites of a chamber, leaving the grooves on the control surface untreated. A distilled-water paste was used in preference to dry material to avoid any
reduction in the moisture of the test surface. The experiment was repeated 16 times using L'Aguila females and 15 times with Jacobina females. Larval Rearing Medium. Larval rearing medium was prepared by mixing rabbit feces, potting compost, sand, and Daphnia in equal proportions by weight and autoclaving the mixture. The rabbit feces were collected from a hutch containing a pet rabbit fed on commercial rabbit food, obtained from pet stores, with occasional supplements of green vegetables. A paste of 0.2 g of the larval rearing medium/0.3 ml distilled water was applied to the grooves of the test sites, leaving the control untreated. Ovipositing Jacobina females were offered the choice between the test and control sites 10 times. Washed and Unwashed Larval Rearing Media. To determine if response to the presence of larval rearing medium was mediated by chemical factors or was due to the physical nature of the materials used, the larval rearing medium was washed by a 24-h immersion in hexane, then rinsed three times in hexane, followed by a 48-h immersion in distilled water and a further three rinses in distilled water. Pastes of unwashed and washed larval food (0.2 g/0.3 ml distilled water) were applied to the grooves of the test and control sites, respectively. Oviposition preference of Jacobina females for the test and control sites was tested six times. In a second experiment, a double control test for this bioassay was carried out. A paste of washed larval rearing medium (0.2 g/0.3 ml distilled water) was applied to the test sites of a chamber, leaving the control sites untreated. Oviposition of Jacobina females on the test and control sites was compared six times. Rabbit Feces. To determine if rabbit feces, the most natural component of larval rearing medium, was responsible for the oviposition attractionstimulation effect observed, Jacobina females were introduced into a chamber containing a paste (0.2 g rabbit feces/0.3 ml distilled water) in the grooves of the test sites and untreated control sites. Six replicates of the experiment were carried out. To determine if rabbit feces can elicit a stronger oviposition response than whole larval rearing medium, pastes of 0.05 g feces/0.08 ml distilled water and 0.2 g larval medium/0.3 ml distilled water were applied to the grooves of the test and control sites of a chamber, respectively. Using these conditions, oviposition of Jacobina females on both sites of the chamber was tested six times. Attraction of Gravid Females to Rabbit Feces in an Oviposition Olfactometer. To study oviposition attraction of gravid females to rabbit feces, an olfactometer (Fig. 2) was constructed of five main parts as follows: holding chamber, horizontal passage, vertical passages, test oviposition chamber (Fig. 2, 7), and control oviposition chamber (Fig. 2, 8). The lid of the holding chamber was covered with a fine mesh screen contain-
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
Fig. 1. Design of test and control sites in chamber (11.0 cm diameter, 7.0 cm high) used to study eflFect of colony remains (frass), laboratory larval rearing medium, and rabbit feces on oviposition by L. longipalpis: T, test sites; C, control sites; G, 3-mm-deep grooves on test and control sites; S, shallow groove delineating oviposition sites.
Vol. 29, no. 1
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ELNAIEM & WARD: OVIPOSITION ATTRACTANTS FOR SAND FLIES
ing a central hole plugged with cotton wool (Fig. 2, 1). In the base of the holding chamber a 4.8cm-diameter hole was drilled (Fig. 2, 3) and the base was glued to a slide unit (taken from a WHO standard adult insecticide test kit) (Fig. 2,4). The sliding bar of the unit has two holes, the smaller 1.5 cm in diameter, the larger 4.8 cm in diameter. The smaller hole was covered with fine mesh screen. The slide unit was connected from below to the edges of a 4.8-cm-diameter opening in the middle of the roof of the horizontal passage. The bar then could be placed in one of three positions: first, with the bar completely sealing the chamber; second, with the meshed smaller hole separating the flies from the oviposition sites but allowing odors to pass into the chamber; and, third, with the larger hole coinciding with the openings in the bottom of the holding chamber and the roof of the horizontal passage. The horizontal passage consisted of a plastic tube (4.8 cm diameter, 24 cm long) which opened at both ends into two descending vertical passages 4.8 cm diameter, 7.0 cm long). The ends of the vertical passages were inserted through, and glued to, the edges of 4.8-cm openings in the plastic cover of the test and control oviposition chambers. The oviposition chambers were constructed as described previously. Before use, the olfactometer was disconnected and the plaster bottom of its test and control oviposition chambers were wetted with distilled
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
Fig. 2. Olfactometer used to study the attraction of gravid L. longipalpis females to oviposition surfaces containing rabbit feces: 1, fine-mesh screen containing a central hole plugged with cotton wool; 2, holding chamber (11.5 cm diameter, 7.0 cm high); 3, 4.8-cmdiameter hole in the base of the holding chamber; 4, sliding bar (WHO insecticide test kit); 4.1, closed part of the sliding bar; 4.2,1.5-cm-diameter hole covered by fine-mesh screen; 4.3, 4.8-cm-diameter hole; 5, plastic horizontal passage (4.8 cm diameter, 24 cm high); 6, vertical passage (4.8 cm diameter, 7.0 cm long); 7, test oviposition chamber; 8, control oviposition chamber; 9, 10, 11, 12, oviposition sites A, B, C, and D, respectively.
water and toweled dry. The oviposition sites of the test chamber were labeled units A and B (Fig. 2, 9 and 10). Those of the control chamber were labeled units C and D (Fig. 2, 11 and 12). A paste of 0.2 g rabbit feces/0.3 ml distilled water was applied only in the grooves of unit A (see Fig. 1, TG), leaving unit B of the test chamber and units C and D of the control chamber untreated. The olfactometer then was reconnected, and a group of 50 gravid females was introduced into the holding chamber. The sliding bar at the bottom of the chamber was moved so that the small screened hole coincided with the hole in the base of the chamber. The bar was left in this position for 5 min, exposing the flies to odors emanating from the oviposition chambers. The olfactometer then was placed in a completely dark polystyrene box (30 by 18 by 25 cm) lined with damp laboratory paper toweling, and the sliding bar was retracted completely so that the flies were allowed access to the oviposition chambers. The polystyrene box was kept in the insectary for 3 d. The box then was opened, and the sliding bar was immediately returned to the closed position. The number of females found in test and control chambers and the numbers of eggs laid on units A, B, C, and D were counted. The experiment was repeated six times, using Jacobina females. Comparisons between the number of eggs laid on different oviposition sites and numbers of females found in test and control chambers were made using Wilcoxon's and Mann-Whitney tests, respectively. Bioassay of Rabbit Feces Extracts. To confirm the oviposition attractant—stimulant response to the presence of rabbit feces was mediated or induced (or both) by chemical factors and to investigate appropriate methods for extraction of the compounds involved, extracts of rabbit feces with different solvents and at different concentrations were bioassayed. Soxhlet hexane, hexane immersion, and distilled water immersion extracts were tested. All extracts were prepared initially by the addition of 4 g of rabbit feces to 75 ml of solvent. The hexane soxhlet extraction was carried out at 80°C for 7 h. The hexane and distilled water immersion extractions were made by soaking the rabbit feces in the solvents at room temperature for 48 h and filtering the solutions through single-thickness thimbles (Whatman International Ltd., Maidstone, England). All extracts were concentrated by air evaporation at room temperature to produce 0.00125 g/1 fi\ and then applied on 2-cm-diameter filter paper disks to give 0.025 and 0.1 g/disks. The resulting impregnated filter papers were used 12—24 h after preparation. To bioassay the rabbit feces-impregnated papers, an oviposition choice chamber was constructed as described above (Fig. 1), but no oviposition sites and grooves were delineated. The top of the plaster bottom of the chamber was
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divided with a shallow groove into "test" and "control" surfaces. Immediately before experimentation, the chamber was wetted with distilled water and toweled dry. On each of the test and control surfaces, three adjacent 2-cm-diameter circles were delineated using sharp forceps. The disk containing the rabbit feces extract was pinned on the central circle of the test surface (Fig. 3, 1). Pinned on the opposite central circle of the control was a disk pretreated with 20 /ul of solvent (Fig. 3, 2). On one side of the central circle of both test and control surfaces, two additional blank disks were pinned (Fig. 3, 3). The remaining two circles on both surfaces were left as exposed damp plaster of paris areas surrounded by shallow grooves (Fig. 3, 4). A group of 20 gravid Jacobina females was introduced into the chamber, which was then kept in a completely dark plastic box lined with damp laboratory paper toweling. The box was kept under the insectary conditions for 3 d to allow the females to oviposit. At the end of this period, total numbers of eggs laid on filter paper disks and on the exposed plaster circles of both test and control surfaces were counted. Two concentrations (0.025 and 0.100 g/2-cm-diameter disk) for each type of the three extracts were bioassayed separately six times. Results were analyzed using the Wilcoxon test. Effect of Extracts of Rabbit Feces on Oviposition and Survival of Individually Tubed Females. To investigate the effects of a distilled
Results Under the insectary conditions described, blood-fed L. longipalpis developed a mean of number of 53 eggs per female. Most of the unlaid eggs during the 3-d observation period were retained until the females died, which usually occurred within this period. Oviposition Responses to the Presence of Frass, Larval Rearing Medium, and Rabbit Feces. Females from L'Aguila and Jacobina populations laid significantly more eggs on test sites containing frass than on untreated control sites (Table 1, experiments 1 and 2). A similar response was obtained when Jacobina females were introduced in chambers containing 0.2 g larval rearing medium on test surfaces and untreated controls (Table 1, experiment 3). In chambers containing 0.2 g unwashed larval rearing medium on test oviposition sites and washed rearing medium on the control sites, more eggs were laid on the test than on the control, although the difference was not statistically significant (Table 1, experiment 4). In the double control of this experiment (Table 1, experiment 5), similar numbers of eggs were laid on the sites containing washed larval rearing medium and untreated controls. The experiment testing the effect of the presence of 0.2 g rabbit feces in choice chambers resulted in significantly more eggs laid on the test sites (containing the feces) than on the untreated controls (Table 1, experiment 6). When a similar test was carried out using a chamber containing rabbit feces on the test sites and whole
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Fig. 3. Test and control surfaces in an oviposition choice chamber prepared to investigate effects of rabbit feces extracts on oviposition by L. longipalpis. T, test surface; C, control surface; P, entomological pin used to fix papers in position. 1, 2-cm-diameter filter paper disk coated with rabbit feces extract; 2, 2-cmdiameter filter paper disk coated with the extract solvent; 3, untreated filter paper disk; 4, 2-cm-diameter circle on blank plaster of paris surface.
water extract of rabbit feces on the stimulation of oviposition and the survival of the females during the egg-laying process, the following experiment was conducted. Two days after blood feeding, gravid females were collected in glass vials (2.1 cm diameter, 4.0 cm high) lined at the base with 2-cm-diameter filter paper disks which were impregnated with 0.025 g rabbit feces extracted in distilled water (test group) or left untreated (control group). The filter paper disks of the test and control groups were both wetted with 0.04 ml (five drops) of distilled water applied with a syringe and hypodermic needle through the gauze covering the tubes. The glass vials then were placed in a plastic box lined with damp laboratory paper toweling and kept at the insectary conditions described. Numbers of eggs laid and survival of females were recorded daily for 3-5 d after a blood meal. Females that laid more than the average number of eggs per experiment and were found healthy between days 3 and 5 after the blood-meal were considered as "oviposition survivors." Fifty-one Jacobina females were tested for each of the test and control groups. Results were analyzed using the MannWhitney test.
January 1992
ELNAIEM & WARD: OVIPOSITION ATTRACTANTS FOR SAND FLIES Table 3. Oviposition response of L. longipalpis to rabbit feces extracts applied on test oviposition surface
Table 1. Oviposition of L. longipalpis on surfaces containing frass, larval rearing medium, and rabbit feces Experimental treatments0
No. eggs laid/8 cm2, x± SE Test Control 153.7 ± 171.9 ± 138.6 ± 67.7 ± 56.7 ± 199.7 ± 167.3 ±
8.6 26.1 14.7 16.5 12.2 28.2 6.2 42.2 8.2 46.3 30.3 58.8 4.2 105.0
No. eggs/9.42 cm2 oviposition surface, x ± SE Test Control
Rabbit feces extract
16 15 10 6 6 6 6
0.025 g/50 fil hexane (Soxhlet) 0.100 g/50 /il hexane (Soxhlet) 0.025 g/50 MI hexane 0.100 g/50 /il hexane 0.025 g/50 Ail distilled water 0.100 g/50 Ail distilled water
2.9
KEY WORDS Insecta, Lutzomyia longipalpis, oviposition attractants, oviposition preferences
DESPITE RECENT PROGRESS in the colonization of phlebotomine sandflies, the methods remain laborious and time-consuming. One problem is the high mortality of females before or during oviposition (Killick-Kendrick et al. 1977, Chaniotis 1986) which restricts many basic studies on sandfly biology and their vectorial competence for Leishmania and arboviruses. Like many other insects, oviposition by sandflies probably is controlled through a combination of environmental and physiological factors, of which very little is known. Of the few oviposition studies, most have dealt with the regulation of physical factors such as temperature and relative humidity (Foster et al. 1970, Chaniotis 1986). Recently, Schlein et al. (1990) found that egg laying by Phlebotomies papatasi Scopoli is promoted by many factors including the presence of cow manure. In addition, we have demonstrated evidence for the presence of an oviposition attractant-stimulant pheromone associated with the eggs of Lutzomyia longipalpis Lutz & Neiva (Elnaiem & Ward 1990). In our study, the effect on oviposition by L. longipalpis of old colony remains (frass), fresh larval rearing medium, and rabbit feces is described. The study was designed to determine if the sandfly female lays more eggs on surfaces containing frass or laboratory larval food than on controls. If so, it was considered important to establish which portion of the larval food was responsible for the increased egg laying and
whether the response was mediated or induced by chemical or physical factors or both. Furthermore, we attempted to determine if rabbit feces acted as an oviposition attractant or stimulant and if it affected postoviposition survival. Materials and Methods Sandflies. The two laboratory colonies of L. longipalpis used in this study, "J a c °bina" and "L'Aguila," originated from females collected in Salvador (Brazil) and Tolima (Colombia), respectively, and were maintained using the methods described by Modi & Tesh (1983). Before use in experiments, females that had fed on a hamster anesthetized with a 12 mg/ml solution of sodium pentabarbitone were isolated with approximately equal numbers of males at 95% RH, 27°C, and 12:12 (L:D) photoperiod for 3 d to allow complete defecation and egg maturation. Influence of Frass, Larval Food, and Rabbit Feces on Oviposition by L. longipalpis. The effects of frass, larval food, and rabbit feces on oviposition by L. longipalpis were investigated using oviposition choice chambers. These chambers were constructed by drilling 30 equally spaced 0.5-cm-diameter holes in the base of polymethyl pentane pots (11.0 cm diameter, 7.0 cm high) (Nalgene, BDH Apparatus, BDH Ltd., Speke, Liverpool, England) which then were lined with a 1-cm-thick layer of plaster of paris on the bottom of each pot. Four oviposition sites
0022-2585/92/0005-0012$02.00/0 © 1992 Entomological Society of America
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
J. Med. Entomol. 29(1): 5-12 (1992) ABSTRACT Oviposition preferences of Lutzomyia longipalpis Lutz & Neiva for surfaces containing frass (colony remains), larval rearing medium, and rabbit feces were investigated in the laboratory. In oviposition choice chambers, significantly more eggs were laid on sites containing frass, larval rearing medium, or rabbit feces than on untreated control sites. Experiments using unwashed and washed materials indicated, for the first time, the presence of chemical oviposition attractants or stimulants or both in larval medium (consisting of equal weight proportions of rabbit feces, potting compost, sand, and Daphnia). In an olfactometer, aroma from rabbit feces strongly attracted ovipositing females, and significantly higher numbers of eggs were laid in the olfactometer test chamber containing the rabbit feces than in the untreated control chamber. In comparisons of oviposition responses to hexane and distilled water extracts of rabbit feces, only the water extract was attractive. Similarly, the distilled water extract of rabbit feces proved to be an oviposition stimulant. Females kept individually with filter papers impregnated with the extract of feces oviposited earlier and survived longer than females kept with untreated papers.
JOURNAL OF MEDICAL ENTOMOLOGY
(4 cm 2 area) then were delineated at the sides of the plaster surface of each pot by a 3-mm-deep groove and quartered by a shallow groove. Two of the oviposition sites adjoined each other on one side of the chamber; the two others were situated on the other side. The former were the "T" or test sites, the latter were the "C" or the control sites (Fig. 1). Immediately before use in experiments, the plaster bottom of the chamber was wetted with distilled water, the excess moisture was removed with laboratory paper toweling, and test and control surfaces were treated as described below. A group of 20 gravid females then were introduced into the chamber, and the chamber was transferred to a sealed plastic box lined with damp laboratory paper toweling. The chamber was kept in complete darkness for 3 d under the insectary conditions described above. At the end of this period, numbers of eggs laid on test and control oviposition sites were counted. Eggs laid outside the test and control sites were ignored. The results were analyzed statistically using Wilcoxon's matched-pairs signed-rank test. The treatments of test and control sites were as follows: Frass. Frass was collected from larval rearing pots from which adults had emerged. A paste of 0.2 g frass/0.3 ml distilled water was applied only in the grooves of the two test oviposition sites of a chamber, leaving the grooves on the control surface untreated. A distilled-water paste was used in preference to dry material to avoid any
reduction in the moisture of the test surface. The experiment was repeated 16 times using L'Aguila females and 15 times with Jacobina females. Larval Rearing Medium. Larval rearing medium was prepared by mixing rabbit feces, potting compost, sand, and Daphnia in equal proportions by weight and autoclaving the mixture. The rabbit feces were collected from a hutch containing a pet rabbit fed on commercial rabbit food, obtained from pet stores, with occasional supplements of green vegetables. A paste of 0.2 g of the larval rearing medium/0.3 ml distilled water was applied to the grooves of the test sites, leaving the control untreated. Ovipositing Jacobina females were offered the choice between the test and control sites 10 times. Washed and Unwashed Larval Rearing Media. To determine if response to the presence of larval rearing medium was mediated by chemical factors or was due to the physical nature of the materials used, the larval rearing medium was washed by a 24-h immersion in hexane, then rinsed three times in hexane, followed by a 48-h immersion in distilled water and a further three rinses in distilled water. Pastes of unwashed and washed larval food (0.2 g/0.3 ml distilled water) were applied to the grooves of the test and control sites, respectively. Oviposition preference of Jacobina females for the test and control sites was tested six times. In a second experiment, a double control test for this bioassay was carried out. A paste of washed larval rearing medium (0.2 g/0.3 ml distilled water) was applied to the test sites of a chamber, leaving the control sites untreated. Oviposition of Jacobina females on the test and control sites was compared six times. Rabbit Feces. To determine if rabbit feces, the most natural component of larval rearing medium, was responsible for the oviposition attractionstimulation effect observed, Jacobina females were introduced into a chamber containing a paste (0.2 g rabbit feces/0.3 ml distilled water) in the grooves of the test sites and untreated control sites. Six replicates of the experiment were carried out. To determine if rabbit feces can elicit a stronger oviposition response than whole larval rearing medium, pastes of 0.05 g feces/0.08 ml distilled water and 0.2 g larval medium/0.3 ml distilled water were applied to the grooves of the test and control sites of a chamber, respectively. Using these conditions, oviposition of Jacobina females on both sites of the chamber was tested six times. Attraction of Gravid Females to Rabbit Feces in an Oviposition Olfactometer. To study oviposition attraction of gravid females to rabbit feces, an olfactometer (Fig. 2) was constructed of five main parts as follows: holding chamber, horizontal passage, vertical passages, test oviposition chamber (Fig. 2, 7), and control oviposition chamber (Fig. 2, 8). The lid of the holding chamber was covered with a fine mesh screen contain-
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
Fig. 1. Design of test and control sites in chamber (11.0 cm diameter, 7.0 cm high) used to study eflFect of colony remains (frass), laboratory larval rearing medium, and rabbit feces on oviposition by L. longipalpis: T, test sites; C, control sites; G, 3-mm-deep grooves on test and control sites; S, shallow groove delineating oviposition sites.
Vol. 29, no. 1
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ELNAIEM & WARD: OVIPOSITION ATTRACTANTS FOR SAND FLIES
ing a central hole plugged with cotton wool (Fig. 2, 1). In the base of the holding chamber a 4.8cm-diameter hole was drilled (Fig. 2, 3) and the base was glued to a slide unit (taken from a WHO standard adult insecticide test kit) (Fig. 2,4). The sliding bar of the unit has two holes, the smaller 1.5 cm in diameter, the larger 4.8 cm in diameter. The smaller hole was covered with fine mesh screen. The slide unit was connected from below to the edges of a 4.8-cm-diameter opening in the middle of the roof of the horizontal passage. The bar then could be placed in one of three positions: first, with the bar completely sealing the chamber; second, with the meshed smaller hole separating the flies from the oviposition sites but allowing odors to pass into the chamber; and, third, with the larger hole coinciding with the openings in the bottom of the holding chamber and the roof of the horizontal passage. The horizontal passage consisted of a plastic tube (4.8 cm diameter, 24 cm long) which opened at both ends into two descending vertical passages 4.8 cm diameter, 7.0 cm long). The ends of the vertical passages were inserted through, and glued to, the edges of 4.8-cm openings in the plastic cover of the test and control oviposition chambers. The oviposition chambers were constructed as described previously. Before use, the olfactometer was disconnected and the plaster bottom of its test and control oviposition chambers were wetted with distilled
Downloaded from http://jme.oxfordjournals.org/ at University of Nebraska-Lincoln Libraries on September 2, 2015
Fig. 2. Olfactometer used to study the attraction of gravid L. longipalpis females to oviposition surfaces containing rabbit feces: 1, fine-mesh screen containing a central hole plugged with cotton wool; 2, holding chamber (11.5 cm diameter, 7.0 cm high); 3, 4.8-cmdiameter hole in the base of the holding chamber; 4, sliding bar (WHO insecticide test kit); 4.1, closed part of the sliding bar; 4.2,1.5-cm-diameter hole covered by fine-mesh screen; 4.3, 4.8-cm-diameter hole; 5, plastic horizontal passage (4.8 cm diameter, 24 cm high); 6, vertical passage (4.8 cm diameter, 7.0 cm long); 7, test oviposition chamber; 8, control oviposition chamber; 9, 10, 11, 12, oviposition sites A, B, C, and D, respectively.
water and toweled dry. The oviposition sites of the test chamber were labeled units A and B (Fig. 2, 9 and 10). Those of the control chamber were labeled units C and D (Fig. 2, 11 and 12). A paste of 0.2 g rabbit feces/0.3 ml distilled water was applied only in the grooves of unit A (see Fig. 1, TG), leaving unit B of the test chamber and units C and D of the control chamber untreated. The olfactometer then was reconnected, and a group of 50 gravid females was introduced into the holding chamber. The sliding bar at the bottom of the chamber was moved so that the small screened hole coincided with the hole in the base of the chamber. The bar was left in this position for 5 min, exposing the flies to odors emanating from the oviposition chambers. The olfactometer then was placed in a completely dark polystyrene box (30 by 18 by 25 cm) lined with damp laboratory paper toweling, and the sliding bar was retracted completely so that the flies were allowed access to the oviposition chambers. The polystyrene box was kept in the insectary for 3 d. The box then was opened, and the sliding bar was immediately returned to the closed position. The number of females found in test and control chambers and the numbers of eggs laid on units A, B, C, and D were counted. The experiment was repeated six times, using Jacobina females. Comparisons between the number of eggs laid on different oviposition sites and numbers of females found in test and control chambers were made using Wilcoxon's and Mann-Whitney tests, respectively. Bioassay of Rabbit Feces Extracts. To confirm the oviposition attractant—stimulant response to the presence of rabbit feces was mediated or induced (or both) by chemical factors and to investigate appropriate methods for extraction of the compounds involved, extracts of rabbit feces with different solvents and at different concentrations were bioassayed. Soxhlet hexane, hexane immersion, and distilled water immersion extracts were tested. All extracts were prepared initially by the addition of 4 g of rabbit feces to 75 ml of solvent. The hexane soxhlet extraction was carried out at 80°C for 7 h. The hexane and distilled water immersion extractions were made by soaking the rabbit feces in the solvents at room temperature for 48 h and filtering the solutions through single-thickness thimbles (Whatman International Ltd., Maidstone, England). All extracts were concentrated by air evaporation at room temperature to produce 0.00125 g/1 fi\ and then applied on 2-cm-diameter filter paper disks to give 0.025 and 0.1 g/disks. The resulting impregnated filter papers were used 12—24 h after preparation. To bioassay the rabbit feces-impregnated papers, an oviposition choice chamber was constructed as described above (Fig. 1), but no oviposition sites and grooves were delineated. The top of the plaster bottom of the chamber was
Vol. 29, no. 1
JOURNAL OF MEDICAL ENTOMOLOGY
divided with a shallow groove into "test" and "control" surfaces. Immediately before experimentation, the chamber was wetted with distilled water and toweled dry. On each of the test and control surfaces, three adjacent 2-cm-diameter circles were delineated using sharp forceps. The disk containing the rabbit feces extract was pinned on the central circle of the test surface (Fig. 3, 1). Pinned on the opposite central circle of the control was a disk pretreated with 20 /ul of solvent (Fig. 3, 2). On one side of the central circle of both test and control surfaces, two additional blank disks were pinned (Fig. 3, 3). The remaining two circles on both surfaces were left as exposed damp plaster of paris areas surrounded by shallow grooves (Fig. 3, 4). A group of 20 gravid Jacobina females was introduced into the chamber, which was then kept in a completely dark plastic box lined with damp laboratory paper toweling. The box was kept under the insectary conditions for 3 d to allow the females to oviposit. At the end of this period, total numbers of eggs laid on filter paper disks and on the exposed plaster circles of both test and control surfaces were counted. Two concentrations (0.025 and 0.100 g/2-cm-diameter disk) for each type of the three extracts were bioassayed separately six times. Results were analyzed using the Wilcoxon test. Effect of Extracts of Rabbit Feces on Oviposition and Survival of Individually Tubed Females. To investigate the effects of a distilled
Results Under the insectary conditions described, blood-fed L. longipalpis developed a mean of number of 53 eggs per female. Most of the unlaid eggs during the 3-d observation period were retained until the females died, which usually occurred within this period. Oviposition Responses to the Presence of Frass, Larval Rearing Medium, and Rabbit Feces. Females from L'Aguila and Jacobina populations laid significantly more eggs on test sites containing frass than on untreated control sites (Table 1, experiments 1 and 2). A similar response was obtained when Jacobina females were introduced in chambers containing 0.2 g larval rearing medium on test surfaces and untreated controls (Table 1, experiment 3). In chambers containing 0.2 g unwashed larval rearing medium on test oviposition sites and washed rearing medium on the control sites, more eggs were laid on the test than on the control, although the difference was not statistically significant (Table 1, experiment 4). In the double control of this experiment (Table 1, experiment 5), similar numbers of eggs were laid on the sites containing washed larval rearing medium and untreated controls. The experiment testing the effect of the presence of 0.2 g rabbit feces in choice chambers resulted in significantly more eggs laid on the test sites (containing the feces) than on the untreated controls (Table 1, experiment 6). When a similar test was carried out using a chamber containing rabbit feces on the test sites and whole
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Fig. 3. Test and control surfaces in an oviposition choice chamber prepared to investigate effects of rabbit feces extracts on oviposition by L. longipalpis. T, test surface; C, control surface; P, entomological pin used to fix papers in position. 1, 2-cm-diameter filter paper disk coated with rabbit feces extract; 2, 2-cmdiameter filter paper disk coated with the extract solvent; 3, untreated filter paper disk; 4, 2-cm-diameter circle on blank plaster of paris surface.
water extract of rabbit feces on the stimulation of oviposition and the survival of the females during the egg-laying process, the following experiment was conducted. Two days after blood feeding, gravid females were collected in glass vials (2.1 cm diameter, 4.0 cm high) lined at the base with 2-cm-diameter filter paper disks which were impregnated with 0.025 g rabbit feces extracted in distilled water (test group) or left untreated (control group). The filter paper disks of the test and control groups were both wetted with 0.04 ml (five drops) of distilled water applied with a syringe and hypodermic needle through the gauze covering the tubes. The glass vials then were placed in a plastic box lined with damp laboratory paper toweling and kept at the insectary conditions described. Numbers of eggs laid and survival of females were recorded daily for 3-5 d after a blood meal. Females that laid more than the average number of eggs per experiment and were found healthy between days 3 and 5 after the blood-meal were considered as "oviposition survivors." Fifty-one Jacobina females were tested for each of the test and control groups. Results were analyzed using the MannWhitney test.
January 1992
ELNAIEM & WARD: OVIPOSITION ATTRACTANTS FOR SAND FLIES Table 3. Oviposition response of L. longipalpis to rabbit feces extracts applied on test oviposition surface
Table 1. Oviposition of L. longipalpis on surfaces containing frass, larval rearing medium, and rabbit feces Experimental treatments0
No. eggs laid/8 cm2, x± SE Test Control 153.7 ± 171.9 ± 138.6 ± 67.7 ± 56.7 ± 199.7 ± 167.3 ±
8.6 26.1 14.7 16.5 12.2 28.2 6.2 42.2 8.2 46.3 30.3 58.8 4.2 105.0
No. eggs/9.42 cm2 oviposition surface, x ± SE Test Control
Rabbit feces extract
16 15 10 6 6 6 6
0.025 g/50 fil hexane (Soxhlet) 0.100 g/50 /il hexane (Soxhlet) 0.025 g/50 MI hexane 0.100 g/50 /il hexane 0.025 g/50 Ail distilled water 0.100 g/50 Ail distilled water
2.9
