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Molecular identification of Aedes phoeniciae (Diptera: Culicidae) in rockpools along the northern Israeli coast

2018; Wiley; Volume: 43; Issue: 2 Linguagem: Inglês

10.1111/jvec.12321

1948-7134

Sahar Rosenfeld, Daniele Porretta, Eyal Rahav, Valentina Mastrantonio, Claire Duchet, Leon Blaustein,

Identification and Quantification in Food

Mosquitoes inhabiting rockpools within supra-littoral zones along the Mediterranean coast belong to the Aedes mariae complex (Coluzzi and Sabatini 1968, Coluzzi et al. 1974b, Schaffner et al. 2001, Mastrantonio et al. 2015). The species in the Ae. mariae complex can be responsible for vectoring bird malaria as vectors of Plasmodium relictum (Clements 1999). Furthermore, they are a biting nuisance for humans in areas densely populated by tourists from spring through autumn. Initially described as a unique species, Aedes (Acartomya) mariae (Sergent and Sergent 1903) has been described as the occurrence of three taxa based on slight morphological differences and the polymorphism at the allozymic locus phosphoglucomutase (Pgm, EC 5.4.2.2) (Coluzzi and Sabatini 1968), which includes Aedes (Acartomyia) mariae, Aedes (Acartomyia) zammitii, and Aedes (Acartomyia) phoeniciae (Coluzzi and Sabatini 1968). Their distribution was suggested by Coluzzi and Sabatini (1968) to be contiguous around the Mediterranean coasts with no naturally sympatric areas. The distribution of Ae. mariae is confined to the western Mediterranean, from the coast of Portugal to Italy, Sicily, Algeria, Tunisia, and Morocco; Ae. zammitii is distributed along the central and eastern Mediterranean and Adriatic coasts of Italy, eastern Sicily, Malta, Greece, and the Aegean coast of Turkey; and Ae. phoeniciae is reported to inhabit the eastern coasts of Turkey, Syria, Lebanon, Israel, and Cyprus (Coluzzi and Sabatini 1968, Coluzzi et al. 1974, Mastrantonio et al. 2015). As these species are almost identical morphologically, it is very difficult to study their spatial distribution using microscopic-based taxonomic approaches alone and thus complimentary genetic methods are needed. In the last few years, a number of studies confirmed the identification and distribution of the species of the Ae. mariae complex using molecular markers (Urbanelli et al. 2014, Mastrantonio et al. 2015, Yavasoglu et al. 2016a, Yavasoglu et al. 2016b). Using both nuclear and mitochondrial DNA markers (14 allozymic loci and cytochrome oxidase I and cytochrome oxidase II gene fragments, respectively), Mastrantonio et al. (2015) analyzed 12 populations of the complex, including five populations of Ae. mariae (one from Spain and four from Italy), five populations of Ae. zammitii (one from Italy, one from Crete and two from Turkey) and two populations from Ae. phoeniciae from Cyprus. The occurrence of Ae. mariae in the Balearic Islands was confirmed by Bueno-Marí and Jiménez-Peydró (2011) using mitochondrial cytochrome oxidase gene. The identification and distribution of Ae. zammitii has been confirmed through an extensive sampling along the Aegean coasts of Turkey using mitochondrial NADH dehydrogenase 4 (ND4) gene (Yavasoglu et al. 2016a, Yavasoglu et al. 2016b). The molecular identification of Ae. phoeniciae is limited to populations along the coasts of the Turkey and Cyprus (Mastrantonio et al. 2015, Yavasoglu et al. 2016b). In Israel, Ae. phoeniciae are quite common. The only record that exists is that of the original paper from Coluzzi and Sabatini (1968) who identified larvae from a single location in Israel as Ae. phoeniciae. Contrarily, other reports that were based on morphological identification of adults and larvae suggested that Ae. (Ochlerotatus) caspius and Ae. mariae are found along the Israeli coast (Margalit and Tahori 1970, Kitron and Pener 1986). In this study, we used nuclear allozymic genetic markers to verify which species of the Ae. mariae complex are found along the Israeli Mediterranean Sea coast. To this end, we collected mosquito specimens from five localities along the Israeli coastline: Ackziv, Tel-Shikmona, Neve-Yam, HaBonim, and Nahsholim (Figure 1) during November, 2015 and April, 2016. The individuals were collected as larvae from the supra-littoral rockpools and brought to the laboratory where a subset was reared to adults. Collected samples were identified morphologically using the identification key of Schaffner et al. (2001). In parallel, using standard horizontal starch gel on single individuals following the protocols described by Mastrantonio et al. (2015), 70 individuals (14 individuals per location) were analyzed for the allozymic loci. The loci adenylate kinase (Adk, EC 2.7.4.03), hydroxybutyrate dehydrogenase (Hbdh, EC 1.1.1.30), superoxide dismutase (Sod-1, EC 1.15.1.1), carbonic anhydrase (Ca-1, EC 4.2.1.1), and phosphoglucomutase (Pgm, EC 5.4.2.2) were used to assess whether the sampled mosquitoes belonged to Ae. phoeniciae or to other species within the Ae. mariae complex (Table 1 and Mastrantonio et al. 2015). The GENEPOP version 3.1 (Raymond and Rousset 1995) was used to estimate allele frequencies and deviations from the Hardy-Weinberg equilibrium. Locus Alleles Aedes mariae Aedes zammitii Aedes phoeniciae Adk 90, 100, 105, 112 90, 100, 105 92, 96 Hbdh 85, 90, 100, 110, 115 90, 100, 105 107 Sod-1 100 100 85 Ca-1 96, 100 88, 92, 102 94 Pgm 100, 106, 110 90, 92, 96 98, 106, 114 Our results demonstrated that all the individuals harbored the alleles characteristic of Ae. phoeniciae (Exact Hardy-Weinberg test: p<0.05), which allowed us to discriminate Ae. phoeniciae from Ae. mariae, Ae. zammitii, and Ae. caspius (Porretta et al. 2007). In particular, at the locus Adk, the allele Adk92 was found in the individuals analyzed as expected for Ae. phoeniciae; at the locus Hbdh, only the allele Hbdh107 was found, as well as at the loci Sod-1 and Ca-1, where only the alleles Sod-185 and Ca-194 were found, respectively. Finally, at the locus Pgm, the alleles Pgm106 and Pgm114 were found, both discriminative of Ae. phoeniciae (Table 2) (Mastrantonio et al. 2015). Locus Allele Populations Ackziv Tel-Shikmona Neve-Yam HaBonim Nahsholim Adk 92 1.0 1.0 1.0 1.0 1.0 Hbdh 107 1.0 1.0 1.0 1.0 1.0 Sod-1 85 1.0 1.0 1.0 1.0 1.0 Ca-1 94 1.0 1.0 1.0 1.0 1.0 Pgm 106 0.95 0.97 0.98 0.95 1.0 114 0.05 0.03 0.02 0.05 0.0 While other species such as Ae. caspius might also utilize these rockpools (Margalit and Tahori 1970, Kitron and Pener 1986), all 70 individuals along the five Israeli coastline sites were identified as Ae. phoeniciae. These results contribute to a more comprehensive picture of the spatial distribution map of the Ae. mariae complex along the Mediterranean coast, highlighting again the non-sympatric nature of the species belonging to this complex. The eastern Mediterranean coast is the hottest and most saline area along the Mediterranean basin, exhibiting high daily, seasonal, and interannual amplitudes (Rilov 2016). This may suggest that Ae. phoeniciae is better adapted to changes in temperature and salinity. These findings are in agreement with Sergent and Sergent (1903) who found Ae. mariae larvae in salinities of up to 200 parts per thousand (ppt), while we observed Ae. phoeniciae larvae in rockpools with 280 ppt (not shown). We stress that more genetic identification should be undertaken in order to shed light on the differences between sub-populations and species along with ecological research that may help in understanding the local and global distribution of the Ae. mariae complex. Furthermore, a clear understanding of the distribution and species composition of the complex is essential for control programs and epidemiological studies, especially in warm areas such as the Israeli coast where Ae. phoeniciae individuals prevail. Our current study contributes to the identification of the complex on the Israeli Mediterranean Sea coast. We are most grateful to Valentina Rovelli, Luca Zoccarato, and Martina Mulas for their help. This project was funded by Israel Science Foundation grant 891/2012 awarded to Leon Blaustein. This study is funded partially by an MSc thesis fellowship for Sahar Rosenfeld from Haifa University.