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Study of Culex tritaeniorhynchus and species composition of mosquitoes in a rice field in Greece
Acta Tropica 134 (2014) 66–71
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Study of Culex tritaeniorhynchus and species composition of mosquitoes in a rice field in Greece Ioanna Lytra ∗ , Nikolaos Emmanouel Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75 Iera Odos, GR-11855 Athens, Greece
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Article history: Received 8 February 2014 Accepted 23 February 2014 Available online 5 March 2014 Keywords: Culex tritaeniorhynchus Seasonal abundance Species composition Paddy field Larvae
a b s t r a c t Mosquito species composition and seasonal abundance were studied in a rice field in western Greece over a three-year period (2009–2011). A total of 11,716 larvae and pupae of mosquitoes were recorded, representing seven species, namely Aedes caspius (Pallas), Anopheles hyrcanus (Pallas), Anopheles sacharovi Favre, Culex theileri Theobald, Culex tritaeniorhynchus Giles, Culex pipiens Linnaeus, Uranotaenia unguiculata Edwards and belonging to four genera. Cx. tritaeniorhynchus constituted the most abundant species. It is the second recorded occurrence of this species in Greece, but the first time that a high population of this mosquito species is recorded in the country. In all three years, the total population density of mosquitoes was found to be higher in early August. The number of immatures of all species was found higher in 2009 and 2010 than in 2011, as well was that of the Cx. tritaeniorhynchus adults derived from the rearing of the collected immatures. This regularity is probably due to the lack of water in the rice field in early August 2011. Cx. tritaeniorhynchus was found to be the most abundant species after the rearing of immatures representing 85.1%, 93.5% and 96.1% of the total number of the mosquito adults in 2009, 2010 and 2011, respectively. The rice culturing practices may have affected the seasonal occurrence of mosquito immatures in all of the study years. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In temperate and tropical regions, rice cultivation provides an ideal habitat for vectors of several important human diseases, including malaria (Service, 1989; Lacey and Lacey, 1990). Among irrigated crops, rice is considered to pose the greatest danger to health, because it is grown under flooded conditions (Muturi et al., 2007). In Greece, where approximately 25,000 ha are leased annually for rice cultivation, mosquitoes constitute a major problem, not only for the severe nuisance they cause but also for the potential serious risk they pose to public health. However, the lack of relevant data such as mosquito seasonal abundance and species mapping hinders the establishment of appropriate control programs. Rice is a traditional crop in the area of western Greece, in the rural region of the Delta of Acheloos River, where there are about 1500 ha of land under rice cultivation, while farmers also raise livestock, such as cows and pigs. The same area offers shelter to 32 of the 38 species of predatory birds that live in Europe (Fourniotis, 2012). Although mosquitoes have a nuisance value for the 35,000
∗ Corresponding author. Tel.: +30 2105294576. E-mail addresses: [email protected], [email protected] (I. Lytra). http://dx.doi.org/10.1016/j.actatropica.2014.02.018 0001-706X/© 2014 Elsevier B.V. All rights reserved.
residents living within 15 km of this rural region, no relevant control program has been conducted for decades in this area. The major nuisance mosquito species found in rice field areas of south Europe are Aedes caspius (Pallas), Culex pipiens Linnaeus, Culex modestus Ficalbi, Anopheles sacharovi Favre and Culex theileri Theobald (Bellini et al., 1994; Almeida et al., 2010; Chaskopoulou et al., 2011). In the past, An. sacharovi used to be the most important malaria vector in Greece and continues to be a malaria risk in a considerable part of the country (Samanidou-Voyadjoglou, 2001). Moreover, An. sacharovi was considered to be the principal vector in the region of Lakonia during a local outbreak of malaria in the summer/autumn of 2011 (Kousoulis et al., 2013). Cx. pipiens s.s. was responsible for the 2010 West Nile virus epidemic that occurred in Greece (Gomes et al., 2013). A preliminary survey (Lytra, unpublished data) during the summer months of 2008 conducted in the area revealed the presence of Culex tritaeniorhynchus Giles at a high population density. This species was first recorded in Greece from a few samples taken in a coastal marsh in the area of Marathon, in the Prefecture of Attica (Samanidou and Harbach, 2003). The presence of this species has also been reported in Albania, where it has been accidentally been introduced (Danielová and Adhami, 1960; Adhami, 1997; Samanidou and Harbach, 2003).
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Larvae of Cx. tritaeniorhynchus can be found in various temporary and permanent ground water habitats that are sunlit and contain vegetation, such as ground pools, streams, swamps, shallow marshes, irrigation ditches, rice fields and animal hoof prints (Bram, 1967; Harbach, 1988). The females feed primarily on domestic animals such as cattle and pigs, but in the absence of animal hosts bite man (Bram, 1967). They mainly bite outdoors between sunset and midnight, but may enter in cattle sheds and dwellings and bite man during any time of the night (Gutsevich et al., 1974; Sirivanakarn, 1976). Cx. tritaeniorhynchus is a potential vector of pathogens that cause human diseases. It is the primary vector of Japanese encephalitis (JE) in southern Asia. It has also been found infected with Dengue, Rift Valley fever, Sindbis, Getah and Tembusu viruses, and microfilariae of both Brugia malayi and Wuchereria bancrofti, in many areas of eastern and southeastern Asia (Lacey and Lacey, 1990). JE is the leading cause of viral encephalitis in South East Asia and is endemic in India, China, Japan and all of South East Asia (Das, 2013). Additionally, the management of paddy water strongly influences the transmission of JE (Keiser et al., 2005). The occurrence of JE is largely restricted to rural settings (Self et al., 1973; Solomon et al., 2000). The rice cultivation practices, the domicilliary surroundings with adjacent water bodies, and the high temperature and humidity were found to be the main environmental factors influencing the abundance of the potential mosquito vectors responsible for the transmission of the virus in the rural community in Assam, Northeast India (Khan et al., 1996). The presence of pigs and marsh birds is also crucial in the epidemiology of JE, as the virus is carried by birds and amplified by pigs (Broom et al., 2003). Currently, reliable information regarding the presence, the seasonal appearance or any other biological aspect of Cx. tritaeniorhynchus in Greece and in the other European countries is extremely limited. The present work was designed to provide information on the mosquito fauna of a paddy field in Greece during a whole growing season, as well as to record the seasonal abundance of Cx. tritaeniorhynchus in relation to other mosquito species, native to the region. 2. Materials and methods 2.1. Study area The study was conducted over three successive years (2009, 2010 and 2011) in an irrigated, organically farmed rice field located in Western Greece (38◦ 20 20 N, 21◦ 15 06 E), 15 km west from the town of Messolonghi. The size of the sampling field was approximately 8 ha and separated by low hills into four paddies. No pesticides were applied in the sampling field during the study. Water was provided from Acheloos River through irrigation ditches. A network of irrigation and drainage channels allowed individual water management for each paddy and permitted quick flooding and draining. Water depth ranged from 2 to 15 cm. 2.2. Mosquito collection Samplings of mosquitoes’ larvae and pupae were conducted at 10-day intervals between June and early October, during rice growing seasons. The rice field was completely drained out of water in early October to harvest the rice fields. The samplings were carried out under similar meteorological conditions (clear sunny weather), during morning hours (from 8 a.m. to 11 a.m.). Mosquitoes’ immatures were collected with a standard larval dipper (350 ml, 13 cm diameter) attached with an elongated handle (BioQuip, Rancho Dominguez, CA) bend so as to minimize the escape of immatures alarmed by water ripples or shadows. The
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collector walked on the low hills and took one dipper every eight steps. Each sample consisted of water from five consecutive dippers which were transferred to 1500 ml plastic containers. On each sampling date 20 samples were taken from the rice field. Potential predators of mosquitoes’ immatures were removed from the water before it was transferred to the containers. The samples were transferred to the laboratory in the Agricultural University of Athens, where larvae and pupae were counted and reared to adults in a rearing room at 25–26 ◦ C. Plastic translucent containers of a 500-ml capacity were used as breeders. Finely ground fish food was used in small amounts to feed larvae until pupation. Food ad libitum and sufficient space (over-crowded containers were split to extra ones) were provided to overcome potential competitive interaction between species or intraspecific antagonism. 2.3. Mosquito identification In order to determine the mosquito species composition in the rice field of the study, the collected immatures were reared in the laboratory until adult emergence (Knio et al., 2005). Despite the fact that there was a degree of larval mortality during the rearing technique in the laboratory, this approach is believed to be more reliable and often necessary (Becker et al., 2010) because descriptions and identification keys are usually based on fourth instar larvae which comprise only a small proportion of the collected immatures (Ohba et al., 2013). Emergent adults were collected every day, killed using ethyl acetate and pinned on paper points. Each mosquito had a unique collection number and was identified to species using taxonomic keys, including Harbach (1985), Glick (1992), Darsie and Samanidou-Voyadjoglou (1997), Samanidou-Voyadjoglou and Harbach (2001), Samanidou and Harbach (2003) and Becker et al. (2010). Identification of Cx. tritaeniorhynchus was also performed indicatively, by sequencing method, using BARCODE primers (Folmer et al., 1994) for cytochrome oxydase I (COI) mitochondrial DNA (mtDNA) gene segment. Total DNA was extracted for each insect using the DNeasy Blood and Tissue. Kit (Qiagen) following the manufacturer’s protocol with minor modifications. Fragment of the mtDNA gene segment was amplified by PCR using two sets of primers: the Barcode primers 5-GGTCAACAAATCATAAAGATATTGG-3 and 5-TAAACTTCAGGGTGACCAAAAAATCA-3 (Folmer et al., 1994). The PCR product was purified using the Nucleospin extract II kit (Macherey-Nagel) according to the supplier’s protocol and each sequence was determined via automated sequencing procedure provided by Macrogen Company (Seoul, Korea). The authenticity of the produced mtDNA sequences was verified using Basic Local Alignment Search Tool (BLAST) program of NCBI. 2.4. Data analyses The differences between the total number of the collected immatures of all species and the numbers of the emerged adults of Cx. tritaeniorhynchus during the sampling periods of the three years of the study were determined by the Kruskal–Wallis H-test, followed by Mann–Whitney U-test for pairwise comparisons. The Bonferroni correction was applied to adjust P-values for pairwise comparisons. The analyses were carried out using the statistical package Statistica version 7 (StatSoft Inc., 2004). Furthermore, the spatial distribution of the mosquitoes’ immatures was estimated by calculating the parameters a and b of Taylor’s power law (Taylor, 1961). This law describes the linear regression between variance and sample mean following a logarithmic transformation s = a + b¯x. Thus, s is the variance, the
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I. Lytra, N. Emmanouel / Acta Tropica 134 (2014) 66–71 Table 1 Mean number (±SE) of total mosquitoes’ larvae and pupae and Culex tritaeniorhynchus (adults which emerged from the rearing of the immatures) per five dips, during the three years of the study. Year
Larvae and pupae
Culex tritaeniorhynchus
18.61 ± 1.98a 19.34 ± 2.06a 14.76 ± 2.31b 10.33 2 0.0057
2009 2010 2011 Chi-square d.f. P
9.18 ± 1.15a 9.94 ± 1.08a 6.86 ± 1.18b 13.82 2 0.001
Means of the same column followed by same letter are not differ significantly (Mann–Whitney U-test with Bonferroni correction, adjusted a = 0.017).
The mean number of immatures of all mosquito species was higher in 2009 and 2010 compared with 2011 while no significant differences were observed between the two former years (Table 1). 3.2. Spatial distribution of total number mosquitoes’ immatures The value of the dispersion pattern b of Taylor’s power law was significantly different from 1 for both 2010 and 2011, suggesting an aggregated distribution pattern of the mosquitoes’ immatures, while in 2009 this value did not differ from 1, suggesting a random distribution. Moreover, in all of the cases the y-intercept values were significantly different from 0 and the correlation coefficient values were significantly higher than 0 at the P = 0.05 level (Table 2). 3.3. Species composition and abundance
Fig. 1. Mean number of total mosquitoes’ larvae and pupae and Culex tritaeniorhynchus (adults which emerged from the rearing of the immatures) per sample in each sampling date during the sampling periods of 2009, 2010 and 2011.
y-intercept a is a scaling factor that is dependent on the sampling unit, x¯ is the sample mean and b is the slope of the regression line and constitutes an index of spatial pattern indicating a uniform (b < 1), random (b = 1) or aggregated (b > 1) distribution (Southwood, 1978; Davis, 1994). Moreover, the values of correlation coefficients (r) were calculated as a goodness-of-fit measure of Taylor’s model. The values of a, b and r were tested for departure from 0, 1 and 0 respectively, using the two-tailed t test at n − 2 degrees of freedom and a probability level of P = 0.05 (Snedecor and Cochran, 1980). 3. Results A total of 11,716 immatures of mosquitoes were collected during the three years of the study. Of those, 4095, 4641 and 2980 immatures were collected in 2009, 2010 and 2011, respectively. 3.1. Seasonal occurrence of total immatures The average seasonal trends in terms of the numbers of mosquitoes’ immatures in the rice field during the three years of the study are shown in Fig. 1. All years of the study the immatures’ first appearance and their highest population density were recorded in the middle of June and in early August, respectively. The mosquitoes’ immatures were observed in the field until late September (2009) or early October (2010 and 2011).
Throughout this study, a total of 6363 adult mosquitoes were derived after rearing the immatures, belonging to seven species (An. sacharovi, Anopheles hyrcanus (Pallas), Ae. caspius, Uranotaenia unguiculata Edwards, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus). Ur. unguiculata was only collected during the samplings of 2009. Identification of Cx. tritaeniorhynchus was performed indicatively, by sequencing method. The sequencing of the mtDNA gene studied produced an alignment of about 600 bp. The authenticity of the mtDNA sequences produced was verified as Cx. tritaeniorhynchus. The two haplotypes revealed, were differentiating in one nucleotide and have been deposited to GenBank with accession numbers KC753196 (haplotype 1) and KC753197 (haplotype 2). Cx. tritaeniorhynchus was the most abundant species among the mosquito assemblages, representing 85.1%, 93.5% and 96.1% of the total number of adults obtained from the rearing of immatures in 2009, 2010 and 2011, respectively. The relative abundance of the mosquitoes’ species recorded in each year of the study is depicted in Table 3. Despite the recorded mortality of immatures in the laboratory, the high number of identified adults, over a three years survey, gives adequately a representative picture of mosquito species composition providing also useful information about their seasonal presence in the rice field.
Table 2 Parameter estimates for Taylor’s power law for the mosquitoes’ larvae and pupae during the three-year study. Year
n
a ± S.E.
b ± S.E.
r
2009 2010 2011
11 11 10
1.5436 ± 0.2153* 0.6925 ± 0.1144* 0.8626 ± 0.1216*
1.0522 ± 0.1778 1.5218 ± 0.0955** 1.4334 ± 0.1212**
0.8919* 0.9827* 0.9726*
n: sample size, a: y – intercept value for Taylor’s model, b: slope value for Taylor’s model, r: correlation coefficient value for goodness of fit for Taylor’s model. * Values are significantly different from 0 (two tailed t-test: ˛ = 0.05). ** Values are significantly different from 1 (two tailed t-test: ˛ = 0.05).
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Table 3 Relative abundance (%) of mosquitoes’ species (adults which emerged from the rearing of the larvae and pupae) in each year of the study.
Aedes caspius Anopheles hyrcanus Anopheles sacharovi Culex pipiens Culex theileri Culex tritaeniorhynchus Uranotaenia unguiculata
2009
2010
2011
1.0 1.9 5.9 5.3 0.5 85.1 0.3
4.1 0.2 1.1 0.5 0.6 93.5 0.00
1.3 0.3 0.9 1.3 0.1 96.1 0.00
From mid-July until the end of the samplings in all study years (after the samplings of July 12th, July 24th and July 21st in 2009, 2010 and 2011 respectively) Cx. tritaeniorhynchus was the species found at high relative abundance, on the basis of the derived adults of the mosquitoes’ species. From early June to mid-July the identified species found at high relative abundance were Cx. pipiens for 2009 and both Ae. caspius and Cx. theileri for 2010 and 2011. Even though Anopheles species were constantly present during the sampling period of all three years of the study, their population density was always low based on the sample of adults derived after the larvae rearing. The highest population density for these species was recorded at the end of the growing season in 2009 and 2011 and in late June in 2010. Fig. 2 depicts the relative abundance of the derived adult mosquitoes corresponding to each sampling for each year of the study. An. sacharovi and An. hyrcanus have been reported as Anopheles spp. Ur. unguiculata has been omitted from Fig. 2 as its population density was extremely low. 3.4. Seasonal occurrence of Cx. tritaeniorhynchus The first adults of Cx. tritaeniorhynchus were derived from the larvae and pupae collected on June 30th, July 24th and July 10th for 2009, 2010 and 2011 respectively. The presence of this species was observed until the end of the samplings (early October) every year. Its highest population density (based on the derived adults) corresponded to early August in all years of the study. The seasonal population fluctuation of Cx. tritaeniorhynchus during the threeyear study is shown in Fig. 1. The seasonal abundance of this species followed a similar pattern with that of the total mosquitoes’ immatures. The mean number of Cx. tritaeniorhynchus adults derived from the larvae and pupae collected was higher in 2009 and 2010 compared with 2011, while no significant differences were observed between the two former years (Table 1). 4. Discussion Human activities can greatly affect the occurrence of mosquito populations. Our data demonstrate that the seasonal occurrence of mosquitoes’ immatures was affected by the rice cultivation practices. The first appearance of immatures in mid-June was combined with the introduction of the water in the field after the seeding of the rice. It seems that the drying of the field after the rice sprout that happened approximately fifteen days later resulted in the lower numbers recorded in 2009 and the complete absence of immature mosquitoes in 2010 and 2011. The population of immatures showed an increase after the filling of the field with water. The highest population density of mosquitoes was recorded in early August. This is the period following the rice’s sprouting, which favors mosquitoes’ breeding. This may be accounted for by the fact that the water surface is quite exposed to direct sunlight, which produces ideal conditions for the mosquitoes’ immatures development (Mogi, 1978). Indeed, it has been reported that immature mosquitoes can develop very rapidly at higher water temperature
Fig. 2. Relative abundance (%) of mosquitoes’ species (adults which emerged from the rearing of the larvae and pupae) on each sampling date during the sampling periods of 2009, 2010 and 2011.
(Mogi, 1984). On the contrary, the high density of rice plants renders the access of gravid females to the oviposition sites more difficult (Mogi, 1984). This might be the reason for the lower population of mosquitoes’ immatures recorded in the field from mid-August till the end of the study period. Kant et al. (1992) also mentioned that the negative correlation between larval densities and the growth of the paddy plant could be due to mechanical obstruction in the oviposition of some species, the low-intensity light and the lowered water level in the later stages of the paddy growth. In our study a remarkably lower population of immatures was found between the 3rd and the 13th of August 2011, caused by the lack of water supply due to water management problems. Mogi (1993) found that, in areas with developed irrigation systems and sufficient water, intermittent irrigation may be managed to control vector mosquito populations. The use of intermittent irrigation as an effective method for the control of vector mosquitoes in rice fields has also been recommended by the WHO (1982). The spatial distribution of insects reflects the behavior of individuals and can exhibit three forms: random, aggregated and uniform (Davis, 1994). Aggregated distributions of mosquitoes’ immatures are extremely common in field studies (Service, 1985; Walker et al., 1988; Pitcairn et al., 1994). According to Taylor’s law, the mosquitoes’ immatures found during 2010 and 2011 followed an aggregated spatial distribution. However, during 2009 the
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immatures were distributed randomly among the sampling units, a fact that could be a consequence of their lower occurrence in terms of numbers. The spatial heterogeneity in the distribution of the mosquitoes’ immatures may often arise from habitat heterogeneity, as well as from the propensity of younger instars to congregate with one another (Renshaw et al., 1995). This is the first time the species Cx. tritaeniorhynchus is recorded in western Greece. Moreover, it is the first time that a high population of this mosquito species is recorded anywhere in Greece, as the data of a previous report by Samanidou and Harbach (2003) concern only a few specimens. Furthermore, the presence of this species has been reported in Albania (Danielová and Adhami, 1960; Adhami, 1997). Cx. tritaeniorhynchus is a common species across southern Asia, the Middle East and the Afrotropical Region (Harbach, 1988; Samanidou and Harbach, 2003; Becker et al., 2010). Since surveillance data for the mosquito fauna in Greece are scarce, it is assumed that this species may have been present in the country, though undetected, before 2003. The seasonal and inter-annual variation of Cx. tritaeniorhynchus adults may be related to culturing practices as well as to the density of the rice plants. This is further supported by Mogi’s findings (1984) who mentioned that the active population growth of Cx. tritaeniorhynchus in Japan occurs during a limited period of time, following transplanting, when the irrigated area is greater and most stable and rice plants are short and sparse. Furthermore, Kant et al. (1992) found Cx. tritaeniorhynchus breeding at the later stages of plant growth whereas Reuben (1971) reported that the abundance patterns of this species gradual increase with rice plant growth. Moreover, Takagi et al. (1995) reported that the alternating flooding and draining is an effective method for suppressing the production of Cx. tritaeniorhynchus adults. On the contrary, Heathcote (1970) observed that the population of Cx. tritaeniorhynchus peaks in newly planted or harvested fields, whereas Somboon et al. (1989) recorded that the highest population peak of this species was observed in ploughed fields, followed by a rapid population decrease after transplanting. Finally, according to Mogi and Miyagi (1990) Cx. tritaeniorhynchus may be relatively adaptable to changing rice field phases. In the present study, Cx. tritaeniorhynchus was found to be the predominant species, developing much higher population densities compared to the other recorded mosquito species. Similar results regarding the population densities of Cx. tritaeniorhynchus in rice fields have also been recorded by Mogi (1978) and Lee (1998) (6.28 and 1.56 larvae per dipper, respectively). However, our finding is in contrast to other reports of predominant mosquito species in rice fields of southern Europe (Bellini et al., 1994; Almeida et al., 2010; Chaskopoulou et al., 2011). Bellini et al. (1994) reported that Ae. caspius attained the highest population density during the initial stages of rice crop growth at rice fields in Italy. During this period, the water management practices made the conditions favorable for the oviposition of this species. Moreover, in the same study, the highest density of Cx. pipiens was recorded during the last ten days of June whereas afterwards the population declined progressively until the end of August when the population density of Anopheles species was always low. Our results are in accordance with the former study about the seasonal abundances of the three mosquitoes’ species. Most of the mosquitoes’ species found during the present study are vectors responsible for the transmission of parasitic and viral infections to millions of people worldwide. The species An. sacharovi and Cx. pipiens are also involved in disease outbreaks and epidemics in Greece in recent years (Gomes et al., 2013; Kousoulis et al., 2013). Furthermore, most of these species are economically important mosquitoes that adversely affect tourism and related business interests and have a negative impact on livestock and poultry production (Becker et al., 2010).
Taking into account that Cx. tritaeniorhynchus demonstrated a high population density in the studied rice field and given that this species is the primary vector of JE, the present work shows that the study area is under the potential threat of the emergence of JE. The establishment of Japanese encephalitis virus (JEV) in new ecosystems outside of its current range is difficult (van den Hurk et al., 2009). With the spread of JEV into much of the Indian subcontinent, however, other destinations served by frequent routes of commerce or passenger air travel, such as Africa and Europe, could also be at risk (Weaver and Reisen, 2010). 5. Conclusions The study of the mosquito fauna composition over time in a Greek rice field revealed high population densities of Cx. tritaeniorhynchus. The mosquito sampling findings in combination with the proximity of rice cultivation to the Delta of the Acheloos River and the presence of livestock (pigs and cows) in that rural region render the implementation of effective control measures to minimize vector abundance and potential disease outbreak in this area imperative. Further investigation is required to study the population status of the Cx. tritaeniorhynchus in other regions of Greece, as well as its bionomics in an area far from its origin. Moreover, the risk of emergence of Cx. tritaeniorhynchus related diseases in Greece and Europe should be studied thoroughly. Acknowledgments The authors would like to thank Dr. M. Bouga and V. Evangelou (Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens) for providing the molecular data for the confirmation of the identification of Cx. tritaeniorhynchus specimens. Suggestions and critical comments on the manuscript by Dr. A. Fantinou (Associate Professor in Agricultural University of Athens) and Dr. G. Koliopoulos (Benaki Phytopathological Institute) are also kindly acknowledged. We thank Dr. H. Balfoussia and Dr. A. Martinou for providing valuable language help. Acknowledgments are also extended to the agronomists N. Rizos and S. Antonatos for the fieldwork support and I. Potirakis for the laboratory assistance. This research has been co-financed by the European Union (European Social Fund – ESF) and Greek National funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica. 2014.02.018. These data include Google maps of the most important areas described in this article. References Adhami, J., 1997. Mushkonjat (Diptera: Culicidae) te Shqiperise, tribu Culicini. Revista Mjekesore (1–2), 82–95 (in Albanian). Almeida, A.P.G., Freitas, F.B., Novo, M.T., Sousa, C.A., Rodrigues, J.C., Alves, R., Esteves, A., 2010. Mosquito surveys and West Nile Virus screening in two different areas of Southern Portugal, 2004–2007. Vector Borne Zoonotic Dis. 10, 673–680. ´ D., Zgomba, M., Boase, C., Madon, M., Dahl, C., Kaiser, A., 2010. Becker, N., Petric, Mosquitoes and their Control. Springer-Verlag, Heidelberg, Berlin, 577 pp. Bellini, R., Veronesi, R., Rizzoli, M., 1994. Efficacy of various fish species (Carassius auratus [L.], Cyprinus carpio [L.], Gambusia affinis [Baird and Girard)] in the control of rice field mosquitoes in northern Italy. Bull. Soc. Vector Ecol. 19, 87–99. Bram, R., 1967. Contributions to the mosquito fauna of Southeast Asia. II. The genus Culex in Thailand (Diptera: Culicidae). Contr. Am. Ent. Inst. 2, 1–296. Broom, A., Smith, D., Hall, R., Johansen, C., Mackenzie, J., 2003. Manson’s Tropical Diseases. Saunders, London, pp. 1864pp.
I. Lytra, N. Emmanouel / Acta Tropica 134 (2014) 66–71 Chaskopoulou, A., Latham, M.D., Pereira, R.M., Connelly, R., Bonds, J.S.A., Koehler, P.G., 2011. Efficacy of aerial ultra-low volume applications of two novel waterbased formulations of unsynergized pyrethroids against riceland mosquitoes in Greece. J. Am. Mosq. Control Assoc. 27, 414–422. Danielová, V., Adhami, J., 1960. Mosquitoes of Albania and their medical importance. Cesk. Parasitol. 7, 41–47 (in Russian). Darsie Jr., R., Samanidou-Voyadjoglou, A., 1997. Keys for the identification of the mosquitoes of Greece. J. Am. Mosq. Control Assoc. 13, 247–254. Das, P., 2013. Mosquito vectors of Japanese Encephalitis virus from Northern India. Springer, New Delhi, pp. 144 pp. Davis, P.M., 1994. Handbook of sampling methods for arthropods in agriculture. In: Pedigo, L.P., Buntin, G.D. (Eds.), Statistics for Describing Populations. CRC Press, Boca Raton, Florida, FL, pp. 33–54 (chapter 3). Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Fourniotis, N., 2012. A proposal for impact evaluation of the diversion of the acheloos river, on the acheloos estuary in Western Greece. Int. J. Eng. Sci. Tech. 4, 1793–1802. Glick, J., 1992. Illustrated key to the female Anopheles of southwestern Asia and Egypt (Diptera: Culicidae). Mosq. Syst. 24, 125–153. Gomes, B., Kioulos, E., Papa, A., Almeida, A., Vontas, J., Pinto, J., 2013. Distribution and hybridization of Culex pipiens forms in Greece during the West Nile virus outbreak of 2010. Infect. Genet. Evol. 16, 218–225. Gutsevich, A., Monchadskii, A., Shtakelberg, A., 1974. Fauna SSSR vol. 3(4), Family Culicidae, Leningrad Akad Nauk SSSR Zool. Inst. N S No. 100), 384 pp. Harbach, R., 1985. Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occurring in southwestern Asia and Egypt, with a note to the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosq. Syst. 17, 83–107. Harbach, R., 1988. The mosquitoes of the subgenus Culex in southwestern Asia and Egypt (Diptera: Culicidae). Contr. Am. Ent. Inst. (Ann Arbor). 24, 1–236. Heathcote, O., 1970. Japanese encephalitis in Sarawak: studies on the juvenile mosquito population. Trans. R. Soc. Trop. Med. Hyg. 64, 483–488. Kant, R., Pandey, S., Sharma, R., 1992. Seasonal prevalence and succession of rice field breeding mosquitoes of central Gujarat. J. Commun. Dis. 24, 164–172. Keiser, J., Maltese, M., Erlanger, T., Bos, R., Tanner, M., Singer, B., Utzinger, J., 2005. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities for integrated vector management. Acta Trop. 95, 40–57. Khan, S., Narain, K., Handique, R., Dutta, P., Mahanta, J., Satyanarayana, K., Srivastava, V., 1996. Role of some environmental factors in modulating seasonal abundance of potential Japanese encephalitis vectors in Assam, India. Southeast Asian J. Trop. Med. Public Health 27, 382–391. Knio, K., Markarian, N., Kassis, A., Nuwayri-Salti, N., 2005. A two-year survey on mosquitoes of Lebanon. Parasite 12, 229–235. Kousoulis, A., Chatzigeorgiou, K., Danis, K., Tsoukalas, G., Vakalis, N., Bonovas, S., Tsiodras, S., 2013. Malaria in Lakonia, Greece, then and now: a 2500-year-old pattern. Int. J. Infect. Dis. 17, 8–11. Lacey, L., Lacey, C., 1990. The medical importance of rice land mosquitoes and their control using alternatives to chemical insecticides. J. Am. Mosq. Control Assoc. 6 (Suppl.), 1–93. Lee, D.K., 1998. Effect of two rice culture methods on the seasonal occurrence of mosquito larvae and other aquatic animals in rice fields of Southwestern Korea. J. Vector Ecol. 23, 161–170. Mogi, M., 1978. Population studies on mosquitoes in the rice field area of Nagasaki, Japan, especially on Culex tritaeniorhynchus. Trop. Med. 20, 173–263. Mogi, M., 1984. Mosquito problems and their solution in relation to paddy rice production. Prot. Ecol. 7, 219–240. Mogi, M., 1993. Effect of intermittent irrigation on mosquitoes (Diptera: Culicidae) and larvivorous predators in rice fields. J. Med. Entomol. 30, 309–319.
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Mogi, M., Miyagi, I., 1990. Colonization of rice fields by mosquitoes (Diptera: Culicidae) and larvivorous predators in asynchronous rice cultivation areas in Philippines. J. Med. Entomol. 27, 530–536. Muturi, E., Shililu, J., Gu, W., Jacob, B., Githure, J., Novak, R., 2007. Larval habitat dynamics and diversity of Culex mosquitoes in rice agro-ecosystem in Mwea, Kenya. Am. J. Trop. Med. Hyg. 76, 95–102. Ohba, S., Matsuo, T., Takagi, M., 2013. Mosquitoes and other aquatic insects in fallow field biotopes and rice paddy fields. J. Med. Vet. Entomol. 27, 96–103. Pitcairn, M.J., Wilson, L.T., Washino, R.K., Rejmankova, E., 1994. Spatial patterns of Anopheles freeborni and Culex tarsalis (Diptera: Culicidae) larvae in California rice fields. J. Med. Entomol. 31, 545–553. Renshaw, M., Silver, J.B., Service, M.W., Birley, M.H., 1995. Spatial dispersion patterns of Aedes cantans (Diptera: Culicidae) in temporary woodland pools. Bull. Entomol. Res. 85, 125–133. Reuben, R., 1971. Studies on the mosquitoes of North Arcot District, Madras State, India. Part 5. Breeding places of the Culex vishnui group of species. J. Med. Entomol. 8, 363–366. Samanidou-Voyadjoglou, A., 2001. Malaria vectors in Greece. In: 1st Balkan Conference, Malaria and Mosquito Control, Serres, Greece, pp. 62–65. Samanidou-Voyadjoglou, A., Harbach, R., 2001. Keys to the adult female mosquitoes of Greece. Eur. Mosq. Bull. 10, 13–20. Samanidou, A., Harbach, R., 2003. Culex tritaeniorhynchus Giles, a newly discovered potential vector of arboviruses in Greece. Eur. Mosq. Bull. 16, 15–17. Self, L., Shin, H., Kim, K., Lee, K., Chow, C., Hong, H., 1973. Ecological studies on Culex tritaeniorhynchus as a vector of Japanese encephalitis. Bull. World Health Organ. 49, 41–47. Service, M.W., 1985. Population dynamics and mortalities of mosquito preadults. In: Lounibos, L.P., Rey, J.R., Frank, J.H. (Eds.), Ecology of Mosquitoes: Proceedings of a Workshop. Florida Medical Entomology Laboratory, Vero Beach, pp. 185– 201. Service, M., 1989. Rice. A challenge to health. Parasitol. Today 5, 162–165. Sirivanakarn, S., 1976. Medical entomology studies – III. A revision of the subgenus Culex in the Oriental region (Diptera: Culicidae). Contr. Am. Ent. Inst. 12, 1–272. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, 7th ed. Iowa State University Press, Ames, IA, USA. Solomon, T., Dung, N., Kneen, R., Gainsborough, M., Vaughn, D., Khanh, V., 2000. Japanese encephalitis. J. Neurol. Neurosurg. Psychiatry 68, 405–415. Somboon, P., Wej, C., Khamboonruang, C., Keha, P., Suwanphanit, P., Sukontasan, K., Chaivong, P., 1989. Studies on the Japanese encephalitis vectors in Amphoe Muang, Chiang Mai, northern Thailand. Southeast Asian J. Trop. Med. Public Health 20, 9–17. Southwood, T.R.E., 1978. Ecological Methods with Particular Reference to the Study of Insect Populations, 2nd ed. John Wiley and Sons, New York. StatSoft Inc., 2004. Statistica (Data Analysis Software System), version 7, Tulsa, USA. Takagi, M., Sugiyama, A., Maruyama, K., 1995. Effect of rice culturing practices on seasonal occurrence of Cx. tritaeniorhynchus (Diptera: Culicidae) immatures in three different types of rice-growing areas in central Japan. J. Med. Entomol. 32, 112–117. Taylor, L.R., 1961. Aggregation, variance and the mean. Nature 189, 732–735. van den Hurk, A., Ritchie, S., Mackenzie, J., 2009. Ecology and geographical expansion of Japanese encephalitis virus. Annu. Rev. Entomol. 54, 17–35. Walker, E.D., Merritt, R.W., Wotton, R.S., 1988. Analysis of the distribution and abundance of Anopheles quadrimaculatus (Diptera: Culicidae) larvae in a marsh. Environ. Entomol. 17, 992–999. Weaver, S., Reisen, W., 2010. Present and future of arboviral threats. Antiviral Res. 85, 328–345. World Health Organization (WHO), 1982. Manual on environmental management for mosquito control, vol. 66. WHO Offset Publ., pp. 1–283.
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Study of Culex tritaeniorhynchus and species composition of mosquitoes in a rice field in Greece Ioanna Lytra ∗ , Nikolaos Emmanouel Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75 Iera Odos, GR-11855 Athens, Greece
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Article history: Received 8 February 2014 Accepted 23 February 2014 Available online 5 March 2014 Keywords: Culex tritaeniorhynchus Seasonal abundance Species composition Paddy field Larvae
a b s t r a c t Mosquito species composition and seasonal abundance were studied in a rice field in western Greece over a three-year period (2009–2011). A total of 11,716 larvae and pupae of mosquitoes were recorded, representing seven species, namely Aedes caspius (Pallas), Anopheles hyrcanus (Pallas), Anopheles sacharovi Favre, Culex theileri Theobald, Culex tritaeniorhynchus Giles, Culex pipiens Linnaeus, Uranotaenia unguiculata Edwards and belonging to four genera. Cx. tritaeniorhynchus constituted the most abundant species. It is the second recorded occurrence of this species in Greece, but the first time that a high population of this mosquito species is recorded in the country. In all three years, the total population density of mosquitoes was found to be higher in early August. The number of immatures of all species was found higher in 2009 and 2010 than in 2011, as well was that of the Cx. tritaeniorhynchus adults derived from the rearing of the collected immatures. This regularity is probably due to the lack of water in the rice field in early August 2011. Cx. tritaeniorhynchus was found to be the most abundant species after the rearing of immatures representing 85.1%, 93.5% and 96.1% of the total number of the mosquito adults in 2009, 2010 and 2011, respectively. The rice culturing practices may have affected the seasonal occurrence of mosquito immatures in all of the study years. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In temperate and tropical regions, rice cultivation provides an ideal habitat for vectors of several important human diseases, including malaria (Service, 1989; Lacey and Lacey, 1990). Among irrigated crops, rice is considered to pose the greatest danger to health, because it is grown under flooded conditions (Muturi et al., 2007). In Greece, where approximately 25,000 ha are leased annually for rice cultivation, mosquitoes constitute a major problem, not only for the severe nuisance they cause but also for the potential serious risk they pose to public health. However, the lack of relevant data such as mosquito seasonal abundance and species mapping hinders the establishment of appropriate control programs. Rice is a traditional crop in the area of western Greece, in the rural region of the Delta of Acheloos River, where there are about 1500 ha of land under rice cultivation, while farmers also raise livestock, such as cows and pigs. The same area offers shelter to 32 of the 38 species of predatory birds that live in Europe (Fourniotis, 2012). Although mosquitoes have a nuisance value for the 35,000
∗ Corresponding author. Tel.: +30 2105294576. E-mail addresses: [email protected], [email protected] (I. Lytra). http://dx.doi.org/10.1016/j.actatropica.2014.02.018 0001-706X/© 2014 Elsevier B.V. All rights reserved.
residents living within 15 km of this rural region, no relevant control program has been conducted for decades in this area. The major nuisance mosquito species found in rice field areas of south Europe are Aedes caspius (Pallas), Culex pipiens Linnaeus, Culex modestus Ficalbi, Anopheles sacharovi Favre and Culex theileri Theobald (Bellini et al., 1994; Almeida et al., 2010; Chaskopoulou et al., 2011). In the past, An. sacharovi used to be the most important malaria vector in Greece and continues to be a malaria risk in a considerable part of the country (Samanidou-Voyadjoglou, 2001). Moreover, An. sacharovi was considered to be the principal vector in the region of Lakonia during a local outbreak of malaria in the summer/autumn of 2011 (Kousoulis et al., 2013). Cx. pipiens s.s. was responsible for the 2010 West Nile virus epidemic that occurred in Greece (Gomes et al., 2013). A preliminary survey (Lytra, unpublished data) during the summer months of 2008 conducted in the area revealed the presence of Culex tritaeniorhynchus Giles at a high population density. This species was first recorded in Greece from a few samples taken in a coastal marsh in the area of Marathon, in the Prefecture of Attica (Samanidou and Harbach, 2003). The presence of this species has also been reported in Albania, where it has been accidentally been introduced (Danielová and Adhami, 1960; Adhami, 1997; Samanidou and Harbach, 2003).
I. Lytra, N. Emmanouel / Acta Tropica 134 (2014) 66–71
Larvae of Cx. tritaeniorhynchus can be found in various temporary and permanent ground water habitats that are sunlit and contain vegetation, such as ground pools, streams, swamps, shallow marshes, irrigation ditches, rice fields and animal hoof prints (Bram, 1967; Harbach, 1988). The females feed primarily on domestic animals such as cattle and pigs, but in the absence of animal hosts bite man (Bram, 1967). They mainly bite outdoors between sunset and midnight, but may enter in cattle sheds and dwellings and bite man during any time of the night (Gutsevich et al., 1974; Sirivanakarn, 1976). Cx. tritaeniorhynchus is a potential vector of pathogens that cause human diseases. It is the primary vector of Japanese encephalitis (JE) in southern Asia. It has also been found infected with Dengue, Rift Valley fever, Sindbis, Getah and Tembusu viruses, and microfilariae of both Brugia malayi and Wuchereria bancrofti, in many areas of eastern and southeastern Asia (Lacey and Lacey, 1990). JE is the leading cause of viral encephalitis in South East Asia and is endemic in India, China, Japan and all of South East Asia (Das, 2013). Additionally, the management of paddy water strongly influences the transmission of JE (Keiser et al., 2005). The occurrence of JE is largely restricted to rural settings (Self et al., 1973; Solomon et al., 2000). The rice cultivation practices, the domicilliary surroundings with adjacent water bodies, and the high temperature and humidity were found to be the main environmental factors influencing the abundance of the potential mosquito vectors responsible for the transmission of the virus in the rural community in Assam, Northeast India (Khan et al., 1996). The presence of pigs and marsh birds is also crucial in the epidemiology of JE, as the virus is carried by birds and amplified by pigs (Broom et al., 2003). Currently, reliable information regarding the presence, the seasonal appearance or any other biological aspect of Cx. tritaeniorhynchus in Greece and in the other European countries is extremely limited. The present work was designed to provide information on the mosquito fauna of a paddy field in Greece during a whole growing season, as well as to record the seasonal abundance of Cx. tritaeniorhynchus in relation to other mosquito species, native to the region. 2. Materials and methods 2.1. Study area The study was conducted over three successive years (2009, 2010 and 2011) in an irrigated, organically farmed rice field located in Western Greece (38◦ 20 20 N, 21◦ 15 06 E), 15 km west from the town of Messolonghi. The size of the sampling field was approximately 8 ha and separated by low hills into four paddies. No pesticides were applied in the sampling field during the study. Water was provided from Acheloos River through irrigation ditches. A network of irrigation and drainage channels allowed individual water management for each paddy and permitted quick flooding and draining. Water depth ranged from 2 to 15 cm. 2.2. Mosquito collection Samplings of mosquitoes’ larvae and pupae were conducted at 10-day intervals between June and early October, during rice growing seasons. The rice field was completely drained out of water in early October to harvest the rice fields. The samplings were carried out under similar meteorological conditions (clear sunny weather), during morning hours (from 8 a.m. to 11 a.m.). Mosquitoes’ immatures were collected with a standard larval dipper (350 ml, 13 cm diameter) attached with an elongated handle (BioQuip, Rancho Dominguez, CA) bend so as to minimize the escape of immatures alarmed by water ripples or shadows. The
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collector walked on the low hills and took one dipper every eight steps. Each sample consisted of water from five consecutive dippers which were transferred to 1500 ml plastic containers. On each sampling date 20 samples were taken from the rice field. Potential predators of mosquitoes’ immatures were removed from the water before it was transferred to the containers. The samples were transferred to the laboratory in the Agricultural University of Athens, where larvae and pupae were counted and reared to adults in a rearing room at 25–26 ◦ C. Plastic translucent containers of a 500-ml capacity were used as breeders. Finely ground fish food was used in small amounts to feed larvae until pupation. Food ad libitum and sufficient space (over-crowded containers were split to extra ones) were provided to overcome potential competitive interaction between species or intraspecific antagonism. 2.3. Mosquito identification In order to determine the mosquito species composition in the rice field of the study, the collected immatures were reared in the laboratory until adult emergence (Knio et al., 2005). Despite the fact that there was a degree of larval mortality during the rearing technique in the laboratory, this approach is believed to be more reliable and often necessary (Becker et al., 2010) because descriptions and identification keys are usually based on fourth instar larvae which comprise only a small proportion of the collected immatures (Ohba et al., 2013). Emergent adults were collected every day, killed using ethyl acetate and pinned on paper points. Each mosquito had a unique collection number and was identified to species using taxonomic keys, including Harbach (1985), Glick (1992), Darsie and Samanidou-Voyadjoglou (1997), Samanidou-Voyadjoglou and Harbach (2001), Samanidou and Harbach (2003) and Becker et al. (2010). Identification of Cx. tritaeniorhynchus was also performed indicatively, by sequencing method, using BARCODE primers (Folmer et al., 1994) for cytochrome oxydase I (COI) mitochondrial DNA (mtDNA) gene segment. Total DNA was extracted for each insect using the DNeasy Blood and Tissue. Kit (Qiagen) following the manufacturer’s protocol with minor modifications. Fragment of the mtDNA gene segment was amplified by PCR using two sets of primers: the Barcode primers 5-GGTCAACAAATCATAAAGATATTGG-3 and 5-TAAACTTCAGGGTGACCAAAAAATCA-3 (Folmer et al., 1994). The PCR product was purified using the Nucleospin extract II kit (Macherey-Nagel) according to the supplier’s protocol and each sequence was determined via automated sequencing procedure provided by Macrogen Company (Seoul, Korea). The authenticity of the produced mtDNA sequences was verified using Basic Local Alignment Search Tool (BLAST) program of NCBI. 2.4. Data analyses The differences between the total number of the collected immatures of all species and the numbers of the emerged adults of Cx. tritaeniorhynchus during the sampling periods of the three years of the study were determined by the Kruskal–Wallis H-test, followed by Mann–Whitney U-test for pairwise comparisons. The Bonferroni correction was applied to adjust P-values for pairwise comparisons. The analyses were carried out using the statistical package Statistica version 7 (StatSoft Inc., 2004). Furthermore, the spatial distribution of the mosquitoes’ immatures was estimated by calculating the parameters a and b of Taylor’s power law (Taylor, 1961). This law describes the linear regression between variance and sample mean following a logarithmic transformation s = a + b¯x. Thus, s is the variance, the
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I. Lytra, N. Emmanouel / Acta Tropica 134 (2014) 66–71 Table 1 Mean number (±SE) of total mosquitoes’ larvae and pupae and Culex tritaeniorhynchus (adults which emerged from the rearing of the immatures) per five dips, during the three years of the study. Year
Larvae and pupae
Culex tritaeniorhynchus
18.61 ± 1.98a 19.34 ± 2.06a 14.76 ± 2.31b 10.33 2 0.0057
2009 2010 2011 Chi-square d.f. P
9.18 ± 1.15a 9.94 ± 1.08a 6.86 ± 1.18b 13.82 2 0.001
Means of the same column followed by same letter are not differ significantly (Mann–Whitney U-test with Bonferroni correction, adjusted a = 0.017).
The mean number of immatures of all mosquito species was higher in 2009 and 2010 compared with 2011 while no significant differences were observed between the two former years (Table 1). 3.2. Spatial distribution of total number mosquitoes’ immatures The value of the dispersion pattern b of Taylor’s power law was significantly different from 1 for both 2010 and 2011, suggesting an aggregated distribution pattern of the mosquitoes’ immatures, while in 2009 this value did not differ from 1, suggesting a random distribution. Moreover, in all of the cases the y-intercept values were significantly different from 0 and the correlation coefficient values were significantly higher than 0 at the P = 0.05 level (Table 2). 3.3. Species composition and abundance
Fig. 1. Mean number of total mosquitoes’ larvae and pupae and Culex tritaeniorhynchus (adults which emerged from the rearing of the immatures) per sample in each sampling date during the sampling periods of 2009, 2010 and 2011.
y-intercept a is a scaling factor that is dependent on the sampling unit, x¯ is the sample mean and b is the slope of the regression line and constitutes an index of spatial pattern indicating a uniform (b < 1), random (b = 1) or aggregated (b > 1) distribution (Southwood, 1978; Davis, 1994). Moreover, the values of correlation coefficients (r) were calculated as a goodness-of-fit measure of Taylor’s model. The values of a, b and r were tested for departure from 0, 1 and 0 respectively, using the two-tailed t test at n − 2 degrees of freedom and a probability level of P = 0.05 (Snedecor and Cochran, 1980). 3. Results A total of 11,716 immatures of mosquitoes were collected during the three years of the study. Of those, 4095, 4641 and 2980 immatures were collected in 2009, 2010 and 2011, respectively. 3.1. Seasonal occurrence of total immatures The average seasonal trends in terms of the numbers of mosquitoes’ immatures in the rice field during the three years of the study are shown in Fig. 1. All years of the study the immatures’ first appearance and their highest population density were recorded in the middle of June and in early August, respectively. The mosquitoes’ immatures were observed in the field until late September (2009) or early October (2010 and 2011).
Throughout this study, a total of 6363 adult mosquitoes were derived after rearing the immatures, belonging to seven species (An. sacharovi, Anopheles hyrcanus (Pallas), Ae. caspius, Uranotaenia unguiculata Edwards, Cx. pipiens, Cx. theileri, Cx. tritaeniorhynchus). Ur. unguiculata was only collected during the samplings of 2009. Identification of Cx. tritaeniorhynchus was performed indicatively, by sequencing method. The sequencing of the mtDNA gene studied produced an alignment of about 600 bp. The authenticity of the mtDNA sequences produced was verified as Cx. tritaeniorhynchus. The two haplotypes revealed, were differentiating in one nucleotide and have been deposited to GenBank with accession numbers KC753196 (haplotype 1) and KC753197 (haplotype 2). Cx. tritaeniorhynchus was the most abundant species among the mosquito assemblages, representing 85.1%, 93.5% and 96.1% of the total number of adults obtained from the rearing of immatures in 2009, 2010 and 2011, respectively. The relative abundance of the mosquitoes’ species recorded in each year of the study is depicted in Table 3. Despite the recorded mortality of immatures in the laboratory, the high number of identified adults, over a three years survey, gives adequately a representative picture of mosquito species composition providing also useful information about their seasonal presence in the rice field.
Table 2 Parameter estimates for Taylor’s power law for the mosquitoes’ larvae and pupae during the three-year study. Year
n
a ± S.E.
b ± S.E.
r
2009 2010 2011
11 11 10
1.5436 ± 0.2153* 0.6925 ± 0.1144* 0.8626 ± 0.1216*
1.0522 ± 0.1778 1.5218 ± 0.0955** 1.4334 ± 0.1212**
0.8919* 0.9827* 0.9726*
n: sample size, a: y – intercept value for Taylor’s model, b: slope value for Taylor’s model, r: correlation coefficient value for goodness of fit for Taylor’s model. * Values are significantly different from 0 (two tailed t-test: ˛ = 0.05). ** Values are significantly different from 1 (two tailed t-test: ˛ = 0.05).
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Table 3 Relative abundance (%) of mosquitoes’ species (adults which emerged from the rearing of the larvae and pupae) in each year of the study.
Aedes caspius Anopheles hyrcanus Anopheles sacharovi Culex pipiens Culex theileri Culex tritaeniorhynchus Uranotaenia unguiculata
2009
2010
2011
1.0 1.9 5.9 5.3 0.5 85.1 0.3
4.1 0.2 1.1 0.5 0.6 93.5 0.00
1.3 0.3 0.9 1.3 0.1 96.1 0.00
From mid-July until the end of the samplings in all study years (after the samplings of July 12th, July 24th and July 21st in 2009, 2010 and 2011 respectively) Cx. tritaeniorhynchus was the species found at high relative abundance, on the basis of the derived adults of the mosquitoes’ species. From early June to mid-July the identified species found at high relative abundance were Cx. pipiens for 2009 and both Ae. caspius and Cx. theileri for 2010 and 2011. Even though Anopheles species were constantly present during the sampling period of all three years of the study, their population density was always low based on the sample of adults derived after the larvae rearing. The highest population density for these species was recorded at the end of the growing season in 2009 and 2011 and in late June in 2010. Fig. 2 depicts the relative abundance of the derived adult mosquitoes corresponding to each sampling for each year of the study. An. sacharovi and An. hyrcanus have been reported as Anopheles spp. Ur. unguiculata has been omitted from Fig. 2 as its population density was extremely low. 3.4. Seasonal occurrence of Cx. tritaeniorhynchus The first adults of Cx. tritaeniorhynchus were derived from the larvae and pupae collected on June 30th, July 24th and July 10th for 2009, 2010 and 2011 respectively. The presence of this species was observed until the end of the samplings (early October) every year. Its highest population density (based on the derived adults) corresponded to early August in all years of the study. The seasonal population fluctuation of Cx. tritaeniorhynchus during the threeyear study is shown in Fig. 1. The seasonal abundance of this species followed a similar pattern with that of the total mosquitoes’ immatures. The mean number of Cx. tritaeniorhynchus adults derived from the larvae and pupae collected was higher in 2009 and 2010 compared with 2011, while no significant differences were observed between the two former years (Table 1). 4. Discussion Human activities can greatly affect the occurrence of mosquito populations. Our data demonstrate that the seasonal occurrence of mosquitoes’ immatures was affected by the rice cultivation practices. The first appearance of immatures in mid-June was combined with the introduction of the water in the field after the seeding of the rice. It seems that the drying of the field after the rice sprout that happened approximately fifteen days later resulted in the lower numbers recorded in 2009 and the complete absence of immature mosquitoes in 2010 and 2011. The population of immatures showed an increase after the filling of the field with water. The highest population density of mosquitoes was recorded in early August. This is the period following the rice’s sprouting, which favors mosquitoes’ breeding. This may be accounted for by the fact that the water surface is quite exposed to direct sunlight, which produces ideal conditions for the mosquitoes’ immatures development (Mogi, 1978). Indeed, it has been reported that immature mosquitoes can develop very rapidly at higher water temperature
Fig. 2. Relative abundance (%) of mosquitoes’ species (adults which emerged from the rearing of the larvae and pupae) on each sampling date during the sampling periods of 2009, 2010 and 2011.
(Mogi, 1984). On the contrary, the high density of rice plants renders the access of gravid females to the oviposition sites more difficult (Mogi, 1984). This might be the reason for the lower population of mosquitoes’ immatures recorded in the field from mid-August till the end of the study period. Kant et al. (1992) also mentioned that the negative correlation between larval densities and the growth of the paddy plant could be due to mechanical obstruction in the oviposition of some species, the low-intensity light and the lowered water level in the later stages of the paddy growth. In our study a remarkably lower population of immatures was found between the 3rd and the 13th of August 2011, caused by the lack of water supply due to water management problems. Mogi (1993) found that, in areas with developed irrigation systems and sufficient water, intermittent irrigation may be managed to control vector mosquito populations. The use of intermittent irrigation as an effective method for the control of vector mosquitoes in rice fields has also been recommended by the WHO (1982). The spatial distribution of insects reflects the behavior of individuals and can exhibit three forms: random, aggregated and uniform (Davis, 1994). Aggregated distributions of mosquitoes’ immatures are extremely common in field studies (Service, 1985; Walker et al., 1988; Pitcairn et al., 1994). According to Taylor’s law, the mosquitoes’ immatures found during 2010 and 2011 followed an aggregated spatial distribution. However, during 2009 the
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immatures were distributed randomly among the sampling units, a fact that could be a consequence of their lower occurrence in terms of numbers. The spatial heterogeneity in the distribution of the mosquitoes’ immatures may often arise from habitat heterogeneity, as well as from the propensity of younger instars to congregate with one another (Renshaw et al., 1995). This is the first time the species Cx. tritaeniorhynchus is recorded in western Greece. Moreover, it is the first time that a high population of this mosquito species is recorded anywhere in Greece, as the data of a previous report by Samanidou and Harbach (2003) concern only a few specimens. Furthermore, the presence of this species has been reported in Albania (Danielová and Adhami, 1960; Adhami, 1997). Cx. tritaeniorhynchus is a common species across southern Asia, the Middle East and the Afrotropical Region (Harbach, 1988; Samanidou and Harbach, 2003; Becker et al., 2010). Since surveillance data for the mosquito fauna in Greece are scarce, it is assumed that this species may have been present in the country, though undetected, before 2003. The seasonal and inter-annual variation of Cx. tritaeniorhynchus adults may be related to culturing practices as well as to the density of the rice plants. This is further supported by Mogi’s findings (1984) who mentioned that the active population growth of Cx. tritaeniorhynchus in Japan occurs during a limited period of time, following transplanting, when the irrigated area is greater and most stable and rice plants are short and sparse. Furthermore, Kant et al. (1992) found Cx. tritaeniorhynchus breeding at the later stages of plant growth whereas Reuben (1971) reported that the abundance patterns of this species gradual increase with rice plant growth. Moreover, Takagi et al. (1995) reported that the alternating flooding and draining is an effective method for suppressing the production of Cx. tritaeniorhynchus adults. On the contrary, Heathcote (1970) observed that the population of Cx. tritaeniorhynchus peaks in newly planted or harvested fields, whereas Somboon et al. (1989) recorded that the highest population peak of this species was observed in ploughed fields, followed by a rapid population decrease after transplanting. Finally, according to Mogi and Miyagi (1990) Cx. tritaeniorhynchus may be relatively adaptable to changing rice field phases. In the present study, Cx. tritaeniorhynchus was found to be the predominant species, developing much higher population densities compared to the other recorded mosquito species. Similar results regarding the population densities of Cx. tritaeniorhynchus in rice fields have also been recorded by Mogi (1978) and Lee (1998) (6.28 and 1.56 larvae per dipper, respectively). However, our finding is in contrast to other reports of predominant mosquito species in rice fields of southern Europe (Bellini et al., 1994; Almeida et al., 2010; Chaskopoulou et al., 2011). Bellini et al. (1994) reported that Ae. caspius attained the highest population density during the initial stages of rice crop growth at rice fields in Italy. During this period, the water management practices made the conditions favorable for the oviposition of this species. Moreover, in the same study, the highest density of Cx. pipiens was recorded during the last ten days of June whereas afterwards the population declined progressively until the end of August when the population density of Anopheles species was always low. Our results are in accordance with the former study about the seasonal abundances of the three mosquitoes’ species. Most of the mosquitoes’ species found during the present study are vectors responsible for the transmission of parasitic and viral infections to millions of people worldwide. The species An. sacharovi and Cx. pipiens are also involved in disease outbreaks and epidemics in Greece in recent years (Gomes et al., 2013; Kousoulis et al., 2013). Furthermore, most of these species are economically important mosquitoes that adversely affect tourism and related business interests and have a negative impact on livestock and poultry production (Becker et al., 2010).
Taking into account that Cx. tritaeniorhynchus demonstrated a high population density in the studied rice field and given that this species is the primary vector of JE, the present work shows that the study area is under the potential threat of the emergence of JE. The establishment of Japanese encephalitis virus (JEV) in new ecosystems outside of its current range is difficult (van den Hurk et al., 2009). With the spread of JEV into much of the Indian subcontinent, however, other destinations served by frequent routes of commerce or passenger air travel, such as Africa and Europe, could also be at risk (Weaver and Reisen, 2010). 5. Conclusions The study of the mosquito fauna composition over time in a Greek rice field revealed high population densities of Cx. tritaeniorhynchus. The mosquito sampling findings in combination with the proximity of rice cultivation to the Delta of the Acheloos River and the presence of livestock (pigs and cows) in that rural region render the implementation of effective control measures to minimize vector abundance and potential disease outbreak in this area imperative. Further investigation is required to study the population status of the Cx. tritaeniorhynchus in other regions of Greece, as well as its bionomics in an area far from its origin. Moreover, the risk of emergence of Cx. tritaeniorhynchus related diseases in Greece and Europe should be studied thoroughly. Acknowledgments The authors would like to thank Dr. M. Bouga and V. Evangelou (Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens) for providing the molecular data for the confirmation of the identification of Cx. tritaeniorhynchus specimens. Suggestions and critical comments on the manuscript by Dr. A. Fantinou (Associate Professor in Agricultural University of Athens) and Dr. G. Koliopoulos (Benaki Phytopathological Institute) are also kindly acknowledged. We thank Dr. H. Balfoussia and Dr. A. Martinou for providing valuable language help. Acknowledgments are also extended to the agronomists N. Rizos and S. Antonatos for the fieldwork support and I. Potirakis for the laboratory assistance. This research has been co-financed by the European Union (European Social Fund – ESF) and Greek National funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) – Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica. 2014.02.018. These data include Google maps of the most important areas described in this article. References Adhami, J., 1997. Mushkonjat (Diptera: Culicidae) te Shqiperise, tribu Culicini. Revista Mjekesore (1–2), 82–95 (in Albanian). Almeida, A.P.G., Freitas, F.B., Novo, M.T., Sousa, C.A., Rodrigues, J.C., Alves, R., Esteves, A., 2010. Mosquito surveys and West Nile Virus screening in two different areas of Southern Portugal, 2004–2007. Vector Borne Zoonotic Dis. 10, 673–680. ´ D., Zgomba, M., Boase, C., Madon, M., Dahl, C., Kaiser, A., 2010. Becker, N., Petric, Mosquitoes and their Control. Springer-Verlag, Heidelberg, Berlin, 577 pp. Bellini, R., Veronesi, R., Rizzoli, M., 1994. Efficacy of various fish species (Carassius auratus [L.], Cyprinus carpio [L.], Gambusia affinis [Baird and Girard)] in the control of rice field mosquitoes in northern Italy. Bull. Soc. Vector Ecol. 19, 87–99. Bram, R., 1967. Contributions to the mosquito fauna of Southeast Asia. II. The genus Culex in Thailand (Diptera: Culicidae). Contr. Am. Ent. Inst. 2, 1–296. Broom, A., Smith, D., Hall, R., Johansen, C., Mackenzie, J., 2003. Manson’s Tropical Diseases. Saunders, London, pp. 1864pp.
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