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Differences in response to temperature and density between two strains of the mosquito, Culex pipiens molestus Forskal
Jir Olejncek1 and Ivan Gelbic2
1

Institute of Parasitology, Academy of Sciences of the Czech Republic, Braniovsk 31, 370 05 Cesk Budejovice, CZECH REPUBLIC 2 Institute of Entomology, Academy of Sciences of the Czech Republic, Braniovsk 31, 370 05 Cesk Budejovice, CZECH REPUBLIC Received 29 November 1999; Accepted 14 April 2000

ABSTRACT: Two strains of Culex pipiens molestus of different geographical origin were reared for more than five years under the same laboratory conditions in order to study to what extent the reactions of these strains to constant temperature and density become similar. Lower temperature and higher densities caused not only the prolongation of developmental time of preimaginal stages and increasing mortality of larvae and pupae in both strains but also the extension of the rate between the first and last emerging imago in both sexes of both strains. Absolute time spent in the preimaginal stage was also influenced by genetic factors. Both strains displayed different values, although both strains were for reared in the laboratory under identical conditions. Journal of Vector Ecology 25(2): 136-145. 2000. Keyword Index: Culex molestus, mosquitoes, preimaginal development. INTRODUCTION Prolonged laboratory colonization often results in the selection for insects that are adapted to laboratory conditions (Knop et al. 1987). The rate of insect development can also be influenced by temperature and density. This phenomenon has been studied in several mosquito species (Brust 1967; Hanec and Brust 1967; Madder et al. 1983; Mogi 1992, Rueda et al. 1990; Shelton 1973; Takagi et al. 1996; Trpis and Shemanchuk 1969, 1970; Tsuda et al. 1997). Strains of the same species with wide distributions may also differ in various life functions (Walter and Hacker 1974). The objective of our experiments was to distinguish the extent to which two different populations of the same species from different geographical origins can differ from each other following long-term rearing under the same conditions and if differences might exist in their responses to temperature and density. Two strains of the Culex pipiens complex were chosen because they represent a typical example of species with different vector competence in different parts of their distribution. The principal members of the complex are Culex pipiens s. str., Culex pipiens molestus, and Culex pipiens quinquefasciatus, the taxonomical status of which is still a issue of discussion. The complex has a cosmopolitan distribution, with the distribution of its particular members being more or less directed by climatic conditions with the temperature as the most important factor. One of the possible consequences of global warming is the change in the geographical distribution of some vector species as well as the pathogens they transmit (Lindsay and Birley 1996). Vector competence is also influenced by environmental temperature (Kay et al. 1989, Turell and Lundstrm 1990). Temperature changes can also result in changes in the population density of particular species. Minimal changes in local climate could initiate the replacement of particular populations, which is, within the same species complex, very difficult to distinguish, but may change the epidemiological situation in a given locality. Intraspecific competition is one of the important mechanisms of natural regulation of mosquito populations. Females that mate with older males can produce a smaller number of eggs in rafts (Olejncek 1990). In preimaginal stages, high abundance causes higher mortality and prolongs preimaginal development (Ikeshoji and Mulla 1970 a,b; Mahmood et al. 1997; Nekrasova 1996; Reisen 1975; Renshaw et al. 1993). The rate of larval development can be an important factor in the competition between species and/or between various strains (Tsuji 1989, Tsuji et al. 1990), especially in species with a climate dependent number of generations per year. It is easy to monitor changes in species distribution when the species are

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distinguishable by their morphological characters, but studies dealing with actual or potential changes in distribution of particular strains of the same species (Chevillon et al. 1995) are very rare. This and abovementioned reasons motivated us to study model situations in the laboratory, which could provide information used for field prognoses. MATERIALS AND METHODS Two strains of autogenous Cx. p. molestus were used in these experiments. The strain designed as CmM originated from Mosul, Iraq, and the CmC strain originated from Cuba. The CmC strain was originally imported by Dr. Rettich to the State Health Institute, Prague, as Cx. p. quinquefasciatus, however, after morphological examination (male genitalia, wing venation) and autogeny, we had to consider it as molestus. Strains were reared in the insectary of the Institute of Parasitology, Academy of Sciences of the Czech Republic, for more than five years under the same conditions. Plexiglas boxes (Olejncek 1993) were used within a standard microclimate (22C, 7080% RH, photoperiod 12L:12D). Females were occasionally (about each second generation) fed on laboratory mice. Eggs less than 12 hours old were maintained at 15, 22, or 30C, respectively. After hatching, the larvae were placed in NUNC multicavity plates containing 10 ml tap water at the same temperature (water surface 9.95 cm2, depth 1 cm). Plates containing mosquito larvae were placed in an incubator with a controlled L:D regime. One, five, and ten larvae of the first instar, respectively, were replaced in particular dishes of the plate. Larvae were fed lyophilized yeast Pangamin (Pivovar Brnk, Praha, CZ). The initial amount fed was 0.01g/larva (0.05g/5 l, 0.1g/10 l) and the same amount was added each fifth day (30C), 10th day (22C), and 15th day (15C) to maintain a surplus of food in each dish. Water was added regularly to maintain its constant volume, if necessary. One hundred first-instar larvae were used in the one larva per dish experiments and 200 larvae were used in the other cases. Dishes were monitored daily and dead larvae were removed and counted. Pupae were replaced separately in vials (2 cm in diameter and 6 cm high) covered with cotton plugs for obtaining adults. Time necessary for completing larval and pupal development was measured. Students t-test and correlation coefficient were used for statistical evaluation of results.

Observed mean developmental times, standard error (SE), and mortality for all tested groups are shown in Tables 1 and 2. In all the observed groups, low temperature and high density both caused prolongation of developmental time with lower temperatures causing the biggest differences between the strains tested. In all cases (except for the CmC strain 15C group of five specimens per dish) the average developmental time of females was longer than male developmental time. Larval + pupal mortality increased (except for one sample) with higher density. Mortality in all tested groups was counted as mean of all cavities. Mortality in particular instars for one larva/cavity is shown in Table 4. Total mortality in particular cavities for five and ten larvae/cavity was very variable. The values fluctuated from 0 to 100% (5 larvae/cavity) and from 40 to 100% (10 larvae/cavity). In spite of that, the experimental groups were not numerous, the correlation coefficients (Table 5) show a high correlation between mortality and density. Figures 1 and 2 display the range between the first and the last emerged specimen for each group. The lower area displays the emergence times of males only, the middle one shows the range in which both sexes emerged, and the upper one the range in which females only hatched. In the Cuban strain first males appeared at 30C on day 10, independent of density. Increased density caused slight prolongation of development. The first females appeared at day 13 (T= 30C, low density, one larva per dish), while in higher densities they appeared at day 11. The range between the first and the last imago of the Cuban strain at 30C increased with increasing density. The highest spread between the first and the last emerging imago was at the highest density and lowest temperature. In the abovementioned strain, male and female emergence showed an overlap, contrary to the CmM strain. In this strain, the differences between males and females were more conspicuous. It appeared that suboptimal conditions extended the range between the first and the last emerging imago in both sexes. Except for the abovementioned data, the relation between density and sex ratio and total mortality and sex ratio in all temperature groups was also tested by the same statistical methods. The results did not show any correlations. DISCUSSION Our results showed that while both strains were kept under identical conditions for five years and approximately 60 generations, they maintained some differences in their response to environmental factors.

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Average length of larval and pupal development of Culex molestus (Cuba strain). 30oC/1 one larva per cavity at 30C; 30C/5 five larvae per cavity at 30oC, etc. Average length of development for male and female specimens and for both sexes together (M+F/n), standard errors (SE) and total preimaginal mortality in particular groups.

Culex molestus (Cuba strain) 30C/1 Males Females SE-M SE-F (M+F)/n SE (M+F) Mortality % 11.30 13.58 0.84 0.64 12.16 1.35 57.0 30C/5 12.23 14.06 1.54 2.52 13.2 2.31 73.0 30C/10 13.43 15.45 1.93 3.51 14.41 2.99 84.0 22C/1 15.94 19.25 1.39 1.92 16.57 1.99 70.0 22C/5 17.09 19.00 1.24 0.63 17.69 1.40 73.0 22C/10 22.00 25.67 2.49 0.94 23.65 2.67 78.0 15C/1 36.71 39.67 2.71 0.94 37.6 2.69 68.0 15C/5 58.57 57.33 4.44 3.30 58.2 4.17 89.0 15C/10 55.00 67.00 4.90 7.52 61.0 8.73 93.0

Table 2. Culex molestus (Mosul strain). For explanation, see Table 1. Culex molestus (Mosul) 30C/1 Males Females SE-M SE-F (M+F)/n SE (M+F) Motality % 11.73 13.27 0.75 0.45 12.5 0.99 55.0 30C/5 12.17 14.20 0.37 1.60 13.09 1.5 90.0 30C/10 12.33 15.00 0.47 0.71 13.4 1.43 92.0 22C/1 20.20 23.62 0.54 1.94 21.79 2.19 30.0 22C/5 24.36 31.00 3.72 3.96 27.83 5.08 43.0 22C/10 28.13 37.50 2.71 2.22 32.14 5.28 53.0 15C/1 40.57 47.75 4.50 4.44 43.18 5.65 69.0 15C/5 46.00 46.67 3.95 3.35 46.36 3.65 63.0 15C/10 46.44 50.00 8.46 4.24 47.33 7.78 95.0

Table 3.

Comparison of the developmental time of the strains, males, and females at various temperatures and in various densities using Students t-test. 30C/1 30C/5 30C/10 22C/1 22C/5 22C/10 15C/1 15C/5 15C/10

Males Females

0.185021 0.919122 0.185497 7.81E-12 9.87E-06 0.000158 0.097253 0.000947 0.000284 0.214556 0.906265 0.802973 0.002134 1.34E-05 6.49E-09 0.046941 0.005249 0.031

Except for food accessibility, temperature and density are the most important factors influencing the time required for successful development in mosquitoes (Ikeshoji and Mulla 1970a,b; Reisen 1975, 1995; Rueda et al. 1990; Nekrasova 1990; Mahmood et al. 1997; Mohsen and Al-Saady 1995). Although the differences at 30C were not significant (Table 3), there were

significant differences between both strains at the lower temperatures. The most significant differences were found between samples from 22C, the lower significance of differences of samples in 15C was caused by high dispersion in low temperature. The most conspicuous difference was between the groups of five and ten larvae per dish at the lowest temperature, as

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Preimaginal mortality in particular instars and total mortality in the strains at three experimental temperatures. (1 larva / cavity). Culex molestus [Cuba] Culex molestus [Mosul] 30C larvae per dish mortality % correlation coefficient: 22C larvae per dish mortality % correlation coefficient: 15C larvae per dish mortality % correlation coefficient:

30C larvae per dish mortality % correlation coefficient: 22C larvae per dish mortality % correlation coefficient: 15C larvae per dish mortality % correlation coefficient:

1 57

5 73 0.985

10 84

1 55

5 90 0.858

10 92

1 70

5 73 0.997

10 78

1 30

5 43 0.990

10 53

1 68

5 89 0.906

10 93

1 69

5 63 0.804

10 95

Table 5. Correlation between mortality and number of larvae per cavity. Temp. I CmM 15C 22C 30C 15C 22C 30C 18 8 2 27 6 13 II 0 14 20 20 33 0 Mortality Percent III 6 2 18 11 14 5 IV 27 0 14 5 2 21 P 18 6 1 5 15 18 Total 69 30 55 68 70 57

CmC

15C is lower than is the minimal temperature in Cuba, while in Mosul the winter temperature reaches below 0C and the round-the-year average temperature in Mosul is more than 5C lower than the same in Cuba. The development of the Cuban strain was faster at 22C, however, this was compensated by a higher mortality. Ikeshoji and Mulla (1970b) observed that mosquito larvae can be effective as bacteriostaticum in the rearing water when the larval density is high. In our field experience, the population density of Cx. p. molestus is correlated with the quantity of food available in the given biotope. Very high densities of Cx. p. molestus were observed only in biotopes like septic tanks. In our

case, when larvae were reared in tiny dishes, the influence of this phenomenon could also be relevant, however, as larvae had a slight surplus of food during the experiment. Ikeshoji and Mulla (1970a) found that metabolites produced by older larvae of Cx. p. quinquefasciatus are toxic for the first-instar larvae of not only the same species but also for larvae of some other species. In our experiments larvae in each dish were of the same age, but the highest mortality was observed in the highest density in each case (Figures 3 and 4). Our results, as well as the data from above mentioned authors, suggest that both increasing density and decreasing temperature cause prolongation of

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Figure 1. The range between the first and the last emerged males and females of the CmM strain. The lower area displays the emergence time of males only, the middle one shows the range in which both sexes emerged and the upper one the range in which only females hatched.

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Figure 2. The range between the first and the last emerged males and females of the CmC strain. For explanation see Fig. 1.

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Figure 3. Average length of larval and pupal development (bars) and mortality (lines) of the CmM strain. (30C/1: one larva per cavity at 30C; 30C/5: five larvae per cavity at 30C; etc.)

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Figure 4. Average length of larval and pupal development of the CmC strain. For explanation, see Figure 3.

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development in mosquitoes. Using Figures 1 and 2, it is possible to estimate the appearance of the first imagoes if we know the time of appearance of eggs, the water temperature, and the density. We can also estimate if larvae present in the breeding site belong to the first or subsequent generations, as the number of generations in Cx. pipiens s.l. is more or less dependent on climate during a particular year. Comparing the situation in Figures 1 and 2 we can theoretically expect that two different populations of the same species or even subspecies may coexist in the same locality, the adults of which cannot meet and interbreed if they are shortlived. While prolonging larval development with lower temperatures results in larger adults and larger autogenous egg rafts in Cx. p. molestus (Olejncek and Gelbic 1995), the prolongation by higher density (in Aedes triseriatus - Mahmood et al. 1977) results in smaller adults. Our data indicate that absolute time spent in the preimaginal stage is also influenced by genetic factors. Both strains displayed different lengths of time necessary for preimaginal development independently even though they were reared for more than five years in the laboratory under the same conditions. That is the reason it is necessary to consider that all the values obtained in this and similar published papers are valid only for the particular strain and not for the whole species. Acknowledgments The research was supported by grant GA CR r. c. 206/97/0324 of the Grant Agency of the Czech Republic. The authors are grateful to Mrs. Emilie Beranov for her skilled technical assistance. REFERENCES CITED Brust, R. A. 1967. Weight and development time of different stadia of mosquitoes reared at various constant temperatures. Canad. Entomol. 99: 986993. Chevillon, C., G. Addis, M. Raymond, and A. Marchi. 1995. Population structure in Mediterranean islands and risk of genetic invasion in Culex pipiens L. (Diptera: Culicidae). Biol. J. Linn. Soc. 55: 329343. Hanec, W. and R. A. Brust. 1967. The effect of temperature on the immature stages of Culiseta inornata (Diptera: Culicidae) in the laboratory. Canad. Entomol. 99: 59-64. Ikeshoji, T. and M. Mulla. 1970a. Overcrowding factors of mosquito larvae. J. Econ. Entomol. 63: 90-96.

Ikeshoji, T. and M. Mulla. 1970b. Overcrowding factors of mosquito Larvae. 2. Growth-retarding and bacteriostatic effects of the overcrowding factors of mosquito larvae. J. Econ. Entomol. 63: 17371743. Kay, B. H., I. D. Fanning, and P. Mottram. 1989. The vector competence of Culex annulirostris, Aedes vexans and Aedes alboannulatus for Murray Valley encephalitis virus at different temperatures. Med. Vet. Entomol. 3: 107-112. Knop, N. F., M. Asman, W. K. Reisen, and M. M. Milby. 1987. Changes in the biology of Culex tarsalis (Diptera: Culicidae) associated with colonization under constanting regimes. Environ. Entomol. 16: 405-414. Lindsay, S. W. and M. H. Birley. 1996. Climate change and malaria transmission. Ann. Trop. Med. Parasitol. 90: 573-588. Madder, D. J., G. A. Surgeoner, and B. V. Helson. 1983. Number of generations, egg production, and developmental time of Culex pipiens and Culex restuans (Diptera: Culicidae) in Southern Ontario. J. Med. Entomol. 20: 275-287. Mahmood, F., W. J. Crans, and N. S. Savur. 1997. Larval competition in Aedes triseriatus (Diptera: Culicidae): Effect of density on size, growth, sex ratio and survival. J. Vector Ecol. 22: 90-94. Mogi, M. 1992. Temperature and photoperiod effects on larval and ovarian development of New Zealand strains of Culex quinquefasciatus (Diptera: Culicidae). Ann. Entomol. Soc. Am. 85: 58-66. Mohsen, Z. H. and Al-Saady, M. 1995. Effect of intraand interspecific competition on survival and development of Culex quinquefasciatus and Culex pipiens molestus larvae (Diptera: Culicidae). Jpn. J. Sanit. Zool. 46: 219-222. Nekrasova, L. S. 1990. Ekologiceskij analiz prenaselennosti licinok krovosususchich komarov. [Ecological analysis of overcrowding of larvae of bloodsucking mosquitoes]. Acad. Sci. USSR, Ural. Dpt., Sverdlovsk, 122 pp. (In Russian). Nekrasova, L. S. 1996. After effect of overcrowding larval populations of bloodsucking mosquitoes. Russ. J. Ecol. 27: 207-213. Olejncek, J. 1990: The effect of age on the fertility of males of Culex molestus (Diptera, Culicidae). Folia Parasitol. 37:355-357. Olejncek, J. 1993. Plexiglas box system for mosquito rearing. Bull. Soc. Vector Ecol. 18: 38-39. Olejncek, J. and I. Gelbic. 1995. Changes in development and reproduction in Culex pipiens complex caused by temperature. IX European meeting Society for Vector Ecology, Prague, 4-7

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Sept. 1995. Program and Abstracts pp. 41-42. Reisen, W. K. 1975. Intraspecific competition in Anopheles stephensi Liston. Mosq. News 35: 473482. Reisen, W. K. 1995. Effect of temperature on Culex tarsalis (Diptera: Culicidae) from the Coachella and San Joaquin Valleys of California. J. Med. Entomol. 32: 636-645. Renshaw, M., M. W. Service, and M. H. Birley. 1993. Density-dependent regulation of Aedes cantans (Diptera: Culicidae) in natural and artificial populations. Ecol. Entomol. 18: 223-233. Rueda, L. M., K. J. Patel, R. C. Axtell, and R. E. Stinner. 1990. Temperature-dependent development and survival rates of Culex quinquefasciatus and Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 27: 892-898. Shelton, R. M. 1973. The effect of temperature on development of eight mosquito species. Mosq. News 33: 1-12. Takagi, M., K. Maruyama, and A. Sugiyama. 1996. A laboratory experiment on the larval development of Culex tritaeniorhynchus (Diptera: Culicidae) Giles under different temperatures and densities. Trop. Med. 38: 69-77. Tsuji, N. 1989. Autogenous and anautogenous mosquitoes: The effect of survival rate during

blood feeding. Acta Erudit. Saga Medical School 8: 1-14. Tsuji, A, T. Okazawa, and N. Yamamura. 1990. Autogenous and anautogenous mosquitoes: A mathematical analysis of reproductive strategies. J. Med. Entomol. 27: 446-453. Trpis, M. and J. A. Shemanchuk. 1969. The effect of temperature on preadult development of Aedes flavescens (Diptera: Culicidae). Canad. Entomol. 101: 28-132. Trpis, M. and J. A. Shemanchuk. 1970. Effect of constant temperature on the larval development of Aedes vexans (Diptera: Culicidae). Canad. Entomol. 102: 1048-1051. Tsuda, Y., A. Suzuki, M. Takagi, and Y. Wada. 1997. Different responses to larval crowding between 2 strains of Aedes albopictus (Diptera: Culicidae) from Japan and Thailand. Med. Entomol. Zool. 48: 19-24. Turell, M. J. and J. O. Lundstrm. 1990. Effect of environmental temperature on the vector competence of Aedes aegypti and Ae. taeniorhynchus for Ocklebo virus. Am. J. Trop. Med. Hyg. 43: 543550. Walter, N. M. and C. S. Hacker. 1974. Variation in life table characteristics among three geographic strains of Culex pipiens quinquefasciatus. J. Med. Entomol. 11: 541-550.