Diptera: Psychodidae

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Jan 5, 2007 - females at 23 or 28C to understand the effect of temperature on bloodmeal ... Egg numbers were positively correlated with the amount of hematin excreted in feces of .... As shown by Lawyer et al. .... Colorado, Niwot, CO.


Effect of Temperature on Metabolism of Phlebotomus papatasi (Diptera: Psychodidae) IVANA BENKOVA



Department of Parasitology, Charles University, Vinicna 7, 128 44 Prague, Czech Republic

J. Med. Entomol. 44(1): 150Ð154 (2007)

ABSTRACT Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) is the most important vector of Leishmania major, and previous experiments revealed that Leishmania development in the sand ßy midgut is signiÞcantly affected by temperature. Therefore, we maintained blood-fed P. papatasi females at 23 or 28⬚C to understand the effect of temperature on bloodmeal digestion and developmental times of this sand ßy. At the lower temperature, the metabolic processes were slower and developmental times were longer: defecation, oviposition, and egg hatch started later and took longer to complete. Also, the mortality of blood-fed females was signiÞcantly lower. The defecation of bloodmeal remains was delayed for 12Ð36 h at 23⬚C compared with the group maintained at 28⬚C. Such delay would provide more time for Leishmania to establish the midgut infection and could partially explain the increased susceptibility of P. papatasi to Leishmania major at 23⬚C. In both experimental groups, blood-fed females laid similar numbers of eggs (mean 60 and 70, maximum 104 and 115 per female). Egg numbers were positively correlated with the amount of hematin excreted in feces of ovipositing females. In parallel experiments, autogeny was recorded in 8% of females. The autogenous egg batches were smaller (mean, 12; range, 1Ð39), but they all produced viable larvae. KEY WORDS sand ßy, Phlebotomus, temperature, metabolic processes, Leishmania

Temperature signiÞcantly inßuences arthropod developmental times (Wagner et al. 1991) and other life history events such as survivorship; however, there are few studies documenting the impact of temperature on phlebotomine sand ßies. Theodor (1936) described thermal limits for Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae), and recently, Erisoz-Kasap and Alten (2005) determined the degree-day requirements and developmental zero for this species. Endris et al. (1984) reported the temperature-dependent development rates for immatures of Lutzomyia anthophora (Addis). In bloodsucking insects, the temperature also may affect susceptibility to transmitted pathogens. Benkova et al. (2006) documented a signiÞcant impact of temperature on the vectorial competence of P. papatasi, with those maintained at 23⬚C having a higher infection rate and more intense infections of Leishmania major Yakimoff and Schokhor than those maintained at 28⬚C. In the work presented here, we maintained P. papatasi females at 23 and 28⬚C to document the effect of temperature on their development and digestion. We determined time to defecation, oviposition, and egg hatch as well as the impact of temperature on survivorship, number of eggs laid, egg viability, and autogeny. Additionally, we measured excreted hematin as an indicator of bloodmeal


Corresponding author, e-mail: [email protected]

size, and we examined its relationship with the number of eggs laid. Materials and Methods Colony Maintenance. The colony of P. papatasi originated from ⬇200 blood-fed females collected by our group in Sanliurfa, Turkey, in September 1999. The same colony was recently used to study the effect of different temperature on L. major development in sand ßies (Benkova et al. 2006). The colony was maintained in an insectary at 25⬚C and 60 Ð70% humidity. Breeding techniques described by Modi (1997) and Killick-Kendrick et al. (1977) were modiÞed based on 15 yr of experience of our group with various sand ßy colonies. Brießy, plastic pots with a hole cut in the bottom were lined with plaster of Paris and closed with a Þne gauze. Breeding pots were maintained in plastic boxes, the bottoms of which were lined with a 1-cm-thick layer of sand dampened with distilled water. The plaster soaked up the water and provided females a humid but solid substrate on which to rest and lay eggs. A composted mixture of ground and dried rabbit feces and rabbit pellets served as larval food. The mixture of Þne ground pellets and air-dried feces was spread in a thin layer in plastic trays and composted under aerobic conditions in acrylic cabinets for 3Ð 4 wk at room temperature. The composted food was dried at room

0022-2585/07/0150Ð0154$04.00/0 䉷 2007 Entomological Society of America

January 2007 Table 1.



Main developmental parameters of P. papatasi females fed on mice and maintained at different temperatures

Parameter Survival time Defecation time Oviposition time No. of eggs per female Egg-to-larva development




Mean ⫾ SD



Mean ⫾ SD


60 57 40 38 19

258.2 ⫾ 108.77* 77.05 ⫾ 14.15* 264 ⫾ 52.13* 69.63 ⫾ 23.55  250.11 ⫾ 17.09*

84Ð432 48Ð120 120Ð408 6Ð115 228Ð288

60 48 39 39 16

65.8 ⫾ 80.77* 60 ⫾ 11.07* 179.69 ⫾ 60.97* 60.15 ⫾ 27.82  165.75 ⫾ 10.93*

48Ð372 36Ð84 72Ð264 3Ð104 144Ð192

Start of metabolic processes is indicated in hours post bloodmeal. * Values are signiÞcantly different between the two temperatures, P ⬍ 0.001.   Values are not signiÞcantly different between the two temperatures, P ⬎ 0.05.

temperature, Þnely ground again, and stored until use at ⫺20⬚C. Larvae are checked and supplied with food three times a week. Emerged adults are released from breeding pots into nylon cloth cages (40 cm2) suspended on a steel frame. High humidity (70 Ð100%) was ensured by wrapping cages in plastic bags with wet cotton wool inside. Both sexes have unlimited access to sugar meals (50% solution of brown sugar in distilled water). Once or twice a week, females were fed on mice anesthetized with ketamin-xylazin. One day later, blood-fed females were separated into small cages (20 cm2), and after defecation they were transferred into breeding pots to lay eggs. Effect of Temperature. Female sand ßies of the same age (4 Ð5 d) were fed on anesthetized BALB/c mouse. Females with fully extended abdomens were carefully separated immediately after blood feeding and put individually into 20-ml glass vials for observation and to obtain data on each female separately, including times of defecation and oviposition, number of eggs, and time of hatch. The technique described by Killick-Kendrick and Killick-Kendrick (1991) was adapted. Two pieces of Þlter paper were put into each vial, a round Þlter paper covering the bottom and a folded Þlter paper in a vertical position. The opening was closed with gauze, and a small piece of cotton wool soaked in 50% solution of brown sugar was put on the top of the gauze and changed daily. Vials with females were kept in plastic containers lined with Þlter paper moistened with distilled water. One container with 30 females was kept in an incubator set at 23⬚C, and the other container was kept in an incubator set at 28⬚C. After defecation, when brown spots were noted on the paper or inner surface of the vial, females were encouraged to lay eggs by moistening the folded piece of Þlter paper with a few drops of distilled water by using a syringe with hypodermic needle. Then, females were given free access to a sugar meal. Data on mortality, defecation, oviposition, and egg hatch were obtained every 12 h after the blood feeding. The experiment was repeated twice. To assess the mortality of females kept on sugar meal only and their possible autogeny, groups of females maintained on sugar only were separated at the age of 4 Ð5 d posteclosion (30 for each temperature) and maintained under the same conditions. The experiment was repeated three times.

Assay of Excreted Hematin. The measurement of hematin defecated by females was used to determine the amount of blood ingested by individual females. The method developed by Briegel (1980) for mosquitoes enables females to be kept alive for oviposition. Brießy, blood-fed females were separated individually and treated as mentioned above, except the Þlter paper was not put into the vials before defecation. After defecation, females were transferred for oviposition into another vial with Þlter papers inside. The feces in the Þrst vial was dissolved in 400 ␮l of a 1% lithium carbonate solution. The absorbance of these samples was measured by an ELISA reader (Customized Applications, Inc., Chicago Heights, IL). The concentration of hematin of the samples was derived from the standard curve made of known concentrations of porcine hematin (1Ð20 ␮g/ml) (Sigma-Aldrich, St. Louis, MO). Statistical Analyses. Data obtained from groups maintained at the two different temperatures were compared using nonparametric statistics (MannÐ Whitney U-test) because transformation of the data did not sufÞciently improve normality. We used a nonparametric Spearman rank correlation test to deÞne the relations between the amount of hematin defecated and the number of eggs (STATISTICA, StatSoft 2001). Results and Discussion Basic developmental parameters of P. papatasi groups maintained at 23 and 28⬚C are summarized in Table 1. As expected, at lower temperature, the metabolic processes were slower; defecation, oviposition, and egg hatch started later and took longer to complete. The mortality of blood-fed females was signiÞcantly lower at 23⬚C: P. papatasi females lived ⬇2 d longer than at 28⬚C (Table 1; Fig. 1). However, mortality of females kept solely on sugar meal was similar in both groups (P ⫽ 0.63), ⬇80% of females survived for ⬎6 d (data not shown). P. papatasi is an arid species adapted to a wide range of temperatures (Theodor 1936) and is prevalent even in very dry and hot areas. Free-living sand ßies usually survive for a substantially shorter period than those from laboratory colonies, mainly due to inclement environmental conditions. Schlein and Jacobson (1999) found in a P. papatasi population



Vol. 44, no. 1

Fig. 1. Survival and defecation of blood-fed P. papatasi females and egg hatch. Comparison of females maintained at 23 and at 28⬚C. Data are given in days PBM.

in central Jordan Valley that only ⬇9% of females lived for ⬎6 d, and most of the population consisted of 2Ð 4-d-old sand ßies.

Defecation of blood-fed females maintained at 23⬚C was delayed by 12Ð36 h (Þrst and last female in the group, respectively) compared with females main-

Fig. 2. Number of eggs, oviposition time, and egg hatch time in two groups of P. papatasi females maintained at 23⬚C (A) and 28⬚C (B). Data are given in days PBM. Numbers above bars indicate number of females ovipositing in the same interval.

January 2007


Fig. 3. Positive correlation between number of eggs and content of hematin defecated by P. papatasi females (n ⫽ 24, P ⫽ 0.048, r ⫽ 0.4072). Dashed lines indicate 95% conÞdence intervals.

tained at 28⬚C (Table 1; Fig. 1). This difference is signiÞcant enough to affect the sand ßy susceptibility to Leishmania infection. As shown by Lawyer et al. (1990), sand ßies with fast digestion are poor vectors as ingested Leishmania parasites are defecated from the sand ßy midgut before establishment of infection. Slower digestion of P. papatasi at 23⬚C provides L. major a longer time to establish the midgut infection. The number of eggs laid by blood-fed females was relatively high (mean, 60 and 70 eggs per female); the difference between groups was not signiÞcant (Table 1; Fig. 2). Also, the maximum number of eggs laid by one female (115 eggs) was relatively high compared with data published for other sand ßy colonies including P. papatasi colonies (Harre et al. 2001). Egg numbers, however, ranged widely among the females visually judged as fully engorged. In bloodsucking insects, the number of eggs is frequently correlated with the amount of ingested blood, because proteins gained from blood digestion are used for the synthesis of yolk proteins (for review, see James and Fallon 1996). Therefore, we measured hematin excreted by ovipositing females to determine the amount of blood ingested by females. The batch size showed a positive correlation (P ⬍ 0.05) with the amount of hematin in feces (Spearman r ⫽ 0.4072) (Fig. 3). The average content of defecated hematin was 2.12 ⫾ 0.58 ␮g, and no signiÞcant difference was found between groups of females maintained at different temperatures (P ⫽ 0.37). Developmental times signiÞcantly differed between groups (Fig. 2). The group of females kept at 28⬚C oviposited earlier and over a relatively short period, on days 3Ð12 post-bloodmeal (PBM). In the group maintained at 23⬚C, oviposition was delayed for ⬇3.5 d, and the egg-laying period of the group was extended to 12 d (days 6 Ð18 PBM). As expected, egg development also was slowed by the lower temperature. At 23⬚C, the mean egg hatch time was 250 h (10.5


d) postoviposition, whereas at 28⬚C it was 166 h (7 d). Similar data were recently obtained for P. papatasi by Erisoz-Kasap and Alten (2005); in their colony, the mean egg developmental time was 8.2 d at 25⬚C and 6.76 d at 28⬚C. Early oviposition by some P. papatasi females on day 6 PBM at 23⬚C (Fig. 2) could be explained by autogeny. In control experiments with P. papatasi maintained on sugar only we recorded 7.7% egg-laying females. These autogenous females laid signiÞcantly smaller egg number than the anautogenous females (mean, 11.8; range, 1Ð39). The number of eggs laid by autogenous females and their average time to oviposition did not show any signiÞcant difference depending on temperature (P ⫽ 0.57). All batches were viable and produced larvae. Autogeny is an adaptation of sand ßies for reproduction during periods without a bloodmeal source (Schmidt 1965) and has been described in various species (Johnson 1961, MontoyaLerma 1992, HanaÞ et al. 1999, Kassem and Hassan 2003), including different populations of P. papatasi (Dolmatova 1946, Schmidt 1965). In certain P. papatasi populations from Egypt, autogeny is frequent, and according to El Kammah (1972), it may occur in up to 86% of females. Acknowledgments We thank an anonymous referee for valuable comments. This work was supported by the Ministry of Education of the Czech Republic (projects VZ 21620828 and LC06009).

References Cited Benkova, I., J. Peckova, A. Svarovska, and P. Volf. 2006. The effect of temperature on the protease activity and Leishmania development in sand ßy midgut. ICOPA XII. Glasgow, United Kingdom. Briegel, H. 1980. Determination of uric acid and hematin in a single sample of excreta from blood-fed insects. Experientia 36: 1428. Dolmatova, A. V. 1946. The autogenous development of eggs in Phlebotomus papatasi (Scopoli). Med. Parasitol. 15: 58 Ð 62. El Kammah, K. M. 1972. Frequency of autogeny in wildcaught Egyptian Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). J. Med. Entomol. 9: 294. Endris, R. G., D. G. Young, and J. F. Butler. 1984. The laboratory biology of the sand ßy Lutzomyia anthophora (Diptara: Psychodidae). J. Med. Entomol. 21: 656 Ð 664. Erisoz-Kasap, O., and B. Alten. 2005. Laboratory estimation of degree-day developmental requirements of Phlebotomus papatasi (Diptera: Psychodidae). J. Vector Ecol. 30: 328 Ð333. Hanafi, A. H., J. R. W. Kanour, M. G. Beavers, and E. G. Tetreault. 1999. Colonization and bionomics of the sandßy Phlebotomus kazeruni from Sinai, Egypt. Med. Vet. Entomol. 13: 295Ð298. Harre, J. G., K. M. Dorsey, K. L. Armstrong, J. R. Burge, and K. E. Kinnamon. 2001. Comparative fecundity and survival rates of Phlebotomus papatasi sandßies membrane fed on blood from eight mammal species. Med. Vet. Entomol. 15: 189 Ð196. James, A. A., and A. M. Fallon. 1996. Gene Expression in Vectors, pp. 229 Ð245. In B. J. Beaty and W. C. Marquardt



[eds.], The biology of disease vectors. University Press of Colorado, Niwot, CO. Johnson, P. T. 1961. Autogeny in Panamanian Phlebotomus sandßies (Diptera: Psychodidae). Ann. Entomol. Soc. Am. 54: 116 Ð118. Kassem, H. A., and A. N. Hassan. 2003. Ovarian development and blood-feeding activity in Phlebotomus bergeroti Parrot (Diptera: Psychodidae) from Egypt. Ann. Trop. Med. Parasitol. 97: 521Ð526. Killick-Kendrick, M., and R. Killick-Kendrick. 1991. The initial establishment of sandßy colonies. Parassitologia 33: 315Ð320. Killick-Kendrick, R., A. J. Leany, and P. D. Ready. 1977. The establishment, maintenance and productivity of a laboratory colony of Lutzomyia longipalpis (Diptera: Psychodidae). J. Med. Entomol. 13: 429 Ð 440. Lawyer, P. G., P. M. Ngumbi, C. O. Anjili, S. O. Odongo, Y. B. Mebrahtu, J. I. Githure, D. K. Koech, and C. R. Roberts. 1990. Development of Leishmania major in Phlebotomus duboscqi and Sergentomyia schwetzi (Diptera: Psychodidae). Am. J. Trop. Med. Hyg. 43: 31Ð 43.

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Modi, G. B. 1997. Care and maintenance of phlebotomine sandßy colonies, pp. 21Ð30. In J. M. Crampton, C. B. Beard, and C. Louis [eds.], Molecular biology of insect disease vectors: a methods manual. Chapman & Hall, London, United Kingdom. Montoya-Lerma, J. 1992. Autogeny in the Neotropical sand ßy Lutzomyia lichyi (Diptera, Psychodidae) from Colombia. J. Med. Entomol. 29: 698 Ð 699. Schlein, Y., and R. L. Jacobson. 1999. Sugar meals and longevity of the sandßy Phlebotomus papatasi in an arid focus of Leishmania major in the Jordan Valley. Med. Vet. Entomol. 13: 65Ð71. Schmidt, M. L. 1965. Autogenic development of Phlebotomus papatasi (Scopoli) from Egypt. J. Med. Entomol. 1: 356. Theodor, O. 1936. On the relation of Phlebotomus papatasii to the temperature and humidity of the environment. Bull. Entomol. Res. 27: 653Ð 671. Wagner, T. L., R. L. Olson, and J. L. Willers. 1991. Modeling Arthropod Development time. J. Agric. Entomol. 8: 251Ð 270. Received 29 June 2006; accepted 3 October 2006.